Taxonomic relationships of the [Pasteurella] haemolytica complex as evaluated by DNA-DNA hybridizations and 16S rRNA sequencing with proposal of Mannheimia haemolytica gen. nov., comb. nov., Mannheimia granulomatis comb. nov., Mannheimia glucosida sp. nov., Mannheimia ruminalis sp. nov. and Mannheimia varigena sp. nov.
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The present paper presents the conclusions of a polyphasic investigation of the taxonomy of the trehalose-negative [Pasteurella] haemolytica complex. Clusters previously identified by ribotyping and multilocus enzyme electrophoresis (MEE) have been evaluated by 16S rRNA sequencing and DNA-DNA hybridizations. Results obtained by the different techniques were highly related and indicated that the [P.] haemolytica complex contains distinct genetic and phenotypic groups. At least seven species were outlined, five of which were named. We refrained in formal naming of more groups until additional strains are characterized. Five 16S rRNA clusters were identified corresponding to distinct lineages previously outlined by MEE. Within 16S rRNA cluster I two distinct genotypic groups have been outlined in addition to [P.] haemolytica sensu stricto (biogroup 1). Each of the clusters II, III, IV and V represent at least one new species. The investigations underline that [P.] haemolytica sensu stricto only contains strains that do not ferment L-arabinose even though they are referred to as 'biotype A' of [P.] haemolytica. The five 16S rRNA clusters identified had a common root relative to the other species within the family Pasteurellaceae, and the overall sequence similarity among these five clusters was higher than what is observed within the existing genera of the family. The allocation of the trehalose-negative [P.] haemolytica complex to a new genus seems to be indicated. Based on the polyphasic investigation performed a new genus Mannheimia is proposed for the trehalose-negative [P.] haemolytica complex. At the present stage two previously named species are transferred to this new genus and three new species are described. [P.] haemolytica is reclassified as Mannheimia haemolytica comb. nov., whereas Pasteurella granulomatis, Bisgaard taxon 20 and [P.] haemolytica biovar 3J are reclassified and combined in the species Mannheimia granulomatis comb. nov. Mannheimia glucosida sp. nov. corresponds to [P.] haemolytica biogroups 3A-3H and the beta-glucosidase and meso-inositol-positive strains of [P.] haemolytica biogroup 9. All typable strains within M. glucosida belong to serotype 11. Mannheimia ruminalis sp. nov. consists of strains previously classified as Bisgaard taxon 18 and [P.] haemolytica biogroup 8D. Finally, Mannheimia varigena sp. nov. includes [P.] haemolytica biogroup 6 as well as Bisgaard taxon 15 and Bisgaard taxon 36. The type strains are NCTC 9380T (M. haemolytica), ATCC 49244T (M. granulomatis), CCUG 38457T = P925T (M. glucosida), CCUG 38470T = HPA92T (M. ruminalis) and CCUG 38462T = 177T (M. varigena). (+info)
Characterization of a CACAG pentanucleotide repeat in Pasteurella haemolytica and its possible role in modulation of a novel type III restriction-modification system.
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In a previous study, a recombinant plasmid that contains a CACAG pentanucleotide repeat was isolated from a Pasteurella haemolytica A1 library. Southern hybridization analysis using a (CACAG)5probe indicated the presence of two loci that contain the pentanucleotide repeats on the genome of P.haemolytica A1. Additional hybridization analyses against genomic DNA from related microorganisms indicated that the repeats are only present in P.haemolytica and Pasteurella trehalosi T3. The various serotypes of P.haemolytica werefound to have either one or two of the CACAG repeat-containing loci. Examination of the locus designated Rpt2 by PCR and sequence analysis indicated that the number of CACAG repeats could change upon serial subculture which most likely occurs as a result of DNA slipped-strand mispairing. A plasmid carrying the Rpt2 locus was isolated and characterized. Sequenceanalysis indicated that the CACAG repeats are contained within the 5'-end of a gene that showed homology to mod genes of type III restriction-modification systems. A second open reading frame downstream was identified which showed homology to res genes of type III restriction-modification systems. Both the modification and restriction proteins could be expressed and polypeptides of the expected sizes were detected by SDS-PAGE. Restriction activity could also be detected in crude cytoplasmic extracts of Escherichia coli strains carrying the mod and res genes on recombinant plasmids. (+info)
Detection of haptoglobin in the high-density lipoprotein and the very high-density lipoprotein fractions from sera of calves with experimental pneumonia and cows with naturally occurring fatty liver.
