Genome Sequencing of Actinobacillus Pleuropneumoniae Serotypes 1 and 7

Project Director
Dyer, D. W.

Recipient Organization
UNIV OF OKLAHOMA HEALTH SCIENCES CTR
1100 N. LINDSAY
OKLAHOMA CITY,OK 73104

Performing Department
MICROBIOLOGY & IMMUNOLOGY

Non Technical Summary
In this application, we propose to determine the genome sequence of the important veterinary pathogen Actinobacillus pleuropneumoniae (Ap), a causative agent of severe respiratory disease in swine. Ap strains vary in their virulence for swine, and in the US, serotype 1 strains are among the most virulent. We therefore propose to sequence to completion the genome of Ap strain 4074, which has been widely used for experimental studies on serotype 1 disease. In future studies, we also will perform low-density sequencing on the genome of Ap serotype 7 strain WF83. This low-density genome sequence will then be compared to the sequence of the serotype 1 strain, to begin to understand the genomic basis underlying the differences in virulence between these Ap serotypes. The sequence data will be publicly available on our web site (http://micro-gen.ouhsc.edu) and will be updated at monthly intervals.

Animal Health Component

(N/A)

Research Effort Categories

Basic

100%

Applied

(N/A)

Developmental

(N/A)

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
311	4010	1040	100%

Knowledge Area
311 - Animal Diseases;

Subject Of Investigation
4010 - Bacteria;

Field Of Science
1040 - Molecular biology;

Keywords

swine

respiratory diseases

actinobacillus

bacterial diseases (animals)

artificial chromosomes

data analysis

polymerase chain reaction

dna fragments

Goals / Objectives
Specific aim 1: We will determine the complete nucleotide sequence and annotate the genome of Ap serotype 1 strain 4074.

Project Methods
For full genome sequencing, we will focus on Ap strain 4047, the type strain for serotype 1 organisms. This strain is widely used for studies on the most common serotype found to cause infections in the US. First, we will construct at least one plasmid library in pUC18, with a 2-4kb average insert size, and shotgun sequence this library to approximately 8-fold coverage. During the initial phases of this shotgun sequencing, the data will be carefully screened to ensure that the library is sufficiently random; otherwise, additional libraries will be built. For the 2.4Mb Ap genome, this shotgun sequencing phase will require 38,400 sequences reads, according to Lander-Waterman calculations. Since our colony-to-sequence read efficiency has been approximately 85% (data not shown), this indicates that we will have to pick about 45,176 colonies to obtain 38,400 reads of sufficient quality for assembly. Simultaneously, we will construct a cosmid-sized (ca. 40kb) large-insert library in pBeloBac11 and end-sequence approximately 1000 clones. This BAC data will be assembled with the shotgun data from the pUC library(s), and will form a backbone upon which the shotgun data can be organized for final closure and annotation. The data will be assembled using Phred/Phrap, and the sequence viewed in Consed for assessing data quality and design of closure experiments. Early in the shotgun sequencing phase (ca.3-4-fold coverage), we will begin to resolve problems caused by repeats by using Consed to identify putative repeat regions, and designing combinatorial PCR experiments to isolate these repeats on PCR amplicons. These amplicons will be primer-walked simultaneously with the latter portion of the shotgun sequencing phase. Final closure will rely on PCR and additional primer walking to close gaps, with primer-directed finishing employed to raise sequence quality in low-coverage regions of the genome. The location and exact sequence of repeats such as the rrn operons will be confirmed by isolating PCR fragments that contain each repeat in its entirety, followed by primer walking across the PCR product.

Progress 10/01/01 to 09/30/02

Outputs
During the initial project period, we constructed both small insert (1-2kb and 2-4kb) and large insert (~40kb) plasmid libraries, in pUC18 and pCC1FOS, respectively, of the virulent App serotype 1 strain 4074. Shotgun sequencing on the small insert libraries was completed to 8-fold coverage (24,828 sequence reads, 714.6bp avg. read length, Phrap score, 86.1). We also completed the end-sequencing of approximately 1000 pCC1FOS fosmid clones, which were also incorporated into the database. Closure sequencing commenced immediately. This sequence data was made publicly available on 09/12/2002 on our web site (http://microgen.ouhsc.edu), and has been updated at regular intervals since the initial data release.

Impacts
This project is designed to obtain the genome sequences of two strains of Actinobacillus pleuropneumoniae, an important swine pathogen that has significant economic impact on the pork-producing industry in the US. Whereas serotype 1 strains of this organism appear to be highly virulent, serotype 7 strains appear to have low or no virulence. By obtaining the complete genome sequence of both organisms, we will be able to compare these serotypes and deduce the major genetic differences between the organisms that lead to their ability/inability to cause pleuropneumonia in pigs. This will inevitably lead to a better understanding of the pathogenesis of disease caused by this important veterinary pathogen, and may reveal new targets for vaccine or drug development.

Publications

D.W. Dyer, A.F. Gillaspy, C. Chen, J. Gipson, M. Gipson, A. Hendrickson, G. Barnes, M. Carson, J. Orvis, B. Fenwick. Genome Sequencing of the Swine Pathogen Actinobacillus pleuropneumoniae. Abstract #62, 3rd ASM and TIGR Conference on Microbial Genomes, January 29-February 1, 2003, New Orleans, LA.