A single promoter designated P<i>_mra</i>, was previously identified upstream of the <i>mra</i> region. P<i>_mra</i> was reported to be essential for expression of the first nine genes of the <i>mra</i> region and may contribute to expression <i>fts</i>Q, A, and Z at the distal end of this gene cluster. This study investigates potential mechanisms of transcriptional regulation at P<i>_mra</i>, in addition to the regulation of two newly identified promoters (P<i>_mra</i>2 and P<i>_mra</i>3) in the <i>fru</i>R-<i>yab</i>B intergenic region. Using <i>lacZ</i> reporter strains we determined that transcription from the P<i>_mra</i> promoters was inversely related to growth rate. Promoters P<i>_mra</i>1 and P<i>_mra</i>3 were the major contributors to the transcription of early genes in the <i>mra</i> region. Transcripts originating from these promoters were detected by RT-PCR to span at least as far as <i>fts</i>L. The transcriptional regulator FIS was shown to bind several sites in the intergenic region, including sequences within the promoter regions of both P<i>_mra</i>2 and P<i>_mra</i>3. FIS caused a 3-fold repression of P<i>_mra</i>2 but had little effect on the transcriptional activity of P<i>_mra</i>3. It was noted that presence of the entire intergenic region caused a 4-fold repression of P<i>_mra</i>3, indicating that transcriptional regulators interact with the DNA upstream of this promoter. The 50S subunit ribosomal protein L3 was shown to bind upstream of P<i>_mra</i>3 . P<i>_mra</i>3 was also shown to be repressed by the cellular alarmone ppGpp which is produced as part of the stringent response and during slow growth or carbon starvation. It was not possible to delete P<i>_mra</i>3 from the chromosome suggesting that it may be essential for cell viability.

This study is the first to analyse the genome of <i>A. butzleri </i>NCTC12481. By using one- and two-dimensional Pulsed Field Gel Electrophoresis in combination with Southern hybridisation the <i>A. butzleri </i>genome size was estimated (2.5 Mb) and a physical map was constructed, containing 35 restriction enzyme recognition sequences and 14 gene markers (5 copies of the genes for 16S rRNA and 23S rRNA’ 2 copies for <i>glyA; </i>one for <i>rpoC </i>and <i>rpoB</i>). In the attempt to identify virulence factors of <i>A. butzleri</i>, a homologue of <i>pglF,</i> a gene involved in N-linked protein glycosylation in <i>C. jejuni,</i> was found. Although the genes surrounding <i>pglF </i>in <i>A. butzleri </i>did not reveal a cluster order as in <i>C. jejuni</i>, the intriguing possibility for protein glycosylation in <i>A. butzleri</i> (perhaps involving different mechanisms) remains, since a glycosylated protein was detected by Alcian blue staining of an outer membrane protein extract. Three major proteins of the <i>A. butzleri </i>outer membrane extract were identified as PorA, FlaB and CadF which are known to play a role in pathogenicity in other bacteria. Electron microscopic analysis revealed a polysaccharide capsule on the surface of <i>A. butzleri.</i> <i>In vitro</i> studies of the interaction of <i>A. butzleri </i>with epithelial cultured cells (Caco-2) demonstrated the ability of <i>A. butzleri </i>to attach to and to invade Caco-2 cells. In order to develop a method for transposon mutagenesis of <i>A. butzleri,</i> a Himar1 transposon delivering vector carrying <i>C. jejuni</i> kanamycin resistance gene (<i>aphA </i>type III), was constructed. Attempts to introduce the vector into <i>A. butzleri </i>by electroporation, conjugation or heat shock transformation all failed. The obtained results provide an important foundation for further investigation of this organism and contribute towards broadening the knowledge of this potential foodborne pathogen.

No description available.

