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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, MI 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. Thus, we hypothesize that the EspAC signaling system is fully functional to degrade MrpC specifically within cells destined to become peripheral rods. To understand the basis of differential espA and espC expression, we are investigating the cis and trans regulatory elements that control their expression. We have identified MrpC binding sites in the promoters for espA and espC, and have demonstrated that MrpC is necessary to upregulate both espA and espC expression. We have additionally demonstrated that espC expression appears to be repressed by a second transcription factor, FruA. FruA is produced primarily inside fruiting bodies. Together, these data suggest production of different quiescent states within the M. xanthus biofilm is tuned to environmental conditions by a novel feedback regulatory network motif.

 
 

A Broadly Conserved Deoxycytidine Deaminase Protects Bacteria from Phage Infection

Brian Y. Hsueh1*, Geoffrey B. Severin2, Clinton A. Elg3, Alex J. Wessel1, Christopher R. Rhoades1, Benjamin J. Ridenhour4, Janani Ravi5, Eva M. Top3, and Christopher M. Waters1

1Department of Microbiology and Molecular Genetics, Michigan State University, East Lansing, Michigan, USA, 48824
2Department of Biochemistry and Molecular Biology, Michigan State University, East Lansing, Michigan, USA, 48824
3Department of Biological Sciences, Institute for Bioinformatics and Evolutionary Studies, Bioinformatics and Computational Biology Program, University of Idaho, Moscow, Idaho, USA, 83844
4Department of Mathematics and Statistical Sciences, University of Idaho, Moscow, Idaho, USA, 83844
5Department of Pathobiology and Diagnostic Investigation, Michigan State University, East Lansing, Michigan, USA, 48824

The El Tor biotype of Vibrio cholerae is responsible for perpetuating the longest cholera pandemic in recorded history. The genomic islands VSP-1 and -2 are understudied genetic features that distinguish El Tor from previous pandemic V. cholerae. To understand the role of VSP genes, we calculated the co-occurrence of VSP genes across bacterial genomes. This analysis predicted that the previously uncharacterized gene vc0175, herein renamed anti-viral cytidine deaminase (avcD), is in a gene network with dncV, a cyclic GMP-AMP synthase involved in phage defense. AvcD consists of two domains; a P-loop NTPase and a deoxycytidylate deaminase, both of which are required for the deamination of dCTP and dCMP. We found that homologs of avcD are broadly conserved across the three domains of life. Additionally, AvcD activity is post-translationally inhibited by a unique noncoding RNA named AvcI encoded immediately upstream of the avcD locus, in a manner analogous to Type III toxin-antitoxin systems, and we demonstrate that AvcID protects bacteria from phage infection. Activation of AvcD upon inhibition of transcription or phage infection significantly alters cellular nucleotides by depleting dC substrates and increasing dUMP. Our results show that AvcID protects against bacteriophage infection by combining aspects of two eukaryotic anti-viral strategies; cytosine deamination (e.g., APOBEC) and the depletion of cellular deoxynucleotides (e.g., SAMHD1).

 
 

Exploring the Effects of Prairie Restoration Management on Soil Microbial Carbon Storage

Ellen Badger Hanson*, Lars Brudvig, Kathryn Docherty

Western Michigan University

Agricultural ecosystems are a major contributor to greenhouse gas emissions. One mitigation method involves integrating native prairie vegetation in marginal lands within agroecosystems. However, these restored prairies often do not regain the soil microbial community structure nor soil carbon storage found in untouched remnant prairies. Further study on the mechanisms behind these discrepancies is necessary to restore prairies more effectively for carbon storage. This study leverages a long-term, ongoing experiment at Kellogg Biological Station in southwest Michigan. In Summer 2021, we examined the effects of restoration size and plant seed mix diversity on soil microbial communities and soil carbon. We hypothesized that restorations with high seed mix diversity would have more diverse and heterogeneous microbial communities than those with low, and that microbial community diversity and heterogeneity would increase with restoration size. Initial PLFA data show microbial heterogeneity is positively affected by both plant diversity and restoration size. We further hypothesized that restorations with high seed mix diversity would have more soil carbon than those with low, and that soil carbon would increase with restoration size. Trends in initial microbial biomass carbon data support these hypotheses. This study aims to provide insight to inform better land management strategies.

