Many enteric pathogens are equipped with multiple cell adhesion factors which are important for host tissue colonization and virulence. Y. enterocolitica, a common food-borne pathogen with invasive properties, uses the surface proteins invasin and YadA for host cell binding and entry. In this study, we demonstrate unique cell adhesion and invasion properties of Y. enterocolitica serotype O:3 strains, the most frequent cause of human yersiniosis, and show that these differences are mainly attributable to variations affecting the function and expression of invasin in response to temperature. In contrast to other enteric Yersinia strains, invasin production in O:3 strains is constitutive and largely enhanced compared to other Y. enterocolitica serotypes, in which invA expression is temperature-regulated and significantly reduced at 37°C. Increase of invasin levels is caused by (i) an IS1667 insertion into the invA promoter region, which includes an additional promoter and RovA and H-NS binding sites, and (ii) a P98S substitution in the invA activator protein RovA rendering the regulator less susceptible to proteolysis. Both variations were shown to influence bacterial colonization in a murine infection model. Furthermore, we found that co-expression of YadA and down-regulation of the O-antigen at 37°C is required to allow efficient internalization by the InvA protein. We conclude that even small variations in the expression of virulence factors can provoke a major difference in the virulence properties of closely related pathogens which may confer better survival or a higher pathogenic potential in a certain host or host environment.

Protochlorophyllide (Pchlide)2 a is a metabolite during chlorophyll and bacteriochlorophyll biosynthesis. Two distinct strategies have evolved for the stereo-and regioselective two electron reduction of ring D of Pchlide a to form chlorophyllide (Chlide) a (Fig.

Nitrogenase-like light-independent protochlorophyllide oxidoreductase (DPOR) is involved in chlorophyll biosynthesis. Bacteriochlorophyll formation additionally requires the structurally related chlorophyllide oxidoreductase (COR). During catalysis, homodimeric subunit BchL 2 or ChlL 2 of DPOR transfers electrons to the corresponding heterotetrameric catalytic subunit, (BchNB) 2 or (ChlNB) 2 . Analogously, subunit BchX 2 of the COR enzymes delivers electrons to subunit (BchYZ) 2 . Various chimeric DPOR enzymes formed between recombinant subunits (BchNB) 2 and BchL 2 from Chlorobaculum tepidum or (ChlNB) 2 and ChlL 2 from Prochlorococcus marinus and Thermosynechococcus elongatus were found to be enzymatically active, indicating a conserved docking surface for the interaction of both DPOR protein subunits. Biotin label transfer experiments revealed the interaction of P. marinus ChlL 2 with both subunits, ChlN and ChlB, of the (ChlNB) 2 tetramer. Based on these findings and on structural information from the homologous nitrogenase system, a site-directed mutagenesis approach yielded 10 DPOR mutants for the characterization of amino acid residues involved in protein-protein interaction. Surface-exposed residues Tyr 127 of subunit ChlL, Leu 70 and Val 107 of subunit ChlN, and Gly 66 of subunit ChlB were found essential for P. marinus DPOR activity. Next, the BchL 2 or ChlL 2 part of DPOR was exchanged with electron-transferring BchX 2 subunits of COR and NifH 2 of nitrogenase. Active chimeric DPOR was generated via a combination of BchX 2 from C. tepidum or Roseobacter denitrificans with (BchNB) 2 from C. tepidum. No DPOR activity was observed for the chimeric enzyme consisting of NifH 2 from Azotobacter vinelandii in combination with (BchNB) 2 from C. tepidum or (ChlNB) 2 from P. marinus and T. elongatus, respectively.

A family of versatile promoter-probe plasmids for gene expression analysis was developed based on a modular expression plasmid system (pZ). The vectors contain different replicons with exchangeable antibiotic cassettes to allow compatibility and expression analysis on a low-, midi- and high-copy number basis. Suicide vector variants also permit chromosomal integration of the reporter fusion and stable vector derivatives can be used for in vivo or in situ expression studies under non-selective conditions. Transcriptional and translational fusions to the reporter genes gfpmut3.1, amCyan, dsRed2, luxCDABE, phoA or lacZ can be constructed, and presence of identical multiple cloning sites in the vector system facilitates the interchange of promoters or reporter genes between the plasmids of the series. The promoter of the constitutively expressed gapA gene of Escherichia coli was included to obtain fluorescent and bioluminescent expression constructs. A combination of the plasmids allows simultaneous detection and gene expression analysis in individual bacteria, e.g. in bacterial communities or during mouse infections. To test our vector system, we analyzed and quantified expression of Yersinia pseudotuberculosis virulence genes under laboratory conditions, in association with cells and during the infection process.