Physiology of Mycobacteria (2024)

The publisher's final edited version of this article is available at Adv Microb Physiol

Mycobacterium tuberculosis is a prototrophic, metabolically flexible bacterium that has achieved a spread in the human population that is unmatched by any other bacterial pathogen. The success of M. tuberculosis as a pathogen can be attributed to its extraordinary stealth and capacity to adapt to environmental changes throughout the course of infection. These changes include: nutrient deprivation, hypoxia, various exogenous stress conditions and, in the case of the pathogenic species, the intraphagosomal environment. Knowledge of the physiology of M. tuberculosis during this process has been limited by the slow growth of the bacterium in the laboratory and other technical problems such as cell aggregation. Advances in genomics and molecular methods to analyse the M. tuberculosis genome have revealed that adaptive changes are mediated by complex regulatory networks and signals, resulting in temporal gene expression coupled to metabolic and energetic changes. An important goal for bacterial physiologists will be to elucidate the physiology of M. tuberculosis during the transition between the diverse conditions encountered by M. tuberculosis. This review covers the growth of the mycobacterial cell and how environmental stimuli are sensed by this bacterium. Adaptation to different environments is described from the viewpoint of nutrient acquisition, energy generation and regulation. To gain quantitative understanding of mycobacterial physiology will require a systems biology approach and recent efforts in this area are discussed.

“It is now 100 years since the first mycobacterium was isolated by Hansen (1874). Somewhat ironically, this was the leprosy bacillus, Mycobacterium leprae, which even today is still resisting all attempts to cultivate it in the laboratory. The tubercle bacillus, M. tuberculosis was not discovered until eight years later (Koch, 1882) and this has remained an object of intensive investigation ever since. The widespread interest in the mycobacteria of course stems from the diseases they cause and, lest it be imagined that tuberculosis is a disease which has now been largely conquered and that leprosy is of relatively rare occurrence, current estimates for the number of case of tuberculosis and leprosy in the world today are 20,000,000 and 11,000,000, respectively (Bechelli and Dominguez, 1972). The annual estimated mortality rate is equally dramatic, namely 3,000,000 (World Health Organization, 1974). Also causing unease is the continuing isolation from tubercular patients of strains already resistant to one or more chemotherapeutic agent”.

C. Ratledge (1976).

2.1. Mycobacterium and its close relatives

Mycobacteria are Gram-positive, acid-fast and (G + C) rich (62 – 70%); they are aerobic and rod shaped and mycolic acids are components of their cell walls (for review see Wayne and Kubica, 1986). Other bacteria including Nocardia, Rhodococcus and Corynebacterium have related properties (see Table 1). The genera Corynebacterium, Nocardia and Mycobacterium form a well defined sub -group (CMN) of actinobacteria (Embley and Stackebrandt, 1994; Ventura et al., 2007). Other genera that synthesize mycolic acids include Dietza, Gordona. Skermania, Tsukamurella, Turicella and Williamsia (Gurtler et al., 2004).

2.2. Mycobacterium

The genus Mycobacterium is now known to comprise more than one hundred species which are all closely related as judged by comparison of their 16S rRNA sequences (Tortoli, 2006). This rapid expansion of the genus since 1990 is based on the invention of the Polymerase Chain Reaction (PCR) for the amplification of DNA sequences (Saiki et al., 1985) and the recognition of the value of 16S rRNA sequences as phylogenetic markers (Woese, 1987). These methods were first applied to mycobacteria less than twenty years ago (Stahl and Urbance, 1990; Rogall et al., 1990a; 1990b). Since then, mycobacteria have been found to be widely distributed in the environment. Mycobacteria are opportunistic pathogens, especially in a clinical environment, and their detection is important to treatment of the infections they cause. Mycobacteria also have possible industrial appplications. M. austroafricanuum is able to metabolize methyl tert-butyl ether which is often added to gasoline and could be used to treat contaminated ground water (Maciel et al., 2008).

The earliest studies of mycobacterial genes concerned the rRNA operons (Cox and Katoch, 1986; Bercovier et al., 1986). After a little more than a decade later, the genome sequences of M. tuberculosis H37Rv (Cole et al., 1998) and M. leprae (Cole et al., 2001) were published. Knowledge of these sequences has facilitated very rapid progress in mycobacterial research. The number of published genome sequences has now reached seventeen.

3.1. Population-average cells

Measurements based on cell cultures provide information about population-average cells (Schaechter et al., 1958) and the picture that emerges is that of a virtual cell. A bacterial culture comprises cells of all ages ranging from new born cells of age a = 0 to cells about to divide (age a = 1). The age range of cells comprising a culture remains constant as the number of cells increase (Powell, 1956) during both exponential growth in batch culture and growth in continuous culture (a chemostat). Thus, the virtual cell is representative of the entire age range. DNA, RNA and protein fractions isolated from cell cultures provide information about population-average cells. Suppose, that D, R, P and N_c refer to the mass of DNA, RNA and protein respectively per ml of culture and N_c is the number of cells per ml of culture then m_DNA(av), m_RNA(av) and m_p(av) which are respectively the masses of DNA, RNA and protein per population-average cell are defined in equation (1) – (3).

Furthermore, the synthesis rates such as ω_p(av) the specific protein synthesis rate [see equation (4)].

and ω_RNA(av) the specific RNA synthesis rate [see equation (5)]

describe the population-average cell.

A schematic view of a population-average cell of M. tuberculosis was devised to provide an instantaneous view of the macromolecular composition (m_DNA(av), m_RNA(av) and m_p(av)) and of protein synthesis and other features (Cox and Cook, 2007).

3.2. Cell size and shape

Mycobacterial cells are irregular rods 0.3–0.5 μm in diameter and of variable length (Wayne and Kubica, 1986). The images of cells of M. smegm*tis shown in Fig. 1 show that the increase in cell size is reflected in an increase in length rather than the diameter of the rods (compare left and right panels). For example, any changes in the diameter are small whereas length ranges from 1.5 μm–4.0 μm according to the period of culture. This mode of cell growth provides the cell with a high ratio of surface area to mass, which is favourable for cells that are relatively impervious to aqueous solvents; that is, the number of porin channels per unit surface area is low compared with E. coli. It has been shown that nutrients are taken up by the cell by passive diffusion through the permeability barrier and then actively transported across the cytoplasmic membrane (Nikaido and Rosenberg, 1981; Stephan et al., 2005; please see section 8). Hence, in vitro the rate of passive diffusion can be increased by increasing the concentration of nutrients in the growth medium and hence allow the optimal growth rate to be attained. It is not known if this situation (occasions of ‘feast’) is often encountered in the environment.

3.3. Macromolecular composition

Measurements of amounts of DNA, RNA and proteins per population-average cell at ‘defined’ specific growth rates provide valuable insights into cell metabolism. Ninety per cent of the energy needed for macromolecular synthesis is devoted to protein synthesis, which accounts for approximately one half of the energy needed for cell growth. The incorporation of each amino acid into a polypeptide chain is achieved at the expense of four high-energy phosphate bonds (for review see Cox and Arnstein, 2007). Cell growth requires protein synthesis, which, in turn requires ribosome synthesis. These aspects of cell proliferation are reflected in the ratios DNA:RNA : protein. However, few data are available for mycobacteria largely because of the difficulties encountered in lysing cells. Available data for M. tuberculosis H37Rv and M. bovis BCG are summarized in Table 2. There is little agreement between the three sets of data. The observed differences are probably too large to be accounted for by differences in growth conditions. Technical difficulties are more likely to be responsible for the wide range of reported values. For example, Youmans and Youmans (1968) used the orcinal method to measure RNA content and this reagent also reacts with polysaccharides. Beste et al. (2005) used different methods to lyse cells for measurements of DNA, RNA and protein and failed to use a secondary standard to measure the extents of cell lysis. The data reported by Winder and Rooney (1970) was shown to be consistent with an equation derived by Bremer (1975) relating the growth rate with parameters affecting the rates of synthesis of ribosomes and RNA polymerase in bacteria (Colston and Cox, 1999). Thus, there is a dearth of information about the DNA, RNA and protein contents of mycobacteria.

3.4. Does genome size influence the specific growth rate?

The genome sizes of mycobacterial species obtained by DNA sequencing range from 3,268,203 base-pairs (M. leprae) to 6,988,209 base-pairs (M. smegm*tis mc ²155). Optimum specific growth rates have been reported for M. tuberculosis H37Rv (Wayne, 1994), M. marinum (Clark and Shepard, 1963) and M. smegm*tis mc ²155 (Sander et al., 1996). The range in genome sizes poses the question; ‘How can specific growth rates be related to genome sizes?’ This problem was approached in the following way. E. coli was chosen as the reference species (The Eco_model) because it is widely studied and an extensive data base is available (see Bremer and Dennis, 1996). The properties of E. coli B/r grown at 37°C in a minimal salts medium with succinate as the carbon source were used as its reference state because, under these conditions of growth, the genome is replicated once only during the cell division cycle (Bremer and Dennis, 1996). Previously, it was found that equations developed to relate the specific growth rates of E. coli B/r with its macromolecular composition (Bremer, 1975) also applied to M. bovis BCG (Colston and Cox, 1999). Quantitative relationships relating specific growth rates and macromolecular composition of M. tuberculosis, Streptomyces coelicolor A3(2) and E. coli B/r were used to develop an instantaneous view of macromolecular synthesis (Cox, 2004). Thus, E. coli grown in succinate medium is a suitable model for the other strains considered in this review; namely mycobacteria, corynebacteria and streptomyces, which also replicate their genomes once only during the growth cycle. The model is based on a generation time t_D of 1.67 h, an interval of t_G1= 0.05 h between cell division and the start of DNA replication (S-phase) which continues for τ_g= 1.17 h (the genome is replicated at a rate of ε_DNA= 2.08 × 10⁶ base -pairs h⁻¹ per replication fork); S-phase is followed by the period t_G2= 0.5 h before cell division takes place. It is supposed that if the species under scrutiny were as metabolically active as E. coli B/r growing in succinate medium at 37°C then t_G1, t_G2 and ε_DNA would have the same values as the E. coli B/r model. However, in this model τ_g the period of DNA synthesis is directly proportional to l_g base-pairs, the genome size of the species of interest. The generation time t_D* of the Eco_model is given by equation (6) where E. coli values are shown by the superscript # sign.

Hence, μ* the specific growth rate of the Eco_model may be evaluated. The Eco_index, which is the ratio of the observed and Eco_model derived specific growth rates, provides a measure of the relative metabolic activities of the subject species growing optimally in an appropriate medium and E. coli B/r growing in succinate medium at 37°C.

The model was applied to three mycobacterial species, which have well characterized optimal specific growth rates, and two other species, namely Streptomyces coelicolor A3(2) and Corynebacterium glutamicum ATCC 13032 that have genomes of 8,667,507 and 3,309,401 base-pairs, respectively. The results are shown in Table 3. The Eco_index was found to vary from 0.1 (M. tuberculosis H37Rv) to 1.15 (S. coelicolor A3(2)). Three species grow optimally at 30°C; namely M. marinum strain M, C. glutamicum, and S. coelicolor A3(2). No correction was applied to allow for growth at different temperatures. The results indicate that both M. smegm*tis mc²155 and M. marinum strain M have specific growth rates that are close (74% and 80%, respectively) to the value predicted for the Eco_model. The model for M. smegm*tis mc²155 is based on a DNA replication rate of ε_DNA= 2.08 × 10⁶ base -pairs h⁻¹ per replication fork. The value of ε_DNA= 2.0 × 10⁶ base-pairs h⁻¹ per replication fork can be deduced from an observed S-phase of 1.75 h (Hiriyanna and Ramakrishnan, 1986), which is very close to the rate observed for E. coli B/r grown in succinate medium.

In contrast, the Eco_index of 0.1 derived for M. tuberculosis H37Rv indicates a metabolic activity of 10% of the E. coli B/r model. This expectation is accord with the observed value of ε_DNA = 0.21 × 10⁶ base -pairs h⁻¹ per replication fork deduced from the reported S-phase of 10.33 h (Hiriyanna and Ramakrishnan, 1986), which is close to one tenth of the rate observed for E. coli B/r. To a first approximation, the Eco_index reflects the relative rates of DNA replication, which provides a guide to the relative metabolic activities of the species concerned. If an allowance was made for growth at 30°C, then the Eco_index of M. marinum, C. glutamicum, and S. coelicolor A3(2) would be closer to or even exceed 1.0.