 
 

Histone Acetyltransferases Esa1 and Gcn5 Coordinate with RSC to Regulate Chromatin Structure and Transcription in Saccharomyces cerevisiae

Biernat, Emily*, Werick, Matthew and Govind, Chhabi

Oakland University, Rochester, MI, 48309

Eukaryotic DNA is packaged into chromatin, which consist of nucleosomes. Nucleosomes are formed by wrapping DNA around a histone octamer (H3, H4, H2A, and H2B) and impede all DNA-dependent processes, including gene transcription by RNA Polymerase II (Pol II). This impediment is relieved by chromatin remodelers (CRs) that can slide or eject nucleosomes. CR mutations are found in 20% of human cancers and in many developmental diseases. The RSC complex is the only essential CR in S. cerevisiae and contains many bromodomains (BD). Histone acetyltransferases (HATs) Gcn5 and Esa1 acetylate H3 and H4, and BDs can bind to such acetylated histones. How HATs collaborate with CRs to regulate DNA accessibility in-vivo is poorly understood. We hypothesized that HATs help in recruiting RSC to nucleosomes to regulate chromatin structure and promote transcription. To map RSC at a nucleosome resolution, we used micrococcal nuclease to digest chromatin from wildtype S. cerevisiae and mutants lacking Gcn5, Esa1 or both HATs. These extracts were subjected to chromatin immunoprecipitation of RSC, Pol II, and H3 and the resulting DNA was sequenced. The sequences were aligned to the yeast genome and normalized. H3, RSC and Pol II occupancies were analyzed to determine the impact of HAT mutants on chromatin structure and transcription. We found reduced RSC levels in the coding sequences (CDSs) of highly transcribed genes in the HAT mutants compared to WT. RSC levels at promoters were increased at a genome-wide scale in mutants lacking Gcn5, and this effect was more severe in mutants lacking Esa1 or both HATS. All HAT mutants showed dampened histone eviction and reduced Pol II occupancies, and these defects were most severe in the HAT double mutant. Overall, our data implicate HATs in recruiting RSC to CDSs to promote transcription, as well as suggest that Esa1 is important for dissociation of RSC from promoters and nucleosome depleted regions.

 
 

Is There a Placental Microbiota? A Re-analysis of Published Placental Microbiota Datasets

Jonathan J. Panzer*, Kevin R. Theis

Wayne State University, Detroit, MI 48202

The existence of a human placental microbiota is under debate. Traditionally, the human placenta was considered sterile since microbial colonization was associated with adverse pregnancy outcomes. Yet, recent DNA sequencing studies have reported that the human placenta in uncomplicated pregnancies ending in a term delivery contains a unique microbiota. However, the placental microbiota is reportedly low biomass, and therefore could be confounded by background DNA contamination from extraction kits, PCR reagents, and laboratory environments. If a placental microbiota exists, then patterns or consistency should be evident in the microbial taxa detected and identified in placentas across studies and different populations of pregnant women. Using all publicly available 16S rRNA gene (i.e., a phylogenetic marker gene for bacteria) datasets with sufficient metadata for sample discrimination, the data were re-analyzed for consistency. 16S rRNA gene Amplicon Sequence Variants (ASVs) identified as Lactobacillus, a typical vaginal commensal bacterium, were among the top five relatively abundant ASVs in eight of fifteen studies. However, it was clear that the prevalence of Lactobacillus was driven primarily by vaginal delivery contamination and secondarily by background DNA contamination. After removal of background DNA contaminants by DECONTAM, Lactobacillus ASVs were ranked among the top five relatively abundant ASVs in one of five studies for which data analysis could be restricted to placentas from term cesarean deliveries. A further sub-analysis of six studies with sequence data from the V4 hypervariable region of the 16S rRNA gene showed that the bacterial DNA profiles of placental samples clustered primarily by study origin and mode of delivery, and that the principal bacterial ASVs in placental samples were also prominent in controls for background DNA contamination within these studies. The preponderance of contemporary evidence supports the historical paradigm that the human placenta does not harbor a microbiota.

