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Microbiology and Molecular Biology Reviews, September 2001, p. 422-444, Vol. 65, No. 3
1092-2172/01/$04.00+0   DOI: 10.1128/MMBR.65.3.422-444.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.

Metabolic Context and Possible Physiological Themes of sigma 54-Dependent Genes in Escherichia coli

Larry Reitzer* and Barbara L. Schneider

Department of Molecular and Cell Biology, The University of Texas at Dallas, Richardson, Texas 75083-0688

SUMMARY
INTRODUCTION AND OVERVIEW
    Scope
    Sigma Subunits and Their Function in E. coli
    Unique Features of sigma 54-Dependent Transcription
    Control of sigma 54-Dependent Promoters
COMMON FEATURES OF sigma 54-DEPENDENT PROMOTERS
COMPUTER IDENTIFICATION OF POTENTIAL sigma 54-DEPENDENT PROMOTERS
    Site Identification and the Problem of False Positives
    Predictive Value of the Promoter Scores
    Estimating the Number of sigma 54-Dependent Promoters in E. coli
sigma 54-DEPENDENT GENES OF NITROGEN METABOLISM
    Nitrogen Assimilation and Its Control
        Nitrogen assimilation.
        Control of ammonia assimilation and GS activity: role of glutamine.
    Nitrogen-Regulated (Ntr) Response
        Nitrogen sources.
        Control of the Ntr response by glutamine.
        alpha -Ketoglutarate counters the effects of unmodified PII.
        NRI regulon.
        Nac regulon.
        Why are there two general regulators of the Ntr response?
        Function of the Ntr response.
    glnALG (glnA-ntrBC) Operon
    glnK-amtB Operon
        GlnK: a PII paralog.
        Why have GlnK?
        Product of amtB and ammonia transport.
    nac
    Catabolism of Arginine, Agmatine, Ornithine, Putrescine, and gamma -Aminobutyrate
        astCADBE operon and catabolism of arginine and ornithine.
        GABA and putrescine catabolism and the gabDTPC operon.
        ygjG.
    sigma 54-dependent Amino Acid Transport Systems
        Arginine.
        (i) The three transport systems.
        (ii) Repression by arginine.
        (iii) Transport and activation during nitrogen limitation.
        Histidine.
        Glutamine.
        Glutamate-aspartate.
        ddpXABCDE operon.
        Peptide transport and ompF.
    Potential sigma 54-Dependent Genes That Are Induced by Nitrogen Limitation
sigma 54-DEPENDENT GENES THAT ARE NOT INVOLVED IN NITROGEN METABOLISM
    Formate Catabolic Genes and the FhlA Regulon
        Formate metabolism.
        The four confirmed sigma 54-dependent operons of formate metabolism.
        Hydrogenase 4.
    ato Operon and Acetoacetate Catabolism
    prpBCDE Operon and Propionate Catabolism
    psp Operon and Phage Shock Response
    rtcBA Operon
    zraSR (hydHG), zraP, and the Response to Zn2+ and Pb2+
OTHER GENES WITH HIGHLY RANKED POTENTIAL Esigma 54 BINDING SITES
sigma 54-DEPENDENT ACTIVATORS
PHYSIOLOGICAL FUNCTION OF sigma 54
    Possible Relationship between the sigma 54-Dependent Genes
    Evolutionary Persistence of sigma 54
ACKNOWLEDGMENTS
REFERENCES


SUMMARY
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sigma 54 has several features that distinguish it from other sigma factors in Escherichia coli: it is not homologous to other sigma  subunits, sigma 54-dependent expression absolutely requires an activator, and the activator binding sites can be far from the transcription start site. A rationale for these properties has not been readily apparent, in part because of an inability to assign a common physiological function for sigma 54-dependent genes. Surveys of sigma 54-dependent genes from a variety of organisms suggest that the products of these genes are often involved in nitrogen assimilation; however, many are not. Such broad surveys inevitably remove the sigma 54-dependent genes from a potentially coherent metabolic context. To address this concern, we consider the function and metabolic context of sigma 54-dependent genes primarily from a single organism, Escherichia coli, in which a reasonably complete list of sigma 54-dependent genes has been identified by computer analysis combined with a DNA microarray analysis of nitrogen limitation-induced genes. E. coli appears to have approximately 30 sigma 54-dependent operons, and about half are involved in nitrogen assimilation and metabolism. A possible physiological relationship between sigma 54-dependent genes may be based on the fact that nitrogen assimilation consumes energy and intermediates of central metabolism. The products of the sigma 54-dependent genes that are not involved in nitrogen metabolism may prevent depletion of metabolites and energy resources in certain environments or partially neutralize adverse conditions. Such a relationship may limit the number of physiological themes of sigma 54-dependent genes within a single organism and may partially account for the unique features of sigma 54 and sigma 54-dependent gene expression.


