Catherine Dauga

Evolution of the gyrB gene and the molecular phylogeny of Enterobacteriaceae: a model molecule for molecular systematic studies

Phylogenetic trees showing the evolutionary relatedness of Enterobacteriaceae based upon gyrB and 16S rRNA genes were compared. Congruence among trees of these molecules indicates that the genomes of these species are not completely mosaic and that molecular systematic studies can be carried out. Phylogenetic trees based on gyrB sequences appeared to be more reliable at determining relationships among Serratia species than trees based on 16S rRNA gene sequences. gyrB sequences from Serratia species formed a monophyletic group validated by significant bootstrap values. Serratia fonticola had the most deeply branching gyrB sequence in the Serratia monophyletic group, which was consistent with its atypical phenotypic characteristics. Klebsiella and Enterobacter genera seemed to be polyphyletic, but the branching patterns of gyrB and 16S rRNA gene trees were not congruent. Enterobacter aerogenes was grouped with Klebsiella pneumoniae on the gyrB phylogenetic tree, which supports that this species could be transferred to the Klebsiella genus. Unfortunately, 16S rRNA and gyrB phylogenetic trees gave conflicting evolutionary relationships for Citrobacter freundii because of its unusual gyrB evolutionary process. gyrB lateral gene transfer was suspected for Hafnia alvei. Saturation of gyrB genes was observed by the pairwise comparison of Proteus spp., Providencia alcalifaciens and Morganella morganii sequences. Depending on their level of variability, 16S rRNA gene sequences were useful for describing phylogenetic relationships between distantly related Enterobacteriaceae, whereas gyrB sequence comparison was useful for inferring intra- and some intergeneric relationships.

Phylogeography of Aedes (Stegomyia) aegypti (L.) and Aedes (Stegomyia) albopictus (Skuse) (Diptera: Culicidae) based on mitochondrial DNA variations.

Aedes (Stegomyia) aegypti (l.) and Aedes (Stegomyia) albopictus (Skuse) are the most important vectors of the dengue and yellow-fever viruses. Both took advantage of trade developments to spread throughout the tropics from their native area: A. aegypti originated from Africa and a. albopictus from South-East Asia. We investigated the relationships between A. aegypti and A. albopictus mosquitoes based on three mitochondrial-DNA genes (cytochrome b, cytochrome oxidase I and NADH dehydrogenase subunit 5). Little genetic variation was observed for a. albopictus, probably owing to the recent spreading of the species via human activities. For A. aegypti, most populations from South America were found to be genetically similar to populations from South-East Asia (Thailand and Vietnam), except for one sample from Boa Vista (northern Amazonia), which was more closely related to samples from Africa (Guinea and Ivory Coast). This suggests that African populations of A. aegypti introduced during the slave trade have persisted in Boa Vista, resisting eradication campaigns.

Novel Major Bacterial Candidate Division within a Municipal Anaerobic Sludge Digester

ABSTRACT In a previous study, we analyzed the molecular diversity of Planctomycetales by PCR amplification and sequencing of 16S rRNA clone libraries generated from a municipal wastewater plant, using planctomycete-specific and universal primer sets (R. Chouari, D. Le Paslier, P. Daegelen, P. Ginestet, J. Weissenbach, and A. Sghir, Appl. Environ. Microbiol. 69:7354-7363, 2003). Only a small fraction (4%) of the 16S rRNA gene sequences of the digester clone library corresponded to the Planctomycetales division. Importantly, 85.9% of the digester clone sequences are grouped into two different clusters named WWE1 (81.4% of the sequences) and WWE2 (4.5%) and are distantly affiliated with unidentified bacterial sequences retrieved from a methanogenic reactor community and from a termite gut, respectively. In phylogenetic analysis using 16S rRNA gene sequence representatives of the main phylogenetic bacterial divisions, the two clusters are monophyletic, branch apart from each other, and are distantly related to Planctomycetales and other bacterial divisions. A novel candidate division is proposed for WWE1, while the WWE2 cluster strongly affiliates with the recently proposed Lentisphearae phylum. We designed and validated a 16S rRNA probe targeting WWE1 16S rRNA sequences by both fluorescent in situ hybridization (FISH) and dot blot hybridization (DBH). Results of FISH analysis show that WWE1 representative microorganisms are rods or filamentous shaped, while DBH shows that WWE1 accounts for 12% of the total bacterial rRNA within the anaerobic digester. The remaining 16S rRNA gene sequences are affiliated with Verrucomicrobia or recently described candidate divisions with no known pure culture representatives, such as OD1, BRC1, or NBL-UPA2, making up less than 3.5% of the clone library, respectively. This inventory expands the known diversity of the latter bacterial division-level lineages.