 
 
Poster Presentations
 
 

Characterizing the function of a network of transcription factors that regulates surface attachment in Caulobacter crescentus

Patrick T. McLaughlin*, Aretha Fiebig, Sean Crosson

Michigan State University

Multicellular communities of surface attached bacteria (i.e. biofilms) are known to significantly contribute to the progression of both disease and environmental biofouling. Biofilm formation enhances tolerance of microbes to both physical and chemical stresses, which can make treatment of biofilms difficult. Surface attachment is the initial stage of biofilm formation and is a highly regulated process. The alphaproteobacterium Caulobacter crescentus displays a dimorphic lifestyle that produces two cell types: sessile, stalked cells and planktonic, swarmer cells. Swarmer cells differentiate into stalked cells and secrete a polysaccharide adhesin known as the holdfast, resulting in permanent attachment to solid surfaces. Recently, our group has shown that a multi-kinase signaling network in C. crescentus influences adhesion by activating expression of two XRE-family transcription factors, rtrA and rtrB, and a cryptic transcription factor, rtrC, that repress expression of the holdfast inhibitor hfiA. ChIP-seq and RNA-seq analysis indicate that RtrA and RtrB primarily function as repressors of gene expression, while RtrC functions as both an activator and a repressor, which correlates with the position of RtrC binding within promoter elements. Soft agar motility assays indicate that overexpression of either rtrA, rtrB, or rtrC reduce swarm size compared to an empty vector control, suggesting that rtrABC repress motility through an unknown mechanism. Taken together, these results suggest that RtrABC modulate the transition from planktonic to sessile lifestyle by both promoting adhesion through repression of hfiA expression and inhibiting motility.

 
 

The Myxococcus xanthus specialized biofilm provides additional protection from environmental insults.

Dave Lall* and Penelope I. Higgs

Dept. of Biological Sciences, Wayne State University, Detroit, MI

Environmental microorganisms have evolved a variety of strategies to survive fluctuations in environmental conditions, such as production of biofilms, differentiation into spores, and/or production of dormant cells (persisters). As the climate change crisis leads to more frequent and more extreme environmental fluctuations, it is important to understand the relative effectiveness of each survival strategy. Myxococcus xanthus are ubiquitous predatory soil bacteria that function as a community to collectively feed on other microorganisms and decaying organic matter. When nutrients are scarce, these bacteria differentiate into spores produced inside of fruiting bodies (a specialized biofilm) or into peripheral rods, a non-growing cell. It has been long assumed that the fruiting bodies evolved to facilitate dispersal of the community to more favorable feeding environments. Here, we hypothesized that the fruiting body provides myxospores additional protection from environmental insults. We investigated recovery (outgrowth) of distinct cell types (vegetative cells, dispersed spores, and fruiting bodies) after exposure to desiccation, freezing temperatures, or ultraviolet radiation. Our data suggest spore-filled fruiting bodies provide additional protection from UV irradiation, but not to desiccation. Interestingly, vegetative cells alone efficiently recover from long-term exposure to -20C. Together, these results suggest that M. xanthus has evolved multiple distinct survival strategies to withstand environmental fluctuations.

 
 

A system-level genetic analysis of two component signal transduction systems (TCS) in Brucella ovis uncovers two overlapping TCS

Esther Chen*, Aretha Fiebig, Sean Crosson

Michigan State University

Brucella spp. are facultative intracellular pathogens that are the etiologic agents of brucellosis, a global zoonosis. Brucellosis commonly afflicts a variety of livestock species, where it can cause abortion and sterility. There are currently 12 recognized Brucella species, which have preferences for particular animal hosts. Though Brucella spp. have distinct host preference, the genus is largely monomorphic, sharing over 97% nucleotide identity. The genetic basis of animal host preference is not understood, but it may be influenced by differences in sensory physiology between species. To better understand environmental sensing/signaling mechanisms in the genus Brucella, we have conducted a systematic analysis of two-component signal transduction (TCS) systems in the bovine pathogen, Brucella abortus and the ovine pathogen, Brucella ovis. These species share the same set of TCS genes but have major differences in axenic cultivation requirements and the molecular composition of their cell envelopes. Deletion of all nonessential TCS genes in B. ovis revealed unexpected congruence in the envelope stress phenotypes of strains lacking the cenR response regulator and the previously uncharacterized RR9-HK12 TCS. Surprisingly, the phenotypes of these B. ovis mutants differ from B.abortus strains harboring deletions of cenR and RR9-HK12 orthologs. Our preliminary RNA-seq data provide evidence that CenR and RR9-HK12 regulate a similar set of genes in B. ovis. Specifically, genes with predicted cell envelope function have significantly reduced transcription in both mutant backgrounds. Defects in transcription of cell envelope genes in ΔcenR and ΔRR9-HK12 are correlated with abnormal morphology of these mutants observed by SEM and TEM.

