Pseudomonas aeruginosaPA01 Bacterial Artificial Chromosomes: Strategies for Mapping, Screening, and Sequencing 100 kb Loci of the 5.9 Mb Genome

Abstract

Pseudomonas aeruginosa is an opportunistic bacterial pathogen frequently found in nosocomial infections and is a major cause of morbidity and mortality in patients with cystic fibrosis. To facilitate molecular studies of this organism, we have generated a bacterial artificial chromosome (BAC) library. Genomic DNA was isolated from the prototype strain PAO1, partially digested with HindIII, size selected after pulsed-field gel electrophoresis, and used to construct a BAC library using the pBeloBAC11 vector. DNAs from approximately 850 clones, representing more than 9.5-fold physical coverage of the 5.9-Mb PAO1 genome, were analyzed after SpeI and HindIII digestions and agarose gel electrophoresis. The BAC library had clones with insert fragments ranging from 20 to more than 290 kb. A subset of 264 BACs having inserts> 80 kb, representing > 4 genome equivalents, were rearrayed into 96-well plates, and a clone pooling and PCR screening strategy was developed. The PCR library screening enabled the identification and recovery of BACs containing genes implicated in cell division and in cell wall biosynthesis, as well as a series of known genes mapping to different regions of the PAO1 chromosome. A physical and genetic map was constructed for the 98-kb pMOC5 BAC clone, which spans the entire fts-mur locus. Chromosome walking from each end of the pMOC5 clone placed it within a contig spanning 243 kb. The BAC library and screening resources now allow a PCR-based screening of a P. aeruginosa genomic library for any gene of interest. The restriction fragment analysis of overlapping clones indicated that BAC clones stably maintain and propagate Pseudomonas DNA, providing evidence that the PAO1 BAC library is an appropriate reagent for genome sequencing.

Characterization of the ftsZ cell division gene of Neisseria gonorrhoeae : expression in Escherichia coli and N. gonorrhoeae

“…This gene order was confirmed by PCR analysis (data not shown). In E. coli, H. influenzae and P. aeruginosa, envA is located downstream of ftsZ (Lutkenhaus et al 1980;Fleishmann et al 1995;Dewar et al 1998); however, after a BLAST search of raw sequence data from the N. gonorrhoeae FA1090 genome project, an envA homologue was located elsewhere in the gonococcal genome and was not associated with the dcw cluster.…”

Comparative Sequence Analysis of the Symbiosis Island ofMesorhizobium lotiStrain R7A

“…Ligations were transformed into Escherichia coli DH10B by electroporation as described previously (52). Sequences obtained from plasmid subclones of cosmids which overlapped the SpeI fragments were used as described previously (11) to design PCR primers to screen BAC clones directly from cultures of pooled clones. Selected BAC clones were sized on PFGE gels and used to probe Southern blots of PFGE gels containing genomic SpeI digests of diverse strains of mesorhizobia containing the island, to confirm that they hybridized to island SpeI fragments of the expected size.…”

Extraction of DNA from soil

“…This approach was used to establish libraries of BAC clones containing large DNA fragments from several microorganisms including Methanosarcina thermophila [14], Bacillus cereus [57], Mycobacterium tuberculosis [5] and Pseudomonas aeruginosa [13]. An integrated approach combining centrifugation-based cell separation from soil particles, in plug lysis and pulsed field gel electrophoresis (PFGE) has been successfully applied to non-culturable bacteria from environmental samples [45].…”