3.5. Mycobacterial rrn operons

Classical pathogenic slow growers such as M. tuberculosis were shown to have a single rrn operon per genome whereas classical fast growers such as M. smegm*tis and M. phlei were found to have two (Bercovier et al., 1986; Domenech et al., 1994). The generalization that mycobacteria have either one or two rrn operons per genome has proved to be correct. All the mycobacteria studied to date were found to have one operon (rrnA) located downstream from murA (Gonzalez-y-Merchand et al., 1996, 1997; Helguera-Reppetto et al., 2004; Rivera-Guttiérez et al., 2003): the second operon (rrnB), if present, was found downstream from tyrS (Gonzalez -y-Merchand et al., 1996; Menendez et al., 2002; Predich et al., 1995). A phylogenetic tree based on 16S rRNA sequences was related to the numbers of rrn operons per genome. The combined data supports the view that the ancestral Mycobacterium possessed one rrnA and one rrnB operon and that on two separate occasions an rrnB operon was lost giving rise to two clusters of mycobacteria possessing a single rrn operon (rrnA) per genome (Stadthagen-Gomez et al., 2008). One cluster includes the classical pathogens M. leprae and M. tuberculosis and the other includes M. abscessus and M. chelonae.

4.1. Replication of individual ORFs

The replication of a particular ORF occupies only a small fraction of S-phase. An ORF (ORF₍₀₎) located close to oriC is replicated at the beginning of S-phase when two copies become available for the rest of the cell’s lifetime. In principle it can benefit the cell if an ORF, whose gene product is in high demand, is located near to oriC. Then the newly replicated ORF becomes available at the earliest opportunity; namely the end of G1-phase. In contrast, an ORF (ORF_(t)) located near to ter provides the cell with a newly replicated copy for no longer than the G2-period.

The time during S-phase when ORF_(i) is replicated depends on the position of ORF_(i) within the genome. The positions of rrn operons are variable and the replication coefficient facilitates comparisons between mycobacteria. The replication coefficient γ is defined as the shortest distance (base pairs) of an ORF_(i) from oriC measured either clockwise or counter clockwise divided by one half the length (base-pairs) of the genome. The replication coefficients of mycobacterial rrn operons are greater than 0.5 revealing that they are replicated in the second half of S-phase. In contrast, the replication coefficients of the ten rrn operons of Bacillus subtilis subsp subtilis are close to zero showing they are replicated very early in S-phase (Kunst et al., 1997). The replication coefficient γ_ORF(i) is related to the time τ_ORF(i) when ORF_(i) is replicated in S-phase. In turn, τ_ORF(i) plus the G1 period is equal to the time t_ORF(i) during the cell division cycle when ORF_(i)is replicated. The age a_(i)•R of a cell when ORF_(i) is replicated is obtained by dividing t_ORF(i) by t_D the generation time Once a_{(i) •R} is known the number of copies of ORF_(i) per population-average cell can be calculated (see Appendix A). The limited data available for M. tuberculosis H37Rv shows that the rrnA operon (γ = 0.67) is replicated in the second half of S-phase; leading to 1.36 copies of rrnA per population -average cell. In contrast, ORF₍₀ located close to oriC is present as 1.72 copies per population-average cell, whereas there are 1.20 copies of ORF_(t) located near to ter.

The available data for M. smegm*tis mc²155 show that this bacterium can grow at its optimum rate with a single rrnB operon; this operon, which has a replication coefficient of 0.90, is replicated late in S-phase. Hence, this mutant has 1.18 copies of rrnB per population -average cell. Thus, a single rrn operon that is replicated late in S-phase is sufficient to support mycobacterial growth of 3 h generation time, a rate that is 74% of the Eco_model (see Table 4). Moreover, when the M. tuberculosis rrnA operon was expressed in M. smegm*tis it was competitive with the rrnA and rrnB operons of the host (Verma et al., 1999). By analogy, it is safe to conclude that the single rrnA operon of M. tuberculosis, which is present at 1.36 copies per population-average cell (see above) is also sufficient to support a generation time of 3 h. In other words, the possession of a single rrn operon per genome is not related to the slow growth of the tubercle bacillus. This view is reinforced by the demonstration that E. coli can grow with a specific growth rate of μ = 0.6 h ⁻¹ even after inactivation of six of its seven rrn operons or even after inactivation of all seven operons with a functional operon provided by a plasmid (Asai et al., 1999). Fluorescence spectroscopy revealed that deletion of six or seven rrn operons changed the size and shape of the cells from short and fat to long and thin like mycobacteria.

5.1. Components of mycobacterial ribosomes

The RNA (rRNA) moiety of ribosomes is encoded by classical rRNA (rrn) operons in the order 5′ 16S rRNA, 23S rRNA and 5S rRNA 3′ (Suzuki et al., 1988; Liesack et al., 1990, 1991; Kempsell et al., 1992). The information available for ribosomal proteins (r-proteins) is largely derived from genomic sequences. For example, r-proteins of M. tuberculosis are orthologues of E. coli r-proteins. However, four r-proteins: namely, S14, S18, L28 and L33 (see Table 5) are each encoded by two non identical genes. Not all of the available mycobacterial genomic sequences were found to have these extra genes. When present, the four genes were located as a group encoding r-proteins in the order 5′ L28, L33, S14, S18 3′. This cluster of genes is absent from M. leprae, Mycobacterium species JLS, Mycobacterium species KMS and Mycobacterium species MCS. The extra set of genes encode four proteins which have different amino acid sequences from their standard orthologues; for example, two proteins with non-identical amino acid sequences are identified as orthologues of E. coli L28. The non-identical orthologues of each of the four r-proteins raises the question of the hom*ogeneity of the ribosome fraction. If ribosomes are heterogeneous with respect to their complement of r-proteins is ribosome function affected? Microarray measurements reveal that the additional set of r-proteins are regulated by growth rate in a manner that differs from that of the other r-proteins. Genes encoding r-proteins are usually down regulated as the specific growth rate is decreased. In contrast, genes encoding the additional proteins are upregulated, as shown in Table 6 for S14 of M. tuberculosis grown in batch culture. A comparable result was obtained when M. bovis BCG was grown in continuous culture. A threefold change (from one day to three days) in the specific growth rate led to expression ratios of 0.61 for rps N1 and 6.0 for rps N2 (calculated from supplementary data of Beste et al., 2007b).

5.2. Growth rate control of ribosome synthesis

Except at very low growth rates, the number of ribosomes per cell is dependent on growth rate (growth rate control of ribosome synthesis). This conclusion is based on the finding that the RNA content of cells varies with growth rate (Schaechter et al., 1958; Verma et al., 1999; Paul et al., 2004b). As expected, the r-protein content is also dependent on growth rate. The results of microarray experiments obtained for M. tuberculosis and its close relative M. bovis BCG are presented in Fig. 2. Panel (a) provides an essential control in which the two labelled cDNA were prepared from the same RNA fraction isolated from mid-exponential cells of M. tuberculosis and the distribution of the expression ratios of the standard 50 r-proteins was examined. The mean value of r₍₅₀₎= 1.00 ± 0.10 was obtained, which is a measure of the accuracy of the expression ratios. The second panel refers to cDNA prepared from mutant (dosR minus) and wild type M. tuberculosis; both mutant and wild type were found to grow at the same rate so that the ribosome contents of the two strains would be expected to be identical. In this experiment the mean value of the expression ratios of the standard set of r-proteins was found to be r₍₅₀₎= 0.93 ± 0.16 which is similar to the control shown in panel (a). The third panel refers to M. bovis BCG grown in continuous culture; the specific growth rates of reference and experimental strains were 0.03 h⁻¹ and 0.01 h ⁻¹, respectively. In this experiment, the expression ratios were found to have a mean value of r₍₅₀₎=0.71 ± 0.01, which indicates a lower level of r -protein mRNAs per μg cDNA in the slower growing cells and therefore a lower level of r-proteins (Cox, 2007). This conclusion is reinforced by the comparison shown in Fig. 3, which shows both the expression profiles of all 3475 ORFs investigated and the ORFs encoding the set of 50 r-proteins. The group of 50 r-protein genes form a subset that is downregulated.

6.1. Organization; transcription of rrn operons

The organization of the two rrn operons of mycobacteria are shown in Fig. 4. The rrnA is present in all mycobacteria studied thus far. The characteristic features of rrnA include the numbers and locations of its promoters (Verma et al., 1994; Gonzalez-y-Merchand et al., 1997). This operon has a minimum of two promoters; one promoter (P1) is located within the coding region of the murA gene near to its 3′-end and the other (PCL1) is located near to a conserved sequence motif (CL1). Up to three additional promoters have been found, separated by 80–100 base-pairs, in some species; the rrnA operon of M. smegm*tis has three promoters (P1, P2 and PCL1), M. fortuitum has four promoters (P1, P2, P3 and PCL1) and M. chelonae and M. abscessus each have five (P1, P2, P3, P4 and PCL1). Moreover, transcripts of murA continue their progress to transcribe the rrn operon (Gonzalez-y-Merchand et al., 1999). The contributions of individual promoters to precursor-rRNA synthesis was found to vary according to growth rate (Gonzalez-y-Merchand et al., 1998; Verma et al., 1999). In brief, a single promoter tends to be dominant at high growth rates.

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Figure 4

Organization of the rrnA and rrnB operons of mycobacteria

The operons are defined by the identities of their upstream genes. (a) tsp, transcription starting point; CL1, Conserved Leader sequence 1; HMPR, Hypervariable Mature Promoter Region. The number of promoters in this region ranges from 1 –4, each promoter is separated by 80 –100 base pairs; diagonal hatching, 16S rRNA coding region; ITS1, Internal Transcribed Spacer region 1; V2 region (black shading), variable region of 16S rDNA which serves to identify the species; horizontal hatching, 23S rRNA coding region; ITS2, Internal Transcribed Spacer region 2; vertical hatching, 5S rRNA gene.

(b) SPR, Single Promoter Region. All other abbreviations and shadings are defined in (a).

(c) Nucleotide sequence of the conserved CL2 motif and its NusA and NusB binding sites. The locations of this motif are shown in (a) and (b). Two regions of NusA (KH1 and KH2) are involved in binding to sequence shown in bold (Beuth et al., 2005). The NusB component of the NusB. NusE (S10) dimer is thought to bind to the BoxA motif shown in italics (Das et al., 2008).

In contrast with rrnA, the rrnB operon is regulated by a single (P1) promoter. Transcription of tyrS was found to continue to progress to transcribe the rrnB operon (Gonzalez-y-Merchand et al., 1999). The mechanism of upstream activation of the rrnB operon was investigated (Arnvig et al., 2005). The results were found to follow the model reported for B. subtilis rather than the model for E. coli(Krásný and Gourse, 2004). B. subtilis rRNA promoters are much less dependent on UP elements and do not seem to use upstream-binding factors analogous to Fis. When the genome of a species encodes both rrnA and rrnB, the sequences corresponding to precursor-rRNA are very similar (see for example Sander et al., 1996), but not always identical (Ninet et al., 1996; Reischl et al., 1998). The relatively few differences that may be present are unlikely to affect the principle features of pre-rRNA secondary structure that is processed by RNase to yield 16S rRNA, 23S rRNA and 5S rRNA. Consensus secondary structures were derived for pre-16S rRNA (Ji et al., 1994a) and pre-23S rRNA and pre-5S rRNA (Ji et al., 1994b; 1994c) based on comparisons of mycobacterial sequences all of which are closely related.

6.2. The stringent response

rRNA synthesis is the rate limiting step in ribosome synthesis (Paul et al., 2004a) and bacteria have devised mechanisms that ensure that ribosome synthesis (and hence rRNA synthesis) is efficiently controlled to meet the cell’s need for protein biosynthesis, including the need to survive under starvation conditions (the stringent response, for review see Cashel et al., 1996). Much of our knowledge of the control of rRNA synthesis has come from studies of E. coli. Two small molecules are important regulators of rRNA synthesis in E. coli; nucleotide triphosphates, especially the NTP incorporated at the 5′-ends of the precursor-rRNA transcripts (Barker and Gourse, 2001) and guanosine 5′-diphosphate 3′-diphosphate (ppGpp) and its precursor pppGpp. Two proteins encoded by relA and spoT are implicated in the synthesis of ppGpp, which is synthesised by RelA in response to uncharged tRNAs at the A-site of the ribosome (Hogg et al., 2004). It is thought that ppGpp may bind directly to RNAP (for review see Paul et al., 2004b). A protein DksA is essential for regulation of rRNA synthesis by NTPs and ppGpp (Paul et al., 2004a). The stringent response has been studied in M. tuberculosis H37Rv (Primm et al., 2000). Whereas E. coli ppGpp is synthesized through the activities of RelA and SpoT proteins, the tubercle bacillus has a single protein, Rel_Mtb, to achieve the same effect; that is, this single protein produces ppGpp in response to nutrient starvation. A knockout mutant (Δ rel_Mtb) was produced and was used to show that the Rel_Mtb protein is needed for the long term survival of M. tuberculosis under starvation conditions.

