Analysis of the core bacterial community associated with consumer-ready Eastern oysters (Crassostrea virginica).

Shellfish, such as the Eastern oyster (Crassostrea virginica), are an important agricultural commodity. Previous research has demonstrated the importance of the native microbiome of oysters against exogenous challenges by non-native pathogens. However, the taxonomic makeup of the oyster microbiome and the impact of environmental factors on it are understudied. Research was conducted quarterly over a calendar year (February 2020 through February 2021) to analyze the taxonomic diversity of bacteria present within the microbiome of consumer-ready-to-eat live Eastern oysters. It was hypothesized that a core group of bacterial species would be present in the microbiome regardless of external factors such as the water temperature at the time of harvest or post-harvesting processing. At each time point, 18 Chesapeake Bay (eastern United States) watershed aquacultured oysters were acquired from a local grocery store, genomic DNA was extracted from the homogenized whole oyster tissues, and the bacterial 16S rRNA gene hypervariable V4 region was PCR-amplified using barcoded primers prior to sequencing via Illumina MiSeq and bioinformatic analysis of the data. A core group of bacteria were identified to be consistently associated with the Eastern oyster, including members of the phyla Firmicutes and Spirochaetota, represented by the families Mycoplasmataceae and Spirochaetaceae, respectively. The phyla Cyanobacterota and Campliobacterota became more predominant in relation to warmer or colder water column temperature, respectively, at the time of oyster harvest.

Development of a Controlled Laboratory-scale Inoculation System to Study Vibrio parahaemolyticus-oyster Interactions.

Prevalence of seafood-borne gastroenteritis caused by the human pathogen Vibrio parahaemolyticus is increasing globally despite current preventative measures. The United States Centers for Disease Control have designated V. parahaemolyticus as a reportable emerging human pathogen. The Eastern oyster (Crassostrea virginica) is a natural reservoir of the bacterium in marine environments, but little is actually known regarding interactions between oysters and V. parahaemolyticus. Therefore, a laboratory-scale Biosafety Level-2 (BSL2) inoculation system was developed wherein Chesapeake Bay region oysters harvested during summer or winter months, were exposed to the clinical RIMD2210633 strain carrying a chloramphenicol-selective marker (VP RIMDmC). Homogenized whole oyster tissues were spread on selective and differential agar medium to measure viable VP RIMDmC levels. Endogenous Vibrio spp. cell numbers were significantly reduced followed chloramphenicol treatment and this likely contributed to higher VP RIMDmC oyster-associated levels, especially using winter-harvested animals. Summer-harvested oysters had significantly higher existing Vibrio levels and a lower level of artificial oyster-associated VP RIMDmC. Thus, the pre-existing microbiome appears to afford some protection from an external V. parahaemolyticus challenge. Overall, this system successfully enabled controlled manipulation of parameters influencing V. parahaemolyticus-oyster interactions and will be useful in safely testing additional pertinent environmental variables and potential mitigation strategies.

The Transcription Factor Lrp of Pantoea stewartii subsp. stewartii Controls Capsule Production, Motility, and Virulence Important for in planta Growth.

The bacterial phytopathogen Pantoea stewartii subsp. stewartii causes leaf blight and Stewart's wilt disease in susceptible corn varieties. A previous RNA-Seq study examined P. stewartii gene expression patterns during late-stage infection in the xylem, and a Tn-Seq study using a P. stewartii mutant library revealed genes essential for colonization of the xylem. Based on these findings, strains with in-frame chromosomal deletions in the genes encoding seven transcription factors (NsrR, IscR, Nac, Lrp, DSJ_00125, DSJ_03645, and DSJ_18135) and one hypothetical protein (DSJ_21690) were constructed to further evaluate the role of the encoded gene products during in vitro and in planta growth. Assays for capsule production and motility indicate that Lrp plays a role in regulating these two key physiological outputs in vitro. Single infections of each deletion strain into the xylem of corn seedlings determined that Lrp plays a significant role in P. stewartii virulence. In planta xylem competition assays between co-inoculated deletion and the corresponding complementation or wild-type strains as well as in vitro growth curves determined that Lrp controls functions important for P. stewartii colonization and growth in corn plants, whereas IscR may have a more generalized impact on growth. Defining the role of essential transcription factors, such as Lrp, during in planta growth will enable modeling of key components of the P. stewartii regulatory network utilized during growth in corn plants.

