Source: http://mi-asm.org/Meetings/Spring/spring11-19/Spring17meeting/Abstracts%20Received.htm
Timestamp: 2019-04-21 06:47:13+00:00

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Clostridium difficile (Cd) is an anaerobic gram-positive pathogen that is the leading cause of nosocomial bacterial infection globally. Cd infection (CDI) typically occurs after ingestion of infectious spores by a patient that has been treated with broad-spectrum antibiotics. While CDI is a toxin-mediated disease, transmission and pathogenesis are dependent on the ability to produce viable spores. These spores must become metabolically active (germinate) in order to cause disease. Cd spore germination occurs when spores encounter bile salts and other co-germinants within the small intestine, however, the germination signaling cascade is unclear. Here we describe a signaling role for Ca2+ during Cd spore germination and provide direct evidence that intestinal Ca2+ coordinates with bile salts to stimulate germination. Endogenous Ca2+ (released from within the spore) and a putative AAA+ ATPase, encoded by Cd630_32980, are both essential for taurocholate-glycine induced germination in the absence of exogenous Ca2+. However, environmental Ca2+ replaces glycine as a co-germinant and circumvents the need for endogenous Ca2+ fluxes. Cd630_32980 is dispensable for colonization in a murine model of Cd infection and ex vivo germination in mouse ileal contents. Calcium-depletion of the ileal contents prevented mutant spore germination and reduced WT spore germination by 90%, indicating that Ca2+ present within the gastrointestinal tract plays a critical role in Cd germination, colonization, and pathogenesis. These data provide a biological mechanism that may provide an explanation why individuals with inefficient intestinal calcium absorption (e.g., vitamin D deficiency, proton pump inhibitor use) are more prone to CDI and suggest that modulating free intestinal calcium is a potential strategy to curb the incidence of CDI.
MacCready, Joshua S.*, Schossau, Jory, Osteryoung, Katherine W., and Ducat, Daniel C. Michigan State University, East Lansing, MI, 48824 (all authors).
The oscillatory Min system of Escherichia coli defines the cell division plane by regulating the site of FtsZ-ring formation and represents one of the best-understood examples of emergent protein self-organization in nature. The oscillatory patterns of the Min-system proteins MinC, MinD and MinE (MinCDE) are strongly dependent on the geometry of membranes they bind. Complex internal membranes within cyanobacteria could disrupt this self-organization by sterically occluding or sequestering MinCDE from the plasma membrane. Here, we show using fluorescent microscopy that the Min system in the cyanobacterium Synechococcus elongatus PCC 7942 oscillates from pole-to-pole despite the potential spatial constraints imposed by their extensive thylakoid network. Moreover, reaction-diffusion simulations predict robust oscillations in modeled cyanobacterial cells provided that thylakoid network permeability is maintained to facilitate diffusion, and suggest that Min proteins require preferential affinity for the plasma membrane over thylakoids to correctly position the FtsZ ring. Our results provide the first direct evidence for Min oscillation outside of E. coli and have broader implications for Min-system function in bacteria and organelles with internal membrane systems.
FDA Approved Salicylanilide Anthelmintic Drugs Closantel and Oxyclozanide Enhance Tobramycin Killing of Pseudomonas aeruginosa Biofilms.
Cystic fibrosis, biofilms, antimicrobial resistance, drug repurposing and development The most important clinical obstacle in cystic fibrosis (CF) is treatment failure due to biofilms. Biofilms are a community of sessile cells enmeshed in a thick gel matrix that leads to thousands of times more resistance to antibacterial therapies, macrophages, and neutrophils. A hallmark of CF is a defective mucociliary transport system that results in dry mucus and clogged airways, creating an environment that is ideal for colonization by Pseudomonas aeruginosa. Central to this pathogen’s success is its biofilm mode of growth within the lungs, which are essentially impossible to eradicate with current antibacterial therapies, leading to immune complex-mediated chronic inflammation, neutrophilic tissue damage, decreased lung function, and ultimately premature death in CF patients. Here, based on a previous high throughput screen of 6,090 compounds from four drug repurposing libraries, we determined that the anthelmintic drugs closantel and oxyclozanide significantly enhance the killing, dispersal, and onset of action of the first-line CF drug, tobramycin. Our results show that 500 µM tobramycin in combination with 100 µM closantel and oxyclozanide killed 86% and 92% of the cells in 24-hr old biofilms, respectively. Whereas, tobramycin killed only 59% of the cells alone and oxyclozanide and closantel essentially have no activity on their own. Crystal violet staining showed that oxyclozanide and closantel in combination with tobramycin, significantly reduced biofilm biomass, indicating dispersal. Time killing studies demonstrated that the onset of action of tobramycin shortened from approximately 5-hrs to 1-hr when tested in combination with either oxyclozanide or closantel. Further, these anthelmintics enhanced several antipseudomonal antibiotics and are effective against several CF clinical isolates, including a tobramycin resistant isolate. The repurposing of FDA approved drugs provides greater clinical safety, costs savings, and accelerated deployment, making it an ideal approach for the development of new therapies.
Community behaviors in bacteria (i.e. biofilms) contribute to disease progression and environmental biofouling. Defining the regulatory mechanisms which drive these behaviors is essential to derive effective inhibitors. Myxococcus xanthus is an excellent model organism for multicellular behavior. These bacteria can enter a developmental program in which cells differentiate into one of three distinct fates: 1) spore filled fruiting bodies, 2) programmed cell death, or 3) a persistor-like state. MrpC, a CRP/FNR family global transcriptional factor, is necessary for coordinating appropriate multicellular development and cell fate segregation. Autoregulation of MrpC is critical for its function, but the mechanism of autoregulation is unknown. Using both a fluorescent transcriptional reporter and qPCR, we observed that mrpC is more highly expressed in a ?mrpC mutant compared to the wild type. These results suggest that MrpC functions as a negative autoregulator, contrary to what has been previously published. Using bioinformatic analyses, five putative MrpC binding sites were defined in the promoter region of mrpC. Specific binding of MrpC to each of the 5 putative sites was analyzed in vitro with electrophoretic mobility shift assays. To understand the in vivo role of each binding site in regulating mrpC transcription, fluorescent reporters that contain the mrpC promoter with mutations in the MrpC binding sites were constructed and analyzed throughout the developmental program. Together, our results suggest MrpC mediates negative autoregulation via direct biding to its own promoter such that it both competes with an enhancer binding complex and modulates transcriptional initiation.
Lactic acid microorganisms have the ability to ferment carbohydrates and synthesize lactic acid, ethanol, and CO2. Lactic acid bacteria can produce 2 moles of lactic acid per 1 mole of hexose sugar if it is a homofermentative, or it can produce 1 mole of lactic acid per 1 mole of hexose sugar if it is a heterofermentative. The aim of this study is to quantitatively measure and compare the amount of lactic acid production by homofermentative and heterofermentative lactic acid bacteria. Lactococcus lactis (ATCC 11454) and Leuconostoc mesenteroides (ATCC 8293) cultures were prepared three weeks before performing the experiment. Then, five tubes with 3 ml of Benedict’s reagent, each containing a culture, 10% glucose, and water, were boiled to test for complete consumption of glucose. After evaporating the remaining CO2 from culture, titration method, with 1% of phenolphthalein as an indicator, was used to determine the volume of 0.1N NaOH required to reach the endpoint for each of culture. Results show a green color change, which indicates the consumption of glucose and production of lactic acid by both strains of bacteria. However, there was a 1:1 ratio in production. The experiment was successfully performed and the quantitative measurements reasonable. Lactococcus lactis produced at least 1 mole of lactic acid instead of 2 moles, which is an indication that glucose was not fully consumed. However, lactic acid was produced by both strains, which elucidates the diversity of the metabolic activities among lactic acid bacteria.
Clostridium difficile (Cd) is a gram-positive, anaerobic spore forming bacterium that can cause severe intestinal infection. Cd is responsible for almost half a million cases of infection (CDI) in the United States alone, with approximately 29,000 deaths in a single year. Spores from feces found in healthcare settings facilitate the spread of infection, but the germination of these spores is what allows the infection to occur within the individual. The use of antibiotics disrupts the resistance of Cd colonization within the intestines by killing the healthy bacteria that usually limit its growth. Though the complete mechanism of germination in Cd is not completely understood, we have found that the presence of bile salts, glycine, and calcium together allow for the germination of Cd spores. In this work, two high-throughput assays were used to test the effects of specific inhibitors and environmental factors on Cd spore germination. We identified chemical inhibitors of BHIS+Taurocholate (Tc) induced germination which include chloropromazine (CPZ), EGTA, and A23187. CPZ is a chemical inhibitor of calcium dependent kinases, and incubation of Cd630 spores with CPZ and BHIS +Tc resulted in a reduction in germination. EGTA is a calcium chelator and resulted in complete inhibition of germination when incubated with Cd630 spores. A23187 is a calcium ionophore, and also resulted in complete inhibition of spore germination with incubation of Cd630 spores. The effect of pH on germination of Cd spores was also measured. We determined that the optimal pH for germination was approximately 7.4. By testing these inhibitors and varying environmental conditions such as pH, some details about the mechanism of germination may be revealed. Finding small molecule inhibitors specific for spore germination would be exceptionally useful for the prevention of recurrent CDI, as it would help prohibit the infection from occurring without disrupting the recovering intestinal microbiota.
