Evelyne Turlin

A tale of two oxidation states: bacterial colonization of arsenic-rich environments.

Microbial biotransformations have a major impact on contamination by toxic elements, which threatens public health in developing and industrial countries. Finding a means of preserving natural environments—including ground and surface waters—from arsenic constitutes a major challenge facing modern society. Although this metalloid is ubiquitous on Earth, thus far no bacterium thriving in arsenic-contaminated environments has been fully characterized. In-depth exploration of the genome of the β-proteobacterium Herminiimonas arsenicoxydans with regard to physiology, genetics, and proteomics, revealed that it possesses heretofore unsuspected mechanisms for coping with arsenic. Aside from multiple biochemical processes such as arsenic oxidation, reduction, and efflux, H. arsenicoxydans also exhibits positive chemotaxis and motility towards arsenic and metalloid scavenging by exopolysaccharides. These observations demonstrate the existence of a novel strategy to efficiently colonize arsenic-rich environments, which extends beyond oxidoreduction reactions. Such a microbial mechanism of detoxification, which is possibly exploitable for bioremediation applications of contaminated sites, may have played a crucial role in the occupation of ancient ecological niches on earth.

The structural and functional organization of H‐NS‐like proteins is evolutionarily conserved in Gram‐negative bacteria

The structural gene of the H‐NS protein, a global regulator of bacterial metabolism, has been identified in the group of enterobacteria as well as in closely related bacteria, such as Erwinia chrysanthemi and Haemophilus influenzae. Isolated outside these groups, the BpH3 protein of Bordetella pertussis exhibits a low amino acid conservation with H‐NS, particularly in the N‐terminal domain. To obtain information on the structure, function and/or evolution of H‐NS, we searched for other H‐NS‐related proteins in the latest databases. We found that HvrA, a trans‐activator protein in Rhodobacter capsulatus, has a low but significant similarity with H‐NS and H‐NS‐like proteins. This Gram‐negative bacterium is phylogenetically distant from Escherichia coli. Using theoretical analysis (e.g. secondary structure prediction and DNA binding domain modelling) of the amino acid sequence of H‐NS, StpA (an H‐NS‐like protein in E. coli ), BpH3 and HvrA and by in vivo and in vitro experiments (e.g. complementation of various H‐NS‐related phenotypes and competitive gel shift assay), we present evidence that these proteins belong to the same class of DNA binding proteins. In silico analysis suggests that this family also includes SPB in R. sphaeroides, XrvA in Xanthomonas oryzae and VicH in Vibrio cholerae. These results demonstrate that proteins structurally and functionally related to H‐NS are widespread in Gram‐negative bacteria.

The PhoP-PhoQ Two-Component Regulatory System of Photorhabdus luminescens Is Essential for Virulence in Insects

Photorhabdus luminescens is a symbiont of entomopathogenic nematodes. Analysis of the genome sequence of this organism revealed a homologue of PhoP-PhoQ, a two-component system associated with virulence in intracellular bacterial pathogens. This organism was shown to respond to the availability of environmental magnesium. A mutant with a knockout mutation in the regulatory component of this system (phoP) had no obvious growth defect. It was, however, more motile and more sensitive to antimicrobial peptides than its wild-type parent. Remarkably, the mutation eliminated virulence in an insect model. No insect mortality was observed after injection of a large number of the phoP bacteria, while very small amounts of parental cells killed insect larvae in less than 48 h. At the molecular level, the PhoPQ system mediated Mg(2+)-dependent modifications in lipopolysaccharides and controlled a locus (pbgPE) required for incorporation of 4-aminoarabinose into lipid A. Mg(2+)-regulated gene expression of pbgP1 was absent in the mutant and was restored when phoPQ was complemented in trans. This finding highlights the essential role played by PhoPQ in the virulence of an entomopathogen.

Pleiotropic role of quorum-sensing autoinducer 2 in Photorhabdus luminescens.

