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27 Jul 2003 12:20 AR AR195-MI57-07.tex AR195-MI57-07.sgm LaTeX2e(2002/01/18) P1: IKH 10.1146/annurev.micro.57.030502.090820 Annu. Rev. Microbiol. 2003. 57:155–76 doi: 10.1146/annurev.micro.57.030502.090820 c 2003 by Annual Reviews. All rights reserved Copyright ° First published online as a Review in Advance on May 1, 2003 NITROGEN ASSIMILATION AND GLOBAL REGULATION IN ESCHERICHIA COLI Annu. Rev. Microbiol. 2003.57:155-176. Downloaded from arjournals.annualreviews.org by WIB6332 - University of Saarland on 01/04/10. For personal use only. Larry Reitzer Department of Molecular and Cell Biology, The University of Texas at Dallas, Richardson, Texas 75080-0688; email: [email protected] Key Words Ntr response, σ 54, Lrp, ppGpp, metabolic integration ■ Abstract Nitrogen limitation in Escherichia coli controls the expression of about 100 genes of the nitrogen regulated (Ntr) response, including the ammonia-assimilating glutamine synthetase. Low intracellular glutamine controls the Ntr response through several regulators, whose activities are modulated by a variety of metabolites. Ntr proteins assimilate ammonia, scavenge nitrogen-containing compounds, and appear to integrate ammonia assimilation with other aspects of metabolism, such as polyamine metabolism and glutamate synthesis. The leucine-responsive regulatory protein (Lrp) controls the synthesis of glutamate synthase, which controls the Ntr response, presumably through its effect on intracellular glutamine. Some Ntr proteins inhibit the expression of some Lrp-activated genes. Guanosine tetraphosphate appears to control Lrp synthesis. In summary, a network of interacting global regulators that senses different aspects of metabolism integrates nitrogen assimilation with other metabolic processes. CONTENTS INTRODUCTION . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . PHYSIOLOGICAL CONTEXT . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Nitrogen Sources . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Ammonia Assimilation and Intracellular Nitrogen Donors . . . . . . . . . . . . . . . . . . . Quantitative Nitrogen Requirements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . NITROGEN LIMITATION AND THE NTR RESPONSE . . . . . . . . . . . . . . . . . . . . . . Proteins Required for General Nitrogen-Limited Growth . . . . . . . . . . . . . . . . . . . . . Regulation of GS Activity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . The Ntr Regulatory Cascade . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Differential Expression of Ntr Genes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Functions of the Ntr Response and Nac . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . THE σ 54 REGULON: ITS FUNCTION AND RELATION TO THE NTR RESPONSE . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Properties of σ 54 -Dependent Genes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . The Functions of σ 54 -Dependent Genes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . METABOLIC INTEGRATION: NITROGEN ASSIMILATION AND CARBON METABOLISM . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0066-4227/03/1013-0155$14.00 156 156 156 156 157 159 159 160 161 164 165 166 166 167 167 155 27 Jul 2003 12:20 156 AR AR195-MI57-07.tex AR195-MI57-07.sgm LaTeX2e(2002/01/18) P1: IKH REITZER Lrp . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Cyclic AMP . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . EINtr -NPr-EIINtr . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . GUANOSINE TETRAPHOSPHATE, AMINO ACID SYNTHESIS, AND AMMONIA ASSIMILATION . . . . . . . . . . . . . . . . . . . . . . . . . . CONCLUDING REMARKS: INTERACTIONS BETWEEN GLOBAL REGULATORS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 167 168 168 170 171 Annu. Rev. Microbiol. 2003.57:155-176. Downloaded from arjournals.annualreviews.org by WIB6332 - University of Saarland on 01/04/10. For personal use only. INTRODUCTION A group of scientists, which included Lavoisier, gave the name azote (without life) to the major inert component of air—a name that is still recognizable in many nitrogen-containing compounds. The name nitrogen was derived from niter (potassium nitrate), a not-so-inert component of gunpowder. Nitrogen is present in many intracellular metabolites and can be assimilated from inorganic or organic sources. Its assimilation from inorganic sources requires reduction to ammonia, followed by incorporation into intracellular metabolites. The appropriate distribution of nitrogen among various pathways usually involves specific or local regulatory mechanisms, such as endproduct inhibition or endproduct-mediated transcriptional control. However, a few global regulators control the expression of genes from several pathways and thereby coordinate metabolism. This review focuses on nitrogen assimilation in Escherichia coli, its regulation, and the role of global regulators in this regulation. A major theme of this review is the integration of nitrogen assimilation with other aspects of metabolism. PHYSIOLOGICAL CONTEXT Nitrogen Sources Bacteria assimilate a variety of inorganic nitrogen sources, but Escherichia coli assimilates only ammonia aerobically. The ability to assimilate particular organic nitrogen sources also depends on the organism. Organic nitrogen sources are usually monomeric units of macromolecules (e.g., amino acids or nucleobases) or compounds derived from them (e.g., agmatine or putrescine). E. coli can assimilate nitrogen from adenine, adenosine, agmatine, L- and D-alanine, allantoin (anaerobically), γ -aminobutyrate, ammonia, arginine, asparagine, aspartate, cytidine, cytosine, glucosamine, glutamine, glutamate, glycine, ornithine, proline, putrescine, L- and D-serine, threonine, and a few other compounds (76). Ammonia supports the fastest growth rate and is therefore considered the preferred nitrogen source for E. coli. Ammonia Assimilation and Intracellular Nitrogen Donors The primary products of ammonia assimilation are glutamate and glutamine, the major intracellular nitrogen donors. Glutamine provides nitrogen for purines, 27 Jul 2003 12:20 AR AR195-MI57-07.tex AR195-MI57-07.sgm LaTeX2e(2002/01/18) REGULATION OF NITROGEN METABOLISM P1: IKH 157 pyrimidines, asparagine (when intracellular ammonia is low), tryptophan, histidine, glucosamine, p-aminobenzoate, and arginine (via carbamoyl phosphate). Glutamate donates nitrogen for most of the E. coli transaminases. Glutamate dehydrogenase (GDH) or glutamine synthetase (GS) assimilates the bulk of ammonia in E. coli (75). Annu. Rev. Microbiol. 