 
 

Role of Shigella flexneri DGCs in Pathogenesis

Ojha Ruchi*, Koestler Benjamin

Western Michigan University, Kalamazoo, Michigan 49006

Shigella flexneri is a gram-negative human pathogen that causes bacillary dysentery. This bacterium targets the colonic epithelium, resulting in bloody diarrhea. There is no vaccine for the prevention or treatment of Shigella infection, and antibiotic resistance is on the rise for Shigella, making it a high priority target for antibacterial therapy development. Before Shigella initiates infection in the colon, it transits through the small intestine where it is exposed to bile. Previous studies have shown that Shigella utilizes bile salt exposure to promote biofilm formation and increase the expression of adhesins. Shigella also utilizes bile salt exposure as a signal to modulate virulence gene expression and intensify infection; however, the mechanism underpinning the regulation of biofilm formation is largely unknown.

Cyclic di-guanosine monophosphate (c-di-GMP), one among the bacterial nucleotide-based signalling systems known, regulates many behavioral changes in bacteria in response to changing environmental conditions, including biofilm formation. The levels of this second messenger are determined by two classes of enzymes: diguanylate cyclases (DGC) and phosphodiesterases (PDE). In many bacteria, high intercellular levels of c-di-GMP levels promote biofilm formation, while low levels promote motility. Shigella flexneri encodes 4 DGCs, namely dgcP, dgcC, dgcI and dgcF; however, there have been no studies examining the role of c-di-GMP signaling in Shigella.

Here, we want to study if the virulence phenotypes in S. flexneri are mediated by c-di-GMP levels. To answer this question, we expressed a Vibrio cholerae DGC, VCA0956 in S. flexneri. Our results suggest that overexpression of an active DGC increases c-di-GMP levels, biofilm formation and decreased virulence. Our results show that increased c-di-GMP levels corresponds to decreased survival to acid shock in S. flexneri. We further characterized each of S. flexneri’s native DGCs by studying their role in biofilm formation and virulence. As DGCs regulate biofilm formation in other enteric and pathogenic bacteria, we hypothesize that knocking out individual DGC will show significant decrease in biofilm formation and increased virulence. Our result show that knocking out individual DGCs reduce S. flexneri biofilm, dgcC and dgcF regulate invasion capacity and dgcF mutant also regulate plaque phenotype.

There is still a knowledge gap on the scope of c-di-GMP signaling in S. flexneri pathogenesis, as it is involved in many functions; with this study, we will characterize the role of DGCs in S. flexneri pathogenesis.

 
 

TodK, an unusual histidine kinase, controls biofilm formation of Myxococcus xanthus by inactivating a key transcriptional regulator, MrpC

Mataczynski, Christopher*, Glaser, Maike, Higgs, Penelope I.

Wayne State University, Detroit, MI 48202

Approximately 80% of bacteria on Earth’s surface exist in a biofilm. Biofilms are a detriment in healthcare, food, and marine industries because they are recalcitrant to chemical and physical treatment methods. To develop control mechanisms, it is important to understand biofilm regulatory mechanisms. Myxococcus xanthus is a model organism to investigate environmental biofilms. The transcription factor, MrpC, is essential to induce M. xanthus biofilm formation. Here, we demonstrate a new level of regulatory control on MrpC by an unusual histidine kinase, TodK. We demonstrate that overproduction of TodK prevents biofilm production but does not significantly perturb MrpC accumulation. However, accumulation of two downstream targets of MrpC are not induced. We hypothesize that TodK inactivates MrpC by post translational modification of MrpC, and we are currently analyzing MrpC by mass spectrometry. Together, these results suggest a novel mechanistic strategy that could be exploited for biofilm control.

 
 

Novel Activity of the Legionella pneumophila Effector Protein LegK7 on Eukaryotic Cell Growth

