A transcription factor from caulophage φCbK modulates expression of biofilm and cell lysis regulators in Caulobacter crescentus

Maeve McLaughlin

Michigan State University

Bacteria commonly form multicellular communities known as biofilms. Biofilm formation often enhances resistance to both physical and chemical stresses, which can pose challenges to antimicrobial treatment of bacterial infections. As an alternative to antibiotics, bacteriophage (i.e. viruses that infect bacteria), are increasingly being used as an alternative to treatment of recalcitrant bacterial infections. Bacteriophages have been reported to disrupt biofilms by infecting and lysing the bacteria within but, in some cases, bacteriophage can trigger biofilm formation. The Alphaproteobacterium, Caulobacter crescentus, provides an outstanding model system to study bacteriophage-biofilm interactions. C. crescentus secretes a polar adhesin known as the holdfast that is required for formation of surface-associated and pellicle biofilms. An ensemble of four paralogous XRE-family transcription factors directly regulate holdfast development in response to diverse environmental cues by directly controlling transcription of the holdfast inhibitor, hfiA. These paralogous transcription factors also directly inhibit the gafYZ system, which activates a cluster of chromosomally encoded phage-like proteins that induce cell lysis. I have discovered that the pilitropic caulophage φCbK encodes an XRE transcription factor, gp216. This gene is homologous to the C. crescentus XRE-family regulators and is highly expressed early during phage infection. Gp216 binds the promoters and represses expression of the holdfast regulator, hfiA, and the cell lysis regulators, gafYZ. Using ChIP-seq, I observed that gp216 bound dozens of other regions within the C. crescentus genome that overlapped with the Caulobacter XRE binding sites. Furthermore, gp216 physically interacts with the C. crescentus XRE proteins, suggesting that this viral protein directly interfaces with host transcriptional networks during infection. These data support a model in which gp216 expression after φCbK infection promotes biofilm formation and prevents abortive infection (i.e. pre-mature cell lysis) during viral replication.

S-nitrosoglutathione (GSNO) ameliorates severity of experimental Staphylococcus aureus endophthalmitis

Susmita Das*¹, Zeeshan Ahmad¹, Sukhvinder Singh¹, Sneha Singh¹, Shailendra Giri² and Ashok Kumar¹

¹Department of Ophthalmology, Visual and Anatomical Sciences, Wayne State University School of Medicine
²Department of Neurology, Henry Ford Hospital

Purpose: Bacterial endophthalmitis remain one of the major complications of ocular surgeries and trauma. The current treatment involves intravitreal injections of antibiotics which control bacterial growth but not inflammation. We adapted integrative transcriptomics and metabolomics approach to identify immunomodulatory pathways altered during endophthalmitis. The aim of this study is to investigate the role of GSNO, whose levels were drastically reduced in Staphylococcus aureus (SA) infected mouse retina.

Methods: Endophthalmitis was induced by intravitreal injections of S. aureus in C57BL/6 mice. Both prophylactic and therapeutic efficacy of GSNO was determined by giving GSNO via intravitreal, oral or a combination routes. Disease progression was monitored by eye exam using microscopy, bacterial burden enumeration, assessment of inflammatory mediators and ERG analyses. For in vitro studies, human retinal cultured cells were treated with GSNO prior to SA infection. The expression of inflammatory mediators was performed by qPCR and ELISA. Cultured retinal cells were evaluated for viability post GSNO treatment with SA infection by live staining with 7AAD. Human RPE cells (ARPE-19) were used to determine the effect of GSNO on outer blood-retinal barrier (BRB) integrity in response to SA infection by assessing the expression of tight junction proteins and fluorescein isothiocyanate (FITC)-dextran transepithelial permeability assay. The induction of nitrosative stress was measured by INOS expression both in vitro as well as in vivo after challenge with SA in presence or absence of GSNO.