6.3. Antitermination of rrn transcription

The size of the pre-rRNA transcript of the rrnA operon of M. tuberculosis extending from the start site of the PCL1 promoter to the intrinsic termination site is 5,550 nucleotides. As transcription proceeds, the nascent pre-rRNA transcript will recruit r-proteins and undergo processing by nucleases. Synthesis of rRNA is the rate-limiting step in ribosome synthesis. Thus the cell is required to synthesise long pre-rRNA transcripts when ribosomes are needed. The strategy employed is to modify the RNAP complex with antitermination factors to prevent premature transcription of the rrn operon. A feedback mechanism is achieved if a ribosomal protein plays a role in antitermination. Particular sequence motifs first identified in phage-λ were recognized as elements in the upstream regions of rrn operons so that related nomenclatures (Nus (N utilizing substances) proteins and nut sequences) are used for both phage-λ and rrn operons. Transcription of the rrn operon requires the formation of a stable RNAP elongation complex that is able to safely ignore transcription termination signals. The properties of the antitermination complex identified for the N protein system of bacteriophage-λ (Mogridge et al., 1995) provides the model for the composition of the antitermination complex needed to obtain full length transcripts of precursor-rRNA. The proteins identified in the phage-λ system are N, NusA, NusB, NusE (which is identical with ribosomal protein S10) and NusG. With one exception, the same proteins are needed to obtain full-length bacterial precursor-rRNA transcripts; all the Nus proteins are needed, but the cellular equivalent of the N protein has not yet been identified (Condon et al., 1995).

The nut sequence elements are found in both the leader and ITS1 regions of rDNA. This duplication of nut sequences suggests that the antitermination complex is formed at the leader region, dispersed after the 16S rRNA gene is transcribed by competition for nut sequences by a complementary strand of RNA located downstream from the 3′-end of the 16S rRNA gene and the complex is reformed before the 23S rRNA gene is transcribed and dispersed after the transcription of the 23S rRNA gene is completed (Ji et al., 1994c). The mycobacterial nut site within the leader region of rDNA comprises a 26 nucleotide sequence (the CL2 motif), which is present in all the mycobacteria examined so far (Gonzalez-y-Merchand et al., 1997; Menendez et al., 2002); the rRNA sequence is 5′-UGUUGUUUGAGAACUCAAUAGUGUGU-3′ [see Fig. 4(c)]. A similar, but not identical sequence is found in the ITS1 region.

Insights into the mechanisms of rrn antitermination have been advanced through physico-chemical and structural studies. A high-affinity interaction between the CL2 region of M. tuberculosis and its cognate NusA was reported (Arnvig et al., 2004). The crystal structure of the NusA component was determined (Gopal et al., 2001a). The X-ray structure of the NusA-RNA complex (Beuth et al., 2005) revealed that the RNA sequence bound by NusA was 5′-AGAACUCAAUA-3′ region of the CL2 sequence [see Fig. 4(c)]. Nodwell and Greenblatt (1993) first proposed that a complex NusB and NusE (S10) interacted with the nut sequence element BoxA. The mycobacterial BoxA sequence occurs at the 5′-end of the CL2 motif [see Fig. 4(c)].

The M. tuberculosis NusB structure was determined by X -ray crystallography (Gopal et al., 2000) and interaction between NusB and S10 was also demonstrated (Gopal et al., 2001b). The structure of E. coli NusB in solution was deterined by Altieri et al., (2000). NusB binds to Box A sequence directly, but its affinity for Box A is increased by interaction with NusE (S10), as shown by Das et al. (2008). This study strengthens the view that the ternary complex of Box A RNA/NusB/NusE (S10) interacts with the RNA polymerase complex and stabilizes it to overcome termination signals.

7.1. In vitro cell free systems

Translation of mRNA into protein is a complex process requiring many of the cell’s resources including initiation, elongation, termination factors and the consumption of energy; the formation of each peptide bond requires the hydrolysis of four high energy phosphate bonds. Studies of mycobacterial protein synthesis were neglected until Böttger and colleagues reconstituted an in vitro protein synthesis system comprising M. smegm*tis ribosomes and the complete range of factors synthesized in vitro from the parent genes. Fully functional hybrid ribosomes were constructed by modifying M. smegm*tis 16S rRNA to form bacterial eukaryotic hybrids. The hybrids were formed by replacing the bacterial decoding site (helix 44) with a eukaryotic counterpart. The drug sensitivities of the hybrid ribosomes were dependent on the origin of the eukaryotic sequence (Hobbie et al., 2007). The M. smegm*tis in vitro protein synthesizing system was then used to compare the rates of reaction of individual steps in protein synthesis with the hom*ologous reaction in E. coli. It was found that the elemental reaction rates of initiation and elongation were remarkably similar with M. smegm*tis and E. coli components. The reconstituted translation system from individual purified M. smegm*tis components is an alternative to that from E. coli to study mechanisms of translation and to test the action of antibiotics against Gram-positive bacteria (Bruell et al., 2008).

7.2. Post-translational modification of proteins

A number of proteins possess an unusual feature termed an intein. The primary transcript contains an insertion sequence that is translated. The amino acid sequence corresponding to the intron sequence is termed an intein. ‘Inteins are self-splicing elements that exist as in-frame protein fusions with two flanking sequences called exteins’ (Perler et al., 1994). The intein splices itself out of primary translational product leaving the two exteins which constitute the mature protein. The first mycobacterial intein recognised was found to be RecA of M. tuberculosis(Davis, 1991). Since then inteins were identified in three other proteins; namely, DnaB, GyrAand Pps1. Fourteen mycobacterial species are now known to have one or more intein-containing proteins (seehttp://bioinfo.weizmann.ac.il/~pietro/inteins/). The precise function of inteins is still a matter for speculation. However, the properties of inteins have been widely examined; including X-ray crystallographic and mutational studies (Van Roey et al., 2007).

8.1. Permeability barriers in mycobacterial cell envelopes

Nutrient uptake mechanisms obviously depend on the permeability barriers imposed by the cell envelope. Therefore, it is necessary to review the current status of the research about the cell envelope of mycobacteria. The terms are defined as follows (Beveridge, 1995; Beveridge and Kadurugamuwa, 1996; Graham et al., 1991): The cell wall consists of the periplasm, the peptidogly can layer and, for gram-negative bacteria, the outer membrane. The periplasmic space represents an extracytoplasmic compartment confined between the plasma membrane and an outer structure (outer membrane versus a peptidoglycan-teichoic acid-protein network for gram-negative and gram-positive bacteria, respectively). The periplasm is composed mostly of soluble components. The cell envelope comprises the inner membrane and the cell wall. The components of the cell envelopes of both gram-positive and gram-negative bacteria have recently been visualized by cryo-electron microscopy (Matias et al., 2003; Matias and Beveridge, 2005; Matias and Beveridge, 2006).

In microbiology textbooks, mycobacteria are classified as gram-positive bacteria. However, it is well documented that mycobacteria, unlike other gram-positive bacteria, have evolved a very complex cell wall, comprising a peptidoglycan-arabinogalactan polymer with covalently bound mycolic acids of considerable size (up to 90 carbon atoms), a large variety of extractable lipids (Barry et al., 1998; Daffé and Draper, 1998) and pore-forming proteins (Niederweis, 2003). Most of the mycobacterial lipids are constituents of the cell envelope, which provides an extraordinarily efficient permeability barrier to noxious compounds, rendering mycobacteria intrinsically resistant to many drugs (Brennan and Nikaido, 1995). Due to the paramount medical importance of M. tuberculosis, the ultrastructure of mycobacterial cell envelopes has been intensively studied for decades by electron microscopy. To account for the extraordinary efficiency of the mycobacterial cell wall as a permeability barrier, Minnikin originally proposed a model in which the mycolic acids are covalently bound to the peptidoglycan–arabinogalactan co-polymer and form the inner leaflet of an asymmetrical bilayer (Minnikin, 1982). Other lipids extractable by organic solvents were thought to form the outer leaflet of this outer bilayer. X-ray diffraction studies of mycobacterial cell walls showed that the mycolic acids are oriented parallel to each other and perpendicular to the plane of the cell envelope (Nikaidoet al, 1993). This provided experimental support for some fundamental aspects of the Minnikin model. Mutants and treatments affecting mycolic acid biosynthesis and the production of extractable lipids showed an increase of cell wall permeability and a drastic decrease of virulence, underlining the importance of the integrity of the cell wall for intracellular survival of M. tuberculosis (Barry et al., 1998). These indirect structural, biochemical and genetic data are consistent with the existence of an outer lipid bilayer as proposed by Minnikin (1982). However, this model faced criticism mainly because electron microscopy of mycobacteria, in particular thin sections thereof, never showed evidence for an additional, outer lipid bilayer (Daffé and Draper, 1998; Draper, 1998). Recently, we have visualized the envelope of intact M. smegm*tis and M. bovis BCG cells by cryo-electron tomography and have provided, for the first time, direct and final evidence for the existence of this structure (Hoffmann et al., 2007; Hoffmann et al., 2008). An important result was that cell envelopes of M. smegm*tis and M. bovis BCG a re very similar both in appearance and in dimensions. Thus, it makes sense to name this unusual structure “mycobacterial outer membrane” (Hoffmann et al., 2008). Cryo-electron tomography and cryo-electron microscopy of ultrathin sections clearly showed that the mycobacterial outer membrane is a bilayer structure. A mycolic acid-deficient mutant of Corynebacterium glutamicum, a close relative of mycobacteria, did not produce an outer membrane demonstrating that mycolic acids are an essential component of the outer membrane in these bacteria (Hoffmann et al., 2008). The results of this study were subsequently confirmed (Zuber et al., 2008) and are summarized in Fig. 5. Biologically very important consequences of these findings are that mycobacteria must have proteins that functionalize the periplasm and the outer membrane.

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Figure 5

Transport processes across the mycobacterial cell envelope

Schematic representation of the mycobacterial cell envelope consisting of the inner membrane (IM) and the cell wall. This representation is based on cryo electron micrographs (Hoffmann et al., 2007; Hoffmann et al., 2008). Mycolic acids are covalently linked to the arabinogalactan (AG) – peptidoglycan (PG) copolymer and are an essential component of the inner leaflet of the outer membrane (OM). Extractable lipids are represented in black. The porin MspA mediates the uptake of small and hydrophilic nutrients such as sugars (Stephan et al., 2005) and phosphates (Wolschendorf et al., 2007) across the outer membrane of M. smegm*tis. The MspA channel is 9.6 nm long (Faller et al., 2004). Approximately 7 nm of the MspA surface are inaccessible to hydrophilic reagents (Mahfoud et al., 2006). Hydrophobic compounds are assumed to diffuse directly across the outer membrane. The dimensions are approximately to scale.

8.2. Transport across mycobacterial outer membranes

The utilization of many solutes such as carbohydrates, alcohols, carboxy acids, fatty acids and amino acids by mycobacteria was originally examined as early as 1951 (Edson, 1951). It is therefore striking that the identity of many transporters for essential nutrients of M. tuberculosis and other mycobacteria are still unknown despite considerable progress in the development of genetic methods for mycobacteria (Kana and Mizrahi, 2004; Machowski et al., 2005). Recently, significant progress has been made to understand the uptake of some nutrients both in M. smegm*tis and M. tuberculosis. For example, proteins were identified and characterized that are involved in uptake of several nutrients such as phosphate (Gebhard et al., 2006; Peirset al., 2005; Vyas et al., 2003; Webb, 2003; Wolschendorfet al., 2007), sulphate (Wooff et al., 2002) and some amino acids (Seth and Connell, 2000; Talaue et al., 2006). The current knowledge about nutrient uptake across both inner and outer membranes in mycobacteria was recently summarized (Niederweis, 2008a; Niederweis, 2008b) and is updated here. A putative link between outer membrane transport and growth rate of mycobacteria has recently been discussed (Cox and Cook, 2007).

