A versatile element for gene addition in bacterial chromosomes.

The increasing interest in genetic manipulation of bacterial host metabolic pathways for protein or small molecule production has led to a need to add new genes to a chromosome quickly and easily without leaving behind a selectable marker. The present report describes a vector and four-day procedure that enable site-specific chromosomal insertion of cloned genes in a context insulated from external transcription, usable once in a construction series. The use of rhamnose-inducible transcription from rhaBp allows regulation of the inserted genes independently of the commonly used IPTG and arabinose strategies. Using lacZ as a reporter, we first show that expression from the rhamnose promoter is tightly regulatable, exhibiting very low leakage of background expression compared with background, and moderate rhamnose-induced expression compared with IPTG-induced expression from lacp. Second, the expression of a DNA methyltransferase was used to show that rhamnose regulation yielded on-off expression of this enzyme, such that a resident high-copy plasmid was either fully sensitive or fully resistant to isoschizomer restriction enzyme cleavage. In both cases, growth medium manipulation allows intermediate levels of expression. The vehicle can also be adapted as an ORF-cloning vector.

Evolution of bacterial phosphoglycerate mutases: non-homologous isofunctional enzymes undergoing gene losses, gains and lateral transfers.

BACKGROUND: The glycolytic phosphoglycerate mutases exist as non-homologous isofunctional enzymes (NISE) having independent evolutionary origins and no similarity in primary sequence, 3D structure, or catalytic mechanism. Cofactor-dependent PGM (dPGM) requires 2,3-bisphosphoglycerate for activity; cofactor-independent PGM (iPGM) does not. The PGM profile of any given bacterium is unpredictable and some organisms such as Escherichia coli encode both forms. METHODS/PRINCIPAL FINDINGS: To examine the distribution of PGM NISE throughout the Bacteria, and gain insight into the evolutionary processes that shape their phyletic profiles, we searched bacterial genome sequences for the presence of dPGM and iPGM. Both forms exhibited patchy distributions throughout the bacterial domain. Species within the same genus, or even strains of the same species, frequently differ in their PGM repertoire. The distribution is further complicated by the common occurrence of dPGM paralogs, while iPGM paralogs are rare. Larger genomes are more likely to accommodate PGM paralogs or both NISE forms. Lateral gene transfers have shaped the PGM profiles with intradomain and interdomain transfers apparent. Archaeal-type iPGM was identified in many bacteria, often as the sole PGM. To address the function of PGM NISE in an organism encoding both forms, we analyzed recombinant enzymes from E. coli. Both NISE were active mutases, but the specific activity of dPGM greatly exceeded that of iPGM, which showed highest activity in the presence of manganese. We created PGM null mutants in E. coli and discovered the ΔdPGM mutant grew slowly due to a delay in exiting stationary phase. Overexpression of dPGM or iPGM overcame this defect. CONCLUSIONS/SIGNIFICANCE: Our biochemical and genetic analyses in E. coli firmly establish dPGM and iPGM as NISE. Metabolic redundancy is indicated since only larger genomes encode both forms. Non-orthologous gene displacement can fully account for the non-uniform PGM distribution we report across the bacterial domain.

Cassette-like variation of restriction enzyme genes in Escherichia coli C and relatives.

A surprising result of comparative bacterial genomics has been the large amount of DNA found to be present in one strain but not in another of the same species. We examine in detail one location where gene content varies extensively, the restriction cluster in Escherichia coli. This region is designated the Immigration Control Region (ICR) for the density and variability of restriction functions found there. To better define the boundaries of this variable locus, we determined the sequence of the region from a restrictionless strain, E.coli C. Here we compare the 13.7 kb E.coli C sequence spanning the site of the ICR with corresponding sequences from five E.coli strains and Salmonella typhimurium LT2. To discuss this variation, we adopt the term 'framework' to refer to genes that are stable components of genomes within related lineages, while 'migratory' genes are transient inhabitants of the genome. Strikingly, seven different migratory DNA segments, encoding different sets of genes and gene fragments, alternatively occupy a single well-defined location in the seven strains examined. The flanking framework genes, yjiS and yjiA, display approximately normal patterns of conservation. The patterns observed are consistent with the action of a site-specific recombinase. Since no nearby gene codes for a likely recombinase of known families, such a recombinase must be of a new family or unlinked.