Intracellular growth attenuator A (IgaA) was identified as a Salmonella enterica regulator limiting bacterial growth inside fibroblasts. Genetic evidence further linked IgaA to repression of the RcsCDB regulatory system, which responds to envelope stress. How IgaA attenuates this system is unknown. Here, we present genome expression profiling data of S. enterica serovar Typhimurium igaA mutants grown at high osmolarity and displaying exacerbated Rcs responses. Transcriptome data revealed that IgaA attenuates gene expression changes requiring phosphorylated RcsB (RcsBϳP) activity. Some RcsB-regulated genes, yciGFE and STM1862 (pagO)-STM1863-STM1864, were equally expressed in wild-type and igaA strains, suggesting a maximal expression at low levels of RcsBϳP. Other genes, such as metB, ypeC, ygaC, glnK, glnP, napA, glpA, and nirB, were shown for the first time and by independent methods to be regulated by the RcsCDB system. Interestingly, IgaA-deficient strains with reduced RcsC or RcsD levels exhibited different Rcs responses and distinct virulence properties. spv virulence genes were differentially expressed in most of the analyzed strains. spvA expression required RcsB and IgaA but, unexpectedly, was also impaired upon stimulation of the RcsC3 RcsD3RcsB phosphorelay. Overproduction of either RcsB ؉ or a nonphosphorylatable RcsB(D56Q) variant in strains displaying low spvA expression unveiled that both dephosphorylated RcsB and RcsBϳP are required for optimal spvA expression. Taken together, our data support a model with IgaA attenuating the RcsCDB system by favoring the switch of RcsBϳP to the dephosphorylated state. This role of IgaA in constantly fine-tuning the RcsBϳP/RcsB ratio may ensure the proper expression of important virulence factors, such as the Spv proteins.

We describe a transcriptomic study of the effect of hha and ydgT mutations in Salmonella enterica serovar Typhimurium. A large number of genes showing altered expression are located in AT-rich horizontally acquired DNA sequences. Many of these genes have also been reported to be targets for H-NS. As Hha and YdgT interact with H-NS, our findings strongly suggest that Hha and/or YdgT must form complexes with H-NS when they silence these DNA regions.

IgaA is a Salmonella enterica membrane protein that attenuates the response of the RcsCDB signalling system to envelope stress. This protein is essential unless the RcsCDB system is inactivated, suggesting that IgaA may constantly adjust the magnitude of the response. Such a functional link is also supported by the concurrence of the igaA and rcsD-rcsB-rcsC loci in genomes of enteric bacteria and the selection of spontaneous mutations in the RcsCDB system following IgaA deprivation. However, the exact nature of the spontaneous mutations rendering IgaA dispensable remains undefined. In this work, we examined how the transduction of an igaA null allele affects the status of the RcsCDB system. Loss of RcsCDB response was registered iñ 90 % of the IgaA-defective clones, which failed to produce the capsule material positively regulated by this system. About half of these non-mucoid clones suppressed the loss of IgaA with large deletions encompassing variable regions of the rcsD-rcsB-rcsC locus. Unexpectedly, mucoid transductants were also reproducibly obtained and indicated the capacity of S. enterica to retain a functional RcsCDB system in the absence of IgaA. Decreased levels of either RcsC or RcsD were shown in 'mucoid' clones lacking IgaA and displaying low responsiveness to stimuli. Taken together, these data demonstrate that the stability and responsiveness of the RcsCDB system relies on its attenuator IgaA. The type of suppressions found also support a model with IgaA controlling the level of signal flowing through RcsC and RcsD.