Identification of soil bacteria capable of utilizing a corn ethanol fermentation byproduct.

A commercial corn ethanol production byproduct (syrup) was used as a bacterial growth medium with the long-term aim to repurpose the resulting microbial biomass as a protein supplement in aquaculture feeds. Anaerobic batch reactors were used to enrich for soil bacteria metabolizing the syrup as the sole nutrient source over an eight-day period with the goal of obtaining pure cultures of facultative organisms from the reactors. Amplification of the V4 variable region of the 16S rRNA gene was performed using barcoded primers to track the succession of microbes enriched for during growth on the syrup. The resulting PCR products were sequenced using Illumina MiSeq protocols, analyzed via the program QIIME, and the alpha-diversity was calculated. Seven bacterial families were the most prevalent in the bioreactor community after eight days of enrichment: Clostridiaceae, Alicyclobacillaceae, Ruminococcaceae, Burkholderiaceae, Bacillaceae, Veillonellaceae, and Enterobacteriaceae. Pure culture isolates obtained from the reactors, and additional laboratory stock strains, capable of facultative growth, were grown aerobically in microtiter plates with the syrup substrate to monitor growth yield. Reactor isolates of interest were identified at a species level using the full 16S rRNA gene and other biomarkers. Bacillus species, commonly used as probiotics in aquaculture, showed the highest biomass yield of the monocultures examined. Binary combinations of monocultures yielded no apparent synergism between organisms, suggesting competition for nutrients instead of cooperative metabolite conversion.

Discovery of Pantoea stewartii ssp. stewartii genes important for survival in corn xylem through a Tn-Seq analysis.

The bacterium Pantoea stewartii ssp. stewartii causes Stewart's wilt disease in corn. Pantoea stewartii is transmitted to plants via corn flea beetles, where it first colonizes the apoplast causing water-soaked lesions, and then migrates to the xylem and forms a biofilm that blocks water transport. Bacterial quorum sensing ensures that the exopolysaccharide production necessary for biofilm formation occurs only at high cell density. A genomic-level transposon sequencing (Tn-Seq) analysis was performed to identify additional bacterial genes essential for survival in planta and to provide insights into the plant-microbe interactions occurring during wilt disease. A mariner transposon library of approximately 40 000 mutants was constructed and used to inoculate corn seedlings through a xylem infection model. Cultures of the library grown in Luria-Bertani (LB) broth served as the in vitro pre-inoculation control. Tn-Seq analysis showed that the number of transposon mutations was reduced by more than 10-fold for 486 genes in planta compared with the library that grew in LB, suggesting that they are important for xylem survival. Interestingly, a small set of genes had a higher abundance of mutants in planta versus in vitro conditions, indicating enhanced strain fitness with loss of these genes inside the host. In planta competition assays retested the trends of the Tn-Seq data for several genes, including two outer membrane proteins, Lon protease and two quorum sensing-associated transcription factors, RcsA and LrhA. Virulence assays were performed to check for correlation between growth/colonization and pathogenicity. This study demonstrates the capacity of a Tn-Seq approach to advance our understanding of P. stewartii-corn interactions.

Integrated downstream regulation by the quorum-sensing controlled transcription factors LrhA and RcsA impacts phenotypic outputs associated with virulence in the phytopathogen Pantoea stewartii subsp. stewartii.

Pantoea stewartii subsp. stewartii is a Gram-negative proteobacterium that causes leaf blight and Stewart's wilt disease in corn. Quorum sensing (QS) controls bacterial exopolysaccharide production that blocks water transport in the plant xylem at high bacterial densities during the later stage of the infection, resulting in wilt. At low cell density the key master QS regulator in P. stewartii, EsaR, directly represses rcsA, encoding an activator of capsule biosynthesis genes, but activates lrhA, encoding a transcription factor that regulates surface motility. Both RcsA and LrhA have been shown to play a role in plant virulence. In this study, additional information about the downstream targets of LrhA and its interaction with RcsA was determined. A transcriptional fusion assay revealed autorepression of LrhA in P. stewartii and electrophoretic mobility shift assays (EMSA) using purified LrhA confirmed that LrhA binds to its own promoter. In addition, LrhA binds to the promoter for the RcsA gene, as well as those for putative fimbrial subunits and biosurfactant production enzymes in P. stewartii, but not to the flhDC promoter, which is the main direct target of LrhA in Escherichia coli. This work led to a reexamination of the physiological function of RcsA in P. stewartii and the discovery that it also plays a role in surface motility. These findings are broadening our understanding of the coordinated regulatory cascades utilized in the phytopathogen P. stewartii.