Clostridium difficile (Cd) is a gram positive, spore-forming bacterium that causes colitis, or inflammation of the large intestine, and leads to severe gastrointestinal disease. Cd infection (CDI) most commonly occurs after broad-spectrum antibiotic treatment has disrupted the intestinal microbiome, allowing for Cd colonization. In recent years Cd infections have been steadily increasing, resulting in significant nosocomial infections in hospitals worldwide (1). CDI occurs when a susceptible host ingests infectious spores. While CDI is described as a toxin-mediated disease, its capacity for transmission and, therefore, disease is dependent on the ability to produce viable spores. Understanding the process of sporulation is important in identifying new targets to prevent transmission of Cd. We have created a mutant ATPase associated with a putative type II secretion system theorized to be essential for sporulation. This mutant yielded spores deficient in DPA, signifying a disruption in the transport of DPA across the outer membrane during sporulation. Characterization of the entire type II secretion system may lead to the development of new strategies to prevent sporulation and therefore transmission of Cd.
Even though Bacillus subtilis is the most well studied Gram-positive model organism, approximately 40% of its genes have no known function. To understand the functionality of uncharacterized genes important for DNA repair and cell cycle regulation in B. subtilis we chose a genome-wide mutagenesis approach. Transposon-insertion mutagenesis followed by deep sequencing (Tn-seq) is a high throughput method used to identify genes required for a particular process or mitigating the stress of a treatment. In an effort to more fully understand the genes required for mitigating the stress from a variety of DNA damaging agents, a library of approximately 140,000 Mariner transposon insertions was created in B. subtilis. We grew the library in triplicate for three serial passages in the presence or absence of hydroxyurea (HU). HU blocks DNA synthesis by inhibiting ribonucleotide diphosphate reductase (RNR), which prevents conversion of rNDPs into dNDPs and therefore blocks the production of dNTPs. By inhibiting RNR in a transposon library across three time points, we can screen for nonessential, uncharacterized genes involved in DNA repair, cell cycle regulation, and perhaps genome stability. Gene candidates will be later analyzed using genetic and biochemical methods to uncover molecular function. We also monitored RecA-GFP focus formation using microscopy to pinpoint when RecA-GFP responded to HU. Since RecA is involved in stabilizing replication forks and repairing DNA breaks during the SOS response, localization of RecA-GFP was observed at the replication fork and used as a proxy for HU-induced replication fork stress. At these time points, RNA-seq was performed to understand the transcriptional responses to stalled replication following HU challenge. From this approach, we expect to identify characterized genes involved in DNA repair (such as recN, addA, and ruvB) and identify unknown genes important for DNA repair and cell cycle csurvival to HU and a broad spectrum of DNA-damaging agents.
Alhabeil, Jamal A.*1, Zora, Jonathan S.1, Thomson, Joshua J.1, Canals, Albert2,3, Pieretti, Simone 2,3, Pérez, Rosa 2,3, Coll, Miquel 2,3, and Krukonis, Eric S.
Cholera, an acute diarrheal disease caused by Vibrio cholerae, is estimated to cause over 100,000 deaths each year. Two key virulence factors, cholera toxin and toxin co-regulated pilus, are directly regulated by ToxT. ToxT synthesis requires activation of the toxT promoter by ToxR in conjunction with TcpP, which stimulates RNA polymerase (RNAP). We previously defined two ToxR-binding sites within the toxT promoter, but our recent crystallographic studies of ToxR bound to the toxT promoter identified three other ToxR-binding sites. Based on these structural findings, ToxR mutant proteins predicted to impact DNA binding were generated. ToxR residues W64, D72, T77 and R84 when mutated to alanine (or proline for D72) resulted in ToxR molecules unable to activate either of two ToxR-dependent promoters, toxT or ompU. To assess DNA-binding activity, the DNA-binding and transactivation domains of these ToxR mutants were purified and used in electrophoretic mobility shift assays (EMSAs). While all four mutants were strongly defective for promoter activation, only ToxR-R84A was completely defective for DNA binding. ToxR-W64A, and ToxR-T77A showed intermediate levels of DNA binding and ToxR-D72P bound DNA like wild-type ToxR. T77 and R84 lie within the DNA recognition helix of ToxR while W64 lies upstream of the transactivation loop of ToxR and D72 lies within the transactivation loop. The fact that ToxR-D72P maintains DNA-binding, but fails to activate both the toxT and ompU promoters suggests this substitution may alter presentation of the transactivation loop for interaction with RNAP. This finding implies ToxR and TcpP may both interact with RNAP on the toxT promoter or ToxR interacts with TcpP via its transactivation loop. In addition, EMSA analysis supports the structurally-based identification of additional promoter-proximal ToxR-binding sites on the toxT promoter. Future studies will more specifically define the DNA sequences required for ToxR binding and regulation of V. cholerae virulence. Presented at the Midwest Microbial Pathogenesis Conference 2016 at the University of Illinois at Urbana-Champaign.
b: Iowa State University, Ames, Iowa, 50011.
Gene expression in methanotrophs has been shown to be affected by the availability of a variety of metals, most notably copper-regulating expression of alternative forms of methane monooxygenase. A copper-binding compound, or chalkophore, called methanobactin plays a key role in copper uptake in methanotrophs. Methanobactin is a ribosomally synthesized and posttranslationally modified peptide (RiPP) with two heterocyclic rings with an associated thioamide for each ring, formed from X-Cys dipeptide sequences that bind copper. The gene coding for the precursor polypeptide of methanobactin, mbnA, is part of a gene cluster, but the role of other genes in methanobactin biosynthesis is unclear. To begin to elucidate the function of these genes, we constructed an unmarked deletion of mbnABCMN in Methylosinus trichosporium OB3b and then homologously expressed mbnABCM using a broad-host-range cloning vector to determine the function of mbnN, annotated as coding for an aminotransferase. Methanobactin produced by this strain was found to be substantially different from wild-type methanobactin in that the C-terminal methionine was missing and only one of the two oxazolone rings was formed. Rather, in place of the N-terminal 3-methylbutanoyl-oxazolone-thioamide group, a leucine and a thioamide-containing glycine (Gly-?) were found, indicating that MbnN is used for deamination of the N-terminal leucine of methanobactin and that this posttranslational modification is critical for closure of the N-terminal oxazolone ring in M. trichosporium OB3b. These studies provide new insights into methanobactin biosynthesis and also provide a platform for understanding the function of other genes in the methanobactin gene cluster.
4Department of Fisheries and Wildlife, College of Agriculture and Natural Resources, Michigan State University, East Lansing, MI, USAF.
Spartansii strain T16T, the emerging virulent fish pathogen, was isolated from a disease outbreak in hatchery-reared Chinook salmon (Oncorhynchus tshawytscha) fingerlings. To gain an insight into its genomic contents, structure and potential pathogenesis factors, the comparative genome analyses were performed using the environmental and virulent Flavobacterium genomes. F. spartansii shared low average nucleotide identity (ANI) to those well-known virulent flavobacterial species such as F. columnare and F. psychrophilum, indicating that it is a new emerging fish pathogen. The genome in F. spartansii strain T16T has a length of 5,359,952 bp, a GC-content 35.7%, and 4,422 predicted protein-coding sequences. The pan-genome analysis revealed open pan-genome nature of Flavobacterium species. Flavobacterium core genome analysis showed that the number of shared genes decreased with the addition of the input genomes and is predicted to converge against 1182 genes. At least 9 genomic islands (GIs) were predicted to be presented in F. spartansii genome. Furthermore, the well-known virulence genes, including those encoding type IX secretion system (T9SS), proteases, adhesion, gliding motility and outer membrane proteins (Omp), were found in the genome of F. spartansii strain T16T. F. spartansii was resistant to penicillin and tetracycline, which agreed with their related antimicrobial genes present in the genome. However, it was susceptible to ampicillin (<10 ?g) though several beta-lactamases (class C and B) were found. The Flavobacterium-E. coli shuttle plasmid pCP29 and transposon pHimar Em1 were successfully conjugatively transferred into F. spartansii strain T16T, indicating this strain was amenable to the genetic manipulation. Introduction of the GFP-expressing plasmid Fj29 led to strong fluorescence production in Flavobacterium cells, which will contribute to elucidate the flavobacterial pathogenesis mechanisms.