Omega-3 polyunsaturated fatty acids (PUFAs), such as eicosapentaenoic acid (EPA, 20:5ω3) and docosahexaenoic acid (DHA, 22:6ω3) play a role in the modulation and prevention of human diseases, in particular cardiovascular diseases. The omega-3 family is found mainly in fish, of which wild stocks are becoming limited. Therefore production of omega-3 PUFAs by marine microbes may provide an alternative source of such componds. The diversity of marine microbes was studied using 16S/18S rRNA gene sequencing of different marine biota with 1500 bacterial strains and 50 microalgae were isolated. The diversity of culturalbe microorganisms inhabiting Mid-Atlantic Ridge (MAR) non-vent sediments was examined for the first time in this area with findings of high diversily of Gram-positive strains, good production of squalene by an unusual strain Bacillus sp. MAR089 and the highest yield of EPA ever recovered from strain Shewanella sp. MAR441. North Sea sponge associated Vibrio sp. strain NSP560 produced considerable levels of EPA, whereas no PUFAs producers were found from tropic Caribbean marine sponge associated bacteria. Photobacterium sp. strain MA665, isolated from the coast of North Sea, was described for the first time of this genus and could be cultured easily under atmospheric conditions with appreciable levels of EPA -1 with up to 25 % of total fatty acids (TFA) (or 10.6 mg g in dried cell). Strain MAR441 was identified as a new species, designated as Shewanella dovemarina sp. T nov. (Type strain MAR441 ). The level of EPA production of strain MAR441 has been optimized by varying fermentation conditions, and 15-25 % EPA of TFA (or 17-30 mg -1 g in dried cell) could be achieved with 40 % improvement. In order to understand the PUFAs biosynthesis pathways and better predict the maximum EPA production, EPA gene clusters (pfaA, pfaB, pfaC, pfaD and pfaE) were cloned and sequenced from the following three species Shewanella, Vibrio and Photobacterium. Great potential was found in marine algae Phaeodactylum tricornutum strain M7 with lipid content of 10 % in dry wt biomass and 22-30 % EPA of TFA when it was cultured outdoors under local weather conditions in UK. Under anaerobic conditions, strain MAR441 contained less -2 amount of EPA and produced electricity of ~100 mW m . Enhanced electricity production using artificial consortia of estuarine bacteria grown as biofilms was -2 observed with power generation of ~200 mW m . In conclusion, bacteria taxonomic resolution based on complete cell fatty acid composition is possible and marine microbes with considerable production of EPA could be potential candidates for industrial production of PUFAs.

In this work a range of recombination mutants were screened for their ability to carry out successful recombinational repair of SbcCD-generated double-strand breaks a the site of a long chromosomal palindrome. The results obtained suggest that the components of both the RecB and RecF pathways are required for successful recombinational repair of SbcCD-generated breaks at the site of the palindrome. This breakage and repair process does not seem to involve replication fork breakage. In the absence of SbcCD recombination still occurs, apparently through the RecF gap-recombination pathway. Once again, this process appears to avoid replication fork collapse. In the absence of recombination, the RecQ helicase was found to be essential to the viability of <i>sbcC</i> cells possessing the palindrome. This suggests that RecQ is involved in a pathway allowing replicative bypass of secondary structure, probably through helicase unwinding of the secondary structure. Using an <i>xerC</i> mutant deficient in the resolution of chromosome dimers, the relationship between recombination at the site of the palindrome and crossing-over was investigated. It was observed that double-strand break repair at the site of the palindrome is associated with crossing-over whereas single-strand gap recombination is not. Using UV irradiation of cells deficient for excision repair it was demonstrated that the association of double-strand break repair, but not single-strand gap repair, with crossing over is a genera phenomenon. The observation that in P1 transduction mainly cross-over products are observed supports the idea that these effects are the result of a rule governing the resolution of Holliday junctions. The random resolution of Holliday junctions in <i>ruvABC</i> mutants suggests that this rule operates through the RuvABC Holliday junction resolution complex.

No description available.

