Cultivation of hard-to-culture subsurface mercury-resistant bacteria and discovery of new merA gene sequences.

Mercury-resistant bacteria may be important players in mercury biogeochemistry. To assess the potential for mercury reduction by two subsurface microbial communities, resistant subpopulations and their merA genes were characterized by a combined molecular and cultivation-dependent approach. The cultivation method simulated natural conditions by using polycarbonate membranes as a growth support and a nonsterile soil slurry as a culture medium. Resistant bacteria were pregrown to microcolony-forming units (mCFU) before being plated on standard medium. Compared to direct plating, culturability was increased up to 2,800 times and numbers of mCFU were similar to the total number of mercury-resistant bacteria in the soils. Denaturing gradient gel electrophoresis analysis of DNA extracted from membranes suggested stimulation of growth of hard-to-culture bacteria during the preincubation. A total of 25 different 16S rRNA gene sequences were observed, including Alpha-, Beta-, and Gammaproteobacteria; Actinobacteria; Firmicutes; and Bacteroidetes. The diversity of isolates obtained by direct plating included eight different 16S rRNA gene sequences (Alpha- and Betaproteobacteria and Actinobacteria). Partial sequencing of merA of selected isolates led to the discovery of new merA sequences. With phylum-specific merA primers, PCR products were obtained for Alpha- and Betaproteobacteria and Actinobacteria but not for Bacteroidetes and Firmicutes. The similarity to known sequences ranged between 89 and 95%. One of the sequences did not result in a match in the BLAST search. The results illustrate the power of integrating advanced cultivation methodology with molecular techniques for the characterization of the diversity of mercury-resistant populations and assessing the potential for mercury reduction in contaminated environments.

Recovery of GFP-labeled bacteria for culturing and molecular analysis after cell sorting using a benchtop flow cytometer.

Exciting opportunities exist for the application of simple fluorescence-activated cell sorting (FACS) to microbiology. The technology is widely available, but critical reports on the efficiency of cell sorting using benchtop instruments are lacking. It is vital that single cell sorting be of the highest purity possible. If purity is compromised detrital material or unwanted cells will be captured along with target cells of interest. Here, the isolation of fluorescent bacteria using a benchtop FACSCalibur-sort flow cytometer is described. The efficiency and purity of isolated cells was determined using fluorescence microscopy, culturing, and molecular analysis. To achieve high purity it was essential that the total event rate did not exceed 300 cells per second. This instrument was capable of recovering >55% sorted Escherichia coli cells, coupled with a purity exceeding 99%. However, the purity of recovered cells was substantially reduced (<25%) when the event rate increased. Cell sorting onto polycarbonate membranes did not reduce the ability of E. coli to form colonies, and sorting of ~1000 E. coli cells was sufficient for 16S rDNA amplification. Additionally, as few as 100 isolated Erwinia sp. carrying the gfp gene were amplified using seminested PCR targeting the single copy gfp gene. With such low numbers of bacteria being required for molecular identification, FACS can be achieved without the requirement for high-speed droplet cell sorters.

Presence of N-acyl homoserine lactones in soil detected by a whole-cell biosensor and flow cytometry.

Quorum sensing enables bacteria to regulate expression of certain genes according to population density. N-acyl homoserine lactone (AHL)-based quorum sensing is known to be widespread among gram-negative bacteria. Several bacterial whole-cell biosensors for AHL detection have been developed and some were used in in situ studies of AHL production. From these studies our knowledge of the significance of quorum sensing in various environments has been improved. However, very little is known about production of AHLs in soil environments. In the present study, an approach for detecting AHL production in bulk soil was developed. A whole-cell biosensor based on the regulatory region of the lux-operon from Vibrio fischeri fused to gfp was constructed, resulting in a luxR-PluxI-gfpmut3*-fusion in the high copy plasmid, pAHL-GFP. Escherichia coli MC4100 harboring pAHL-GFP responded to the AHL-compound N-octanoyl homoserine lactone (OHL) by expressing green fluorescence. In situ application of E. coli MC4100/pAHL-GFP was tested by adding OHL in different concentrations to sterile soil microcosms. E. coli MC4100/pAHL-GFP were incubated in the soil microcosms and extracted by an improved Nycodenz-extraction method optimized for flow cytometry. The presence of induced cells was then verified by single-cell analysis by flow cytometry. OHL concentrations between 0.5 and 50 nmol per g soil were detected. When introducing the AHL-producing Serratia liquefaciens to soil microcosms, expression of green fluorescent protein was induced in E. coli MC4100/pAHL-GFP. Thereby, the ability of this strain to detect excretion of AHLs by S. liquefaciens in sterile soil was shown. The use of an improved extraction method and a whole-cell biosensor combined with flow cytometry analysis proved to be promising tools in future studies of AHL production by microbial populations in soil environments.