INTRODUCTION AND OVERVIEW
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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). sigma 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 sigma 54 and sigma 54-dependent activation, including recent ones (14, 28, 97, 110, 113, 148, 151). However, discussions of sigma 54 function usually consider genes from several organisms and therefore remove sigma 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 sigma 54-dependent genes from a single organism, Escherichia coli. The most meaningful discussion of sigma 54 function requires a complete set of sigma 54-dependent genes. Two sources provide information on likely sigma 54-dependent promoters: a recent DNA microarray analysis of transcripts present during nitrogen-limited growth (176) and computer analysis of potential sigma 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 sigma 54-dependent genes. The central thesis of this review is that the sigma 54-dependent genes of E. coli have only a few metabolic themes and that these themes may be related.

Sigma Subunits and Their Function in E. coli

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

Unique Features of sigma 54-Dependent Transcription

The sigma  factors in E. coli are homologous to sigma 70, except for sigma 54 (110). Not surprisingly, sigma 54-dependent transcription has several distinctive features (reviewed in reference 28). Core RNA polymerase (E) complexed to a sigma 70-like factor can be sufficient for open promoter complex formation. In contrast, Esigma 54 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 sigma 54 (see "Physiological function of sigma 54" below).

The activators of Esigma 54-dependent genes are unusual. (The individual E. coli activators are discussed in a separate section.) Unlike most eubacterial transcriptional activators, the sigma 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 Esigma 54 from these binding sites. This interaction sometimes requires a DNA bending protein. The DNA bending proteins that participate in sigma 54-dependent gene expression in E. coli are integration host factor (IHF) and ArgR, the arginine repressor (72, 103). When transcription from a sigma 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 sigma 54-dependent activators is an essential ATPase activity (166).

Control of sigma 54-Dependent Promoters

The most important control of sigma 54-dependent genes is through modulation of the activator's ATPase activity. The sigma 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 sigma 54 activity, either by ligand binding or by covalent modification, are not known in E. coli. Furthermore, the intracellular level of sigma 54 is apparently constant (87), and expression of rpoN, which specifies sigma 54, appears to be constitutive (33). Strain W3110 contains about 700 molecules of sigma 70 per cell and 110 molecules of sigma 54, whereas strain MC4100 may contain only about 285 molecules of sigma 70 and as few as 13 to 22 molecules of sigma 54 (87). The low level of sigma 54 could have regulatory implications. For example, it is possible that different sigma 54-dependent operons compete for limiting sigma 54. This possibility is even more plausible if there is some physiological relationship between the sigma 54-dependent genes. One purpose of this review is to explore this issue.


COMMON FEATURES OF sigma 54-DEPENDENT PROMOTERS
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There are 11 sigma 54-dependent promoters for which the transcription start site has been determined in vivo, in vitro, or both. These promoters precede astCADBE, fdhF, glnALG, glnHPQ, hycABCDEFGHI, hydN-hypF, hypABCDE-fhlA, prpBCDE, pspABCDE, rtcBA, and ygjG. It is virtually a certainty that atoDAEB, glnK-amtB, nac, zraP, and zraSR (hydHG) have a sigma 54-dependent promoter, since their expression absolutely requires sigma 54 and their promoter regions contain an easily recognizable site for Esigma 54. Also, argT-hisJQMP has a verified sigma 54-dependent promoter in Salmonella enterica serovar Typhimurium, and nitrogen limitation induces these genes in E. coli, which suggests that this operon possesses a sigma 54-dependent promoter.

We will use the promoters for these 17 operons to characterize the sigma 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 sigma 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 Esigma 54 in relation to the binding sites for the sigma 54-dependent activators (when known) and the nearest upstream structural gene.

                              
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TABLE 1.   E. coli operons with confirmed sigma 54-dependent promoters



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FIG. 1.   sigma 54-dependent genes in E. coli. The known sigma 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.


