INTRODUCTION AND OVERVIEW

Top Previous Next References

Scope

The dissociable sigma subunits of RNA polymerase are responsible for specific binding to DNA and are therefore important determinants of differential gene expression (68, 170). ⁵⁴ was discovered during an analysis of glutamine synthetase and nitrogen assimilation in enteric bacteria (71, 77). Subsequent studies have confirmed its role in nitrogen assimilation but have also shown that it is involved in a variety of seemingly unrelated functions, such as carbon source utilization, certain fermentation pathways, flagellar synthesis, and bacterial virulence (97, 151). There have been several reviews about ⁵⁴ and ⁵⁴-dependent activation, including recent ones (14, 28, 97, 110, 113, 148, 151). However, discussions of ⁵⁴ function usually consider genes from several organisms and therefore remove ⁵⁴ from the metabolic or physiological context of a single organism. The primary purpose of this review is to redress this imbalance and to discuss the functions of the known ⁵⁴-dependent genes from a single organism, Escherichia coli. The most meaningful discussion of ⁵⁴ function requires a complete set of ⁵⁴-dependent genes. Two sources provide information on likely ⁵⁴-dependent promoters: a recent DNA microarray analysis of transcripts present during nitrogen-limited growth (176) and computer analysis of potential ⁵⁴-dependent promoters, which are readily identified from the completed nucleotide sequence of the E. coli genome (23). Results of these analyses are integrated into the discussion of the function of the known ⁵⁴-dependent genes. The central thesis of this review is that the ⁵⁴-dependent genes of E. coli have only a few metabolic themes and that these themes may be related.

E. coli has seven  subunits, and each has a distinct function. ⁷⁰ is considered the primary sigma factor. Core RNA polymerase (E) associated with ⁷⁰ initiates transcription of housekeeping genes (68). E⁷⁰ also initiates the transcription of nonessential genes that are induced in specialized environments. ^S has been called either an alternative sigma factor or a second primary sigma factor (69). Although E^S binds the same sequences as E⁷⁰ (unpublished results cited in reference 69), ^S is considered a general stress factor since it is associated with a variety of growth-impairing stresses: nutrient depletion, oxidative stress, high temperature, high osmolarity, acidic pH, or exposure to ethanol (69). ³² and ^E are also associated with stress. ³² is required for the response to damage of cytoplasmic proteins, which is most commonly associated with heat shock, and ^E controls the response to extracytoplasmic or extreme heat stress (174). ^FecI and ²⁸ (FliA) are required for synthesis of the ferric citrate transporter and flagella, respectively (8, 115). As mentioned above, ⁵⁴ is usually associated with nitrogen assimilation.

The  factors in E. coli are homologous to ⁷⁰, except for ⁵⁴ (110). Not surprisingly, ⁵⁴-dependent transcription has several distinctive features (reviewed in reference 28). Core RNA polymerase (E) complexed to a ⁷⁰-like factor can be sufficient for open promoter complex formation. In contrast, E⁵⁴ catalyzes strand separation only with the help of a distinct class of transcriptional activators. A consequence of this property is that transcription can be completely turned off. Such absolute control may account for the evolutionary persistence of ⁵⁴ (see "Physiological function of ⁵⁴" below).

The activators of E⁵⁴-dependent genes are unusual. (The individual E. coli activators are discussed in a separate section.) Unlike most eubacterial transcriptional activators, the ⁵⁴-dependent activators bind to sites that are effective regardless of distance and orientation (28, 29, 131). In this respect, the activator binding sites are analogous to eukaryotic enhancers, and the activators are often called enhancer binding proteins. However, the analogy to eukaryotic enhancer binding proteins is not precise, since the bacterial proteins do not enhance basal transcription but, instead, are absolutely required for any transcription. The activators interact with E⁵⁴ from these binding sites. This interaction sometimes requires a DNA bending protein. The DNA bending proteins that participate in ⁵⁴-dependent gene expression in E. coli are integration host factor (IHF) and ArgR, the arginine repressor (72, 103). When transcription from a ⁵⁴-dependent promoter does not require a DNA bending protein, DNA curvature facilitates the interaction between the activator and RNA polymerase (32). Another distinctive feature of ⁵⁴-dependent activators is an essential ATPase activity (166).

