Mercuric reductase genes (merA) and mercury resistance plasmids in High Arctic snow, freshwater and sea-ice brine.

Bacterial reduction in Hg(2+) to Hg(0) , mediated by the mercuric reductase (MerA), is important in the biogeochemical cycling of Hg in temperate environments. Little is known about the occurrence and diversity of merA in the Arctic. Seven merA determinants were identified among bacterial isolates from High Arctic snow, freshwater and sea-ice brine. Three determinants in Bacteriodetes, Firmicutes and Actinobacteria showed < 92% (amino acid) sequence similarity to known merA, while one merA homologue in Alphaproteobacteria and 3 homologues from Betaproteobacteria and Gammaproteobacteria were > 99% similar to known merA's. Phylogenetic analysis showed the Bacteroidetes merA to be part of an early lineage in the mer phylogeny, whereas the Betaproteobacteria and Gammaproteobacteria merA appeared to have evolved recently. Several isolates, in which merA was not detected, were able to reduce Hg(2+) , suggesting presence of unidentified merA genes. About 25% of the isolates contained plasmids, two of which encoded mer operons. One plasmid was a broad host-range IncP-α plasmid. No known incompatibility group could be assigned to the others. The presence of conjugative plasmids, and an incongruent distribution of merA within the taxonomic groups, suggests horizontal transfer of merA as a likely mechanism for High Arctic microbial communities to adapt to changing mercury concentration.

Diversity and characterization of mercury-resistant bacteria in snow, freshwater and sea-ice brine from the High Arctic.

It is well-established that atmospheric deposition transports mercury from lower latitudes to the Arctic. The role of bacteria in the dynamics of the deposited mercury, however, is unknown. We characterized mercury-resistant bacteria from High Arctic snow, freshwater and sea-ice brine. Bacterial densities were 9.4 × 10(5), 5 × 10(5) and 0.9-3.1 × 10(3) cells mL(-1) in freshwater, brine and snow, respectively. Highest cultivability was observed in snow (11.9%), followed by freshwater (0.3%) and brine (0.03%). In snow, the mercury-resistant bacteria accounted for up to 31% of the culturable bacteria, but <2% in freshwater and brine. The resistant bacteria belonged to the Alpha-, Beta- and Gammaproteobacteria, Firmicutes, Actinobacteria, and Bacteriodetes. Resistance levels of most isolates were not temperature dependent. Of the resistant isolates, 25% reduced Hg(II) to Hg(0). No relation between resistance level, ability to reduce Hg(II) and phylogenetic group was observed. An estimation of the potential bacterial reduction of Hg(II) in snow suggested that it was important in the deeper snow layers where light attenuation inhibited photoreduction. Thus, by reducing Hg(II) to Hg(0), mercury-resistant bacteria may limit the supply of substrate for methylation processes and, hence, contribute to lowering the risk that methylmercury is being incorporated into the Arctic food chains.

Physiological osmotic induction of Leptospira interrogans adhesion: LigA and LigB bind extracellular matrix proteins and fibrinogen.

Transmission of leptospirosis occurs through contact of mucous membranes and abraded skin with freshwater contaminated by pathogenic Leptospira spp. Exposure to physiological osmolarity induces leptospires to express high levels of the Lig surface proteins containing imperfect immunoglobulin-like repeats that are shared or differ between LigA and LigB. We report that osmotic induction of Lig is accompanied by 1.6- to 2.5-fold increases in leptospiral adhesion to immobilized extracellular matrix and plasma proteins, including collagens I and IV, laminin, and especially fibronectin and fibrinogen. Recombinant LigA-unique and LigB-unique repeat proteins bind to these same host ligands. We found that the avidity of LigB in binding fibronectin is comparable to that of the Staphylococcus aureus FnBPA D repeats. Both LigA- and LigB-unique repeats interact with the amino-terminal fibrin- and gelatin-binding domains of fibronectin, which are also recognized by fibronectin-binding proteins mediating the adhesion of other microbial pathogens. In contrast, repeats common to both LigA and LigB do not bind these host proteins, and nonrepeat sequences in the carboxy-terminal domain of LigB show only weak interaction with fibronectin and fibrinogen. A functional role for the binding activity of LigA and LigB is suggested by the ability of the recombinants to inhibit leptospiral adhesion to fibronectin by 28% and 21%, respectively. The binding of LigA and LigB to multiple ligands present in different tissues suggests that these adhesins may be involved in the initial colonization and dissemination stages of leptospirosis. The characterization of the Lig adhesin function should aid the design of Lig-based vaccines and serodiagnostic tests.