8.3. Porin-mediated diffusion of hydrophilic solutes

Porins are defined as non-specific protein channels in bacterial outer membranes which enable the influx of hydrophilic solutes (Nikaido, 2003). Channel-forming proteins that are functionally similar toporins of gram-negative bacteria have been observed in many mycobacteria (Niederweis, 2003) and other closely related genera such as corynebacteria. MspA was discovered as the first porin of M. smegm*tis (Niederweis et al., 1999). Deletion of mspA reduced the outer membrane permeability of M. smegm*tis towards cephaloridine and glucose nine -and four -fold, respectively (Stahl et al., 2001). These results show that MspA is the major general diffusion pathway for hydrophilic solutes in M. smegm*tis. Consecutive deletions of the two porin genes mspA and mspC reduced the number of pores by 15 -fold compared to wild-type M. smegm*tis. The loss of porins lowered the permeability for glucose by 75-fold and, concomitantly, the growth rate of M. smegm*tis on plates and in liquid medium dropped drastically (Stephan et al., 2005). This showed for the first time that the porin-mediated influx of hydrophilic nutrients limited the growth rate of porin mutants. However, it is unknown which and how many nutrients are in low supply in the M. smegm*tis porin mutants. Since MspA could not be express ed in M. smegm*tis above wild-type levels (Stephan et al., 2005), it is also not clear whether the influx of nutrients really limits the growth rate of wild-type mycobacteria as suggested earlier (Jarlier and Nikaido, 1990). The fact that the lack of the MspA and MspC porins also caused a reduced uptake of phosphates and slower growth on low-phosphate plates (Wolschendorf et al., 2007) indeed suggested that the slow uptake of essential hydrophilic nutrients other than the carbon source may also contribute to the slow growth of M. smegm*tis porin mutants.

The crystal structure of MspA represents the first crystal structure of any mycobacterial outer membrane protein and revealed an octameric goblet-like conformation with a single central channel of 10 nm in length (Faller et al., 2004). This structure is different from that of the trimeric porins of Gram-negative bacteria, which have one pore per monomer and are approximately 5 nm long (Koebnik et al., 2000). Its structural features define MspA as the founding member of a new class of outer membrane proteins. The crystal structure also revealed that the constriction zone of MspA consists of 16 aspartates (D90/D91). Thus, the zone of MspA with the smallest diameter is highly negatively charged (Faller et al., 2004). This most likely explains the previously observed preference of MspA for cations (Kartmann et al., 1999). The MspA pore provides an example of how outer membrane transport proteins can contribute to the selectivity of mycobacteria towards particular nutrients.

The existence of channel-forming proteins in M. tuberculosis and in M. bovis BCG has been demonstrated (Kartmann et al., 1999; Lichtinger et al., 1999; Senaratne et al., 1998). Uptake of serine, but not of glycine, was reduced in the ompATb mutant compared to wild-type M. tuberculosis. This was interpreted as proof that OmpATb is a porin consistent with its apparent channel-forming activity in vitro (Raynaud et al., 2002b). However, the overall permeability of the outer membrane of M. tuberculosis was reduced at pH 5.5 compared to pH 7.2, although the levels of OmpATb in the outer membrane were strongly increased (Raynaud et al., 2002b). Considering these contradicting results, it is doubtful that OmpATb has significant porin function in M. tuberculosis (Niederweis, 2003). The observation that a central domain of approximately 150 amino acids is sufficient for the channel activity of OmpATb in vitro does not contribute to the understanding of its biological functions (Molle et al., 2006).

Rv1698 was annotated as a protein of unknown function and was predicted as an outer membrane protein of M. tuberculosis (Lee et al., 2008). Lipid bilayer experiments of purified protein and uptake experiments in a porin mutant of M. smegm*tis demonstrated that Rv1698 is a channel-forming membrane protein (Siroy et al., 2008). Its surface accessibility was shown by protease experiments in whole cells (Song et al., 2008). A hom*olog of Rv1698 exists in all mycolic acid containing bacteria. Hence, Rv1698 represents the first member of a new class of channel proteins specific for mycolic acid-containing outer membranes.

8.4. Direct diffusion of hydrophobic solutes through the cell membranes

Hydrophobic molecules, in particular nonelectrolytes, can easily diffuse through phospholipid bilayers. However, the lipopolysaccharide-containing outer membrane of Gram-negative bacteria constitutes a considerable permeability barrier which does not allow the penetration of even extremely hydrophobic β-lactam antibiotics (Nikaido et al., 1983). The lipids in mycobacterial cell walls are likely to be organized in a very unusual, asymmetric bilayer (Nikaido et al., 1993). Differential scanning calorimetry showed that the lipids in mycobacterial cell walls have very high phase transition temperatures in the range of 60 – 70 °C. This is suggestive of a lipid domain of extremely low fluidity (Liu et al., 1995). Isolated cell walls of corynebacteria, which contain much shorter corynemycolic acids, displayed a much lower temperature transition, suggesting that the fluidity of this lipid bilayer is mainly determined by the mycolic acids. Since direct diffusion of a hydrophobic molecule through a lipid membrane requires that it is dissolved in the lipid phase, the permeability of a particular membrane is directly correlated with its fluidity. This has been demonstrated directly by Nikaido and co-workers (Andersson et al., 1996). It is concluded that the mycobacterial outer membrane presents a strong permeability barrier for hydrophobic molecules. On the other hand, there is emerging evidence that fatty acids rather than carbohydrates might be the dominant carbon source of M. tuberculosis after the onset of the immune response. This includes the requirement for isocitrate lyase for growth and persistence of M. tuberculosis in macrophages and in mice (McKinney et al., 2000), and the induction of expression of genes encoding enzymes involved in the β-oxidation of fatty acids in macrophages (Schnappinger et al., 2003) and mice (Dubnau et al., 2005; Timmet al., 2003). However, it is unknown how fatty acids are transported across the outer membrane of mycobacteria.

9.1. Transporters of carbohydrates

It has been widely documented that M. smegm*tis can grow on many carbon sources such as polyols, pentoses and hexoses (Edson, 1951; Franke and Schillinger, 1944; Izumori et al., 1976). A recent comprehensive analysis of carbohydrate uptake systems revealed that the soil bacterium M. smegm*tis has 28 putative carbohydrate transporters (Titgemeyer et al., 2007). The majority of sugar transport systems (19/28) in M. smegm*tis belongs to the ATP-binding cassette (ABC) transporter family (Fig. 6A). M. smegm*tis further possesses one putative glycerol facilitator of the major intrinsic protein (MIP) family, four sugar permeases of the major facilitator super family (MFS) of which one was assigned as a glucose transporter, and one galactose permease of the sodium solute super family (SSS). Thus, inner membrane transport systems for polyols, pentoses and hexoses are predicted to exist in M. smegm*tis (Fig. 6A). This bioinformatic prediction was proven recently by the discovery of glucose permease GlcP (Msmeg_4182) from M. smegm*tis mc²155. Upon overexpression of this protein in a glucose-negative mutant of E. coli it was shown that the strain was capable of glucose fermentation in addition to increased uptake of glucose (Pimentel-Schmitt et al., 2008). Therefore, GlcP is a first known example of a high-affinity sugar permease (K_mof 19 μM) found in mycobacteria.

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Figure 6

Transporters for uptake of carbohydrates across the inner membrane of M.smegm*tis and M. tuberculosis.

Shown are the transport proteins of the ATP-binding cassette (ABC, red), phosphotransferase system (PTS, yellow), major facilitator super family (MFS, green), major intrinsic protein family (MIP, dark blue) and the sodium solute super family (SSS, blue). The derived putative substrates are inferred from in silico analyses in combination with experimental data (Titgemeyer et al., 2007). Systems are denoted by the protein name instead of their locus tags (MSMEG_XXXX) if the substrates were experimentally verified or were predicted with a high likelihood. Note that the outer membranes of M.smegm*tis and M.tuberculosis were omitted for clarity reasons. The figure was taken from Niederweis (2008b) with permission.

M. smegm*tis does not grow on lactose, maltose and sucrose as a sole carbon source (Titgemeyer et al., 2007). Franke and Schillinger obtained the same result for lactose and maltose, but observed respiration of M. smegm*tis in the presence of sucrose (Franke and Schillinger, 1944). M. smegm*tis has at least three inner membrane transport systems with significant similarities to other bacterial disaccharide transporters (Niederweis, 2008b). However, the substrate specificities of the transporters encoded by msmeg0501-0508 and msmeg0509-0517 are not known. Growth of bacteria on disaccharides as sole carbon sources requires enzymes, which cleave the disaccharide and release the monosaccharides for further utilization. The absence of proteins similar to known bacterial β-D-galactosidases (LacZ of E. coli, BgaB of B. circulans, MbgA of B. megaterium, LacA S. coelicolor) provides a molecular explanation for the inability of M.s megmatis to utilize lactose as a sole carbon source. By contrast, M. smegm*tis has six hom*ologs (MSMEG3191, 3577, 4901, 4902, 4685, 6477) of MalL of B. subtilis, which hydrolyzes maltose, longer maltodextrines up to maltohexose, isomaltose and sucrose (Schönert et al., 1999), and of the cytoplasmic trehalase TreC of E.coli, which cleaves trehalose-6-phosphate (Rimmele and Boos, 1994). It is conceivable that these enzymes are used in trehalose metabolism considering the unusual importance of trehalose in mycobacteria (Murphy et al., 2005; Woodruff et al., 2004) and the observation that trehalose was the only disaccharide which was used by M. smegm*tis as a sole carbon source. However, it cannot be excluded that some of the enzymes with similarities to TreC and MalF have roles in pathways distinct from trehalose metabolism.

Bioinformatic analysis of the genome of M. tuberculosis H37Rv revealed four ABC-type transporters and one permease of the MFS class for carbohydrates (Titgemeyer et al., 2007)(Fig. 6B). These ABC transporters have been described earlier in a global analysis of the M. tuberculosis genome (Braibant et al., 2000; Content et al., 2005). It is obvious that M. tuberculosis is poorly equipped with carbohydrate transport systems in comparison to M. smegm*tis. Two of the operons, the lpgY sugABC and the uspABC operons, are highly conserved between the two species. The proteins of the ABC^Sug and of the ABC ^Usp systems share between 62% and 80% similar amino acids, compared to only 25 to 30% similar amino acids for the ABC^Ugp and the Rv2038c/Rv2039c/R v2040c/Rv2041c systems. The similarities of all four ABC systems to known transporters outside the genus Mycobacterium is so low (< 25%) that substrates of these transporters cannot be predicted (Titgemeyer et al., 2007).

The ABC^Sug sugar transport system was predicted to be essential for virulence of M. tuberculosis in mice based on transposon site hybridization (TraSH) experiments (Sassetti et al., 2003). Previously, it was suggested that this permease may transport maltose or maltodextrins (Borich et al., 2000; Braibant et al., 2000). However, both the similarities of ABC^Sug and of the corresponding substrate binding protein LpgY to the maltose transporters and periplasmic maltose binding proteins MalE of E. coli and S. coelicolor are very low (< 25%). Thus, it is questionable whether maltose is the substrate of ABC^Sug. This conclusion is supported by the fact that neither M. smegm*tis, which has a highly similar ABC^Sug system, nor M. tuberculosis (Edson, 1951) grow on maltose as a sole carbon source. It has to be noted that similar uncertainties exist about the substrate specificities of the four other carbohydrate uptake system of M. tuberculosis including the ABC^Usp transporter which was proposed to transport sn-glycerol-3-phosphate based on low protein similarities (Braibant et al., 2000; Content et al., 2005). The SugI transporter of the MFS class shows distant sequence similarity to the glucose permease GlcP (28%) of S. coelicolor and to the galacto se (GalP, 24%) and arabinose (AraE, 24%) transporters of E. coli. Thus, the SugI system may transport a monosaccharide.

Glycerol is used as the standard carbon source to grow M. tuberculosis, however no uptake system is known or apparent by sequence similarity (Titgemeyer et al., 2007). Since M. tuberculosis grows with a generation time of 24 hours and it has been shown that glycerol can directly diffuse through lipid membranes both in vitro(Paula et al., 1996) and in vivo(Eze and McElhaney, 1981), it is conceivable that the rate of glycerol intake by passive diffusion may be sufficient for growth. Incoming glycerol would then be converted by glycerol kinase (GlpK) to glycerol-3-phosphate to enter the route of central carbon metabolism (Fig. 6B). M. tuberculosis has one putative glycerol kinase that shows a high similarity to the two glycerol kinases of M. smegm*tis (MSMEG_6759, 77% and MSMEG_6756, 57%) and to the two glycerol kinases from S. coelicolor SCO0509 (75%) and SCO1660 (59%).

9.2. Comparison of inner membrane sugar transporters of M. smegm*tis and M. tuberculosis

The analysis of the carbohydrate uptake proteins in the genomes of M. smegm*tis and M. tuberculosis confirms the very early phenotypical observations that the saprophytic mycobacteria have a much broader spectrum of substrates, which they can use as sole carbon and energy sources (Edson, 1951). It is striking that the genome of M. tuberculosis has only five permeases for carbohydrate uptake compared to 28 of M. smegm*tis. This suggests that the phagosome does not provide an environment rich in diverse sugars. The tantalizing conclusion is that an experimental analysis of the substrate specificity of the inner membrane carbohydrate transporters of M. tuberculosis may reveal the carbon sources available in the phagosome of human macrophages and/or in other cellular hideouts of M. tuberculosis inside the human body.