The most important control of ⁵⁴-dependent genes is through modulation of the activator's ATPase activity. The ⁵⁴-dependent activators usually contain a regulatory domain that controls ATPase activity. Several mechanisms control the interaction of the regulatory domain with the ATP binding domain: phosphorylation, interaction with a low-molecular-weight ligand, or interaction with one or several regulatory proteins (148). Some control mechanisms are extremely complex, such as that for PspF (discussed below). Variations in ⁵⁴ activity, either by ligand binding or by covalent modification, are not known in E. coli. Furthermore, the intracellular level of ⁵⁴ is apparently constant (87), and expression of rpoN, which specifies ⁵⁴, appears to be constitutive (33). Strain W3110 contains about 700 molecules of ⁷⁰ per cell and 110 molecules of ⁵⁴, whereas strain MC4100 may contain only about 285 molecules of ⁷⁰ and as few as 13 to 22 molecules of ⁵⁴ (87). The low level of ⁵⁴ could have regulatory implications. For example, it is possible that different ⁵⁴-dependent operons compete for limiting ⁵⁴. This possibility is even more plausible if there is some physiological relationship between the ⁵⁴-dependent genes. One purpose of this review is to explore this issue.

We will use the promoters for these 17 operons to characterize the ⁵⁴-dependent promoters in E. coli (Table 1). These promoters suggest an apparent consensus of aaN₃TGGCAcN₆TGCNNt, where small letters indicate two to five mismatches, capital letters denote zero or one mismatch, and underlined bases have no mismatches. By similar criteria, the consensus derived from 186 promoters from a variety of organisms is N₅tGGcacN₅ttGC (14), which is similar but not identical to the apparent E. coli consensus. Figure 1 shows the locations of the ⁵⁴-dependent operons within the context of neighboring genes, the size of the transcripts, and the direction of expression. Figure 2 shows the binding sites for E⁵⁴ in relation to the binding sites for the ⁵⁴-dependent activators (when known) and the nearest upstream structural gene.

View this table: [in this window] [in a new window]   TABLE 1.   E. coli operons with confirmed 54-dependent promoters

View larger version (33K): [in this window] [in a new window]   FIG. 1.   54-dependent genes in E. coli. The known 54-dependent operons and their transcripts are shown in the context of neighboring genes. The open and solid boxes indicate counterclockwise and clockwise transcription, respectively. If the gene has been assigned a function, this is indicated by the gene name underneath. Boxes without gene names indicate that the gene has an unknown function. The sizes of the genes and their intergenic regions are to scale.

View larger version (21K): [in this window] [in a new window]   FIG. 2.   Regulatory regions of the characterized 54-dependent genes. The following features are shown for each of the 54-dependent promoters: binding sites for activators, DNA bending proteins (when required), and RNA polymerase. Solid boxes indicate that the binding site has been demonstrated. A hatched box indicates a confirmed binding site, but binding is weak. An open box signifies a proposed binding site. The relative location and orientation of the nearest upstream gene are also shown. All diagrams are to the same scale, except for the hyp operon.