2003.57:155-176. Downloaded from arjournals.annualreviews.org by WIB6332 - University of Saarland on 01/04/10. For personal use only. α-ketoglutarate + NH3 + NADPH → glutamate + NADP+ (GDH) glutamate + ATP + NH3 → glutamine + ADP + PO−2 4 (GS) + glutamine + α-ketoglutarate + NADPH → 2 glutamate + NADP (GOGAT) If GS assimilates ammonia, then glutamate synthase (GOGAT) catalyzes glutamate formation. E. coli contains both the GDH- and the GS-dependent pathways, and they are not physiologically equivalent. The ATP-consuming GS-GOGAT pathway in E. coli is used in energy-rich environments, whereas the GDH pathway is employed in energy-limited (presumably nitrogen-rich) environments (39, 40). The GS-GOGAT pathway is more appropriate for ammonia assimilation in a nitrogen-limited environment, since GS has a much lower Km for ammonia than GDH. Ammonia assimilation by the GS-GOGAT pathway accounts for an astonishing 15% of the cell’s ATP requirement, which is calculated from the fact that synthesis of 1 g of E. coli requires 40 mmoles of carbon, about 11 mmoles of nitrogen, and 72 mmoles of ATP (56). [GS is highly conserved (52), which is consistent with the possibility that it assimilates ammonia in most microbial organisms. Nonetheless, it appears that alanine dehydrogenase assimilates ammonia in Rhizobium leguminosarum bacteroids, which subsequently secrete alanine to the pea plant (1).] Quantitative Nitrogen Requirements Once ammonia is assimilated, it must be distributed in the correct ratios to a variety of compounds. The biosynthetic requirement of each nitrogen-containing metabolite (how much must be synthesized) can be calculated from the chemical composition of E. coli (the amount of each component that would be obtained from complete hydrolysis of whole cells) (Table 1). The difference between the biosynthetic requirement and the cellular composition can be quantitatively large. For example, 1 g of E. coli contains an estimated 250 µmoles of glutamate in protein. However, 810 µmoles of glutamate are required for arginine, glutamine, proline, the polyamines, and peptidoglycan synthesis. Furthermore, glutamate is the nitrogen donor for at least 1 nitrogen for 11 amino acids and the polyamines, which requires 7108 µmoles of glutamate. Therefore, the biosynthetic requirement for glutamate is 8168 µmoles. Three observations are worth noting from these calculations. First, over one third of a cell’s nitrogen is present in guanine nucleotides (1142 µmoles of nitrogen, or 11%), arginine (1124 µmoles, or 11%), adenine nucleotides (948 µmoles, or 9%), and lysine (652 µmoles, or 6%). Second, more carbon skeletons must be synthesized for aspartate and serine than for other amino acids (Table 1, footnote d). 27 Jul 2003 12:20 158 AR AR195-MI57-07.tex AR195-MI57-07.sgm LaTeX2e(2002/01/18) P1: IKH REITZER TABLE 1 Cellular composition and biosynthetic requirements for nitrogena Annu. Rev. Microbiol. 2003.57:155-176. Downloaded from arjournals.annualreviews.org by WIB6332 - University of Saarland on 01/04/10. For personal use only. Compositionb C for other compoundsc alanine 488 arginine 281 asparagine 229 aspartate 229 1565 1794 cysteine 87 153 240 250 810 1060 250 glutamate glutamine 250 glycine 582 histidine isoleucine 55 C skeleton subtotald N donatione µmoles Ng 543 543 488 281 281 1124 229 458 2772 229 229 418 Totalf 1000 979 240 87 7108 8168 250 10226 10476 500 1000 582 90 90 90 270 276 276 276 276 leucine 428 428 428 428 lysine 326 28 354 354 652 methionine 146 7 153 153 146 phenylalanine 176 176 176 176 proline 210 210 210 210 serine 205 1409 1614 1614 205 threonine 241 276 517 517 241 tryptophan 54 54 54 108 tyrosine 131 131 131 131 valine 402 402 402 402 AMP + dAMP 190 948 GMP + dGMP 228 1142 CMP + dCMP 151 454 UMP + dTMP 161 321 Other compoundsh 380 455 All units are µmoles per g dry weight of E. coli grown in glucose-ammonia minimal medium. It is assumed that glutamate is made by the GS-glutamate synthase route. a b Chemical composition taken from (65), which should be consulted for assumptions. For the amino acids the content from proteins is presented. c This refers to syntheses that require the carbon skeleton of the amino acid, e.g., methionine synthesis requires aspartate. d This number is the amount of the carbon skeleton that must be synthesized. e After nitrogen donation it is not necessary to resynthesize the carbon skeleton, which is why this number is not contained within the previous column. For example, 7108 µmoles of α-ketoglutarate for glutamate synthesis come from deamination of glutamate, and only 1060 µmoles come from citric acid cycle components. f This column shows the biosynthetic requirement, i.e., the total µmoles of each amino acid synthesized per g E. coli. g This number is the number of nitrogen atoms in each compound times the amount in the composition column (footnote b). The sum of nitrogen content is about 10,300 µmoles of nitrogen atoms per gram. h These include putrescine, spermidine, ethanolamine, glucosamines, and cell wall components. The amounts for each of these components have been presented (65). 27 Jul 2003 12:20 AR AR195-MI57-07.tex AR195-MI57-07.sgm LaTeX2e(2002/01/18) REGULATION OF NITROGEN METABOLISM P1: IKH 159 Annu. Rev. Microbiol. 