Use of electrochemical DNA biosensors for rapid molecular identification of uropathogens in clinical urine specimens.

We describe the first species-specific detection of bacterial pathogens in human clinical fluid samples using a microfabricated electrochemical sensor array. Each of the 16 sensors in the array consisted of three single-layer gold electrodes-working, reference, and auxiliary. Each of the working electrodes contained one representative from a library of capture probes, each specific for a clinically relevant bacterial urinary pathogen. The library included probes for Escherichia coli, Proteus mirabilis, Pseudomonas aeruginosa, Enterocococcus spp., and the Klebsiella-Enterobacter group. A bacterial 16S rRNA target derived from single-step bacterial lysis was hybridized both to the biotin-modified capture probe on the sensor surface and to a second, fluorescein-modified detector probe. Detection of the target-probe hybrids was achieved through binding of a horseradish peroxidase (HRP)-conjugated anti-fluorescein antibody to the detector probe. Amperometric measurement of the catalyzed HRP reaction was obtained at a fixed potential of -200 mV between the working and reference electrodes. Species-specific detection of as few as 2,600 uropathogenic bacteria in culture, inoculated urine, and clinical urine samples was achieved within 45 min from the beginning of sample processing. In a feasibility study of this amperometric detection system using blinded clinical urine specimens, the sensor array had 100% sensitivity for direct detection of gram-negative bacteria without nucleic acid purification or amplification. Identification was demonstrated for 98% of gram-negative bacteria for which species-specific probes were available. When combined with a microfluidics-based sample preparation module, the integrated system could serve as a point-of-care device for rapid diagnosis of urinary tract infections.

An Escherichia coli MG1655 lipopolysaccharide deep-rough core mutant grows and survives in mouse cecal mucus but fails to colonize the mouse large intestine.

The ability of E. coli strains to colonize the mouse large intestine has been correlated with their ability to grow in cecal and colonic mucus. In the present study, an E. coli MG1655 strain was mutagenized with a mini-Tn5 Km (kanamycin) transposon, and mutants were tested for the ability to grow on agar plates with mouse cecal mucus as the sole source of carbon and nitrogen. One mutant, designated MD42 (for mucus defective), grew poorly on cecal-mucus agar plates but grew well on Luria agar plates and on glucose minimal-agar plates. Sequencing revealed that the insertion in MD42 was in the waaQ gene, which is involved in lipopolysaccharide (LPS) core biosynthesis. Like "deep-rough" E. coli mutants, MD42 was hypersensitive to sodium dodecyl sulfate (SDS), bile salts, and the hydrophobic antibiotic novobiocin. Furthermore, its LPS core oligosaccharide was truncated, like that of a deep-rough mutant. MD42 initially grew in the large intestines of streptomycin-treated mice but then failed to colonize (<10(2) CFU per g of feces), whereas its parent colonized at levels between 10(7) and 10(8) CFU per g of feces. When mouse cecal mucosal sections were hybridized with an E. coli-specific rRNA probe, MD42 was observed in cecal mucus as clumps 24 h postfeeding, whereas its parent was present almost exclusively as single cells, suggesting that clumping may play a role in preventing MD42 colonization. Surprisingly, MD42 grew nearly as well as its parent during growth in undiluted, highly viscous cecal mucus isolated directly from the mouse cecum and, like its parent, survived well after reaching stationary phase, suggesting that there are no antimicrobials in mucus that prevent MD42 colonization. After mini-mariner transposon mutagenesis, an SDS-resistant suppressor mutant of MD42 was isolated. The mini-mariner insertion was shown to be in the bipA gene, a known regulator of E. coli surface components. When grown in Luria broth, the LPS core of the suppressor mutant remained truncated; however, the LPS core was not truncated when the suppressor mutant was grown in the presence of SDS. Moreover, when the suppressor mutant was grown in the presence of SDS and fed to mice, it colonized the mouse large intestine. Collectively, the data presented here suggest that BipA may play a role in E. coli MG1655 LPS core biosynthesis and that because MD42 forms clumps in intestinal mucus, it is unable to colonize the mouse large intestine.