Childhood trajectories of anxiousness and disruptiveness explain the association between early-life adversity and attempted suicide.

BACKGROUND: Suicidal behavior is frequently associated with a history of childhood abuse yet it remains unclear precisely how early life adversity may increase suicide risk later in life. As such, our aim was to examine whether lifetime trajectories of disruptiveness and anxiousness trait dysregulation explain the association between childhood adversity and suicidal behavior; and moreover, to test the potential modifying effects of mental disorders on these associations. METHOD: A sample of 1776 individuals from a prospective school-based cohort followed longitudinally for over 22 years was investigated. We tested the influence of disruptiveness and anxiousness trajectories from age 6 to 12 years on the association between childhood adversity (i.e. sexual and physical abuse) and history of suicide attempts (SA) using logistic regression models. Both adolescent externalizing and internalizing Axis I disorders and gender were tested as potential modifiers of these associations. RESULTS: Four distinct longitudinal trajectories were identified for both disruptiveness and anxiousness. The high disruptiveness trajectory accounted for the association between childhood adversity and SA, but only for females. The high anxiousness trajectory also explained the association between adversity and SA; however, in this case it was not sex but mental disorders that influenced the potency of the mediating effect. More specifically, anxiousness fully explained the effect of adversity on SA in the presence of externalizing disorders, whereas in the absence of these disorders, this effect was significantly attenuated. CONCLUSIONS: This study provides evidence that both disruptiveness and anxiousness play an important role in explaining the relationship between childhood adversity and SA.

The 2-aminoethylphosphonate-specific transaminase of the 2-aminoethylphosphonate degradation pathway.

The 2-aminoethylphosphonate transaminase (AEPT; the phnW gene product) of the Salmonella enterica serovar Typhimurium 2-aminoethylphosphonate (AEP) degradation pathway catalyzes the reversible reaction of AEP and pyruvate to form phosphonoacetaldehyde (P-Ald) and L-alanine (L-Ala). Here, we describe the purification and characterization of recombinant AEPT. pH rate profiles (log V(m) and log V(m)/K(m) versus pH) revealed a pH optimum of 8.5. At pH 8.5, K(eq) is equal to 0.5 and the k(cat) values of the forward and reverse reactions are 7 and 9 s(-1), respectively. The K(m) for AEP is 1.11 +/- 0.03 mM; for pyruvate it is 0.15 +/- 0.02 mM, for P-Ald it is 0.09 +/- 0.01 mM, and for L-Ala it is 1.4 +/- 0.03 mM. Substrate specificity tests revealed a high degree of discrimination, indicating a singular physiological role for the transaminase in AEP degradation. The 40-kDa subunit of the homodimeric enzyme is homologous to other members of the pyridoxalphosphate-dependent amino acid transaminase superfamily. Catalytic residues conserved within well-characterized members are also conserved within the seven known AEPT sequences. Site-directed mutagenesis demonstrated the importance of three selected residues (Asp168, Lys194, and Arg340) in AEPT catalysis.

Homogeneous expression of the P(BAD) promoter in Escherichia coli by constitutive expression of the low-affinity high-capacity AraE transporter.

Genes placed under the control of the arabinose-inducible araBAD promoter (P(BAD)) of Escherichia coli are expressed in an all-or-none fashion, in which the percentage of induced cells in the population, rather than the degree of induction in individual cells, varies with the concentration of arabinose in the culture medium. Previous work showed that all-or-none gene expression from P(BAD) was due to the arabinose-dependent expression of the gene encoding the low-affinity high-capacity transporter (araE), and that expression of heterologous genes from P(BAD) in individual cells could be regulated by placing the araE gene under control of an arabinose-independent promoter. Based on these results, two expression systems were developed to allow regulatable control of genes under control of P(BAD). In one system, the native araE promoter on the chromosome was replaced by constitutive promoters of different strengths. In the second system, the araE gene under control of the same constitutive promoters was placed on a medium-copy plasmid. Both systems allow regulatable expression of a plasmid-borne P(BAD)-controlled heterologous gene and a homogeneous population of cells over a wide range of arabinose concentrations. While the degree of induction varied slightly with the strength of the constitutive promoter, expression was affected most by the arabinose concentration.