Strategy for the detection of Helicobacter species by amplification of 16S rRNA genes and identification of H. felis in a human gastric biopsy

The aim of the present work was to develop polymerase chain reactions (PCRs) based on the conserved nucleotide sequence of the 16S rRNA gene for detection of bacteria of the Helicobacter genus in human antral biopsy samples. The assay for Helicobacter spp was developed by amplifying a 399-bp 16S rRNA gene sequence specific to the genus Helicobacter. The identity of the amplicon was confirmed by hybridization with an internal probe and by restriction by endonuclease VspI showing two expected fragments of 295 and 104 base pairs. A total of 65 dyspeptic patients from France and New Caledonia were screened for Helicobacter spp infection through the use of the following diagnostic assays on biopsy specimens collected through endoscopy: direct detection of bacteria in histological sections by Giemsa and Warthin Starry staining, urease test and bacterial isolation, PCR for Helicobacter pylori ureC/glmM gene, and PCR targeted to 16S rRNA genes. The 16S rRNA gene PCR assay was able to detect down to 680 bacterial cells, as assessed by agarose gel electrophoresis, and down to 4 bacterial cells by hybridization of amplicon with the internal probe. The 16S rRNA PCR test was 100% specific and sensitive; results obtained with this test were in agreement with the visualization of bacteria by histology. Urease test and culture were 86.4% and 22.7% sensitive, and 96.5 and 100% specific, respectively. The H. pylori ureC/glmM gene-based PCR was 100% specific and only 95.4% sensitive, since one biopsy from a Melanesian patient contained a Helicobacter strain other than H. pylori. For this Melanesian patient, a branch-specific PCR targeting the epsilon branch of Proteobacteria was used to amplify a 967-bp amplicon. This amplicon was sequenced and matched with the H. felis sequence. This was confirmed using an H. felis-specific urease PCR test.

A Crypt-Specific Core Microbiota Resides in the Mouse Colon

ABSTRACT In an attempt to explore the microbial content of functionally critical niches of the mouse gastrointestinal tract, we targeted molecular microbial diagnostics of the crypts that contain the intestinal stem cells, which account for epithelial regeneration. As current evidence indicates, the gut microbiota affects epithelial regeneration; bacteria that are likely to primarily participate in this essential step of the gut, microbiota cross talk, have been identified. We show in this article that only the cecal and colonic crypts harbor resident microbiota in the mouse and that regardless of the line and breeding origin of these mice, this bacterial population is unexpectedly dominated by aerobic genera. Interestingly, this microbiota resembles the restricted microbiota found in the midgut of invertebrates; thus, the presence of our so-called “crypt-specific core microbiota” (CSCM) in the mouse colon potentially reflects a coevolutionary process under selective conditions that can now be addressed. We suggest that CSCM could play both a protective and a homeostatic role within the colon. This article is setting the bases for such studies, particularly by providing a bona fide—and essentially cultivable—crypt microbiota of reference. IMPORTANCE Metagenomic typing of the whole-gut luminal microbiome was recently provided, revealing great opportunities for physiological and physiopathological analysis of the host-microbiota interface. On this basis, it appears increasingly important to analyze which niches of the gut exposed to a particular microbiota are of major functional importance, specifically focusing on the crypt, which accounts for permanent epithelial renewal, and to analyze how this microbiota compares to its luminal counterpart in composition and quantity. Crypt-specific core microbiotas may show themselves as important elements regarding crypt protection and homeostasis of its functions. Metagenomic typing of the whole-gut luminal microbiome was recently provided, revealing great opportunities for physiological and physiopathological analysis of the host-microbiota interface. On this basis, it appears increasingly important to analyze which niches of the gut exposed to a particular microbiota are of major functional importance, specifically focusing on the crypt, which accounts for permanent epithelial renewal, and to analyze how this microbiota compares to its luminal counterpart in composition and quantity. Crypt-specific core microbiotas may show themselves as important elements regarding crypt protection and homeostasis of its functions.

Multi-genome identification and characterization of chlamydiae-specific type III secretion substrates: the Inc proteins

BackgroundChlamydiae are obligate intracellular bacteria that multiply in a vacuolar compartment, the inclusion. Several chlamydial proteins containing a bilobal hydrophobic domain are translocated by a type III secretion (TTS) mechanism into the inclusion membrane. They form the family of Inc proteins, which is specific to this phylum. Based on their localization, Inc proteins likely play important roles in the interactions between the microbe and the host. In this paper we sought to identify and analyze, using bioinformatics tools, all putative Inc proteins in published chlamydial genomes, including an environmental species.ResultsInc proteins contain at least one bilobal hydrophobic domain made of two transmembrane helices separated by a loop of less than 30 amino acids. Using bioinformatics tools we identified 537 putative Inc proteins across seven chlamydial proteomes. The amino-terminal segment of the putative Inc proteins was recognized as a functional TTS signal in 90% of the C. trachomatis and C. pneumoniae sequences tested, validating the data obtained in silico. We identified a macro domain in several putative Inc proteins, and observed that Inc proteins are enriched in segments predicted to form coiled coils. A surprisingly large proportion of the putative Inc proteins are not constitutively translocated to the inclusion membrane in culture conditions.ConclusionsThe Inc proteins represent 7 to 10% of each proteome and show a great degree of sequence diversity between species. The abundance of segments with a high probability for coiled coil conformation in Inc proteins support the hypothesis that they interact with host proteins. While the large majority of Inc proteins possess a functional TTS signal, less than half may be constitutively translocated to the inclusion surface in some species. This suggests the novel finding that translocation of Inc proteins may be regulated by as-yet undetermined mechanisms.