ABSTRACT Bacterial virulence is an integrative process that may involve quorum sensing. In this work, we compared by global expression profiling the wild-type entomopathogenic Photorhabdus luminescens subsp. laumondii TT01 to a luxS-deficient mutant unable to synthesize the type 2 quorum-sensing inducer AI-2. AI-2 was shown to regulate more than 300 targets involved in most compartments and metabolic pathways of the cell. AI-2 is located high in the hierarchy, as it controls the expression of several transcriptional regulators. The regulatory effect of AI-2 appeared to be dose dependent. The luxS-deficient strain exhibited decreased biofilm formation and increased type IV/V pilus-dependent twitching motility. AI-2 activated its own synthesis and transport. It also modulated bioluminescence by regulating the synthesis of spermidine. AI-2 was further shown to increase oxidative stress resistance, which is necessary to overcome part of the innate immune response of the host insect involving reactive oxygen species. Finally, we showed that the luxS-deficient strain had attenuated virulence against the lepidopteran Spodoptera littoralis. We concluded that AI-2 is involved mainly in early steps of insect invasion in P. luminescens.

Enhanced structural and functional genome elucidation of the arsenite-oxidizing strain Herminiimonas arsenicoxydans by proteomics data

The arsenite-oxidizing strain Herminiimonas arsenicoxydans proteome was investigated with gel electrophoresis and tandem mass spectrometry analyses. The comparison of experimental and theoretical M(r) and pI, as well as that of peptide sequences identified by MS and predicted protein sequences, allowed the correction of five protein annotations. More importantly, the functional analysis of SDS- and 2D-PAGE proteome maps obtained in the presence of arsenic, combined with partial transcriptomic results indicate that H. arsenicoxydans expressed genes and proteins required not only for arsenic detoxification or stress response but also involved in motility, exopolysaccharide synthesis, phosphate import or energetic metabolism. This study provides therefore new insights into the adaptation processes of H. arsenicoxydans in response to arsenic.

Sulfur-limitation-regulated proteins in Bacillus subtilis: a two-dimensional gel electrophoresis study

Little is known about the genes and enzymes involved in sulfur assimilation in Bacillus subtilis, or about the regulation of their expression or activity. To identify genes regulated by sulfur limitation, the authors used two- dimensional (2D) gel electrophoresis to compare the proteome of a wild-type strain grown with either sulfate or glutathione as sole sulfur source. A total of 15 proteins whose synthesis is modified under these two conditions were identified by matrix-assisted laser desorption/ionization time of flight (MALDI TOF) mass spectrometry. In the presence of sulfate, an increased amount of proteins involved in the metabolism of C(1) units (SerA, GlyA, FolD) and in the biosynthesis of purines (PurQ, Xpt) and pyrimidines (Upp, PyrAA, PyrF) was observed. In the presence of glutathione, the synthesis of two uptake systems (DppE, SsuA), an oxygenase (SsuD), cysteine synthase (CysK) and two proteins of unknown function (YtmI, YurL) was increased. The changes in expression of the corresponding genes, in the presence of sulfate and glutathione, were monitored using slot-blot analyses and lacZ fusions. The ytmI gene is part of a locus of 12 genes which are co-regulated in response to sulfur availability. This putative operon is activated by a LysR-like regulator, YTLI: This is the first regulator involved in the control of expression in response to sulfur availability to be identified in B. subtilis.

Staphylococcus aureus FepA and FepB Proteins Drive Heme Iron Utilization in Escherichia coli

EfeUOB-like tripartite systems are widespread in bacteria and in many cases they are encoded by genes organized into iron-regulated operons. They consist of: EfeU, a protein similar to the yeast iron permease Ftrp1; EfeO, an extracytoplasmic protein of unknown function and EfeB, also an extracytoplasmic protein with heme peroxidase activity, belonging to the DyP family. Many bacterial EfeUOB systems have been implicated in iron uptake, but a prefential iron source remains undetermined. Nevertheless, in the case of Escherichia coli, the EfeUOB system has been shown to recognize heme and to allow extracytoplasmic heme iron extraction via a deferrochelation reaction. Given the high level of sequence conservations between EfeUOB orthologs, we hypothesized that heme might be the physiological iron substrate for the other orthologous systems. To test this hypothesis, we undertook characterization of the Staphylococcus aureus FepABC system. Results presented here indicate: i) that the S. aureus FepB protein binds both heme and PPIX with high affinity, like EfeB, the E. coli ortholog; ii) that it has low peroxidase activity, comparable to that of EfeB; iii) that both FepA and FepB drive heme iron utilization, and both are required for this activity and iv) that the E. coli FepA ortholog (EfeO) cannot replace FepA in FepB-driven iron release from heme indicating protein specificity in these activities. Our results show that the function in heme iron extraction is conserved in the two orthologous systems.