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In addition to the lipoprotein-deficient d > 1.25 fraction, haptoglobin was detected in the high-density lipoprotein (HDL) and the very high-density lipoprotein (VHDL) fractions from sera of calves with experimental pneumonia and cows with naturally occurring fatty liver. It was not found in the chylomicrons, very low-density lipoprotein and low-density lipoprotein fractions. Washing of the HDL fraction did not decrease the haptoglobin concentration. Transferrin and immunoglobulin G were immunoblotted to examine the possibility of contamination of the lipoprotein fractions by the d > 1.25 fraction. The two serum proteins were detected only in the d > 1.25 fraction, not in any lipoprotein fractions. The distribution pattern of haptoglobin in the lipoprotein fractions was distinct from that of serum albumin. Concentrations of haptoglobin in the HDL fractions from pneumonic sera were largely proportional to those in whole sera. Cholesteryl ester concentrations were decreased in sera from calves with pneumonia, as in cows with fatty liver. A protein immunologically related to hemoglobin was also detected in particular in the VHDL fractions from sera of both groups. These results suggest that haptoglobin or a complex with the hemoglobin-like protein may have a role or roles related to the lipid metabolism. (+info)
Regulation of capsular polysialic acid biosynthesis by temperature in Pasteurella haemolytica A2.
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The capsular polysaccharide of Pasteurella haemolytica A2 consists of a linear polymer of N-acetylneuraminic acid (Neu5Ac) with alpha(2-8) linkages. The production of this polymer is strictly regulated by the growth temperature and above 40 degrees C no production is detected. Analysis of the enzymatic activities directly involved in its biosynthesis reveals that Neu5Ac lyase, CMP-Neu5Ac synthetase and polysialyltransferase are involved in this regulation. Very low activities were found in P. haemolytica grown at 43 degrees C (at least 25 times lower than those observed when the growth temperature was 37 degrees C). The synthesis of these enzymes increased rapidly when bacteria grown at 43 degrees C were transferred to 37 degrees C and decreased dramatically when cells grown at 37 degrees C were transferred to 43 degrees C. These findings indicate that the cellular growth temperature regulates the synthesis of these enzymes and hence the concentration of the intermediates necessary for capsular polysaccharide genesis in P. haemolytica A2. (+info)
CRITICA: coding region identification tool invoking comparative analysis.
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Gene recognition is essential to understanding existing and future DNA sequence data. CRITICA (Coding Region Identification Tool Invoking Comparative Analysis) is a suite of programs for identifying likely protein-coding sequences in DNA by combining comparative analysis of DNA sequences with more common noncomparative methods. In the comparative component of the analysis, regions of DNA are aligned with related sequences from the DNA databases; if the translation of the aligned sequences has greater amino acid identity than expected for the observed percentage nucleotide identity, this is interpreted as evidence for coding. CRITICA also incorporates noncomparative information derived from the relative frequencies of hexanucleotides in coding frames versus other contexts (i.e., dicodon bias). The dicodon usage information is derived by iterative analysis of the data, such that CRITICA is not dependent on the existence or accuracy of coding sequence annotations in the databases. This independence makes the method particularly well suited for the analysis of novel genomes. CRITICA was tested by analyzing the available Salmonella typhimurium DNA sequences. Its predictions were compared with the DNA sequence annotations and with the predictions of GenMark. CRITICA proved to be more accurate than GenMark, and moreover, many of its predictions that would seem to be errors instead reflect problems in the sequence databases. The source code of CRITICA is freely available by anonymous FTP (rdp.life.uiuc.edu in/pub/critica) and on the World Wide Web (http:/(/)rdpwww.life.uiuc.edu). (+info)
Lipopolysaccharide complexes with Pasteurella haemolytica leukotoxin.