2003.57:155-176. Downloaded from arjournals.annualreviews.org by WIB6332 - University of Saarland on 01/04/10. For personal use only. Both amino acids provide carbon and nitrogen for other amino acids and nucleotides. These properties distinguish aspartate and serine from other amino acids and might provide a metabolic basis for the specificity of the major chemotactic receptors. Third, the biosynthetic requirement for glycine (1000 µmoles) is almost equal to that for C1-derivatives in purine, thymine, and methionine synthesis (1104 µmoles). This stoichiometry necessarily couples nucleotide and amino acid synthesis and may obviate the need for more complex regulatory mechanisms. It is conceivable that the amazingly complex regulation directed at the glycine cleavage enzyme (38) is sufficient for fine-tuning C1 unit synthesis. NITROGEN LIMITATION AND THE NTR RESPONSE Proteins Required for General Nitrogen-Limited Growth Growth in a minimal medium with a single organic nitrogen source is slower than that with ammonia. Such growth affects the expression of about 100 genes. These collective changes are considered the nitrogen-regulated (Ntr) response. Loss of a few metabolic enzymes can prevent the utilization of a variety of nitrogen sources. This section considers those enzymes. GLUTAMINE SYNTHETASE Nitrogen-limited growth generally requires both enzymes of the GS-GOGAT pathway of ammonia assimilation. GS catalyzes the only reaction of glutamine formation, and its complete loss results in glutamine auxotrophy (75). Basal glnA (GS-encoding) expression from the glnAp1 promoter (which occurs in a glnG mutant) is sufficient for growth with ammonia and a few nitrogen sources (aspartate and putrescine), but not with other single nitrogen sources (A. Kiupakas & L. Reitzer, unpublished observation). Expression of the glnALG (or glnA-ntrBC) operon from the glnAp2 promoter is required for optimal GS synthesis and for high levels of two important regulators of the Ntr response, nitrogen regulator II (NRII, also called NtrB) and nitrogen regulator I (NRI, also called NtrC), which are required for maximal GS synthesis. GLUTAMATE SYNTHASE The gltBD operon codes for the two subunits of GOGAT (75). The proposals that the E. coli gltBD operon also contains gltF (23) and that GltF regulates the Ntr response (23, 24) have not withstood close scrutiny (35, 36). gltBD mutants are pleiotrophically defective in nitrogen source utilization and are said to have an Asm− phenotype (76). E. coli and Klebsiella pneumoniae mutants can still utilize a few nitrogen sources either because they can readily generate glutamate (e.g., asparagine, aspartate, glutamate, glutamine) or because they generate ammonia so rapidly that GDH can synthesize glutamate (e.g., D-serine) (93). Two different explanations can account for the Asm− phenotype (35). The glutamine-excess hypothesis proposes that GS assimilates ammonia into glutamine, which accumulates in the absence of GOGAT (the major glutaminemetabolizing enzyme) and prevents Ntr gene expression. The products of Ntr genes 27 Jul 2003 12:20 160 AR AR195-MI57-07.tex AR195-MI57-07.sgm LaTeX2e(2002/01/18) P1: IKH REITZER Annu. Rev. Microbiol. 2003.57:155-176. Downloaded from arjournals.annualreviews.org by WIB6332 - University of Saarland on 01/04/10. For personal use only. would normally generate glutamate independent of both GOGAT and GDH (e.g., arginine catabolism). Alternatively, the glutamate-starvation hypothesis proposes that glutamate starvation prevents Ntr induction (35). A mechanism by which this occurs is not stated. A hybrid hypothesis is that glutamine accumulation prevents Ntr gene expression until glutamate starvation stops metabolism and growth. In any case, the arguments are complex and subject to several untested assumptions (35). It is possible that the explanation for the Asm− phenotype involves still other factors, such as an indirect effect of glutamate starvation. ASNB, NADE, AND THE AMIDOTRANSFERASES Defects in several nonassimilatory enzymes can also have pleiotropic effects in nitrogen source utilization. Enteric bacteria have ammonia- and glutamine-dependent asparagine synthetases, AsnA and AsnB, respectively. A K. aerogenes asnB mutant fails to grow in ammoniarestricted nitrogen-limited environments because AsnA is insufficient for asparagine synthesis (77). NAD synthetases in E. coli and other bacteria are often, but not always, ammonia dependent (19, 82). A nadE (formerly nit) mutant with diminished NAD synthetase fails to grow in ammonia-restricted environments (15, 82). Glutamine-dependent amidotransferases with defective glutamine binding or amide transferase can inefficiently use ammonia as an alternative substrate (61). Mutants with such defective amidotransferases fail to grow with organic nitrogen sources (L. Reitzer, unpublished observation). In summary, mutants with altered enzymes that require a high concentration of ammonia are generally defective in utilization of organic nitrogen sources. Regulation of GS Activity GS synthesizes glutamine and assimilates ammonia, and the ratio of these functions depends on the environment. If GDH assimilates ammonia, then glutamine’s amide provides 25% of cellular nitrogen and the function of GS is primarily anabolic. If the GS-GOGAT pathway assimilates ammonia, then glutamine’s amide provides almost 100% of cellular nitrogen and the assimilatory function is quantitatively three times more important. FEEDBACK INHIBITION The anabolic function of GS is controlled by cumulative feedback inhibition by alanine, glycine, serine, adenosine monophosphate (AMP), carbamoyl phosphate, cytidine triphosphate (CTP), glucosamine-6-phosphate, histidine, and tryptophan (28). These nine compounds are competitive inhibitors that bind to the glutamate or nucleotide substrate site (28, 57, 58). The last six require glutamine for their synthesis and can be considered products of glutamine metabolism. Although serine and glycine synthesis do not require glutamine, these two amino acids are still excellent indicators of glutamine sufficiency. Nucleotide synthesis accounts for 74% of the glutamine requirement (ignoring glutamate synthesis), which implies that nucleotide sufficiency indicates glutamine sufficiency. Nucleotide synthesis also accounts for 42% of the glycine biosynthetic 27 Jul 2003 12:20 AR AR195-MI57-07.tex AR195-MI57-07.sgm LaTeX2e(2002/01/18) REGULATION OF NITROGEN METABOLISM P1: IKH 161 Annu. Rev. Microbiol. 