Synthesis of the stationary-phase sigma factor sigma s is positively regulated by ppGpp.

Strains of Escherichia coli which lack detectable guanosine 3',5'-bispyrophosphate (ppGpp) display a pleiotropic phenotype that in some respects resembles that of rpoS (katF) mutants. This led us to examine whether ppGpp is a positive regulator of sigma s synthesis. sigma s is a stationary-phase-specific sigma factor that is encoded by the rpoS gene. We found that a ppGpp-deficient strain is defective in sigma s synthesis as cells enter stationary phase in a rich medium, as judged by immunoblots. Under more-defined conditions we found that the stimulation of sigma s synthesis following glucose, phosphate, or amino acid starvation of wild-type strains is greatly reduced in a strain lacking ppGpp. The failure of ppGpp-deficient strains to synthesize sigma s in response to these starvation regimens could indicate a general defect in gene expression rather than a specific dependence of rpoS expression on ppGpp. We therefore tested the effect of artificially elevated ppGpp levels on sigma s synthesis either with mutations that impair ppGpp decay or by gratuitously inducing ppGpp synthesis with a Ptac::relA fusion. In both instances, we observed enhanced sigma s synthesis. Apparently, ppGpp can activate sigma s synthesis under conditions of nutrient sufficiency as well as during entry into stationary phase. This finding suggests that changes in ppGpp levels function both as a signal of imminent stationary phase and as a signal of perturbations in steady-state growth.     

Mecillinam resistance in Escherichia coli is conferred by loss of a second activity of the AroK protein.

Mecillinam, a beta-lactam antibiotic specific to penicillin-binding protein 2 (PBP 2) in Escherichia coli, blocks cell wall elongation and, indirectly, cell division, but its lethality can be overcome by increased levels of ppGpp, the nucleotide effector of the stringent response. We have subjected an E. coli K-12 strain to random insertional mutagenesis with a mini-Tn10 element. One insertion, which was found to confer resistance to mecillinam in relA+ and relA strains, was mapped at 75.5 min on the E. coli map and was located between the promoters and the coding sequence of the aroK gene, which codes for shikimate kinase 1, one of two E. coli shikimate kinases, both of which are involved in aromatic amino acid biosynthesis. The mecillinam resistance conferred by the insertion was abolished in a delta relA delta spoT strain completely lacking ppGpp, and it thus depends on the presence of ppGpp. Furthermore, the insertion increased the ppGpp pool approximately twofold in a relA+ strain. However, this increase was not observed in relA strains, although the insertion still conferred mecillinam resistance in these backgrounds, showing that mecillinam resistance is not due to an increased ppGpp pool. The resistance was also abolished in an ftsZ84(Ts) strain under semipermissive conditions, and the aroK::mini-Tn10 allele partially suppressed ftsZ84(Ts); however, it did not increase the concentration of the FtsZ cell division protein. The insertion greatly decreased or abolished the shikimate kinase activity of AroK in vivo and in vitro. The two shikimate kinases of E. coli are not equivalent; the loss of AroK confers mecillinam resistance, whereas the loss of Arol, does not. Furthermore, the ability of the aroK mutation to confer mecillinam resistance is shown to be independent of polar effects on operon expression and of effects on the availability of aromatic amino acids or shikimic acid. Instead, we conclude that the AroK protein has a second activity, possibly related to cell division regulation, which confers mecillinam sensitivity. We were able to separate the AroK activities mutationally with an aroK mutant allele lacking shikimate kinase activity but still able to confer mecillinam sensitivity.     