Several generalizations can be made concerning these promoters. First, all the known ⁵⁴-dependent promoters are located outside the structural genes. This does not necessarily mean that an authentic binding site for E⁵⁴ will not be found within a gene, but it does mean that such binding sites will be the exception. Second, the activator binding sites are also outside the structural genes. The most spectacular example is the FhlA binding site for the hyp operon in the hyp-hyc regulatory region. The entire hycA gene is located between the binding sites for FhlA and RNA polymerase (Fig. 2). Even though the FhlA binding site for the hyp operon is within the hyc operon, it is not within a structural gene. Instead, it is completely located within the 130-base intergenic region between hycA and hycB. Third, the activator binding sites are almost always a significant distance from the adjacent upstream gene and therefore do not apparently interfere with expression of these genes (Fig. 2). The only exception is the binding site for PspF, which activates the pspABCDE operon. The PspF sites for activation overlap with the promoter for the pspF gene (Fig. 2), and it has been proposed that this has physiological significance (see "psp operon and phage shock response" below). The average size of the intergenic region that contains a known ⁵⁴-dependent promoter is 267 ± 106 bases, with a range from 148 to 507 bases. This distance is apparently large enough for binding sites for both E⁵⁴ and an activator. Fourth, the distance from the 3' end of the E⁵⁴ binding site to the nucleotides coding for the initiation codon is, on average, 50 bases. Such a short distance reduces the potential for RNA secondary structures or protein binding regions near the translational start site and therefore reduces the potential for translational control. Finally, the average A+T content of the 50 bases just upstream from the E⁵⁴ binding site is 70%, and no upstream region has an AT content less than 50%. Two rationales for such a bias can be suggested. Some ⁵⁴-dependent promoters require a DNA bending protein. Both of the DNA bending proteins that facilitate the activation of ⁵⁴-dependent genes in E. coli, IHF and ArgR, have an AT-rich consensus sequence. Alternately, ⁵⁴-dependent promoters that do not require a DNA bending protein often have an intrinsic bend between the activator and RNA polymerase binding sites (32), and this may favor AT-rich regions. In either case, the requirement for long-range protein-protein interactions appears to bias the base composition of DNA just upstream from the E⁵⁴ binding site.

Despite the reasonably uniform properties of the known ⁵⁴-dependent promoters, there is little useful information for promoter prediction. For uncharacterized promoters, the activator binding sites will probably not be known, and there may be uncertainty in the size of intergenic regions if the protein coding regions have been misidentified (which is not uncommon). A more useful predictor of potential promoters is based on computer identification of binding sites for E⁵⁴, which is described in the next section.

COMPUTER IDENTIFICATION OF POTENTIAL ⁵⁴-DEPENDENT PROMOTERS

Top Previous Next References

Site Identification and the Problem of False Positives

The most meaningful analysis of the physiological function of ⁵⁴ requires a comprehensive set of ⁵⁴-dependent genes. One method to help determine the complete set of such promoters is a computer analysis of potential ⁵⁴-dependent promoters from the completed E. coli genome sequence. We used the SeqScan program (B. T. Nixon, Department of Biochemistry and Molecular Biology, Pennsylvania State University [http://www.bmb.psu.edu/seqscan]) for this analysis. This program uses 86 ⁵⁴-dependent promoters from several organisms to define a consensus sequence that does not differ substantially from that derived from the 17 known E. coli promoters (Table 2). The program uses a weighted matrix to give sites a score from 0 to 100, and then reports sites with a score higher than 60. 

View this table: [in this window] [in a new window]   TABLE 2.   Comparison of consensus sequence for 54-dependent promoters from known E. coli promoters and the SeqScan programa

The SeqScan program identified approximately 8,000 potential ⁵⁴-dependent promoters from the E. coli genome, or about 1 for every 600 bases. Clearly, there are a number of false positives. We can use the properties of the known ⁵⁴-dependent promoters to eliminate many of these false positives. We eliminated all the sites within structural genes, which reduced the number of potential sites over 30-fold. The 17 known ⁵⁴-dependent promoters were in the set of 213 intergenic sites in which the potential promoter transcribed a gene in the correct direction. We found that 121 potential intergenic E⁵⁴ binding sites transcribe genes in the wrong orientation. Table 3 and its footnotes list all of the intergenic sites that could potentially transcribe a gene in the correct direction.

View this table: [in this window] [in a new window]   TABLE 3.   Computer ranking of intergenic 54 sitesa

The distribution of scores for properly and improperly oriented sites is shown in Fig. 3. There are only 2 incorrectly oriented sites (1.6%), and there are 25 correctly oriented sites (11.7%) with a score of at least 76. Of the 25 high-scoring correctly oriented sites, 18 (72%) are known ⁵⁴-dependent promoters or are induced by nitrogen limitation. Together, these results imply that properly oriented high-scoring sites are not common and are likely to contain an authentic ⁵⁴-dependent promoter. In contrast, only 7 (3%) of 213 sites with a score lower than 76 are known or likely (i.e., nitrogen limitation-induced) ⁵⁴-dependent promoters. Therefore, scores below 76 are a reasonably good, but not infallible, indicator that the site is not a promoter. Despite the correlation with promoter scores, the promoter ranking is not an exact indicator of promoter strength. For example, the nac promoter has a higher score than the ⁵⁴-dependent promoter of the glnALG operon, even though the latter is stronger (54). Nonetheless, and this point cannot be emphasized too much, the computer program recognizes all known E⁵⁴ binding sites, which implies that the failure to detect such a site is a reliable indicator that such a site does not exist.