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FIG. 2.   Regulatory regions of the characterized sigma 54-dependent genes. The following features are shown for each of the sigma 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 sigma 54-dependent promoters are located outside the structural genes. This does not necessarily mean that an authentic binding site for Esigma 54 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 sigma 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 Esigma 54 and an activator. Fourth, the distance from the 3' end of the Esigma 54 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 Esigma 54 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 sigma 54-dependent promoters require a DNA bending protein. Both of the DNA bending proteins that facilitate the activation of sigma 54-dependent genes in E. coli, IHF and ArgR, have an AT-rich consensus sequence. Alternately, sigma 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 Esigma 54 binding site.

Despite the reasonably uniform properties of the known sigma 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 Esigma 54, which is described in the next section.


COMPUTER IDENTIFICATION OF POTENTIAL sigma 54-DEPENDENT PROMOTERS
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Site Identification and the Problem of False Positives

The most meaningful analysis of the physiological function of sigma 54 requires a comprehensive set of sigma 54-dependent genes. One method to help determine the complete set of such promoters is a computer analysis of potential sigma 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 sigma 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. 

                              
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TABLE 2.   Comparison of consensus sequence for sigma 54-dependent promoters from known E. coli promoters and the SeqScan programa

The SeqScan program identified approximately 8,000 potential sigma 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 sigma 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 sigma 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 Esigma 54 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.

                              
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TABLE 3.   Computer ranking of intergenic sigma 54 sitesa

Predictive Value of the Promoter Scores

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 sigma 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 sigma 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) sigma 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 sigma 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 Esigma 54 binding sites, which implies that the failure to detect such a site is a reliable indicator that such a site does not exist.


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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.

Estimating the Number of sigma 54-Dependent Promoters in E. coli

There are 17 sigma 54-dependent promoters which either have been verified by direct evidence or whose expression requires sigma 54; there are 7 other operons which are induced by nitrogen limitation (often associated with sigma 54), appear to require a sigma 54-dependent activator, and for which computer analysis suggests the presence of an appropriately located sequence for a sigma 54-dependent promoter: b1012-b1006, chaC, ddpXABCDE, gltIJKL, potFGHI, yeaGH, and yhdWXYZ. Assuming that all of these genes have a functional sigma 54-dependent promoter (which is unlikely) and that a few have been missed for various reasons (e.g., misidentified open reading frames), we estimate that E. coli contains about 30 sigma 54-dependent promoters.


sigma 54-DEPENDENT GENES OF NITROGEN METABOLISM
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Of the 17 known sigma 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 sigma 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 and Its Control

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.

Ammonia can be considered the focal point of nitrogen assimilation. There are two routes of ammonia assimilation (Fig. 4). For the first pathway, glutamate dehydrogenase assimilates ammonia and synthesizes glutamate. For the second pathway, glutamine synthetase (GS) assimilates ammonia, and glutamate synthase synthesizes glutamate. The former pathway is often associated with the presence of ammonia, and the latter pathway is associated with low ammonia levels or growth with a nitrogen source other than ammonia, since the Km for ammonia for glutamate dehydrogenase is about 20-fold higher than that for GS. However, the most important difference between the two pathways appears to be that the former does not consume ATP but the latter does. Helling has shown that the glutamate dehydrogenase pathway is physiologically advantageous during carbon- and energy-limited growth while the GS-glutamate synthase pathway is used whenever energy is readily available (66, 67). (The energy difference between the ammonia assimilation pathways can be calculated, and it is significant. A 1-g amount of E. coli requires the synthesis of about 57,000 µmol of ATP and contains about 10,500 µmol of nitrogen. The ATP requirement for glutamine synthesis depends on the pathway of ammonia assimilation. If glutamate dehydrogenase assimilates ammonia, the cell requires about 2,300 µmol of glutamine and a corresponding amount of ATP for its synthesis. If the GS-glutamate synthase route assimilates ammonia, glutamine is also the precursor for glutamate and the cell must synthesize an additional 8,070 µmol of glutamine and ATP, or 14% more energy.)


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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 sigma 54, is beyond the scope of the review. Instead, we will focus on the control of GS activity.