Results: Our data showed that GSNO (1µg/eye) treatment significantly reduced bacterial burden and levels of pro-inflammatory mediators when administered prophylactically. The oral administration of GSNO (1mg/kg) for 48h also exerted protective effects as evidenced by a gradual decline in bacterial counts and reduction in intraocular inflammation. In vitro studies showed attenuation of SA induced inflammatory mediators in cultured retinal cells. Oral GSNO treatment also exhibited synergy with intravitreal vancomycin injection in bacteria-infected eyes. Interestingly, GSNO treatment reduced bacteria-induced Muller glial cell death indicating cytoprotection. Moreover, GSNO strengthened the outer BRB as evidenced by preserved ZO-1 staining and reduced transepithelial permeability in response to S. aureus infection. INOS expression was significantly reduced under GSNO treatment post infection, suggesting a rescue in nitrosative stress environment.

Conclusion: Our study revealed that GSNO administration exert antibacterial, anti-inflammatory and cytoprotective properties in the eye. Thus, GSNO can be used as an immunomodulatory therapeutic to ameliorate bacterial endophthalmitis.

Vibrio cholerae modulates cyclic di-GMP in response to zinc via a horizontally acquired genomic island

Aathmaja Anandhi Rangarajan¹*, Marissa K. Malleck¹, Geoffrey B. Severin^1,2, and Christopher M. Waters¹

Department of Microbiology and Molecular Genetics, Michigan State University, East Lansing, MI, USA¹
Department of Microbiology and Immunology, University of Michigan, Ann Arbor, MI, USA²

Cyclic-di GMP is an important signaling molecule that controls several important biological processes including biofilm formation, motility and protein secretion. The intracellular level of cyclic-di GMP is regulated by diguanylate cyclases (DGCs) that synthesize cyclic-di GMP from GTP and phosphodiesterases (PDE) that degrade cyclic-di GM. Ongoing 7^th pandemic Vibrio cholerae El Tor strains have acquired two genomic islands, VSP-1 and 2, that are hypothesized to be important for their pathogenicity and adaptability.  However, the functions of several genes in these genomic islands are unknown. Recently it has been shown that genes vc0512-vc0515 in the VSP-2 island are repressed by zinc via the Zurrepressor and are induced by transcriptional activator VerA (vc0513). This region includes the predicted cyclic-di GMP phosphodiesterase, vc0515.

In this study, we delineated role of zinc in the regulation of phosphodiesterase VC0515 present in VSP-2 island. We have shown that VC0515 is an active PDE and the mutation of the active site ELL-AAA rendered the protein inactive. We then determined that the intracellular concentrations of cyclic-di GMP in a Δzur mutant was lower than the WT and ΔverA mutant owing to increased expression of VC0515. In line with the decreased cyclic-di GMP levels, the Δzur mutant had higher motility and decreased biofilm formation. Addition of zinc to a ΔznuABC importer mutant repressed the vc0512-vc0515 operon, increasing cyclic-di GMP and biofilm formation.  We also analyzed the presence of vc0512-vc0515 in several El Tor strains of V. cholerae and found them to be prevalent in the 7^th pandemic strains underlining the evolutionary significance of these genes. Our results demonstrate that the current pandemic of V. cholerae has acquired a new regulatory system to alter cyclic di-GMP and biofilms in response to zinc.

Understanding the role of mitochondrial pyruvate carrier (MPC) proteins in the adaptive mitochondrial response to endoplasmic reticulum stress (ERS) in Saccharomyces cerevisiae

Orozco-Quime, Michelle*¹ and Chang, Amy¹

¹University of Michigan, Ann Arbor, MI 48109

The endoplasmic reticulum (ER) is a vital organelle responsible for secretory protein synthesis, folding, and modification, as well as lipid synthesis. Disruptions in ER function can lead to widespread ramifications on cell health, so cellular responses to ERS are critical in maintaining homeostasis. The unfolded protein response (UPR) is a conserved transcriptional response to ERS that can be observed in eukaryotes ranging from budding yeast to mammals. While the UPR is the major pathway of the ER stress response, a multitude of other pathways and organelles have been suggested to play a role in the mitigation of ER stress. Through the use of DHE staining, oxygen consumption assays and other microscopy methods, the ER and mitochondria have been shown to work together to alleviate ER stress through the modulation of pyruvate using the mitochondrial pyruvate carrier (MPC) proteins embedded in the mitochondrial membrane in the model organism Saccharomyces cerevisiae. This is accompanied by an increase in MPC1 levels following ER stress and followed by increases in cellular respiration and upregulation of OXPHOS subunits. This delivery system is also sufficient in rescuing the ERS response in peroxisome deficient mutants, adding to the current knowledge of the intersectionality of organelles in the ERS response.

