Austin Vanwyk, Anne Spain
Ferris State University
Horizontal Gene Transfer in the Presence of AntisepticsAntibiotic resistance among bacteria is arguably one of the more troublesome topics in medicine today. Genes for resistance can be transferred between bacteria, and it has been found that environmental stressors such as antibiotics can increase the rate at which transfer occurs. However, little research has been done to see what effect antiseptics may have on transfer of resistances. Chlorhexidine was selected as the antiseptic to be used in this research because of its wide spread use in hospitals. Door handles and other regularly touched surfaces from clinical and non-clinical sources were swabbed and then plated, using a type of agar for gram-positive bacteria and another for gram-negative. Ten gram-negative and 20 gram-positive isolates were obtained and assays were done to determine their resistance to six different antibiotics. Four-gram positive isolates had resistance to the antibiotic ceftriaxone. To assess gene transfer, multiple combinations of ceftriaxone resistant gram-positive bacteria and susceptible gram-negative bacteria were grown together with varying levels of chlorhexidine, the hospital antiseptic. The cultures were incubated for 24 hours then plated on agar containing ceftriaxone. The gram-positive isolates were able to grow on the ceftriaxone plates; however no gram-negative isolates were. This indicates that no resistance transfer occurred, though, more trials could yield different results, preliminary data supports that chlorhexidine does not induce transfer of antibiotic resistance genes.
Sandra D. Olenic *1 and Lee Kroos
11Michigan State University, East Lansing, MI 48824
Bacillus subtilis is a Gram-positive bacterium that undergoes sporulation during starvation. During sporulation, ? factors are activated in a controlled spatiotemporal manner that coordinates much of the gene expression. Activation of Pro-?K is controlled by regulated intramembrane proteolysis (RIP). RIP of Pro-?K is catalyzed by the intramembrane metalloprotease SpoIVFB and removes the prosequence, releasing active ?K into the mother cell cytoplasm. RIP is a poorly understood process that is conserved in both eukaryotes and prokaryotes. In order to study RIP of Pro-?K, Escherichia coli was engineered to coexpress SpoIVFB with Pro-?K and two inhibitory proteins, BofA and SpoIVFA. Coexpression of SpoIVFB and Pro-?K in E. coli allowed accurate and abundant RIP. The additional expression of both BofA and SpoIVFA from the same plasmid resulted in total inhibition of RIP. An alignment of 70 BofA homologs shows conservation is only present in the second transmembrane region and in the C-terminal end, suggesting that these parts could be crucial for inhibitory function. Deletions in the C-terminal parts of either BofA or SpoIVFA greatly hinder inhibition. Half of the alanine substitutions to conserved residues in BofA hindered inhibition in the E. coli model. In order to confirm the E. coli model, we created B. subtilis strains with three BofA variants (N48A, N61A, and T64A), and inhibition was also hindered. Preliminary data from affinity purification assays suggests that BofA and SpoIVFA interact with SpoIVFB and Pro-?K. This data suggests that all four proteins form a complex that prevents Pro-?K processing. Functional Cys-less derivatives of SpoIVFA and Pro-?K with mono-Cys versions of SpoIVFB and BofA were created for disulfide crosslinking studies. Crosslinks formed between C46 in the TMS of BofA to C44 and C135 near the active site of SpoIVFB. These results begin to elucidate a mechanism of intramembrane metalloprotease inhibition that could have implications for related RIP events that govern bacterial pathogenesis and other processes impacting human health.