Conditional-replication, integration, excision, and retrieval plasmid-host systems for gene structure-function studies of bacteria.

We have developed a series of powerful and versatile conditional-replication, integration, and modular (CRIM) plasmids. CRIM plasmids can be replicated at medium or high copy numbers in different hosts for making gene (or mutant) libraries. They can be integrated in single copies into the chromosomes of Escherichia coli and related bacteria to study gene function under normal physiological conditions. They can be excised from the chromosome, e.g., to verify that phenotypes are caused by their presence. Furthermore, they can be retrieved singly or en masse for subsequent molecular analyses. CRIM plasmids are integrated into the chromosome by site-specific recombination at one of five different phage attachment sites. Integrants are selected as antibiotic-resistant transformations. Since CRIM plasmids encode different forms of resistance, several can be used together in the same cell for stable expression of complex metabolic or regulatory pathways from diverse sources. Following integration, integrants are stably maintained in the absence of antibiotic selection. Each CRIM plasmid has a polylinker or one of several promoters for ectopic expression of the inserted DNA. Their modular design allows easy construction of new variants with different combinations of features. We also report a series of easily curable, low-copy-number helper plasmids encoding all the requisite Int proteins alone or with the respective Xis protein. These helper plasmids facilitate integration, excision ("curing"), or retrieval of the CRIM plasmids.

Dual transcriptional regulation of the Escherichia coli phosphate-starvation-inducible psiE gene of the phosphate regulon by PhoB and the cyclic AMP (cAMP)-cAMP receptor protein complex.

We have shown that the Escherichia coli phosphate-starvation-inducible psiE gene is regulated by both phosphate and the carbon source by using both lacZ and chloramphenicol acetyltransferase gene (cat) fusions. Yet, under all conditions tested, a single transcriptional start site lying 7 bp downstream of a predicted -10 region was revealed by primer extension analysis. DNase I footprinting showed that the PhoB transcriptional-activator protein protects two predicted pho boxes lying upstream of and near the -35 promoter region. Similar analysis showed that the cyclic AMP (cAMP)-cAMP receptor protein (cAMP-CRP) complex binds a region that overlaps with the downstream pho box. These results, together with measurements of the in vivo psiE promoter activity under various conditions, show that expression of the psiE gene is under direct positive and negative control by PhoB and cAMP-CRP, respectively.

One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products.

We have developed a simple and highly efficient method to disrupt chromosomal genes in Escherichia coli in which PCR primers provide the homology to the targeted gene(s). In this procedure, recombination requires the phage lambda Red recombinase, which is synthesized under the control of an inducible promoter on an easily curable, low copy number plasmid. To demonstrate the utility of this approach, we generated PCR products by using primers with 36- to 50-nt extensions that are homologous to regions adjacent to the gene to be inactivated and template plasmids carrying antibiotic resistance genes that are flanked by FRT (FLP recognition target) sites. By using the respective PCR products, we made 13 different disruptions of chromosomal genes. Mutants of the arcB, cyaA, lacZYA, ompR-envZ, phnR, pstB, pstCA, pstS, pstSCAB-phoU, recA, and torSTRCAD genes or operons were isolated as antibiotic-resistant colonies after the introduction into bacteria carrying a Red expression plasmid of synthetic (PCR-generated) DNA. The resistance genes were then eliminated by using a helper plasmid encoding the FLP recombinase which is also easily curable. This procedure should be widely useful, especially in genome analysis of E. coli and other bacteria because the procedure can be done in wild-type cells.

