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In vivo expression of the mannose-resistant fimbriae of Photorhabdus temperata K122 during insect infection.

ABSTRACT: Photorhabdus temperata K122 is an entomopathogenic bacterium symbiotically associated with nematodes of the family Heterorhabditidae: Surface fimbriae are important for the colonization of many pathogenic bacteria, and here we report the nucleotide sequence and analysis of the expression of a 12-kbp fragment encoding the mannose-resistant fimbriae of P. temperata (mrf). The mrf gene cluster contains 11 genes with an organization similar to that of the mrp locus from Proteus mirabilis. mrfI (encoding a putative recombinase) and mrfA (encoding pilin), the first gene in an apparent operon of nine other genes, are expressed from divergent promoters. The mrfI-mrfA intergenic region contains inverted repeats flanking the mrfA promoter. This region was shown to be capable of inversion, consistent with an ON/OFF regulation of the operon. In in vitro liquid cultures, both orientations were detected. Nevertheless, when we analyzed the expression of all of the genes in the mrf locus by semiquantitative reverse transcription-PCR during infection of Galleria mellonella (greater wax moth) larvae, expression of mrfA was not detected until 25 h postinfection, preceding the death of the larvae at 32 h. In contrast, mrfJ (a putative inhibitor of flagellar synthesis) was expressed throughout infection. Expression of mrfI was also detected only late in infection (25 to 30 h), indicating a possible increase in inversion frequency at this stage. In both in vitro liquid cultures and in vivo larval infections, the distal genes of the operon were expressed at substantially lower levels than mrfA. These results indicate the complex regulation of the mrf cluster during infection.

SUBMITTER: Meslet-Cladiere LM

PROVIDER: S-EPMC321496 | biostudies-literature | 2004 Feb

REPOSITORIES: biostudies-literature

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In vivo expression of the mannose-resistant fimbriae of Photorhabdus temperata K122 during insect infection.

Meslet-Cladiere L M LM Pimenta A A Duchaud E E Holland I B IB Blight M A MA

Journal of bacteriology 20040201 3

Photorhabdus temperata K122 is an entomopathogenic bacterium symbiotically associated with nematodes of the family Heterorhabditidae: Surface fimbriae are important for the colonization of many pathogenic bacteria, and here we report the nucleotide sequence and analysis of the expression of a 12-kbp fragment encoding the mannose-resistant fimbriae of P. temperata (mrf). The mrf gene cluster contains 11 genes with an organization similar to that of the mrp locus from Proteus mirabilis. mrfI (enco ...[more]

PMID: 14729685

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Project description:BackgroundPhotorhabdus and Xenorhabdus are Gram-negative, phylogenetically related, enterobacteria, forming mutualism with the entomopathogenic nematodes Heterorhabditis and Steinernema, respectively. The mutualistic bacteria living in the intestines of the nematode infective juveniles are pathogenic to the insect upon release by the nematodes into the insect hemolymph. Such a switch needs activation of genes that promote bacterial virulence. We studied in vivo gene expression in Photorhabdus temperata and Xenorhabdus koppenhoeferi upon infection of the white grub Rhizotrogus majalis using selective capture of transcribed sequences technique.ResultsA total of 40 genes in P. temperata and 39 in X. koppenhoeferi were found to be upregulated in R. majalis hemolymph at 24 h post infection. Genomic presence or upregulation of these genes specific in either one of the bacterium was confirmed by the assay of comparative hybridization, and the changes of randomly selected genes were further validated by quantitative real-time PCR. The identified genes could be broadly divided into seven functional groups including cell surface structure, regulation, virulence and secretion, stress response, intracellular metabolism, nutrient scavenging, and unknown. The two bacteria shared more genes in stress response category than any other functional group. More than 60% of the identified genes were uniquely induced in either bacterium suggesting vastly different molecular mechanisms of pathogenicity to the same insect host. In P. temperata lysR gene encoding transcriptional activator was induced, while genes yijC and rseA encoding transcriptional repressors were induced in X. koppenhoeferi. Lipopolysaccharide synthesis gene lpsE was induced in X. koppenhoeferi but not in P. temperata. Except tcaC and hemolysin related genes, other virulence genes were different between the two bacteria. Genes involved in TCA cycle were induced in P. temperata whereas those involved in glyoxylate pathway were induced in X. koppenhoeferi, suggesting differences in metabolism between the two bacteria in the same insect host. Upregulation of genes encoding different types of nutrient uptake systems further emphasized the differences in nutritional requirements of the two bacteria in the same insect host. Photorhabdus temperata displayed upregulation of genes encoding siderophore-dependent iron uptake system, but X. koppenhoeferi upregulated genes encoding siderophore-independent ion uptake system. Photorhabdus temperata induced genes for amino acid acquisition but X. koppenhoeferi upregulated malF gene, encoding a maltose uptake system. Further analyses identified possible mechanistic associations between the identified gene products in metabolic pathways, providing an interactive model of pathogenesis for each bacterium species.ConclusionThis study identifies set of genes induced in P. temperata and X. koppenhoeferi upon infection of R. majalis, and highlights differences in molecular features used by these two closely related bacteria to promote their pathogenicity in the same insect host.

Project description:An entomopathogenic bacterium, Photorhabdus temperata subsp. temperata, is mutualistic to its host nematode, Heterorhabditis megidis. The infective juvenile nematodes enter target insects through natural openings and release the symbiotic bacteria into the insect hemocoel. The released bacteria suppress the insect immune responses and cause septicemia through their secondary metabolites. GameXPeptide (GXP) is one of the common secondary metabolites of most Photorhabdus species and is produced by the catalytic activity of a specific non-ribosomal peptide synthetase called GxpS encoded by the gxpS gene. This study confirmed gxpS to be encoded in the P. temperata temperata genome and analyzed its expression during bacterial growth. LC-MS/MS analysis of the bacterial culture broth contained at least four different GXPs (GXP-A to GXP-D), in which GXP-A was the most abundant. To investigate GXP synthesis following gxpS expression, the gxpS promoter of P. temperata temperata was replaced with an inducible arabinose promoter by homologous recombination. The gxpS transcript levels in the mutant were altered by the addition of l-arabinose. Without the inducer, the gxpS transcript level was significantly lower compared to the wild type and produced significantly lower amounts of the four GXPs. The addition of the inducer to the mutant significantly increased gxpS expression and produced significantly higher levels of the four GXPs compared to the wild type. The metabolite extracts obtained from wild-type and mutant bacteria showed differential immunosuppressive activities according to their GXP contents against the cellular and humoral immune responses of a lepidopteran insect, Spodoptera exigua. Interestingly, the gxpS-mutant bacteria showed less insecticidal activity compared to the wild type, whereas the addition of GXP to the mutant significantly restored insecticidal activity. These results suggest that the gxpS gene encoded in P. temperata temperata is responsible for the production of at least four different GXPs, which play crucial roles in bacterial virulence.

Dataset Information

In vivo expression of the mannose-resistant fimbriae of Photorhabdus temperata K122 during insect infection.

Publications

In vivo expression of the mannose-resistant fimbriae of Photorhabdus temperata K122 during insect infection.

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