ppGpp-dependent stationary phase induction of genes on Salmonella pathogenicity island 1

We have examined expression of the genes on Salmonella pathogenicity island 1 (SPI1) during growth under the physiologically well defined standard growth condition of Luria-Bertani medium with aeration. We found that the central regulator hilA and the genes under its control are expressed at the onset of stationary phase. Interestingly, the two-component regulatory genes hilC/hilD, sirA/barA, and ompR, which are known to modulate expression from the hilA promoter (hilAp) under so-called "inducing conditions" (Luria-Bertani medium containing 0.3 m NaCl without aeration), acted under standard conditions at the stationary phase induction level. The induction of hilAp depended not on RpoS, the stationary phase sigma factor, but on the stringent signal molecule ppGpp. In the ppGpp null mutant background, hilAp showed absolutely no activity. The stationary phase induction of hilAp required spoT but not relA. Consistent with this requirement, hilAp was also induced by carbon source deprivation, which is known to transiently elevate ppGpp mediated by spoT function. The observation that amino acid starvation elicited by the addition of serine hydroxamate did not induce hilAp in a RelA(+) SpoT(+) strain suggested that, in addition to ppGpp, some other alteration accompanying entry into the stationary phase might be necessary for induction. It is speculated that during the course of infection Salmonella encounters various stressful environments that are sensed and translated to the intracellular signal, ppGpp, which allows expression of Salmonella virulence genes, including SPI1 genes.     

Co-ordination of Legionella pneumophila virulence with entry into stationary phase by ppGpp

Legionella pneumophila survives in aquatic environments, but replicates within amoebae or the alveolar macrophages of immunocompromised individuals. Here, the signal transduction pathway that co-ordinates L. pneumophila virulence expression in response to amino acid depletion was investigated. To facilitate kinetic and genetic studies, a phenotypic reporter of virulence was engineered by fusing flaA promoter sequences to a gene encoding green fluorescent protein. When subjected to amino acid depletion, L. pneumophila accumulated ppGpp and converted from a replicative to a virulent state, as judged by motility and sodium sensitivity. ppGpp appeared to initiate this response, as L. pneumophila induced to express the Escherichia coli RelA ppGpp synthetase independently of nutrient depletion accumulated ppGpp, exited the exponential growth phase and expressed flaAgfp, motility, sodium sensitivity, cytotoxicity and infectivity, five traits correlated with virulence. Although coincident with the stationary phase, L. pneumophila virulence expression appeared to require an additional factor: mutant Lp120 accumulated ppGpp and acquired two stationary phase traits but none of six virulence phenotypes analysed. We propose that, when nutrients are limiting, ppGpp acts as an alarmone, triggering the expression of multiple traits that enable L. pneumophila to escape its spent host, to survive and disperse in the environment and to re-establish a protected intracellular replication niche.     

Isolation of an Escherichia coil strain mutant unable to form biofilm on polystyrene and to adhere to human pneumocyte cells: involvement of tryptophanase

Escherichia coli adherence to biotic and abiotic surfaces constitutes the first step of infection by promoting colonization and biofilm formation. The aim of this study was to gain a better understanding of the relationship between E. coli adherence to different biotic surfaces and biofilm formation on abiotic surfaces. We isolated mutants defective in A549 pneumocyte cells adherence, fibronectin adherence, and biofilm formation by random transposition mutagenesis and sequential passages over A549 cell monolayers. Among the 97 mutants tested, 80 were decreased in biofilm formation, 8 were decreased in A549 cells adherence, 7 were decreased in their adherence to fibronectin, and 17 had no perturbations in either of the three phenotypes. We observed a correlation between adherence to fibronectin or A549 cells and biofilm formation, indicating that biotic adhesive factors are involved in biofilm formation by E. coli. Molecular analysis of the mutants revealed that a transposon insertion in the tnaA gene encoding for tryptophanase was associated with a decrease in both A549 cells adherence and biofilm formation by E. coli. The complementation of the tnaA mutant with plasmid-located wild-type tnaA restored the tryptophanase activity, epithelial cells adherence, and biofilm formation on polystyrene. The possible mechanism of tryptophanase involvement in E. coli adherence and biofilm formation is discussed.     