Identification of Bacillus subtilis sigma-dependent genes that provide intrinsic resistance to antimicrobial compounds produced by Bacilli

Bacillus subtilis produces many antibiotics of varying structures and specificity. Here we identify a prominent role for sigma(W), an extracytoplasmic function (ECF) sigma factor, in providing intrinsic resistance to antimicrobial compounds produced by other Bacilli. By using a panel of B. subtilis mutants disrupted for each of the 30 known sigma(W)-dependent operons we identified resistance genes for at least three different antimicrobial compounds. The ydbST and fosB genes contribute to resistance to antimicrobial compound(s) produced by B. amyloliquefaciens FZB42, the yqeZyqfAB operon provides resistance to the SPbeta prophage-encoded bacteriocin sublancin, and the yknWXYZ operon and yfhL provide resistance to the antimicrobial peptide SdpC. YfhL encodes a paralogue of SdpI, a membrane protein that provides immunity to SdpC. In competition experiments, we identify sigma(W) as a key factor in allowing B. subtilis to resist antibiotic killing and encroachment by competing strains. Together with the previous observation that sigma(W) provides inducible resistance against the Streptomyces antibiotic fosfomycin, these studies support the notion that sigma(W) controls an antibiosis regulon important in the microbial ecology of soil bacteria.     

Underproduction of sigma 70 mimics a stringent response. A proteome approach

When Escherichia coli cells enter stationary phase due to carbon starvation the synthesis of ribosomal proteins is rapidly repressed. In a DeltarelA DeltaspoT mutant, defective in the production of the alarmone guanosine tetraphosphate (ppGpp), this regulation of the levels of the protein synthesizing system is abolished. Using a proteomic approach we demonstrate that the production of the vast majority of detected E. coli proteins are decontrolled during carbon starvation in the DeltarelA DeltaspoT strain and that the starved cells behave as if they were growing exponentially. In addition we show that the inhibition of ribosome synthesis by the stringent response can be qualitatively mimicked by artificially lowering the levels of the housekeeping sigma factor, sigma(70). In other words, genes encoding the protein-synthesizing system are especially sensitive to reduced availability of sigma(70) programmed RNA polymerase. This effect is not dependent on ppGpp since lowering the levels of sigma(70) gives a similar but less pronounced effect in a ppGpp(0) strain. The data is discussed in view of the models advocating for a passive control of gene expression during stringency based on alterations in RNA polymerase availability.     

Impact of antimicrobial usage on antimicrobial resistance in commensal Escherichia coli strains colonizing broiler chickens

Escherichia coli strains isolated from commercial broilers and an experimental flock of chickens were screened to determine phenotypic expression of antimicrobial resistance and carriage of drug resistance determinants. The goal of this study was to investigate the influence of oxytetracycline, sarafloxacin, and enrofloxacin administration on the distribution of resistance determinants and strain types among intestinal commensal E. coli strains isolated from broiler chickens. We detected a high prevalence of resistance to drugs such as tetracycline (36 to 97%), sulfonamides (50 to 100%), and streptomycin (53 to 100%) in E. coli isolates from treated and untreated flocks. These isolates also had a high prevalence of class 1 integron carriage, and most of them possessed the streptomycin resistance cassette, aadA1. In order to investigate the contribution of E. coli strain distribution to the prevalence of antimicrobial resistance and the resistance determinants, isolates from each flock were DNA fingerprinted by enterobacterial repetitive intergenic consensus sequence (ERIC) PCR. Although very diverse E. coli strain types were detected, four ERIC strain types were present on all of the commercial broiler farms, and two of the strains were also found in the experimental flocks. Each E. coli strain consisted of both susceptible and antimicrobial agent-resistant isolates. In some instances, isolates of the same E. coli strain expressed the same drug resistance patterns although they harbored different tet determinants or streptomycin resistance genes. Therefore, drug resistance patterns could not be explained solely by strain prevalence, indicating that mobile elements contributed significantly to the prevalence of resistance.     

Comparison of DeltarelA strains of Escherichia coli and Salmonella enterica serovar Typhimurium suggests a role for ppGpp in attenuation regulation of branched-chain amino acid biosynthesis

