The alkylation response protein AidB is localized at the new poles and constriction sites in Brucella abortus

<p>Abstract</p> <p>Background</p> <p><it>Brucella abortus </it>is the etiological agent of a worldwide zoonosis called brucellosis. This alpha-proteobacterium is dividing asymmetrically, and PdhS, an essential histidine kinase, was reported to be an old pole marker.</p> <p>Results</p> <p>We were interested to identify functions that could be recruited to bacterial poles. The <it>Brucella </it>ORFeome, a collection of cloned predicted coding sequences, was placed in fusion with yellow fluorescent protein (YFP) coding sequence and screened for polar localizations in <it>B. abortus</it>. We report that AidB-YFP was systematically localized to the new poles and at constrictions sites in <it>B. abortus</it>, either in culture or inside infected HeLa cells or RAW264.7 macrophages. AidB is an acyl-CoA dehydrogenase (ACAD) homolog, similar to <it>E. coli </it>AidB, an enzyme putatively involved in destroying alkylating agents. Accordingly, a <it>B. abortus aidB </it>mutant is more sensitive than the wild-type strain to the lethality induced by methanesulphonic acid ethyl ester (EMS). The exposure to EMS led to a very low frequency of constriction events, suggesting that cell cycle is blocked during alkylation damage. The localization of AidB-YFP at the new poles and at constriction sites seems to be specific for this ACAD homolog since two other ACAD homologs fused to YFP did not show specific localization. The overexpression of <it>aidB</it>, but not the two other ACAD coding sequences, leads to multiple morphological defects.</p> <p>Conclusions</p> <p>Data reported here suggest that AidB is a marker of new poles and constriction sites, that could be considered as sites of preparation of new poles in the sibling cells originating from cell division. The possible role of AidB in the generation or the function of new poles needs further investigation.</p

Phosphotransferase-dependent accumulation of (p)ppGpp in response to glutamine deprivation in Caulobacter crescentus

The alarmone (p)ppGpp is commonly used by bacteria to quickly respond to nutrient starvation. Although (p)ppGpp synthetases such as SpoT have been extensively studied, little is known about the molecular mechanisms stimulating alarmone synthesis upon starvation. Here, we describe an essential role of the nitrogen-related phosphotransferase system (PTS(Ntr)) in controlling (p)ppGpp accumulation in Caulobacter crescentus. We show that cells sense nitrogen starvation by way of detecting glutamine deprivation using the first enzyme (EI(Ntr)) of PTS(Ntr). Decreasing intracellular glutamine concentration triggers phosphorylation of EI(Ntr) and its downstream components HPr and EIIA(Ntr). Once phosphorylated, both HPr∼P and EIIA(Ntr)∼P stimulate (p)ppGpp accumulation by modulating SpoT activities. This burst of second messenger primarily impacts the non-replicative phase of the cell cycle by extending the G1 phase. This work highlights a new role for bacterial PTS systems in stimulating (p)ppGpp accumulation in response to metabolic cues and in controlling cell cycle progression and cell growth

Identification of the essential Brucella melitensis porin Omp2b as a suppressor of Bax-induced cell death in yeast in a genome-wide screening.

BACKGROUND: Inhibition of apoptosis is one of the mechanisms selected by numerous intracellular pathogenic bacteria to control their host cell. Brucellae, which are the causative agent of a worldwide zoonosis, prevent apoptosis of infected cells, probably to support survival of their replication niche. METHODOLOGY/PRINCIPAL FINDINGS: In order to identify Brucella melitensis anti-apoptotic effector candidates, we performed a genome-wide functional screening in yeast. The B. melitensis ORFeome was screened to identify inhibitors of Bax-induced cell death in S. cerevisiae. B. melitensis porin Omp2b, here shown to be essential, prevents Bax lethal effect in yeast, unlike its close paralog Omp2a. Our results based on Omp2b size variants characterization suggest that signal peptide processing is required for Omp2b effect in yeast. CONCLUSION/SIGNIFICANCE: We report here the first application to a bacterial genome-wide library of coding sequences of this "yeast-rescue" screening strategy, previously used to highlight several new apoptosis regulators. Our work provides B. melitensis proteins that are candidates for an anti-apoptotic function, and can be tested in mammalian cells in the future. Hypotheses on possible molecular mechanisms of Bax inhibition by the B. melitensis porin Omp2b are discussed