Complete Genome Assembly of Pantoea stewartii subsp. stewartii DC283, a Corn Pathogen.

The phytopathogen Pantoea stewartii subsp. stewartii DC283 causes Stewart's wilt disease in corn after transmission from the corn flea beetle insect vector. Here, we report that the complete annotated genome of P. stewartii DC283 has been fully assembled into one circular chromosome, 10 circular plasmids, and one linear phage.

Analysis of the in planta transcriptome expressed by the corn pathogen Pantoea stewartii subsp. stewartii via RNA-Seq.

Pantoea stewartii subsp. stewartii is a bacterial phytopathogen that causes Stewart's wilt disease in corn. It uses quorum sensing to regulate expression of some genes involved in virulence in a cell density-dependent manner as the bacterial population grows from small numbers at the initial infection site in the leaf apoplast to high cell numbers in the xylem where it forms a biofilm. There are also other genes important for pathogenesis not under quorum-sensing control such as a Type III secretion system. The purpose of this study was to compare gene expression during an in planta infection versus either a pre-inoculum in vitro liquid culture or an in vitro agar plate culture to identify genes specifically expressed in planta that may also be important for colonization and/or virulence. RNA was purified from each sample type to determine the transcriptome via RNA-Seq using Illumina sequencing of cDNA. Fold gene expression changes in the in planta data set in comparison to the two in vitro grown samples were determined and a list of the most differentially expressed genes was generated to elucidate genes important for plant association. Quantitative reverse transcription PCR (qRT-PCR) was used to validate expression patterns for a select subset of genes. Analysis of the transcriptome data via gene ontology revealed that bacterial transporters and systems important for oxidation reduction processes appear to play a critical role for P. stewartii as it colonizes and causes wilt disease in corn plants.

Using a Concept Inventory to Reveal Student Thinking Associated with Common Misconceptions about Antibiotic Resistance.

Misconceptions, also known as alternate conceptions, about key concepts often hinder the ability of students to learn new knowledge. Concept inventories (CIs) are designed to assess students' understanding of key concepts, especially those prone to misconceptions. Two-tiered CIs include prompts that ask students to explain the logic behind their answer choice. Such two-tiered CIs afford an opportunity for faculty to explore the student thinking behind the common misconceptions represented by their choice of a distractor. In this study, we specifically sought to probe the misconceptions that students hold prior to beginning an introductory microbiology course (i.e., preconceptions). Faculty-learning communities at two research-intensive universities used the validated Host-Pathogen Interaction Concept Inventory (HPI-CI) to reveal student preconceptions. Our method of deep analysis involved communal review and discussion of students' explanations for their CI answer choice. This approach provided insight valuable for curriculum development. Here the process is illustrated using one question from the HPI-CI related to the important topic of antibiotic resistance. The frequencies with which students chose particular multiple-choice responses for this question were highly correlated between institutions, implying common underlying misconceptions. Examination of student explanations using our analysis approach, coupled with group discussions within and between institutions, revealed patterns in student thinking to the participating faculty. Similar application of a two-tiered concept inventory by general microbiology instructors, either individually or in groups, at other institutions will allow them to better understand student thinking related to key concepts in their curriculum.

Development, Validation, and Application of the Microbiology Concept Inventory.