Baxter Grand Valley State University, Allendale, MI, 49401.
Pathogenicity islands are clusters of virulence genes that a pathogen gains from exogenous sources during evolution. Salmonella species invade intestinal epithelial cells via forced uptake by utilizing a type 3 secretion system (T3SS) and its effector proteins, which are mostly encoded in a genetic region called Salmonella pathogenicity island 1 (SPI1). SPI1 is intensively regulated according to various environmental signals. hilE, a gene outside SPI1, encodes a major repressor of SPI1, whose surrounding genetic region show characteristics of a novel pathogenicity island. To investigate this putative pathogenicity island, polar mutations in open reading frames (ORFs) around hilE were made. These mutations’ effects on motility, adherence, invasion, and gene expression are being investigated via various assays. One ORF has been observed to show potential inhibition of SPI1 expression under non-inducing environmental conditions. Future work for this project includes continuing the work of characterizing the role these ORFs have on Salmonella virulence.
Copy number variations (CNVs) are duplications or deletions of large genomic regions. CNVs have been linked to diseases such as heart defects and autism. However, little is known about how they arise. Evidence shows CNV locations are correlated with the locations of human common fragile sites. Common fragile sites are areas of the genome that are prone to breaking under replication stress. To investigate the formation of CNVs, we created yeast strains containing human fragile site FRA3B or FRAXB, and a reporter cassette containing genes for resistance to copper and formaldehyde. This cassette allows us to screen for the formation of CNVs at the fragile site in yeast cells under stress.
Background: The signaling networks underlying the transition between motile and biofilm lifestyles in V. cholerae have been under intense investigation and it appears that c-di-GMP signaling plays a central role in controlling phenotypic switching. However, isogenic populations often exhibit phenotypic diversity owing to stochastic molecular fluctuations and bi-stability in the regulatory networks. Phenotypic diversity can be beneficial as a bet-hedging or division of labor strategy, but in V. cholerae, the role and regulation of phenotypic diversity in different environments has not yet been characterized.Method: We used video-microscopy and multiple-particle tracking to characterize single-cell motile behavior. Swimming speed and reversion frequency were calculated from individual trajectories to measure phenotypic distributions. We quantified cell-to-cell variability in the activity of the vpsT promoter (VpsT is a transcription factor controlling biofilm formation) using a fluorescent reporter to examine the origin of phenotypic diversity in the regulatory network. We tested different V. cholerae mutants in different growth conditions to identify factors that are involved in shaping phenotypic distributions.Results: Single-cell tracking revealed that motile and non-motile cells coexist during the exponential growth phase in all the conditions we tested. However, all cells become motile when the population enters the stationary growth phase. Mutations in the quorum sensing system (QS) had little effects on the distributions of motility phenotypes. On the other hand, the observed bimodality of the vpsT promoter activity in the wild-type strain is lost when the QS is perturbed. A constitutively active (?luxO) QS upregulates vpsT, whereas a constitutively inactive (?hapR) QS downregulates its activity.Conclusion: Exponentially growing cells are phenotypically diverse even in conditions that are known to promote either motility or biofilm formation. Motility phenotype does not appear to have a perfect inverse correlation with the transcriptional activity of genes that promote biofilm formation.
1 The Water Quality, Environmental and Molecular Microbiology Laboratory, Department of Fisheries and Wildlife, Michigan State University East Lansing, MI 48824, USA.
2 Enteric Pathogens and Water Research Laboratory, Institute of Primate Research, P.O Box 24481, 00502 Karen, Nairobi, Kenya.
AbstractGlobally rotavirus (RV) causes severe diarrhea in infants and young children where group A RV causes the most severe cases. Rotavirus is a double stranded (ds) ribonucleic acid (RNA), non-enveloped virus with segmented genome and unlike other enteric viruses that have a single stranded RNA. The detection, identification and quantification of RV in environmental samples is cumbersome and its recovery requires collection and concentration of a large volume sample. Molecular detection methods such as quantitative polymerase chain reaction (qPCR) and conventional RT-PCR have been used to detect, identify and characterize RV in both clinical and environmental samples. New methods developed for poliovirus surveillance in sewage has yet to be evaluated for RV which will be needed as the vaccine for RV is further distributed. The goal of this study was i) to evaluate droplet digital PCR (ddPCR) as a tool to detect and quantify RV in untreated sewage and compare the RV concentrations from different geographical settings and ii) to optimize the sampling preparation method that can be able to quantify RNA viruses in untreated sewage. In this study, untreated sewage samples (grab samples) were collected from lagoons in Kenya (5 L, n=10) in the summer of 2015, from a US Lagoon (10 L, n=10) in the summer of 2016 and effluent samples from the US (10 L, n=18) from a wastewater treatment plant (WWTP) for a period of 12 month (August 2015-July 2016) as part of a study on virus removal through wastewater treatment. The samples collected from Kenya lagoon (5 L) and from US lagoon (7.5 L) were concentrated using an adsorption-elution method (ViroCap), while 2 L of US lagoon and 2 L of WWTP samples were concentrated using was concentrated using polyethylene glycol (PEG) method with ultracentrifugation so as to compare the volume of sample needed to yield higher RV concentration. Nucleic acid for RV (dsRNA) was extracted using commercially available kit (QIAamp Viral RNA Mini Kit) and RV was detected and quantified using ddPCR.Rotavirus was detected in all the samples accounting for 100% (10/10) in the Kenya and US lagoons and 100% (18/18) in the US WWTP. Rotavirus was detected in the Kenya Lagoons at a mean concentration of 1.09E+05 ± 1.90E+05 (range 3.24E+03 to 5.84E+05) and a geometric mean of 2.71E+04 genome copies/L. In the US lagoon, the samples concentrated using ViroCap had a mean of 7.68E+02 ± 9.41E+02 (range 7.20E+01 to 2.83E+03) genome copies/L, while the one concentrated using PEG had a mean of 1.48E+04 ± 8.97E+03 (range 2.76E+03 to 2.81E+04) genome copies/L. The mean difference of RV concentration between the 2 viral concentration methods (ViroCap Vs PEG) in the US lagoon samples were statistically significant p<0.05. The US WWTP had a mean concentration of 4.92E+05 ± 8.19E+05 (range 5.04E+03 to 2.74E+06 and a geometric mean of 1.31E+05 genome copies/L.In conclusion, the droplet digital PCR is less cumbersome and a promising technology for detection, identification and quantitation of RV in untreated sewage and therefore minimizing the time needed for developing a standard curve used for qPCR.
Trypanosoma brucei is a disease causing parasite that lives in mammalian hosts and is transmitted by the bite of a tsetse fly. This salivarian trypanosome is exclusively extracellular in the vertebrate host and can continuously replicate within the bloodstream for months, escaping the host’s immune response by switching surface glycoproteins. During growth in the glucose-rich bloodstream, the mitochondrion is down-regulated and lacks a functional electron transport chain. This unique life cycle leads to two main problems: 1) the lack of selection for genes involved in mitochondrial energy production and 2) repeated population bottlenecks, each time they undergo an antigenic switch. These conditions should lead to the rapid accumulation of mutations in the mitochondrial genes required for survival in the insect vector. In our analyses of the gRNA transcriptome, we have identified alternative terminal gRNAs (the last gRNA in the editing cascade) for the several transcripts that generate transcripts using different open reading frames. Subsequent analyses of the pan-edited mitochondrial genes indicate that several contain extended dual ORFs, and thus could be dual-coding. We hypothesize that RNA editing is a unique mechanism that can be used to gain access to multiple ORFs, and that the overlapping of genetic information helps combat deleterious genetic drift that would occur during growth under non-selective conditions. Using Illumina deep sequencing, we have identified alternative forms of CR3 which align its start codon with two different ORFs, and we have identified the alternative gRNAs that direct these edits.
ToxR is the master transcription factor that upregulates virulence gene expression in Vibrio cholerae and is required to cause the human disease cholera in the intestine. ToxR responds to various environmental signals, such as bile acids found in the intestine. It was previously shown that synthetic human bile (SHB), a heterogeneous mixture of six bile acids found in humans that aid in digestion, increases cyclic di-GMP (c-di-GMP) levels in V. cholerae through the repression of the phosphodiesterase, vc1295, which degrades c-di-GMP. We identified four putative ToxR binding sites and two transcription start sites in the vc1295 promoter. Two of the ToxR binding sites flank the 5’ transcriptional start site named P1. We hypothesized that P1 is the transcription start site responsible for regulating vc1295 transcription through binding of ToxR to the promoter region in a SHB-dependent manner. We found that deletion of P1 abolished vc1295 expression, while deletion of the 3’ transcription start site had no effect, supporting our hypothesis. Moreover, the cytoplasmic, transmembrane, and periplasmic domains of ToxR were required for repression of vc1295 by SHB. Electro-mobility shift assays showed that ToxRS-expressing membrane vesicles did not bind to the promoter of vc1295 in vitro although they did interact with the promoter of toxT, a gene known to be directly regulated by ToxR. This suggests that either ToxRS binds to the promoter of vc1295 differently than the promoter of toxT, which cannot be recapitulated in vitro, or that ToxRS does not directly regulate vc1295. We further found that vc1295 repression is observed to be an additive effect of all six individual bile acids as no individual bile acid drove significant repression of vc1295. Together, these results suggest that in response to bile, ToxRS controls c-di-GMP levels through the repression of vc1295 in V. cholerae, showing that modulating c-di-GMP is part of the ToxRS regulon.
