Genetic Analysis of Bacterial Gene Variations in Sulfidic Springs and the Influence on Geochemistry

Culture-independent methods have revolutionized environmental microbiology and geomicrobiology studies and next-generation sequencing and metagenomics techniques continue to reveal the vast genetic diversity of microorganisms. But, these approaches provide comparatively little quantitative information about the roles that naturally occurring microbial gene variations play in local biogeochemical cycling. The goal of this study was to identify how the physical distribution and genetic diversity of microbial genes within a habitat impact environmental geochemistry by examining the biogeography of 16S rRNA genes and bacterial sulfur oxidation (Sox) genes in terrestrial sulfidic springs. 16S rRNA gene pyrosequences were obtained from microbial mats inhabiting eight sulfidic springs in the United States. Pyrosequences were clustered to determine occurrence patterns and the results found that most of the 16S rRNA genes were unique to the springs from which they were retrieved. The occurrence of microbial populations at each spring correlated to specific geochemical conditions, particularly among putative sulfur-oxidizing bacterial groups. Closer examination of a single outflow channel from Rattlesnake Spring in Oklahoma (USA) using twenty-five samples revealed that the distribution of microbial populations were locally distributed and correlated to geochemical conditions. However, cluster-based genetic analysis from this site indicated that inherent biases associated with point sampling affect interpretations of microbial biogeographic patterns. Evaluation of soxB genes from the Rattlesnake Spring outflow channel indicated that the occurrences and distribution patterns of the genes correlated to geochemical conditions within the spring, like the 16S rRNA genes. However, analysis of the translated soxB gene amino acid sequences suggested that the SoxB enzymes could be functionally different. The spatial distribution of the soxB gene variants along the outflow channel could indicate niche partitioning among the bacteria that oxidize reduced sulfur compounds within the spring because the genes could be optimized to specific geochemical conditions and/or reduced sulfur substrates. The identification of gene variations that potentially encode for functionally different enzymes implies that it may be inappropriate to infer function based on DNA or amino acid sequence similarity

Biogeographic congruency among bacterial communities from terrestrial sulfidic springs

Terrestrial sulfidic springs support diverse microbial communities by serving as stable conduits for geochemically diverse and nutrient-rich subsurface waters. Microorganisms that colonize terrestrial springs likely originate from groundwater, but may also be sourced from the surface. As such, the biogeographic distribution of microbial communities inhabiting sulfidic springs should be controlled by a combination of spring geochemistry and surface and subsurface transport mechanisms, and not necessarily geographic proximity to other springs. We examined the bacterial diversity of seven springs to test the hypothesis that occurrence of taxonomically similar microbes, important to the sulfur cycle, at each spring is controlled by geochemistry. Complementary Sanger sequencing and 454 pyrosequencing of 16S rRNA genes retrieved five proteobacterial classes, and Bacteroidetes, Chlorobi, Chloroflexi, and Firmicutes phyla from all springs, which suggested the potential for a core sulfidic spring microbiome. Among the putative sulfide-oxidizing groups (Epsilonproteobacteria and Gammaproteobacteria), up to 83% of the sequences from geochemically similar springs clustered together. Abundant populations of Hydrogenimonas-like or Sulfurovum-like spp. (Epsilonproteobacteria) occurred with abundant Thiothrix and Thiofaba spp. (Gammaproteobacteria), but Arcobacter-like and Sulfurimonas spp. (Epsilonproteobacteria) occurred with less abundant gammaproteobacterial populations. These distribution patterns confirmed that geochemistry rather than biogeography regulates bacterial dominance at each spring. Potential biogeographic controls were related to paleogeologic sedimentation patterns that could control long-term microbial transport mechanisms that link surface and subsurface environments. Knowing the composition of a core sulfidic spring microbial community could provide a way to monitor diversity changes if a system is threatened by anthropogenic processes or climate change

Transport of E. coli D21g with runoff water under different solution chemistry conditions and surface slopes

Tracer and indicator microbe runoff experiments were conducted to investigate the influence of solution chemistry on the transport, retention, and release of Escherichia coli D21g. Experiments were conducted in a chamber (2.25 m long, 0.15 m wide, and 0.16 m high) packed with ultrapure quartz sand (to a depth of 0.10 m) that was placed on a metal frame at slopes of 5.6%, 8.6%, or 11.8%. Runoff studies were initiated by adding a step pulse of salt tracer or D21g suspension at a steady flow rate to the top side of the chamber and then monitoring the runoff effluent concentrations. The runoff breakthrough curves (BTCs) were asymmetric and exhibited significant amounts of concentration tailing. The peak concentration levels were lower and the concentration tailing was higher with increasing chamber slope because of greater amounts of exchange with the sand and/or extents of physical nonequilibrium (e.g., water flow in rills and incomplete mixing) in the runoff layer. Lower amounts of tailing in the runoff BTC and enhanced D21g retention in the sand occurred when the solution ionic strength (IS) was 100 mM NaCl compared with 1 mM NaCl, due to compression of the double layer thickness which eliminated the energy barrier to attachment. Retained cells were slowly released to the runoff water when the IS of the runoff water was reduced to deionized water. The amount and rate of cell release was greatest at the highest chamber slope, which controlled the amount of exchange with the sand and/or the extent of physical nonequilibrium in the runoff layer, and the amount of retained cells. The observed runoff BTCs were well described using a transient storage model, but fitted parameters were not always physically realistic. A model that accounted for the full coupling between flow and transport in the runoff and sand layers provided useful information on exchange processes at the sand surface, but did not accurately describe the runoff BTCs which were influenced by physical nonequilibrium in the runoff layer

Physicochemical Factors That Favor Conjugation of an Antibiotic Resistant Plasmid in Non-growing Bacterial Cultures in the Absence and Presence of Antibiotics

Horizontal gene transfer (HGT) of antibiotic resistance genes has received increased scrutiny from the scientific community in recent years owing to the public health threat associated with antibiotic resistant bacteria. Most studies have examined HGT in growing cultures. We examined conjugation in growing and non-growing cultures of E. coli using a conjugative multi antibiotic and metal resistant plasmid to determine physiochemical parameters that favor horizontal gene transfer. The conjugation frequency in growing and non-growing cultures was generally greater under shaken than non-shaken conditions, presumably due to increased frequency of cell collisions. Non-growing cultures in 9.1 mM NaCl had a similar conjugation frequency to that of growing cultures in Luria-Bertaini broth, whereas those in 1 mM or 90.1 mM NaCl were much lower. This salinity effect on conjugation was attributed to differences in cell-cell interactions and conformational changes in cell surface macromolecules. In the presence of antibiotics, the conjugation frequencies of growing cultures did not increase, but in non-growing cultures of 9.1 mM NaCl supplemented with Cefotaxime the conjugation frequency was as much as nine times greater than that of growing cultures. The mechanism responsible for the increased conjugation in non-growing bacteria was attributed to the likely lack of penicillin-binding protein 3 (the target of Cefotaxime), in non-growing cells that enabled Cefotaxime to interact with the plasmid and induce conjugation. Our results suggests that more attention may be owed to HGT in non-growing bacteria as most bacteria in the environment are likely not growing and the proposed mechanism for increased conjugation may not be unique to the bacteria/plasmid system we studied