Search

Salmonella enterica polymyxin B (PM) resistance is modulated mainly by substitutions of the acyl chains and the phosphate groups on the lipid A moiety of lipopolysaccharide. These modifications are mediated by genes under the control of the PmrA/PmrB and PhoP/PhoQ two-component regulatory systems. In this study, a deletion in the gene encoding the alternative σ54 factor, rpoN, was shown to increase PM resistance without affecting protamine sensitivity. The results presented here showed that the increased polymyxin resistance observed in the Δ rpoN mutant occurs through a PmrA/PhoP-independent pathway. Downregulation of one or more genes belonging to the RpoN regulon may provide an additional mechanism of defence against membrane-permeabilizing antimicrobial peptides that helps the pathogen to survive in different environments. [ABSTRACT FROM AUTHOR]

Aims: To characterize the luxO gene in fish pathogen Vibrio alginolyticus MVP01 and investigate its roles in regulation of extracellular products (ECP) and siderophore production. Methods and Results: The luxO gene was cloned from V. alginolyticus MVP01. Genetic analysis revealed that it encoded a protein with high similarity to other LuxO homologues. The luxO in-frame deletion mutant and rpoN null mutant were constructed with suicide plasmids. We demonstrated that sole deletion in LuxO increased the secretion of extracellular protease and haemolytic products, but decreased siderophore production for V. alginolyticus MVP01. Mutants with null rpoN displayed significantly enhanced protease level and siderophore production while notable reduction in haemolytic activities of ECP. Conclusions: Vibrio alginolyticus harbours functional luxO gene that regulates the secretion of extracellular protease and haemolytic materials as well as siderophore production in either σ54 dependent or independent manners. Significance and Impact of the Study: The current study demonstrated that V. alginolyticus MVP01 produces extracellular protease and haemolytic activity material as well as siderophore, which may be characteristics of the virulence of the strain. Revelations that secretion of these products is under the regulation of LuxO and σ54 as well as the potential quorum sensing systems in V. alginolyticus MVP01 will expedite the understanding of vibriosis pathogenesis. [ABSTRACT FROM AUTHOR]

Listeria monocytogenes is able to grow under conditions of high osmolarity. We constructed a deletion mutant of rpoN, encoding the alternative sigma factor RpoN, and analyzed its response to osmotic stress. In a minimal medium with 4% NaCl and 1 mM betaine, the mutant showed a similar growth to that of the parental strain, EGD. In the same medium with 4% NaCl and 1 M carnitine, the growth rate of the mutant was greatly reduced, when the optical density at 600 nm (OD600) at the starting point of growth, was 0.15. However, when growth of the culture was started at an OD600 of 0.025, the growth of the mutant was similar to that of EGD. The mutant's expression of two betaine transporter genes, betL and gbuB, and the carnitine transporter gene opuCA, was osmotically induced at a level similar to EGD, and its rate of carnitine uptake was similar to that of EGD. These results suggest that the growth defect from the rpoN mutant is caused not by the transcriptional regulation of opuCA or by a decrease in carnitine uptake, but possibly by larger amounts of carnitine being needed for growth of the mutant in minimal medium when NaCl is present. [ABSTRACT FROM AUTHOR]

Regulation of medium-chain-length polyhydroxyalkanoate biosynthesis in Pseudomonas aeruginosa and Pseudomonas putida was studied conducting PHA accumulation experiments and transcriptional analysis of PHA biosynthesis genes with wild type strains and rpoN-negative mutants. In P. putida PHA accumulation was RpoN-independent, whereas in P. aeruginosa PHA accumulation was RpoN-dependent. Transcriptional analysis applying reverse transcriptase-polymerase chain reaction showed strong induction of phaG, encoding the transacylase, under nitrogen starvation in P. putida KT2440 and the respective rpoN-negative mutant, indicating an RpoN-independent regulation of phaG. No transcription of phaG and no PHA accumulation was detected in the rpoN-negative mutant of P. aeruginosa neither from gluconate nor from octanoate as carbon source. Alginate-overproducing mutant P. aeruginosa FRD1 showed strongly decreased PHA accumulation from gluconate but no difference in phaC1 (encoding the PHA synthase) transcription, indicating that alginate biosynthesis competes with PHA biosynthesis regarding acetyl-CoA as precursor for both biopolymers. Transcription of phaF and phaI-F was nitrogen independent. [Copyright &y& Elsevier]