Staphylococcus aureus is a Gram positive pathogen that is a leading cause of skin and soft tissue infections, pneumonia, endocarditis, and bacteremia in the United States. S. aureus poses a difficult clinical challenge because of its ability to rapidly develop resistance to antibiotics. One mechanism by which S. aureus resists antimicrobial therapies is by adapting a slow growing, respiration arrested state called the small colony variant (SCV) phenotype. To identify the metabolic pathways that support the respiration-arrested growth of SCVs, we performed a genetic screen using a defined transposon mutant library. The respiration inhibitor, zinc protoporphyrin (ZnPPIX) was used to arrest respiration and the mutants that displayed a significant growth reduction in the presence of ZnPPIX were isolated for further analysis. One mutant impaired for respiration-arrested growth mapped to the gene ispA. Heme biosynthesis mutants are SCVs commonly isolated from patients persistently colonized with S. aureus. To determine if ispA is required for the SCV phenotype associated with heme biosynthesis mutants, the ispA transposon was transduced into ?hemA mutant background. The ?hemA ispA double mutant exhibited a reduced colony size compared to the parental ?hemA SCV. The reduced growth phenotype occurs in both solid and liquid media. The double mutant is also more susceptible to oxidative stress than the parental SCV strain. These results demonstrate that ispA-dependent metabolic pathways support the growth of SCVs. Future studies will identify the specific ispA pathways that assist the transition to respiration-arrested growth of S. aureus.
The genus Caldicellulosiruptor are extremely thermophilic, anaerobic plant biomass degraders. Some species, like Caldicellulosiruptor bescii have been demonstrated as exceptional plant biomass degraders, making it a candidate for consolidated bioprocessing of biofuels. However, development of this ability mandates a better understanding of the mechanisms C. bescii uses to facilitate adherence and deconstruction of plant biomass. Bioinformatic analysis of the genome from C. bescii predicted that it possesses a type IV pilus operon (Athe_1872 - Athe_1886). Type IV pili are extracellular filaments with a demonstrated role in adherence and motility in other bacteria. From this operon, the annotated hypothetical protein Athe_1880, was predicted to be the major pilin, based on amino acid sequence similarity to other major pilins. Our working hypothesis is that based on the proximity of the type IV pilus operon to secreted cellulases in the genome, the type IV pilus plays a role in adherence to plant biomass. In order to confirm a role for the predicted major pilin, Athe_1880, recombinant, soluble protein (Athe_1880T) was produced by truncating the hydrophobic domain. Qualitative binding assays verified that Athe_1880T binds to representative plant polysaccharides such as crystalline cellulose and xylan. Xylan appears to be the main inducer of expression of Athe_1880 as immunoblots detected the highest presence of Athe_1880 on cell surfaces in comparison to plant polysacharides pectin, crystalline cellulose and glucomannan. Quantitative binding assays determined that Athe_1880T has an affinity for crystalline cellulose and xylan (say more once we have more data). Based on this precursory data we propose that the protein Athe_1880T plays a limited role in cell adherence to xylan, and presumedly plant biomass.
The asymmetric cell division in Caulobacter crescentus results in two functionally and morphologically different cell types; motile swarmer and sessile stalked cells. During the cell cycle, about 20% of cellular mRNAs are cell-cycle regulated. While much of this is due to a transcriptional regulatory circuit that controls the cell cycle timing of transcription of 57% of these promoters, the role of mRNA decay remains unknown. Recent reports showed that the major mRNA turnover nuclease, RNase E, has cell cycle-regulated protein levels suggesting a role for mRNA decay in the cell cycle-regulation of mRNA levels. To explore this possibility, we developed a conditional expression system that allowed us to control the protein levels of RNase E by addition of xylose. Using this system we find that alteration of the RNE protein levels (by depletion or overexpression) leads to deregulation of the cell cycle. Additionally, we found the catalytic activity of RNase E is required to avoid cell cycle defects. Using RNA-Seq in RNase E depletion and overexpression conditions we found a large number of RNase E substrates to be known cell cycle-regulated mRNAs, including mRNAs for cell cycle transcriptional master regulators. These observations strongly suggest that RNA decay through RNE plays a role in C. crescentus cell cycle-regulation, potentially through a rewiring of the cell cycle transcriptional regulatory circuit.
Onion thrips, Thrips tabaci Lindeman, is a primary pest insect of onions (Allium cepa), directly affecting the United States’ onion industry. Feeding by onion thrips results in the destruction of epidermal tissue, often leaving silvery leaf spots that turn into white blotches along the leaves. Onion thrips are known vectors of Pantoea ananatis (Serrano) Mergaert, causal agent of center rot in onions. Infection with these bacteria produces white streaks with water-soaked margins along the length of the leaves; these lesions turn necrotic and may progress into the onion bulb, leading to bulb rot during long-term storage. However, the role of thrips feeding injury on progression of bacterial infection in onions has not been investigated. Our objectives were to 1) determine the role of onion thrips feeding injury in facilitating the development of bacterial center rot, 2) investigate the relationship between thrips abundance on plants and disease development and, 3) observe morphological changes in onion tissue from onion thrips feeding and P. ananatis infection. Three experiments were conducted in which individually caged onions were infested with varying numbers of thrips per plant and inoculated seven days later with a liquid culture of P. ananatis (~1 × 108 CFU/ml). Thrips feeding injury, bacterial disease severity and various growth parameters were measured 7 and 8 days following inoculation. Onions infested with thrips and inoculated with P. ananatis had a higher proportion of necrotic tissue and bacterial symptoms were more severe with increasing thrips numbers. Under fluorescence microscopy, we were able to visualize a GFP-tagged strain of P. ananatis colonizing areas of the onion leaf tissue damaged by thrips. In the absence of thrips injury, P. ananatis was able to colonize plants using existing wounds or natural openings, but when thrips were present, P. ananatis used their feeding sites as additional entry points, resulting in greater progression of infection and disease. Overall, our results demonstrate that wounds caused by onion thrips feeding enhance bacterial center rot development by providing entry sites for P. ananatis.
Abstract Text: Background. Lake Sturgeon (Acipenser fulvescens), one of the longest-lived and oldest species of Great Lakes fish, is threatened or endangered and high egg mortality is thought to be a major cause. To gain a better understanding of the relationship between egg mortality and microbial activity we have investigated the bacterial communities of sturgeon eggs from streams using cultivation independent and dependent approaches.Methods. We have isolated 380 bacteria from the surface of sturgeon eggs (2-3 days after spawning) using selective (Pseudomonas isolation agar; PIA) and non-selective (R2A) media. Isolates were identified with comparative sequence analysis of 16S rRNA and screened for virulence factors (extracellular protease and hemolysin) and ability to form biofilm. In addition Illumina sequencing of 16S rRNA genes was used to profile the complete community.Results. Comparative analysis of 16S rRNA sequences identified 90% of the bacterial isolates (340) to genus level and 10% (39) to species level (18 genera and 7 species). Extracellular proteases and ß-hemolysins were detected in 62.6% and 48.5% of the isolates, respectively. Biofilm formation varied among species as follows; Aeromonas (214 strains; 56.8% positive for biofilm), Pseudomonas (59 strains; 15.6%), Rhodoferax (26 strains; 6.9%), Flavobacterium (14 strains; 3.7%), and Serratia (6 strains; 1.6%). Media supplemented with exogenous protein increased biofilm formation in Aeromonas. Pseudomonas and Rhodoferax biofilm was not stimulated by exogenous protein. Illumina sequencing of bacterial communities from river water and eggs revealed complex but different communities. The Aeromonas population was only 1.3% of the water community but represented up to 26.5% of the egg community. Albidiferax and Flavobacterium were the most abundant genera on the egg.Conclusions. The number of fish pathogens (Aeromonas and Flavobacterium) associated with sturgeon eggs from streams was surprising to us and suggests that in natural environments sturgeon eggs are rapidly colonized by putative pathogens. This may impact successful recruitment of the species.
Wang, Ce*, Hakim, Pusparanee* & Vecchiarelli, Anthony G.