If we are to teach effectively, tools are needed to measure student learning. A widely used method for quickly measuring student understanding of core concepts in a discipline is the concept inventory (CI). Using the American Society for Microbiology Curriculum Guidelines (ASMCG) for microbiology, faculty from 11 academic institutions created and validated a new microbiology concept inventory (MCI). The MCI was developed in three phases. In phase one, learning outcomes and fundamental statements from the ASMCG were used to create T/F questions coupled with open responses. In phase two, the 743 responses to MCI 1.0 were examined to find the most common misconceptions, which were used to create distractors for multiple-choice questions. MCI 2.0 was then administered to 1,043 students. The responses of these students were used to create MCI 3.0, a 23-question CI that measures students' understanding of all 27 fundamental statements. MCI 3.0 was found to be reliable, with a Cronbach's alpha score of 0.705 and Ferguson's delta of 0.97. Test item analysis demonstrated good validity and discriminatory power as judged by item difficulty, item discrimination, and point-biserial correlation coefficient. Comparison of pre- and posttest scores showed that microbiology students at 10 institutions showed an increase in understanding of concepts after instruction, except for questions probing metabolism (average normalized learning gain was 0.15). The MCI will enable quantitative analysis of student learning gains in understanding microbiology, help to identify misconceptions, and point toward areas where efforts should be made to develop teaching approaches to overcome them.

Concept Inventory Development Reveals Common Student Misconceptions about Microbiology.

Misconceptions, or alternative conceptions, are incorrect understandings that students have incorporated into their prior knowledge. The goal of this study was the identification of misconceptions in microbiology held by undergraduate students upon entry into an introductory, general microbiology course. This work was the first step in developing a microbiology concept inventory based on the American Society for Microbiology's Recommended Curriculum Guidelines for Undergraduate Microbiology. Responses to true/false (T/F) questions accompanied by written explanations by undergraduate students at a diverse set of institutions were used to reveal misconceptions for fundamental microbiology concepts. These data were analyzed to identify the most difficult core concepts, misalignment between explanations and answer choices, and the most common misconceptions for each core concept. From across the core concepts, nineteen misconception themes found in at least 5% of the coded answers for a given question were identified. The top five misconceptions, with coded responses ranging from 19% to 43% of the explanations, are described, along with suggested classroom interventions. Identification of student misconceptions in microbiology provides a foundation upon which to understand students' prior knowledge and to design appropriate tools for improving instruction in microbiology.

Analyzing the Transcriptomes of Two Quorum-Sensing Controlled Transcription Factors, RcsA and LrhA, Important for Pantoea stewartii Virulence.

The Gram-negative proteobacterium Pantoea stewartii subsp. stewartii causes wilt disease in corn plants. Wilting is primarily due to bacterial exopolysaccharide (EPS) production that blocks water transport in the xylem during the late stages of infection. EsaR, the master quorum-sensing (QS) regulator in P. stewartii, modulates EPS levels. At low cell densities EsaR represses or activates expression of a number of genes in the absence of its acyl homoserine lactone (AHL) ligand. At high cell densities, binding of AHL inactivates EsaR leading to derepression or deactivation of its direct targets. Two of these direct targets are the key transcription regulators RcsA and LrhA, which in turn control EPS production and surface motility/adhesion, respectively. In this study, RNA-Seq was used to further examine the physiological impact of deleting the genes encoding these two second-tier regulators. Quantitative reverse transcription PCR (qRT-PCR) was used to validate the regulation observed in the RNA-Seq data. A GFP transcriptional fusion reporter confirmed the existence of a regulatory feedback loop in the system between LrhA and RcsA. Plant virulence assays carried out with rcsA and lrhA deletion and complementation strains demonstrated that both transcription factors play roles during establishment of wilt disease in corn. These efforts further define the hierarchy of the QS-regulated network controlling plant virulence in P. stewartii.

Chlorinated phenol-induced physiological antibiotic resistance in Pseudomonas aeruginosa.

Pseudomonas aeruginosa is a ubiquitous environmental bacterium and an opportunistic pathogen with the ability to rapidly develop multidrug resistance under selective pressure. Previous work demonstrated that upon exposure to the environmental contaminant pentachlorophenol (PCP), P. aeruginosa PAO1 increases expression of multiple multidrug efflux pumps, including the MexAB-OprM pump. The current study describes increases in the antibiotic resistance of PAO1 upon exposure to PCP and other chlorinated organics, including triclosan. Only exposure to chlorinated phenols induced the mexAB-oprM-mediated antibiotic-resistant phenotype. Thus, chlorinated phenols have the potential to contribute to transient phenotypic increases of antibiotic resistance that are relevant when both compounds are present in the environment.

