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The pOV plasmid isolated from the Pasteurella multocida strain PMOV is a new plasmid, and its molecular characterization is important for determining its gene content and its replicative properties in Pasteurellaceae family bacteria. Antimicrobial resistance mediated by the pOV plasmid was tested in bacteria. Purified pOV plasmid DNA was used to transform E. coli DH5α and Gallibacterium anatis 12656-12, including the pBluescript II KS(−) plasmid DNA as a control for genetic transformation. The pOV plasmid was digested with Eco RI for cloning fragments into the pBluescript II KS(−) vector to obtain constructs and to determine the full DNA sequence of pOV. The pOV plasmid is 13.5 kb in size; confers sulfonamide, streptomycin and ampicillin resistance to P. multocida PMOV; and can transform E. coli DH5α and G. anatis 12656-12. The pOV plasmid was digested for the preparation of chimeric constructs and used to transform E. coli DH5α, conferring resistance to streptomycin (plasmid pSEP3), ampicillin (pSEP4) and sulfonamide (pSEP5) on the bacteria; however, similar to pBluescript II KS(−), the chimeric plasmids did not transform G. anatis 12656-12. A 1.4 kb fragment of the streptomycin cassette from pSEP3 was amplified by PCR and used to construct pSEP7, which in turn was used to interrupt a chromosomal DNA locus of G. anatis by double homologous recombination, introducing str A- str B into the G. anatis chromosome. The pOV plasmid is a wide-range, low-copy-number plasmid that is able to replicate in some gamma-proteobacteria. Part of this plasmid was integrated into the G. anatis 12656-12 chromosome. This construct may prove to be a useful tool for genetic studies of G. anatis. • The plasmid pOV can replicate independently from chromosomal DNA in several Pasteurellaceae bacterial species and in E. coli. • The plasmid belongs to IncX incompatibility group. • pOV sequence analysis revealed a novel integron arrangement. • A chimeric plasmid can to transform G. anatis and to recombine within the chromosome. [ABSTRACT FROM AUTHOR]

Gallibacterium anatis is a member of the Pasteurellaceae family and is an opportunistic pathogen that causes gallibacteriosis in chickens. Stress plays a relevant role in promoting the development of pathogenicity in G. anatis. Epinephrine (E) and norepinephrine (NE) are relevant to stress; however, their effects on G. anatis have not been elucidated. In this work, we evaluated the effects of E and NE on the growth, biofilm formation, expression of adhesins, and proteases of two G. anatis strains, namely, the hemolytic 12656-12 and the nonhemolytic F149T biovars. E (10 μM/mL) and NE (30 and 50 μM/mL) increased the growth of G. anatis 12656-12 by 20 % and 25 %, respectively. E did not affect the growth of F149T, whereas 40 μM/mL NE decreased bacterial growth by 25 %. E and NE at a dose of 30–50 μM/mL upregulated five fibrinogen adhesins in the 12565-12 strain, whereas no effect was observed in the F149T strain. NE increased proteolytic activity in both strains, whereas E diminished proteolytic activity in the 12656-12 strain. E and NE reduced biofilm formation (30 %) and increased Congo red binding (15 %) in both strains. QseBC is the E and NE two-component detection system most common in bacteria. The qse C gene, which is the E and NE receptor in bacteria, was identified in the genomic DNA of the 12565-12 and F149T G. anatis strains via PCR amplification. Our results suggest that QseC can detect host changes in E and NE concentrations and that catecholamines can modulate the expression of several virulence factors in G. anatis. [Display omitted] • Epinephrine (E) and Norepinephrine (NE) promote the Gallibacterium anatis 12656-12 growth but just NE of the F149T strain. • E and NE induce adhesins differential expression in the G. anatis 12656-12 strain, without affect G. anatis F149T strain. • Norepinephrine increases the secreted proteolytic activity of G. anatis. • E and NE diminished biofilm formation and Congo red binding in G. anatis. • Informed consent statement not applicable. [ABSTRACT FROM AUTHOR]

Mannheimia haemolytica is an opportunistic agent of the respiratory tract of bovines, a member of the Pasteurellaceae family, and the causal agent of fibrinous pleuropneumonia. This bacterium possesses different virulence factors, allowing it to colonize and infect its host. The present work describes the isolation and characterization of a serine protease secreted by M. haemolytica serotype 1. This protease was isolated from M. haemolytica cultured media by precipitation with 50 % methanol and ion exchange chromatography on DEAE-cellulose. It is a 70-kDa protease able to degrade sheep and bovine fibrinogen or porcine gelatin but not bovine IgG, hemoglobin, or casein. Mass spectrometric analysis indicates its identity with protease IV of M. haemolytica. The proteolytic activity was active between pH 5 and 9, with an optimal pH of 8. It was stable at 50 °C for 10 min but inactivated at 60 °C. The sera of bovines with chronic or acute pneumonia recognized this protease. Still, it showed no cross-reactivity with rabbit hyperimmune serum against the secreted metalloprotease from Actinobacillus pleuropneumoniae , another member of the Pasteurellaceae family. M. haemolytica secreted proteases could contribute to the pathogenesis of this bacterium through fibrinogen degradation, a characteristic of this fibrinous pleuropneumonia. [Display omitted] • Mannheimia haemolytica expresses a 70 kDa secreted serine protease at 37 °C or 39 °C. • This serine protease degrades sheep fibrinogen but not bovine IgG neither hemoglobin nor casein. • This 70 kDa protease is immune recognized by sera from bovines suffering acute or chronic pneumonia. • This protease was identified as protease IV. [ABSTRACT FROM AUTHOR]