9.3. Transporters of lipids

Several lines of evidence strongly suggest that M. tuberculosis switches from a carbohydrate to a fat diet after the onset of the adaptive immune response. (i) Biochemical studies suggest that in chronically infected lung tissues, fatty acids might be a major source of carbon and energy for M. tuberculosis (Wheeler et al., 1990). (ii) During the first 10 days of infection of mice, M. tuberculosis requires the sugar transporter SugAB for survival (Sassetti et al., 2003). Thereafter, enzymes such as isocitratelyase and malatesynthases are essential for virulence (McKinney et al., 2000). This indicates that lipids are the major carbon and energy source of M. tuberculosis because the glyoxylate shunt is required for running the citric acid cycle on acetate which is produced by degradation of lipids through β-oxidation. (iii) M. tuberculosis possesses four genes encoding putative phospholipases C, plcA, plcB, plcC and plcD. These genes are required for virulence of M. tuberculosis in mice (Raynaudet al., 2002a). The fact that the phospholipases C are attached to the cell wall by lipid anchors argues for a role of these enzymes in the controlled release of fatty acids likely from phospholipids of the phagosomal membrane. However, while several proteins have been identified in M. tuberculosis involved in the transport of lipids from the cytoplasm to the outer membrane (Jackson et al., 2007), the proteins involved in the transport of lipids across the outer membrane are yet unknown. By contrast, the mechanisms that govern the uptake of exogenous fatty acids are well established in E. coli (Dirusso and Black, 2004). When the cell encounters long chain fatty acids in the environment, these ligands bind to outer membrane protein FadL and via a ligand-induced conformational shift within the protein are transported into the periplasmic space. The more acidified environment of the periplasmic space promotes the formation of uncharged fatty acid molecules, which partition into and flip across the inner membrane. Within the cytosol, the acyl-CoA synthase FadD partitions into the inner membrane, where it functions in the vectorial esterification of the long chain fatty acids (Dirusso and Black, 2004). Consistent with the importance of lipid uptake, M. tuberculosis possesses numerous hom*ologs of FadD proteins (Trivedi et al., 2004). Moreover, an 82-gene cluster (Rv3492c-Rv3574) responsible for catabolism of cholesterol including at least one set of genes that encode all enzymes necessary to perform the full cycle of β-oxidation and a multicomponent cholesterol uptake system consisting of mce4ABCDEF and an ABC-transporter supAB has been found in M. tuberculosis (Van der Geize et al., 2007). It was shown that Mce4 forms the major cholesterol import system of M. tuberculosis, which can be used by the cell for both carbon and energy production as a primary carbon source (Pandey and Sassetti, 2008). Furthermore, mutation in mce4 operon leads to attenuation during chronic phage of infection in mouse spleen and lungs and in activated, but not resting macrophages, suggesting that uptake of lipids function as a major carbon source at the later stage of M. tuberculosis infection (Joshi et al., 2006; Pandey and Sassetti, 2008; Sassetti et al., 2003).

9.4. Transporters of phosphorus-containing solutes

Phosphorus is indispensable for energy supply, for the biosynthesis of nucleic acids and phospholipids, and many other cellular processes. While inorganic phosphate is the preferred source of phosphorous, many bacteria can also take up organic phosphates and release phosphate by the action of periplasmic phosphatases such as PhoA. Gram-negative bacteria employ sophisticated transport mechanisms to acquire phosphorus containing nutrients from the environment. E.coli uses four phosphate transport systems Pst, Pit, GlpT and UhpT to translocate inorganic phosphate across the inner membrane (van Veen, 1997). Part of the Pst system is the periplasmic protein PstS, which binds and transfers phosphate to the transmembrane components PstA and PstC. PstB hydrolyzes ATP and delivers energy for phosphate translocation across the inner membrane by PstA/PstC. Pst systems from gram-negative bacteria bind and transport phosphate with binding constants and apparent transport K_m values in the submicromolar range. M. tuberculosis contains several copies of the genes encoding the Pst system (Braibant et al., 1996). Two Pst components, PstS1 and PstS2, have been shown to be virulence factors in M. tuberculosis (Peirs et al., 2005; Sassetti et al., 2003). Further, M. tuberculosis contains two genes, pitA and pitB, which encode putative constitutive inorganic phosphate transporters (Content et al., 2005a). The physiological role of the Pit transporters is unclear.

The single pstSCAB operon of the fast-growing M. smegm*tis encodes a high-affinity Pst system with an apparent K_mvalue of 40 μM phosphate. A second high-affinity phosphate uptake system of M. smegm*tis is encoded by the phnDCE operon (Gebhard et al., 2006). However, even a phnD pstS double mutant did not show a reduced phosphate uptake suggesting the presence of a third high-affinity phosphate uptake system of M. smegm*tis (Gebhard et al., 2006). Considering the presence of three high-affinity phosphate uptake systems, which are inducible at low phosphate concentration in M. smegm*tis, it is unclear why a pstB mutant showed a reduced phosphate transport (Bhatt et al., 2000). Taken together, these results underline the importance of phosphate uptake for mycobacteria. The transcriptional profiles of M. tuberculosis and Salmonellaenterica in infected macrophages revealed that the proteins involved in inorganic phosphate transport are up-regulated (Eriksson et al., 2003; Schnappinger et al., 2003) indicating that phosphate levels inside phagosomes of macrophages are indeed limited. Consistent with this conclusion, genes encoding efficient phosphate transport systems were found to be essential for the survival of M. tuberculosis in macrophages and mice (Rengarajan et al., 2005; Sassetti et al., 2003). However, it is unknown how inorganic or organic phosphates cross the outer membrane of M. tuberculosis. Since direct diffusion of phosphates through model lipid membranes is extremely slow (permeability coefficient of the mono anion: 5 × 10⁻¹² cm/s (Chakrabarti and Deamer, 1992)) it appears likely that slowly growing mycobacteria also use outer membrane pore proteins for uptake of phosphate. Indeed, the existence of a porin with anion specificity has been demonstrated (Lichtinger et al., 1999). This porin still awaits discovery.

9.5. Transporters of sulfur-containing solutes

Sulfur is essential in cells for biological activities such as translation initiation and maintenance of the redox potential. Transposon insertions in the cysA and subI genes of M. bovis BCG yielded methionine auxotrophs. These mutants were resistant to chromate and did not take up sulfate. These results identified the products of the genes cysTWA and subI as components of a sulphate permease and indicated that this transporter is the sole sulphate transporter of M. bovis BCG (McAdam et al., 1995; Wooff et al., 2002). The sensitivity of sulphate uptake to azide and 1,3-dicyclohexylcarbodiimide are characteristic of ABC transporters. Survival of the cysA and subI mutants in mice was not different from wild-type M.bovis BCG. This indicated that, in the host, methionine may be a more important sulfur source than sulphate for growth of the M. tuberculosis complex. This may also explain how M. leprae remains a pathogen, despite being a natural methionine auxotroph because of its loss of cysTWA for sulphate transport (Wood, 1995). An alternative explanation may be that other putative sulphate transporters such as the predicted sulphate permease (SulP) of M. tuberculosis are induced in vivo and compensate for the loss of the CysTWA transporter (Content et al., 2005a). To this end, it was shown that overexpression of SulP protein Rv1739c in E. coli leads to increased uptake of sulfate by intact cells with its maximum at pH 6.0 (Zolotarev et al., 2008). However, to prove the function of this protein as a sulfate transporter of M. tuberculosis, construction of knock-out mutant and sulfate uptake experiments are required.

9.6. Transporters of nitrogen-containing solutes

Nitrogen is an essential component of nearly all complex macromolecules in a bacterial cell, such as proteins, nucleic acids and cell wall components. Ammonium is the preferred nitrogen source of many bacteria. In enteric bacteria, diffusion of uncharged ammonia (NH₃) through the cytoplasmic membrane into the cell is sufficient to support growth in the presence of high amounts of ammonium (NH₄+) in the growth medium. Only when diffusion across the cell envelope becomes limiting for growth is the ammonium transporter AmtB synthesized. As in enteric bacteria, mycobacteria possess hom*ologues of AmtB (Nolden et al., 2001). However, no biochemical data are available for ammonium uptake by mycobacteria and the role of AmtB in this process. Two ammonium transporters, AmtB (Msmeg_2425) and Amt1 (Msmeg_6259), have been proposed to take up ammonium in M. smegm*tis based on their transcriptional regulation by GlnR, a key regulator of nitrogen control in mycobacteria, and RT-PCR analysis under nitrogen limited conditions (Amon et al., 2008). However, direct evidence on involvement of amtB and amt1 into transport of ammonium in M. smegm*tis is missing.

Nitric oxide (NO) is generated in large amounts within the macrophages and restricts the growth of M. tuberculosis. Nitrate can be produced by oxidation of nitric oxide and is an alternative source of nitrogen for bacteria within the human host. Early work in E. coli had suggested that narK was involved only in nitrite export (Rowe et al., 1994), and so the hom*ologous narK2 in M. tuberculosis was annotated as a “nitrite extrusion protein”. More recent work with an E. coli narK narU double mutant indicated that the two proteins could transport nitrate into and nitrite out of the cell (Clegg et al., 2002; Jia and Cole, 2005). In M. tuberculosis, four genes, narK1 through narK3 and narU are hom*ologous to narK and narU. Since M. tuberculosis is unable to reduce nitrite, which could accumulate to toxic levels, it must be exported out of the cell. The M. tuberculosis narK2 was shown to complement this E. coli double mutant, supporting a role for narK2 in nitrate reduction by coding for a transporter of nitrate into and nitrite out of the cell (Sohaskey and Wayne, 2003). Nitrate reduction by M. tuberculosis is regulated by control of nitrate transport into the cell by NarK2. It is proposed that NarK2 senses the redox state of the cell, possibly by monitoring the flow of electrons to cytochrome oxidase, and adjusts its activity so that nitrate is transported under reducing, but not under oxidizing, conditions (Sohaskey, 2005). Inhibition of nitrate transport by oxygen has been documented in other bacteria (Moir and Wood, 2001). It is intriguing that M. tuberculosis, classified as an obligate aerobe, should have such intricate control of an anaerobic enzyme system. Transcription of narK2 is controlled by DosR/DevR, which responds to oxygen (O₂), nitric oxide (NO), and carbon monoxide (CO) (Kumar et al., 2008; Ohno et al., 2003; Voskuil et al., 2003). Both the transcription of the narK2 gene and the activity of NarK2 are controlled by similar signals (Sohaskey, 2005).

9.7. Transporters of amino acids

Many microorganisms use amino acids as a source of energy and/or nitrogen, and also for biosynthetic purposes. It was shown early in seminal papers by Yabu that D-amino acids are taken up rapidly by mycobacteria while the L-forms are transported at a much lower rate (Yabu, 1967; Yabu, 1970; Yabu, 1971). These results can be attributed to the specificity of the inner membrane transporters for the natural form of amino acids. It was also found early that M. tuberculosis cannot utilize amino acids to support metabolism in contrast to saprophytic mycobacteria (Edson, 1951).

Nevertheless, some amino acids are taken up by M. tuberculosis and M. bovis BCG (Seth and Connell, 2000). In particular, uptake of arginine was examined because arginine plays also an important role in the cellular immune response as the substrate of the inducible nitric oxide synthase (iNOS) which generates nitric oxide to kill bacterial and parasitic pathogens in macrophages (Chan et al., 2001; Nathan and Shiloh, 2000). Thus, competition between the pathogen and macrophages for arginine has been suggested to contribute to the outcome of infection (Mills, 2001). Not surprisingly, M. tuberculosis has several genes encoding putative L-arginine uptake transporters: Rv0522, Rv1979c, Rv1999c, Rv2320c and Rv3253c (Cole et al., 1998). Transport of L-arginine, but not of L-lysine and L-ornithine, was reduced by 70% in a mutant of M. bovis BCG lacking the gene hom*ologous to rv0522. This identified Rv0522 (GabP) as an arginine transporter of M. tuberculosis (Seth and Connell, 2000). The remaining 30% of L-arginine transport activity and the uptake of other cationic amino acids by the mutant are likely mediated by other amino acid permeases.

Some amino acids such as proline and glycine betaine are important in regulating the osmotic pressure in bacterial cells (Wood et al., 2001). Recently, it was demonstrated that the ABC transporter ProXVWZ imports glycine betaine and protects M. tuberculosis against osmotic stress and thereby provides a growth adavantage both in vitro and in macrophages (Price et al., 2008).