Subodh Samrat and Haidong Gu
Department of Biological Science, Wayne State University
Infected cell protein 0 (ICP0) of herpes simplex virus 1(HSV-1) is an immediate early protein containing an RING-type E3 ubiquitin ligase, which targets multiple cellular restrictive factors for proteosomal degradation. ICP0 contains a canonical nuclear localization sequence (NLS) and it executes the main counter-defense actions in the nucleus. Interestingly, late in HSV-1 infection ICP0 is found solely in the cytoplasm, suggesting that a nucleus-to-cytoplasm translocation occurs after its nuclear functions are completed. In this report, we found that the C-terminal 35 amino acids of ICP0 contains a cis-element required for ICP0 cytoplasmic translocation in late infection. Deletion of the 35 amino acids confined ICP0 inside the nucleus throughout the infection whereas deletion of the last 18 amino acids led to a partial translocation. However, restitution of the last 35 residues back to an ICP0 mutant lacking the C-terminal 107 amino acids did not bring the mutant ICP0 into the cytoplasm late in infection, suggesting that the C-terminus 35 amino acids are essential but not sufficient for ICP0 nuclear to cytoplasmic translocation. In addition, we found that ICP0 bearing an inactive RING finger was completely restricted within the nucleus of nonpermissive HEL cells, whereas in the permissive U2OS cells it was successfully translocated to the cytoplasm. These results suggest that the RING finger domain per se is not required for the translocation process, but the viral ability to proceed in DNA replication and late protein expression is necessary for the translocation. Besides late viral proteins, certain cellular proteins also play as trans factors to stimulate the nucleus to cytoplasmic translocation of ICP0.
Ribose Metabolism in Bacteroides thetaiotaomicron Plays an Important Role In Vivo in a Diet-Dependent Manner, and may Represent a Nutrient Niche Exploited by Commensals and Pathogens
Author Block: Robert W.P. Glowacki*1, Yunus E. Tuncil2, Nicholas A. Pudlo1, Eric C. Martens1
1Univ. of Michigan, Ann Arbor, MI, 2Purdue Univ., West Layfette, IN
Abstract Body:Gut bacteria of the prominent Bacteroidetes phylum devote large portions of their genome towards host and dietary carbohydrate degradation through partially homologous gene clusters, Polysaccharide Utilization Loci (PULs). Here, we describe a new PUL for metabolism of ribose-containing substrates, the ribose utilization system, rus. Previous data shows the rus of Bacteroides thetaiotaomicron (Bt) to be upregulated in vivo in absence of dietary fiber, suggesting rus responds to a host-derived nutrient. We used Bt as a model organism to examine function and mechanism of ribose utilization via rus. In vivo studies used 6-8 week old germfree Swiss-Webster mice maintained on a standard lab diet or custom dietary fiber-free diet. As measured by qPCR, growth on ribose induced expression of the Bt rus and homologous loci of several other species. Our results support the hypothesis that Bt metabolizes ribose-containing compounds through rus-encoded functions. Deletion of a putative regulator or one or both of two ribokinases, completely eliminates growth, while deletion of other genes has little effect. Interestingly, to metabolize nucleosides and ADP-ribose, a low amount of inducing ribose is required. In vivo, the ?rus strain is outcompeted by wild-type Bt, only in mice fed the standard high-fiber diet. A similar defect was observed in mice fed the fiber-free diet supplemented with water containing 1% ribose, but not RNA or nucleosides. Comparative genomic analysis, growth and gene expression studies, revealed the ability to catabolize ribose is partially, but broadly represented in the Bacteroidetes and reveal several rus loci with variable enzymatic potential. We conclude ability to catabolize ribose within Bacteroidetes is conferred via the rus PUL. Further, rus activation allows access to otherwise inaccessible nutrients such as nucleosides, although the critical molecules foraged in vivo are unknown. Ability to catabolize ribose in vivo confers a competitive advantage, and ribose may represent a nutrient niche exploited by both commensals and pathogens. This abstract was previously presented at the 2017 ASM general meeting.
