Biological Nitrogen Fixation is critical for ecosystem productivity. Select members of Bacteria and Archaea express a nitrogenase enzyme complex that reduces atmospheric nitrogen to ammonia. Several nitrogen fixing bacteria form symbiotic associations with plants, but free-living diazotrophs also contribute a substantial amount of nitrogen to ecosystems. The aim of this study was to isolate and characterize free-living diazotrophs in arid lands of South Dakota Badlands. Samples were obtained from sod tables and the surrounding base in spring and fall. Diazotrophs were isolated on solid nitrogen free medium (NFM) under hypoxic conditions, and their16S rRNA and nifH genes sequenced. nifH was also amplified directly from soil DNA extracts. The 16S rRNA gene data indicated a diversity of putative free-living diazotrophs across 4 phyla (Actinomycetes, Proteobacteria, Bacteroidetes, and Firmicutes), but ∼50% of these clustered with Streptomyces. These Streptomyces isolates grew in liquid NFM in an ammonia-depleted environment. Only 5 of these yielded a nifH gene product using the PolF/PolR primer set. Four of these aligned with nifH of the cyanobacteria Scytonema and Nostoc, and the other one aligned with nifH of Bradyrhizobium. Six selected Streptomyces isolates, three of which were nifH positive by PCR, all indicated N incorporation, providing strong support of nitrogen fixation. All nifH amplicons from soil DNA extract resembled Cyanobacteria. This is the first known report of diazotrophic Streptomyces, other than the thermophilic, autotrophic S. thermoautotrophicus. nifH genes of these Streptomyces were related to those from Cyanobacteria. It is possible that the cyanobacteria-like nifH amplicons obtained from soil DNA were associated with Streptomyces.

Escherichia coli is widely considered to not survive for extended periods outside the intestines of warm-blooded animals; however, recent studies demonstrated that E. coli strains maintain populations in soil and water without any known fecal contamination. The objective of this study was to investigate whether the niche partitioning of E. coli occurs between cattle and their pasture. We attempted to clarify whether E. coli from bovine feces differs phenotypically and genotypically from isolates maintaining a population in pasture soil over winter. Soil, bovine fecal, and run-off samples were collected before and after the introduction of cattle to the pasture. Isolates (363) were genotyped by uidA and mutS sequences and phylogrouping, and evaluated for curli formation (Rough, Dry, And Red, or RDAR). Three types of clusters emerged, viz. bovine-associated, clusters devoid of cattle isolates and representing isolates endemic to the pasture environment, and clusters with both. All isolates clustered with strains of E. coli sensu stricto, distinct from the cryptic species Clades I, III, IV, and V. Pasture soil endemic and bovine fecal populations had very different phylogroup distributions, indicating niche partitioning. The soil endemic population was largely comprised of phylogroup B1 and had a higher average RDAR score than other isolates. These results indicate the existence of environmental E. coli strains that are phylogenetically distinct from bovine fecal isolates, and that have the ability to maintain populations in the soil environment.

Enterohaemorrhagic Escherichia coli, such as serotype O157:H7, are a leading cause of food-associated outbreaks. While the primary reservoir is associated with cattle, plant foods have been associated as sources of human infection. E. coli is able to grow in the tissue of food plants such as spinach. While fecal contamination is the primary suspect, soil has been underestimated as a potential reservoir. Persistence of bacterial populations in open systems is the product of growth, death, predation, and competition. Here we report that E. coli O157:H7 can grow using the soluble compounds in soil, and characterize the effect of soil growth on the stationary phase proteome. E. coli 933D (stxII−) was cultured in Soil Extracted Soluble Organic Matter (SESOM) and the culturable count determined for 24d. The proteomes of exponential and stationary phase populations were characterized by 2D gel electrophoresis and protein spots were identified by MALDI-TOF mass spectrometry. While LB controls displayed a death phase, SESOM grown population remained culturable for 24d, indicating an altered physiological state with superior longevity. This was not due to decreased cell density on entry to stationary phase as 24 h SESOM populations concentrated 10-fold retained their longevity. Principal component analysis showed that stationary phase proteomes from SESOM and LB were different. Differences included proteins involved in stress response, motility, membrane and wall composition, nutrient uptake, translation and protein turnover, and anabolic and catabolic pathways, indicating an altered physiological state of soil-grown cells entering stationary phase. The results suggest that E. coli may be a soil commensal that, in absence of predation and competition, maintains stable populations in soil.

Enterohaemorrhagic Escherichia coli such as serotype O157:H7 are a leading cause of food-associated outbreaks. While the primary reservoir is associated with cattle, plant foods have been associated as sources of human infection. E. coli is able to grow in the tissue of food plants such as spinach. While fecal contamination is the primary suspect, soil has been underestimated as a potential reservoir. Persistence of bacterial populations in open systems is the product of growth, death, predation, and competition. Here we report that E. coli O157:H7 can grow using the soluble compounds in soil, and characterize the effect of soil growth in the stationary phase proteome.

Escherichia coli comprises diverse strains with a large accessory genome, indicating functional diversity and the ability to adapt to a range of niches. Specific strains would display greatest fitness in niches matching their combination of phenotypic traits. Given this hypothesis, we sought to determine whether E. coli in a peri-urban pond and associated cattle pasture display niche preference. Samples were collected from water, sediment, aquatic plants, water snails associated with the pond, as well as bovine feces from cattle in an adjacent pasture. Isolates (120) were obtained after plating on Membrane Lactose Glucuronide Agar (MLGA). We used the uidA and mutS sequences for all isolates to determine phylogeny by maximum likelihood, and population structure through gene flow analysis. PCR was used to allocate isolates to phylogroups and to determine the presence of pathogenicity/virulence genes (stxI, stxII, eaeA, hlyA, ST, and LT). Antimicrobial resistance was determined using a disk diffusion assay for Tetracycline, Gentamicin, Ciprofloxacin, Meropenem, Ceftriaxone, and Azithromycin. Our results showed that isolates from water, sediment, and water plants were similar by phylogroup distribution, virulence gene distribution, and antibiotic resistance while both snail and feces populations were significantly different. Few of the feces isolates were significantly similar to aquatic ones, and most of the snail isolates were also different. Population structure analysis indicated three genetic backgrounds associated with bovine, snail, and aquatic environments. Collectively these data support niche preference of E. coli isolates occurring in this ecosystem.