Using small molecules to identify host-cellular pathways for Brucella infection

Thomas Kim

Michigan State University

Brucellosis is a highly contagious zoonosis caused by exposure to animal products contaminated with bacteria of the genus Brucella. In addition to the burden it inflicts on human health, brucellosis causes devasting losses to the livestock industry and small-scale livestock holders in many areas of the world. Current understanding of Brucella infection and macrophage trafficking have been characterized through the identification of bacterial virulence factors, however, the host-factors required for infection remain elusive. The identification of host-cellular processes involved in Brucella infection is critical for the development of novel host-targeted therapeutic interventions. Host-directed therapy is a treatment strategy that aims to target essential host-cellular processes that are required for pathogen survival. By targeting the host rather than the bacteria itself, antimicrobial resistance is less likely to develop, mitigating the development of multi-drug resistance. We have used a pharmacologic approach to identify possible host-cellular processes that are involved in Brucella infection. We screened 8000 small molecules in a tissue culture infection model involving the non-human pathogen Brucella ovis, a close relative of the zoonotic Brucella species in THP-1 macrophage-like cells. We engineered a constitutively luminescent strain of B. ovis to monitor infection in a high-throughput fashion. Our screening efforts resulted in 147 primary hits. Multiple hits were FDA approved drugs that target Ca2+ homeostasis, many of which are clinically indicated for cardiovascular diseases. Our follow up studies have confirmed that the Ca2+ targeting small molecules inhibit Brucella ovis intracellularly in our THP-1 infection model. These preliminary data show that FDA approved Ca2+ modulators are an effective host-directed small molecule in vitro. Moreover, these data provide evidence that Ca2+ homeostasis plays an important role in Brucella pathogenesis in the macrophage.

Metagenomic Sequencing Reveals Vaginal Microbiome Signatures Unique to Preterm Birth

Jonathan J. Panzer*^,1, Roberto Romero², Andrew D. Winters¹, Tinnakorn Chaiworapongsa¹, Stanley M. Berry¹, Nardhy Gomez-Lopez¹, Adi L. Tarca¹, Kevin R. Theis¹

¹ Wayne State University, Detroit, MI 48201
² Pregnancy Research Branch, Bethesda, MD, 20892

Preterm birth is the leading cause of neonatal morbidity and mortality worldwide. A substantial subset of preterm births is caused by maternal and/or fetal inflammatory responses to bacteria that ascended from the vagina into the amniotic cavity. Thus, the vaginal microbiome serves as a primary source of pathogenic bacteria for the amniotic cavity, yet the factors and mechanisms leading to microbial ascension and invasion are not understood. Herein, we present the largest longitudinal study of the vaginal microbiome in the context of pregnancy ending in spontaneous preterm or term delivery (N=771 women, n=2855 samples). All subjects were sampled at least three times (median = 4) from 8 to 36+6 weeks gestation. Metagenomic sequencing was performed to characterize the microbiome both taxonomically and functionally, utilizing a custom Kraken2 database and HUMAnN3, respectively. Differentially abundant functional pathways were identified with MaAsLin2. The vaginal microbiome of women who ultimately delivered preterm had significantly higher microbial diversity than those who delivered at term. More specifically, those women who experienced preterm prelabor rupture of membranes (PPROM) before 34 weeks gestation (i.e., early PPROM) exhibited significantly higher alpha diversity throughout gestation than their term control counterparts. Predictive modelling of early PPROM yielded an AUC of 0.721 (0.629, 0.812) when based on the maximum Chao1 richness from a subject’s samples collected between 8- and 28-weeks gestation. Taxonomically stratified functional pathways revealed significant decreases in Lactobacillus crispatus and Lactobacillus iners purine nucleotide de novo biosynthesis and significant increases in carbohydrate biosynthesis and metabolism of largely Prevotella species among subjects ultimately experiencing early PPROM. Therefore, we conclude that alterations in the vaginal microbiome are strongest in subjects experiencing early PPROM. Future work should focus on elucidating how the vaginal microbiome contributes to the incidence of early PPROM in pregnancy.