Quagliato, Sydney M.* and Lee, Pei-Chung

Wayne State University, Detroit, Michigan 48202

Legionella pneumophila is a bacterial pathogen that causes Legionnaires’ disease, a fatal pneumonia that poses high risk to immunocompromised individuals. L. pneumophila thrives in freshwater environments and aerosolizes into particles that humans can inhale, allowing for infection into lung macrophages. This pathogen uses an intelligent type IV secretion system to secrete over 300 effector proteins that hijack host signaling pathways allowing L. pneumophila to replicate within its hosts. One important virulence factor is the effector protein, LegK7, that has a kinase domain at its N-terminus for phosphorylating the Mob1 scaffold protein in the host Hippo signaling pathway. The highly conserved Hippo pathway regulates cell differentiation and apoptosis in eukaryotic hosts. This study aims to characterize functional domains of LegK7 and its effects on eukaryotic pathways. We performed a yeast growth assay in which Saccharomyces cerevisiae expresses different LegK7 variants. Full length LegK7 significantly inhibited yeast growth, but interestingly the catalytic mutant still inhibited yeast growth, suggesting that domains other than the kinase domain are essential for the novel inhibitory activity of LegK7. We also revealed partial inhibition of cell growth when expressing the first 530 a.a. of LegK7 and demonstrated inhibition effects comparable to the full-length LegK7 when the first 730 a.a. are expressed. Structural homology analyses predicted two tetratricopeptide repeats in a.a. 534-603 and 451-523 of LegK7 that might mediate interactions with host proteins. Together, these data suggest a new functional domain between a.a. 450 to 730 of LegK7 with inhibitory activity on eukaryotic cell growth. Dissecting the functional domains of LegK7 and their ability to hijack a conserved eukaryotic signaling pathway allows us to better understand infection strategies used by the pathogen.

 
 

A Novel Reporter System for Monitoring Type 4 Effector Secretion in Legionella pneumophila

St. Louis, Brendyn, M.*, Lee, Pei-Chung

Wayne State University, Detroit, Michigan 48202

Legionella pneumophila is a bacterial pathogen that infects humans through inhalation of contaminated water droplets and invades alveolar macrophages causing severe respiratory illness. L. pneuomophila secretes more than 300 effector proteins into host cells through a dot/icm Type 4 secretion system (T4SS) to modulate intracellular trafficking, replication, and subsequent evasion of host immune response. This secretion system is inactive in effector secretion until the bacteria is in direct contact with a host cell, indicating that an unidentified protein, termed the “gatekeeper protein,” controls activation of the T4SS. Here, we sought to develop a reporter system in L. pneumophila, using the T4SS effector protein, SidJ, and the flagellar regulatory protein, FlgM. In the bacterium, secretion of FlgM leads to expression of flagellin. We attempt to fuse sidJ with flgM on the chromosome by allelic exchange to produce a SidJ-FlgM fusion protein that is predicted to be secreted by the T4SS. We will transform the bacterium with a plasmid carrying a GFP reporter under control of the flagellin promoter. This system would allow us to identify bacteria with active T4SS based on secretion of SidJ-FlgM and activation of flagellin-GFP reporter expression. Currently, we have constructed a sidJ-flgM fusion gene on the allelic exchange plasmid, pSR47s. Subsequently, the construct was delivered into L. pneumophila through triparental conjugation and colonies with the sidJ-flgM fusion gene were successfully selected for. Using this reporter system, we will identify mutants that secrete effectors in the absence of a host cell, indicating a mutation in the gatekeeper protein, and allowing us to identify the specific gatekeeper protein. This reporter system will also provide a novel method for real-time tracking of effector secretion during L. pneumophila infection.

 
 

Characterizing regulatory regions in a major transcription factor impacting biofilm formation in the bacteria Myxococcus xanthus

Kreinbring, Lindsey*; Mataczynski, C; McLaughlin, P; Adwada, M; Gretzinger A, and Higgs, Penelope I.

Wayne State University, Detroit, Michigan 48202

Biofilms are complex multicellular communities of microorganisms. Biofilms confer tolerance to physical and chemical treatments including antibiotics. Myxococcus xanthus is a Gram-negative, environmental bacterial species that produces a specialized biofilm consisting of spore-filled fruiting bodies. MrpC, a Crp/Fnr family transcription factor is necessary to direct fruiting body formation, and misaccumulation of MrpC has drastic effects on the timing of fruiting body production and fruiting body morphology. MrpC accumulation is controlled by transcriptional and post-transcriptional regulation, including proteolytic turnover. A genetic screen has identified two potential protease recognition sites within the mrpC gene. Site-directed mutagenesis has confirmed a single substitution within MrpC leads to premature accumulation of MrpC and accelerated production of fruiting bodies.