The carboxysome is the best characterized organelle in bacteria, shown to fix more than 25% of the Earth’s carbon, improve the photosynthetic efficiency of plants, and act as a bioreactor for metabolic engineering and biomedical applications. Our aim is to understand the molecular mechanisms governing the intricate subcellular organization of carboxysomes required for their faithful inheritance in a cell population. Little is known about the players and the mechanisms governing this organizing system aside from the requirement of a ParA-type ATPase, which are typically known for DNA positioning in vivo. Here we attempt to define and characterize the ParA-type system responsible for Carboxysome positioning in Synechococcus elongatus by comparing and contrasting with the known features of ParA-mediated DNA positioning. Our preliminary finds suggest that, like DNA cargo, a ParA-mediated transport system spatially organizes carboxysomes over the nucleoid region of the cell.
Transcriptional Repressor Tup1p.The ability to change between budding yeast form growth and filamentous morphologies is an important virulence factor in the opportunistic fungal pathogen Candida albicans. A key regulator of this process is the transcriptional repressor Tup1p. A strain lacking this protein grows only as filamentous pseudohyphae and is more sensitive to numerous environmental stresses, including elevated temperature and osmotic stress. Tup1p interacts with one of its corepressors, Nrg1p, through a C-terminal WD-40 domain, but little is known about the contributions of other domains to its function. To address this question, we have attempted to rescue the mutant strain with truncated versions of Tup1p. We have been able to partially restore the response to morphology signals and resistance to some stresses. This highlights the importance of previously uncharacterized N-terminal protein interactions to the function of Tup1p. We are currently constructing strains expressing tagged versions of these proteins to allow purification and identification of Tup1p binding partners.
The co-repressor Nrg1p is a key regulator of cell morphology in the opportunistic fungal pathogen Candida albicans. Expression of many genes is activated during hyphal growth as a result of relief of Nrg1p-mediated repression, but the functions of some of these genes are not yet known. Two such genes are orf19.2302 and orf19.6705. Orf19.2302 encodes a Candida-specific protein whose only homologue is a hypothetical protein in the closely related species Candida dubliniensis. In a strain where we deleted both copies of this gene we found that in cell culture medium RPMI-1640, cells showed increased adhesion compared to a wild-type strain. During an infection, cell adhesion is important for both biofilm formation and interaction with host cells. Although loss of this gene does not appear to influence biofilm formation in vitro, we found that it resulted in attenuated virulence in the Galleria model of systemic candidiasis. The gene orf19.6705 is predicted to encode a member of the cell membrane guanyl nucleotide exchange factor protein family and could therefore play a role in how C. albicans successfully sense and adapts to its varied surroundings. When we deleted both copies of this gene we found that the adhesive properties of hyphae were altered with increased cell aggregation in some media, and decreased aggregation in other media. This medium-specific phenotype was also reflected in biofilm formation, where deletion of the gene resulted in increased biofilm only in some conditions. Taken together, this suggests that the protein can activate or repress signalling pathways depending on the stimulus. In the Galleria model of systemic candidiasis, the deletion strain also shows attenuated virulence. We are continuing to examine the growth of these strains under different conditions to better understand their role in filamentation.
Authors and Addresses: McKindles, Katelyn M.* and Angell, Michael G.
Abstract Text:Every year, Lake Erie and other bodies of water experience blooms of mixed algal species, known as Harmful Algal Blooms (HABs). Of particular concern in this region is the cyanobacteria Microcystis aeruginosa, which is a species that has the potential to produce hepatotoxins. As the organisms in the HAB deplete the available nutrients, the bloom declines. To address the potential role of cyanophages on the decline of HABs in this nutrient limited context, we use a specific host-phage system: Ma-LMM01 (phage) and M. aeruginosa strain NIES298 (host), both of which originate from a eutrophic lake in Japan. Both control cells and phage-infected cells were inoculated into standard media (CB media) or phosphorus-limited CB media. Growth of the host cells was monitored using spectrophotometer readings (600nm), while phage (genome) replication was quantified using real-time PCR (qPCR). The infection under nutrient replete conditions alters the growth rate of the cyanobacterial population (from 0.226 uninfected to 0.197 infected, p<0.01), but infection under phosphorous limited stress shows an even greater decrease the growth rate of the host (from 0.222 uninfected to 0.169 infected, p<0.001). Additionally, phage replication reaches the same ratio in both the nutrient replete and phosphorus-limited media, suggesting that the nutrient limitation does not alter phage replication ability, just the host’s. One possible explanation for the effect on the host under phosphorous stress is that the phage has a homolog gene to phoH, which is an ATPase induced under phosphate stress that may allow the phage to steal phosphate from its host to ensure its own replication. This data supports the hypothesis that phage infection has a greater negative effect on M. aeruginosa growth under phosphorous stress than nutrient replete conditions.
Both the physical and financial burdens of urinary tract infections (UTIs) are staggering. In the U.S. alone, UTIs result in an estimated societal cost of $3.5 billion. Urinary tract infections (UTIs) are primarily caused by uropathogenic Escherichia coli (UPEC) and 1 in 40 women experience recurrent UTIs. Women experiencing at least two UTIs per year are frequently given antibiotics prophylactically. Not surprisingly, the rates of resistance to these antibiotics in UPEC strains have steadily risen over the past few decades, highlighting the need for new antibiotic scaffolds and therapeutic strategies to treat UTIs. Iron, an essential bacterial nutrient used as a co-factor in many biological processes, is restricted in the host environment. Thus, iron acquisition may serve as a vulnerable target for E. coli. Notably, the primary site of UPEC infection, the bladder, has dramatically lower iron levels than the sera. It is not surprising then that UPEC strains deficient in iron acquisition are attenuated. To identify novel scaffolds and validate bacterial iron acquisition as a viable therapeutic target, we screened 33,000 marine microbial-derived natural product extracts against an unmodified UPEC clinical isolate. This ensured that active hits are not susceptible to Gram-negative efflux pumps. We identified 204 natural product extracts that reduce wildtype UPEC growth in low iron by over 90% without chelating iron or impacting bacterial viability in iron-replete medium. From these hits, we purified a novel family of cyclic natural products that inhibit UPEC growth at nanomolar concentrations. Current studies are underway to identify the target of these molecules. The isolation of multiple compounds with similar structures provides an excellent platform for exploring the structure-activity relationships of these compounds with their bacterial targets. In summary, we have identified a novel scaffold that inhibits the growth of wildtype UPEC in low iron conditions. These studies will ultimately inform the development of antimicrobial therapies that target iron homeostasis in UPEC and other Gram-negative bacteria.
Developing Methods to Study E. cloacae Colonization Mechanisms Enterobacter cloacae is a rod shaped facultatively anaerobic Gram-negative bacterium belonging to the Enterobacteriaceae family. Some E. cloacae strains have acquired resistance to a majority of the available antibiotics including carbapenems, making E. cloacae one of the most dangerous human pathogens currently causing nosocomial infections. E. cloacae is an opportunistic pathogen and common commensal bacterium from the human gastrointestinal (GI) tract. It is only pathogenic however, when able to invade the bloodstream and colonize sterile organs. The mechanisms by which E. cloacae does this are still largely unknown. The goal of my project is to develop genetic tools for the study of the molecular mechanisms required for E. cloacae pathogenesis and colonization. I am working to apply a method of genome engineering called recombineering to E. cloacae. Recombineering utilizes a recombination system called RED from phage lambda and enables very precise, efficient, and rapid insertion or deletion of genomic DNA. By expressing the RED system from a plasmid in E. cloacae along with small fragments of DNA to be introduced into the genome, I will construct mutations in specific E. cloacae genes to assess their role in colonization outside of the GI tract.
A defining characteristic of the bacterial lifestyle is the formation of a biofilm; a community of cells living together, usually with the aid of a secreted extracellular matrix. This matrix gives the biofilm a regular structure and protects the cells from damage by antibiotics, predation, shear and oxidative stresses. The extracellular matrix produced by *E. coli* and many other enteric bacteria primarily consists of protein and carbohydrate polymers, called curli and cellulose, respectively. Curli and cellulose are coordinately-regulated by a master biofilm transcription factor, CsgD. We found that CsgD expression is dependent on the diguanylate cyclase YfiN and its associated redox-sensitive repressor YfiR. YfiR interacts with YfiN and prevents YfiN dimerization and cyclic-di-GMP production. YfiR is stabilized by a disulfide bond, but the conditions that destabilize YfiR to promote YfiN activity, as well as how YfiN affects CsgD expression are unknown. Using a urinary tract isolate of *E. coli* called UTI89, we found that interruption of the cysteine biosynthesis pathway resulted in dysregulation of extracellular matrix production that could be suppressed by deletion of *yfiR*. Furthermore, changes in the redox state of the periplasm through diminished respiration altered matrix components and rugose biofilm morphology. Again, deleting *yfiR* could restore production of matrix components and the rugose morphology. Cysteine auxotrophy may be linked to hyper-oxidation as mutants show resistance to mecillinam, though sensitivity can be restored with supplementation of cysteine or glutathione. Mutations in the YfiN GGDEF domain are currently being constructed to test the hypothesis that c-di-GMP production is required for *csgD *transcription. We would also like to identify how c-di-GMP impacts CsgD protein levels since there is no predicted ci-di-GMP binding site in CsgD. One possibility that is currently being tested is the role of MlrA, a putative c-di-GMP-binding protein that regulates *csgD* transcription. Collectively, these results clarify the role of redox chemistry on the development of the biofilm extracellular matrix.