View larger version (18K): [in this window] [in a new window]   FIG. 3.   Distribution of scores with properly and improperly oriented sites. The open bars indicate sites that are oriented toward the 3' end of a gene; the solid bars indicate sites that are oriented toward the 5' end.

Of the 17 known ⁵⁴-dependent promoters, 7 are involved in nitrogen metabolism. In addition, microarray analysis has identified 7 other genes that are induced by nitrogen limitation (176), and these have an appropriately placed potential ⁵⁴-dependent promoter. This section discusses the 14 operons that are involved in nitrogen metabolism and the functions of their products. To understand the metabolic context of these proteins, it is first necessary to discuss nitrogen assimilation and the response to nitrogen deprivation. Because there have recently been some major changes in our understanding of these topics, the next sections summarize our current knowledge.

Nitrogen assimilation. Glutamate and glutamine are the major intracellular nitrogen donors, and they provide about 75 and 25% of the cell's nitrogen, respectively (calculated from numbers presented in reference 116). Nitrogen assimilation must therefore result in the synthesis of these two nitrogen donors.

View larger version (8K): [in this window] [in a new window]   FIG. 4.   Pathways of ammonia assimilation. GDH, glutamate dehydrogenase.

Control of ammonia assimilation and GS activity: role of glutamine. Ammonia assimilation involves the regulation of three enzymes. Little is known about the regulation of glutamate dehydrogenase activity or synthesis in E. coli (127). The leucine-responsive regulatory protein (Lrp) controls the synthesis of glutamate synthase (53). A discussion of the function of Lrp and Lrp-dependent regulation, which does not require ⁵⁴, is beyond the scope of the review. Instead, we will focus on the control of GS activity.

View larger version (24K): [in this window] [in a new window]   FIG. 5.   Regulation of GS activity and the Ntr response. The pathways are shown for conditions of nitrogen excess (high glutamine) (top) and nitrogen limitation (low glutamine) with partial GlnK uridylylation (bottom). The open arrow in the bottom panel is meant to indicate that only partial uridylylation occurs. It is assumed that partial uridylylation occurs either during nitrogen limitation or during the transition to steady-state nitrogen-limited growth. The T-like symbol indicates an inhibition.

Nitrogen sources. Ammonia is considered the preferred nitrogen source for E. coli grown in a minimal medium because ammonia supports the fastest growth and its presence prevents the synthesis of several proteins of nitrogen metabolism (reviewed in references 127 and 129). In place of ammonia, E. coli and related organisms can utilize a small number of nitrogen sources, usually amino acids, nucleosides, nucleobases, and a few inorganic nitrogen sources, e.g., nitrite and nitrate, which are reduced to ammonia. Steady-state growth on alternate nitrogen sources is slower and is said to be nitrogen limited. Catabolism of the alternate nitrogen sources must produce ammonia for the synthesis of glutamine, one of the intracellular nitrogen donors. For growth with a nitrogen source that cannot transfer its nitrogen to glutamate by transamination (e.g., adenosine), ammonia becomes an obligatory intermediate for all cellular nitrogen. In these situations, GS is the primary enzyme of ammonia and nitrogen assimilation (Fig. 4). Nitrogen-limited growth results in maximal synthesis of GS and also induces proteins that transport and catabolize several nitrogen sources. The coordinated response to nitrogen limitation is called the nitrogen-regulated (Ntr) response.