CdsZ represses conversion formation and infectious progeny in the developmental cycle of Chlamydia trachomatis.

Srishti Baid* and P. Scott Hefty

Department of Molecular Biosciences, University of Kansas, Lawrence, KS

Chlamydia trachomatis is a worldwide public health challenge as the primary cause of bacterial sexually transmitted infections and blindness. These are obligate intracellular bacteria that are maintained through a biphasic developmental cycle that includes conversions between distinct infectious elementary bodies and non-infectious, replicative reticulate bodies. The ability of the organism to infect and cause disease is dependent on the precise regulation of conversion and replication processes. The regulatory factors and mechanisms that control the conversion processes of the developmental cycle are still being discovered and characterized. Through protein structure crystallization, a protein of unknown function, CT398, was characterized and named CdsZ (Barta et al.,2015). This was followed by co-immunoprecipitation experiments, which revealed an interaction of CdsZ with an alternative chlamydial sigma factor, σ⁵⁴ and components of RNA polymerase machinery. To evaluate the function of CdsZ, RNA seq analysis, and associated gene expression analyses were performed following CdsZ overexpression indicating that it represses a subset of late gene expression. Other phenotypic and morphologic analyses indicate that overexpression has a negative effect on growth and the key developmental cycle process of conversion. Future studies will be focused on the potential mechanism of repression as well as those that contribute to de-repression.

Characterization of a secreted biofilm inhibitor in Myxococcus xanthus

Mataczynski, Christopher R.* and Higgs, Penelope I.

Wayne State University, Detroit, Michigan 48202

Biofilms are microbial communities which pose a health and biofouling risk, leading to a multitrillion dollar economic loss yearly. Complete removal of biofilms is often difficult because of an increased tolerance to physical and chemical treatments. Therefore, elucidation of the molecular mechanisms controlling biofilm formation is key to identification of therapeutics that prevent biofilm formation. We use the specialized biofilm produced by the bacterium Myxococcus xanthus as a model for biofilm formation processes. Nutrient limitation induces M. xanthus to aggregate into mounds and then differentiate into spores. We have recently discovered that spent media from a strain overproducing a biofilm regulatory protein, TodK, contains an inhibitory factor that prevents biofilm formation in wild-type strains, but does not dissolve pre-formed biofilms. We have demonstrated that the inhibitory factor is functional after DNase treatment and after boiling at 99^oC for 10 minutes, suggesting eDNA or proteins are not the components. We have also ruled out the trivial explanation that the spent media contains nutrients released by lysing cells. Instead, we hypothesize TodK regulates secretion of a small inhibitory factor that prevents biofilm formation when nutrients are replete. We are currently investigating whether the inhibitory factor can prevent biofilms in other species, suggesting a universal biofilm inhibitor.

Discovery of new phage defense systems in Vibrio cholerae

Jasper B. Gomez* and Christopher M. Waters

Michigan State University

Over a century ago, Felix d' Herelle began using bacterial viruses which he termed "bacteriophages" to treat bacterial infections; such "phage therapy" has gained new interest due to the emergence of multi-drug resistant bacterial pathogens. Phage therapy has many advantages such as the ability to lyse specific bacteria without disrupting the host microbiota. Although phages can infect and lyse bacteria, bacteria have evolved a myriad of molecular defense systems to protect against phage infection. Although many phage defense mechanisms have recently been identified, other mechanisms of phage defense have yet to be discovered. The bacterial pathogen Vibrio cholerae is intimately linked to phage predation both during environmental persistence and infection in humans, and three novel phage defense systems have been recently discovered in this bacterium. I hypothesized that V. cholerae encodes additional novel phage defense systems. To identify these systems, I screened a V. cholerae cosmid genomic library in Escherichia coli for segments of V. cholerae's genome that protected E. coli from T2 phage infection. Two unique cosmids, each encoding approximately 25kB of V. cholerae DNA, that do not encode the known phage defense systems, were found to protect E. coli from T2 infection. Transposon and deletion mutagenesis on one of the cosmids revealed two distinct phage defense systems, one which resembles a novel restriction/modification system and the second which encodes the previously described but unstudied Zorya system. My studies will uncover the molecular mechanisms by which these systems protect bacteria from phage infection, increasing our understanding of the evolution and ecology of V. cholerae while highlighting important mechanisms by which bacteria can resist phage therapy.