 
 

Envelope integrity protein EipA is important for viability and cell separation in Brucella ovis

Melene A. Alakavuklar1*, Julien Herrou2, Aretha Fiebig1, and Sean Crosson1

1Department of Microbiology and Molecular Genetics, Michigan State University, East Lansing, Michigan, USA, 48824
2CNRS - Institut de Microbiologie de la Méditerranée, Laboratoire de Chimie Bactérienne, Marseille cedex 13420, France

The cell envelope provides an important barrier for bacterial cells from their environment and contributes to cell integrity and rigidity. In the alphaproteobacterial pathogenic clade Brucella, one factor that promotes envelope integrity is the Domain of Unknown Function (DUF) DUF1134 protein EipA. EipA localizes to the periplasm and is important for envelope stress resistance and virulence. EipA has a unique β-barrel structure; however, the molecular function of EipA remains unknown. In the zoonotic pathogen Brucella abortus, deletion of eipA is synthetically sick with disruption of genes involved in synthesis of the O-polysaccharide. In the naturally rough strain Brucella ovis, which lacks the O-polysaccharide, eipA is essential for viability. Depletion of eipA in B. ovis results in the formation of chained cells which fail to separate following cell division. We have preliminary data that indicate mutations in a predicted glycosyl transferase suppress the essentiality of eipA, restoring viability and cell separation to the eipA depletion strain. The mechanism of suppression is unknown and under current investigation. Fusion of EipA to mCherry reveals that EipA-mCherry may localize polarly and to the site of cell constriction prior to cell division. Future directions include analysis of the EipA-mCherry localization dynamics throughout the cell cycle and discovery of the molecular interaction partners of EipA. DUF1134 is found in many alphaproteobacterial species. Ectopic expression of putative eipA orthologs from Agrobacterium tumefaciens and Caulobacter crescentus partially cross-complements the B. ovis eipA depletion strain viability defect, suggesting a possible conservation of EipA function among the class Alphaproteobacteria.

 
 

Randomly barcoded transposon sequencing reveals a conserved gene pair with a functional role in iron acquisition

Hernandez-Ortiz, Sergio*

Michigan State University

Iron acquisition is necessary for cell growth and survival. Though iron is relatively abundant, it forms insoluble Fe3+ aggregates in the environment, and is tightly sequestered by plant and animal hosts. Many mechanisms of iron acquisition have been identified in bacteria, but our understanding of this process is incomplete, particularly in soil and aquatic ecosystems. Caulobacter crescentus is a bacterium common in freshwater and soil. To identify C. crescentus genes involved in iron acquisition, we cultivated a pool of randomly barcoded (RB) transposon insertion strains in complex broth containing 0.3 mM of the cation chelating agent EDTA. Our RB-TnSeq screen showed that insertions in an adjacent pair of genes, a TonB dependent transporter (CCNA_00028) and a predicted 2OG-Fe(II) oxygenase (CCNA_00027), result in diminished growth in broth containing EDTA. These genes are repressed by the iron regulatory protein, Fur, and co-conserved in several bacterial orders but have not been characterized. We generated in-frame deletions strains of each gene (ΔCCNA_00028 and ΔCCNA_00027) and measured their growth in M2 defined medium with limited iron, and in medium containing EDTA. Low iron conditions, and EDTA restricted growth of ΔCCNA_00028 and ΔCCNA_00027 strains on solid media. The growth defect of each mutant strain was complemented by iron supplementation, and by ectopic expression of the missing gene. We conclude that both CCNA_00028 and CCNA_00027 have a functional role in iron acquisition. Current studies focus on testing the hypothesis that these genes function in siderophore-dependent iron uptake.

 
 

Discovery of new phage defense systems in Vibrio cholerae

Jasper B. Gomez* and Christopher M. Waters

Michigan State University

Since the discovery of antibiotics, bacterial infections have quickly evolved resistance making them increasingly difficult to treat. Thus, alternate methods to combat resistance has been a critical public health goal. An important method to combat resistance is phage therapy, which utilizes lytic phage to eradicate infections. Phage therapy has been used for almost a century and has renewed interest due to phages ability to lyse and target specific bacteria, avoiding the broad disruption to the microbiome caused by antibiotics. Although phages can infect and lyse bacteria, bacteria have evolved a myriad of defense mechanisms to protect against phage infection. Such defense mechanisms are readily transferred amongst bacteria by horizontal gene transfer and could offer mechanisms of resistance to phage therapy approaches. Thus, it is important to identify phage defense systems and understand the mechanisms by which they function. Although many phage defense mechanisms have been identified, other mechanisms of phage defense have yet to be studied. I therefore searched for new phage defense systems in the bacterial pathogen Vibrio cholerae, which has two known phage defense systems. I hypothesized that V. cholerae could encode other novel phage defense systems. To determine whether V. cholerae carries multiple phage defense systems, I screened a V. cholerae cosmid genomic library in E. coli to identify segments of V. cholerae’s genome that protected E. coli from T2 phage infection. My research identified two unique cosmids, each encoding approximately 25 kB of V. cholerae DNA, that protect E. coli from T2 infection. Neither of these regions have been implicated in phage defense, suggesting they encode novel defense systems that have not been studied. I have now isolated transposon mutants of one of these cosmids that have lost defense and am currently sequencing these transposon insertions to determine the genetic basis of this phage defense. My studies will lead to the identification of new V. cholerae phage defense mechanisms that will increase our understanding of the evolution and ecology of V. cholerae while highlighting important mechanisms by which bacteria can resist phage therapy.