-Ketoglutarate counters the effects of unmodified P_II. Even though recent studies have suggested that the ratio of glutamine to -ketoglutarate does not regulate UTase-UR activity, -ketoglutarate does affect P_II activity (83-85). -Ketoglutarate counteracts the effects of unmodified P_II (present when glutamine levels are high and nitrogen is in excess) and therefore stimulates glnALG expression and increases GS activity. In other words, the ratio of glutamine (via UTase-UR) to -ketoglutarate (via P_II) appears to control nitrogen assimilation during relative nitrogen sufficiency. This leaves the question whether there is a mechanism to coordinate carbon and nitrogen metabolism during nitrogen-limited growth when P_II is uridylylated. It will be suggested elsewhere in this review that such coordination might be a function of GlnK, a P_II-like protein.

NR_I regulon. NR_I directly or indirectly controls the vast majority of Ntr genes. It is known to activate the expression of glnALG (GS and Ntr regulators), astCADBE (arginine catabolism), glnK-amtB (an alternate P_II and an ammonia transporter), nac (a ⁷⁰-dependent transcriptional activator), and glnHPQ (glutamine transport) in E. coli. Several lines of evidence also suggest that it controls the expression of argT-hisJMPQ (arginine and histidine transport) and gltIJKL (glutamate-aspartate transport). In addition to these genes, microarray analysis suggests that NR_I might also activate b1012-b1006 (possibly for pyrimidine catabolism), chaC (calcium transport), ddpXABCDE (D-alanine-D-alanine metabolism), potFGHI (putrescine transport), yeaGH (unknown function), ygjG (a transaminase), and yhdWXYZ (amino acid transport) (176). In addition to activation, NR_I represses the two minor promoters, glnAp₁ and glnLp, of the glnALG operon.

Nac regulon. There are two majors regulators of the Ntr response: NR_I and Nac. NR_I activates ⁵⁴-dependent promoters, while Nac activates ⁷⁰-dependent promoters. Nac is homologous to LysR (114). Unlike LysR, Nac apparently does not bind a ligand, which implies that it is constitutively active (16, 63).

Why are there two general regulators of the Ntr response? The main question concerning Nac is why there is a second Ntr regulator. Clearly, it is not necessary, since S. enterica serovar Typhimurium lacks it (114). We suggest that Nac-dependent control is important physiologically and serves a different function from NR_I-dependent control. NR_I-dependent genes respond to general nitrogen limitation, i.e., to intracellular glutamine, and not to specific induction mechanisms. The only known exception is the ast operon, which requires arginine-specific induction. In contrast, many Nac-dependent genes require both general and specific regulation: pyrimidines control the codBA operon by a complex process called reiterative transcription (126); GABA controls gab operon expression via the GabC repressor (Ruback and Reitzer, unpublished); and histidine and HutC control the hut operons in K. aerogenes (16). Nac may permit specific regulation, which may be difficult for ⁵⁴-dependent promoters. This is illustrated in Fig. 6, which shows the regulatory sites for the single hutUH promoter of K. aerogenes. Other rationales for Nac have been proposed (16).

View larger version (10K): [in this window] [in a new window]   FIG. 6.   Binding sites for regulatory proteins at the hutUH promoter.

The glnALG operon codes for GS, NR_II, and NR_I, respectively (127, 129). All three products of this operon are required for nitrogen assimilation and the Ntr response (discussed above). ⁵⁴ and NR_I~P are required for transcription from the major promoter, glnAp₂, which has a score of 83.3. Nitrogen limitation activates glnA expression 11- to 24-fold, but appears to have little effect on glnL or glnG transcription (176). However, direct measurements of NR_I indicate that nitrogen limitation induces NR_I synthesis 14-fold (128). Minor promoters, glnAp₁ and glnLp, ensure basal synthesis of the important products of this operon (130, 157).

Nitrogen limitation and NR_I are required for expression of the glnK-amtB operon (10, 160). Although the transcription start site has not been directly determined, the promoter has a score of 90.2 and potential binding sites for both E⁵⁴ and NR_I (Fig. 2) (160). In addition, nitrogen limitation increases the glnK transcript at least 10-fold (176). Therefore, this operon undoubtedly contains an authentic ⁵⁴-dependent promoter. Both products of the operon contribute to the response to nitrogen limitation.