2003.57:155-176. Downloaded from arjournals.annualreviews.org by WIB6332 - University of Saarland on 01/04/10. For personal use only. requirement, and for most of the consumption of C1 derivatives. Therefore, high serine and glycine probably indicate nucleotide and glutamine sufficiency. Alanine is more difficult to rationalize as a GS inhibitor. ADENYLYLATION Covalent adenylylation is one mechanism that controls the assimilatory function of GS. Each of the 12 subunits of GS can be adenylylated on a tyrosine residue, which inactivates one subunit and enhances the susceptibility of the other subunits to cumulative feedback inhibition (87). In other words, adenylylation determines the extent to which GS is anabolic or assimilatory. Loss of the adenylylation system is a problem during the transition from a nitrogen-limited to an ammonia-containing (nitrogen-rich) environment, when fully induced unadenylylated GS rapidly depletes intracellular glutamate (53, 54). Such a loss is not a problem for nitrogen-sufficient cells (other mechanisms regulate GS synthesis) or for cells in which GS is normally not adenylylated (during nitrogen limitation or during the transition to steady-state nitrogen-limited growth). The Ntr Regulatory Cascade THE CENTRAL ROLE OF GLUTAMINE Nitrogen limitation increases GS specific activity, GS synthesis, and Ntr gene expression, whereas nitrogen sufficiency decreases all three. Several regulators and environmental factors control this coordinated response, which has been extensively reviewed (3, 11, 60, 69, 70, 75, 87). Measurements of intracellular glutamine and studies with highly purified uridylyltransferase (UTase)/uridylyl-removing enzyme (UR), the first enzyme of the Ntr regulatory cascade, suggest that the absolute concentration of glutamine controls the regulatory cascade (44, 46, 47). High glutamine stimulates UR activity, which removes uridine monophosphate (UMP) groups from PII-UMP and GlnK-UMP, whereas low glutamine stimulates UTase activity, which uridylylates PII and GlnK. Glutamine apparently binds to a single site on UTase/UR, from which it controls both activities (46). FUNCTIONAL REDUNDANCY: PII AND GLNK PII and GlnK have 67% amino acid identity and similar biochemical activities (5–7, 94). The phenotypes of glnB and glnK mutants, which lack PII and GlnK, respectively, differ only slightly from a wild-type strain (6). In contrast, a glnB glnK double mutant has serious metabolic problems, which result from overexpression of Ntr genes (6, 12). Furthermore, placing glnK under the glnB promoter and vice versa do not result in appreciable phenotypic differences (5). On the other hand, several observations suggest different functions for PII and GlnK. First, the properties of the two purified regulators with respect to Ntr control are not identical. Second, they are not synthesized at the same time. Third, during nitrogen-limited growth GlnK is more abundant than PII. Fourth, GlnK and PII form heterotrimers whose properties can differ from the homotrimers (33, 96). Finally, GlnK can have activities that PII does not have (26). 27 Jul 2003 12:20 Annu. Rev. Microbiol. 2003.57:155-176. Downloaded from arjournals.annualreviews.org by WIB6332 - University of Saarland on 01/04/10. For personal use only. 162 AR AR195-MI57-07.tex AR195-MI57-07.sgm LaTeX2e(2002/01/18) P1: IKH REITZER THE FUNCTION OF NTR REGULATORS IN DIFFERENT ENVIRONMENTS The functions of these regulators must be considered in the context of nitrogen-rich and nitrogen-limited environments and in the transitions between them (summarized in Figure 1). In nitrogen-rich (i.e., ammonia-containing) minimal medium, GS is adenylylated, glnA is expressed at a basal level, and other Ntr genes are not expressed. PII is in excess over GlnK because PII synthesis is constitutive, while glnK is not expressed (6, 17, 95). PII is mostly deuridylylated (33) and stimulates the adenylylating activity of adenylyltransferase (ATase) and dephosphorylation of NRI (via NRII), which prevents induction of Ntr genes. α-Ketoglutarate at physiological concentrations binds to unmodified PII and interferes with its interaction with ATase and NRII. Therefore, α-ketoglutarate is a signal of carbon sufficiency and relative nitrogen limitation, but only to the extent that PII is not uridylylated (i.e., in cells with high glutamine). During the transition to nitrogen-limited growth PII is in excess over GlnK and is rapidly uridylylated. PII-UMP stimulates deadenylylation of GS and fails to interact with NRII, which results in phosphorylation of NRI (3, 69). The initial level of NRI ∼ P is sufficient for activation of glnA, but not for other Ntr operons (4, 80). In the absence of NRII-stimulated dephosphorylation acetyl phosphate probably contributes to NRI phosphorylation (31). During steady-state nitrogen-limited growth the concentrations of two important regulators, GlnK and NRI, are higher. It can be deduced that GlnK becomes more abundant than PII on the basis of immunological assays and the predominance of GlnK homotrimers and (GlnK)2(PII)1 heterotrimers over (GlnK)1(PII)2 heterotrimers and PII homotrimers (26, 96). Both PII and GlnK are readily uridylylated in vivo and in vitro, and the heterotrimers are presumably rapidly uridylylated (7, 33, 94, 96). Purified GlnK-UMP slowly deadenylylates GS compared with purified PII-UMP (96). Deadenylylation in a strain with only GlnK-UMP might be a little slower than deadenylylation in a strain with both GlnK-UMP and PII-UMP, although this cannot be stated with certainty because cells with different levels of GS (because of their prior growth) were compared (94). The rate of deadenylylation suggests that the PII subunits in purified uridylylated heterotrimers are active (96). Because GlnK can substitute for PII and mediate Ntr induction (6), it can be deduced that GlnK-UMP, like PII-UMP, fails to interact with NRII, which results in net phosphorylation of NRI. Acetyl phosphate is not required for optimal glnA expression but is required for arginine −−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−→ Figure 1 The Ntr regulatory cascade: regulators and effectors. Regulation is considered for (a) steady-state growth in ammonia-containing minimal medium, (b) the transition from nitrogen-rich to nitrogen-limited growth, (c) steady-state nitrogenlimited growth, and (d) the transition from nitrogen-limited to nitrogen-rich growth. Abbreviations: ATase-A, adenylylating ATase; ATase-D, deadenylylating ATase; and αKG, α-ketoglutarate. Annu. Rev. Microbiol. 2003.57:155-176. Downloaded from arjournals.annualreviews.org by WIB6332 - University of Saarland on 01/04/10. For personal use only. 27 Jul 2003 12:20 AR AR195-MI57-07.tex AR195-MI57-07.sgm LaTeX2e(2002/01/18) REGULATION OF NITROGEN METABOLISM P1: IKH 163 27 Jul 2003 12:20 Annu. Rev. Microbiol. 