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First chromosomal restriction map of Actinobacillus pleuropneumoniae and localization of putative virulence-associated genes.

ABSTRACT: Combined physical and genetic maps of the genomes of Actinobacillus pleuropneumoniae AP76 (serotype 7 clinical isolate) and of A. pleuropneumoniae ATCC 27088 (serotype 1 reference strain) were constructed by using the restriction endonucleases ApaI, AscI, NotI, and SalI. The chromosome sizes as determined by the addition of estimated fragment sizes were 2.4 Mbp, and both maps had a resolution of approximately 100 kbp. The linkages between the ApaI, AscI, NotI, and SalI fragments and their relative positions were determined by (i) fragment excision and redigestion and (ii) partial digests of defined fragments and Southern blot using end-standing probes. The single SalI site within the chromosome of strain A. pleuropneumoniae AP76 was defined as position 1 of the map; for the map of A. pleuropneumoniae ATCC 27088, the corresponding SalI site was chosen. Putative virulence-associated genes (apx, omlA, sodA, tbpBA, ureC, and a repeat element) and housekeeping genes (glyA, metJ, recA, and rhoAP) were positioned on the physical maps and located on the ApaI and NotI fragments of A. pleuropneumoniae serotype reference strains.

SUBMITTER: Oswald W

PROVIDER: S-EPMC93915 | biostudies-literature | 1999 Jul

REPOSITORIES: biostudies-literature

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First chromosomal restriction map of Actinobacillus pleuropneumoniae and localization of putative virulence-associated genes.

Oswald W W Konine D V DV Rohde J J Gerlach G F GF

Journal of bacteriology 19990701 14

Combined physical and genetic maps of the genomes of Actinobacillus pleuropneumoniae AP76 (serotype 7 clinical isolate) and of A. pleuropneumoniae ATCC 27088 (serotype 1 reference strain) were constructed by using the restriction endonucleases ApaI, AscI, NotI, and SalI. The chromosome sizes as determined by the addition of estimated fragment sizes were 2.4 Mbp, and both maps had a resolution of approximately 100 kbp. The linkages between the ApaI, AscI, NotI, and SalI fragments and their relati ...[more]

PMID: 10400571

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Project description:The capsular polysaccharide (CP) of Actinobacillus pleuropneumoniae is required for virulence of the bacteria in swine. However, a molecular investigation of whether the type or quantity of CP affects A. pleuropneumoniae virulence has not been reported. To initiate this investigation, a DNA region downstream of conserved genes required for CP export in A. pleuropneumoniae serotype 1 was cloned and sequenced. Three open reading frames, designated cps1A, cps1B, and cps1C, were identified that had amino acid homology to bacterial carbohydrate biosynthesis genes. A kanamycin resistance cassette (Kan(r)) was inserted into a 750-bp deletion spanning cps1AB or into a 512-bp deletion in cps1B only, and the constructs were cloned in a suicide vector. The Kan(r) gene was then transferred into the chromosome of strain 4074 by homologous recombination to produce strain 4074Deltacps1N and strain 4074Deltacps1B, respectively. Strain 4074Deltacps1N produced no detectable CP, but strain 4074Deltacps1B made 15% of the serotype 1 CP made by the parent strain, 4074, as determined by enzyme-linked immunosorbent assay and precipitation of free CP. The cps1ABC genes of strain 4074 and the cps5ABC and cps5ABCDE genes of serotype 5a strain J45 were cloned into the shuttle vector pLS88 and electroporated into 4074Deltacps1N to produce 4074Deltacps1N(pABcps101), 4074Deltacps1N(pJMLcps53), and 4074Deltacps1N(pABcps55), respectively. Strain 4074Deltacps1N(pABcps101) produced about 33% of the serotype 1 CP produced by strain 4074. Strains 4074Deltacps1N(pJMLcps53) and 4074Deltacps1N(pABcps55) produced serotype 5a CP in similar quantity or in fourfold excess, respectively, to that produced by strain 4074. With intratracheal challenge in pigs at similar dosages, the order of virulence of strains producing serotype 1 CP (assessed by mortality, lung consolidation, hemorrhage, and fibrinous pleuritis) was the following: strain 4074 > strain 4074Deltacps1N(pABcps101) > or = strain 4074Deltacps1N > strain 4074Deltacps1B. Strain 4074Deltacps1N(pJMLcps53) was less virulent than strain 4074Deltacps1N(pABcps55). However, both strains produced serotype 5a CP in similar or greater quantities than was observed for production of serotype 1 CP by the parent strain, 4074, but were less virulent than the parent strain. Therefore, the amount of serotype 1 or 5a CP produced by isogenic strains of A. pleuropneumoniae correlated with the virulence of the bacteria in pigs. However, virulence was also influenced by the type of CP produced or by its mechanism of expression.