GlnK: a P_II paralog. The existence of a P_II paralog was first suspected because of the rapid deadenylylation of GS in an E. coli glnB (P_II-encoding) mutant (160). The gene coding for this protein was cloned and called glnK (160). A glnK mutant has only a subtle phenotype (10). It has higher basal expression of an Ntr gene (glnK itself) in an ammonia-containing medium (even though the GlnK concentration should be low) and lower expression of an Ntr gene (again glnK) in a nitrogen-limiting medium. The mutant also has less of a lag during the transition to growth with arginine as a nitrogen source. This might result from higher basal expression of the ast operon, whose products degrade arginine. The phenotype of a glnB glnK double mutant, which lacks both P_II and GlnK, is more dramatic. It fails to grow in a nitrogen-rich minimal medium. The reason for this lethality is not known with certainty, but it may be related to uncontrolled phosphorylation of NR_I, which has been suggested to cause inappropriate overexpression of an Ntr gene (10). An alternate explanation is NR_I-dependent overexpression of a ⁵⁴-dependent gene that is not normally regulated by NR_I.

Why have GlnK? The lower induction of an Ntr gene and higher basal expression in a glnK mutant suggest that GlnK sharpens the response to nitrogen availability. Perhaps the responsiveness of GlnK to -ketoglutarate partially explains this effect. In this case, GlnK essentially restores the coordination of carbon and nitrogen metabolism (the responsiveness to the ratio of -ketoglutarate to glutamine) that is lost when P_II is completely uridylylated, i.e., during nitrogen-limited growth, and is no longer responsive to -ketoglutarate. A second function for GlnK has been found, but not in E. coli. GlnK is required for control of NifL, which inhibits the activity of NifA in Klebsiella species (65). NifA is the transcriptional activator required for expression of the nitrogenase gene cluster in Klebsiella species. Uridylylated and nonuridylylated GlnK can relieve repression. It is not known how GlnK mediates shutoff of the nif genes when ammonia is added, but it may interact with other proteins.

Product of amtB and ammonia transport. The second gene of the operon, amtB, codes for an ammonia transporter. None of the phenotypes of the glnK mutant could be attributed to polar effects on amtB expression (10). An amtB mutant of S. enterica serovar Typhimurium has only a subtle phenotype (149). It is unable to utilize a low concentration of ammonia if the pH is less than 7. This phenotype implied that uncharged NH₃, not NH₄⁺, is transported. It was proposed that AmtB did not concentrate ammonia but only facilitated equilibrium across the membrane. This mechanism of transport might have significant physiological implications, which are discussed in "Physiological Function of ⁵⁴" (below).

Although E. coli, S. enterica serovar Typhimurium, and K. aerogenes are closely related, they differ in their ability to utilize certain nitrogen sources and in the regulation of some genes of nitrogen metabolism. For example, nitrogen limitation strongly represses glutamate dehydrogenase in K. aerogenes but not in E. coli (16, 127). The transcriptional regulator Nac accounts for many of these differences in regulation. Nac has been most extensively studied from K. aerogenes but has also been studied from E. coli. In contrast, S. enterica serovar Typhimurium lacks Nac (114).

The nac operon is monocistronic (114, 145). Transcription initiated from the K. aerogenes nac promoter requires ⁵⁴ and NR_I~P (16, 54, 106). Nitrogen limitation induces E. coli nac (114, 176), and computer analysis indicates a likely binding site for E⁵⁴ with a score of 85.2, which is very high. These results suggest that NR_I and E⁵⁴ are also required for E. coli nac expression. Nac negatively modulates its own synthesis in K. aerogenes by interfering with the interaction between NR_I and RNA polymerase (55, 114), and results from the DNA microarray analysis are consistent with such regulation in E. coli (176).

Arginine (via agmatine) and ornithine are both precursors for putrescine, which can be metabolized to GABA, and then to succinate (Fig. 7). Nitrogen limitation induces enzymes of GABA catabolism (175). Therefore, it was reasonable to propose that Ntr regulators affect the catabolism of all of these compounds, and some evidence is consistent with this regulation (147). However, recent studies with mutants containing targeted gene disruptions have indicated unsuspected pathways and a surprising complexity and redundancy of pathways and regulators. Only the enzymes of arginine and GABA catabolism require ⁵⁴, while the enzymes of putrescine catabolism may not.

