|
|
|
|
|
|
Variation between Pathogenic Serovars within Salmonella Pathogenicity Islands. P. Amavisit, 2003.Although four of the five Salmonella pathogenicity islands (SPIs) have been characterized in detail for Salmonella enterica serovar Typhimurium, and the fifth has been characterized for Salmonella enterica serovar Dublin, there have been limited studies to examine them in detail in a range of pathogenic serovars of S . enterica . The aim of this study was to examine these regions, shown to be crucial in virulence, in pathogenic serovars to identify any major deletions or insertions that may explain variation in virulence and provide further understanding of the elements involved in the evolution of these regions . Multiple strains of each of the 13 serovars were compared by Southern blot hybridization using a series of probes that together encompassed the full length of all five SPIs . With the exception of serovar Typhimurium, all strains of the same serovar were identical in all five SPIs . Those serovars that differed from serovar Typhimurium in SPI-1 to SPI-4 and from serovar Dublin in SPI-5 were examined in more detail in the variant regions by PCR, and restriction endonuclease digestion and/or DNA sequencing . While most variation in hybridization patterns was attributable to loss or gain of single restriction endonuclease cleavage sites, three regions, in SPI-1, SPI-3, and SPI-5, had differences due to major insertions or deletions . In SPI-1 the avrA gene was replaced by a 200-base fragment in three serovars, as reported previously . In SPI-5, two serovars had acquired an insertion with similarity to the pagJ and pagK genes between pipC and pipD. In SPI-3 the genes sugR and rhuM were deleted in most serovars and in some were replaced by sequences that were very similar to either the Escherichia coli fimbrial operon, flanked by two distinct insertion sequence elements, or to the E . coli retron phage  R73 .
The distribution of these differences suggests that there have been a
number of relatively recent horizontal transfers of genes into S .
enterica and that in some cases the same event has occurred in
multiple lineages of S . enterica. Thus, it seems that
insertion sequences and retron phages are likely to be involved in
continuing evolution of the pathogenicity islands of pathogenic
Salmonella serovars .
Chloromethane-Induced Genes Define a Third C1 Utilization Pathway in Methylobacterium chloromethanicum CM4. Alex Studer, 2002.Methylobacterium chloromethanicum CM4 is an aerobic  -proteobacterium capable of growth with chloromethane as the sole carbon and energy source . Two proteins, CmuA and CmuB, were previously purified and shown to catalyze the dehalogenation of chloromethane and the vitamin B12-mediated transfer of the methyl group of chloromethane to tetrahydrofolate . Three genes located near cmuA and cmuB, designated metF, folD and purU and encoding homologs of methylene tetrahydrofolate (methylene-H4folate) reductase, methylene-H4folate dehydrogenase-methenyl-H4folate cyclohydrolase and formyl-H4folate hydrolase, respectively, suggested the existence of a chloromethane-specific oxidation pathway from methyl-tetrahydrofolate to formate in strain CM4 . Hybridization and PCR analysis indicated that these genes were absent in Methylobacterium extorquens AM1, which is unable to grow with chloromethane . Studies with transcriptional xylE fusions demonstrated the chloromethane-dependent expression of these genes . Transcriptional start sites were mapped by primer extension and allowed to define three transcriptional units, each likely comprising several genes, that were specifically expressed during growth of strain CM4 with chloromethane . The DNA sequences of the deduced promoters display a high degree of sequence conservation but differ from the Methylobacterium promoters described thus far . As shown previously for purU, inactivation of the metF gene resulted in a CM4 mutant unable to grow with chloromethane . Methylene-H4folate reductase activity was detected in a cell extract of strain CM4 only in the presence of chloromethane but not in the metF mutant . Taken together, these data provide evidence that M . chloromethanicum CM4 requires a specific set of tetrahydrofolate-dependent enzymes for growth with chloromethane .