Cloning and characterization of the UDP-sugar hydrolase gene (ushA) of Enterobacter aerogenes IFO 12010.

A bacterial alkaline phosphatase (BAP, the phoA gene product) is primarily responsible for the hydrolysis of the substrates 5-bromo-4-chloro-3-indolylphosphate-p-toluidine (XP) and p-nitrophenyl phosphate (pNPP). Using these substrates and an E. coli phoA mutant, we have cloned Enterobacter aerogenes genes conferring an XP(+) phenotype. Two types of clones were identified based on phenotypic tests and DNA sequences. One of them is a E. aerogenes phoA gene (XP(+), pNPP(+)) as expected; surprisingly the other one was found to be a ushA gene (XP(+), pNPP(-)), which encodes an UDP (uridine 5'-diphosphate)-sugar hydrolase. The E. aerogenes ushA gene shares high sequence identity with ushA of E. coli and the mutationally silent ushA0 gene of Salmonella typhimurium at both the nucleotide (over 79%) and amino acid (over 93%) levels. Expression of the E. aerogenes ushA gene in E. coli produced high level of UDP-sugar hydrolase, as confirmed by TLC (thin layer chromatography) analysis together with a presence of a strong band due to a XP hydrolysis on a polyacrylamide gel.

Multiple factors independently regulate hilA and invasion gene expression in Salmonella enterica serovar typhimurium.

HilA activates the expression of Salmonella enterica serovar Typhimurium invasion genes. To learn more about regulation of hilA, we isolated Tn5 mutants exhibiting reduced hilA and/or invasion gene expression. In addition to expected mutations, we identified Tn5 insertions in pstS, fadD, flhD, flhC, and fliA. Analysis of the pstS mutant indicates that hilA and invasion genes are repressed by the response regulator PhoB in the absence of the Pst high-affinity inorganic phosphate uptake system. This system is required for negative control of the PhoR-PhoB two-component regulatory system, suggesting that hilA expression may be repressed by PhoR-PhoB under low extracellular inorganic phosphate conditions. FadD is required for uptake and degradation of long-chain fatty acids, and our analysis of the fadD mutant indicates that hilA is regulated by a FadD-dependent, FadR-independent mechanism. Thus, fatty acid derivatives may act as intracellular signals to regulate hilA expression. flhDC and fliA encode transcription factors required for flagellum production, motility, and chemotaxis. Complementation studies with flhC and fliA mutants indicate that FliZ, which is encoded in an operon with fliA, activates expression of hilA, linking regulation of hilA with motility. Finally, epistasis tests showed that PhoB, FadD, FliZ, SirA, and EnvZ act independently to regulate hilA expression and invasion. In summary, our screen has identified several distinct pathways that can modulate S. enterica serovar Typhimurium's ability to express hilA and invade host cells. Integration of signals from these different pathways may help restrict invasion gene expression during infection.

In vivo characterization of the type A and B vancomycin-resistant enterococci (VRE) VanRS two-component systems in Escherichia coli: a nonpathogenic model for studying the VRE signal transduction pathways.

Escherichia coli reporter strains modeling the high-level type A and B vancomycin resistances of Enterococcus faecium BM4147 and Ent. faecalis have been developed to study the respective VanR-VanS two-component regulatory systems. PvanH-, PvanRa-, PvanY-, and PvanRb-lacZ fusions report on expression from the vancomycin-resistant enterococci promoters of the type A vanRSHAXYZ and type B vanRSYWHBX gene clusters. These strains also express from single-copy chromosomal genes vanRa, vanRb, or vanRSb behind their respective promoter (PvanRa or PvanRb) or vanSa or vanSb behind the rhamnose-inducible PrhaB. Results show that activation (phosphorylation) of the response regulator VanRa by its sensor kinase VanSa leads to transcriptional activation of both PvanH and PvanRa. Additionally, VanRb activates its cognate promoters PvanY and PvanRb, although this occurs only in the absence of VanSb and presumably is caused by VanRb phosphorylation by an unidentified endogenous E. coli kinase. Thus, VanSb interferes with activation of VanRb, probably by acting as a phospho-VanRb phosphatase. Although both VanRa and VanRb activate their cognate promoters, neither activates the heterologous PvanR, PvanH, or PvanY, arguing against the interchangeability of type A and B two-component regulatory switches in vancomycin-resistant enterococci. VanRa also is activated by the nonpartner kinase PhoR. Because this occurs in the absence of its inducing signal (Pi limitation), PhoR autophosphorylation apparently is regulated in vivo. Furthermore, the activation of VanRa caused by cross talk from PhoR in the absence of a signal allows distinction of cross talk from crossregulation as the latter, but not the former, responds to environmental cues.

