| Porcine contagious pleuropneumonia(PCP),caused by Actinobacillus pleuropneumoniae(APP),is a porcine respiratory infectious disease leading to severe economic losses in the swine industry worldwide.APP presents at least 15 serotypes with differences in their virulence and geographic distribution and thus has caused difficulties in developing safe and effective vaccines as well as easy means of diagnosis. Fully understanding of the molecular pathogenesis of APP will contribute to develop new tools to prevent and control this disease.In this study,we have identified an mreBCD operon from the APP genomes,which is a potential target for new antibiotic drugs.At the same time,four signature-tagged mutagenesis(STM) pools were constructed,which contained 216 transposol mutants.1.Structure analysis of mre operon of A.pleuropneumoniaeThe mre operon is an important operon which is in charge of modurating the cell shape in rod bacteria.The sequences of amino acid,assembly way,structure,function and tertiary structure of MreB,coded by mreB of the mre operon,is highly similar to the actin filaments of the eukaryote.MreB was considered to be involved in the formation and maintenance of cell shape,and essential for chromosome separation and survival in some species of bacteria.In this study,the presence of mre operon was identified from the contigs of the gemomes of A.pleuropneumoniae by bioinformatics teconology.The mre operons showed a similar genomic organization in different bacteria,consisting of B,C and D genes.The mre operon was cloned and sequenced from the genomic DNA of A. pleuropneumoniae strains of serotypes 1,3 and 5.BLAST analysis revealed that they are almost identical among the three strains representing serotypes 1,3 and 5.They also show 79%and 82%of homology to that of Escherichia coli and Haemophilus ducreyi,respectively.These results indicated that the rare operon is highly conserved in rod bacteria.2.Functional characterization of mre operon ofA.pleuropneumoniae.Based on the sequences of the rare operon in serotype 1 of A.pleuropneumoniae, the complete coding sequences of mreB and mreCD genes,and the 5'-end fragment encoding the first 66 amino acids of MreB were separately cloned by PCR amplification.The PCR products were separately subcloned into a suicide plasmid under the control of an L-arabinose inducible promoter.The resultant recombinant plasmids were then electroporated into A.pleuropneumoniae,strain 4074 of serotype 1. The transformants of single cross-over were screened on the TSA plates with or without L-arabinose.The growth characterastics and morphologic changes were observed.The results showed that the bacteria turn small and round because of the absence of the mreB and mreCD genes,whereas the bacteria which over-express the mreB and mreCD genes grow longer and thicker.However,both deletion and overproduction strains grew more slowly than the wildtype.The above changes were more obvious in mreB mutants compared with the mreCD mutants.Our data demonstrated that the mreBCD operon is necessary to keep the rod shape and normal growth of A.pleuropneumoniae.It might be a potential target for antibacterials.3.Construction of STM mutants of A.pleuropneumoniae.STM is a powerful approach to identify genes associated with virulence of the pathogenic bacteria through negative selection.In this study,we compared the efficiency of biparental and triparental mating strategies and established a sophisticated biparental mating method to construct A.pleuropneumoniae mutants.Fouty-eight mini-Tn10 based plasmids carrying different tag sequences(pLOF/Tag1-48) were transformed into E.coli S 17-1λpir.The transformants were separately mated with A. pleuropneumoniae reference strain 4074 of serotype 1.A total of 216 mutant strains have been obtained using 4 different tags(Tag7,Tag8,Tag11 and Tag12) by the biparental mating.This work will contribute to the construction of a STM bank and functional genomics study ofA.pleuropneumoniae.
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