Recombination proteins differently control the acquisition of homeologous DNA during Bacillus subtilis natural chromosomal transformation

In naturally competent Bacillus subtilis cells the acquisition of closely related genes occurs via homology-directed chromosomal transformation (CT), and its frequency decreases log-linearly with increased sequence divergence (SD) up to 15%. Beyond this and up to 23% SD the interspecies boundary prevails, the CT frequency marginally decreases, and short (<10-nucleotides) segments are integrated via homology-facilitated micro-homologous integration. Both poorly known CT mechanisms are RecA-dependent. Here we identify the recombination proteins required for the acquisition of interspecies DNA. The absence of AddAB, RecF, RecO, RuvAB or RecU, crucial for repair-by-recombination, does not affect CT. However, inactivation of dprA, radA, recJ, recX or recD2 strongly interfered with CT. Interspecies CT was abolished beyond ~8% SD in ΔdprA, ~10% in ΔrecJ, ΔradA, ΔrecX and 14% in ΔrecD2 cells. We propose that DprA, RecX, RadA/Sms, RecJ and RecD2 help RecA to unconstrain speciation and gene flow. These functions are ultimately responsible for generating genetic diversity and facilitate CT and gene acquisition from bacteria of the same genus.

Natural transformation of the filamentous cyanobacterium Phormidium lacuna

Research for biotechnological applications of cyanobacteria focuses on synthetic pathways and bioreactor design, while little effort is devoted to introduce new, promising organisms in the field. Applications are most often based on recombinant work, and the establishment of transformation can be a risky, time-consuming procedure. In this work we demonstrate the natural transformation of the filamentous cyanobacterium Phormidium lacuna and insertion of a selection marker into the genome by homologous integration. This is the first example for natural transformation of a member of the order Oscillatoriales. We found that Phormidium lacuna is polyploid, each cell has about 20-100 chromosomes. Transformed filaments were resistant against up to 15 mg/ml of kanamycin, and the high resistance feature allowed for rapid segregation into all chromosomes. Formerly, natural transformation in cyanobacteria has been considered a rare and exclusive feature of a few unicellular species. Our finding suggests that natural competence is more distributed among cyanobacteria than previously thought. This is supported by bioinformatic analyses which show that all protein factors for natural transformation are present in the majority of the analyzed cyanobacteria.

Allelic Exchange in Actinomyces oris with mCherry Fluorescence Counterselection

ABSTRACT Described here is a method for facile generation of markerless gene deletion mutants of Actinomyces oris. Homologous integration of a nonreplicative vector carrying a gene exchange cassette into the bacterial chromosome was selected for by using mCherry fluorescence and resistance to kanamycin. Completion of allelic replacement was counterselected for by using loss of fluorescence.

The akuBKU80 Mutant Deficient for Nonhomologous End Joining Is a Powerful Tool for Analyzing Pathogenicity in Aspergillus fumigatus

ABSTRACT To increase the frequency of homologous recombination, we inactivated the KU80 homologue in Aspergillus fumigatus (named akuB KU80). Homologous integration reached about 80% for both calcineurin A (calA) and polyketide synthase pksP (alb1) genes in the akuB KU80 mutant to 3 and 5%, respectively, when using a wild-type A. fumigatus strain. Deletion of akuB KU80 had no influence on pathogenicity in a low-dose murine infection model.