View larger version (24K): [in this window] [in a new window]   FIG. 7.   Metabolic relationships between ornithine, arginine, putrescine, and GABA. A thick black arrow indicates that nitrogen limitation induces the enzyme indicated. A reaction catalyzed by two (or more) enzymes is indicated by two arrows. The genes that specify the enzymes are shown when they are known. A dashed arrow indicates that the gene has yet to be identified.

astCADBE operon and catabolism of arginine and ornithine. The five-step arginine succinyltransferase pathway catabolizes arginine (144). The pathway is named after the first reaction, which is the succinylation of the -amino group of arginine. The astCADBE operon codes for the proteins of the pathway (144). Disruption of the operon in E. coli prevents growth with arginine as a nitrogen source and impairs but does not eliminate growth with ornithine (60, 144). It has been proposed that AstC (which catalyzes the deamination of succinylornithine) is one of at least two transaminases that can deaminate ornithine, which generates an intermediate of proline catabolism (144). The identity of the second transaminase is unknown.

GABA and putrescine catabolism and the gabDTPC operon. E. coli can utilize GABA as a nitrogen source (H. Kasbarian, S. Ruback, and L. Reitzer, unpublished results), although earlier studies indicated otherwise (50). A mutant with a gabDT deletion cannot utilize GABA as a nitrogen source (Ruback and Reitzer, unpublished). The distance between genes suggests the existence of a gabDTPC operon. Furthermore, transcript analysis indicates the presence of only one promoter for these genes, a promoter just upstream from gabD. Each gene of the putative operon has been implicated in GABA catabolism. GabT is a transaminase that deaminates GABA to succinic semialdehyde. GabD is an NADP-specific succinic semialdehyde dehydrogenase that oxidizes succinic semialdehyde to succinate. (An NAD-dependent succinic semialdehyde dehydrogenase is specified by the sad gene, and GABA or a product of GABA metabolism induces its synthesis [49].) GabP specifies a GABA permease (92). GabC appears to be a specific repressor since a deletion of gabC stimulates growth with GABA as a nitrogen source (Kasbarian et al., unpublished). Either nitrogen limitation or entry into stationary phase activates gab operon expression (12, 175; Kasbarian et al., unpublished). Expression of the gab operon requires Nac during nitrogen-limited growth, and Nac is required to reconstitute transcription in vitro (H. Kasbarian, A. Kiupakis, S. Ruback, and L. Reitzer, unpublished results). ^S is required for expression during stationary phase.

ygjG. Nitrogen limitation results in a three- to fivefold increase in the levels of steady-state ygjG transcripts (176). Expression of this gene requires ⁵⁴, and the transcription start site has been determined (A. Kiupakis, R. Ye, and L. Reitzer, unpublished result). The score for this promoter is 70.7, which is low for an authentic ⁵⁴-dependent promoter. The gene specifies a putative -transaminase which either removes the amino group from compounds with terminal primary amines (e.g., putrescine or ornithine), or adds amino groups to compounds with an aldehyde group (e.g., N-acetylglutamic semialdehyde, an intermediate in ornithine formation). Putrescine and compounds metabolized to putrescine activate ygiG expression, which suggests a possible role in putrescine catabolism. However, a mutant with a disruption of ygjG grows normally with putrescine as a nitrogen source, which suggests that YgjG is a redundant transaminase (C. Pybus and L. Reitzer, unpublished observation).

(i) The three transport systems. E. coli has three characterized arginine transport systems (described below), while S. enterica serovar Typhimurium has at least two (98). An early study suggested the presence of three periplasmic arginine binding proteins in E. coli (136). No strain has been constructed with mutations in all the characterized systems. Therefore, it is conceivable that there are other transport systems.