2003.57:155-176. Downloaded from arjournals.annualreviews.org by WIB6332 - University of Saarland on 01/04/10. For personal use only. 164 AR AR195-MI57-07.tex AR195-MI57-07.sgm LaTeX2e(2002/01/18) P1: IKH REITZER utilization (and therefore for the ast operon) and presumably for other Ntr genes (31, 81). During the transition from a nitrogen-limited to a nitrogen-replete environment transcription of glnA, an Ntr gene, is rapidly abolished (78), and GS is rapidly adenylylated (94). GlnK is in excess over PII. Purified GlnK-UMP is deuridylylated 10 times slower than PII-UMP (7). However, because purified GlnK effectively stimulates NRII-mediated dephosphorylation of NRI (7), the accumulation of deuridylylated GlnK must be sufficient to prevent glnA expression (94). However, it is not clear whether GlnK stimulates GS adenylylation in vivo. The activity of purified GlnK in stimulating adenylylation is 40 times less than that of PII (7, 94). It is more likely that GlnK-independent ATase activity is sufficient for GS adenylylation (7, 33) because glutamine stimulates the adenylylating activity of ATase and nitrogen-limited cells without either GlnK or PII can rapidly adenylylate GS (6). In addition to these properties GlnK associates with AmtB, an ammonia transporter/facilitator, and inhibits its activity (26). PII also associates with AmtB but does not inhibit its activity (26). Therefore, ammonia transport should be impaired during the transition to an ammonia-containing, nitrogen-rich environment. These properties have interesting implications. If AmtB and GlnK are made stoichiometrically, then AmtB is in excess over PII. AmtB has the potential to sequester PII and GlnK or to allosterically affect their activities. If so, AmtB might regulate nitrogenase synthesis in K. pneumoniae, in which GlnK controls the activity of NifL (37, 45). In summary, glutamine ultimately controls the activities and interactions of the individual regulators of the Ntr regulatory cascade. Considering the importance of glutamine, it is probably not surprising that synthesis of the two glutamine-sensing regulators, UTase/UR and ATase, is constitutive. In contrast, nitrogen limitation controls the synthesis of the other regulators. Glutamine also determines whether other signals are sensed. In a high-glutamine environment α-ketoglutarate modifies the activities of unuridylylated PII and GlnK, and the products of glutamine metabolism inhibit GS. In a low-glutamine environment acetyl phosphate contributes to Ntr gene expression. Until recently, it was thought that a single protein, UTase/UR, sensed the ratio of glutamine to α-ketoglutarate. Such regulation permits the possibility of a high ratio (apparent nitrogen excess) with insufficient glutamine for numerous glutamine-dependent enzymes (functional nitrogen deficiency). Sensing the absolute concentration of glutamine avoids this problem. Instead, the ratio of glutamine to α-ketoglutarate is important only when glutamine is high and there is sufficient glutamine to drive the glutamine-dependent reactions. Differential Expression of Ntr Genes SEQUENTIAL EXPRESSION Several mechanisms permit differential expression of Ntr genes. The transition to nitrogen limitation induces Ntr genes sequentially. The low NRI ∼ P concentration initially present during the transition to nitrogenlimited growth is sufficient for expression of the glnA operon (80). However, a 27 Jul 2003 12:20 AR AR195-MI57-07.tex AR195-MI57-07.sgm LaTeX2e(2002/01/18) REGULATION OF NITROGEN METABOLISM P1: IKH 165 higher NRI concentration is required for nac, glnK, the astCADBE operon, and probably other Ntr operons (4). Annu. Rev. Microbiol. 2003.57:155-176. Downloaded from arjournals.annualreviews.org by WIB6332 - University of Saarland on 01/04/10. For personal use only. NITROGEN SOURCE–DEPENDENT CONTROL Growth with different nitrogen sources induces the glnA operon equally well (84). However, this is not the case for the activation of astCADBE, gabDTPC, and patA/ygjG and the repression of gltBD [(35, 50, 83); C. Pybus, B. Schneider & L. Reitzer, unpublished observations]. The basis for this differential regulation is not known. PREVENTION OF OVEREXPRESSION Three distinct mechanisms prevent Ntr gene overexpression. Low NRI activates the glnAp2 promoter of the glnA operon, and high NRI partially inhibits the activation (86). The binding of NRI to low-affinity sites apparently mediates this modulation (8). NRI activates nac expression, and then Nac impairs this activation (32). Finally, a glnK mutant overexpresses several Ntr genes during nitrogen starvation, which apparently reduces viability (13). This suggests that one function of GlnK is to prevent such overexpression. Functions of the Ntr Response and Nac Microarray analysis, computer analysis of σ 54-dependent genes, and other studies suggest that the vast majority of Ntr genes have been identified and that nitrogen limitation affects about 100 genes. This section considers the functions of these genes. The functions of individual Ntr genes within specific physiological contexts (e.g., argT-dependent arginine transport within the context of other arginine transport systems in E. coli) have been reviewed (74). AMMONIA ASSIMILATION The central role of glutamine in regulation of the Ntr response suggests that an important physiological function of the Ntr response is to maintain intracellular glutamine and obviously to control ammonia assimilation. Several regulatory mechanisms suggest the importance of this function. First, GS is synthesized before other Ntr proteins. Second, different mechanisms prevent overexpression of glnA and other Ntr genes. Finally, nitrogen source–dependent regulation affects expression of Ntr genes, except for glnA. SCAVENGING Zimmer et al. proposed that numerous Ntr transport systems scavenge nitrogen-containing compounds (100). Nitrogen limitation induces transport systems for several amino acids (D-alanine, arginine, aspartate, glutamate, glutamine, glycine, histidine, lysine, ornithine, and D-serine), peptides (D-alanylD-alanine, dipeptides, and oligopeptides), polyamines and related compounds (putrescine, spermidine, and γ -aminobutyrate), cytosine, and nucleosides. Scavenging obviously spares the need to assimilate ammonia. METABOLIC INTEGRATION Six Ntr operons, astCADBE, codBA, ddpXABCDE, gabDTPC, patA (ygjG), and ydcSTUVW, contain genes for catabolic enzymes. It could be argued that these enzymes contribute to scavenging. However, this would 27 Jul 2003 12:20 Annu. Rev. Microbiol. 