Characterizing Short-term Spread of Methicillin-resistant Staphylococcus aureus And Vancomycin-resistant Enterococcus in Community Living Centers

Clement, Tasmine*, Cassone, Marco, Pirani, Ali, Snitkin, Evan, Mody, Lona

University of Michigan - Ann Arbor, Michigan, 48104

The spread of multidrug resistant organisms (MDROs) such as Staphylococcus aureus and vancomycin-resistant Enterococcus (VRE) in community living centers can cause significant mortality for patients. More research is needed to understand how to utilize genomic and epidemiological data to identify unappreciated pathways mediating transmission. Our study included genome sequencing of patient and environmental samples from 6 patients within the VA Ann Arbor Healthcare System from April 19 - June 17, 2021. Single nucleotide variants (SNVs) were identified by mapping reads and calling variants against strain-specific reference genomes. We used ape v5.6-2 in R v4.2.2 to analyze and infer evolution, acquisition, and transmission events based on pairwise single-nucleotide variant (SNV) distances.

A total of 76 samples from 5 patients tested positive for MRSA (19) and VRE (57). Patients 1 & 3 were co-colonized with MRSA and VRE. For patients 1 and 3, MDROswere found on both patient and environmental samples. 73% of sample pairs had less than 20 SNVs while 23% had more than 20 SNVs, indicating that the independent acquisition of multiple strains is common.

The distribution of pairwise SNV distances within each patient suggests intra-host variation that may be obscured when using traditional methods of sequencing and analyzing genomes derived from single isolates colonizing a patient. Incorporating genomic data from multiple isolates and from varied environmental sources within community living centers may allow for an increased understanding of drivers of endemicity and lead to improved infection prevention interventions for the vulnerable populations in congregate settings.

Unveiling the Biological Potential of the Human Oral Microbiome

Maribel EK Okiye^1,2,3, Michelle A Velez⁵, Jim Sugai⁵, Janet Kinney⁶, William Gianobbile⁶, Ashootosh Tripathi^2,4, and David H Sherman^1,3,4

¹Department of Chemistry, University of Michigan, Ann Arbor, MI; ²Department of Computational Medicine and Bioinformatics, University of Michigan, Ann Arbor, MI; ³Life Sciences Institute, University of Michigan, Ann Arbor, MI; ⁴Department of Medicinal Chemistry, College of Pharmacy, University of Michigan, Ann Arbor, MI; ⁵Department of Dental Hygiene, School of Dentistry, University of Michigan, Ann Arbor MI; ⁶Department of Periodontics and Oral Medicine, School of Dentistry, University of Michigan, Ann Arbor, MI

The human oral microbiome typically contains over 700 different microbial species. These interactions can shape the microenvironment throughout the human body, as these interactions are paramount to maintaining oral and overall systemic health. Recent advances in technology, such as next-generation sequencing (NGS), have revealed the complexities of the oral microbiome, linking dysbiosis of the oral microbiome with several chronic ailments such as cardiovascular disease, diabetes, and rheumatoid arthritis. However, the role of microbial secondary metabolites in oral and systemic disease progression remains poorly understood. We conducted a metabolomics study on the human salivary secondary metabolome during the progression of early-stage periodontal disease (gingivitis). In this study, we sought to assess the changes in the oral secondary metabolome during disease progression by emulating dysbiosis of the oral microbiome through a twenty-one-day induction of gingivitis in twenty human subjects. Our study identifies three secondary metabolites, cyclo(L-Val-L-Pro) and cyclo(L-Pro-L-4-hydroxy-Phe), with regulatory properties for bacterial biofilm formation and inflammatory marker secretion, indicating a specialized role for secondary metabolites in oral health maintenance. Surprisingly, we also uncovered a previously unknown metabolic lag that occurs during dysbiosis recovery of the oral cavity, which suggests either a lingering presence of signaling molecules for pathogenic microbe proliferation or a total oral metabolome modification following microenvironmental stress in the oral cavity. This work represents a high-resolution metabolomic landscape for understanding oral health during gingivitis that opens new opportunities for combating progressive periodontal disease and sepsis due to the translocation of oral microbes in the human body.