Gene Structure, Organization, Expression, and Potential Regulatory Mechanisms of Arginine Catabolism in Enterococcus faecalis. Belén Barcelona-Andrés, 2002.Although Enteroccus faecalis is the paradigm for biochemical studies on the arginine deiminase (ADI) pathway of fermentative arginine catabolism, little genetic information exists on this pathway in this organism . We fill this important gap by characterizing, in an 8,228-bp region cloned from a  gt11 genomic library of E . faecalis, a five-gene cluster forming a transcriptional unit (revealed by Northern blots and primer extension in E . faecalis) that corresponds to the ADI operon . Four additional genes in the opposite DNA strand and one in the same DNA strand are also identified . Studies on the protein products, including heterologous expression and/or sequence comparisons, allow us to ascertain or propose functions for all but 1 of the 10 genes . The ADI operon genes, arcABCRD, encode, respectively, ADI, ornithine transcarbamylase, carbamate kinase, a putative Crp/Fnr-type regulator (ArcR), and a putative ornithine-arginine antiporter (ArcD) . Arginine induces the expression of arcABCRD, most likely by means of two homologous ArgR/AhrC-type regulators encoded by two genes, argR1 and argR2, that precede arcABCRD in each DNA strand and that are transcribed monocistronically, their transcription being influenced differentially by glucose and arginine . Potential ArgR1/ArgR2 (double and single) binding sequences are found in the promoter regions of arcA and of argR1/argR2 themselves . In addition, putative binding sequences for ArcR and for CcpA are found, respectively, in the argR1/argR2 and arcA promoter regions . Of the three other genes identified, two form a transcriptional unit and encode a putative metal-sensitive transcriptional regulator (ArsR) and a cysteine protease .
Electrophoretic Mobility of Bacillus subtilis Knockout Mutants with and without Flagella. Shujiro Okuda, 2003.Mutants of Bacillus subtilis 168 strain were obtained by inactivation of a specific gene by homologous recombination with the plasmid pMutinT3 . The cell surface properties of these strains were characterized by measuring the electrophoretic mobility of the cells as a function of pH and ionic strength . The surface properties were different for the strains possessing flagella on their cells and strain FlgB, having no flagellum, due to knockout of the corresponding gene . The cell surface properties of the strains possessing flagella become similar to those of strain FlgB after acid treatment . It was confirmed that the acid treatment degraded the flagella without causing any apparent structural change on the cell surface via observations made using atomic force microscopy, transmission electron microscopy, and scanning electron microscopy . These results indicate that the flagella are a key factor influencing cell surface properties . aadA Confers Streptomycin Resistance in Borrelia burgdorferi. Kristi L. Frank, 2003.To enhance genetic manipulation of the Lyme disease spirochete Borrelia burgdorferi, we assayed the aadA gene for the ability to confer resistance to the antibiotics spectinomycin and streptomycin . Using the previously described pBSV2 as a backbone, a shuttle vector, termed pKFSS1, which carries the aadA open reading frame fused to the B . burgdorferi flgB promoter was constructed . The hybrid flgB promoter-aadA cassette confers resistance to spectinomycin and streptomycin in both B . burgdorferi and Escherichia coli . pKFSS1 has a replication origin derived from the 9-kb circular plasmid and can be comaintained in B . burgdorferi with extant shuttle vector pCE320, which has a replication origin derived from a 32-kb circular plasmid, or pBSV2, despite the fact that pKFSS1 and pBSV2 have the same replication origin . Our results demonstrate the availability of a new selectable marker and shuttle vector for genetically dissecting B . burgdorferi at the molecular level . A Gene Cluster for Chlorate Metabolism in Ideonella dechloratans. Helena Danielsson Thorell, 2003.Chlorate reductase has been isolated from the chlorate-respiring bacterium Ideonella dechloratans, and the genes encoding the enzyme have been sequenced . The enzyme is composed of three different subunits and contains molybdopterin, iron, probably in iron-sulfur clusters, and heme b . The genes (clr) encoding chlorate reductase are arranged as clrABDC, where clrA, clrB, and clrC encode the subunits and clrD encodes a specific chaperone . Judging from the subunit composition, cofactor content, and sequence comparisons, chlorate reductase belongs to class II of the dimethyl sulfoxide reductase family . The clr genes are preceded by a novel insertion sequence (transposase gene surrounded by inverted repeats), denoted ISIde1 . Further upstream, we find the previously characterized gene for chlorite dismutase (cld), oriented in the opposite direction . Chlorate metabolism in I . dechloratans starts with the reduction of chlorate, which is followed by the decomposition of the resulting chlorite to chloride and molecular oxygen . The present work reveals that the genes encoding the enzymes catalyzing both these reactions are in close proximity .
|
© 2005
Transgalactic Ltd (manufacturer of Bioscreen C software) |
Privacy Statement | P.O. Box
1393, 00101 Helsinki, Finland,
Last modified: May 25, 2005
| ||||||