CsgA dependent activation of FruA is critical for the expressions of the sporulation genes during commitment in Myxocccus xanthus
Shreya Saha1, Pintu Patra2, Oleg Igoshin2, and Lee Kroos1
1Michigan State University, East Lansing, MI 488242Rice University, Houston, TX 77005
Upon starvation M. xanthus cells aggregate and mature into fruiting bodies as some cells differentiate to spores. MrpC is a starvation-responsive transcription factor, whereas another transcription factor, FruA, responds to cell positional information conveyed by C-signaling. FruA activity has been proposed to be regulated post-translationally by csgA encoded C-signal.FruA and MrpC cooperatively regulate transcription of the dev operon. DevS negatively autoregulates dev transcription and DevI overexpression inhibits spore formation. The exo and nfs operon products are outputs of the network that are critical for sporulation. Previous work has determined 24-30 h poststarvation (PS) to be the critical period for commitment to spore formation. We are taking a systematic, quantitative approach to elucidate the network dynamics in terms of gene expression before and during the commitment period (i.e., 18-30 h PS). The levels of dev, nfs, and exo transcripts increase 3-, 4-, and 20-fold, respectively, coinciding with spore formation. Two other genes that have been shown to be critical for spore formation, Mxan_3259 and Mxan_5372, increase 75- and 4-fold, respectively, during commitment.The csgA mutant shows a 3-fold decrease in FruA protein and 20-fold reduction in dev transcript level compared to wild type during commitment. Notably, the 20-fold decrease in dev transcripts cannot be accounted for by the 3-fold decrease of FruA, neither by the instability of dev transcripts in the csgA mutant. Computational modeling shows that cooperative binding of MrpC and FruA cannot account for the decrease in dev. Together, the results are consistent with the model that C-signaling activates FruA, which in turn activates dev transcription. Our result shows that nfs, exo, and Mxan_3259 transcript levels are elevated at 18 h PS in a fruA mutant as compared with wild type. Hence, FruA negatively regulates these genes early, whereas activates nfs and exo later during commitment, but not Mxan_3259. Further, we find that exo and Mxan_3259 transcripts are significantly higher in an mrpC mutant than the fruA mutant at 18 h PS, suggesting that MrpC exerts negative regulation independent of FruA. In contrast, MrpC appears to positively regulate nfs independent of FruA. Finally, both dev and Mxan_5372 transcripts are low in both mrpC and fruA mutants as compared with wild type. While expression of these two genes is consistent with the observation from the mathematical model that C-signaling activates FruA which in turn activates transcription, our results suggest MrpC together with both unactivated and activated FruA regulates expression of the sporulation genes during commitment.
Role of ATP in Regulated Intramembrane Proteolysis of Pro-?K by SpoIVFB during Bacillus subtilis sporulation
Daniel Parrell, Sabyasachi Halder, Michael Feig and Lee Kroos
Departments of Microbiology and Molecular Genetics, and Biochemistry and Molecular Biology, Michigan State University, East Lansing, Michigan, USA
Bacillus subtilis endospore formation provides an excellent model system to study signaling and gene regulatory mechanisms. During sporulation asymmetric cell division and an engulfment process produce a mother cell and a forespore. After the forespore becomes engulfed, a signaling pathway from the forespore relieves inhibition of the intramembrane metalloprotease SpoIVFB, by two other proteins forming a complex with SpoIVFB in the outer forespore membrane. Active SpoIVFB cleaves membrane-associated Pro-?K, releasing it into the mother cell to direct cell-type-specific transcription. Intramembrane metalloproteases like SpoIVFB are broadly conserved and control many important signaling pathways, however a better understanding of their regulatory mechanisms is needed. SpoIVFB has a CBS domain that binds ATP and is required for SpoIVFB to cleave Pro-?K in vitro. A number of amino acid substitutions for CBS domain residues have been made. The effects of these changes will be described in vivo using Pro-?K cleavage and sporulation assays, as well as in vitro using biochemical assays. ATP binding to the CBS domain has been measured using an assay based on binding of a fluorescent ATP analog (TNP-ATP). The effects of amino acid substitutions on ATP binding will be presented. We also present an ATP-binding assay that utilizes proximity of SpoIVFB and Pro-?K residues near the active site and different length chemical cross-linkers to demonstrate a conformational change of the complex upon ATP binding. CBS domains commonly regulate activity of proteins via conformational changes. A model for an ATP-induced conformational change in SpoIVFB that positions Pro-?K for cleavage will be discussed in the context of a structural homology model. By studying regulated intramembrane proteolysis in B. subtilis, we hope to elucidate fundamental principles of how these broadly conserved proteins function in signaling pathways that govern sporulation and many other important processes.Also presented at ASM Microbe 2017 and the 2017 Molecular Genetics of Bacteria and Phages meeting.
Characterizing autoregulation of a cell fate determination protein in Myxococcus xanthus
Patrick T. McLaughlin, Vidhi Bhardwaj, Penelope I. Higgs
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 and development, 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 competes with the enhancer binding complex required for transcriptional activation. Additionally, MrpC-mediate negative autoregulation plays a more significant role in regulating the timing of development, than in regulating cell fate segregation.
