Distribution and Characterization of Salmonella enterica Isolates from Irrigation Ponds in the Southeastern United States

Irrigation water has been implicated as a likely source of produce contamination by Salmonella enterica. Therefore, the distribution of S. enterica was surveyed monthly in irrigation ponds (n = 10) located within a prime agricultural region in southern Georgia and northern Florida. All ponds and 28.2% of all samples (n = 635) were positive for Salmonella, with an overall geometric mean concentration (0.26 most probable number [MPN]/liter) that was relatively low compared to prior reports for rivers in this region. Salmonella peaks were seasonal; the levels correlated with increased temperature and rainfall (P < 0.05). The numbers and occurrence were significantly higher in water (0.32 MPN/liter and 37% of samples) than in sediment (0.22 MPN/liter and 17% of samples) but did not vary with depth. Representative isolates (n = 185) from different ponds, sample types, and seasons were examined for resistance to 15 different antibiotics; most strains were resistant to streptomycin (98.9%), while 20% were multidrug resistant (MDR) for 2 to 6 antibiotics. DiversiLab repetitive extragenic palindromic-element sequence-based PCR (rep-PCR) revealed genetic diversity and showed 43 genotypes among 191 isolates, as defined by >95% similarity. The genotypes did not partition by pond, season, or sample type. Genetic similarity to known serotypes indicated Hadar, Montevideo, and Newport as the most prevalent. All ponds achieved the current safety standards for generic Escherichia coli in agricultural water, and regression modeling showed that the E. coli level was a significant predictor for the probability of Salmonella occurrence. However, persistent populations of Salmonella were widely distributed in irrigation ponds, and the associated risks for produce contamination and subsequent human exposure are unknown, supporting continued surveillance of this pathogen in agricultural settings.     

Metabolic Adaptations of Azospirillum brasilense to Oxygen Stress by Cell-to-Cell Clumping and Flocculation

The ability of bacteria to monitor their metabolism and adjust their behavior accordingly is critical to maintain competitiveness in the environment. The motile microaerophilic bacterium Azospirillum brasilense navigates oxygen gradients by aerotaxis in order to locate low oxygen concentrations that can support metabolism. When cells are exposed to elevated levels of oxygen in their surroundings, motile A. brasilense cells implement an alternative response to aerotaxis and form transient clumps by cell-to-cell interactions. Clumping was suggested to represent a behavior protecting motile cells from transiently elevated levels of aeration. Using the proteomics of wild-type and mutant strains affected in the extent of their clumping abilities, we show that cell-to-cell clumping represents a metabolic scavenging strategy that likely prepares the cells for further metabolic stresses. Analysis of mutants affected in carbon or nitrogen metabolism confirmed this assumption. The metabolic changes experienced as clumping progresses prime cells for flocculation, a morphological and metabolic shift of cells triggered under elevated-aeration conditions and nitrogen limitation. The analysis of various mutants during clumping and flocculation characterized an ordered set of changes in cell envelope properties accompanying the metabolic changes. These data also identify clumping and early flocculation to be behaviors compatible with the expression of nitrogen fixation genes, despite the elevated-aeration conditions. Cell-to-cell clumping may thus license diazotrophy to microaerophilic A. brasilense cells under elevated oxygen conditions and prime them for long-term survival via flocculation if metabolic stress persists.     

A generalist brood parasite modifies use of a host in response to reproductive success

Avian obligate brood parasites, which rely solely on hosts to raise their young, should choose the highest quality hosts to maximize reproductive output. Brown-headed cowbirds (Molothrus ater) are extreme host generalists, yet female cowbirds could use information based on past reproductive outcomes to make egg-laying decisions thus minimizing fitness costs associated with parasitizing low-quality hosts. We use a long-term (21 years) nest-box study of a single host, the prothonotary warbler (Protonotaria citrea), to show that local cowbird reproductive success, but not host reproductive success, was positively correlated with the probability of parasitism the following year. Experimental manipulations of cowbird success corroborated that female cowbirds make future decisions about which hosts to use based on information pertaining to past cowbird success, both within and between years. The within-year pattern, in particular, points to local cowbird females selecting hosts based on past reproductive outcomes. This, coupled with high site fidelity of female cowbirds between years, points to information use, rather than cowbird natal returns alone, increasing parasitism rates on highly productive sites between years.     