Expanding Thermophile Diversity and Searching for Evidence of Microbial Scouts in the Soils Overlying a Long Burning Subterranean Coal Mine Fire.
Abstract: Thermophiles are frequently found in temperate soils, despite being unable to reproduce at temperatures below 40°C. We investigated microbial thermophile diversity in temperate soils that are impacted by the coal mine fire in Centralia, PA, which ignited in 1962 and continues to burn along near-surface coal seams. The fire exacts a press disturbance on the overlying soil microbial communities by increasing temperatures and depositing coal combustion pollutants. As the fire moves through the coal seam, previously affected soils recover, providing a chronosequence of disturbance history. We sampled Centralia soils along the chronosequence and used metagenome sequencing to interrogate microbial communities for taxa and genes that are responsive to increased temperature. We found that thermophilic archaea in Centralia predominately belong to Crenarchaeota and Parvarchaeota, and bacteria belong to Acidobacteria and Proteobacteria. KEGG orthologs’ abundances across the gradient indicated increases in genomes containing trehalose metabolism and autotrophic carbon fixation pathways. Overall, our findings suggest soils overlying the coalmine fire harbor novel thermophile phylogenetic and functional diversity.
Myxococcus xanthus is an excellent model system for studying multicellular behavior in bacteria. Under nutrient limitation, M. xanthus swarms enter a developmental program in which cells segregate into at least three distinct fates: aggregation into fruiting bodies filled with spores, programmed cell death, or formation of a persister-like state. Cell fate segregation requires MrpC, which is a member of the Crp/Fnr family of transcriptional regulators. MrpC was previously reported to be activated by proteolytic processing into a truncated isoform (MrpC2). It was also suggested that two eukaryotic-like Ser/Thr kinases, Pkn8 and Pkn14 phosphorylate MrpC (MrpC~P) to prevent production of MrpC2. We have reinvestigated the in vivo role of MrpC isoforms by generating mutants that produce only MrpC2 (mrpC?1-25) or lack the putative TTSS phosphorylation motif (mrpCAAAA). Both mrpC mutants fail to aggregate or to sporulate. To further investigate the role of MrpC~P, we demonstrated that Pkn14, but a not a kinase-inactive mutant (Pkn14K48N), autophosphorylates in vitro and phosphorylates MrpC, but not MrpCAAAA or MrpC2. A strain producing inactive Pkn14 (Pkn14K48N) phenocopies the wild type phenotype, but when generated in a background in which Pkn8 is also inactivated (pkn8K116N), sporulation efficiency in the double mutant is reduced to 35%. Together, these results suggest that MrpC2 is not the active isoform. Rather, MrpC~P is necessary to induce both aggregation into fruiting bodies and sporulation. We propose that unknown kinases phosphorylate MrpC to induce aggregation into fruiting bodies, and that Pkn8 and Pkn14 redundantly phosphorylate MrpC to induce sporulation.
Colonization of the human gut requires a multifaceted behavioral response from V. cholerae involving the transition between biofilm and motile lifestyles. The secondary messenger, cyclic diguanylate (c-di-GMP), is central to the regulation of motility, biofilm formation, and virulence in V. cholerae. However, the molecular mechanisms underlying the control of single-cell phenotypes remain largely uncharacterized. We propose that phenotypic diversity, that is the stochastic generation of distinct phenotypes within a clonal population, plays an important role during infection. Our preliminary results show that both biofilm-like aggregates and motile cells can be found in V. cholerae populations grown in homogeneous conditions. We investigate the role of c-di-GMP by perturbing signaling pathways that control phenotypic switching. We manipulated the intracellular concentrations of c-di-GMP directly using inducible diguanylate cyclases and phosphodiaesterases. V. cholerae’s motile behavior was characterized using single-cell tracking by measuring swimming speed, directional persistence, and the probability of changing direction. Biofilm formation was measured using microtiter plate crystal violet staining.
Campylobacter jejuni is one of the most common bacterial pathogens to cause enteritis in the United States with two million cases recorded annually. C. jejuni can discriminate and take up its own DNA from the external environment through a process called natural transformation. Some genes necessary for transformation are phylogenetically related to those that encode proteins for a specific secretion system (type II) found in other virulent bacteria, such as Vibrio cholerae. A type II secretion system is a complex mechanism frequently used by infectious bacteria to secrete proteins that enhance fitness in the host. These proteins are commonly toxins or degradative enzymes. My work is to test our hypothesis that C. jejuni uses its transformation system for both DNA uptake and protein secretion. I am using wild type C. jejuni 81-176 and mutant ctsE (which lacks a key component of the transformation system) to examine supernatants and determine if wild type cells can secrete proteins that are not secreted in the ctsE mutant. Any such proteins will be examined for their roles in the biology and pathogenicity of C. jejuni.
AbstractAdenovirus infection often causes asymptomatic infection in immunocompetent individuals but can affect many organ systems in immunocompromised individuals. Study of Murine Adenovirus 1 (MAV-1) as a model in mice was used to distinguish cause of increased CD40 surface expression after 9 hours of infection in mouse macrophages (MH-S cells); the same time it takes early gene 3 (E3) to be expressed. Increased CD40 expression can be related to encephalitis in mice and E3 is known to contain regions related to immunoregulation of host cell. Using qPCR to quantitate expression, PCR to amplify expressed genes, and transient fluorescently tagged siRNA; this project was designed to study the link between E3 RNA expression and CD40 surface protein expression. Recent studies observed time-course infection of MAV-1 in MH-S cells and silencing of E3 in cells infected with MAV-1 at two different multiplicity of infection’s (m.o.i.). Silencing of E3 was observed by PCR analysis of E3 expression in silenced cells and the corresponding Hexon DNA expression (viral amplification) and Hexon RNA expression (infection progression). Future studies will observe CD40 surface expression in E3 silenced cells by flow cytometry.
Curli are extracellular amyloid fibers produced by Escherichia coli (E. coli) and other enteric bacteria that are critical for biofilm formation and host colonization. Biofilms contribute to resistance against antibiotics and the host immune system, thus understanding curli biogenesis has direct implications on the abrogation of bacterial biofilms and the development of new antimicrobial therapy. Although the putative functions of each of the curli subunits and assembly proteins has been determined, there remain large gaps in our understanding of the protein-protein interactions between the assembly and subunit proteins underlying curli biogenesis. Here, we propose to perform single molecule microscopy employing differentially fluorescently tagged curli proteins to assess both the timing and spatial distribution of curli proteins during curli assembly. We have engineered several fluorescently-tagged constructs of a pore-forming lipoprotein CsgG and a chaperon-like accessory protein CsgE to be analyzed by this technique. We have generated a construct wherein CsgE is C-terminally tagged with both a 6x Histidine tag and PAmCherry. To ensure that tagging of CsgE did not interfere with its required function in curli biogenesis, E. coli MC4100 ?csgE cells were grown on YESCA-Congo red plates with and without plasmid-associated CsgE-his-PAmCherry. We found that the fusion protein fully complements curli transport and assembly. We have also generated 2 distinct fluorescently-tagged CsgG constructs based on the known crystal structure of the CsgG nonamer. We will similarly tag the other Csg proteins, so that they can be imaged in super-resolution fluorescent microscopy experiments. Once these fluorescent constructs have been fully validated, we will use them in the experiments to learn the location, order and timing of the assembly of the large curli protein export machinery in living cells.Our single-molecule analysis will reveal details about the dynamic interactions that lead to efficient curli biogenesis.
1School of Biology, 2Center for Bioinformatics and Computational Genomics, and 4School of Civil and Environmental Engineering, Georgia Institute of Technology, Atlanta, GA 30332, USA. 3Center for Microbial Ecology, and 5Department of Microbiology and Molecular Genetics, Michigan State University, East Lansing, MI 48824, USA.