Identifying CBASS Phage Defense Activation in Vibrio cholerae El Tor

Ram Sanath-Kumar*, Soo Hun Yoon, Kaylee Wilburn, & Christopher M. Waters

Michigan State University

Cholera is a serious threat, causing 1.4 to 4.3 million cases every year worldwide especially in developing countries². Vibrio cholerae El Tor differs from the previous pandemic causing classical biotype through its two pathogenicity islands VSP-1 and VSP-2 that offer an advantage in virulence and its epidemiological success². Transposon mutagenesis was done on VSP-1 which isolated an abortive cyclic-oligonucleotide based antiphage signaling system dubbed CBASS. CBASS is not unique to Vibrio cholerae, with it being a well-conserved system in all bacteria phyla and found near 65.5% of other microbial defense systems¹. Better understanding of the mechanism and gene relationships may reveal novel bacteria control methods amid rising drug resistant microbes. In addition, as a well-conserved phage defense mechanism, further exploitation of this system may prove imperative in growing areas such as phage therapy. One area of exploitation is the initiation of the defense system to induce cell death. CBASS is activated by activation of its dinucleotide cyclase through folate level changes during phage infection. The proposed activation comes from prior protein work indicating CBASS’s dinucleotide cyclase is regulated by folate⁶. Work done, found CBASS defense being specific to phages T2, T4, and T5 all which carry viral gene products for dihydrofolate reductases (DHFR)³ that manipulates host folate levels. The proposed activation mechanism was tested through viability assays of phage T5 DHFR expression along with a DHFR phage knockout testing and mass spectrometry of folate levels within the cell during infection.

Environmental Monitoring of Listeria spp. in Controlled Atmosphere Apple Storage Facilities

De'Anthony Morris*, Department of Food Science and Human Nutrition, Michigan State University; Erik Diaz-Santiago, Department of Food Science and Human Nutrition, Michigan State University; Elliot T. Ryser, Department of Food Science and Human Nutrition, Michigan State University; and Teresa M. Bergholz, Department of Food Science and Human Nutrition, Michigan State University

Background: Since the infamous caramel apple outbreak of 2014 that sickened 36 individuals with listeriosis, research regarding the relationship between apples and Listeria monocytogenes has increased. Previous studies have suggested Listeria spp. can be introduced onto apples in apple packinghouses via workers and equipment, subsequently spread throughout the processing environment, and persist even after attempts of thorough cleaning and sanitization. Prior to packing, apples may be held for months in controlled atmosphere (CA) storage rooms. There is a need to investigate if Listeria spp. are present in CA storage rooms throughout the storage season.

Purpose: Determine prevalence of Listeria spp. in CA storage facilities over two years

Methods: For the 2021-2022 season, 3 CA rooms each at 2 storage facilities were sampled. For the 2022-2023 season, 3 CA rooms each at 3 storage facilities were sampled. 10 zone 3 locations in each CA room were swabbed. Samples were stored at 4°C after sampling and processed within 24 hours. Swabs were enriched in Less-Plus media at a 1:10 w/v ratio and incubated at 37°C for 18-20 hours. Following enrichment, samples were evaluated for the presence of Listeria spp using the Listeria ANSR assay. Presumptive positives were confirmed by plating onto Neogen Listeria Chromogenic Harlequin media.

Results: During the 21-22 storage season, 2 CA rooms at two separate storage facilities had a 15% (9/60) and 18% (11/60) positive rate of Listeria spp. for the pre-storage and post-storage sampling period, respectively. During the 2022 pre-storage season, 4 CA rooms across 3 storage facilities had a 10% (9/90) positive rate of Listeria spp.

Significance: These data will contribute to a QMRA that will inform apple growers and processors of risks associated with Listeria monocytogenes in CA rooms, allowing growers to improve apple handling practices and weigh the benefits of sourcing capital to improve infrastructure.

