ABSTRACT Testing for the causative agent of coronavirus disease 2019 (COVID-19), severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has been crucial in tracking disease spread and informing public health decisions. Wastewater-based epidemiology has helped to alleviate some of the strain of testing through broader, population-level surveillance, and has been applied widely on college campuses. However, questions remain about the impact of various sampling methods, target types, environmental factors, and infrastructure variables on SARS-CoV-2 detection. Here, we present a data set of over 800 wastewater samples that sheds light on the influence of a variety of these factors on SARS-CoV-2 quantification using droplet digital PCR (ddPCR) from building-specific sewage infrastructure. We consistently quantified a significantly higher number of copies of virus per liter for the target nucleocapsid 2 (N2) compared to nucleocapsid 1 (N1), regardless of the sampling method (grab vs composite). We further show some dormitory-specific differences in SARS-CoV-2 abundance, including correlations to dormitory population size. Environmental variables like precipitation and temperature show little to no impact on virus load, with the exception of higher temperatures for grab sample data. We observed significantly higher gene copy numbers of the Omicron variant than the Delta variant within ductile iron pipes but no difference in nucleocapsid abundance (N1 or N2) across the three different sewage pipe types in our data set. Our results indicate that contextual variables should be considered when interpreting wastewater-based epidemiological data. IMPORTANCE Testing for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of coronavirus disease 2019 (COVID-19), has been crucial in tracking the spread of the virus and informing public health decisions. SARS-CoV-2 viral RNA is shed by symptomatic and asymptomatic infected individuals, allowing its genetic material to be detected and quantified in wastewater. Here, we used wastewater-based epidemiology to measure SARS-CoV-2 viral RNA from several dormitories on the Appalachian State University campus and examined the impact of sampling methods, target types, environmental factors, and infrastructure variables on quantification. Changes in the quantification of SARS-CoV-2 were observed based on target type, as well as trends for the quantification of the Delta and Omicron variants by sampling method. These results highlight the value of applying the data-inquiry practices used in this study to better contextualize wastewater sampling results.

Biofilm formation in the human intestinal pathogen Vibrio cholerae is in part regulated by norspermidine, spermidine and sper-mine. V. cholerae senses these polyamines through a signalling pathway consisting of the periplasmic protein, NspS, and the integral membrane c- di- GMP phosphodiesterase MbaA. NspS and MbaA belong to a proposed class of novel signalling systems composed of periplasmic ligand-binding proteins and membrane- bound c- di- GMP phosphodiesterases containing both GGDEF and EAL domains. In this signal transduction pathway, NspS is hypothesized to interact with MbaA in the periplasm to regulate its phosphodiesterase activity. Polyamine binding to NspS likely alters this interaction, leading to the activation or inhibition of biofilm formation depending on the polyamine. The purpose of this study was to determine the amino acids important for NspS function. We performed random mutagenesis of the nspS gene, identified mutant clones deficient in biofilm formation, determined their responsiveness to norspermidine and mapped the location of these residues onto NspS homology models. Single mutants clustered on two lobes of the NspS model, but the majority were found on a single lobe that appeared to be more mobile upon norspermidine binding. We also identified residues in the putative ligand-binding site that may be important for norspermidine binding and interactions with MbaA. Ultimately, our results provide new insights into this novel signalling pathway in V. cholerae and highlight differences between periplasmic binding proteins involved in transport versus signal transduction.

The aquatic bacterium and human intestinal pathogen, Vibrio cholerae, senses and responds to a variety of environment-specific cues to regulate biofilm formation. Specifically, the polyamines norspermidine and spermidine enhance and repress V. cholerae biofilm formation, respectively. These effects are relevant for understanding V. cholerae pathogenicity and are mediated through the periplasmic binding protein NspS and the transmembrane bis-(3-5) cyclic diguanosine monophosphate (c-di-GMP) phosphodiesterase MbaA. However, the levels of spermidine required to inhibit biofilm formation through this pathway are unlikely to be encountered by V. cholerae in aquatic reservoirs or within the human host during infection. We therefore hypothesized that other polyamines in the gastrointestinal tract may control V. cholerae biofilm formation at physiological levels. The tetramine spermine has been reported to be present at nearly 50 m concentrations in the intestinal lumen. Here, we report that spermine acts as an exogenous cue that inhibits V. cholerae biofilm formation through the NspS-MbaA signaling system. We found that this effect probably occurs through a direct interaction of spermine with NspS, as purified NspS protein could bind spermine in vitro. Spermine also inhibited biofilm formation by altering the transcription of the vps genes involved in biofilm matrix production. Global c-di-GMP levels were unaffected by spermine supplementation, suggesting that biofilm formation may be regulated by variations in local rather than global c-di-GMP pools. Finally, we propose a model illustrating how the NspS-MbaA signaling system may communicate exogenous polyamine content to the cell to control biofilm formation in the aquatic environment and within the human intestine.