Microbiological Quality and Antibiotic Resistance of Shrimp Retailed in Rochester, MI
Mamo, Andrina, V*, Walia, Satish
Oakland University, Rochester, MI 48309
In 2009, the United States imported 589,670 metric tons of farmed shrimp worth more than $6 billion from Asia. Antibiotics are used in the shrimp ponds to stimulate growth and to retard the incidence of diseases caused by overcrowded, factory farm conditions. The indiscriminate use of these antibiotics may select bacteria resistant to multiple antibiotics, and such bacteria may transfer their antibiotic resistance determinants to pathogenic bacteria. The aim of the study was to investigate the prevalence and the antimicrobial resistance patterns of gram-negative bacteria isolated from shrimp retailed in Rochester, Michigan. In this preliminary study, a total of 16 shrimp samples were collected from retail grocery stores located in Rochester, MI. Out of 16 samples, 63% showed positive growth for Vibrio spp. as indicated by growth on TCBS agar. In this study eighty-three gram-negative bacterial isolates were tested for antibiotic susceptibility against six antibiotics (gentamicin [GEM], tetracycline [TET], ampicillin [AMP], ciprofloxacin [CIP], chloramphenicol [CHL], and ceftazidime [CEF]) by Kirby-Bauer method of disk diffusion. Bacteria showed high rates of resistance against ampicillin (58%), while a moderate rate was observed for gentamicin (35%) and intermediate resistance towards tetracycline (20%) and ciprofloxacin (20%). A low resistance rate was recorded against chloramphenicol (11%). Ceftazidime clavulanic acid resistance was found in 25% of the samples. Seven percent of the isolates were resistant to 4 antibiotics, displaying 3 different patterns. 22 out of 83 samples (27%) were resistant to a combination of 3 or more antibiotics. Only one isolate, AM 100, was resistant to 5 antibiotics. (GEM, TET, AMP, CHL, CEF). The heterotrophic bacterial count on Tryptic Soy Agar plates were ranging from 7.8 x 102 CFU/g to 4.18 x 103 CFU/g, meeting the microbiological quality standards for human consumption. The results of this study revealed the ubiquitous nature of Vibrio spp. in shrimp at retail. To reduce the potential risk of Vibrio infections due to handling or consumption of undercooked seafood, good manufacturing practice as well as safe handling and processing should be encouraged.
Staphylococcus aureus Resists Fatty Acid Synthase Inhibitors via the Degradation of Human Low-density Lipoprotein.
Phillip C. Delekta1*, John Shook1, Martha H. Mulks1 & Neal D. Hammer1
1Department of Microbiology & Molecular Genetics, Michigan State University, East Lansing, MI 48824.
The Gram-positive pathogen, Staphylococcus aureus is a major threat to global health because of its ability to colonize many vertebrate organs and the increasing prevalence of antibiotic resistance. Treatment of staphylococcal disease is challenging due to the numerous strategies S. aureus utilizes to resist antimicrobials. For example, S. aureus and many other Gram-positive pathogens resist small molecule inhibition of fatty acid synthesis II (FASII) by incorporating exogenous fatty acids from the host environment. Notably, the source(s) of host-derived fatty acids and the mechanism by which S. aureus incorporates exogenous fatty acids remains poorly defined. Low-density lipoprotein (LDL) particles are the major carriers of lipids in vertebrates and function to deliver lipids throughout the host. Composed partly of cholesterol esters and triglycerides, LDL particles represent a potentially rich source of exogenous fatty acids for S. aureus to resist the effects of FASII inhibitors. We demonstrate that S. aureus counters FASII inhibition when grown in the presence of purified human LDL particles. Loss of lipase activity leads to increased sensitivity to the FASII inhibitor, triclosan. Similarly, lipase-deficient clinical isolates also demonstrate greater sensitivity to triclosan compared to lipase-positive isolates cultured in the presence of LDL particles. These results support a model whereby S. aureus lipase activity liberates fatty acids from host LDLs, an abundant in vivo source of lipids.