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The presence of lipopolysaccharide (LPS) in gram-negative bacterial repeats-in-toxin (RTX) toxin preparations, as well as the harsh conditions required to remove it, suggests that LPS may complex with RTX toxins. Concentrated culture supernatant (CCS) preparations of the RTX toxin Pasteurella haemolytica leukotoxin (LKT) contained LKT and LPS as the most prominent components, with LKT and LPS constituting approximately 30 and 50% of the density of the silver-stained fraction on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), respectively. CCS LKT contained 3.69 +/- 0.46 mg of LPS per mg of protein, which was estimated to indicate an LPS/LKT molar ratio of approximately 60:1. Subjection of the CCS LKT to preparative SDS-PAGE resulted in separation of LPS from LKT as detected by silver-stained analytical SDS-PAGE; however, the LKT fraction (SDS-PAGE LKT) contained significant endotoxin activity as detected by the Limulus amebocyte lysate assay. Subjection of the SDS-PAGE LKT to a second preparative SDS-PAGE run resulted in a reduction of the LPS/LKT molar ratio to 1:20. The target cell specificity of LKT for bovine leukocytic cells was retained by the SDS-PAGE LKT, and isolated LPS at comparable concentrations to that in CCS LKT exhibited no leukolytic activity. Addition of isolated LPS back to SDS-PAGE LKT resulted in reconstitution of an LPS-LKT complex. Immediately following reconstitution of the LPS-LKT complex, there was minimal change in leukolytic activity of the complex, but following 9.5 h at temperatures from -135 to 37 degrees C, the LPS-LKT complex exhibited increased leukolytic activity and thermal stability compared to SDS-PAGE LKT. Therefore, it appears that LPS complexes with LKT, resulting in enhanced and stabilized leukolytic activity. (+info)
Cloning and characterization of the gene encoding Pasteurella haemolytica FnrP, a regulator of the Escherichia coli silent hemolysin sheA.
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A Pasteurella haemolytica A1 gene was identified from a recombinant library clone that expressed hemolysis in host Escherichia coli cells. The gene, designated fnrP, had sequence identity to E. coli fnr, a global transcriptional regulator of genes required for conversion to anaerobic growth. FnrP complemented anaerobic deficiencies of a fnr-null mutant strain of E. coli and increased expression of the Fnr-dependent, anaerobic terminal reductase gene, frdA. FnrP was purified, identified by immunoblotting, and shown to be nonhemolytic. When FnrP was expressed in E. coli DeltasheA, a null mutant of the cryptic hemolysin SheA, the transformants were nonhemolytic, indicating that FnrP activates this silent hemolysin. (+info)
Conservation of expression and N-terminal sequences of the Pasteurella haemolytica 31-kilodalton and Pasteurella trehalosi 29-kilodalton periplasmic iron-regulated proteins.
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This study examined the conservation of expression of a 31-kDa iron-regulated protein by serotypes of Pasteurella haemolytica and Pasteurella trehalosi associated with pasteurellosis of cattle and sheep. A polyclonal antibody prepared against the purified 31-kDa periplasmic iron-regulated protein from P. haemolytica serotype A1 showed that all P. haemolytica serotypes expressed similar 31-kDa proteins with identical N-terminal sequences, whereas P. trehalosi serotypes expressed immunologically different 29-kDa proteins with a different N-terminal sequence. Antibody to the 31-kDa iron-regulated protein was a useful tool to distinguish similarities and differences of the iron-regulated proteins of P. haemolytica and P. trehalosi. (+info)