Sylviane Derzelle

The genome sequence of the entomopathogenic bacterium Photorhabdus luminescens.

Photorhabdus luminescens is a symbiont of nematodes and a broad-spectrum insect pathogen. The complete genome sequence of strain TT01 is 5,688,987 base pairs (bp) long and contains 4,839 predicted protein-coding genes. Strikingly, it encodes a large number of adhesins, toxins, hemolysins, proteases and lipases, and contains a wide array of antibiotic synthesizing genes. These proteins are likely to play a role in the elimination of competitors, host colonization, invasion and bioconversion of the insect cadaver, making P. luminescens a promising model for the study of symbiosis and host-pathogen interactions. Comparison with the genomes of related bacteria reveals the acquisition of virulence factors by extensive horizontal transfer and provides clues about the evolution of an insect pathogen. Moreover, newly identified insecticidal proteins may be effective alternatives for the control of insect pests.

Identification, Characterization, and Regulation of a Cluster of Genes Involved in Carbapenem Biosynthesis in Photorhabdus luminescens

ABSTRACT The luminescent entomopathogenic bacterium Photorhabdus luminescens produces several yet-uncharacterized broad-spectrum antibiotics. We report the identification and characterization of a cluster of eight genes (named cpmA to cpmH) responsible for the production of a carbapenem-like antibiotic in strain TT01 of P. luminescens. The cpm cluster differs in several crucial aspects from other car operons. The level of cpm mRNA peaks during exponential phase and is regulated by a Rap/Hor homolog identified in the P. luminescens genome. Marker-exchange mutagenesis of this gene in the entomopathogen decreased antibiotic production. The luxS-like signaling mechanism of quorum sensing also plays a role in the regulation of the cpm operon. Indeed, luxS, which is involved in the production of a newly identified autoinducer, is responsible for repression of cpm gene expression at the end of the exponential growth phase. The importance of this carbapenem production in the ecology of P. luminescens is discussed.

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.

Tool for quantification of staphylococcal enterotoxin gene expression in cheese.

ABSTRACT Cheese is a complex and dynamic microbial ecosystem characterized by the presence of a large variety of bacteria, yeasts, and molds. Some microorganisms, including species of lactobacilli or lactococci, are known to contribute to the organoleptic quality of cheeses, whereas the presence of other microorganisms may lead to spoilage or constitute a health risk. Staphylococcus aureus is recognized worldwide as an important food-borne pathogen, owing to the production of enterotoxins in food matrices. In order to study enterotoxin gene expression during cheese manufacture, we developed an efficient procedure to recover total RNA from cheese and applied a robust strategy to study gene expression by reverse transcription-quantitative PCR (RT-qPCR). This method yielded pure preparations of undegraded RNA suitable for RT-qPCR. To normalize RT-qPCR data, expression of 10 potential reference genes was investigated during S. aureus growth in milk and in cheese. The three most stably expressed reference genes during cheese manufacture were ftsZ, pta, and gyrB, and these were used as internal controls for RT-qPCR of the genes sea and sed, encoding staphylococcal enterotoxins A and D, respectively. Expression of these staphylococcal enterotoxin genes was monitored during the first 72 h of the cheese-making process, and mRNA data were correlated with enterotoxin production.

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.

Proteome analysis of Streptococcus thermophilus grown in milk reveals pyruvate formate-lyase as the major upregulated protein.

ABSTRACT We investigated the adaptation to milk of Streptococcus thermophilus LMG18311 using a proteomic approach. Two-dimensional electrophoresis of cytosolic proteins were performed after growth in M17 medium or in milk. A major modification of the proteome concerned proteins involved in the supply of amino acids, like the peptidase PepX, and several enzymes involved in amino acid biosynthesis. In parallel, we observed the upregulation of the synthesis of seven enzymes directly involved in the synthesis of purines, as well as formyl-tetrahydrofolate (THF) synthetase and serine hydroxy-methyl transferase, two enzymes responsible for the synthesis of compounds (THF and glycine, respectively) feeding the purine biosynthetic pathway. The analysis also revealed a massive increase in the synthesis of pyruvate formate-lyase (PFL), the enzyme which converts pyruvate into acetyl coenzyme A and formate. PFL has been essentially studied for its role in mixed-acid product formation in lactic acid bacteria during anaerobic fermentation. However, formate is an important methyl group donor for anabolic pathway through the formation of folate derivates. We hypothesized that PFL was involved in purine biosynthesis during growth in milk. We showed that PFL expression was regulated at the transcriptional level and that pfl transcription occurred during the exponential growth phase in milk. The complementation of milk with formate or purine bases was shown to reduce pfl expression, to suppress PFL synthesis, and to stimulate growth of S. thermophilus. These results show a novel regulatory mechanism controlling the synthesis of PFL and suggest an unrecognized physiological role for PFL as a formate supplier for anabolic purposes.

H-NS and H-NS-like proteins in Gram-negative bacteria andtheir multiple role in the regulation of bacterial metabolism

In Escherichia coli, the H-NS protein plays an important role in the structure and the functioning of bacterial chromosome. A homologous protein has also been identified in several enteric bacteria and in closely related organisms such as Haemophilus influenzae. To get information on their structure and their function, we identified H-NS-like proteins in various microorganisms by different procedures. In silico analysis of their amino acid sequence and/or in vivo experiments provide evidence that more than 20 proteins belong to the same class of regulatory proteins. Moreover, large scale technologies demonstrate that, at least in E. coli, the loss of motility in hns mutants results from a lack of flagellin biosynthesis, due to the in vivo repression of flagellar gene expression. In contrast, several genes involved in adaptation to low pH are strongly induced in a H-NS deficient strain, resulting in an increased resistance to acidic stress. Finally, expression profiling and phenotypic analysis suggest that, unlike H-NS, its paralogous protein StpA does not play any role in these processes.

Improved Adaptation to Cold-Shock, Stationary-Phase, and Freezing Stresses in Lactobacillus plantarum Overproducing Cold-Shock Proteins

ABSTRACT We have investigated the effect of overproducing each of the three cold shock proteins (CspL, CspP, and CspC) in the mesophilic lactic acid bacterium Lactobacillus plantarum NC8. CspL overproduction transiently alleviated the reduction in growth rate triggered by exposing exponentially growing cells to cold shock (8°C), suggesting that CspL is involved in cold adaptation. The strain overproducing CspC resumed growth more rapidly when stationary-phase cultures were diluted into fresh medium, indicating a role in the adaptation and recovery of nutritionally deprived cells. Overproduction of CspP led to an enhanced capacity to survive freezing.

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.

Cold Shock Induction of the cspL Gene in Lactobacillus plantarum Involves Transcriptional Regulation

Fragments of the cspL promoter region were fused to the gusA reporter and reintroduced into Lactobacillus plantarum cells, either on multicopy plasmids or through single-copy chromosomal integration. beta-Glucuronidase activity and primer extension data demonstrate that the cspL promoter is induced in response to cold shock and that multicopy constructs quench the induction of the resident cspL gene.