(ii) Repression by arginine. Arginine represses all three E. coli transport systems (41, 137, 169). A possible mechanism of repression would involve ArgR, which mediates arginine repression for the enzymes of arginine synthesis. A computer analysis of ArgR sites in E. coli identified two sites in the art operon: one preceding artP and one preceding artJ (111). However, a missense mutation in argR had no effect on the kinetically detectable arginine transport systems (39). Instead of ArgR, ArgP and ArgK have been proposed to mediate arginine repression. ArgP is a transcriptional regulator required for synthesis of ArgK, which is required for arginine transport. Mutations in argP and argK affect both the ArgT and AbpS systems (40, 41, 137). Only one gene separates argP and argK, but they appear to be independently expressed. (argK is currently not listed in either GenBank or the latest E. coli genetic map. However, argK is ygfD, also called b2918 in GenBank.) ArgP is a LysR-type regulator that activates argK expression in the absence of arginine (37). ArgP complexed with arginine fails to activate argK expression and represses its own synthesis. ArgK has an ATPase activity that is apparently required for transport activity (158). ArgK also phosphorylates the periplasmic ArgT and AbpS (36), although this phosphorylation is not required for transport (38). ArgP is the previously characterized IciA, an inhibitor of the initiation of DNA replication (37, 154). An iciA mutant has no obvious phenotype, except for difficulty during dilution into fresh growth medium (154). It is conceivable that ArgP/IciA is a sensor of amino acid sufficiency that couples DNA synthesis with metabolism in some environments.

(iii) Transport and activation during nitrogen limitation. Nitrogen limitation induces ArgT in E. coli and S. enterica serovar Typhimurium, and this induction does not require arginine (98, 176). The genetics and regulation of arginine transport have been studied in S. enterica serovar Typhimurium, and it is assumed that they will be similar in E. coli. In S. enterica serovar Typhimurium, loss of ArgT reduced but did not eliminate the binding of arginine to periplasmic proteins and an argT mutant grew normally with arginine as a nitrogen source, which implies a second transport system in S. enterica serovar Typhimurium during nitrogen-limited growth (98).

Glutamine. Glutamine transport has been studied in both E. coli and S. enterica serovar Typhimurium. The kinetically dominant system requires GlnH, a high-affinity glutamine-specific binding protein in the periplasm (162). The glnHPQ operon specifies GlnH and two membrane proteins, which presumably interact with GlnH (119). Loss of GlnH unmasks a low-affinity glutamate-inhibitable glutamine transport system in E. coli (162) but has no effect on growth in S. enterica serovar Typhimurium (98). These results suggest a second glutamine transport system. Kinetic assays of glutamine transport also suggest two transport systems (11, 168). It is possible that the glutamate-inhibitable system requires the periplasmic glutamate-aspartate binding protein (see the next section), which also binds glutamine (167).

Glutamate-aspartate. Schellenberg and Furlong defined five transport systems for glutamate and aspartate in E. coli by a combination of genetic and biochemical experiments (140). There are no studies with gene fusions; therefore, the regulation and functions of the individual systems are poorly understood.

ddpXABCDE operon. Microarray analysis shows that general nitrogen limitation (i.e., no specific induction) induces the ddpXABCDE operon 52- to 60-fold, which is more than any other operon. Expression appears to require NR_I (176). A potential ⁵⁴-dependent promoter with a score of 76.6 precedes the operon. DdpX is a zinc-containing D-alanyl-D-alanine dipeptidase, while the other products of the operon appear to code for components of a dipeptide permease (100). There are two sources of D-alanyl-D-alanine: it is an intermediate in peptidoglycan synthesis, and it may be released during cross-linking of two diaminopimelic acids. Peptidoglycan cross-linking occurs in the stationary phase (156). Entry into the stationary phase induces the ddpXABCDE operon, and this induction requires ^S (100). It is not known whether peptidoglycan remodeling also occurs in nitrogen-limited cultures. It has been proposed that the function of the ddpXABCDE products is to scavenge D-alanyl-D-alanine (176).

Peptide transport and ompF. E. coli, S. enterica serovar Typhimurium, and other gram-negative bacteria digest peptides intracellularly after their passage through the outer membrane and transport via periplasmic binding protein-dependent transport systems. Nitrogen limitation activates the expression of dppABCDF and oppABCDF (176). The products of these operons are the major peptide transporters in E. coli and S. enterica serovar Typhimurium (120), and the periplasmic components of these systems, DppA and OppA, are among the most abundant proteins in the periplasm (1, 70, 121). In addition to its function as a transport protein, DppA is required for chemotaxis to peptides (