Tools for high efficiency genetic manipulation of the human pathogen Penicillium marneffei.

Penicillium marneffei is an opportunistic pathogen of humans and displays a temperature dependent dimorphic transition. Like many fungi, exogenous DNA introduced by DNA mediated transformation is integrated randomly into the genome resulting in inefficient gene deletion and position-specific effects. To enhance successful gene targeting, the consequences of perturbing components of the non-homologous end joining recombination pathway have been examined. The deletion of the KU70 and LIG4 orthologs, pkuA and ligD, respectively, dramatically enhanced the observed homologous recombination frequency leading to efficient gene deletion. While ΔpkuA was associated with reduced genetic stability over-time, ΔligD represents a suitable recipient strain for downstream applications and combined with a modified Gateway™ system for the rapid generation of gene deletion constructs, this represents an efficient pipeline for characterizing gene function in P. marneffei.

Evolution of microcin V and colicin Ia plasmids in Escherichia coli.

Survey results and genotypic characterization of Escherichia coli strains demonstrate that the bacteriocins colicin Ia and microcin V coassociate in a strain more often than would be expected by chance. When these two bacteriocins co-occur, they are encoded on the same conjugative plasmid. Plasmids encoding colicin Ia and microcin V are nonrandomly distributed with respect to the genomic background of the host strain. Characterization of microcin V and colicin Ia nucleotide variation, together with the backbone of plasmids encoding these bacteriocins, indicates that the association has evolved on multiple occasions and involves the movement of the microcin V operon, together with the genes iroNEDCB and iss, onto a nonrandom subset of colicin Ia plasmids. The fitness advantage conferred on cells encoding both colicin Ia and microcin V has yet to be determined.