Annick Jacq

Global analysis of extracytoplasmic stress signaling in Escherichia coli.

The Bae, Cpx, Psp, Rcs, and σE pathways constitute the Escherichia coli signaling systems that detect and respond to alterations of the bacterial envelope. Contributions of these systems to stress response have previously been examined individually; however, the possible interconnections between these pathways are unknown. Here we investigate the dynamics between the five stress response pathways by determining the specificities of each system with respect to signal-inducing conditions, and monitoring global transcriptional changes in response to transient overexpression of each of the effectors. Our studies show that different extracytoplasmic stress conditions elicit a combined response of these pathways. Involvement of the five pathways in the various tested stress conditions is explained by our unexpected finding that transcriptional responses induced by the individual systems show little overlap. The extracytoplasmic stress signaling pathways in E. coli thus regulate mainly complementary functions whose discrete contributions are integrated to mount the full adaptive response.

The secD locus of E. coli codes for two membrane proteins required for protein export.

Cold‐sensitive mutations in the secD locus of Escherichia coli result in severe defects in protein export at the non‐permissive temperature of 23 degrees C. DNA sequence of a cloned fragment that includes the secD locus reveals open reading frames for seven polypeptide chains. Both deletions and TnphoA insertions in this clone have been used in maxicell and complementation studies to define the secD locus and its products. The secD mutations fall into two complementation groups, defining genes we have named secD and secF. These two genes comprise an operon, the first case of two genes involved in the export process being co‐transcribed. The DNA sequence of the two genes along with alkaline phosphatase fusion analysis indicates that they code for integral proteins of the cytoplasmic membrane. We suggest that these two proteins may form a complex in the membrane which acts at late steps in the export process.

Experimental discovery of small RNAs in Staphylococcus aureus reveals a riboregulator of central metabolism

Using an experimental approach, we investigated the RNome of the pathogen Staphylococcus aureus to identify 30 small RNAs (sRNAs) including 14 that are newly confirmed. Among the latter, 10 are encoded in intergenic regions, three are generated by premature transcription termination associated with riboswitch activities, and one is expressed from the complementary strand of a transposase gene. The expression of four sRNAs increases during the transition from exponential to stationary phase. We focused our study on RsaE, an sRNA that is highly conserved in the bacillales order and is deleterious when over-expressed. We show that RsaE interacts in vitro with the 5′ region of opp3A mRNA, encoding an ABC transporter component, to prevent formation of the ribosomal initiation complex. A previous report showed that RsaE targets opp3B which is co-transcribed with opp3A. Thus, our results identify an unusual case of riboregulation where the same sRNA controls an operon mRNA by targeting two of its cistrons. A combination of biocomputational and transcriptional analyses revealed a remarkably coordinated RsaE-dependent downregulation of numerous metabolic enzymes involved in the citrate cycle and the folate-dependent one-carbon metabolism. As we observed that RsaE accumulates transiently in late exponential growth, we propose that RsaE functions to ensure a coordinate downregulation of the central metabolism when carbon sources become scarce.

An assessment of the role of intracellular free Ca2+ in E. coli

We have previously proposed that fluctuations in Ca(2+) levels should play an important role in bacteria as in eukaryotes in regulating cell cycle events (Norris et al., J. Theor. Biol. 134 (1998) 341-350). This proposal implied the presence of Ca(2+) uptake systems in bacteria, cell cycle mutants simultaneously defective in Ca(2+)-homeostasis, and perturbation of cell cycle processes when cellular Ca(2+) levels are depleted. We review the properties of new cell cycle mutants in E. coli and B. subtilis resistant to inhibitors of calmodulin, PKC or Ca(2+)-channels; the evidence for Ca(2+)-binding proteins including Acp and FtsZ; and Ca(2+)-transporters. In addition, the effects of EGTA and verapamil (a Ca(2+) channel inhibitor) on growth, protein synthesis and cell cycle events in E. coli are described. We also describe new measurements of free Ca(2+)-levels, using aequorin, in E. coli. Several new cell cycle mutants were obtained using this approach, affecting either initiation of DNA replication or in particular cell division at non-permissive temperature. Several of the mutants were also hypersensitive to EGTA and or Ca(2+). However, none of the mutants apparently involved direct alteration of a drug target and surprisingly in some cases involved specific tRNAs or a tRNA synthetase. The results also indicate that the expression of several genes in E. coli may be regulated by Ca(2+). Cell division in particular appears very sensitive to the level of cell Ca(2+), with the frequency of division clearly reduced by EGTA and by verapamil. However, whilst the effect of EGTA was clearly correlated with depletion of cellular Ca(2+) including free Ca(2+), this was not the case with verapamil which appears to change membrane fluidity and the consequent activity of membrane proteins. Measurement of free Ca(2+) in living cells indicated levels of 200-300 nM, tightly regulated in wild type cells in exponential phase, somewhat less so in stationary phase, with apparently La(2+)-sensitive PHB-polyphosphate complexes involved in Ca(2+) influx. The evidence reviewed increasingly supports a role for Ca(2+) in cellular processes in bacteria, however, any direct link to the control of cell cycle events remains to be established.