9.8. Transporters of inorganic cations

Metal ions such as Fe²⁺/Fe³⁺, Cu⁺/Cu²⁺ and Zn ²⁺ play structural and catalytic roles in metalloenzymes. Genome analysis of M. tuberculosis revealed 28 genes encoding a broad repertoire of putative metal ion transporters. They comprise eight families of secondary active transporters and three families of primary active transporters, including twelve ‘P’ type ATPases, and represent approximately one quarter of all transporters in this organism. Potential metal ion specificities include K⁺, Na⁺, Cu²⁺, Cd²⁺, Zn²⁺, Mn²⁺, Mg²⁺, Ca²⁺, Co²⁺, Ni²⁺, Fe^2+/3+, Hg²⁺, AsO₂^- and AsO₄^3-. Seventeen of these transporters are also encoded as complete open reading frames in Mycobacterium leprae, suggesting a role in intracellular survival. The properties of these transporters, including the NRAMP orthologue MntH which transports manganese ions in other bacteria, has been reviewed recently (Agranoff and Krishna, 2004; Content et al., 2005). Here we summarize only the new findings for the uptake of iron by M. tuberculosis.

Throughout the living world, iron is contained in the active centers of most redox enzymes. Because iron occurs in the insoluble Fe³⁺form under oxic conditions (10⁻⁹M Fe ³⁺in soil and water) (Ratledge and Dover, 2000), proteins and siderophores with high binding affinity are required to make Fe³⁺biologically available. M. tuberculosis produces salicylate-containing siderophores named mycobactins. The more polar form (carboxymycobactin) is released into the medium, whereas the less polar form (mycobactin) remains cell-associated (Ratledge and Dover, 2000). Upon binding by siderophores, Fe³⁺is transported into the bacterium and released from the siderophore, possibly by reduction. In most bacteria, Fe³⁺-siderophore complexes bind to specific receptor proteins on the cell surface and are actively transported into the cytoplasm by specialized proteins that belong to the family of ATP-binding cassette (ABC) transporters (Braun and Killmann, 1999). It is suggested that three proteins, IrtA, IrtB and Rv2895c, are mainly involved in iron homeostasis in M. tuberculosis(Farhana et al., 2008). The ABC transporter IrtAB is required by M. tuberculosis to replicate in iron-deficient medium and to use carboxymycobactin as an iron source, indicating that IrtAB is involved in the transport of this compound (Rodriguez, 2006). Further evidence suggested that IrtA is a carboxymycobactin exporter based on in vitro reconstitution experiments, while the two-component IrtB-Rv2895c system functions as importer of Fe³⁺-carboxymycobactin. A knockout mutant of msmeg_6554, the irtA hom*ologue in M. smegm*tis, displayed an impaired siderophore export that is restored upon complementation with M. tuberculosis irtA (Farhana et al., 2008). Moreover, deletion of the irtAB genes in M. tuberculosis reduced its ability to survive in macrophages and in the lungs of mice. However, the lack of irtAB does not completely eliminate replication of M. tuberculosis in iron-deficient conditions, which indicates that other transporters can partially compensate for the lack of IrtAB (Rodriguez and Smith, 2006). Given the importance of iron, this is not surprising because pathogenic bacteria often have multiple pathways for iron acquisition. The genome of M. tuberculosis does not reveal other obvious siderophore transporters (Rodriguez, 2006). However, there are numerous ABC transporters, the substrates of which are unknown; therefore, one or more of these could contribute to iron uptake.

10.1 Sensing nutrient availability

10.2. Oxygen and redox state

Mycobacteria are obligate aerobes and as such have to possess mechanisms to detect the ambient oxygen tension to enable them to adapt to changes in oxygen availability by adjusting their metabolism accordingly. Furthermore, gradual depletion of oxygen has been implicated in entry of M. tuberculosis into non -replicating persistence and latency (Wayne and Sohaskey, 2001). The mechanisms by which mycobacteria sense oxygen have been extensively studied, and the two major sensory systems known to date, the DosT/DosS/DosR system and WhiB3, are discussed below.

10.3. Exogenous stress conditions

11.1. Mycobacterial metabolism in vitro

A large body of detailed literature is available on the physiology of mycobacterial metabolism (Edson, 1951; Ramakrishnan et al., 1972; Ratledge, 1976; Ratledge, 1982; Wheeler and Blanchard, 2005). During in vitro growth of M. tuberculosis, various carbon and energy sources feed into central metabolic pathways where they are oxidized to pyruvate and ultimately CO₂. In mycobacteria, the primary route of glucose conversion to pyruvate is the Embden-Meyerhof-Parnas (EMP) pathway. Approximately 70% of the glucose is directed down the EMP pathway and the remaining glucose (30%) flows into the pentose phosphate pathway to generate C₅ and C₄sugars and reducing power in the form of NADPH (Ratledge, 1976). No other pathways exist for sugar catabolism in pathogenic mycobacteria. The preferred carbon source for mycobacteria is glycerol and the metabolic route for its degradation is well established in these bacteria (for reviews see (Ratledge, 1976; Wheeler and Blanchard, 2005)). The metabolism of glycerol involves the phosphorylation of glycerol to form glycerol-3-phosphate via glycerol kinase, followed by the oxidation of this intermediate to dihydroxyacetone phosphate via glycerol-3-phosphate dehydrogenase. This triose phosphate is further metabolised via the EMP pathway and the Krebs cycle. Mycobacterium bovis lacks the ability to utilize glycerol unless pyruvate is provided, and this defect has been shown to be due to a single nucleotide polymorphism in the gene pykA encoding for pyruvate kinase (Keating et al., 2005). The Krebs cycle in M. tuberculosis appears a typical because α-ketoglutarate dehydrogenase is not present and is replaced by the following reaction scheme: α-ketoglutarate is first converted to succinyl semialdehyde by a α-ketoglutarate decarboxylase and then oxidized to succinate by a succinyl semialdehyde dehydrogenase coupled to NADP reduction (Tian et al., 2005).

In general, the complete oxidation of one mole of glycerol yields 7 moles of reduced pyridine nucleotide (NADH) or equivalent (FADH₂). Six moles of NADH are generated by glycerol-3-phosphate dehydrogenase, glyceraldehyde-3-phosphate dehydrogenase, pyruvate dehydrogenase, isocitrate dehydrogenase, succinyl semialdehyde dehydrogenase and malate dehydrogenase and one mole of FADH₂(via succinate dehydrogenase). At present, it has not been experimentally determined how much energy (e.g. moles of ATP) is produced per mole of glycerol utilized by mycobacterial species. Hypothetical calculations based on a mycobacterial respiratory chain that has three proton translocating sites when NADH is used as the endogenous electron donor (Prasada-Reddy et al., 1975), and assuming one mole ATP is synthesized per three protons translocated by the ATP synthase would suggest that the oxidation of one mole NADH leads to the synthesis of 2 moles of ATP. Oxidation of one mole of FADH₂ would lead to the synthesis of approximately 1 mole of ATP. In addition, 3 moles of ATP (or an energetically equivalent nucleotide) are produced via substrate level phosphorylation (via phosphoglycerate kinase, pyruvate kinase and succinyl-CoA synthetase) and one of these is consumed by glycerol kinase. Based on these calculations, the theoretical ATP yield would be 15 moles of ATP produced per mole of glycerol consumed. In E. coli and Bacillus megaterium, the complete oxidation of one mole of glycerol yields 14 moles ofATP (Downs and Jones, 1975; Farmer and Jones, 1976).

The energetics of mycobacterial growth has been studied in glycerol-limited continuous culture (Beste et al., 2005). In the slow growing M. bovis BCG, at both low (0.01 h⁻¹) and high (0.03 h⁻¹) dilution rates, the cell yield or Y_glycerol was 27 g [dry weight] cells/mol glycerol utilized and 33 g [dry weight] cells/mol glycerol utilized respectively (Beste et al., 2005). Under similar growth conditions in continuous culture, the fast growing non-pathogenic saprophyte M. smegm*tis exhibits a Y_glycerol of 30 to 40 g [dry weight] cells/mol glycerol utilized over the dilution rate range (0.02 to 0.15 h⁻¹) (S.L. Tran and G.M. Cook, unpublished data). The glycerol consumption rate for maintenance functions (m_glycerol) is 0.28 mmoles of glycerol/h/ g [dry weight] cells (S.L. Tran and G.M. Cook, unpublished data), a value that is comparable with E. coli. Beste et al. (2007b) have studied the effect of growth rate on gene expression of M. bovis BCG in carbon -limited continuous culture at dilution rates of 0.01 h⁻¹(t _d= 69 h) and 0.03 h⁻¹(t _d= 23 h). At low growth rate, there was a down regulation of genes involved in aerobic respiration, TCA cycle enzymes and the ATP synthase, despite the oxygen tension being maintained at 70–100 %, but consistent with a decreased oxygen demand due to a low growth rate. The authors reported that there was some overlap with the transcriptional profile of M. bovis BCG to growth rate and both Wayne’s model of persistence (Muttucumaru et al., 2004) and macrophage infection (Schnappinger et al., 2003), butlittle correlation with the response of M. tuberculosis to oxygen limitation in chemostat culture (Bacon et al., 2004). These data suggest that adaptation to slow growth rate in carbon-limited conditions is an important trigger for gene expression in both the Wayne model of persistence and growth of M. tuberculosis in macrophages (Beste et al., 2007b).

11.2. Mycobacterial metabolism in vivo

The now widely accepted view for in vivo metabolism of M. tuberculosis is that the bacterium switches its intermediary metabolism from carbon sources such as glucose and glycerol to fatty acids and host lipids during the course of infection (Munoz-Elias and McKinney, 2005; Munoz-Elias and McKinney, 2006; Munoz-Elias et al., 2005; Munoz-Elias et al., 2006; Schnappinger et al., 2003; Pandey and Sassetti, 2008). This concept was first supported by the sequencing of the M. tuberculosis genome, which revealed a large repertoire of genes for lipid degradation (Cole et al., 1998). A central role for fatty acid metabolism is also implied by the considerable duplication of genes involved in lipid metabolism in mycobacterial genomes (Cole et al., 1998; Cole et al., 2001; Fleischmann et al., 2002; Garnier et al., 2003). Further studies have shown an induction of genes in the phagosomal environment that encode for enzymes that are required for the biochemical activation and β-oxidation of fatty acids (Schnappinger et al., 2003). Breakdown products of fatty acids are further metabolised via the Krebs cycle and the carbon conserving (no loss of CO₂) glyoxylate shunt (McKinney et al., 2000; Schnappinger et al., 2003). M. tuberculosis has two copies of the gene ( icl1 and icl2) that encodes the enzyme isocitrate lyase, a key enzyme in the glyoxylate shunt that converts isocitrate to succinate and glyoxylate. Recent work has unequivocally shown that a double knockout mutant (icl1 icl2) of M. tuberculosis is unable to establish an infection in mice due to its inability to metabolise host-derived lipids (Munoz-Elias and McKinney, 2005). The methylcitrate cycle is important for the metabolism of propionyl-CoA in M. tuberculosis, an intermediate of odd chain number fatty acid breakdown, but paradoxically is not essential for persistence in mice (Munoz-Elias et al., 2006).

Two recent studies have shown that cholesterol, a major component of host cell membranes, is essential for M. tuberculosis persistence in the lungs of chronically infected animals and for growth within the IFN-γ-activated macrophages that predominate at this stage of infection (Gatfield and Pieters, 2000; Pandey and Sassetti, 2008). Pandey and Sassetti (2008) demonstrate that both M. tuberculosis and M. smegm*tis utilize cholesterol, but the mechanism of energy generation during growth on cholesterol is unknown. Kendall et al. (2007) have identified a transcriptional repressor, KstR, that controls the expression of a significant number of genes involved in lipid metabolism in both M. tuberculosis and M. smegm*tis.

