No description available.

No description available.

No description available.

No description available.

No description available.

It is generally accepted that bacteria belonging to the Atopobium cluster are predominant members of the human gastrointestinal (GI) microbiota with Collinsella and Eggerthella species isolated from human faeces. However, a relative paucity of information is available regarding the composition of this population of the human GI microbiota and its role in host health. The overall aim of this study was to examine the diversity and dynamics of the Atopobium cluster in the faecal microbiota of healthy human adults and to further characterize members of this population, including differentiation of Eggerthella lenta and related isolates from human clinical and faecal samples.

Yersinia enterocolitica is a widespread human gastrointestinal pathogen which causes stomach cramps, nausea, vomiting and diarrhea It possesses an N-acylhomoserine lactone (AHL)-dependent quorum sensing system (QS) composed of a LuxRI homologue pair, YenR and YenI and a second ' orphan' LuxR homologue, YeaR. Its virulence weaponry, which includes the adhesin YadA, a type three secretion system (T3SS) and the Yersinia outer protein (Yap) effectors, is carried by the 70kb plasmid pYVe. When subcultured at 37°C a population of Y enterocolitica will, over time, lose the virulence plasmid and this was more pronounced in the QS mutants. In a recent microarray study the QS regulan of Y. enterocolitica whas shown to regu late a number of virulence, plasmid replication and partition genes. This study aimed to investigate the molecular mechanisms which underlie these observations in order to understand in more detail the relationship between QS. virulence plasmid stability and pathogenesis of Y. enterocoiitica 808 1. To successfully study the QS-associated plasmid loss phenotype it was first necessary to label pYVe to allow easy selection and visualization of plasmid positive ceUs. p YVe was therefore labelled with a combined gentamicin resistance and gfp cassette (GG I). Plasmid loss studies were then conducted on the Y. enleroco/ilica GG I parent and QS mutants and revealed that in the absence of gentamicin the GG 1 insertion actually increased plasmid stabi lity. Further analys is of the region into which GG I had inserted indicated that no apparent open reading frame was disrupted. Because DNA topology plays an important role in virulence plasmid gene regulation in Y enrerocolitica an analysis of the DNA secondary structure around the insertion point of GG I was conducted and revealed that upon insertion the GG 1 cassette had changed the DNA secondary structure of the region from curved to more linear. lux-based transcriptional fusion to the promoter of repA and spyA. two genes which playa central role in plasmid replication and partition, suggested that QS does not regulate their transcription and might rather act at the post-transcriptional level. To further characterize the role of QS on Y. enterocolitica pathogenic lifestyle phenotypical analysis were conducted. Swarming motility of Y. enteroco/itica is known to be QS controlled and this study extended these observations in demonstrating that YenR is directly involved in swarming control, as it activates motility in the absence of AHLs (low cell density) and represses motility the presence of AHLs (high cell density). Adhesion and invasion to Caco-2 cells and Yop production were tested in Y.

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No description available.

Methanotrophic bacteria convert methane to methanol using a methane monooxygenase enzyme (MMO). Two types of MMO exist: a membrane bound enzyme (PMMO) and a cytoplasmic enzyme (sMMO). A system for the site-directed mutagenesis of residues in the active site of sMMO has recently been developed that uses a sMMO-minus strain of Methylosinus trichosporium OB3b as the expression host (Smith et al., 2002. Appl. Environ. Microbiol. 68:5265-5273); this strain, designated Mutant F, was created by disrupting the mmoX gene by marker-exchange mutagenesis. In this study a Ms. trichosporium OB3b strain was created in which all the sMMO structural genes were deleted or disrupted. This mutant was designated Ms. trichosporium SMDM. The recombinant expression of sMMO was performed in Ms. trichosporium SMDM using the same sMMO expression plasmid used for Ms. trichosporium Mutant F. The effect of sMMO expression in the absence of the enigmatic mmoD gene was investigated. Preliminary results indicate that mmoD is required for active expression of sMMO. The sMMO genes from Methylocella silvestris BL2T were sequenced and conjugated into Ms. trichosporium SMDM on a broad host range plasmid. No expression of Methylocella silvestris BL2 T sMMO was detected in Ms. trichosporium SMDM. A new system for the mutagenesis of the Ms. trichosporium OB3b sMMO a-subunit was created. Chimaeric sMMO mutants were created by introducing gene sequence from the alkene monooxygenase enzyme of Rhodococcus corallin us into the mmoX. The chimaeric sMMO enzymes appeared to be unstable in Ms. trichosporium Mutant F. An attempt was made to improve the stability of sMMO mutants in Ms. trichosporium Mutant F by disrupting the gene encoding the Lon protease. The Ms. trichosporium OB3b Ion gene was cloned and sequenced and attempts were made to disrupt the Ms. trichosporium OB3b Ion by marker-exchange mutagenesis. A mutant was not obtained, suggesting that Lon may be essential for vegetative growth of Ms. trichosporium OB3b. The diversity of sMMO in several environmental samples was investigated using PCR. The objective was to isolate novel mmoX sequences from uncultivated methanotrophs that could be used to design sMMO mutagenesis experiments. New PCR primers targeting the mmoX were developed. The primers were used to generate libraries from a blanket bog peat (UK) and from cave water (Romania). A group of sequences that did not cluster with the mmoX of any cultivated methanotroph was obtained from the cave water. The use of a PCR independent approach to clone methanotroph genes from environmental samEles was also investigated. This was performed by developing a method to clone 1 C-DNA from a stable isotope probing experiment with 13CH4 into a BAC vector. A library of 2300 clones was generated. Greater than 95 % of plasmids analysed contained inserts, which ranged in size from approximately 10 - 30 kb. The library was screened for mxaF, mmoX and pmoA by colony hybridization. A clone (15 kb) containing pmoA was completely sequenced. Other genes encoding proteins with (potential) roles in methylotrophy were contained on the clone, includingpmoC, pmoB, folP, folK, mptG and moxF.