Point mutations in the transmembrane domain of DjlA, a membrane-linked DnaJ-like protein, abolish its function in promoting colanic acid production via the Rcs signal transduction pathway

DjlA is a novel DnaJ‐like protein localized to the inner membrane of Escherichia coli through the single transmembrane domain (TMD) found at the N‐terminus. The overproduction of DjlA activates expression of the cps operon, controlling synthesis and export of the extracellular polysaccharide colanic acid via the Rcs/B two‐component signal transduction pathway. We now show that both the TMD and the J‐region are essential for the induction of cps expression observed with the overproduction of DjlA. Furthermore, we describe the isolation and characterization of different point mutations in the TMD that completely or partially block the induction of cps expression associated with overproduction of DjlA. These mutations were shown not to affect the localization, stability or topology of the mutant DjlA proteins. We propose that these mutations are affecting specific interactions between the TMD of DjlA and its substrate protein(s), for example RcsC, the membrane sensor kinase partner of the Rcs/B signal transduction pathway.

A novel DnaJ‐like protein in Escherichia coli inserts into the cytoplasmic membrane with a Type III topology

We describe a novel Escherichia coli protein, DjlA, containing a highly conserved J‐region motif, which is present in the DnaJ protein chaperone family and required for interaction with DnaK. Remarkably, DjlA is shown to be a membrane protein, localized to the inner membrane with the unusual Type III topology (N‐out, C‐in). Thus, DjlA appears to present an extremely short N‐terminus to the periplasm and has a single transmembrane domain (TMD) and a large cytoplasmic domain containing the C‐terminal J‐region. Analysis of the TMD of DjIA and recently identified homologues in Coxiella burnetti and Haemophilus influenzae revealed a striking pattern of conserved glycines (or rarely alanine), with a four‐residue spacing. This motif, predicted to form a spiral groove in the TMD, is more marked than a repeating glycine motif, implicated in the dimerization of TMDs of some eukaryotic proteins. This feature of DjlA could represent a promiscuous docking mechanism for interaction with a variety of membrane proteins. DjlA null mutants can be isolated but these appear rapidly to accumulate suppressors to correct envelope and growth defects. Moderate (10‐fold) overproduction of DjlA suppresses a mutation in FtsZ but markedly perturbs cell division and cell‐envelope growth in minimal medium. We propose that DjlA plays a role in the correct assembly, activity and/or maintenance of a number of membrane proteins, including two‐component signal‐transduction systems.

The emergence of Vibrio pathogens in Europe: ecology, evolution, and pathogenesis (Paris, 11-12th March 2015).

Global change has caused a worldwide increase in reports of Vibrio-associated diseases with ecosystem-wide impacts on humans and marine animals. In Europe, higher prevalence of human infections followed regional climatic trends with outbreaks occurring during episodes of unusually warm weather. Similar patterns were also observed in Vibrio-associated diseases affecting marine organisms such as fish, bivalves and corals. Basic knowledge is still lacking on the ecology and evolutionary biology of these bacteria as well as on their virulence mechanisms. Current limitations in experimental systems to study infection and the lack of diagnostic tools still prevent a better understanding of Vibrio emergence. A major challenge is to foster cooperation between fundamental and applied research in order to investigate the consequences of pathogen emergence in natural Vibrio populations and answer federative questions that meet societal needs. Here we report the proceedings of the first European workshop dedicated to these specific goals of the Vibrio research community by connecting current knowledge to societal issues related to ocean health and food security.