11.3. Recycling of reducing equivalents and electron transport

A crucial feature in the adaptation of any bacterium to alternative energy sources and changing environmental parameters (e.g. oxygen tension) is the balance of oxidative and reductive reactions in the metabolic scheme. During mycobacterial growth, both in vitro and in vivo, substrate oxidation leads to the formation of reduced cytoplasmic electron carriers, e.g. NADH, FADH₂ or ferredoxin. The oxidation of NADH or equivalent by aerobicbacteria is critical for continuous metabolic flux, and in the absence of a fermentative metabolism, NADH oxidation will be carried out primarily by membrane-bound NADH dehydrogenases. NADH dehydrogenase is the first component of the respiratory chain and transfers electrons from NADH to quinones (e.g. ubiquinone or menaquinone). Weinstein et al. (2005) have identified genes for two classes of NADH:menaquinone oxido reductases in the genome of M. tuberculosis. NDH-1 is encoded by the nuoABCDEFGHIJKLMN operon and as this dehydrogenase transfers electrons to menaquinone, it conserves energy by translocating protons across the membrane to generate a proton motive force (Δμ_H+) (Fig. 7). The second class is NDH-2, a non-proton translocating NADH dehydrogenase that does not conserve energy and is present in two copies (ndh and ndhA) in M. tuberculosis(Weinstein et al., 2005). Mutagenesis studies have established that both NDH-1 and ndhA are dispensable for growth in vitro (Sassetti et al., 2003), but the lack of a viable strain with a disrupted or deleted ndh suggests that it is essential for growth despite the presence of ndhA(McAdam et al., 2002; Sassetti et al., 2003; Weinstein et al., 2005). Furthermore, deleterious point mutations in ndh of M. smegm*tis are pleiotropic, conferring temperature -sensitive growth arrest and multiple amino acid auxotrophy. Some mutants also have a 25-fold reduced NADH dehydrogenase activity, implying NDH-2 is the primary enzyme responsible for NADH oxidation and it is essential for the viability of M. smegm*tis(Miesel et al., 1998; Vilcheze et al., 2005).

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Figure 7

Schematic diagram outlining the electron transport chain components and ATP synthase of Mycobacterium tuberculosis growing under aerobic (top panel) and hypoxic conditions (bottom panel)

MK = menaquinone, MKH₂= menaquinol.

In M. tuberculosis, no energetic role for the non-essential type I NADH:menaquinone oxido reductases has been established and the persistence of M. tuberculosis in an in vitro Wayne model was shown not to be compromised in a nuo operon deletion mutant (Rao et al., 2008). Velmurugan et al. (2007) have demonstrated a role for nuo (i.e. the NuoG subunit) in the ability of M. tuberculosis to inhibit macrophage apoptosis. The nuo operon has been lost from the genome of M. leprae with a single nuoN pseudogene remaining (Cole et al., 2001). M. smegm*tis also contains genes for a type I NADH:menaquinone oxido reductases (viz. nuoA-N), but enzyme assays for NDH-I activity in M. smegm*tis failed to detect the enzyme suggesting it is not expressed (Miesel et al., 1998). Several studies have reported that nuo is down-regulated in M. tuberculosis during mouse lung infection (Shi et al., 2005), survival in macrophages (Schnappinger et al., 2003), in both NRP-1 (1% oxygen saturation) and NRP-2 (0.06% oxygen saturation) relative to aerated mid-log growth (Shi et al., 2005), and upon starvation in vitro (Betts et al., 2002). The transcription of ndh is also down-regulated in M. tuberculosis during mouse lung infection, but transcript levels for ndh peak (are induced) during NRP-2 in vitro demonstrating that the pattern of ndh regulation is different between in vivo and in vitro conditions (Schnappinger et al., 2003). These data are in contrast to E. coli where NuoA-N (NDH-1) is usually associated with anaerobic respiratory pathways (e.g. fumarate) and non-coupling dehydrogenases such as NDH-2 are synthesized aerobically (Unden and Bongaerts, 1997). Moreover, E. coli mutants lacking NDH -I have a competitive disadvantage in stationary phase (Zambrano and Kolter, 1993). Some interesting questions arise from these observations. The first is “why do mycobacteria use type II NADH dehydrogenases to recycle NADH when they could continue to use the energy conserving (Δμ_H+ generating) NDH-1?” One potential explanation is that because NDH-2 are non-proton translocating, they will not be impeded by a high Δμ_H+ which could ultimately slow down glycolytic flux due to back pressure on the system. This mechanism is akin to a “relief valve” that would allow for a higher metabolic flux and ultimately higher rates of ATP synthesis at the expenses of low energetic efficiency of the respiratory chain. Secondly, why is ndh an essential gene when mycobacteria could also use ndh2 or nuo? The fact that ndh is essential implies that mycobacteria do not have another mechanism to recycle NADH during normal aerobic growth. Alternatively, this is the only NADH dehydrogenase that is operating under these growth conditions and the activity of this enzyme and subsequent electron transfer (and proton translocation) to oxygen is what fuels the essential F₁F_o-ATP synthase of mycobacteria (Tran and Cook, 2005).

The mechanisms used by mycobacteria to recycle NADH under either hypoxia or anaerobic conditions remain unknown. Compounds that target either the F₁F_o-ATP synthase or NDH-2 are bactericidal towards hypoxic non-replicating M. tuberculosis suggesting that the respiratory chain is essential for the recycling of NADH under these conditions (Rao et al., 2008).

11.4. Aerobic respiratory pathways in mycobacteria

During aerobic respiration, electrons from the oxidation of either NADH (via NADH:menaquinone oxidoreductases) or succinate (via succinate dehydrogenase sdhABCD) flow into the menaquinone_(oxid)-menaquinol_(red)pool (Fig. 7). In mycobacteria, menaquinol can transfer electrons either directly to a cytochrome bd-type menaquinol oxidase (encoded by cydABCD) or through a cytochrome c pathway (Boshoff and Barry, 2005; Kana et al., 2001; Matsoso et al., 2005). The cytochrome bd branch is the bioenergetically less efficient branch (non-proton translocating) and is synthesized at low oxygen tensions (Kana et al., 2001). The cytochrome c pathway consists of a menaquinol-cytochrome c oxidoreductase termed the bc₁complex (encoded by the qcrCAB operon) and an aa₃-type cytochrome c oxidase (encoded by ctaBCDE) belonging tothe haem-copper respiratory oxidase family (Boshoff and Barry, 2005; Matsoso et al., 2005). The cytochrome c oxidase functions as a proton pump and may form a “supercomplex” with menaquinol cytochrome c oxidoreductase (Matsoso et al., 2005). Support for this hypothesis comesfrom the observation that such supercomplexes have been reported in other actinomycetes such as C. glutamicum(Niebisch and Bott, 2003). In addition to cytochrome bd and an aa₃-type cytochrome c oxidase, the M. smegm*tis respiratory chain has been proposed to contain a third possible respiratory branch terminating in the YthAB (bd-type) menaquinol oxidase (Kana et al., 2001). The bc₁-aa₃ pathway is the major respiratory route in mycobacteria under standard aerobic culturing conditions (Matsoso et al., 2005). Matsoso et al. (2005) have demonstrated that disruption of this pathway in M. smegm*tis is accompanied by a constitutive upregulation of the cytochrome bd-type menaquinol oxidase. In M. tuberculosis, the bc₁-aa₃ pathway is essential for growth suggesting an inability of this bacterium to adapt in a manner analogous to M. smegm*tis. The aa₃ branch is also proposed to contain two ctaD alleles in M. smegm*tis vers us the one in M. tuberculosis, suggesting alternate isoforms of cytochrome c oxidase in M. smegm*tis(Kana et al., 2001).

11.5. Generation of an electrochemical gradient of protons (Δμ_H+) in mycobacteria

When growing aerobically at an external pH of 7.0, M. smegm*tis and M. bovis BCG generate an Δμ_H+ of approximately −180 mV which appears to be of a significant magnitude to drive proton-coupled bioenergetic processes (eg. ATP synthesis, solute transport, etc) (Rao et al., 2001). Growth of mycobacteria is sensitive tothe electrogenic proton translocator carbonyl cyanide m-chlorophenylhydrazone (CCCP) demonstrating that a Δμ_H+ is indispensable for normal growth. Moreover, growth is inhibited by the F-type ATP synthase inhibitor NN′,-dicyclohexylcarbodiimide (DCCD) further supporting the role of the Δμ_H+ in driving ATP synthesis by the membrane-bound F₁F_O-ATP synthase. While proton translocation via the respiratory chain generates the Δμ_H+ during respiration with oxygen as the terminal electron acceptor, it is not clear how the Δμ_H+ is established in the absence of oxygen under anaerobic growth conditions. Anaerobic bacteria are able to generate a significant Δμ_H+ (−100 mV) using their membrane-bound F₁F_o-ATP synthase in the ATP hydrolysis direction (Dimroth and Cook, 2004). The ATPase activity (proton pumping) of the enzyme is fuelled by ATP produced by substrate level phosphorylation. The F₁F_O-ATP synthase of M. tuberculosis appears to have latent ATPase activity when purified suggesting that the enzyme may not function in the ATP hydrolysis direction (Higashi et al., 1975). Whether the enzyme is also latent in actively growing cells is not known and therefore the potential exists for this enzyme to function as a primary proton pump in the absence of oxygen and a functional respiratory chain to generate the Δμ_H+. To our knowledge, the effect of DCCD on anaerobic cultures of mycobacteria has not been determined to address this hypothesis. Rao et al. (2008) have reported that hypoxic, non -replicating M. tuberculosis generate a total proton motive force of −113 mV, −73 mV of electrical component (Δψ) and −41 mV of ZΔpH. The addition of thioridazine, a compound that targets NDH-2, results in dissipation of the electrical potential and significant cell death suggesting that NADH is an important electron donor for the generation of the Δψ under hypoxic conditions. The addition of R207910 (TMC207), a specific inhibitor of the F₁F_o-ATP synthase was bactericidal against hypoxic, non-replicating M. tuberculosis, but had no effect on the Δψ (Raoet al., 2008), an observation consistent with the latent ATPase activity of this enzyme (see above).

A potential solution to the generation of a Δμ_H+ under hypoxia is the use of a respiratory nitrate reductase (Fig. 7). Breakdown of nitric oxide in mammalian tissue would provide a source of nitrate that could be used as an alternative electron acceptor. A transport system for the exchange of nitrate and nitrite into and out of the cell are present in the genome of M. tuberculosis (e.g. NarK2) (Cole, 1998). M. tuberculosis contains genes (narGHJI) that encode for a putative membrane-bound molybdenum-containing nitrate reductase complex similar to the corresponding narGHJI operon of E. coli. Moreover, the narGHJI operon of M. tuberculosis is able to functionally complement a nar mutant of E. coli to grow on glycerol and reduce nitrate anaerobically (Sohaskey and Wayne, 2003). Importantly however, the expression of narGHJI operon in M.tuberculosis is not upregulated in response to either hypoxia or stationary phase (Sohaskey and Wayne, 2003). Furthermore, Sohaskey and Wayne (2003) demonstrate that overexpression of recombinant M. tuberculosis nitrate reductase in either M. tuberculosis or M. smegm*tis (low nitrate reductase) does not confer the ability of these cells to grow anaerobically ie. no growth of either species is observed with nitrate anaerobically even though the nitrate reductase activity of whole cells increases (Sohaskey and Wayne, 2003). The genome of M. tuberculosis also lacks orthologs of FNR which, in combination with NarL, are responsible for the transcriptional activation of the narGHJI operon anaerobically in E. coli (Unden and Bongaerts, 1997). A putative NarL (Rv0884c) has been indentifed in M. tuberculosis but the promoter of the narGHJI lacks consensus -like binding sites for this regulatory protein. Based on these observations, it is apparent that this enzyme does not support anaerobic growth of mycobacteria and therefore the role of this enzyme in the physiology of mycobacteria is unclear. Given the proposed membrane-bound location of the enzyme and the proton-pumping activity of the E. coli enzyme, perhaps the primary role of the mycobacterial enzyme is to generate a Δμ_H+ when the concentration of oxygen is low, and hence its activity increases but not its expression. An alternative role for nitrate reductase may be maintaining the redox balance of the cell during conditions of hypoxia. Sohaskey (2008) has reported that exogenously supplied nitrate has no effect on long-term persistence during gradual oxygen depletion, but played an important role during rapid adaptation to hypoxia (< 18 h). This effect required a functional nitrate reductase, suggesting that nitrate reduction may play a role in protecting cells during sudden changes in oxygen concentration leading to disruption of aerobic respiration. Sohaskey (2005) proposes a role for NarK2 in sensing the redox state of the cell such that nitrate is transported into the cell under reducing, but not oxidizing conditions.

