Sample sequencing of a selected region of the genome of Erwinia carotovora subsp. atroseptica reveals candidate phytopathogenicity genes and allows comparison with Escherichia coli The GenBank accession numbers for the 424 sequences determined in this work are BH614193 to BH614616.

Abstract

Genome sequencing is making a profound impact on microbiology. Currently, however, only one plant pathogen genome sequence is publicly available and no genome-sequencing project has been initiated for any species of the genus Erwinia, which includes several important plant pathogens. This paper describes a targeted sample sequencing approach to study the genome of Erwinia carotovora subsp. atroseptica (Eca), a major soft-rot pathogen of potato. A large insert DNA (approx. 115 kb) library of Eca was constructed using a bacterial artificial chromosome (BAC) vector. Hybridization and endsequence data revealed two overlapping BAC clones that span an entire hrp gene cluster. Random subcloning and one-fold sequence coverage (S 200 kb) across these BACs identified 25 (89 %) of 28 hrp genes predicted from the orthologous hrp cluster of Erwinia amylovora. Regions flanking the hrp cluster contained orthologues of known or putative pathogenicity operons from other Erwinia species, including dspEF (E. amylovora), hecAB and pecSM (E. chrysanthemi), sequences similar to genes from the plant pathogen Xylella fastidiosa, including haemagglutinin-like genes, and sequences similar to genes involved in rhizobacterium-plant interactions. Approximately 10 % of the sequences showed strongest nucleotide similarities to genes in the closely related model bacterium and animal pathogen Escherichia coli. However, the positions of some of these genes were different in the two genomes. Approximately 30 % of sequences showed no significant similarity to any database entries. A physical map was made across the genomic region spanning the hrp cluster by hybridization to the BAC library and to digested BAC clones, and by PCR between sequence contigs. A multiple genome coverage BAC library and one-fold sample sequencing are an effective combination for extracting useful information from important regions of the Eca genome, providing a wealth of candidate novel pathogenicity genes for functional analyses. Other genomic regions could be similarly targeted.

The phage abortive infection system, ToxIN, functions as a protein–RNA toxin–antitoxin pair

“…Eca 1043 (21) and 1039 (37) were grown at 25°C, E. coli DH5␣ (Gibco/BRL) was grown at 37°C, and S. marcescens Db11 (38) was grown at 30°C in Luria broth (LB) at 300 rpm or on LB agar (LBA) containing 1.5% (w v Ϫ1 ) agar, and growth (OD600) was measured as described previously (39). When required, LB was supplemented with the following antibiotics: kanamycin 50 g mL Ϫ1 , spectinomycin (Sp) 50 g mL Ϫ1 , ampicillin (Ap) 100 g mL Ϫ1 , and tetracycline 35 g mL Ϫ1 .…”

Structure, Function, and Regulation of the Aldouronate Utilization Gene Cluster from Paenibacillus sp. Strain JDR-2

“…An equal volume of 2% low-melting-point agarose was added to the cell suspension, and the cell-agarose mix was poured into 800-l plug molds. Plugs were further processed as described previously (1).…”

A novel non-protein-coding infection-specific gene family is clustered throughout the genome of Phytophthora infestans

“…The UPGMA clustering method was used to align different fingerprints. Insert DNA from BAC clones was subcloned into vector pGEM-3Z (digested with BamHI and alkaline phosphatase; Promega), as described by Bell et al (2002), following partial digestion with Sau3AI (Promega). A total of 1152 recombinant transformed clones were selected, and stored in 384-well microtitre plates in freezing medium containing ampicillin, at 270 uC, until needed.…”