Myxococcus xanthus Responds Ultrasensitively to the Addition of Nutrients during DevelopmentY Hoang* and Lee Kroos
Department of Biochemistry and Molecular Biology, Michigan State University, East Lansing, MI 48824 USA
Upon depletion of nutrients, Myxococcus xanthus cells begin to aggregate to form mounds. The differentiation of rod-shaped cells inside mounds into spherical stress-resistant spores results in the maturation of fruiting bodies. Some cells can persist outside fruiting bodies as peripheral rods while the majority of the population undergoes lysis. The development can be perturbed by the addition of nutrients (CTTYE medium) before the critical period of commitment to form spores. Nutrient addition triggers the rapid degradation of MrpC, a key transcription factor in the sporulation network (1). To further investigate the effect of nutrient addition on development, a two-fold dilution series of CTTYE medium was added to starving cells at 18 h, and sonication-resistant spores were measured at 30, 36, and 42 h. M. xanthus exhibited an ultrasensitive response indicated by a steep increase in spore numbers at 12.5% CTTYE as compared with 25%. The molecular response to these two nutrient concentrations was compared by systematically measuring transcript and protein levels of sporulation network components. While the mrpC transcript, fruA transcript, and FruA protein levels are similar in response to 12.5 and 25% CTTYE addition, the MrpC protein levels correlated with the number of sonication-resistant spores. The MrpC protein levels decreased significantly by one hour post-addition of nutrients, but the levels rebounded more in the presence of 12.5% CTTYE than 25%. The difference in MrpC levels was greatest at 27 h, when fruiting bodies also showed more disaggregation of cells in response to 25% CTTYE addition than to 12.5%. The critical period for commitment to sporulation under normal conditions is between 24 and 30 h post-starvation (1). Treatment of cells with chloramphenicol, which is an inhibitor of protein synthesis, showed that turnover of MrpC at 27 h is similar after addition of 12.5% or 25% CTTYE at 18 h. Altogether, the results suggest that the ultrasensitive response to nutrient addition during development may be regulated at the level of MrpC translation. Although the MrpC levels and the numbers of sonication-resistant spores are different in response to 12.5% and 25% CTTYE, the transcript levels of downstream components of the sporulation network (dev, exo, nfs, MXAN_5372, MXAN_3259) are similar. These genes are known to be important for the maturation of spores, so we will measure the numbers of heat- and sonication-resistant spore capable of germination. Surprisingly, the transcript levels of these genes are high at 30 h, after adding 100% CTTYE at 18 h, but no spores formed. Perhaps these genes aid recovery during resumption of growth.1. Rajagopalan R, Kroos L. 2014. Nutrient-regulated proteolysis of MrpC halts expression of genes important for commitment to sporulation during Myxococcus xanthus development. J. Bacteriol. 196:2736-2747.
Proximity to the promoter and terminator regions regulates the transcription enhancement potential of an intron
Neha Agarwal, Katherine Dwyer, Alisa Gega, Athar Ansari
Department of Biological Sciences, Wayne State University, Detroit, MI 48202
One of the evolutionarily conserved features of introns is their ability to enhance expression of the genes that harbor them. Introns have been shown to regulate gene expression at the transcription as well as post-transcription level. The general perception is that the promoter-proximal intron is most efficient in enhancing gene expression and the effect diminishes with the increase in distance from the promoter. Here we show that the intron regains its positive influence on gene expression with the increase in proximity to the terminator region. To get an insight into the role of position of an intron on gene expression we inserted ACT1 intron into four different positions within INO1 gene. RT-PCR analysis revealed that the transcription of INO1 was maximum in the construct with a promoter-proximal intron and decreased with the increase in distance of the intron from the promoter. The transcription activation potential, however, was partially restored when the intron was placed in the vicinity of the terminator region. Similar results were obtained with IMD4 gene. We have previously demonstrated that the promoter-proximal intron stimulates transcription by affecting promoter directionality through looping-mediated recruitment of termination factors in the vicinity of the promoter region. Here we show that the terminator-proximal intron affected transcription in two ways; first through regulation of termination step of transcription, and second through the enhancement of transcription directionality. On the basis of these results we propose that the proximity to both the promoter and the terminator regions affects the transcription regulatory potential of an intron in budding yeast.
