| 1. |
Christensen H,
Kuhnert P,
Olsen JE,
Bisgaard M,
( 2004 ) Comparative phylogenies of the housekeeping genes atpD, infB and rpoB and the 16S rRNA gene within the Pasteurellaceae. PMID : 15388716 : DOI : 10.1099/ijs.0.03018-0 Abstract >>
Phylogenies of housekeeping gene and 16S rRNA gene sequences were compared to improve the classification of the bacterial family Pasteurellaceae and knowledge of the evolutionary relationships of its members. Deduced partial protein sequences of the housekeeping genes atpD, infB and rpoB were compared in 28, 36 and 28 representative taxa of the Pasteurellaceae, respectively. The monophyly of representatives of the genus Gallibacterium was recognized by analysis of all housekeeping genes, while members of Mannheimia, Actinobacillus sensu stricto and the core group of Pasteurella sensu stricto formed monophyletic groups with two out of three housekeeping genes. Representatives of Mannheimia, Actinobacillus sensu stricto, [Haemophilus] ducreyi and [Pasteurella] trehalosi formed a monophyletic unit by analysis of all three housekeeping genes, which was in contrast to the 16S rRNA gene-derived phylogeny, where these taxa occurred at separate positions in the phylogenetic tree. Representatives of the Rodent, Avian and Aphrophilus-Haemophilus 16S rRNA gene groups were weakly supported by phylogenetic analysis of housekeeping genes. Phylogenies derived by comparison of the housekeeping genes diverged significantly from the 16S rRNA gene-derived phylogeny as evaluated by the likelihood ratio test. A low degree of congruence was also observed between the individual housekeeping gene-derived phylogenies. Estimates on speciation derived from 16S rRNA and housekeeping gene sequence comparisons resulted in quite different evolutionary scenarios for members of the Pasteurellaceae. The phylogeny based on the housekeeping genes supported observed host associations between Mannheimia, Actinobacillus sensu stricto and [Pasteurella] trehalosi and animals with paired hooves.
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2. |
Korczak B,
Christensen H,
Emler S,
Frey J,
Kuhnert P,
( 2004 ) Phylogeny of the family Pasteurellaceae based on rpoB sequences. PMID : 15280320 : DOI : 10.1099/ijs.0.03043-0 Abstract >>
Sequences of the gene encoding the beta-subunit of the RNA polymerase (rpoB) were used to delineate the phylogeny of the family Pasteurellaceae. A total of 72 strains, including the type strains of the major described species as well as selected field isolates, were included in the study. Selection of universal rpoB-derived primers for the family allowed straightforward amplification and sequencing of a 560 bp fragment of the rpoB gene. In parallel, 16S rDNA was sequenced from all strains. The phylogenetic tree obtained with the rpoB sequences reflected the major branches of the tree obtained with the 16S rDNA, especially at the genus level. Only a few discrepancies between the trees were observed. In certain cases the rpoB phylogeny was in better agreement with DNA-DNA hybridization studies than the phylogeny derived from 16S rDNA. The rpoB gene is strongly conserved within the various species of the family of Pasteurellaceae. Hence, rpoB gene sequence analysis in conjunction with 16S rDNA sequencing is a valuable tool for phylogenetic studies of the Pasteurellaceae and may also prove useful for reorganizing the current taxonomy of this bacterial family.
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3. |
Kim P,
Laivenieks M,
McKinlay J,
Vieille C,
Gregory Zeikus J,
( 2004 ) Construction of a shuttle vector for the overexpression of recombinant proteins in Actinobacillus succinogenes. PMID : 15003707 : DOI : 10.1016/j.plasmid.2003.11.003 Abstract >>
To express foreign proteins in Actinobacillus succinogenes, a shuttle vector was constructed based on the Actinobacillus pleuropneumoniae-Escherichia coli shuttle vector, pGZRS-19. We demonstrated that A. succinogenes is transformed by electroporation at reasonably high efficiency, that pGZRS-19 is stable in A. succinogenes, and that the ampicillin resistance gene carried by pGZRS-19 is expressed in A. succinogenes. Three steps were then required to develop our A. succinogenes-E. coli shuttle vector. (i) The constitutively expressed A. succinogenes phosphoenolpyruvate carboxykinase gene, pckA, was cloned and sequenced. (ii) Its promoter region and ribosome-binding site were subcloned into pGZRS-19. (iii) Finally, the ColE1 origin of replication was added to the vector to increase its stability in E. coli. High levels of A. succinogenes phosphoenolpyruvate carboxykinase, E. coli NADP-dependent malic enzyme, and Bacillus subtilis NAD-dependent malic enzyme activities detected in recombinant A. succinogenes strains confirmed that A. succinogenes and foreign proteins could be expressed in A. succinogenes under control of the A. succinogenes pckA promoter carried by pLGZ920. A. succinogenes is sensitive to chloramphenicol and tetracycline. Although not expressed from their own promoters, the Tn9 chloramphenicol and the Tn10 tetracycline resistance genes are expressed under control of the pckA promoter, and they can be used as additional selection markers in A. succinogenes.
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4. |
Adhikary S,
Bisgaard M,
Nicklas W,
Christensen H,
( 2018 ) Reclassification of Bisgaard taxon 5 as Caviibacterium pharyngocola gen. nov., sp. nov. and Bisgaard taxon 7 as Conservatibacter flavescens gen. nov., sp. nov. PMID : 29303698 : DOI : 10.1099/ijsem.0.002558 Abstract >>
A total of 29 strains mainly from guinea pigs were investigated by a polyphasic approach that included previously published data. The strains were classified as Bisgaard taxa 5 and 7 by comparison of phenotypic characteristics and the strains showed typical cultural characteristics for members of family Pasteurellaceae and the strains formed two monophyletic groups based on 16S rRNA gene sequence comparison. Partial rpoB sequence analysis as well as published data on DNA-DNA hybridization showed high genotypic relationships within both groups. A new genus with one species, Caviibacterium pharyngocola gen. nov., sp. nov., is proposed to accommodate members of taxon 5 of Bisgaard, whereas members of taxon 7 are proposed as Conservatibacter flavescens gen. nov., sp. nov. The two genera are clearly separated by phenotype from each other and from existing genera of the family Pasteurellaceae. The type strain of Caviibacterium pharyngocola is 7.3T (=CCUG 16493T=DSM 105478T) and the type strain of Conservatibacter flavescens is 7.4T (=CCUG 24852T=DSM 105479T=HIM 794-7T), both were isolated from the pharynx of guinea pigs.
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