Temporal metagenomic characterization of microbial community structure and nitrogen modification genes within an activated sludge bioreactor system.

IMPORTANCE: Wastewater treatment plays an essential role in minimizing negative impacts on downstream aquatic environments. Microbial communities are known to play a vital role in the wastewater treatment process, particularly in the removal of nitrogen and phosphorus, which can be especially damaging to aquatic ecosystems. There is limited understanding of how these microbial communities may change in response to fluctuating temperatures or how seasonality may impact their ability to participate in the treatment process. The findings of this study indicate that the microbial communities of wastewater are relatively stable both compositionally and functionally across fluctuating temperatures.

Sources of Antibiotic Resistance Genes in a Rural River System.

The increasing prevalence of antibiotic resistance genes (ARGs) in the environment is problematic due to the risk of horizontal gene transfer and development of antibiotic resistant pathogenic bacteria. Using a suite of monitoring tools, this study aimed to investigate the sources of ARGs in a rural river system in Nova Scotia, Canada. The monitoring program specifically focused on the relative contribution of ARGs from a single tertiary-level wastewater treatment plant (WWTP) in comparison to contributions from the upgradient rural, sparsely developed, watershed. The overall gene concentration significantly ( < 0.05) increased downstream from the WWTP, suggesting that tertiary-level treatment still contributes ARGs to the environment. As a general trend, ARG concentrations upstream were found to decrease as proximity to human-impacted areas decreased; however, many ARGs remained above detection limits in headwater river samples, which suggested their ubiquitous presence in this watershed in the absence of obvious pollution sources. Significant correlations with ARGs were found for human fecal marker, and some antibiotics, suggesting that these markers may be useful for prediction and understanding of ARG levels and sources in rural rivers.

Role of O2 in the Growth of Rhizobium leguminosarum bv. viciae 3841 on Glucose and Succinate.

Insertion sequencing (INSeq) analysis of Rhizobium leguminosarum bv. viciae 3841 (Rlv3841) grown on glucose or succinate at both 21% and 1% O2 was used to understand how O2 concentration alters metabolism. Two transcriptional regulators were required for growth on glucose (pRL120207 [eryD] and RL0547 [phoB]), five were required on succinate (pRL100388, RL1641, RL1642, RL3427, and RL4524 [ecfL]), and three were required on 1% O2 (pRL110072, RL0545 [phoU], and RL4042). A novel toxin-antitoxin system was identified that could be important for generation of new plasmidless rhizobial strains. Rlv3841 appears to use the methylglyoxal pathway alongside the Entner-Doudoroff (ED) pathway and tricarboxylic acid (TCA) cycle for optimal growth on glucose. Surprisingly, the ED pathway was required for growth on succinate, suggesting that sugars made by gluconeogenesis must undergo recycling. Altered amino acid metabolism was specifically needed for growth on glucose, including RL2082 (gatB) and pRL120419 (opaA, encoding omega-amino acid:pyruvate transaminase). Growth on succinate specifically required enzymes of nucleobase synthesis, including ribose-phosphate pyrophosphokinase (RL3468 [prs]) and a cytosine deaminase (pRL90208 [codA]). Succinate growth was particularly dependent on cell surface factors, including the PrsD-PrsE type I secretion system and UDP-galactose production. Only RL2393 (glnB, encoding nitrogen regulatory protein PII) was specifically essential for growth on succinate at 1% O2, conditions similar to those experienced by N2-fixing bacteroids. Glutamate synthesis is constitutively activated in glnB mutants, suggesting that consumption of 2-ketoglutarate may increase flux through the TCA cycle, leading to excess reductant that cannot be reoxidized at 1% O2 and cell death. IMPORTANCE: Rhizobium leguminosarum, a soil bacterium that forms N2-fixing symbioses with several agriculturally important leguminous plants (including pea, vetch, and lentil), has been widely utilized as a model to study Rhizobium-legume symbioses. Insertion sequencing (INSeq) has been used to identify factors needed for its growth on different carbon sources and O2 levels. Identification of these factors is fundamental to a better understanding of the cell physiology and core metabolism of this bacterium, which adapts to a variety of different carbon sources and O2 tensions during growth in soil and N2 fixation in symbiosis with legumes.

Metabolic Adaptation of a C-Terminal Protease A-Deficient Rhizobium leguminosarum in Response to Loss of Nutrient Transport.

Post-translational modification expands the functionality of the proteome beyond genetic encoding, impacting many cellular processes. Cleavage of the carboxyl terminus is one of the many different ways proteins can be modified for functionality. Gel-electrophoresis and mass spectrometric-based techniques were used to identify proteins impacted by deficiency of a C-terminal protease, CtpA, in Rhizobium leguminosarum bv. viciae 3841. Predicted CtpA substrates from 2D silver stained gels were predominantly outer membrane and transport proteins. Proteins with altered abundance in the wild type and ctpA (RL4692) mutant, separated by 2D difference gel electrophoresis, were selected for analysis by mass spectrometry. Of those identified, 9 were the periplasmic solute-binding components of ABC transporters, 5 were amino acid metabolic enzymes, 2 were proteins involved in sulfur metabolism, and 1 each was related to carbon metabolism, protein folding and signal transduction. Alterations to ABC-binding-cassette transporters, nutrient uptake efficiency and to amino acid metabolism indicated an impact on amino acid metabolism and transport for the ctpA mutant, which was validated by measured amino acid levels.

Comparison of the Prevalences and Diversities of Listeria Species and Listeria monocytogenes in an Urban and a Rural Agricultural Watershed.