Background. The small subunit ribosomal RNA gene (16S rRNA) has been successfully used to catalogue and study the diversity of microbial species and their communities to date. Nonetheless, several aspects of the rRNA-based studies remain problematic. Most importantly, how to better resolve microbial communities at the levels where the 16S rRNA gene provides inadequate resolution, namely species and finer levels, and how to best catalogue whole-genome diversity and fluidity. Further, the deluge of genomic data in the last three decades has promoted the development of a rapidly growing set of bioinformatics techniques and implementations. However, the lack of standards in genomic analyses complicates data sharing and comparisons across research projects.Methods. Here, we introduce the Microbial Genome Atlas (MiGA), a genomic data management and processing tool integrating best practices in genomic analyses with recent and novel developments in whole-genome-based taxonomy and classification. Data is processed according to presets tuned for isolate genomes, single-cell sequences, metagenome-derived bins, or metagenomes. MiGA features an indexing system based on medoid clustering over sparse matrices of Average Nucleotide and Amino Acid Identity (ANI/AAI) guided by heuristic approximations, enabling the fast classification of query genomes, as well as calculation of pangenome and assessment of horizontal gene transfer among the NCBI included in its database.Conclusions. MiGA represents the genome equivalent of the Ribosomal Database Project and aims to facilitate classification and diversity studies at the genome level. End users can interact with MiGA through the web server (http://microbial-genomes.org), the built-in Command Line Interface (https://github.com/bio-miga/miga) or deploy a virtual image on Amazon Web Services (AWS cloud).
Parkinson’s disease (PD) is a neurodegenerative disease with no cure affecting nearly 1 million people in the United States. The hallmarks of PD include the amyloidogenic aggregation of alpha synuclein (AS), loss of motor function and loss of dopaminergic neurons in the substantia nigra. Symptoms occur late in the course of disease and the current treatment is focused mainly on alleviating symptoms rather that preventing the disease progression. Nearly 80% of PD patients experience gastrointestinal dysfunction, in particular constipation, which may precede the onset of motor symptoms by years. Recent studies have shown that there is a significant difference in the gut microbiota between PD patients and healthy individuals. Coupled with the early gastrointestinal dysfunction, this study has led to the speculations that the gut microbiota may be implicated in PD.Enteric bacteria like E. coli produce extracellular protein fibers called curli which are structurally and biochemically defined as amyloid. Amyloid formation by human proteins is the hallmark of many neurodegenerative disorders, including Parkinson’s disease. However, unlike disease-associated amyloids, the functional amyloids like curli, assist bacteria in attachment to biotic and abiotic surfaces and in invasion of epithelial cells. Bacteria have involved an efficient secretion system and chaperone network comprising of proteins likes CsgC and CsgE which ensure that the major curli fiber subunit, CsgA does not form toxic intracellular amyloid aggregates. CsgA can also form amyloids in vitro, which can be accelerated by adding preformed CsgA fibers, a process called seeding. Similarly, our lab has also been able to show that alpha synuclein aggregation can also be accelerated in vitro by preformed CsgA fibers and inhibited by bacterial proteins like CsgC. Furthermore, a recent study by the Friedland lab at U. Louisville showed in PD model systems that exposure to E. coli producing curli increased the AS aggregation in neuronal cells in the gut and brain. Similarly, using a murine model of PD, we have found in collaboration with Dr. Sarkis Mazmanian’s group that the microbiota is important for the development of PD symptoms. In the wake of these studies and initial studies conducted by our lab, we hypothesize that amyloid producing bacteria in the gut play a key role in human amyloid-associated diseases and we will continue to characterize cross-seeding between microbial amyloids and alpha-synuclein and the consequences of this cross-seeding on the Parkinson’s disease cascade.
1Laboratory of Environmental Microbiology, The University of Hong Kong, Hong Kong SAR, People’s Republic of China. 2Laboratory of Microbial Ecology and Toxicology, Guangdong Academy of Forestry, Guangzhou, People’s Republic of China, 510000.
Abstract Text: Acidification is one of the major climate change threats to soil ecological function. Ammonia oxidizers, initiating the first step of nitrification, are often under stress in acidic soils, due to the limited availability of deprotonated substrate and the potential toxicity of nitric and nitrous acids. Among the intensive studies of nitrification, few have explored extremely acidic forest soils (pH < 4.6). For this work we sampled soils from three natural forests with pH values from 3.66 to 4.58 in Nanling National Nature Reserve, Guangdong, China, to investigate abundances and community structures of ammonia-oxidizing archaea (AOA) and bacteria (AOB) at both genomic DNA and RNA levels. Quantitative analysis of the functional biomarker, subunit of ammonia monooxygenase (amoA) gene and transcript revealed AOA was not only numerically, but also functionally dominant over AOB. The same three AOA lineages, Nitrososphaera, Nitrososphaera sister group and Nitrosotalea were found in both DNA and RNA based phylogenetic studies, suggesting these niche-inhabiting phylotypes were potentially functional. The relative abundances of the three lineages inferred by DNA and RNA, however, differed. For instance, Nitrosotalea was the most abundant AOA but was found to be only the second most functionally important, after the Nitrososphaera sister group. These results further suggest that AOA species do not contribute to nitrification proportionately, possibly due to their differentiated physiological features. Organic matter and exchangeable Al were found to profoundly affect AOA abundance and to shape the community structure. This study contributes to a more comprehensive understanding of nitrification in acidic soils and re-evaluates the relative functional importance of AOA lineages.
The human pathogen Vibrio cholerae causes the diarrheal disease Cholera, which is endemic in certain developing nations. V. cholerae is primarily found in aquatic environments where it forms microbial communities known as biofilms. Adopting a biofilm lifestyle provides protection against environmental stressors, which can indirectly facilitate disease transmission. In many cases, biofilms are produced when concentrations of the second messenger cyclic dimeric GMP (cyclic di-GMP) are high in the cell. In V. cholerae, biofilms increase tolerance to hydrogen peroxide, however the direct role of cyclic di-GMP is not well characterized. Therefore, we hypothesized that cyclic di-GMP played a more direct role in regulating tolerance to hydrogen peroxide. Using hydrogen peroxide turnover assays, luciferase transcriptional reporters, and cell survival assays, we found that cyclic di-GMP increases catalase expression and hydrogen peroxide tolerance in the absence of biofilm formation. Further, we determined these phenotypes were dependent on transcription factors that canonically regulate biofilm formation. These data suggest cyclic di-GMP increases tolerance to hydrogen peroxide through two separate mechanisms: biofilm formation and catalase induction. Biofilms may provide a scaffold to concentrate catalase producing cells, allowing the microbial community to tolerate hydrogen peroxide treatment, a common reactive oxygen species found in aquatic and human host environments.
Cyanobacteria have significant potential for sustainable production of chemical commodities at industrial scale. However, scaled cultivation of cyanobacteria faces significant challenges due to a scarcity of vetted genetic tools and prohibitive costs of harvesting suspended cells from large cultures. We have made significant progress toward creating a synthetic surface display system in model organism Synechococcus elongatus PCC7942 that could be applied to the industrial harvest of cyanobacterial biomass. Specifically, through the expression of a modified outer membrane porin (SomA), we have shown that engineered peptides can be displayed on the surface of living cyanobacteria. In making the heterologous SomA accessible, we showed that genetic ablation of two surface components was required: the O-antigen and a hypothetical surface layer protein. We demonstrated that this display system can be used to bind cyanobacteria to both surface coated agarose beads and engineered Saccharomyces cerevisiae. Expanding upon this system, we are investigating alternative functional domains to be displayed on the surface of S. elongatus to further our research in artificial cell adhesion as well as the generation of whole-cell biocatalysts. This surface display technology in S. elongatus could allow these cyanobacteria to be programmed to bind specific abiotic and/or biotic partners, which has potential applications in both industry (e.g. biomass recovery) and academia (e.g. construction of autotroph/heterotroph consortia). Finally, we discuss ongoing efforts to diversify the passenger domains displayed in order to modulate the surface of cyanobacterial cells for various functions.
During the Deepwater Horizon (DWH) oil spill, the deep water oil plumes enriched for large quantities of the psychrophilic microbe Colwellia psychrerythraea (CP) with respect to the general microbe population. Additionally, in lab-based microcosms that contain oil and other known oil degraders, CP grows in large quantities. However, CP is not known to degrade oil by itself. These findings are indicative of a communal relationship between CP and oil-degrading bacteria. During DWH COREXIT 9500, which is a chemical dispersant, was used to increase the solubility of the escaping oil plume. The problem with COREXIT is that it degrades very slowly and four years later it was still found in quantifiable amounts in the Gulf. Previous experiments qualitatively demonstrated that spent culture medium of CP resulted in increased emulsion layers between oil and media. This finding suggests that CP may produce a surfactant to increase the solubility of oil. This biosurfactant may have similar abilities to solubilize oil like chemical dispersants, but may not have the same persistence as COREXIT. To test the ability of CP to produce compounds that solubilize oil, the solubility of hexadecane (HD) was tested in a variety of different conditions. The conditions tested are as follows: Control (HD with no CP), hexadecane and CP 34H, HD and COREXIT, and HD and CP 34H+COREXIT. The microcosms were held in a 6? water bath shaker. After 7 days, the culture below the surface slick was removed from the microcosms and centrifuged to remove cells. Trichlorobenzene was added to track the extraction efficiency. The spent media was extracted with DCM to recover organic compounds. The organic phase was concentrated down to 1 mL. The extracts was analyzed on a GC equipped with an FID. It was found that conditions with CP were capable of solubilizing oil. The peaks relative to the control microcosm were largest for COREXIT, followed by CP 34H only, CP 34H+COREXIT, then finally the control, which is near 0. One control microcosm was contaminated and experienced highly soluble hexadecane. The reduced efficiency of COREXIT could be attributed to CP 34H degrading components of COREXIT.