The growth recovery of Escherichia coli K-12 and Salmonella enterica serovar Typhimurium DeltarelA mutants were compared after nutritional downshifts requiring derepression of the branched-chain amino acid pathways. Because wild-type E. coli K-12 and S. enterica serovar Typhimurium LT2 strains are defective in the expression of the genes encoding the branch point acetohydroxy acid synthetase II (ilvGM) and III (ilvIH) isozymes, respectively, DeltarelA derivatives corrected for these mutations were also examined. Results indicate that reduced expression of the known global regulatory factors involved in branched-chain amino acid biosynthesis cannot completely explain the observed growth recovery defects of the DeltarelA strains. In the E. coli K-12 MG1655 DeltarelA background, correction of the preexisting rph-1 allele which causes pyrimidine limitations resulted in complete loss of growth recovery. S. enterica serovar Typhimurium LT2 DeltarelA strains were fully complemented by elevated basal ppGpp levels in an S. enterica serovar Typhimurium LT2 DeltarelA spoT1 mutant or in a strain harboring an RNA polymerase mutation conferring a reduced RNA chain elongation rate. The results are best explained by a dependence on the basal levels of ppGpp, which are determined by relA-dependent changes in tRNA synthesis resulting from amino acid starvations. Expression of the branched-chain amino acid operons is suggested to require changes in the RNA chain elongation rate of the RNA polymerase, which can be achieved either by elevation of the basal ppGpp levels or, in the case of the E. coli K-12 MG1655 strain, through pyrimidine limitations which partially compensate for reduced ppGpp levels. Roles for ppGpp in branched-chain amino acid biosynthesis are discussed in terms of effects on the synthesis of known global regulatory proteins and current models for the control of global RNA synthesis by ppGpp.     

A rifampicin resistance mutation in the rpoB gene confers ppGpp-independent antibiotic production in Streptomyces coelicolor A3(2)

In Streptomyces coelicolor A3(2), deletion of relA or a specific mutation in rplK ( relC) results in an inability to synthesize ppGpp (guanosine 5'-diphosphate 3'-diphosphate) and impairs production of actinorhodin. We have found that certain rifampicin-resistant ( rif) mutants isolated from either relA or relC strains regain the ability to produce actinorhodin at the same level as the wild-type strain, although their capacity to synthesize ppGpp is unchanged. These rif mutants were found to have a missense mutation in the rpoB gene that encodes the RNA polymerase beta-subunit. This rpoB mutation was shown to be responsible for the observed changes in phenotype, as demonstrated by gene replacement experiments. Gene expression analysis revealed that the restoration of actinorhodin production in both relA and relC strains is accompanied by increased expression of the pathway-specific regulator gene actII-ORF4, which is normally decreased in the rel mutants. In addition to the restoration of antibiotic production, the rif mutants also exhibited a lower rate of RNA synthesis compared to the parental strain when grown in a rich medium, suggesting that these mutant RNA polymerases behave like "stringent" RNA polymerases. These results indicate that rif mutations can alter gene expression patterns independently of ppGpp. We propose that RNA polymerases carrying particular rif mutations in the beta-subunit can functionally mimic the modification induced by binding of ppGpp.     

Iron limitation induces SpoT-dependent accumulation of ppGpp in Escherichia coli

In Escherichia coli the beta-lactam mecillinam specifically inhibits penicillin-binding protein 2 (PBP2), a peptidoglycan transpeptidase essential for maintaining rod shape. We have previously shown that PBP2 inactivation results in a cell division block and that an increased concentration of the nucleotide ppGpp, effector of the RelA-dependent stringent response, confers mecillinam resistance and allows cells to divide as spheres in the absence of PBP2 activity. In this study we have characterized an insertion mutation which confers mecillinam resistance in wild-type and DeltarelA strains but not in DeltarelADeltaspoT strains, devoid of ppGpp. The mutant has an insertion in the fes gene, coding for enterochelin esterase. This cytoplasmic enzyme hydrolyses enterochelin-Fe(3+) complexes, making the scavenged iron available to the cells. We show that inactivation of the fes gene causes iron limitation on rich medium plates and a parallel SpoT-dependent increase of the ppGpp pool, as judged by the induction of the iron-regulated fiu::lacZ fusion and the repression of the stringently controlled P1(rrnB)::lacZ fusion respectively. We further show, by direct ppGpp assays, that iron starvation in liquid medium produces a SpoT-dependent increase of the ppGpp pool, strongly suggesting a role for iron in the balance of the two activities of SpoT, synthesis and hydrolysis of (p)ppGpp. Finally, we present evidence that ppGpp exerts direct or indirect positive control on iron uptake, suggesting a simple homeostatic regulatory circuit: iron limitation leads to an increased ppGpp pool, which increases the expression of iron uptake genes, thereby alleviating the limitation.     