2003.57:155-176. Downloaded from arjournals.annualreviews.org by WIB6332 - University of Saarland on 01/04/10. For personal use only. 166 AR AR195-MI57-07.tex AR195-MI57-07.sgm LaTeX2e(2002/01/18) P1: IKH REITZER not explain why so few catabolic enzymes are under Ntr control and why nine of them degrade compounds that are associated with polyamine metabolism. The products of astCADBE, gabDTPC, and patA (ygjG) degrade arginine/ornithine, γ -aminobutyrate, putrescine, respectively [(81, 83); B. Schneider, C. Pybus & L. Reitzer, unpublished observations). YdcW degrades γ -aminobutyraldehyde, a product of putrescine catabolism (B. Schneider & L. Reitzer, unpublished observation). In addition to these catabolic enzymes, nitrogen limitation induces two putrescine or polyamine transport systems, potFGHI and ydcSTUVW (100). At least 19 Ntr genes transport putrescine, degrade putrescine, or degrade the precursors of putrescine synthesis. Polyamine levels are correlated to the growth rate (89, 92). These Ntr genes may integrate slower nitrogen-limited growth with polyamine content. Metabolic integration could account for why nitrogen limitation represses both enzymes of glutamate synthesis, the first enzyme of serine synthesis, and AsnC (the activator of asnA) (12, 35, 64, 71). Nitrogen limitation also induces a peptidase that degrades D-alanyl-D-alanine, a precursor for peptidoglycan (100), which may modulate peptidoglycan synthesis. In summary, Ntr genes might integrate slower growth with several major metabolic pathways. Such a function may account for the lethality of Ntr gene overexpression (13). NAC Nac is an Ntr protein that represses the enzymes of glutamate and serine synthesis and AsnC (12, 35, 64, 71). Nac activates codBA (cytosine metabolism), ydcSTUVW (putative putrescine transport and catabolism), nupC (nucleoside transport), gabDTPC (γ -aminobutyrate transport and degradation), dppABCDF (dipeptide transport), and fklB-cycA (100). The products of these genes might modulate the levels of important metabolic intermediates and, by this mechanism, integrate nitrogen assimilation with other aspects of metabolism. If so, then such integration is dispensable (or at least redundant) because an E. coli nac mutant does not have a dramatic phenotype and Salmonella lacks the gene (64). THE σ 54 REGULON: ITS FUNCTION AND RELATION TO THE NTR RESPONSE Properties of σ 54-Dependent Genes Expression of many Ntr genes requires σ 54, which is the only σ factor in E. coli that is not homologous to σ 70 (62). σ 54-dependent transcription has several distinctive features (16, 74). It always requires a transcriptional activator. The activators bind to sites that are analogous to eukaryotic enhancers. The activators hydrolyze ATP and interact with σ 54-containing RNA polymerase. The activator-RNA polymerase interaction requires either a DNA-bending protein, such as integration host factor, or a DNA curvature (20, 42). Because of the absolute requirement for an activator, σ 54-dependent expression can be completely turned off. One advantage of such control is the wide range of activity, from very low to very high expression 27 Jul 2003 12:20 AR AR195-MI57-07.tex AR195-MI57-07.sgm LaTeX2e(2002/01/18) REGULATION OF NITROGEN METABOLISM P1: IKH 167 (PspA and GS, products of σ 54-dependent operons, can be a few percent of E. coli proteins). Perhaps this dynamic range is important for proteins or pathways that must at times consume large amounts of energy or metabolic intermediates. Annu. Rev. Microbiol. 2003.57:155-176. Downloaded from arjournals.annualreviews.org by WIB6332 - University of Saarland on 01/04/10. For personal use only. The Functions of σ 54-Dependent Genes σ 54 is widespread among bacteria and is required for a variety of functions that are not associated with nitrogen assimilation (88). About half of the 25 known or strongly suspected σ 54-dependent operons in E. coli do not specify proteins of nitrogen metabolism (74). FhlA activates five of these operons, which code for proteins associated with formate hydrogen lyase. The other σ 54-dependent operons specify proteins for the phage shock response, zinc tolerance, two products of fatty acid catabolism, propionate and acetoacetate, and an RNA-modifying enzyme of unknown function. The products of these genes are associated with a variety of environmental stresses. FhlA regulon genes are thought to contribute to pH homeostasis during fermentation, which potentially facilitates growth or survival in an acidic, energy-limited, anaerobic environment (14). The phage shock proteins are required for survival in an alkaline environment and, more generally, may maintain the proton motive force during stress (51, 97). Zinc tolerance alleviates the stress of high zinc. Propionate and acetoacetate are also associated with stress. FadR participates in the universal stress response by mediating fatty acid degradation, which would generate propionate and acetoacetate for energy (27, 30). Perhaps the products of these genes alleviate problems associated with certain stresses that make nitrogen assimilation difficult (74). Alternatively, expression of these genes might somehow modulate expression of the σ 54-dependent genes of nitrogen metabolism. A conceivable modulation mechanism is competition for σ 54, which is not an abundant protein (48). It is also possible that these genes have no obligatory association with nitrogen assimilation and that strains with certain genes with the unique properties of σ 54-dependent promoters have a selective advantage. METABOLIC INTEGRATION: NITROGEN ASSIMILATION AND CARBON METABOLISM Ammonia assimilation requires energy and intermediates from central metabolism. Several mechanisms integrate intermediary metabolism with ammonia assimilation. This section considers three mechanisms that have been identified. Lrp The leucine-responsive regulatory protein (Lrp) is a moderately abundant DNAbinding protein (98), but it is not a major nucleoid-binding protein (9, 10). Lrp has several modes of action: It can either activate or repress gene expression; leucine can reverse, enhance, or have no effect on this regulation (68). Guanosine tetraphosphate (ppGpp) controls Lrp synthesis (55), which accounts for its synthesis in amino acid–poor minimal media (25) and during the transition into 27 Jul 2003 12:20 Annu. Rev. Microbiol. 