OpaR controls a network of downstream transcription factors in Vibrio parahaemolyticus BB22OP.

Vibrio parahaemolyticus is an emerging world-wide human pathogen that is associated with food-borne gastroenteritis when raw or undercooked seafood is consumed. Expression of virulence factors in this organism is modulated by the phenomenon known as quorum sensing, which permits differential gene regulation at low versus high cell density. The master regulator of quorum sensing in V. parahaemolyticus is OpaR. OpaR not only controls virulence factor gene expression, but also the colony and cellular morphology associated with growth on a surface and biofilm formation. Whole transcriptome Next Generation sequencing (RNA-Seq) was utilized to determine the OpaR regulon by comparing strains BB22OP (opaR+, LM5312) and BB22TR (∆opaR1, LM5674). This work, using the published V. parahaemolyticus BB22OP genome sequence, confirms and expands upon a previous microarray analysis for these two strains that used an Affymetrix GeneChip designed from the closely related V. parahaemolyticus RIMD2210633 genome sequence. Overall there was excellent correlation between the microarray and RNA-Seq data. Eleven transcription factors under OpaR control were identified by both methods and further confirmed by quantitative reverse transcription PCR (qRT-PCR) analysis. Nine of these transcription factors were demonstrated to be direct OpaR targets via in vitro electrophoretic mobility shift assays with purified hexahistidine-tagged OpaR. Identification of the direct and indirect targets of OpaR, including small RNAs, will enable the construction of a network map of regulatory interactions important for the switch between the nonpathogenic and pathogenic states.

Toward development of an autonomous network of bacteria-based delivery systems (BacteriaBots): spatiotemporally high-throughput characterization of bacterial quorum-sensing response.

Characterization of bacterial innate and engineered cooperative behavior, regulated through chemical signaling in a process known as quorum sensing, is critical to development of a myriad of bacteria-enabled systems including biohybrid drug delivery systems and biohybrid mobile sensor networks. Here, we demonstrate, for the first time, that microfluidic diffusive mixers can be used for spatiotemporally high-throughput characterization of bacterial quorum-sensing response. Using this batch characterization method, the quorum-sensing response in Escherichia coli MG1655, transformed with a truncated lux operon from Vibrio fischeri, in the presence of 1-100 nM exogenous acyl-homoserine lactone molecules has been quantified. This method provides a rapid and facile tool for high-throughput characterization of the quorum-sensing response of genetically modified bacteria in the presence of a wide concentration range of signaling molecules with a precision of ±0.5 nM. Furthermore, the quorum-sensing response of BacteriaBots has been characterized to determine if the results obtained from a large bacterial population can serve as a robust predictive tool for the small bacterial population attached to each BacteriaBot.

Acyl-homoserine lactone recognition and response hindering the quorum-sensing regulator EsaR.

During quorum sensing in the plant pathogen Pantoea stewartii subsp. stewartii, EsaI, an acyl-homoserine lactone (AHL) synthase, and the transcription factor EsaR coordinately control capsular polysaccharide production. The capsule is expressed only at high cell density when AHL levels are high, leading to inactivation of EsaR. In lieu of detailed structural information, the precise mechanism whereby EsaR recognizes AHL and is hindered by it, in a response opposite to that of most other LuxR homologues, remains unresolved. Hence, a random mutagenesis genetic approach was designed to isolate EsaR* variants that are immune to the effects of AHL. Error-prone PCR was used to generate the desired mutants, which were subsequently screened for their ability to repress transcription in the presence of AHL. Following sequencing, site-directed mutagenesis was used to generate all possible mutations of interest as single, rather than multiple amino acid substitutions. Eight individual amino acids playing a critical role in the AHL-insensitive phenotype have been identified. The ability of EsaR* variants to bind AHL and the effect of individual substitutions on the overall conformation of the protein were examined through in vitro assays. Six EsaR* variants had a decreased ability to bind AHL. Fluorescence anisotropy was used to examine the relative DNA binding affinity of the final two EsaR* variants, which retained some AHL binding capability but remained unresponsive to it, perhaps due to an inability of the N-terminal domain to transduce information to the C-terminal domain.