 
 

Biofilms Develop on Disposable Face Masks Under Laboratory Conditions

Snelson, Tamara J.* and Rickard, Alexander H.

University of Michigan School of Public Health, Ann Arbor, Michigan, 48109

The COVID-19 pandemic has led to the wide-spread use of face masks. Prolonged use or reuse of surgical-style face masks could lead to accumulation of biofilm-forming microorganisms on face mask surfaces. The aim of this work was to explore the potential for Methicillin Resistant Staphylococcus aureus (MRSA) or Pseudomonas aeruginosa biofilms to form on face masks over a 5 hour period. MRSA and P. aeruginosa were grown overnight in BHI broth. Cells were washed with PBS and standardized to an O.D of 0.1 at 600 nm and 200 uL were plated onto BHI agar. Sterile hole punched discs of face masks were placed onto the inoculated BHI agar with coverslips acting as a weight, and incubated upright over a 5 hour period. Biofilm development was measured qualitatively by staining the face mask biofilms with Live/Dead stain and imaging using a Leica SPE confocal laser scanning microscope. Quantitative analyses were performed by serial dilution on BHI and calculating CFU/cm2 on the face mask discs. Confocal microscopy demonstrated that face mask discs inoculated with MRSA had rapidly increasing biofilm mass over a 5 hour incubation period while P. aeruginosa also formed biofilms but these were seemingly weakly attached to face mask fibers. For MRSA, the number of colony forming units (CFU/cm2) increased over 5 hours by nearly two orders of magnitude while the number of colony forming units for P. aeruginosa increased by less than one order of magnitude over the same period. In conclusion, biofilms form on face mask material under laboratory conditions using nutrient rich media and differences in the rate of biofilm development between the two organisms were observed. Future work will focus on additional organisms native to either the skin or the mouth and the examination of biofilms on worn face masks used in the real world.

 
 

A genetic link between inorganic nitrogen assimilation and cell cycle control in Caulobacter

Hunter North*, Aretha Fiebig, Sean Crosson

Department of Microbiology and Molecular Genetics, Michigan State University

The essential, conserved CckA-CtrA regulatory system controls polar cell development and cell cycle progression in the class Alphaproteobacteria. We designed a forward genetic selection to identify spontaneous mutations that suppress lethality of temperature sensitive (ts) alleles of cckA and ctrA in the model alphaproteobacterium, Caulobacter crescentus. Point mutations in the DNA-binding region of the predicted nitrogen assimilation regulator NtrC (NtrCL424P and NtrCA446P) fully suppress lethality of cckAts and ctrAts at restrictive temperatures, while an in-frame deletion of ntrC (ΔntrC) weakly suppresses lethality. ΔntrC cannot grow in defined medium with ammonium as the sole nitrogen source, and transcriptome analysis of ΔntrC provides evidence that Caulobacter NtrC regulates nitrogen assimilation via activation of glutamine synthase, as in other bacteria. NtrCL424P and NtrCA446P strains also fail to grow in ammonium-defined medium and have transcriptome profiles that are correlated with ΔntrC, indicating that these suppressing NtrC alleles are loss-of-function with respect to nitrogen assimilation. However, both NtrC point mutations rescue the defect of cckAts in transcription of flagellar assembly genes (flg, fla, flj) and type IV pilus genes (cpa). Our results indicate that activating transcription of flagellar and/or pilus assembly genes via a secondary pathway can overcome loss of the cckA-ctrA system, and provide evidence for a regulatory link between environmental nitrogen assimilation, polar morphogenesis, and cell cycle progression in Caulobacter.

 
 

 
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