The polyamine norspermidine is one of the major polyamines synthesized by Vibrionales and has also been found in various aquatic organisms. Norspermidine is among the environmental signals that positively regulate Vibrio cholerae biofilm formation. The NspS/MbaA signaling complex detects extracellular norspermidine and mediates the response to this polyamine. Norspermidine binding to the NspS periplasmic binding protein is thought to inhibit the phosphodiesterase activity of MbaA, increasing levels of the biofilm-promoting second messenger cyclic diguanylate monophosphate, thus enhancing biofilm formation. V. cholerae can also synthesize norspermidine using the enzyme NspC as well as import it from the environment. Deletion of the nspC gene was shown to reduce accumulation of bacteria in biofilms, leading to the conclusion that intracellular norspermidine is also a positive regulator of biofilm formation. Because V. cholerae uses norspermidine to synthesize the siderophore vibriobactin it is possible that intracellular norspermidine is required to obtain sufficient amounts of iron, which is also necessary for robust biofilm formation. The objective of this study was to assess the relative contributions of intracellular and extracellular norspermidine to the regulation of biofilm formation in V. cholerae. We show the biofilm defect of norspermidine synthesis mutants does not result from an inability to produce vibriobactin as vibriobactin synthesis mutants do not have diminished biofilm forming abilities. Furthermore, our work shows that extracellular, but not intracellular norspermidine, is mainly responsible for promoting biofilm formation. We establish that the NspS/MbaA signaling complex is the dominant mediator of biofilm formation in response to extracellular norspermidine, rather than norspermidine synthesized by NspC or imported into the cell.

The polyamines norspermidine and spermidine are among the environmental signals that regulateVibrio choleraebiofilm formation. The effects of these polyamines are mediated by NspS, a member of the bacterial periplasmic solute binding protein superfamily. Almost all members of this superfamily characterized to date are components of ATP-binding cassette-type transporters involved in nutrient uptake. Consequently, in the current annotation of theV. choleraegenome, NspS has been assigned a function in transport. The objective of this study was to further characterize NspS and investigate its potential role in transport. Our results support a role for NspS in signal transduction in response to norspermidine and spermidine, but not their transport. In addition, we provide evidence that these polyamine signals are processed by c-di-GMP signalling networks in the cell. Furthermore, we present comparative genomics analyses which reveal the presence of NspS-like proteins in a variety of bacteria, suggesting that periplasmic ligand binding proteins may be widely utilized for sensory transduction.

Polyamines play an essential role in biofilm formation of diverse Gram-negative and Gram-positive bacteria. Biosynthetic pathways and transport systems for diverse polyamines have been identified as key components of bacterial biofilm formation.

Vibrio cholerae is the causative agent of the severe enteric disease cholera. To cause cholera the bacterium must be able to synthesize both cholera toxin (CT) and toxin-coregulated pilus (TCP) which mediates autoagglutination and is required for colonization of the small intestine. Only a few environmental signals have been shown to regulate V. cholerae virulence gene expression. Polyamines, which are ubiquitous in nature, and have been implicated in regulating virulence gene expression in other bacteria, have not been extensively studied for their effect on V. cholerae virulence properties. The objective of this study was to test the effect of several polyamines that are abundant in the human intestine on V. cholerae virulence properties. All of the polyamines tested inhibited autoagglutination of V. cholerae O1 classical strain in a concentration dependent manner. Putrescine and cadaverine decreased the synthesis of the major pilin subunit, TcpA, spermidine increased its production, and spermine had no effect. Putrescine and spermidine led to a decrease and increase, respectively, on the relative abundance of TCP found on the cell surface. Spermine led to a small reduction in cholera toxin synthesis whereas none of the other polyamines had an effect. The polyamines did not affect pili bundling morphology, but caused a small reduction in CTXφ transduction, indicating that the TCP present on the cell surface may not be fully functional. We hypothesize the inhibition of autoagglutination is likely to be caused by the positively charged amine groups on the polyamines electrostatically disrupting the pili-pili interactions which mediate autoagglutination. Our results implicate that polyamines may have a protective function against colonization of the small intestine by V. cholerae.