Transcriptome-based analysis of the Pantoea stewartii quorum-sensing regulon and identification of EsaR direct targets.

Pantoea stewartii subsp. stewartii is a proteobacterium that causes Stewart's wilt disease in corn plants. The bacteria form a biofilm in the xylem of infected plants and produce capsule that blocks water transport, eventually causing wilt. At low cell densities, the quorum-sensing (QS) regulatory protein EsaR is known to directly repress expression of esaR itself as well as the genes for the capsular synthesis operon transcription regulator, rcsA, and a 2,5-diketogluconate reductase, dkgA. It simultaneously directly activates expression of genes for a putative small RNA, esaS, the glycerol utilization operon, glpFKX, and another transcriptional regulator, lrhA. At high bacterial cell densities, all of this regulation is relieved when EsaR binds an acylated homoserine lactone signal, which is synthesized constitutively over growth. QS-dependent gene expression is critical for the establishment of disease in the plant. However, the identity of the full set of genes controlled by EsaR/QS is unknown. A proteomic approach previously identified around 30 proteins in the QS regulon. In this study, a whole-transcriptome, next-generation sequencing analysis of rRNA-depleted RNA from QS-proficient and -deficient P. stewartii strains was performed to identify additional targets of EsaR. EsaR-dependent transcriptional regulation of a subset of differentially expressed genes was confirmed by quantitative reverse transcription-PCR (qRT-PCR). Electrophoretic mobility shift assays demonstrated that EsaR directly bound 10 newly identified target promoters. Overall, the QS regulon of P. stewartii orchestrates three major physiological responses: capsule and cell envelope biosynthesis, surface motility and adhesion, and stress response.

Proteomic analysis of the quorum-sensing regulon in Pantoea stewartii and identification of direct targets of EsaR.

The proteobacterium Pantoea stewartii subsp. stewartii causes Stewart's wilt disease in maize when it colonizes the xylem and secretes large amounts of stewartan, an exopolysaccharide. The success of disease pathogenesis lies in the timing of bacterial virulence factor expression through the different stages of infection. Regulation is achieved through a quorum-sensing (QS) system consisting of the acyl-homoserine lactone (AHL) synthase, EsaI, and the transcription regulator EsaR. At low cell densities, EsaR represses transcription of itself and of rcsA, an activator of the stewartan biosynthesis operon; it also activates esaS, which encodes a small RNA (sRNA). Repression or activation ceases at high cell densities when EsaI synthesizes sufficient levels of the AHL ligand N-3-oxo-hexanoyl-L-homoserine lactone to bind and inactivate EsaR. This study aims to identify other genes activated or repressed by EsaR during the QS response. Proteomic analysis identified a QS regulon of more than 30 proteins. Electrophoretic mobility shift assays of promoters of genes encoding differentially expressed proteins distinguished direct targets of EsaR from indirect targets. Additional quantitative reverse transcription-PCR (qRT-PCR) and DNA footprinting analysis established that EsaR directly regulates the promoters of dkgA, glpF, and lrhA. The proteins encoded by dkgA, glpF, and lrhA are a 2,5-diketogluconate reductase, glycerol facilitator, and transcriptional regulator of chemotaxis and motility, respectively, indicating a more global QS response in P. stewartii than previously recognized.

Complete Genome Sequence of Prepandemic Vibrio parahaemolyticus BB22OP.

The number of inflammatory gastroenteritis outbreaks due to the food-borne pathogen Vibrio parahaemolyticus is rising sharply worldwide and in the United States in particular. Here we report the complete, annotated genome sequence of the prepandemic V. parahaemolyticus strain BB22OP and make some initial comparisons to the complete genome sequence for pandemic strain RIMD2210633.

Working together for the common good: cell-cell communication in bacteria.

The 4th ASM Conference on Cell-Cell Communication in Bacteria was held in Miami, FL, from 6 to 9 November 2011. This review highlights three key themes that emerged from the many exciting talks and poster presentations in the area of quorum sensing: sociomicrobiology, signal transduction mechanisms, and interspecies communication.