Double-strand DNA breaks (DSBs) occur during normal cell metabolism and are lethal unless repaired. <i>E. coli </i>repairs DSBs using a pathway that involves homologous recombination. The mechanisms involved in this process were investigated by manipulating the <i>Eco</i>KI restriction-modification system of <i>E. coli</i> so that the restriction activity cleaves chromosomes to produce DSBs. The viability of recombination and repair mutants was measured following the induction of DSBs. The results show that RecG and RuvABC facilitate the survival of DSBs. Surprisingly, RuvABC was able to promote survival even when recombination could not be initiated. Pulsed field gel electrophoresis (PFGE) was carried out on the genomic DNA of mutants exposed to DSBs. This allowed Holliday junctions (HJs) linking the chromosomes of strains lacking RuvABC to be detected. Most significantly, the PFGE phenotype of a <i>recG </i>mutant mirrored that of the wild-type, suggesting that the RecG protein is not involved in the resolution of HJs. The outcome of HJ resolution to form crossover or non-crossover products was also investigated in mutants exposed to DSBs by measuring the effect on viability of inactivating the XerCD/<i>dif </i>system that is involved in chromosome dimer resolution. The deleterious effect of <i>xerC</i> mutations on <i>recG </i>and <i>ruvAC </i>mutants was approximately 10-fold greater than on wild-type. These results prompted an interesting discussion as to how the functions of the products of these genes interact in the cell. Finally, the theory that the product of the essential <i>yqgF </i>gene is an alternative HJ resolvase was investigated. <i>yqgF </i>was placed under the control of an inducible promoter and the effect of depleting YqgF levels on survival of DSBs was measured. No evidence to suggest that YqgF can resolve HJs was found.

No description available.

The content and cellular location of cytochromes in <I>Bacillus azotoformans</I> was examined under aerobic and denitrifying anaerobic conditions. An abundance of cytochromes was expressed. Two cytochromes were induced under aerobic conditions, one of which was only expressed under conditions of high aeration. One cytochrome, a b-type cytochrome, may be induced under denitrifying conditions. Cytochromes common to both pathways appear to be expressed in similar amounts per unit cell weight. Both c- and b-type cytochromes from <I>B. azotoformans</I> appear to be membrane bound, as they were only detected in membrane fractions. Different methods for releasing cytochromes from cell membranes were tried. Only extraction with detergents and mild proteolytic treatment were successful. Four different membrane bound cytochromes from <I>Bacillus azotoformans</I>, designated P1-c552, P2-c551, P3&P4-c555 and P5-c552, were isolated and purified. The bases of classification of Class I cytochromes c are examined and revisions are suggested to the existing classification schemes. The evolutionary implications of the distribution of the proposed cytochrome c groups in the phylogenetic eubacterial tree, based on 16sRNA analyses, are discussed. The past and present ideas about the evolution of the phylum of Gram positive bacteria are presented and discussed in the light of the present sequence information of cytochromes c and other electron transport proteins. The electron transport pathway in the genus <I>Bacillus</I> is compared with its counterparts in distantly related bacteria. The significance of observed similarities and differences, in types of cytochromes and composition, for the elucidation of the evolutionary history of the Gram positive phylum as well as for eubacteria in general, is emphasised.

An analysis of the outer membrane proteins of Enterobacter aerogenes exopolysaccharide producing and non-producing strains was made. Growth of these strains under nutrient limitation or in the presence of membrane active agents altered the ratios of proteins in the outer membrane and the yield of exopolysaccharide. The latter compounds had a substantial effect particularly on the major outer membrane proteins. The cytoplasmic membrane proteins of different strains varied slightly in quantity, unrelated to exopolysaccharide synthesis. Lipopolysaccharide extracted from a number of strains was examined by polyacrylamide gel electrophoresis. Differences were detected between strains but this did not correlate with exopolysaccharide yield or form. The major outer membrane proteins were identified as 34K and 38K polypeptides by SDS-polyacrylamide gel electrophoresis. These proteins exhibited the normal properties associated with the major non-porin and porin proteins of E coli. The former protein was cleaved by 'Pronase' in situ and was heat-modifiable in SDS-containing gel sample buffer. The latter was peptidoglycan associated. Several polypeptides were induced by iron limitation and the citrate-ferric ion receptor protein of the Enterobacter aerogenes strains tested appeared to correspond to an 86K polypeptide. Growth in the presence of chaotropic agents resulted in a significant decrease in the 38K protein and an increase in a polypeptide in the 29K-32K range. Other changes in minor proteins may also have occurred. Two polypeptides, 50K and 58K were apparently associated with encapsulation and slime-formation respectively. A role in exopolysaccharide production was postulated.