Two different but related mechanisms control GS activity: cumulative feedback inhibition by metabolites that require glutamine for their synthesis and covalent adenylylation (127). GS is a dodecamer, and adenylylation inactivates the modified subunit and renders the remaining subunits sensitive to feedback inhibition (127). A major function of adenylylation is to determine the function of GS. When GS is highly adenylylated and subject to cumulative feedback inhibition, its primary function is glutamine synthesis. In this situation, GS is just active enough to supply glutamine but not to supply glutamate. E. coli requires about 2,310 µmol of glutamine for biosyntheses per g (dry weight). In contrast, unadenylylated GS, which is not subject to feedback inhibition, can assimilate enough ammonia to meet all the cell's need for organic nitrogen. In this situation, E. coli needs to synthesize about 10,300 µmol of glutamine per g. A second function of adenylylation is to prevent the depletion of intracellular glutamate during the transition to a nitrogen-rich environment (96).

A cascade of three proteins controls GS adenylylation: the uridylyltransferase (UTase)-uridylyl removing (UR) enzyme, which in turn controls the activities of PII and adenylyltransferase (ATase). It has been a long-standing paradigm that the ratio of alpha -ketoglutarate to glutamine (a sensor of relative carbon-to-nitrogen sufficiency) controls UTase-UR activity and therefore GS adenylylation. This conclusion was based on the properties of partially purified UTase-UR (2), which were not confirmed with purified UTase-UR (83). Furthermore, metabolite measurements suggested that low intracellular glutamine levels might be sufficient to control the response to nitrogen limitation (discussed below), which UTase-UR also controls (79). These results suggest that glutamine is the primary effector of UTase-UR and therefore of GS adenylylation (Fig. 5). Low glutamine levels stimulate UTase activity, which uridylylates PII. PII-UMP interacts with adenylyltransferase, which now removes adenylyl groups from GS and activates GS activity. High glutamine (nitrogen excess) stimulates UR activity, which results in the formation of unmodified PII, whose interaction with adenylyltransferase stimulates adenylylation and reduces GS specific activity. Even though alpha -ketoglutarate does not affect UTase-UR, it does control the activity of unmodified PII (discussed below).


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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-Regulated (Ntr) Response

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.

Control of the Ntr response by glutamine. The two proteins that control GS adenylylation, UTase-UR and PII, also control the Ntr response. High glutamine levels (nitrogen sufficiency) stimulate UR activity, which prevents uridylylation of PII. Unmodified PII interacts with nitrogen regulator II (NRII, also called NtrB) and stimulates the dephosphorylation of nitrogen regulator I (NR1, also called NtrC). The net effect is low expression of the glnALG operon and failure to activate Ntr genes. Low glutamine levels (nitrogen limitation) result in the formation of PII-UMP, which is unable to interact with NRII. In this situation, NRII phosphorylates itself and transfers the activated phosphate to NRI. NRI-P then activates the expression of the glnALG operon and other Ntr genes.

alpha -Ketoglutarate counters the effects of unmodified PII. Even though recent studies have suggested that the ratio of glutamine to alpha -ketoglutarate does not regulate UTase-UR activity, alpha -ketoglutarate does affect PII activity (83-85). alpha -Ketoglutarate counteracts the effects of unmodified PII (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 alpha -ketoglutarate (via PII) 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 PII is uridylylated. It will be suggested elsewhere in this review that such coordination might be a function of GlnK, a PII-like protein.

NRI regulon. NRI 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 PII and an ammonia transporter), nac (a sigma 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 NRI 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, NRI represses the two minor promoters, glnAp1 and glnLp, of the glnALG operon.

Nac regulon. There are two majors regulators of the Ntr response: NRI and Nac. NRI activates sigma 54-dependent promoters, while Nac activates sigma 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).

Nac has been most intensively studied in Klebsiella aerogenes, where it activates genes for histidine, proline, urea, and D-alanine catabolism and represses glutamate dehydrogenase (16, 80, 106). It does not regulate the same genes in E. coli. E. coli lacks hut and ure operons, and Nac does not regulate the E. coli dad operon (16, 109). Nac deficiency in E. coli results in a slight derepression of glutamate dehydrogenase synthesis, slightly slower growth with cytosine as the nitrogen source, and slightly faster growth with arginine (114). The effect on arginine utilization is undoubtedly indirect, since synthesis of arginine catabolic enzymes does not require Nac (114) (see "astCADBE operon and catabolism of arginine and ornithine" below). Microarray analysis suggests Nac-dependent induction of b1440-1444 (probably for putrescine transport), codBA (cytosine metabolism), dppABCDF (dipeptide transport), fklB-cycA (D-alanine, D-serine, and glycine transport), gabDTP (gamma -aminobutyrate [GABA] metabolism), nupC (nucleoside transport), ompF (outer membrane protein F), oppABCD (oligopeptide transport), yedL (unknown function), and yhiE (unknown function) (176). Nac-dependent control has been directly verified for the gab operon (S. Ruback and L. Reitzer, unpublished observation) but not for the other genes.