The role of endogenous electron acceptors may also fuel the generation of a membrane potential (Δψ) in the absence of exogenous acceptors. Mycobacteria utilize menaquinone/menaquinol as a conduit between electron-donating and -accepting reactions. Menaquinone has a lower midpoint redox potential (E_m= −74 mV) compared to ubiquinone (E_m= +113 mV) and is ideally poised to donate electrons to fumarate during anaerobic conditions (Cecchini et al., 2002). Fumarate reductase (encoded by frdABCD) is present in M. tuberculosis and has been shown to be upregulated during carbon starvation and oxygen depletion (Betts et al., 2002) and in macrophages (Schnappinger et al., 2003), suggesting a role for this enzyme in persistence (Fig. 7). A recent study suggests that fumarate may be an important endogenous electron acceptor for energy production and maintenance of redox balance (oxidation of NADH to NAD⁺) in hypoxic nonreplicating mycobacteria, but this remains to be experimentally validated (Rao et al., 2008). Interestingly, the use of fumarate as an electron acceptor in E. coli requires complex I, and expression of the nuo operon is stimulated by the presence of fumarate (Unden and Bongaerts, 1997).

11.6. ATP synthesis by mycobacteria

In M. tuberculosis, ATP is synthesized via substrate level phosphorylation and oxidative phosphorylation using the membrane-bound F₁F_o-ATP synthases (encoded by the atpBEFHAGDC operon). No atpI gene is present in the operon, but an ORF (i.e. Rv1303) potentially may play this role being located immediately upstream of the atp locus. The F₁F_o-ATP synthase catalyzes ATP synthesis by utilizing the electrochemical gradient of protons to generate ATP from ADP and inorganic phosphate (P_i) and operates under conditions of a high Δμ_H+ and low intracellular ATP. The enzyme is also capable of working as an ATPase, hydrolyzing ATP to pump protons from the cytoplasm to the outside of the cell and operates under conditions of high intracellular ATP and an overall low Δμ_H+. In M. smegm*tis, the atp operon has been shown to be constitutively expressed and an increase in expression is noted upon growth with non-fermentable substrates that are strictly coupled to oxidative phosphorylation (e.g. succinate) compared to fermentable carbon sources like glucose (Tran and Cook, 2005). The F₁F_o-ATP synthase in M. tuberculosis and M. smegm*tis has been shown to be essential for growth (Sassetti et al., 2003; Tran and Cook, 2005). In other bacteria, the F₁F_o-ATP synthase has been shown to be dispensable for growth on fermentable carbon sources (Friedl et al., 1983; Santana et al., 1994), where increased glycolytic flux can compensate for the loss of oxidative phosphorylation. This strategy does not appear to be exploited by M. smegm*tis: the F₁F_o-ATP synthase is essential for growth even on fermentable substrates, suggesting that ATP production from substrate level phosphorylation alone, despite increased glycolytic flux, may be insufficient to sustain growth of these bacteria. This would be reflected by an extraordinarily high value for the amount of ATP required to synthesize a mycobacterial cell, a possibility that requires further investigation. Alternatively, or in conjunction with a high ATP demand for growth, the ATP synthase may be an obligatory requirement for the oxidation of NADH by providing a sink for translocated protons during NADH oxidation coupled to oxygen reduction. Such strict coupling would imply that mycobacteria do not support uncoupled respiration, either they lack a conduit for proton re-entry in the absence of the F₁F_o-ATP synthase or they are unable to adjust the proton permeability of the cytoplasmic membrane to allow a futile cycle of protons to operate. In this context, the cytoplasmic membrane of M. smegm*tis is extremely impermeable to protons (Tran et al., 2005).

Several new anti-tubercular drugs have recently been reported (Andries et al., 2005; Weinstein et al., 2005). It is particularly noteworthy that both classes of drugs target oxidative phosphorylation in mycobacteria. The first class, diarylquinolines, have beenshown to target the F ₁F_o-ATP synthase to inhibit ATP synthesis by the enzyme (Andries et al., 2005; Koul et al., 2007). Given the essential role of this enzyme for mycobacterial growth (Tran and Cook, 2005), even on fermentable carbon sources, the ATP synthase would appear to represent a valid target. Genome sequencing of both M. tuberculosis and M. smegm*tis mutants that are resistant to diarylquinolines (i.e. R207910) revealed that the target of these compounds is the oligomeric c ring (encoded by atpE) of the enzyme. Purified c ring from M. smegm*tis binds R207910 with a K _D of 500 nm, and modelling studies suggest that R207910 blocks proton transfer by the enzyme (de Jonge et al., 2007). The second class, phenothiazine analogs, target the NADH: menaquinone oxidoreductase (Weinstein et al., 2005) suggesting that NADH oxidation via aerobic respiration coupled to oxidative phosphorylation is essential for the growth of mycobacteria.

11.7. Other mechanisms to recycle NADH during hypoxia

Hydrogenases are found in a wide variety of microorganisms and catalyse the reversible conversion of H₂to 2H ⁺and 2e⁻ thus enabling the bacterium to utilize H ₂ as a source of reducing equivalents. Conversely, some bacteria use the enzyme to reduce protons to H₂ thereby releasing the reducing equivalents. Hydrogen evolution is a mechanism commonly employed by anaerobic bacteria to recycle reducing equivalents obtained from anaerobic degradation of organic substrates. However, even strictly aerobic bacteria have been shown to produce H₂ under anaerobic conditions (Kuhn et al., 1984). It has been shown that M. smegm*tis, among other mycobacterial species, can oxidize molecular hydrogen in the presence of carbon monoxide, implying that M. smegm*tis expresses a functional hydrogenase (King, 2003). No studies have reported the ability of mycobacteria to produce hydrogen, but analysis of the M. tuberculosis genome reveals the presence of a gene cluster (viz. Rv0082 to Rv0087) encoding components of a putative hydrogenase and formate hydrogen lyase that is upregulated during infection of human macrophage-like THP-1 cells (Fontan et al., 2008). Moreover, the transcription of the early genes of the hycP/hycQ containing operon was shown to be upregulated during anaerobic adaptation in several studies (Bacon et al., 2004; Park et al., 2003; Sherman et al., 2001; Voskuil et al., 2003; Voskuil et al., 2004). The putative hydrogenase gene cluster of M. tuberculosis shows hom*ology to components of hydrogenase 4 and 3 complex of Escherichia coli, the latter of which has been shown to catalyze hydrogen evolution at acidic pH (Mnatsakanyan et al., 2004). It is tempting to propose that the acidic, hypoxic and lipid-rich environment in the macrophages might require the expression of a hydrogenase complex in M.tuberculosis to help with the recycling of reducing equivalents under these conditions. The requirement for a low potential redox carrier in hydrogen production could be satisfied by reactions that are coupled to ferredoxins and not NADH. For example, M. tuberculosis contains a putative pyruvate:ferredoxin oxidoreductase (Rv2454c/Rv2455c) that would catalyse the oxidation of pyruvate to acetyl-CoA with reduced ferredoxins acting as the electron donor for hydrogen production. The role of hydrogen in the cycling of reducing equivalents in mycobacteria during hypoxia warrants investigation.

11.8. Control of energy generation in mycobacteria

In bacteria there is often a trade off between obtaining the maximum energy yield from a substrate and the maximum flux (rate of ATP production) or growth rate (Pfeiffer et al., 2001). A cell that uses a pathway with a high yield and low rate (e.g. respirers) can produce more ATP from a given amount of substrate compared to a bacterium that produces ATP at a higher rate but a lower yield (e.g. fermenters). The growth yield of bacterial cultures has been used to estimate the efficiency of energy generation during respiration or fermentation. Mycobacteria would appear to adopt the first strategy where the cells grow slowly using oxidative phosphorylation to generate large amounts of ATP at a slow rate. Furthermore, this strategy appears to be employed by both fast growing and slow growing mycobacteria; the growth yield on glycerol is comparable between M. smegm*tis and M. bovis BCG at similar growth rates (see above).

In order for mycobacteria to utilize oxygen efficiently and obtain the maximum growth yield on a particular carbon and energy source, there must be co-ordinate regulation of terminal respiratory oxidase expression. For example, In E. coli cytochrome bo (K_m for oxygen in the micromolar range) and cytochrome bd(K _mfor oxygen in the nanomolar range) (D’Mello et al., 1995; D’Mello et al., 1996) are coordinately regulated by the ArcBA system and FNR (Cotter et al., 1997). Cytochrome bo is synthesized at high oxygen tension (optimal between 15 to 100% air saturation) and repressed as the oxygen concentrations decreases (Tseng et al., 1994). This coincides with the induction of cytochrome bd at 7% air saturation, which is turned off (FNR-mediated repression) once the cells enter anaerobiosis (Cotter et al., 1997; Tseng et al., 1994). E. coli also uses non -coupling dehydrogenases (NDH-2) during aerobic growth that allow a fast metabolic flux (fast growth rate) and switches to coupling dehydrogenases (NDH-1) during anaerobic growth with fumarate (Unden and Bongaerts, 1997).

Mycobacteria also adopt regulation of oxidase expression to match oxygen supply. Under conditions of low oxygen tension (ca. 1 % air saturation), cytochrome bd is induced in M. smegm*tis as the transition to anaerobiosis is approached (Kana et al., 2001), a value that is 10-fold lower than that observed in E. coli (ca. 10% air saturation). In M. tuberculosis, cytochrome bd is upregulated in the early stages of NRP-1 (i.e. decreasing oxygen) (Voskuil et al., 2004). A strategy that appears to be invoked by mycobacteria is one of down-regulation or a slowing of metabolism as cells enter NRP-1 and NRP-2. Transcriptional analysis of M. tuberculosis in the macrophage (phagosomal environment) has revealed that NDH-1, menaquinol-cytochrome c oxidoreductase and the ATP synthase are all down regulated when compared to cells growing exponentially suggesting the reduced need for energy generation during bacteriostasis i.e., the growth state of intraphagosomal M. tuberculosis(Schnappinger et al., 2003). Consistent with these observations is the repression of these operons during starvation. In contrast, fumarate reductase, nitrate reductase and NDH-2 are all upregulated under these conditions (Schnappinger et al., 2003). Whilst these proteins do not appear to contribute to increased energy production, it has been suggested that they may play a pivotal role in the recycling of NAD⁺ as a result of β-oxidation of fatty acids (Schnappinger et al., 2003). Furthermore, the induction of ald, encoding L-alanine dehydrogenase may also contribute to the recycling of NAD⁺ through the reductive amination of pyruvate to alanine and subsequent oxidation of NADH.

While various components of the oxidative phosphorylation machinery are down-regulated (e.g. ATP synthase) during persistence or dormancy, this does not mean they are entirely absent. Koul et al. (2008) demonstrate that R207910 kills dormant M. tuberculosis as effectively as aerobically grown M. tuberculosis with the same target specificity. Moreover, the authors show that dormant M. tuberculosis do indeed have residual ATP synthase activity.

12.1. Large-scale omics analyses and computational analyses of such data

12.2. Towards obtaining a system understanding through mathematical modeling

Through systems biology omics analyses, as well as the traditional approaches to biology, molecular components and the interactions between these components can be identified. In order to obtain an understanding about how these interactions actually result in system behaviour, i.e. how a certain environment makes a cell express a certain phenotype, the mere knowledge about biological components and their interactions has to be quantitatively integrated into mathematical models and analyzed by computational means (Kitano 2002; Stelling 2004). This approach is typically referred to as the bottom-up branch of systems biology (Bruggeman and Westerhoff, 2007). For M. tuberculosis, two different modelling approaches have been applied to study bacterial metabolism and physiology:

12.3. Outlook and challenges for systems biology with M. tuberculosis

Systems biology as a new biological discipline not only provides novel large-scale measurement techniques, but ultimately aims to achieve quantitative understanding of biological systems. Due to the complexity of biological systems, such understanding can most likely only be achieved by using mathematical models that can quantitatively and dynamically describe molecular interactions. In the mycobacteria field, only recent first efforts along these lines have been made.

It should be noted that mathematical models, by means of computational simulations, can generate novel hypotheses about the modeled biological system. These hypotheses can then be experimentally tested. Unfortunately, in the papers outlined above very little experimental follow-up has been performed to test the derived hypotheses. In order to realize the full potential of systems biology for mycobacterial research, two communities, i.e. those with the computational expertise and mycobacterial biologists, will need to work together to harvest the potential inherent to the systems biology modeling and computational analyses.

n_c(a) =n_c(a=0) +e^-a•;n2

(1A)

nc(a)=nc(a=0)a=0∫a=0e-a•;n2da

(2A)

n_c =0.72 n_c(a=0)

(3A)

a_r(ORFi) =(t_G1 +γ_ORF(i)•τ_g)/t_D

(4A)

nORF(i)/c=(nORF(i)g/nc){nc(a=0)0∫ar(ORFi)e-a•ln2•da+2nc(a=0)ar(ORFi)∫a=1e-a•ln2•da}

(5A)

n_ORF(i)/c =(n_ORF(i)/g/(0.72ln2)){[e^-a•ln2]_a^ar(ORFi) +2[e^-a•ln2]¹_ar(ORFi)}

(6A)

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