The planctomycetes are a phylogenetically distinct group of bacteria, widespread in aquatic and terrestrial environments. Their cell walls lack peptidoglycan and their compartmentalised cells undergo a yeast-like budding cell division process. Many bacteria regulate a subset of their genes by an enhancer-dependent mechanism involving the alternative sigma factor σ54 (RpoN, σN) in association with σ54-dependent transcriptional activators known as enhancer-binding proteins (EBPs). The σ54-dependent regulon has previously been studied in several groups of bacteria, but not in the planctomycetes. We wished to exploit the recently published complete genome sequence of Pirellula species strain 1 to predict and analyse the σ54-dependent regulon in this interesting group of bacteria. The genome of Pirellula species strain 1 encodes one homologue of σ54, and 16 σ54-dependent EBPs, including 10 two-component response regulators and a homologue of Escherichia coli RtcR. Two EBPs contain forkhead-associated domains, representing a novel protein domain combination not previously observed in bacterial EBPs and suggesting a novel link between the enhancer-dependent regulon and ‘eukaryotic-like’ protein phosphorylation in bacterial signal transduction. We identified several potential σ54-dependent promoters upstream of genes and operons including two homologues of csrA, which encodes the global regulator CsrA, and rtcBA, encoding a RNA 3′-terminal phosphate cyclase. Phylogenetic analysis of EBP sequences from a wide range of bacterial taxa suggested that planctomycete EBPs fall into several distinct clades. Also the phylogeny of the σ54 factors is broadly consistent with that of the host organisms. These results are consistent with a very ancient origin of σ54 within the bacterial lineage. The repertoire of functions predicted to be under the control of the σ54-dependent regulon in Pirellula shares some similarities (e.g. rtcBA) as well as exhibiting differences with that in other taxonomic groups of bacteria, reinforcing the evolutionarily dynamic nature of this regulon. [Copyright &y& Elsevier]

The rhl quorum sensing (QS) circuit of Pseudomonas aeruginosa is known to regulate the expression of a number of virulence factors. This study investigates the regulation of rhlI, encoding the auto-inducer synthase RhlI responsible for the synthesis of N-butryl-L-homoserine lactone (BHL). A putative RpoN binding site was located upstream, in the promoter region of rhlI. Utilising a rhlI-lacZ transcriptional reporter, we demonstrate that under certain media conditions RpoN is a positive regulator of rhlI transcription. Measurements of BHL in extracted supernatant showed that the transcriptional patterns were reflected in the BHL levels, which were reduced in the rpoN mutant. Elastase and pyocyanin, known to be regulated by the rhl QS circuit, were shown to be reduced in a RpoN deficient strain. However, exogenous addition of BHL to the rpoN mutant did not restore these phenotypes suggesting that other regulatory factors apart from BHL are involved. Consistent with other rhl regulated phenotypes, we found that a rpoN mutant strain forms a biofilm that is different from that of the wild-type but similar to that displayed by a rhlI mutant. [Copyright &y& Elsevier]

Helicobacter pylori possesses a gene (HP0326/JHP309) homologous to neuA of other bacteria, encoding a cytidyl monophosphate-N-acetylneuraminic acid synthetase-homologous enzyme in its N-terminal portion. We analysed the function of this gene, which is controlled by a flagellar class 2 σ54 promoter, in flagellar biosynthesis. HP0326/JHP309 actually represents a bicistronic operon consisting of a neuA and a flmD-like putative glycosyl transferase gene. An isogenic flmD mutant synthesized basal bodies but no filaments, was non-motile, and expressed severely reduced amounts of a FlaA flagellin of reduced molecular mass. FlaA flagellin was found to be glycosylated in its exported form within the flagellar filament, but not inside the cytoplasm. Glycosylated FlaA was not detectable in the flmD mutant. Together with other genes in the H. pylori genome, a proposed function of the neuA/flmD gene products could be to provide a pathway for glycosylation of flagellin and other extracytoplasmic molecules during type III secretion. [Copyright &y& Elsevier]