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 NRI-dependent control. NRI-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 sigma 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).


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FIG. 6.   Binding sites for regulatory proteins at the hutUH promoter.

Function of the Ntr response. It is likely that most of the sigma 54-dependent Ntr genes have been identified using results from a microarray analysis and the complementary computer analysis of potential sigma 54-dependent promoters. The Ntr genes can be divided into a few categories: GS, regulators, transport proteins, and catabolic enzymes.

Most Ntr genes specify transport proteins: amtB (ammonia), argT-hisJMPQ (arginine, lysine, ornithine, and histidine), b1006 (uracil?), b1440-b1444 (putrescine?), codB (cytosine), cycA (D-alanine, D-serine, and glycine), ddpXABCDE (D-alanyl-D-alanine), dppABCDE (dipeptides), gabP (GABA), glnHPQ (glutamine), gltIJKL (glutamate-aspartate), nupC (nucleosides), oppABCD (oligopeptides), potFGHI (putrescine), and yhdWXYZ (amino acids?). It has been suggested that a major function of the Ntr response is scavenging (
176). However, Ntr proteins do not scavenge all amino acids. There are no Ntr-dependent transport systems for the aromatic amino acids, the branched-chain amino acids, threonine, methionine, or cysteine. An explanation for this pattern of expression may be that E. coli does not readily use these amino acids as nitrogen sources, which implies that their nitrogens are not readily available. In other words, E. coli generally has Ntr-dependent transport systems only for amino acids that can readily provide nitrogen for glutamate and glutamine synthesis.

Another class of Ntr genes specify enzymes for catabolic pathways. There are very few Ntr catabolic pathways, and unlike the transport genes, optimal synthesis usually requires specific induction. It should be noted that these catabolic pathways are not the major amino acid catabolic pathways, i.e., those that degrade amino acids that can be converted in one or two steps to intermediates of central metabolism, such as aspartate, glutamate, glutamine, serine, alanine, and glycine. An explanation for this observation is not apparent.

In summary, the function of the Ntr response is nitrogen assimilation when the intracellular glutamine level is low. This explains all the major aspects of the Ntr response: the regulation of GS activity and synthesis by glutamine, the regulation of the Ntr response by glutamine, and the reason why there are so many Ntr transport systems that scavenge nitrogenous compounds that have readily utilizable nitrogen.

glnALG (glnA-ntrBC) Operon

The glnALG operon codes for GS, NRII, and NRI, respectively (127, 129). All three products of this operon are required for nitrogen assimilation and the Ntr response (discussed above). sigma 54 and NRI~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 NRI indicate that nitrogen limitation induces NRI synthesis 14-fold (128). Minor promoters, glnAp1 and glnLp, ensure basal synthesis of the important products of this operon (130, 157).

glnK-amtB Operon

Nitrogen limitation and NRI 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 Esigma 54 and NRI (Fig. 2) (160). In addition, nitrogen limitation increases the glnK transcript at least 10-fold (176). Therefore, this operon undoubtedly contains an authentic sigma 54-dependent promoter. Both products of the operon contribute to the response to nitrogen limitation.

GlnK: a PII paralog. The existence of a PII paralog was first suspected because of the rapid deadenylylation of GS in an E. coli glnB (PII-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 PII 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 NRI, which has been suggested to cause inappropriate overexpression of an Ntr gene (10). An alternate explanation is NRI-dependent overexpression of a sigma 54-dependent gene that is not normally regulated by NRI.

Purified GlnK and PII have similar activities, but the regulation of these activities is different (9, 58, 160, 161). However, the physiological relevance of many differences has not been established and is sometimes refuted by mutant phenotypes. The only safe basis for discussing the relevant properties of GlnK is when they account for the phenotype of mutants. One aspect of the mutant phenotype is the higher basal expression of an Ntr gene in a nitrogen-rich environment, which implies that GlnK suppresses this expression. One property of purified GlnK that accounts for this suppression is the relatively slow uridylylation of GlnK compared to that of PII (9). This property is accentuated by the formation of GlnK-PII heterotrimers (58, 161) and the inactivation of PII-UMP by GlnK in such heterotrimers (161). The net effect is enhanced dephosphorylation of NRI~P and lower expression of Ntr genes. The second aspect of the phenotype of a glnK mutant is lower induced expression of an Ntr gene in a nitrogen-limited environment, which implies that GlnK stimulates Ntr expression. This is consistent with one property of purified GlnK. Although GlnK can efficiently stimulate the dephosphorylation of NRI~P via NRII, alpha -ketoglutarate is more efficient in inhibiting the activity of GlnK than of PII (9).

