TraA is required for megaplasmid conjugation in Rhodococcus erythropolis AN12.

Pulsed-field gel electrophoresis (PFGE) revealed three previously uncharacterized megaplasmids in the genome of Rhodococcus erythropolis AN12. These megaplasmids, pREA400, pREA250, and pREA100, are approximately 400, 250, and 100kb, respectively, based on their migration in pulsed-field gels. Genetic screening of an AN12 transposon insertion library showed that two megaplasmids, pREA400, and pREA250, are conjugative. Mobilization frequencies of these AN12 megaplasmids to recipient R. erythropolis SQ1 were determined to be approximately 7x10(-4) and 5x10(-4) events per recipient cell, respectively. It is known for other bacterial systems that a relaxase encoded by the traA gene is required to initiate DNA transfer during plasmid conjugation. Sequences adjacent to the transposon insertion in megaplasmid pREA400 revealed a putative traA-like open reading frame. A targeted gene disruption method was developed to generate a traA mutation in AN12, which allowed us to address the role of the traA gene product for Rhodococcus megaplasmid conjugation. We found that the AN12 traA mutant is no longer capable of transferring the pREA400 megaplasmid to SQ1. Furthermore, we confirmed that the conjugation defect was specifically due to the disruption of the traA gene, as pREA400 megaplasmid conjugation defect is restored with a complementing copy of the traA gene.

Bacterial communities in industrial wastewater bioreactors.

Wastewater bioreactors have been used to treat domestic and industrial waste for nearly a century. Development of molecular tools such as PCR and DNA microarrays have enabled identification and characterization of some of the microbes in these bioreactors; however, molecular characterization of the microbes is still in its infancy, and only a few of the molecular tools have been applied to improving performance of wastewater bioreactors at the commercial level. Several new plasmids and enzymes have been isolated from wastewater bioreactors. There is enormous opportunity to use the microbes from wastewater for industrial bioprocesses.

Genetic organization of a plasmid from an industrial wastewater bioreactor.

Pseudomonas strain CT14 was isolated from activated sludge. Strain CT14 contained a 55, 216 bp plasmid that was characterized by sequence analysis. The plasmid had a modular structure with 51 open reading frames (ORFs) that were distributed between two clearly demarcated domains. Domain I primarily contained genes for plasmid-related functions and a novel origin of replication. Domain II bore evidence of extensive transposition and recombination. Domain II contained several genes from a meta-cleavage pathway for aromatic rings. These genes appeared to have been recruited from different hosts. This observation suggests that sequencing pCT14 may have revealed an intermediate stage in the evolution of a new assemblage of meta-cleavage pathway genes.

Inhibition of Vorticella microstoma stalk formation by wheat germ agglutinin.

Fluorescently labeled conjugates of wheat germ agglutinin and concanavalin A stained the contractile stalk but not the cell body of Vorticella microstoma trophonts. Binding of the fluorescent conjugants did not noticeably alter the activity of the trophonts. However, unconjugated wheat germ agglutinin prevented free swimming telotrochs from adhering to a glass surface and deploying a contractile stalk during differentiation into trophonts. These observations indicated that the stalk, the material that binds the stalk to surfaces, and the precursors for these components have saccharide residues in common.