Homologs of the vancomycin resistance D-Ala-D-Ala dipeptidase VanX in Streptomyces toyocaensis, Escherichia coli and Synechocystis: attributes of catalytic efficiency, stereoselectivity and regulation with implications for function.

BACKGROUND: Vancomycin-resistant enterococci are pathogenic bacteria that have altered cell-wall peptidoglycan termini (D-alanyl-D-lactate [D-Ala-D-lactate] instead of D-alanyl-D-alanine [D-Ala-D-Ala]), which results in a 1000-fold decreased affinity for binding vancomycin. The metallodipeptidase VanX (EntVanX) is key enzyme in antibiotic resistance as it reduces the cellular pool of the D-Ala-D-Ala dipeptide. RESULTS: A bacterial genome search revealed vanX homologs in Streptomyces toyocaensis (StoVanX), Escherichia coli (EcoVanX), and Synechocystis sp. strain PCC6803 (SynVanX). Here, the D,D-dipeptidase catalytic activity of all three VanX homologs is validated, and the catalytic efficiencies and diastereoselectivity ratios for dipeptide cleavage are reported. The ecovanX gene is shown to have an RpoS (sigma(s))-dependent promoter typical of genes turned on in stationary phase. Expression of ecovanX and an associated cluster of dipeptide permease genes permitted growth of E. coli using D-Ala-D-Ala as the sole carbon source. CONCLUSIONS: The key residues of the EntVanX active site are strongly conserved in the VanX homologs, suggesting their active-site topologies are similar. StoVanX is a highly efficient D-Ala-D-Ala dipeptidase; its gene is located in a vanHAX operon, consistent with a vancomycin-immunity function. StoVanX is a potential source for the VanX found in gram-positive enterococci. The catalytic efficiencies of D-Ala-D-Ala hydrolysis for EcoVanX and SynVanX are 25-fold lower than for EntVanX, suggesting they have a role in cell-wall turnover. Clustered with the ecovanX gene is a putative dipeptide permease system that imports D-Ala-D-Ala into the cell. The combined action of EcoVanX and the permease could permit the use of D-Ala-D-Ala as a bacterial energy source under starvation conditions.

Phosphate-independent expression of the carbon-phosphorus lyase activity of Escherichia coli.

On the basis of mutational analysis, the genes for phosphonate uptake and degradation in Escherichia coli were shown to be organized in a 10.9-kb operon of 14 genes (named phnC to phnP) and induced by phosphate (P(i)) starvation [Metcalf and Wanner (1993) J Bacteriol 175: 3430-3442]. The repression of phosphonate utilization by P(i) has hindered both the biochemical characterization of the carbon-phosphorus (C-P) lyase activity and the development of improved methods for phosphonate biodegradation in biotechnology. We have cloned the genes phnG to phnP (associated with C-P lyase activity) with the lac promoter to provide expression of C-P lyase in the presence of P(i). A number of strains lacking portions of the phn operon have been constructed. In vivo complementation of the strains, in which phnC to phnP (including both Pn transport and catalysis genes) or phnH to phnP (including only catalysis genes) was deleted, with plasmids carrying various fragments of the phn operon revealed that the expression of phnC-phnP gene products is essential to restore growth on minimal medium with phosphonate as the sole phosphorus source, while phnG-phnM gene products are required for C-P lyase activity as assessed by in vivo methane production from methylphosphonic acid. The minimum size of the DNA required for the whole-cell C-P lyase activity has been determined to be a 5.8-kb fragment, encompassing the phnG to phnM genes. Therefore, there is no requirement for the phn CDE-encoded phosphate transport system, suggesting that cleavage of the C-P bond may occur on the outer surface of the inner membrane of E. coli cells, releasing the carbon moiety into the periplasm. These data are in agreement with the observation that phosphonates cannot serve as the carbon source for E. coli growth.