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 alpha -ketoglutarate partially explains this effect. In this case, GlnK essentially restores the coordination of carbon and nitrogen metabolism (the responsiveness to the ratio of alpha -ketoglutarate to glutamine) that is lost when PII is completely uridylylated, i.e., during nitrogen-limited growth, and is no longer responsive to alpha -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 sigma 54" (below).

nac

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

Catabolism of Arginine, Agmatine, Ornithine, Putrescine, and gamma -Aminobutyrate

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 sigma 54, while the enzymes of putrescine catabolism may not.


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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 alpha -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.

Expression of the astCADBE operon in E. coli and S. enterica serovar Typhimurium requires either nitrogen limitation or entry into stationary phase (12, 60, 103, 144). There are two transcription start sites, which are separated by five bases (A. Kiupakis and L. Reitzer, unpublished results). Expression from the Ntr promoter requires NRI~P and sigma 54, while expression from the other promoter requires Esigma S. SeqScan gives the sigma 54-dependent promoter a score of 85.5. Some evidence suggests that transcription from one promoter prevents transcription from the other (60). One unusual feature of the ast operon in E. coli is that ArgR binds to the region between the Esigma 54 and NRI binding sites and has been proposed to stimulate the interaction between the two proteins (103). ArgR is required for optimal transcription of the E. coli ast operon but is not absolutely necessary (Kiupakis and Reitzer, unpublished). In contrast, ArgR appears to be required for the S. enterica serovar Typhimurium ast operon (103). The ast operon contains the only known E. coli NRI-dependent promoter that also requires specific induction. Microarray analysis indicates that general nitrogen limitation (i.e., without arginine induction) increases ast transcription 7- to 11-fold (176), which is consistent with the results of a direct assay of the gene products (144). Arginine induces the enzymes three- to fourfold further (144), which is consistent with in vitro results (Kiupakis and Reitzer, unpublished). Another unusual aspect of ast expression is that a strain with a glnL (ntrB) deletion cannot utilize arginine as a nitrogen source (L. Reitzer, unpublished observation) but can still activate glnA expression, albeit not as rapidly (130). Expression of the ast operon requires phosphorylation of NRI by both NRII and small phosphodonors (B. L. Schneider, D. Fewell, and L. J. Reitzer, unpublished observation).

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). sigma S is required for expression during stationary phase.

GABA (via gamma -aminobutyraldehyde) is a presumed intermediate in putrescine catabolism. Unexpectedly, a strain with a deletion of gabDT grew normally with putrescine or agmatine as a nitrogen source (Kasbarian et al., unpublished). Even more surprising is the observation that an rpoN mutant (sigma 54 deficient) grew normally with putrescine as a nitrogen source (Kiupakis and Reitzer, unpublished). These results would suggest that nitrogen limitation is not required for induction of putrescine catabolic genes, but this is not the case. Microarray analysis suggests that nitrogen limitation activates the expression of two different putrescine transport operons: potFGHI (five- to sixfold) and b1440-1444 (five- to sevenfold) (176). In addition to these transport systems, E. coli possesses two sigma 54-independent transport systems, the products of potABCD (which preferentially transport spermidine but also transport putrescine) and of potE (78). The genes of putrescine catabolism and the physiological function of the four transport systems have yet to be established.

ygjG. Nitrogen limitation results in a three- to fivefold increase in the levels of steady-state ygjG transcripts (176). Expression of this gene requires sigma 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 sigma 54-dependent promoter. The gene specifies a putative omega -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).

sigma 54-dependent Amino Acid Transport Systems

More than half of the genes activated by nitrogen limitation in E. coli code for transport systems. Such activation usually does not require specific induction. Such regulation combined with the observation that mutational inactivation of the genes for sigma 54-dependent amino acid transport system does not prevent growth on the respective amino acid suggests a scavenging function. In this section, the sigma 54-dependent amino acid transport systems are considered within the context of the multiple transport systems for these amino acids.

Arginine.

(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.