Structural Insight into the Antibiotic Resistance Mechanism of the VraFG-GraRSX Module in Staphylococcus aureus

Ellen C. Bennett, Natasha L. George*, Benjamin J. Orlando

Michigan State University

Many Firmicutes utilize Bce modules to resist multiple antimicrobial peptides. The Bce module is a membrane-bound complex that consists of an ATP-binding cassette (ABC) transporter and a two-component system. Upon sensing antimicrobial peptides, the conformational switch of the ABC transporter is suggested to activate the two-component system which then upregulates genes that aid in antimicrobial resistance. In Staphylococcus aureus, the VraFG-GraRSX module provides resistance to a variety of antimicrobial peptides, including vancomycin and peptides produced by the innate immune system. VraFG is the ABC transporter and GraRS is the two-component system. However, unlike most Bce modules, the complex in S. aureus involves a fifth protein of unknown function, GraX. The goal of this investigation is to determine the function of GraX and how it fits into the Bce module. First, the five component system from S. aureus strain JE2 will be expressed and purified. Then, cryo-electron microscopy will be used to determine how GraX fits into the complex architecture. After resolving the structure, mutational and biochemical experiments will be performed to reveal the function of GraX and how it interacts with the rest of the protein.

Abstract

Hunter North*

Michigan State University

In Alphaproteobacteria, the conserved, often essential CckA-CtrA regulatory system controls polar cell development and cell cycle progression. To gain insight into factors that influence this critical regulatory circuit, we selected for spontaneous mutations that suppress the lethality of cckA and ctrA temperature sensitive (ts) alleles in the Alphaproteobacterium, Caulobacter crescentus. We identified several classes of mutants, one of which includes non-synonymous mutations in the DNA-binding region of NtrC, a conserved transcriptional regulator of nitrogen assimilation in bacteria. Two identified alleles, ntrC^L424P and ntrC^A446P, fully suppress lethality of cckA^ts and ctrA^ts at restrictive temperatures, while an in-frame deletion of ntrC (∆ntrC) partially suppresses lethality. Several lines of evidence support a role for NtrC in regulation of nitrogen assimilation in Caulobacter, similar to other bacteria: ∆ntrC cannot grow when ammonium is the sole nitrogen source, and our transcriptomic and ChIP-seq analyses provide evidence that Caulobacter NtrC regulates nitrogen assimilation via activation of glutamine synthetase (glnA). Additionally, our analyses demonstrate that Caulobacter NtrC regulates key cell cycle regulators (e.g., mucR1, hdaA). The C. crescentus ntrC^L424P and ntrC^A446P mutants fail to utilize ammonium as a sole nitrogen source and have transcriptional profiles similar to ∆ntrC, indicating that these ntrC alleles are loss-of-function with respect to nitrogen assimilation. However, both ntrC^L424P and ntrC^A446P rescue the defect of cckA^ts in transcription of flagellar assembly genes (flg, fla, fli, flj), unlike ∆ntrC. Our results indicate that DNA-binding mutations in NtrC can restore flagellar transcription and other developmental defects of cckA^ts and ctrA^ts mutants and provide evidence for a regulatory link between environmental nitrogen assimilation, polar morphogenesis, and cell cycle progression in Caulobacter.

Impact of chlorinated water tempering on enteric pathogens reduction and resulting flour quality

Yawei Lin¹, Senay Simsek², Teresa M. Bergholz¹

¹Department of Food Science and Human Nutrition, Michigan State University
²Department of Food Science, Purdue University

Introduction: Outbreaks of enteric pathogens linked to wheat flour have led the wheat milling industry to seek solutions addressing this food safety concern. Chlorinated water at 400-500ppm has been used in the flour milling industry as a tempering aid to control growth of yeast and mold in tempering bins. However, the effectiveness of chlorinated water on inactivating enteric pathogens on wheat kernels remained unknown.

Purpose: Quantify pathogen reduction on wheat kernels and changes in flour quality when chlorine is added to wheat tempering water.

Method: Five strains of Shiga-toxin producing Escherichia coli (STEC) and two strains of Salmonella were inoculated onto hard red spring wheat at 7 log CFU/g and stored at room temperature for 1-month.Inoculated wheat was temperedwith four concentrations (0, 400, 800, 1200ppm) of chlorinated water (pH 6.5). The effect of chlorine was determined by calculating the change in cell density for each level using the response at 0ppm as a reference. Uninoculated wheat tempered with chlorinated water was used to measure flour quality parameters such as gluten functionality.