EGTA induces the synthesis in Escherichia coli of three proteins that cross-react with calmodulin antibodies

Escherichia coli mutants, (verA, dilA) specifically resistant to the Ca2+ channel inhibitors verapamil and diltiazem, respectively, are hypersensitive to EGTA and BAPTA. We have shown, using 1‐D and 2‐D gel electrophoresis, that the synthesis of at least 25 polypeptides in the mutants was enhanced by treatment with Ca2+ chelators and the synthesis of at least 11 polypeptides was repressed. This pattern of induction was not observed in heat‐ or SDS‐treated cells and therefore does not appear to be a general stress response. The majority of the induced proteins are low molecular weight, extremely heat stable and acidic, characteristic properties of calmodulin. Moreover, of the major induced species, three with apparent molecular masses of 12, 18, and 34kDa all cross‐reacted with polyclonal and monoclonal antibodies to eukaryote calmodulins or calerythrin, a heat‐resistant Ca2+‐binding protein from Saccharo‐polyspora erythraea. The verA, dilA mutants. In being hypersensitive to EGTA and to the Ca2+ ionophore A23187 + Ca2+, may be defective in the regulation of the level of free intracellular Ca2+.

pH and monovalent cations regulate cytosolic free Ca2+ in E. coli

The results here show for the first time that pH and monovalent cations can regulate cytosolic free Ca(2+) in E. coli through Ca(2+) influx and efflux, monitored using aequorin. At pH 7.5 the resting cytosolic free Ca(2+) was 0.2-0.5 microM. In the presence of external Ca(2+) (1 mM) at alkaline pH this rose to 4 microM, being reduced to 0.9 microM at acid pH. Removal of external Ca(2+) caused an immediate decrease in cytosolic free Ca(2+) at 50-100 nM s(-1). Efflux rates were the same at pH 5.5, 7.5 and 9.5. Thus, ChaA, a putative Ca(2+)/H(+)exchanger, appeared not to be a major Ca(2+)-efflux pathway. In the absence of added Na(+), but with 1 mM external Ca(2+), cytosolic free Ca(2+) rose to approximately 10 microM. The addition of Na(+)(half maximum 60 mM) largely blocked this increase and immediately stimulated Ca(2+) efflux. However, this effect was not specific, since K(+) also stimulated efflux. In contrast, an increase in osmotic pressure by addition of sucrose did not significantly stimulate Ca(2+) efflux. The results were consistent with H(+) and monovalent cations competing with Ca(2+) for a non-selective ion influx channel. Ca(2+) entry and efflux in chaA and yrbG knockouts were not significantly different from wild type, confirming that neither ChaA nor YrbG appear to play a major role in regulating cytosolic Ca(2+) in Escherichia coli. The number of Ca(2+) ions calculated to move per cell per second ranged from <1 to 100, depending on conditions. Yet a single eukaryote Ca(2+) channel, conductance 100 pS, should conduct >6 million ions per second. This raises fundamental questions about the nature and regulation of Ca(2+) transport in bacteria, and other small living systems such as mitochondria, requiring a new mathematical approach to describe such ion movements. The results have important significance in the adaptation of E. coli to different ionic environments such as the gut, fresh water and in sea water near sewage effluents.

A single regulatory gene is sufficient to alter Vibrio aestuarianus pathogenicity in oysters

Oyster diseases caused by pathogenic vibrios pose a major challenge to the sustainability of oyster farming. In France, since 2012 a disease affecting specifically adult oysters has been associated with the presence of Vibrio aestuarianus. Here, by combining genome comparison, phylogenetic analyses and high-throughput infections of strains isolated before or during the recent outbreaks, we show that virulent strains cluster into two V. aestuarianus lineages independently of the sampling dates. The bacterial lethal dose was not different between strains isolated before or after 2012. Hence, the emergence of a new highly virulent clonal strain is unlikely. Each lineage comprises nearly identical strains, the majority of them being virulent, suggesting that within these phylogenetically coherent virulent lineages a few strains have lost their pathogenicity. Comparative genomics allowed the identification of a single frameshift in a non-virulent strain. This mutation affects the varS gene that codes for a signal transduction histidine-protein kinase. Genetic analyses confirmed that varS is necessary for infection of oysters and for a secreted metalloprotease expression. For the first time in a Vibrio species, we show here that VarS is a key factor of pathogenicity.