Regulatory role of UvrY in adaptation of Photorhabdus luminescens growth inside the insect.

We report global expression profiling of a uvrY-deficient mutant of Photorhabdus luminescens. We found that the regulator moiety of the two-component regulatory system BarA/UvrY regulated more than 500 target genes coding for functions involved in the synthesis of major compartments and metabolic pathways of the cell. This regulation appeared to be in part indirect as UvrY affected the expression of several regulators. Indeed, the flagellum biosynthesis transcription activator FlhC and the flagella regulon were induced in the absence of UvrY, leading to a hyperflagellated phenotype and an increase in motility and biofilm formation. Two major regulatory systems were also altered: the type 2 quorum-sensing inducer AI-2 was activated by UvrY, and the CsrA regulator function appeared to be repressed by the increase of the small-untranslated RNA csrB, the CsrA activity inhibitor TldD and the chaperonin GroESL. Both through and independently of these systems, UvrY regulated oxidative stress resistance; bioluminescence; iron, sugar and peptide transport; proteases; polyketide synthesis enzymes and nucleobases recycling, related to insect degradation and assimilation by bacteria. As a consequence, the uvrY-deficient strain exhibited a decreased killing of insect cells and a reduced growth on insect cells culture, suggesting a UvrY role in the adaptation of P. luminescens inside the insect.

Protoporphyrin (PPIX) efflux by the MacAB-TolC pump in Escherichia coli

In most organisms, heme biosynthesis is strictly controlled so as to avoid heme and heme precursor accumulation, which is toxic. Escherichia coli regulates heme biosynthesis by a feedback loop involving heme‐induced proteolytic cleavage of HemA, glutamyl‐tRNA reductase, which is the first enzyme in the heme biosynthetic pathway. We show here that heme homeostasis can be disrupted by overproduction of YfeX, a cytoplasmic protein that captures iron from heme that we named deferrochelatase. We also show that it is disrupted by iron chelation, which reduces the intracellular iron concentration necessary for loading iron into protoporphyrin IX (PPIX, the immediate heme precursor). In both cases, we established that there is an increased PPIX concentration and we demonstrate that this compound is expelled by the MacAB‐TolC pump, an efflux pump involved in E. coli and Salmonella for macrolide efflux. The E. coli macAB and tolC mutants accumulate PPIX and are sensitive to photo‐inactivation. The MacAB‐TolC pump is required for Salmonella typhimurium survival in macrophages. We propose that PPIX is an endogenous substrate of the MacAB‐TolC pump in E. coli and S. typhimurium and that this compound is produced inside bacteria when natural heme homeostasis is disrupted by iron shortage, as happens when bacteria invade the mammalian host.

Mutants of Escherichia coli producing pyrroloquinoline quinone.

In glucose minimal medium a PTS- strain of Escherichia coli [delta (ptsH ptsI crr)] could grow slowly (doubling time, d = 10 h). When the population reached 5 x 10(6) to 2 x 10(7) cells ml-1, mutants growing rapidly (d = 1.5 h) appeared and rapidly outgrew the initial population. These mutants (EF mutants) do not use a constitutive galactose permease for glucose translocation. They synthesize sufficient pyrroloquinoline quinone (PQQ) to yield a specific activity of glucose dehydrogenase (GDH) equivalent to that found in the parent strain grown in glucose minimal medium supplemented with 1 nM-PQQ. Membrane preparations containing an active GDH oxidized glucose to gluconic acid, which was also present in the culture supernatant of EF strains in glucose minimal medium. Glucose utilization is the only phenotypic trait distinguishing EF mutants from the parent strain. Glucose utilization by EF mutants was strictly aerobic as expected from a PQQ-dependent catabolism. The regulation of PQQ production by E. coli is discussed.