Overproduced Brucella abortus PdhS-mCherry forms soluble aggregates in Escherichia coli, partially associating with mobile foci of IbpA-YFP

<p>Abstract</p> <p>Background</p> <p>When heterologous recombinant proteins are produced in <it>Escherichia coli</it>, they often precipitate to form insoluble aggregates of unfolded polypeptides called inclusion bodies. These structures are associated with chaperones like IbpA. However, there are reported cases of "non-classical" inclusion bodies in which proteins are soluble, folded and active.</p> <p>Results</p> <p>We report that the <it>Brucella abortus </it>PdhS histidine kinase fused to the mCherry fluorescent protein forms intermediate aggregates resembling "non-classical" inclusion bodies when overproduced in <it>E. coli</it>, before forming "classical" inclusion bodies. The intermediate aggregates of PdhS-mCherry are characterized by the solubility of PdhS-mCherry, its ability to specifically recruit known partners fused to YFP, suggesting that PdhS is folded in these conditions, and the quick elimination (in less than 10 min) of these structures when bacterial cells are placed on fresh rich medium. Moreover, soluble PdhS-mCherry foci do not systematically colocalize with IpbA-YFP, a marker of inclusion bodies. Instead, time-lapse experiments show that IbpA-YFP exhibits rapid pole-to-pole shuttling, until it partially colocalizes with PdhS-mCherry aggregates.</p> <p>Conclusion</p> <p>The data reported here suggest that, in <it>E. coli</it>, recombinant proteins like PdhS-mCherry may transit through a soluble and folded state, resembling previously reported "non-classical" inclusion bodies, before forming "classical" inclusion bodies. The dynamic localization of IbpA-YFP foci suggests that the IbpA chaperone could scan the <it>E. coli </it>cell to find its substrates.</p

Functional Characterization of the Incomplete Phosphotransferase System (PTS) of the Intracellular Pathogen Brucella melitensis

Background: In many bacteria, the phosphotransferase system (PTS) is a key player in the regulation of the assimilation of alternative carbon sources notably through catabolic repression. The intracellular pathogens Brucella spp. possess four PTS proteins (EI Ntr, NPr, EIIA Ntr and an EIIA of the mannose family) but no PTS permease suggesting that this PTS might serve only regulatory functions

Genome-wide analysis of Brucella melitensis genes required throughout intranasal infection in mice

Brucellae are facultative intracellular Gram-negative coccobacilli that chronically infect various mammals and cause brucellosis. Human brucellosis is among the most common bacterial zoonoses and the vast majority of cases are attributed to B .melitensis .Using transposon sequencing (Tn-seq) analysis, we showed that among 3369 predicted genes of the B .melitensis genome, 861 are required for optimal growth in rich medium and 186 additional genes appeared necessary for survival of B .melitensis in RAW 264.7 macrophages in vitro .As the mucosal immune system represents the first defense against Brucella infection, we investigated the early phase of pulmonary infection in mice. In situ analysis at the single cell level indicates a succession of killing and growth phases, followed by heterogenous proliferation of B .melitensis in alveolar macrophages during the first 48 hours of infection. Tn-seq analysis identified 94 additional genes that are required for survival in the lung at 48 hours post infection. Among them, 42 genes are common to RAW 264.7 macrophages and the lung conditions, including the T4SS and purine synthesis genes. But 52 genes are not identified in RAW 264.7 macrophages, including genes implicated in lipopolysaccharide (LPS) biosynthesis, methionine transport, tryptophan synthesis as well as fatty acid and carbohydrate metabolism. Interestingly, genes implicated in LPS synthesis and β oxidation of fatty acids are no longer required in Interleukin (IL)-17RA -/- mice and asthmatic mice, respectively. This demonstrates that the immune status determines which genes are required for optimal survival and growth of B .melitensis in vivo .info:eu-repo/semantics/publishe