Insights into the mechanism of catalysis by the P-C bond-cleaving enzyme phosphonoacetaldehyde hydrolase derived from gene sequence analysis and mutagenesis.

Phosphonoacetaldehyde hydrolase (phosphonatase) catalyzes the hydrolysis of phosphonoacetaldehyde to acetaldehyde and inorganic phosphate. In this study, the genes encoding phosphonatase in Bacillus cereus and in Salmonella typhimurium were cloned for high-level expression in Escherichia coli. The kinetic properties of the purified, recombinant phosphonatases were determined. The Schiff base mechanism known to operate in the B. cereus enzyme was verified for the S. typhimurium enzyme by phosphonoacetaldehyde-sodium borohydride-induced inactivation and by site-directed mutagenesis of the catalytic lysine 53. The protein sequence inferred from the B. cereus phosphonatase gene was determined, and this sequence was used along with that from the S. typhimurium phosphonatase gene sequence to search the primary sequence databases for possible structural homologues. We found that phosphonatase belongs to a novel family of hydrolases which appear to use a highly conserved active site aspartate residue in covalent catalysis. On the basis of this finding and the known stereochemical course of phosphonatase-catalyzed hydrolysis at phosphorus (retention), we propose a mechanism which involves Schiff base formation with lysine 53 followed by phosphoryl transfer to aspartate (at position 11 in the S. typhimurium enzyme and position 12 in the B. cereusphosphonatase) and last hydrolysis at the imine C(1) and acyl phosphate phosphorus.

Use of new methods for construction of tightly regulated arabinose and rhamnose promoter fusions in studies of the Escherichia coli phosphate regulon.

Escherichia coli genes regulated by environmental inorganic phosphate (Pi) levels form the phosphate (Pho) regulon. This regulation requires seven proteins, whose synthesis is under autogenous control, including response regulator PhoB, its partner, histidine sensor kinase PhoR, all four components of the Pi-specific transport (Pst) system (PstA, PstB, PstC, and PstS), and a protein of unknown function called PhoU. Here we examined the effects of uncoupling PhoB synthesis and PhoR synthesis from their normal controls by placing each under the tight control of the arabinose-regulated P(araB) promoter or the rhamnose-regulated P(rhaB) promoter. To do this, we made allele replacement plasmids that may be generally useful for construction of P(araB) or P(rhaB) fusions and for recombination of them onto the E. coli chromosome at the araCBAD or rhaRSBAD locus, respectively. Using strains carrying such single-copy fusions, we showed that a P(rhaB) fusion is more tightly regulated than a P(araB) fusion in that a P(rhaB)-phoR+ fusion but not a P(araB)-phoR+ fusion shows a null phenotype in the absence of its specific inducer. Yet in the absence of induction, both P(araB)-phoB+ and P(rhaB)-phoB+ fusions exhibit a null phenotype. These data indicate that less PhoR than PhoB is required for transcriptional activation of the Pho regulon, which is consistent with their respective modes of action. We also used these fusions to study PhoU. Previously, we had constructed strains with precise delta phoU mutations. However, we unexpectedly found that such delta phoU mutants have a severe growth defect (P. M. Steed and B. L. Wanner, J. Bacteriol. 175:6797-6809, 1993). They also readily give rise to compensatory mutants with lesions in phoB, phoR, or a pst gene, making their study particularly difficult. Here we found that, by using P(araB)-phoB+, P(rhaB)-phoB+, or P(rhaB)-phoR+ fusions, we were able to overcome the extremely deleterious growth defect of a Pst+ delta phoU mutant. The growth defect is apparently a consequence of high-level Pst synthesis resulting from autogenous control of PhoB and PhoR synthesis in the absence of PhoU.