2003.57:155-176. Downloaded from arjournals.annualreviews.org by WIB6332 - University of Saarland on 01/04/10. For personal use only. 168 AR AR195-MI57-07.tex AR195-MI57-07.sgm LaTeX2e(2002/01/18) P1: IKH REITZER stationary phase (41, 90). Lrp affects the expression of about 10% of the genes in E. coli (43, 90). Several reviews and a recent microarray analysis consider the broader functions of Lrp (18, 67, 68, 90). Lrp has been implicated in several aspects of the Ntr response. Lrp activates gltBD, which means that Lrp is required for the Ntr response (29). Lrp also controls the metabolism of alanine, serine, and glycine, which are feedback inhibitors of GS. Furthermore, Nac, an Ntr protein, represses two Lrp-activated operons, serA and gltBD (12, 35). Finally, evidence pieced together from different sources suggests that Lrp and Ntr regulators might independently activate ompF, oppABCDF, ydcSTUVW, and yeaGH (68, 90, 100). In summary, Lrp controls Ntr gene expression, and regulators of the Ntr response modulate the expression of some Lrp-regulated genes. The effects of Lrp without leucine (Figure 2, top panel) and with leucine (Figure 2, bottom panel) must be considered separately in order to understand Lrp’s function in ammonia assimilation. Lrp without leucine activates synthesis of GOGAT and pyridine nucleotide transhydrogenase, which together provide two of the three substrates for ammonia assimilation. Lrp without leucine represses two serine deaminases and alanine dehydrogenase. The net effect would appear to favor ammonia assimilation (and amino acid synthesis) over ammonia generation (amino acid degradation). The addition of leucine moderately reduces GOGAT and pyridine nucleotide transhydrogenase, but increases the amino acid degradative enzymes. To the extent that nitrogen is transferred to serine and alanine via reversible transaminations, leucine will stimulate net amino acid catabolism. Lrp appears to be one factor that coordinates ammonia assimilation and amino acid catabolism in an amino acid–poor environment. This hypothesis is supported by the observation that an lrp mutant has enhanced amino acid degradation (101). It has been proposed that Lrp mediates the transitions between feast and famine. For the former, amino acid catabolic functions are appropriate; while for the latter, amino acid biosynthetic functions (including ammonia assimilation) are more appropriate (18, 66). If Lrp coordinates ammonia assimilation with amino acid catabolism, then leucine is an indicator of amino acid sufficiency. Alanine may also contribute to this regulation because alanine also binds Lrp [(59) and references cited therein]. Cyclic AMP Cyclic AMP bound to its receptor protein interferes with expression from the glnAp2 promoter of the glnA operon and from other Ntr promoters (91). It is possible that the primary effect of high cyclic AMP is at the glnALG operon and that lowering its expression prevents synthesis of sufficient NRI to activate other Ntr genes. EINtr-NPr-EIINtr EINtr (PtsP), NPr (PtsO), and EIIANtr (PtsN) are paralogs of sugar phosphotransferase transport proteins in E. coli and other organisms, which might also 27 Jul 2003 12:20 AR AR195-MI57-07.tex AR195-MI57-07.sgm LaTeX2e(2002/01/18) 169 Annu. Rev. Microbiol. 2003.57:155-176. Downloaded from arjournals.annualreviews.org by WIB6332 - University of Saarland on 01/04/10. For personal use only. REGULATION OF NITROGEN METABOLISM P1: IKH Figure 2 Lrp and nitrogen assimilation. The information is from Newman et al. (68). The filled arrows and numbers indicate activation, whereas the open arrows and numbers indicate repression. The top panel compares the effects on the indicated pathways or enzymes between strains with and without Lrp, both without added leucine. The bottom panel compares the effects of a wild-type strain with and without leucine. SerineEXT is external serine. 27 Jul 2003 12:20 Annu. Rev. Microbiol. 2003.57:155-176. Downloaded from arjournals.annualreviews.org by WIB6332 - University of Saarland on 01/04/10. For personal use only. 170 AR AR195-MI57-07.tex AR195-MI57-07.sgm LaTeX2e(2002/01/18) P1: IKH REITZER contribute to coordination of carbon and nitrogen metabolism. Purified EINtr, NPr, and EIINtr form a phosphorelay, which accepts phosphate from phosphoenolpyruvate (72, 73). EINtr-NPr-EIINtr phosphorylates sugars 1000 times slower than EIHPr-EII systems, which suggests that the former has a regulatory function, not a transport function (73). EINtr contains a GAF domain, which regulates some σ 54dependent activators, and therefore suggests regulatory potential (2, 79). Furthermore, Bradyrhizobium japonicum EINtr interacts with an aspartokinase isozyme, which also suggests regulatory potential (49). Genetic evidence suggests that EINtr and EIINtr, but not NPr, have a regulatory function. An E. coli ptsN mutant (EIINtr deficient) grew less well with organic nitrogen sources in carbon-limited media, which suggests positive regulation (72). The mutant grew normally with glucose as the carbon source and expressed at least one Ntr gene normally (72). In contrast, a K. pneumoniae ptsN mutant had increased Ntr gene expression, which suggests negative regulation (63). An E. coli ptsP (EINtr deficient) has not been isolated; but loss of EINtr in other organisms prevents the expression of important genes (49, 85). ptsO and ptsN are members of the putative rpoN-yhbH-ptsN-yhbJ-ptsO operon, whereas ptsP is unlinked (72, 73). yhbH, yhbJ, and ptsO in E. coli, and their homologs in P. putida, do not appear to contribute to regulation (21, 72). A model for how the EINtr-NPr-EIINtr system might affect gene expression is not obvious from these results, and none has been presented. GUANOSINE TETRAPHOSPHATE, AMINO ACID SYNTHESIS, AND AMMONIA ASSIMILATION Slower growth and elevated ppGpp activate lrp expression, which in turn is required for the Ntr response (29, 55). Therefore, the factors that regulate ppGpp synthesis are important in the control of nitrogen metabolism. (Where it is stated that ppGpp controls something, what is meant is that ppGpp appears to mediate this control. The intent is not to exclude potential indirect effects, such as diminished GTP, or other potential mechanisms of growth rate control.) Two factors control the ppGpp concentration: tRNA aminoacylation and energy source availability (22). In general, ppGpp stimulates amino acid synthesis and inhibits nucleotide synthesis and nucleobase salvage (22). This pattern suggests a preference for amino acid synthesis over nucleotide synthesis, which is appropriate with diminished tRNA aminoacylation. Nitrogen availability determines which pathways of amino acid synthesis are affected. In a glucose-ammonia minimal medium, ppGpp0 strains, which cannot synthesize ppGpp, absolutely require arginine, glycine, histidine, leucine, phenylalanine, serine, threonine, and valine and have weak or strain-dependent requirements for lysine, methionine, and tyrosine (99). These strains do not require alanine, asparagine, aspartate, cysteine, glutamate, glutamine, or proline (99). In a nitrogen-limited environment, ppGpp via Lrp is required for glutamate synthesis, induction of the Ntr response, and ammonia assimilation, and therefore the synthesis of all the amino acids. 