The gene for glycine betaine transmethylase (gbt) was identified in Pseudomonas aeruginosa strain Fildes III by biochemical, physiological, and molecular approaches. Based on sequence analysis, the knockout gene corresponded to an open reading frame (ORF) named PA3082 in the genome of P. aeruginosa PAO1. The translated product of this ORF displayed similarity to transferases of different microorganisms. Mutation in gbt blocked the utilization of choline and glycine betaine as carbon and nitrogen sources.Choline and its degradation product glycine betaine (N,N,Ntrimethylglycine) support the growth of Pseudomonas aeruginosa in media of iso-osmolarity, serving as both carbon and nitrogen sources (7). Additionally, in a hyperosmolar medium, P. aeruginosa utilizes choline not only as the sole carbon and nitrogen source but also as an osmoprotectant, accumulating glycine betaine as the main osmolite compound (6). Thus, glycine betaine plays a central role in P. aeruginosa, since it is catabolized to allow cellular growth and is accumulated at a high intracellular concentration to confer an osmoprotection action. The latter condition suggests that the accumulation of glycine betaine may occur by a decreased activity of glycine betaine transmethylase (EC 2.1.1.5) (GBT), the enzyme responsible for the demethylation of glycine betaine to N,Ndimethylglycine (DMG), as was reported for Rhizobium meliloti (9). The genes involved in the osmoprotection of P. aeruginosa (betA, betB, betI,and betT1, encoding choline dehydrogenase, betaine aldehyde dehydrogenase, a putative regulatory protein, and a choline transporter, respectively) had been localized from nucleotides 6047363 to 6053193 of the PAO1 complete genome (10), but those involved in glycine betaine degradation have not yet been localized. In this study, we report the identification of a genomic region that encodes a product required in the catabolic pathway of choline, specifically in the demethylation of glycine betaine to DMG.Isolation and characterization of the TN5::751 mutant of P. aeruginosa. To identify the locus required for the utilization of glycine betaine as a carbon and nitrogen source in P. aeruginosa, strain PRS (a streptomycin-resistant [Str r ] derivative of the wild-type strain Fildes III) was subjected to random mutagenesis with the Tn5::751 transposon, encoding kanamycin resistance (Km r ) and trimethoprim resistance (Tp r ) (8). Approximately 3,200 colonies capable of growing in nutritive medium with streptomycin, kanamycin, and trimethoprim (1,000, 250, and 500 g ⅐ ml Ϫ1 , respectively) were screened for growth on iso-osmolar high-phosphate basal salt medium (HPi-BSM) (7) containing succinate plus NH 4 Cl or on choline as a carbon and nitrogen source. One strain that did not grow on choline was selected and designated ALS-96. Studies of growth on plates, subsequently confirmed to occur in liquid media ( Table 1), showed that this strain was not capable of growing on either choline or glycine betaine under iso-osmolar or hyperosmolar conditions. Nev...

PrtA is the major secreted metalloprotease of Previous reports implicate PrtA in the pathogenic capacity of this bacterium. PrtA is also clinically used as a potent analgesic and anti-inflammatory drug, and its catalytic properties attract industrial interest. Comparatively, there is scarce knowledge about the mechanisms that physiologically govern PrtA expression in In this work, we demonstrate that PrtA production is derepressed when the bacterial growth temperature decreases from 37°C to 30°C. We show that this thermoregulation occurs at the transcriptional level. We determined that upstream of , there is a conserved motif that is directly recognized by the CpxR transcriptional regulator. This feature is found along strains irrespective of their isolation source, suggesting an evolutionary conservation of CpxR-dependent regulation of PrtA expression. We found that in s, the CpxAR system is more active at 37°C than at 30°C. In good agreement with these results, in a mutant background, is derepressed at 37°C, while overexpression of the NlpE lipoprotein, a well-known CpxAR-inducing condition, inhibits PrtA expression, suggesting that the levels of the activated form of CpxR are increased at 37°C over those at 30°C. In addition, we establish that PrtA is involved in the ability of to develop biofilm. In accordance, CpxR influences the biofilm phenotype only when bacteria are grown at 37°C. In sum, our findings shed light on regulatory mechanisms that fine-tune PrtA expression and reveal a novel role for PrtA in the lifestyle of We demonstrate that metalloprotease PrtA expression is transcriptionally thermoregulated. While strongly activated below 30°C, its expression is downregulated at 37°C. We found that in , the CpxAR signal transduction system, which responds to envelope stress and bacterial surface adhesion, is activated at 37°C and able to downregulate PrtA expression by direct interaction of CpxR with a binding motif located upstream of the gene. Moreover, we reveal that PrtA expression favors the ability of to develop biofilm, irrespective of the bacterial growth temperature. In this context, thermoregulation along with a highly conserved CpxR-dependent modulation mechanism gives clues about the relevance of PrtA as a factor implicated in the persistence of on abiotic surfaces and in bacterial host colonization capacity.

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