Genetic and transmission analysis of Helicobacter pylori strains within a family.

Point mutations, intragenic recombination, and introduction of foreign alleles enhanced strain diversity within the family.

Presence of Active Aliphatic Amidases in Helicobacter Species Able To Colonize the Stomach

ABSTRACT Ammonia production is of great importance for the gastric pathogen Helicobacter pylori as a nitrogen source, as a compound protecting against gastric acidity, and as a cytotoxic molecule. In addition to urease, H. pylori possesses two aliphatic amidases responsible for ammonia production: AmiE, a classical amidase, and AmiF, a new type of formamidase. Both enzymes are part of a regulatory network consisting of nitrogen metabolism enzymes, including urease and arginase. We examined the role of the H. pylori amidases in vivo by testing the gastric colonization of mice with H. pylori SS1 strains carrying mutations in amiE and/or amiF and in coinfection experiments with wild-type and double mutant strains. A new cassette conferring resistance to gentamicin was used in addition to the kanamycin cassette to construct the double mutation in strain SS1. Our data indicate that the amidases are not essential for colonization of mice. The search for amiE and amiF genes in 53 H. pylori strains from different geographic origins indicated the presence of both genes in all these genomes. We tested for the presence of the amiE and amiF genes and for amidase and formamidase activities in eleven Helicobacter species. Among the gastric species, H. acinonychis possessed both amiE and amiF, H. felis carried only amiF, and H. mustelae was devoid of amidases. H. muridarum, which can colonize both mouse intestine and stomach, was the only enterohepatic species to contain amiE. Phylogenetic trees based upon the sequences of H. pylori amiE and amiF genes and their respective homologs from other organisms as well as the amidase gene distribution among Helicobacter species are strongly suggestive of amidase acquisition by horizontal gene transfer. Since amidases are found only in Helicobacter species able to colonize the stomach, their acquisition might be related to selective pressure in this particular gastric environment.

Halogenase Genes in Nonribosomal Peptide Synthetase Gene Clusters of Microcystis (Cyanobacteria): Sporadic Distribution and Evolution

Cyanobacteria of the genus Microcystis are known to produce secondary metabolites of large structural diversity by nonribosomal peptide synthetase (NRPS) pathways. For a number of such compounds, halogenated congeners have been reported along with nonhalogenated ones. In the present study, chlorinated cyanopeptolin- and/or aeruginosin-type peptides were detected by mass spectrometry in 17 out of 28 axenic strains of Microcystis. In these strains, a halogenase gene was identified between 2 genes coding for NRPS modules in respective gene clusters, whereas it was consistently absent when the strains produced only nonchlorinated corresponding congeners. Nucleotide sequences were obtained for 12 complete halogenase genes and 14 intermodule regions of gene clusters lacking a halogenase gene or containing only fragments of it. When a halogenase gene was found absent, a specific, identical excision pattern was observed for both synthetase gene clusters in most strains. A phylogenetic analysis including other bacterial halogenases showed that the NRPS-related halogenases of Microcystis form a monophyletic group divided into 2 subgroups, corresponding to either the cyanopeptolin or the aeruginosin peptide synthetases. The distribution of these peptide synthetase gene clusters, among the tested Microcystis strains, was found in relative agreement with their phylogeny reconstructed from 16S–23S rDNA intergenic spacer sequences, whereas the distribution of the associated halogenase genes appears to be sporadic. The presented data suggest that in cyanobacteria these prevalent halogenase genes originated from an ancient horizontal gene transfer followed by duplication in the cyanobacterial lineage. We propose an evolutionary scenario implying repeated gene losses to explain the distribution of halogenase genes in 2 NRPS gene clusters that subsequently defines the seemingly erratic production of halogenated and nonhalogenated aeruginosins and cyanopeptolins among Microcystis strains.

Molecular evidence of host influences on the evolution and spread of human tapeworms

The taeniasis/cysticercosis complex is included in the list of neglected zoonotic diseases by the World Health Organization due to its significant impact on public health in tropical areas. Cysticercosis is still endemic in many regions of Asia, Africa and Latin America. Long absent in Europe and in other developed countries, cysticercosis has recently re‐emerged in the United States and Canada, due to immigration, travel and local transmission. This has encouraged the use of molecular data to understand better the influence of animal and human hosts on the emergence and spread of Taenia species.