27 Jul 2003 12:20 AR AR195-MI57-07.tex AR195-MI57-07.sgm LaTeX2e(2002/01/18) REGULATION OF NITROGEN METABOLISM P1: IKH 171 Annu. Rev. Microbiol. 2003.57:155-176. Downloaded from arjournals.annualreviews.org by WIB6332 - University of Saarland on 01/04/10. For personal use only. CONCLUDING REMARKS: INTERACTIONS BETWEEN GLOBAL REGULATORS A hierarchy of global regulators affects the pathways of nitrogen metabolism. Elevated ppGpp stimulates several pathways of amino acid synthesis and inhibits pathways of nucleotide synthesis. ppGpp activates the synthesis of Lrp, which may fine-tune the balance between amino acid synthesis and degradation. Lrp positively controls the synthesis of GOGAT, which affects the level of glutamine, the most important effector of the Ntr response. Finally, the regulators of the Ntr response control ammonia assimilation. Numerous environmental factors modify the activities of these global regulators. The interplay of these environmental factors is both complex and subtle. For example, carbon limitation through growth rate control increases ppGpp, which via Lrp is required for induction of the Ntr response. However, cyclic AMP interferes with this induction by an unknown mechanism. Another example is the effect of impaired protein synthesis by uncharged tRNA, which increases ppGpp and Lrp. Lrp, through its control of glutamate synthase, induces the Ntr response, which stimulates expression of polyamine catabolic enzymes. Diminished polyamines complete the regulatory circuit, which should impair protein synthesis. Both of these examples show mechanisms by which global regulators and their effectors integrate ammonia assimilation with other aspects of metabolism. E. coli Lrp has limited sequence similarity with homologous proteins in other organisms and is not a global regulator in at least one organism (34). Perhaps the entire global regulatory network that controls nitrogen metabolism is also not conserved, but instead has evolved in enteric and related bacteria in response to environments with large and rapid fluctuations in nutrient availability. ACKNOWLEDGMENTS This work was supported in part by grant MCB-0077904 from the National Science Foundation. The Annual Review of Microbiology is online at http://micro.annualreviews.org LITERATURE CITED 1. 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CONTENTS FRONTISPIECE, Julian Davies GATHERING NO MOSS, Julian Davies MOLECULAR PATHOGENICITY OF THE ORAL OPPORTUNISTIC PATHOGEN ACTINOBACILLUS ACTINOMYCETEMCOMITANS, Brian Henderson, Sean P. Nair, John M. Ward, and Michael Wilson BRUCELLA STATIONARY-PHASE GENE EXPRESSION AND VIRULENCE, R. Martin Roop II, Jason M. Gee, Gregory T. Robertson, John M. Richardson, Wai-Leung Ng, and Malcolm E. Winkler HOW BACTERIA ASSEMBLE FLAGELLA, Robert M. Macnab A SALVAGE PATHWAY FOR PROTEIN SYNTHESIS: TMRNA AND TRANS-TRANSLATION, Jeffrey H. Withey and David I. Friedman ASSEMBLY DYNAMICS OF THE BACTERIAL CELL DIVISION PROTEIN FTSZ: POISED AT THE EDGE OF STABILITY, Laura Romberg and Petra Anne Levin NITROGEN ASSIMILATION AND GLOBAL REGULATION IN ESCHERICHIA COLI, Larry Reitzer ON THE TRAIL OF A CEREAL KILLER: EXPLORING THE BIOLOGY OF MAGNAPORTHE GRISEA, Nicholas J. Talbot BACTERIAL MEMBRANE LIPIDS: WHERE DO WE STAND? John E. Cronan SPATIAL AND TEMPORAL CONTROL OF DIFFERENTIATION AND CELL CYCLE PROGRESSION IN CAULOBACTER CRESCENTUS, Nora Ausmees and Christine Jacobs-Wagner BACTERIAL MOTILITY ON A SURFACE: MANY WAYS TO A COMMON GOAL, Rasika M. Harshey TRANSPOSABLE ELEMENTS IN FILAMENTOUS FUNGI, Marie-Josée Daboussi and Pierre Capy BACTERIOPHAGE-INDUCED MODIFICATIONS OF HOST RNA POLYMERASE, Sergei Nechaev and Konstantin Severinov VACCINIA VIRUS MOTILITY, Geoffrey L. Smith, Brendan J. Murphy, and Mansun Law vi xii 1 29 57 77 101 125 155 177 203 225 249 275 301 323 P1: FRK July 27, 2003 15:23 Annual Reviews AR195-FM Annu. Rev. Microbiol. 2003.57:155-176. Downloaded from arjournals.annualreviews.org by WIB6332 - University of Saarland on 01/04/10. For personal use only. CONTENTS vii MEASLES VIRUS 1998–2002: PROGRESS AND CONTROVERSY, Glenn F. Rall 343 THE UNCULTURED MICROBIAL MAJORITY, Michael S. Rappé and Stephen J. Giovannoni 369 PATHWAYS OF OXIDATIVE DAMAGE, James A. Imlay GENE ORGANIZATION: SELECTION, SELFISHNESS, AND SERENDIPITY, Jeffrey G. Lawrence MULTIPLE SIGMA SUBUNITS AND THE PARTITIONING OF BACTERIAL TRANSCRIPTION SPACE, Tanja M. Gruber and Carol A. Gross NATURAL SELECTION AND THE EMERGENCE OF A MUTATION PHENOTYPE: AN UPDATE OF THE EVOLUTIONARY SYNTHESIS CONSIDERING MECHANISMS THAT AFFECT GENOME VARIATION, Lynn Helena Caporale ARCHAEAL DNA REPLICATION: EUKARYAL PROTEINS IN A BACTERIAL CONTEXT, Beatrice Grabowski and Zvi Kelman MOLECULAR GENETICS OF MYCOBACTERIUM TUBERCULOSIS PATHOGENESIS, Josephine E. Clark-Curtiss and Shelley E. Haydel THE BACTERIAL RECA PROTEIN AS A MOTOR PROTEIN, Michael M. Cox DNA MISMATCH REPAIR: MOLECULAR MECHANISMS AND BIOLOGICAL FUNCTION, Mark J. Schofield and Peggy Hsieh KAPOSI’S SARCOMA–ASSOCIATED HERPESVIRUS IMMUNOEVASION AND TUMORIGENESIS: TWO SIDES OF THE SAME COIN? Patrick S. Moore and Yuan Chang THE SECRET LIVES OF THE PATHOGENIC MYCOBACTERIA, Christine L. Cosma, David R. Sherman, and Lalita Ramakrishnan BACTERIAL BIOFILMS: AN EMERGING LINK TO DISEASE PATHOGENESIS, Matthew R. Parsek and Pradeep K. Singh INDEXES Subject Index Cumulative Index of Contributing Authors, Volumes 53–57 Cumulative Index of Chapter Titles, Volumes 53–57 ERRATA An online log of corrections to Annual Review of Microbiology chapters (if any, 1997 to the present) may be found at http://micro.annualreviews.org/ 395 419 441 467 487 517 551 579 609 641 677 703 739 742