Enhanced biofilm formation and/or cell viability by polyamines through stimulation of response regulators UvrY and CpxR in the two-component signal transducing systems, and ribosome recycling factor

We have reported that polyamines increase cell viability at the stationary phase of cell growth through translational stimulation of ribosome modulation factor, and SpoT and RpoZ proteins involved in the synthesis and function of ppGpp in Escherichia coli. Since biofilm formation is also involved in cell viability, we looked for proteins involved in biofilm formation and cell viability whose synthesis is stimulated by polyamines at the level of translation. It was found that the synthesis of response regulators UvrY and CpxR in the two-component signal transducing systems and ribosome recycling factor (RRF) was increased by polyamines at the level of translation. Polyamine stimulation of the synthesis of UvrY and RRF was dependent on the existence of the inefficient initiation codons UUG and GUG in uvrY and frr mRNA, respectively; and polyamine stimulation of CpxR synthesis was dependent on the existence of an unusual location of a Shine-Dalgarno (SD) sequence in cpxR mRNA. Biofilm formation and cell viability in the absence of polyamines was increased by transformation of modified uvrY and cpxR genes, and cell viability by modified frr gene whose translation occurs effectively without polyamines. The results indicate that polyamines are necessary for both biofilm formation and cell viability.     

Significance of rpoS during maturation of Escherichia coli biofilms

Presence of starved, stationary phase-like zones in biofilms seems to be an important factor for biofilm formation. In this study, roles of rpoS gene in the formation of Escherichia coli biofilms were investigated. E. coli MG1655 wild type (WT) and rpoS mutant (DeltarpoS) strains were used to compare biofilm formation capacity and global gene expression. Even though the DeltarpoS strain could attach and form microcolonies on glass surfaces, it could not establish mature biofilms. DNA microarray analysis revealed that WT biofilms (WBF) showed similar pattern of gene expression with WT planktonic stationary phase, whereas DeltarpoS biofilms (MBF) showed similar pattern of gene expression with WT planktonic exponential phase. Genes involved in energy metabolism (atpIBEFHAG, atpC, cydAB) and flagella synthesis (flgB, flgC, flhD, fliA, fliC, fliY) showed increased expression in the MBF, but not in the WBF. Moreover, genes involved in stress responses (blc, cspG, dinD poxB, wcaF, wcaI, and yfcF) showed increased expression in the WBF compared to the MBF. These results suggested that the rpoS gene contributed in maturation of E. coli biofilms through regulation of global gene expression including energy metabolism, motility, and stress responses.     

The relA/spoT-Homologous Gene in Streptomyces coelicolor Encodes Both Ribosome-Dependent (p)ppGppSynthesizing and -Degrading Activities

Streptomyces coelicolor (p)ppGpp synthetase (Rel protein) belongs to the RelA and SpoT (RelA/SpoT) family, which is involved in (p)ppGpp metabolism and the stringent response. The potential functions of the rel gene have been examined. S. coelicolor Rel has been shown to be ribosome associated, and its activity in vitro is ribosome dependent. Analysis in vivo of the active recombinant protein in well-defined Escherichia coli relA and relA/spoT mutants provides evidence that S. coelicolor Rel, like native E. coli RelA, is functionally ribosome associated, resulting in ribosome-dependent (p)ppGpp accumulation upon amino acid deprivation. Expression of an S. coelicolor C-terminally deleted Rel, comprised of only the first 489 amino acids, catalyzes a ribosome-independent (p)ppGpp formation, in the same manner as the E. coli truncated RelA protein (1 to 455 amino acids). An E. coli relA spoT double deletion mutant transformed with S. coelicolor rel gene suppresses the phenotype associated with (p)ppGpp deficiency. However, in such a strain, a rel-mediated (p)ppGpp response apparently occurs after glucose depletion, but only in the absence of amino acids. Analysis of ppGpp decay in E. coli expressing the S. coelicolor rel gene suggests that it also encodes a (p)ppGpp-degrading activity. By deletion analysis, the catalytic domains of S. coelicolor Rel for (p)ppGpp synthesis and degradation have been located within its N terminus (amino acids 267 to 453 and 93 to 397, respectively). In addition, E. coli relA in an S. coelicolor rel deletion mutant restores actinorhodine production and shows a nearly normal morphological differentiation, as does the wild-type rel gene, which is in agreement with the proposed role of (p)ppGpp nucleotides in antibiotic biosynthesis.     

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.     

Effect of polyamines on synthesis and degradation of guanosine 5'-diphosphate 3'-diphosphate

The effect of polyamines on the in vitro and in vivo synthesis and degradation on guanosine 5'-diphosphate 3'-diphosphate (ppGpp) has been studied in Escherichia coli. The presence of 2 mM spermidine lowered the optimal Mg2+ concentration for ppGpp formation from 17 mM to 11 mM. The formation of ppGpp in the presence of 2 mM spermidine and 11 mM Mg2+ was about 15% greater than that in the presence of 17 mM Mg2+. At a concentration of less than 11 mM Mg2+, spermidine was found to stimulate ppGpp formation greatly. Putrescine did not cause any effect. When a polyamine-requiring mutant of E. coli (EWH319) was starved for an amino acid by the addition of valine, spermidine stimulated ppGpp formation. The degradation of ppGpp was not influenced significantly by polyamines.     