Involvement of ppGpp, ribosome modulation factor, and stationary phase-specific sigma factor sigma(S) in the decrease in cell viability caused by spermidine.

Accumulation of spermidine in Escherichia coli causes a decrease in cell viability at the late stationary phase of cell growth. The mechanism underlying this effect has been studied. Spermidine accumulation caused an increase in the level of ppGpp and a decrease in ribosome modulation factor (RMF) and stationary phase-specific sigma factor sigma(S), both of which are believed to be involved in cell viability. Transformation of E. coli with the gene for stringent factor, which synthesizes ppGpp, also caused a significant decrease in the levels of RMF and sigma(S) factor and a decrease in cell viability. The results strongly suggest that the accumulation of ppGpp is also involved in the decrease in cell viability and that the sigma(S) factor assists the function of RMF in cell viability.     

Toxic effects of high levels of ppGpp in Escherichia coli are relieved by rpoB mutations.

A controversy has surrounded the questions of whether or not guanosine tetraphosphate (ppGpp) is a specific inhibitor of bacterial rRNA and tRNA synthesis, especially during normal exponential growth, and whether the RNA polymerase is the target of ppGpp action. To answer these questions, a pBR322-derived plasmid, pKT28, was constructed that carries the Escherichia coli relA gene encoding a ppGpp synthetase under control of the lacUV5 promoter. The plasmid was used to transform the ppGpp reporter strain VH271 in which expression of beta-galactosidase from an rrnB P1 promoter is inhibited by ppGpp. In the presence of high concentrations of lac inducer, bacteria of the transformed strain accumulate ppGpp with the result that synthesis of rRNA and beta-galactosidase is inhibited and growth ceases. At low concentrations of inducer, growth is only reduced and cells form small white colonies on X-gal indicator plates. After continued incubation, these colonies form blue sectors of faster growing mutant cells. Phage P1 transduction experiments showed that these mutants have mutations cotransducing with rpoB, the gene encoding the beta-subunit of RNA polymerase. One particular mutant strain, KT13, had acquired partial resistance to ppGpp inhibition of rRNA synthesis. The mutation in this strain was cloned by in vivo recombination into an rpoB plasmid. The presence of this plasmid conferred increased resistance to overproduction of ppGpp. These results suggest that ppGpp is a specific inhibitor of rRNA synthesis, even in the absence of amino acid starvation, and that RNA polymerase is involved as the target of ppGpp action.     

Stringent control during carbon starvation of marine Vibrio sp. strain S14: molecular cloning, nucleotide sequence, and deletion of the relA gene.

In order to evaluate the role of the stringent response in starvation adaptations of the marine Vibrio sp. strain S14, we have cloned the relA gene and generated relaxed mutants of this organism. The Vibrio relA gene was selected from a chromosomal DNA library by complementation of an Escherichia coli delta relA strain. The nucleotide sequence contains a 743-codon open reading frame that encodes a polypeptide that is identical in length and highly homologous to the E. coli RelA protein. The amino acid sequences are 64% identical, and they share some completely conserved regions. A delta relA::kan allele was generated by replacing 53% of the open reading frame with a kanamycin resistance gene. The Vibrio relA mutants displayed a relaxed control of RNA synthesis and failed to accumulate ppGpp during amino acid limitation. During carbon and energy starvation, a relA-dependent burst of ppGpp synthesis concomitant with carbon source depletion and growth arrest was observed. Also, in the absence of the relA gene, there was an accumulation of ppGpp during carbon starvation, but this was slower and smaller than that which occurred in the stringent strains, and it was preceded by a marked decrease in the [ATP]/[ADP] ratio. In both the wild-type and the relaxed strains, carbon source depletion caused an immediate decrease in the size of the GTP pool and a block of net RNA accumulation. The relA mutation did not affect long-term survival or the development of resistance against heat, ethanol, and oxidative stress during carbon starvation of Vibrio sp. strain S14.