Characterization of multiple novel aerobic polychlorinated biphenyl (PCB)-utilizing bacterial strains indigenous to contaminated tropical African soils

Contaminated sites in Lagos, Nigeria were screened for the presence of chlorobiphenyl-degrading bacteria. The technique of continual enrichment on Askarel fluid yielded bacterial isolates able to utilize dichlorobiphenyls (diCBs) as growth substrates and six were selected for further studies. Phenotypic typing and 16S rDNA analysis classified these organisms as species of Enterobacter, Ralstonia and Pseudomonas. All the strains readily utilized a broad spectrum of xenobiotics as sole sources of carbon and energy. Growth was observed on all monochlorobiphenyls (CBs), 2,2′-, 2,3-, 2,4′-, 3,3′- and 3,5-diCB as well as di- and trichlorobenzenes Growth was also sustainable on Askarel electrical transformer fluid and Aroclor 1221. Time-course studies using 100 ppm of 2-, 3- or 4-CB resulted in rapid exponential increases in cell numbers and CB transformation to respective chlorobenzoates (CBAs) within 70 h. Significant amounts of chloride were recovered in culture media of cells incubated with 2-CB and 3-CB, suggesting susceptibilities of both 2- and 3-chlorophenyl rings to attack, while the 4-CB was stoichiometrically transformed to 4-CBA. Extensive degradation of most of the congeners in Aroclor 1221 was observed when isolates were cultivated with the mixture as a sole carbon source. Aroclor 1221 was depleted by a minimum of 51% and maximum of 71%. Substantial amounts of chloride eliminated from the mixture ranged between 15 and 43%. These results suggest that some contaminated soils in the tropics may contain exotic micro-organisms whose abilities and potentials are previously unknown. An understanding of these novel strains therefore, may help answer questions about the microbial degradation of polychlorinated biphenyls (PCBs) in natural systems and enhance the potential use of bioremediation as an effective tool for cleanup of PCB-contaminated soils.     

A novel scheme to assess factors involved in the reproducibility of DNA-microarray data

Background  

In research laboratories using DNA-microarrays, usually a number of researchers perform experiments, each generating possible sources of error. There is a need for a quick and robust method to assess data quality and sources of errors in DNA-microarray experiments. To this end, a novel and cost-effective validation scheme was devised, implemented, and employed.     

Listening in on bacteria: acyl-homoserine lactone signalling

Bacterial cell-to-cell signalling has emerged as a new area in microbiology. Individual bacterial cells communicate with each other and co-ordinate group activities. Although a lot of detail is known about the mechanisms of a few well-characterized bacterial communication systems, other systems have been discovered only recently. Bacterial intercellular communication has become a target for the development of new anti-virulence drugs.     

Phosphorus limitation enhances biofilm formation of the plant pathogen Agrobacterium tumefaciens through the PhoR-PhoB regulatory system

The plant pathogen Agrobacterium tumefaciens forms architecturally complex biofilms on inert surfaces. Adherence of A. tumefaciens C58 was significantly enhanced under phosphate limitation compared to phosphate-replete conditions, despite slower overall growth under low-phosphate conditions. Replacement of Pi with sn-glycerol-3-phosphate and 2-aminoethylphosphonate yielded similar results. The increase in surface interactions under phosphate limitation was observed in both static culture and continuous-culture flow cells. Statistical analysis of confocal micrographs obtained from the flow cell biofilms revealed that phosphate limitation increased both the overall attached biomass and the surface coverage, whereas the maximum thickness of the biofilm was not affected. Functions encoded on the two large plasmids of A. tumefaciens C58, pTiC58 and pAtC58, were not required for the observed phosphate effect. The phosphate concentration at which increased attachment was observed triggered the phosphate limitation response, controlled in many bacteria by the two-component regulatory system PhoR-PhoB. The A. tumefaciens phoB and phoR orthologues could only be disrupted in the presence of plasmid-borne copies of the genes, suggesting that this regulatory system might be essential. Expression of the A. tumefaciens phoB gene from a tightly regulated inducible promoter, however, correlated with the amount of biofilm under both phosphate-limiting and nonlimiting conditions, demonstrating that components of the Pho regulon influence A. tumefaciens surface interactions.     

A simple screening protocol for the identification of quorum signal antagonists

Quorum sensing (QS) is a mechanism by which diverse microorganisms can control specific processes in response to population density. A relatively well-known form of QS among Proteobacteria involves production and subsequent response to acylated homoserine lactones (AHLs). Quorum sensing inhibition (QSI), targeting AHL-dependent signaling, has been reported as a strategy for the control of biofilm formation used by several marine organisms. We developed a simple soft agar overlay protocol, based on pigmentation inhibition, to rapidly screen for the presence of potential QSI by bacteria and plants. For bacterial screens, test organisms are first streaked onto their appropriate media and incubated overnight. For plant screens, the plant material (leaf, stem, flower, etc.) is placed onto LB agar. The bacterial growth or plant samples are then covered with an overlay of LB soft agar containing an inoculum of either Pseudomonas aureofaciens 30-84 or Chromobacterium violaceum ATCC 12472 (indicator cultures) and then incubated overnight. These indicator bacteria regulate pigment production by N-hexanoyl-HSL (C6-HSL) QS and are readily inhibited by AHL analogues and other antagonists. QSI is indicated by the lack of pigment production of the indicator culture in the vicinity of the test sample. Growth inhibition of the indicator culture indicates possible antibiotic production. Two different biosensor organisms based on derivatives of Agrobacterium tumefaciens and C. violaceum, capable of detecting a range of AHLs were used to determine whether QSI is due to the production of interfering AHLs competing with the C6-HSL regulation of C. violaceum and P. aureofaciens pigment production. This simple protocol will facilitate the screening of multiple organisms for the production of potential antifouling compounds.     

Resource and competitive dynamics shape the benefits of public goods cooperation in a plant pathogen

Cooperative benefits depend on a variety of ecological factors. Many cooperative bacteria increase the population size of their groups by making a public good available. Increased local population size can alleviate the constraints of kin competition on the evolution of cooperation by enhancing the between-group fitness of cooperators. The cooperative pathogenesis of Agrobacterium tumefaciens causes infected plants to exude opines--resources that provide a nearly exclusive source of nutrient for the pathogen. We experimentally demonstrate that opines provide cooperative A. tumefaciens cells a within-group fitness advantage over saprophytic agrobacteria. Our results are congruent with a resource-consumer competition model, which predicts that cooperative, virulent agrobacteria are at a competitive disadvantage when opines are unavailable, but have an advantage when opines are available at sufficient levels. This model also predicts that freeloading agrobacteria that catabolize opines but cannot infect plants competitively displace the cooperative pathogen from all environments. However, we show that these cooperative public goods also promote increased local population size. A model built from the Price Equation shows that this effect on group size can contribute to the persistence of cooperative pathogenesis despite inherent kin competition for the benefits of pathogenesis.