Foods and related processing environments are commonly contaminated with the pathogenic Listeria monocytogenes. To investigate potential environmental reservoirs of Listeria spp. and L. monocytogenes, surface water and point source pollution samples from an urban and a rural municipal water supply watershed in Nova Scotia, Canada, were examined over 18 months. Presumptive Listeria spp. were cultured from 72 and 35% of rural and urban water samples, respectively, with 24% of the positive samples containing two or three different Listeria spp. The L. innocua (56%) and L. welshimeri (43%) groups were predominant in the rural and urban watersheds, respectively. Analysis by the TaqMan assay showed a significantly (P < 0.05) higher prevalence of L. monocytogenes of 62% versus 17% by the culture-based method. Both methods revealed higher prevalences in the rural watershed and during the fall and winter seasons. Elevated Escherichia coli (≥ 100 CFU/100 ml) levels were not associated with the pathogen regardless of the detection method. Isolation of Listeria spp. were associated with 70 times higher odds of isolating L. monocytogenes (odds ratio = 70; P < 0.001). Serogroup IIa was predominant (67.7%) among the 285 L. monocytogenes isolates, followed by IVb (16.1%), IIb (15.8%), and IIc (0.4%). L. monocytogenes was detected in cow feces and raw sewage but not in septic tank samples. Pulsotyping of representative water (n = 54) and local human (n = 19) isolates suggested genetic similarities among some environmental and human L. monocytogenes isolates. In conclusion, temperate surface waters contain a diverse Listeria species population and could be a potential reservoir for L. monocytogenes, especially in rural agricultural watersheds.

Characterizing spatial structure of sediment E. coli populations to inform sampling design.

Escherichia coli can persist in streambed sediments and influence water quality monitoring programs through their resuspension into overlying waters. This study examined the spatial patterns in E. coli concentration and population structure within streambed morphological features during baseflow and following stormflow to inform sampling strategies for representative characterization of E. coli populations within a stream reach. E. coli concentrations in bed sediments were significantly different (p = 0.002) among monitoring sites during baseflow, and significant interactive effects (p = 0.002) occurred among monitoring sites and morphological features following stormflow. Least absolute shrinkage and selection operator (LASSO) regression revealed that water velocity and effective particle size (D 10) explained E. coli concentration during baseflow, whereas sediment organic carbon, water velocity and median particle diameter (D 50) were important explanatory variables following stormflow. Principle Coordinate Analysis illustrated the site-scale differences in sediment E. coli populations between disconnected stream segments. Also, E. coli populations were similar among depositional features within a reach, but differed in relation to high velocity features (e.g., riffles). Canonical correspondence analysis resolved that E. coli population structure was primarily explained by spatial (26.9­31.7 %) over environmental variables (9.2­13.1 %). Spatial autocorrelation existed among monitoring sites and morphological features for both sampling events, and gradients in mean particle diameter and water velocity influenced E. coli population structure for the baseflow and stormflow sampling events, respectively. Representative characterization of streambed E. coli requires sampling of depositional and high velocity environments to accommodate strain selectivity among these features owing to sediment and water velocity heterogeneity.

Effect of hillslope position and manure application rates on the persistence of fecal source tracking indicators in an agricultural soil.

The influence of liquid dairy manure (LDM) application rates (12.5 and 25 kL ha) and soil type on the decay rates of library-independent fecal source tracking markers (host-associated and mitochondrial DNA) and persistent (>58 d) population structure was examined in a field study. The soils compared were an Aquic Haplorthod and a Typic Haplorthod in Nova Scotia, Canada, that differed according to landscape position and soil moisture regime. Soil type and LDM application rate did not influence decay rates (0.045-0.057 d). population structure, in terms of the occurrence of abundance of strain types, varied according to soil type ( = 0.012) but did not vary by LDM application rate ( = 0.121). Decay of ruminant-specific (BacR), bovine-specific (CowM2), and mitochondrial DNA (AcytB) markers was analyzed for 13 d after LDM application. The decay rates of BacR were greater under high-LDM application rates (0.281-0.358 d) versus low-LDM application rates (0.212-0.236 d) but were unaffected by soil type. No decay rates could be calculated for the CowM2 marker because it was undetectable within 6 d after manure application. Decay rates for AcytB were lower for the Aquic Haplorthod (0.088-0.100 d), with higher moisture status compared with the Typic Haplorthod (0.135 d). Further investigation into the decay of fecal source tracking indicators in agricultural field soils is warranted to assess the influence of soil type and agronomic practice on the differential decay of relevant markers and the likelihood of transport in runoff.

Molecular typing techniques to characterize the development of a lactic acid bacteria community on vacuum-packaged beef.

The development of a community of lactic acid bacteria from vacuum-packaged beef was investigated during a 6-week storage trial at 2 degrees C. The lactic acid bacteria population was monitored by using molecular techniques to identify a random sample of isolates at biweekly intervals during the storage trial. The polymerase chain reaction and a randomly amplified polymorphic DNA technique were used to identify and distinguish populations of lactic acid bacteria that developed during the storage trial. At week 0, the population of lactic acid bacteria was 3.5 log cfu/120 cm2 and by week 6, the population reached a maximum of 7.6 log cfu/120 cm2. A sampling from the week 0 population indicated a mixed community of Lactobacillus curvatus, Lactobacillus sakei and Leuconostoc spp. However, the sampling from week 6 indicated the population composition had changed to one where a single Leuconostoc strain predominated. This strain demonstrated antagonism towards the growth of other lactic acid bacteria isolated during the study. Additionally, the strain inhibited the growth of foodborne pathogens Escherichia coli O157:H7 and Listeria monocytogenes. DNA sequence data from the 16S rRNA gene suggested that the isolate may be a Leuconostoc gelidum strain.