Only one of the three E. coli arginine transport systems requires sigma 54 for its synthesis. It contains the periplasmic ArgT protein, also called the LAO protein, which binds lysine, ornithine, and arginine with high affinity (136). The argT gene and its product have been extensively studied in S. enterica serovar Typhimurium but not in E. coli. ArgT interacts with HisP of S. enterica serovar Typhimurium (98). HisJ, the periplasmic histidine binding protein, also interacts with HisP (5). An S. enterica serovar Typhimurium hisP mutant grows much more slowly with arginine as the nitrogen source than an argT mutant does, which suggests that HisP also interacts with an arginine binding periplasmic protein other than ArgT (98).

sigma 54-independent arginine transport systems have been studied only in E. coli. One system contains AbpS, also called the arginine-ornithine protein, which binds arginine and ornithine in the periplasm with lower affinity than the LAO protein does (34). Early reports refer to this protein as a low-affinity, arginine-specific protein (41, 136). The abpS gene has been approximately mapped to min 63.5 of the most recent E. coli map (35). AbpS has been purified, and its size and amino acid composition have been determined (36). However, no gene near min 63 specifies a protein with the published amino acid composition. The nearest matches to this amino acid composition in the E. coli genome, in descending order, are the products of artI, artJ, and hisJ, which are located at min 19.4, 19.4, and 52.3, respectively. It is possible that the sequenced MG1655 does not contain abpS. The second sigma 54-independent system consists of the artPIQM-artJ operons, which are at min 19 of the E. coli chromosome (169). ArtJ is a periplasmic protein that binds arginine but not ornithine (169). ArtI is another putative periplasmic binding protein, but it does not detectably bind any amino acid. Mutants with mutations in the ArtJ system do not exist, but overexpression increases arginine transport, which is consistent with a proposed function in arginine transport (169). Promoters precede artP and artJ, and neither appears to require sigma 54 (169).

(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).

argT is adjacent to the hisJQMP operon, which codes for components of a histidine transport system. Transcript mapping in wild-type S. enterica serovar Typhimurium, not in an extensively studied dhuA1 mutant which appears to have a mutationally created promoter, shows a sigma 54-dependent promoter immediately preceding argT but not immediately preceding hisJ (6). This is consistent with expression studies with reporter gene fusions, which failed to identify an Ntr promoter preceding hisJ (142, 143). The potential sigma 54-dependent promoter preceding argT in E. coli has a score of 72.3. There is only one binding site for NRI, and it is in the argT-hisJ intercistronic region and not upstream from argT (6). The function of this site is not clear. It does not appear to be necessary for expression, which would imply that NRI activates the argT promoter without binding to DNA (143). There is precedent for NRI-dependent transcription that does not require a DNA binding site (131, 171).

Histidine. The genes and regulation of the HisJ transport system were discussed in the preceding section because of their relation to arginine transport. It is not known whether there are other histidine transport systems.

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).

Expression of glnHPQ requires nitrogen limitation but not glutamine (18, 98, 168). Nitrogen limitation increases the production of glnHPQ transcripts five- to ninefold (176). The operon contains two promoters (118). Transcription from the downstream promoter, glnHp2, requires sigma 54 and NR1 and is enhanced by IHF (44). The promoter has a score of 80.7. The factors that control the upstream promoter, glnHp1, have not been examined.

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.

Nitrogen limitation induces a periplasmic protein that binds both glutamate and aspartate in S. enterica serovar Typhimurium (98). Such a protein has been purified and characterized from E. coli (167). The closest match in the entire SWISS-PROT database with the published amino acid composition of the glutamate-aspartate binding protein is gltI (also called ybeJ) from E. coli. The gltI product was the only protein in E. coli with the correct pI, size, and number of cysteines. gltI might be part of a gltIJKL operon. Despite the annotation of gltIJKL as part of a glutamate-aspartate transport system, no published evidence supports this possibility (17).

There are no potential sigma 54-dependent promoters preceding gltI with a score greater than 60. However, the gene preceding gltI specifies an IS5 transposase, and the promoter region for the transposase gene contains a possible sigma 54-dependent promoter with a score of 88, which is very high. Microarray analysis indicates a three- to sixfold increase of gltI transcription during nitrogen limitation, and NRI-dependent activation (176). These results suggest that the sigma 54-dependent promoter preceding the transposase gene can initiate gltIJKL transcription.

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 NRI (176). A potential sigma 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 sigma 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 (