Structure-based redesign of corepressor specificity of the Escherichia coli purine repressor by substitution of residue 190.

Guanine or hypoxanthine, physiological corepressors of the Escherichia coli purine repressor (PurR), promote formation of the ternary PurR-corepressor-operator DNA complex that functions to repress pur operon gene expression. Structure-based predictions on the importance of Arg190 in determining 6-oxopurine specificity and corepressor binding affinity were tested by mutagenesis, analysis of in vivo function, and in vitro corepressor binding measurements. Replacements of Arg190 with Ala or Gln resulted in functional repressors in which binding of guanine and hypoxanthine was retained but specificity was relaxed to permit binding of adenine. X-ray structures were determined for ternary complexes of mutant repressors with purines (adenine, guanine, hypoxanthine, and 6-methylpurine) and operator DNA. These structures indicate that R190A binds guanine, hypoxanthine, and adenine with nearly equal, albeit reduced, affinity in large part because of a newly made compensatory hydrogen bond between the rotated hydroxyl side chain of Ser124 and the exocyclic 6 positions of the purines. Through direct and water-mediated contacts, the R190Q protein binds adenine with a nearly 75-fold higher affinity than the wild type repressor while maintaining wild type affinity for guanine and hypoxanthine. The results establish at the atomic level the basis for the critical role of Arg190 in the recognition of the exocyclic 6 position of its purine corepressors and the successful redesign of corepressor specificity.

Transcriptional regulation of the Enterococcus faecium BM4147 vancomycin resistance gene cluster by the VanS-VanR two-component regulatory system in Escherichia coli K-12.

An Escherichia coli K-12 model system was developed for studying the VanS-VanR two-component regulatory system required for high-level inducible vancomycin resistance in Enterococcus faecium BM4147. Our model system is based on the use of reporter strains with lacZ transcriptional and translational fusions to the PvanR or PvanH promoter of the vanRSHAX gene cluster. These strains also express vanR and vanS behind the native PvanR promoter, the arabinose-inducible ParaB promoter, or the rhamnose-inducible PrhaB promoter. Our reporter strains have the respective fusions stably recombined onto the chromosome in single copy, thereby avoiding aberrant regulatory effects that may occur with plasmid-bearing strains. They were constructed by using allele replacement methods or a conditionally replicative attP plasmid. Using these reporter strains, we demonstrated that (i) the response regulator VanR activates PvanH, but not PvanR, expression upon activation (phosphorylation) by the partner kinase VanS, the noncognate kinase PhoR, or acetyl phosphate, indicating that phospho-VanR (P-VanR) is a transcriptional activator; (ii) VanS interferes with activation of VanR by PhoR or acetyl phosphate, indicating that VanS also acts as a P-VanR phosphatase; and (iii) the conserved, phosphate-accepting histidine (H164) of VanS is required for activation (phosphorylation) of VanR but not for deactivation (dephosphorylation) of P-VanR. Similar reporter strains may be useful in new studies on these and other interactions of the VanS-VanR system (and other systems), screening for inhibitors of these interactions, and deciphering the molecular logic of the signal(s) responsible for activation of the VanS-VanR system in vivo. Advantages of using an E. coli model system for in vivo studies on VanS and VanR are also discussed.

Altered recognition mutants of the response regulator PhoB: a new genetic strategy for studying protein-protein interactions.