The Sorrento wetland hosts several Fe-and Mn-rich seeps that are reported to have appeared after the area was disturbed by recent attempts at development. Culture-independent and culture-based analyses were utilized to characterize the microbial community at the main site of the Fe and Mn seep. Several bacteria capable of oxidizing Mn(II) were isolated, including members related to the genera Bacillus, Lysinibacillus, Pseudomonas, and Leptothrix, but none of these were detected in clone libraries. Most probable number assays demonstrated that seep and wetland sites contained higher numbers of culturable Mn-oxidizing microorganisms than an upstream reference site. When compared with quantitative real time PCR (qPCR) assays of total bacteria, MPN analyses indicated that less than 0.01% of the total population (estimated around 10(9) cells/g) was culturable. Light microscopy and fluorescence in situ hybridization (FISH) images revealed an abundance of morphotypes similar to Fe-and Mn-oxidizing Leptothrix spp. and Gallionella spp. in seep and wetland sites. FISH allowed identification of Leptothrix-type sheath-forming organisms in seep samples but not in reference samples. Gallionella spp. and Leptothrix spp. cells numbers were estimated using qPCR with a novel primer set that we designed. Results indicated that numbers of Gallionella, Leptothrix or total bacteria were all significantly higher at the seep site relative to the reference site (where Gallionella was below detection). Interestingly, numbers of Leptothrix in the seep site were estimated at only 10(7) cells/g and were not statistically different in the late summer versus the late winter, despite dramatic changes in sheath abundance (as indicated by microscopy). qPCR also indicated that Gallionella spp. may represent up to 10% (3 x 108 cells/g) of the total bacteria in seep samples. These data corroborate clone library data from samples taken in October 2008, where 11 SSU rRNA sequences related to Gallionella spp. were detected out of 77 total sequences (roughly 10-15%), and where Leptothrix sequences were not detected. Analysis of this SSU rRNA clonal library revealed that a diverse microbial community was present at seep sites. At a 3% difference cutoff, 30 different operational taxonomic units were detected out of 77 sequences analyzed. Dominant sequence types clustered among the beta-and gamma-Proteobacteria near sequences related to the genera Ideonella, Rhodoferax, Methylotenera, Methylobacter, and Gallionella. Overall, results suggest that high metal concentrations at the seep sites have enriched for Fe-and Mn-oxidizing bacteria including organisms related to Gallionella and Leptothrix species, and that members of these genera coexist within a diverse microbial community.

Biofilm formation in Vibrio cholerae is in part regulated by norspermidine, a polyamine synthesized by the enzyme carboxynorspermidine decarboxylase (NspC). The absence of norspermidine in the cell leads to a marked reduction in V. cholerae biofilm formation by an unknown mechanism. In this work, we show that overexpression of nspC results in large increases in biofilm formation and vps gene expression as well as a significant decrease in motility. Interestingly, increased NspC levels do not lead to increased concentrations of norspermidine in the cell. Our results show that NspC levels inversely regulate biofilm and motility and implicate the presence of an effective feedback mechanism maintaining norspermidine homeostasis in V. cholerae. Moreover, we provide evidence that NspC and the norspermidine sensor protein, NspS, provide independent and distinct inputs into the biofilm regulatory network.

SUMMARY Biofilms are communities of microorganisms that live attached to surfaces. Biofilm formation has received much attention in the last decade, as it has become clear that virtually all types of bacteria can form biofilms and that this may be the preferred mode of bacterial existence in nature. Our current understanding of biofilm formation is based on numerous studies of myriad bacterial species. Here, we review a portion of this large body of work including the environmental signals and signaling pathways that regulate biofilm formation, the components of the biofilm matrix, and the mechanisms and regulation of biofilm dispersal.

Vibrio cholerae, the causative agent of the devastating diarrheal disease cholera, can form biofilms on diverse biotic and abiotic surfaces. Biofilm formation is important for the survival of this organism both in its natural environment and in the human host. Development of V. cholerae biofilms are regulated by complex regulatory networks that respond to environmental signals. One of these signals, norspermidine, is a polyamine that enhances biofilm formation via the NspS/MbaA signaling system. In this work, we have investigated the role of the polyamine spermidine in regulating biofilm formation in V. cholerae. We show that spermidine import requires PotD1, an ortholog of the periplasmic substrate-binding protein of the spermidine transport system in Escherichia coli. We also show that deletion of the potD1 gene results in a significant increase in biofilm formation. We hypothesize that spermidine imported into the cell hinders biofilm formation. Exogenous spermidine further reduces biofilm formation in a PotD1-independent, but NspS/MbaA-dependent, manner. Our results suggest that polyamines affect biofilm formation in V. cholerae via multiple pathways involving both transport and signaling networks.