Common beta-lactamases inhibit bacterial biofilm formation

Beta-lactamases, which evolved from bacterial penicillin-binding proteins (PBPs) involved in peptidoglycan (PG) synthesis, confer resistance to beta-lactam antibiotics. While investigating the genetic basis of biofilm development by Pseudomonas aeruginosa, we noted that plasmid vectors encoding the common beta-lactamase marker TEM-1 caused defects in twitching motility (mediated by type IV pili), adherence and biofilm formation without affecting growth rates. Similarly, strains of Escherichia coli carrying TEM-1-encoding vectors grew normally but showed reduced adherence and biofilm formation, showing this effect was not species-specific. Introduction of otherwise identical plasmid vectors carrying tetracycline or gentamicin resistance markers had no effect on biofilm formation or twitching motility. The effect is restricted to class A and D enzymes, because expression of the class D Oxa-3 beta-lactamase, but not class B or C beta-lactamases, impaired biofilm formation by E. coli and P. aeruginosa. Site-directed mutagenesis of the catalytic Ser of TEM-1, but not Oxa-3, abolished the biofilm defect, while disruption of either TEM-1 or Oxa-3 expression restored wild-type levels of biofilm formation. We hypothesized that the A and D classes of beta-lactamases, which are related to low molecular weight (LMW) PBPs, may sequester or alter the PG substrates of such enzymes and interfere with normal cell wall turnover. In support of this hypothesis, deletion of the E. coli LMW PBPs 4, 5 and 7 or combinations thereof, resulted in cumulative defects in biofilm formation, similar to those seen in beta-lactamase-expressing transformants. Our results imply that horizontal acquisition of beta-lactamase resistance enzymes can have a phenotypic cost to bacteria by reducing their ability to form biofilms. Beta-lactamases likely affect PG remodelling, manifesting as perturbation of structures involved in bacterial adhesion that are required to initiate biofilm formation.     

A novel methyltransferase catalyzes the methyl esterification of trans-aconitate in Escherichia coli

We have identified a new type of S-adenosyl-L-methionine-dependent methyltransferase in the cytosol of Escherichia coli that is expressed in early stationary phase under the control of the RpoS sigma factor. This enzyme catalyzes the monomethyl esterification of trans-aconitate at high affinity (Km = 0.32 mM) and cis-aconitate, isocitrate, and citrate at lower velocities and affinities. We have purified the enzyme to homogeneity by gel-filtration, anion-exchange, and hydrophobic chromatography. The N-terminal amino acid sequence was found to match that expected for the o252 open reading frame at 34.57 min on the E. coli genomic sequence whose deduced amino acid sequence contains the signature sequence motifs of the major class of S-adenosyl-L-methionine-dependent methyltransferases. Overexpression of the o252 gene resulted in an overexpression of the methyltransferase activity, and we have now designated it tam for trans-aconitate methyltransferase. We have generated a knock-out strain of E. coli lacking this activity, and we find that its growth and stationary phase survival are similar to that of the parent strain. We demonstrate the endogenous formation of trans-aconitate methyl ester in extracts of wild type but not tam- mutant cells indicating that trans-aconitate is present in E. coli. Since trans-aconitate does not appear to be a metabolic intermediate in these cells but forms spontaneously from the key citric acid cycle intermediate cis-aconitate, we suggest that its methylation may limit its potential interference in normal metabolic pathways. We have detected trans-aconitate methyltransferase activity in extracts of the yeast Saccharomyces cerevisiae, whereas no activity has been found in extracts of Caenorhabditis elegans or mouse brain.     

Guanosine 3',5'-bispyrophosphate (ppGpp) synthesis in cells of Escherichia coli starved for Pi.

Cells of Escherichia coli which enter a phase of starvation for Pi induce the synthesis of the nucleotide guanosine 3',5'-bispyrophosphate (ppGpp). This induction is relA independent but depends on the spoT gene product. A mutant unable to produce ppGpp is impaired in the expression of two genes which belong to the pho regulon, a defect which is dependent on the product of spoT. We suggest that ppGpp is essential for the proper induction of the genes which belong to the pho regulon.