Bacillus anthracis—a Gram-positive, spore-forming bacterium—causes anthrax, a highly lethal disease with high bacteremia titers. Such rapid growth requires ample access to nutrients, including iron. However, iron access is heavily restricted in mammals requiring B. anthracis to use petrobactin (PB), an iron-scavenging small molecule known as a siderophore. PB biosynthesis within and import into the bacterium have been well studied but PB export is unclear. We identified the RND-type transporter daxH as a putative PB exporter in B. anthracis Sterne. Deleting daxH abrogated export of intact PB, which instead accumulated inside the cell. However, ?daxH virulence in mice was not attenuated. Per gallium inhibition assays—where gallium is supplemented for import by PB and measured by growth inhibition—iron import appears to be uninhibited. Indeed, ?daxH demonstrates wildtype levels of 3,4-dihydroxybenzoate, a fragment of PB. We hypothesize that the PB accumulated within the cell is degraded and the fragments are exported through a different protein at concentrations high enough to allow iron transport. Our findings also suggest that PB fragments are sufficient for B. anthracis virulence in mice. This is the first report of in vivo functionality of siderophore fragments and of a functional siderophore exporter in B. anthracis.
Antibiotic resistance Staphylococcus aureus strains cause several life threatening infections. Medical science needs new drug treatment options, but these options are slow to develop because 50% of the S. aureus genome is hypothetical. This work used a publically available microarray dataset to identify statistically significant, differentially expressed genes between Methicillin sensitive and resistant Staphylococcus aureus strains whose protein products were hypothetical in National Center for Biotechnology Information and UniProt databases. Computational algorithms characterized the physiochemical features, cellular location, homologs, and potential binding partners for these proteins. This approach identified two down-regulated hypothetical proteins, SACOL0456 and SACOL1723, as possible acetyl-transferases. Both hypothetical proteins had similar instability and aliphatic indices and theoretical isoelectric points. They have similar solubility scores and have the same cytoplasmic localization score. Neither protein had transmembrane regions. Most PSI_BLAST identified homologs were other hypothetical proteins though SACOL0456 did match with an N-acetyltransferase from another Staphylococcus species. Two domain recognition algorithms could not identify domains in either protein. Phyre2 produces great quality models, which Verify3D showed had 35% and 45% resides, for SACOL0456 and SACOL1723, respectively, modeled with high confidence (3D-1D score >0.2). While 3DLigand was unable to predict bound ligands due to insufficient homologous structures, STITCH predicted with moderate confidence that SACOL1723 may bind 50S ribosomal proteins. The scientific literature links acetyltransferases to resistance to several antibiotics including chloramphenicol, a ribosome inhibitor. Researchers need more laboratory work to determine the exact function of these possible acetyltransferases along with their specific role in antibiotic resistance.
Bacillus anthracis is a gram-positive, spore-forming bacterium, and is the causative agent of the disease anthrax in humans and other animals. Like many disease-causing bacteria, B. anthracis growth relies on many nutritional factors, one of which is iron. However, iron is tightly sequestered within the host. B. anthracis uses a siderophore, petrobactin, to steal iron from its host during infection. The petrobactin biosynthesis operon (asbA-F) is upregulated in response to host conditions including low-iron, however, how other host factors such as oxidative stress, temperature, or CO2 regulate the operon is unclear. To study asb regulation, we created two reporter constructs to measure transcription and translation of the asb operon by gfp florescence under different conditions. To model the low-iron conditions of the human body, we grew both spores and vegetative cells (VCs) in iron-depleted media with 1% inosine (IDM) at 37°C. We then tested the effect of different oxidative stress sources including: paraquat (Pq), hydrogen peroxide (H2O2), and sodium hypochlorite (NaClO). Our preliminary data suggests that paraquat regulates asb differently than other oxidative stress conditions. We also saw that within the Pq condition, there are marked differences between VC transcription and translation, with asb translation dampened despite transcription upregulation. In contrast, both were induced by Pq when starting as spores. In the H2O2 and NaClO conditions, we found that oxidative stress seemed to consistently decrease asb operon transcription and translation, which is unlike the increase observed with Pq. These data indicate a complex relationship between oxidative stress conditions and asb operon regulation that we will study further using gfp fluorescence under different asb promoter constructs.
Candida albicans is the 4th most frequent hospital acquired infection worldwide and is known for targeting already compromised patients. It can grow as yeast cells, pseudohyphae, hyphae, or within a biofilm. The ability to form both hyphae and biofilms has been fundamentally linked to the disease-causing potential of this organism and the way it grows is intimately related to the how it senses and reacts to its surrounding environment. One of the environmental factors known to stimulate filamentation which also happens to be human body temperature is growth at 37 degrees Celsius.Previous experiments studying protein changes in Candida albicans during filamentation at 37 degrees Celsius revealed a subset of proteins showing discordance between the levels of different peptides quantified from within the same protein. Deeper analysis suggested specific regions of these proteins with different quantitative differences when compared to the peptides from other regions of the same protein. The data from the discordance peptides suggested that that proteins may be degraded by a unique mechanism distinct from ubiquitin dependent protein degradation.To further study this observation, we constructed two different strains to study two proteins that represented this discordance, C4_00700c_a and Smt3. The strains expressed Myc-tagged versions of these proteins to facilitate Western Blot analysis. This analysis showed varying levels of the individual protein at 37 and 30 degrees Celsius at time points of 1 and 3 hours, paralleling the observations from previous experiments. An inhibitor of ubiquitin dependent protein degradation was used to test the method by which these proteins were degrading. Our data correlated with the findings from the previous experiments. Analysis of filamentation in these strains demonstrated the importance of one of these proteins to filamentation. Further investigation should help shed light on this mechanism of regulating filament formation.
Indole is an important small signaling molecule produced by the gut commensal microbiota. Several studies have been shown that indole and its derivatives play modulate pathogen behavior through effects on motility, chemotaxis, antibiotic resistance and secretion of virulence factors. Indole regulates pathogenesis of intestinal pathogens Salmonella Typhimurium and EHEC by inhibiting quorum sensing regulatory protein expression. We are analyzing the effects of indole and its derivatives on Campylobacter jejuni, a major agent of gastroenteritis in developing countries. We demonstrate that indole, but not the related tryptophan, strongly reduces motility of C. jejuni in semisolid agar at low concentrations. At higher concentrations, indole significantly reduces growth of the C.jejuni. Indole treatment also induces resistance against oxidative stress from H2O2. We conclude that indole can regulate C.jejuni. motility is an important colonization trait of this microbe.
Single molecule analysis of DNA through molecular combing is a promising method of analyzing replication fork progression and its perturbations. Molecular combing utilizes a vinyl silane coated cover slip which is pulled at a constant rate through the meniscus of solubilized DNA. The technique has been successfully used to study DNA replication in humans. In contrast, this type of analysis of DNA replication in the model organism Saccharomyces cerevisiae (budding yeast) is challenging. We have developed a protocol that aims to make the technique more accessible to researchers using yeast. To visualize DNA, we modified yeast strains, added halogenated thymidines, labeled the DNA with antibodies, and imaged using a fluorescent microscope. During this process, we identified the most effective pH for attachment and stretching of DNA. Additionally, we determined the most effective amount of time for DNA denaturation permitting antibody attachment while maintaining DNA integrity. Minute differences in pH or denaturation time are critical to combing quality.
Fungi and bacteria that colonize dead organic matter are often important components detritivore diets. This is particularly true for freshwater ecosystems where the degree of microbial colonization increases detritus palatability. Detritivore feeding undoubtedly influences microbial communities. Much of what is known regarding consumer effects on freshwater microbial communities is derived from herbivore-autotroph interactions. Studying heterotrophic microbial community responses to feeding pressure has recently become more feasible due to advancements in DNA sequencing technology. The objective of this study was to assess changes in leaf-associated bacterial and fungal communities as they develop with and without feeding pressure by Aedes triseriatus larvae. We exposed early and late stage microbial communities to varying larval densities (0, 15, 20, 30, 40 larvae/g leaf). We sequenced the V4 region of the bacterial/archeal 16s gene and the eukaryotic ITS1 region. Bacterial diversity and evenness increased with community development. Feeding pressure constrained bacterial communities, increasing evenness. In contrast, feeding pressure maintained evenness of fungal communities to pre-larval addition levels.

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