Mechanisms of Human Erythrocytic Bioactivation of Nitrite

Nitrite signaling likely occurs through its reduction to nitric oxide (NO). Several reports support a role of erythrocytes and hemoglobin in nitrite reduction, but this remains controversial, and alternative reductive pathways have been proposed. In this work we determined whether the primary human erythrocytic nitrite reductase is hemoglobin as opposed to other erythrocytic proteins that have been suggested to be the major source of nitrite reduction. We employed several different assays to determine NO production from nitrite in erythrocytes including electron paramagnetic resonance detection of nitrosyl hemoglobin, chemiluminescent detection of NO, and inhibition of platelet activation and aggregation. Our studies show that NO is formed by red blood cells and inhibits platelet activation. Nitric oxide formation and signaling can be recapitulated with isolated deoxyhemoglobin. Importantly, there is limited NO production from erythrocytic xanthine oxidoreductase and nitric-oxide synthase. Under certain conditions we find dorzolamide (an inhibitor of carbonic anhydrase) results in diminished nitrite bioactivation, but the role of carbonic anhydrase is abrogated when physiological concentrations of CO₂ are present. Importantly, carbon monoxide, which inhibits hemoglobin function as a nitrite reductase, abolishes nitrite bioactivation. Overall our data suggest that deoxyhemoglobin is the primary erythrocytic nitrite reductase operating under physiological conditions and accounts for nitrite-mediated NO signaling in blood.     

The DNA Damage Response and Checkpoint Adaptation in Saccharomyces cerevisiae: Distinct Roles for the Replication Protein A2 (Rfa2) N-Terminus

In response to DNA damage, two general but fundamental processes occur in the cell: (1) a DNA lesion is recognized and repaired, and (2) concomitantly, the cell halts the cell cycle to provide a window of opportunity for repair to occur. An essential factor for a proper DNA-damage response is the heterotrimeric protein complex Replication Protein A (RPA). Of particular interest is hyperphosphorylation of the 32-kDa subunit, called RPA2, on its serine/threonine-rich amino (N) terminus following DNA damage in human cells. The unstructured N-terminus is often referred to as the phosphorylation domain and is conserved among eukaryotic RPA2 subunits, including Rfa2 in Saccharomyces cerevisiae. An aspartic acid/alanine-scanning and genetic interaction approach was utilized to delineate the importance of this domain in budding yeast. It was determined that the Rfa2 N-terminus is important for a proper DNA-damage response in yeast, although its phosphorylation is not required. Subregions of the Rfa2 N-terminus important for the DNA-damage response were also identified. Finally, an Rfa2 N-terminal hyperphosphorylation-mimetic mutant behaves similarly to another Rfa1 mutant (rfa1-t11) with respect to genetic interactions, DNA-damage sensitivity, and checkpoint adaptation. Our data indicate that post-translational modification of the Rfa2 N-terminus is not required for cells to deal with “repairable” DNA damage; however, post-translational modification of this domain might influence whether cells proceed into M-phase in the continued presence of unrepaired DNA lesions as a “last-resort” mechanism for cell survival.     

Gastrin-stimulated Gα13 Activation of Rgnef Protein (ArhGEF28) in DLD-1 Colon Carcinoma Cells

The guanine nucleotide exchange factor Rgnef (also known as ArhGEF28 or p190RhoGEF) promotes colon carcinoma cell motility and tumor progression via interaction with focal adhesion kinase (FAK). Mechanisms of Rgnef activation downstream of integrin or G protein-coupled receptors remain undefined. In the absence of a recognized G protein signaling homology domain in Rgnef, no proximal linkage to G proteins was known. Utilizing multiple methods, we have identified Rgnef as a new effector for Gα₁₃ downstream of gastrin and the type 2 cholecystokinin receptor. In DLD-1 colon carcinoma cells depleted of Gα₁₃, gastrin-induced FAK Tyr(P)-397 and paxillin Tyr(P)-31 phosphorylation were reduced. RhoA GTP binding and promoter activity were increased by Rgnef in combination with active Gα₁₃. Rgnef co-immunoprecipitated with activated Gα₁₃Q226L but not Gα₁₂Q229L. The Rgnef C-terminal (CT, 1279–1582) region was sufficient for co-immunoprecipitation, and Rgnef-CT exogenous expression prevented Gα₁₃-stimulated SRE activity. A domain at the C terminus of the protein close to the FAK binding domain is necessary to bind to Gα₁₃. Point mutations of Rgnef-CT residues disrupt association with active Gα₁₃ but not Gα_q. These results show that Rgnef functions as an effector of Gα₁₃ signaling and that this linkage may mediate FAK activation in DLD-1 colon carcinoma cells.