Project description:BackgroundComparative analysis of gene expression among serotypes within a species can provide valuable information on important differences between related genomes. For the pig lung pathogen Actinobacillus pleuropneumoniae, 15 serotypes with a considerable variation in virulence potential and immunogenicity have been identified. This serotypic diversity can only partly be explained by amount of capsule and differences in the RTX toxin genes in their genomes. Iron acquisition in vivo is an important bacterial function and in pathogenic bacteria, iron-limitation is often a signal for the induction of virulence genes. We used a pan-genomic microarray to study the transcriptional response to iron restriction in vitro in six serotypes of A. pleuropneumoniae (1, 2, 3, 5b, 6, and 7), representing at least two levels of virulence.ResultsIn total, 45 genes were significantly (p < 0.0001) up-regulated and 67 genes significantly down-regulated in response to iron limitation. Not previously observed in A. pleuropneumoniae was the up-regulation of a putative cirA-like siderophore in all six serotypes. Three genes, recently described in A. pleuropneumoniae as possibly coding for haemoglobin-haptoglobin binding proteins, displayed significant serotype related up-regulation to iron limitation. For all three genes, the expression appeared at its lowest in serotype 3, which is generally considered one of the least virulent serotypes of A. pleuropneumoniae. The three genes share homology with the hmbR haemoglobin receptor of Neisseria meningitidis, a possible virulence factor which contributes to bacterial survival in rats.ConclusionsBy comparative analysis of gene expression among 6 different serotypes of A. pleuropneumoniae we identified a common set of presumably essential core genes, involved in iron regulation. The results support and expand previous observations concerning the identification of new potential iron acquisition systems in A. pleuropneumoniae, showing that this bacterium has evolved several strategies for scavenging the limited iron resources of the host. The combined effect of iron-depletion and serotype proved to be modest, indicating that serotypes of both moderate and high virulence at least in vitro are reacting almost identical to iron restriction. One notable exception, however, is the haemoglobin-haptoglobin binding protein cluster which merits further investigation.

Project description:Actinobacillus pleuropneumoniae is the pathogen of porcine contagious pleuropneumoniae, a highly contagious respiratory disease of swine. Although the genome of A. pleuropneumoniae was sequenced several years ago, limited information is available on the genome-wide transcriptional analysis to accurately annotate the gene structures and regulatory elements. High-throughput RNA sequencing (RNA-seq) has been applied to study the transcriptional landscape of bacteria, which can efficiently and accurately identify gene expression regions and unknown transcriptional units, especially small non-coding RNAs (sRNAs), UTRs and regulatory regions. The aim of this study is to comprehensively analyze the transcriptome of A. pleuropneumoniae by RNA-seq in order to improve the existing genome annotation and promote our understanding of A. pleuropneumoniae gene structures and RNA-based regulation. In this study, we utilized RNA-seq to construct a single nucleotide resolution transcriptome map of A. pleuropneumoniae. More than 3.8 million high-quality reads (average length ~90 bp) from a cDNA library were generated and aligned to the reference genome. We identified 32 open reading frames encoding novel proteins that were mis-annotated in the previous genome annotations. The start sites for 35 genes based on the current genome annotation were corrected. Furthermore, 51 sRNAs in the A. pleuropneumoniae genome were discovered, of which 40 sRNAs were never reported in previous studies. The transcriptome map also enabled visualization of 5'- and 3'-UTR regions, in which contained 11 sRNAs. In addition, 351 operons covering 1230 genes throughout the whole genome were identified. The RNA-Seq based transcriptome map validated annotated genes and corrected annotations of open reading frames in the genome, and led to the identification of many functional elements (e.g. regions encoding novel proteins, non-coding sRNAs and operon structures). The transcriptional units described in this study provide a foundation for future studies concerning the gene functions and the transcriptional regulatory architectures of this pathogen.

Dataset Information

First chromosomal restriction map of Actinobacillus pleuropneumoniae and localization of putative virulence-associated genes.

Publications

First chromosomal restriction map of Actinobacillus pleuropneumoniae and localization of putative virulence-associated genes.

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