Two-component regulatory systems require highly specific interactions between histidine kinase (transmitter) and response regulator (receiver) proteins. We have developed a novel genetic strategy that is based on tightly regulated synthesis of a given protein to identify domains and residues of an interacting protein that are critical for interactions between them. Using a reporter strain synthesizing the nonpartner kinase VanS under tight arabinose control and carrying a promoter-lacZ fusion activated by phospho-PhoB, we isolated altered recognition (AR) mutants of PhoB showing enhanced activation (phosphorylation) by VanS as arabinose-dependent Lac+ mutants. Changes in the PhoBAR mutants cluster in a "patch" near the proposed helix 4 of PhoB based on the CheY crystal structure (a homolog of the PhoB receiver domain) providing further evidence that helix 4 lies in the kinase-regulator interface. Based on the CheY structure, one mutant has an additional change in a region that may propagate a conformational change to helix 4. The overall genetic strategy described here may also be useful for studying interactions of other components of the vancomycin resistance and P1 signal transduction pathways, other two-component regulatory systems, and other interacting proteins. Conditionally replicative oriRR6K gamma attP "genome targeting" suicide plasmids carrying mutagenized phoB coding regions were integrated into the chromosome of a reporter strain to create mutant libraries; plasmids encoding mutant PhoB proteins were subsequently retrieved by P1-Int-Xis cloning. Finally, the use of similar genome targeting plasmids and P1-Int-Xis cloning should be generally useful for constructing genomic libraries from a wide array of organisms.

Involvement of the sensor kinase EnvZ in the in vivo activation of the response-regulator PhoB by acetyl phosphate.

Three signalling pathways lead to activation of the phosphate (Pho) regulon by phosphorylation of the response-regulator PhoB in Escherichia coli. One pathway responds to the extracellular inorganic phosphate (PI) level and leads to activation by the Pi sensor kinase, PhoR. The other two pathways are Pi independent and are apparent in the absence of PhoR. One Pi-independent pathway responds to the level of an unknown catabolite and leads to activation by the catabolite regulatory sensor kinase, CreC (originally called PhoM); the other Pi-independent pathway responds to acetyl phosphate and leads to activation by a process requiring acetyl phosphate. Here we show that activation of PhoB by acetyl phosphate can require the sensor kinase EnvZ. Accordingly, we propose that the in vivo activation of PhoB by acetyl phosphate (and perhaps other two-component response-regulators as well) probably always requires a certain kinase that can vary depending upon the growth conditions.

Global analysis of proteins synthesized during phosphorus restriction in Escherichia coli.

The pattern of proteins synthesized in Escherichia coli during steady-state growth in media with ample inorganic phosphate (Pi), upon limitation for Pi (without an alternative phosphorous compound), and during steady-state growth in media containing phosphonate (PHN) as the sole P source was examined by two-dimensional gel electrophoresis. Of 816 proteins monitored in these experiments, all those with differential synthesis rates greater than 2.0 or less than 0.5 upon phosphate limitation (P limitation) or during growth on PHN compared with their rates in the cultures with Pi were classified as belonging to the PL or PHN stimulon, respectively. The PL stimulon included 413 proteins, 208 showing induced synthesis and 205 showing repressed synthesis. The PHN stimulon was smaller: it included 257 proteins; 227 showed induced synthesis and 30 showed repressed synthesis. The overlap of the two stimulons included 137 proteins: most (118) were ones showing induced synthesis. The promoter regions of genes for several of the proteins with induced or repressed synthesis contained sequences which resembled the consensus sequence for PhoB binding. The aggregate mass of proteins responding to P limitation or growth on PHN was 30 to 40% of the cells' total mass. By comparing the proteins responding to P limitation with those responding to growth on PHN, one can speculate which proteins are likely involved in adapting cells to new P sources or in preparing cells to survive stationary phase.