Result: Changes in pathogen density over 18 hours ranged from -1.93 to -0.35 log CFU/g at 400ppm chlorine, and changes at 800ppm and 1200ppm were not significantly (p>0.05) different from those at 400ppm. Significant (p< 0.05) differences in the extent of reduction were observed among strains. However, the impact of chlorinated water in reducing native microbes on wheat kernels was minimal, ranging from -0.69 to -0.22 log CFU/g for all concentrations. No significant (p>0.05) changes occurred in flour quality and gluten functionality during the breadmaking process.

Significance: The data show chlorinated water at concentrations currently used in industry for tempering could reduce enteric pathogen numbers by 0.85 log CFU/g for STEC, and 1.71 log CFU/g for Salmonella, with no significant effects on flour quality and gluten functionality.

Identifying novel phage defense systems in Vibrio cholerae

Elise N. Trost*, Jasper B. Gomez, Chris M. Waters

Michigan State University

Antibiotic resistance has been one of the greatest public health threats since the discovery of antibiotics. Thus, it is important to find alternate methods to combat resistance. Phage therapy is an alternative that has been used for almost a century and has renewed interest due to its ability to target and lyse specific bacteria without disrupting the microbiome. Although bacteriophages are able to lyse bacteria, bacteria have evolved defense mechanisms which protect against phage infection. Thus, it is important we uncover new phage defense systems and understand their underlying mechanisms. To address this we use Vibrio cholerae as a model. V. cholerae is useful because they have two pathogenicity islands (VSP-I and VSP-II) which has two known phage defense systems known as CBASS and AvcID. Due to the discovery of two previous phage defense systems, it is hypothesized that V. cholerae still harbors unknown phage defense sytems. A previous screen of a V. cholerae cosmid library within Escherichia coli showed a 25kb fragment that confers protection against T2 coliphage. This fragment has multiple genes that have yet to be identified. Thus, I screened a transposon mutant library of this cosmid and found transposon mutants that have restored susceptibility to multiple phage. I am currently determining the genetic basis causing phage resistance. Overall, understanding the mechanisms for phage defense can help create more effective phage therapies.

A Novel Signal Transduction System Controls Cell Fate Segregation in the Myxococcus xanthus Specialized Biofilm

Kasto, Shelby*, Schramm, Andreas, Higgs, Penelope I.

Wayne State University, Detroit, Michigan 48202

Biofilms are microbial communities encased in a self-produced extracellular matrix. Cells in the biofilm state are difficult to combat in part because of increased production of quiescent cells, which are highly tolerant to killing agents. Quiescent cells, such as persisters, are mixed within the biofilm which makes their production difficult to study. Myxococcus xanthus produces a specialized biofilm involving spatial segregation of distinct quiescent cells: production of spores packaged into fruiting bodies, and persister-like cells (peripheral rods) arrayed outside the fruiting bodies. The proportion of cells in these two fates depends on environmental conditions. Thus, M. xanthus is an excellent model system to investigate regulatory mechanisms controlling production of distinct quiescent states. M. xanthus cell fate segregation is controlled by MrpC, a transcription factor that is necessary to induce fruiting body formation, and is absent from peripheral rods. EspAC are two sensory kinases that must function together to trigger MrpC degradation. We have observed that EspA and EspC are differentially expressed within the biofilm, with EspA produced in all cells and EspC overrepresented in peripheral rods. We have identified a transcriptional circuit that likely controls differential production of EspA/C within the two cell fates.  Western blot, qPCR and DNA binding assays indicate that MrpC is required to induce expression of EspA and EspC early during biofilm formation. Subsequently, the fruiting body-specific transcription factor FruA represses EspC accumulation.  To further identify the esp gene control elements, we have used promoter pull-down experiments and mass spectrometry to identify unique factors that bind to these promoters. Together, these data suggest production of different quiescent states within the M. xanthus biofilm is tuned to spatial cues by a novel feedback regulatory network motif.