Vibrio cholerae is both an environmental bacterium and a human intestinal pathogen. The attachment of bacteria to surfaces in biofilms is thought to be an important feature of the survival of this bacterium both in the environment and within the human host. Biofilm formation occurs when cell-surface and cell-cell contacts are formed to make a three-dimensional structure characterized by pillars of bacteria interspersed with water channels. In monosaccharide-rich conditions, the formation of the V. cholerae biofilm requires synthesis of the VPS exopolysaccharide. MbaA (locus VC0703), an integral membrane protein containing a periplasmic domain as well as cytoplasmic GGDEF and EAL domains, has been previously identified as a repressor of V. cholerae biofilm formation. In this work, we have studied the role of the protein NspS (locus VC0704) in V. cholerae biofilm development. This protein is homologous to PotD, a periplasmic spermidine-binding protein of Escherichia coli. We show that the deletion of nspS decreases biofilm development and transcription of exopolysaccharide synthesis genes. Furthermore, we demonstrate that the polyamine norspermidine activates V. cholerae biofilm formation in an MbaA- and NspS-dependent manner. Based on these results, we propose that the interaction of the norspermidine-NspS complex with the periplasmic portion of MbaA diminishes the ability of MbaA to inhibit V. cholerae biofilm formation. Norspermidine has been detected in bacteria, archaea, plants, and bivalves. We suggest that norspermidine serves as an intercellular signaling molecule that mediates the attachment of V. cholerae to the biotic surfaces presented by one or more of these organisms.

ABSTRACT Vibrio cholerae is a halophilic facultative human pathogen found in marine and estuarine environments. Accumulation of compatible solutes is important for growth of V. cholerae at NaCl concentrations greater than 250 mM. We have identified and characterized two compatible solute transporters, OpuD and PutP, that are involved in uptake of glycine betaine and proline by V. cholerae. V. cholerae does not, however, possess the bet genes, suggesting that it is unable to synthesize glycine betaine. In contrast, many Vibrio species are able to synthesize glycine betaine from choline. It has been shown that many bacteria not only synthesize but also secrete glycine betaine. We hypothesized that sharing of compatible solutes might be a mechanism for cooperativity in microbial communities. In fact, we have demonstrated that, in high-osmolarity medium, V. cholerae growth and biofilm development are enhanced by supplementation with either glycine betaine or spent media from other bacterial species. Thus, we propose that compatible solutes provided by other microorganisms may contribute to survival of V. cholerae in the marine environment through facilitation of osmoadaptation and biofilm development.

Bacillus subtilis has a more complex mechanism of chemotaxis than does the paradigm organism, Escherichia coli. In order to understand better the role of the novel chemotaxis proteins--CheC, CheD and CheV--mutants in which increasing numbers of the corresponding genes had been deleted were studied as tethered cells and their biases and sometimes durations of counterclockwise (CCW) and clockwise (CW) flagellar rotations in response to addition and removal of the attractant asparagine were observed. The cheC mutant was found to have considerably reduced switching frequency (that is, prolonged CCW and CW rotations) without a significantly different prestimulus CCW bias, compared with wild-type. This result may indicate that in absence of CheC the switch might be in a conformation less resembling the transition state than in presence of CheC. Conversely, the cheB (methylesterase) mutant showed considerably increased switching frequency without affecting CCW bias, compared with wild-type. Removal of all known adaptation systems--the methylation, CheC and CheV systems--resulted in a mutant (cheRBCDV) that still retained some adaptation following the addition of attractant.

We have constructed a phage-displayed library based on the human fibronectin tenth type III domain (FN3) scaffold by randomizing residues in its FG and BC loops. Screening against the SH3 domain of human c-Src yielded six different clones. Five of these contained proline-rich sequences in their FG loop that resembled class I (i.e., +xxPxxP) peptide ligands for the Src SH3 domain. The sixth clone lacked the proline-rich sequence and showed particularly high binding specificity to the Src SH3 domain among various SH3 domains tested. Competitive binding, loop replacement, and NMR perturbation experiments were conducted to analyze the recognition properties of selected binders. The strongest binder was able to pull down full-length c-Src from murine fibroblast cell extracts, further demonstrating the potential of this scaffold for use as an antibody mimetic.