An Investigation into the Potentially Novel Role of Rat1p in the RNA Polymerase II Mediated Transcription Cycle in Budding Yeast
Zuzer Dhoondia* and Athar AnsariWayne State University, Detroit, MI 48202
Transcription mediated by RNAPII yields mRNA that is processed co-transcriptionally by 5? end capping, splicing, and 3? end cleavage/polyadenylation. Rat1p is a chromatin-bound factor and is known to cross-link to the terminator and promoter regions of a gene. We have discovered that Rat1p also cross-links to the intronic region of the gene. Additionally, using RT-PCR analysis, we found accumulation of unspliced mRNA for the selected intron-containing genes in the absence of functionally active Rat1 in the cell in the temperature-sensitive mutant or anchor away strain. Complementation studies revealed that exoribonuclease activity of Rat1 was important for the accumulation of unspliced transcripts. The unspliced transcripts were observed when we analyzed nascent transcription using transcription run-on (TRO) assay. These results suggest a role of Rat1p in either co-transcriptional splicing or co-transcriptional degradation of pre-mRNA. Deletion of Rat1p-interacting partners, Rai1 and Rtt103, however, had no effect on increased levels of unspliced mRNA. We also found that the termination function of Rat1 was not responsible for the increased accumulation of unspliced transcripts. We have purified the Rat1p complex using Tandem Affinity Purification (TAP) approach to look for the presence of any splicing factors. This may help to explain if Rat1p is playing a role in splicing or if it is simply resulting in cotranscriptional degradation of unspliced transcripts. We are also planning to perform an in vitro splicing assay in the presence and absence of Rat1p to further probe the role of Rat1p, if any, in splicing in budding yeast. This will help us in understanding the broader role of Rat1p in transcription and co-transcriptional RNA processing.
Gram Stain DiscrepanciesAguilar, Lisa N* - Detroit, MI 48202
Agotesku, Adam - Detroit, MI 48202
Gram staining is considered a “High Complexity Procedure” by the Clinical Laboratory Improvement Amendments (CLIA) program. Although the goal is to correlate the results of the Gram stain with culture results 100% of the time, the complexity of this procedure makes this a tough goal to reach.We compared the Gram stain results to culture results over a 2 week period. We found that 41% of the total positive cultures did not have an organism detected on the Gram stain upon preliminary examination. In addition, 38% of the positive cultures with no organisms detected on the original smear yielded moderate to heavy bacterial growth, which could indicate a problem with Gram stain preparation and/or interpretation. This study provided us with comprehensive data regarding our Gram stain processing and reading methods, which now allows for target goals to be set for future improvements.
Mitochondrial DNA Damage And Dysfunction Cause Increase In Nuclear Genome Mutation Rate But Not Oxidative Stress Specific
Duong Nguyen*, Allison Macielog, Weilong Hao*
PresenterFor all author's institution/affiliation:Wayne State University Detroit, MI 48202
Mitochondrial DNA damage and dysfunction can lead to nuclear genome instability and cell death due to strong mitochondrial and nuclear genomic interaction. Yeast cells lacking mitochondrial DNA (mtDNA) have been found to be more sensitive to oxidative stress presumably due to defect in an energy-requiring process in the nucleus for detoxification of reactive oxygen species (ROS). Yet, it has not been empirically tested whether ROS damage is the direct cause for nuclear DNA mutations in mitochondrial dysfunction. In this study, we performed accumulated mutation experiment on two Saccharomyces cerevisiae strains (one mtDNA-containing or rho+, and one mtDNA-lacking or rho0) through 100 single-cell bottlenecks. Our results show that the rho0 strain exhibits a genome-wide mutation rate 1.36x higher than the rho+ strain. We then carefully examined and compared their mutation spectra, as ROS damage results in 8-oxoguanine (8-oxoG) and ultimately leads to G:C to T:A transversions. However, no significant difference was evident between the mutation spectra of the rho0 and rho+ strains. Instead, mutation rates in the rho0 strain have increased for all substitution types. Thus, ROS damage is unlikely the direct cause of nuclear genome mutation in mtDNA damage and dysfunction. Our study not only highlights the impact of mtDNA defect on nuclear genome stability, but also opens the door for comprehensive studies on the molecular and genetic mechanisms underlying both mtDNA and nuclear DNA mutations.
Analysis of +1 Frameshift mutation in Yeast Mitochondrial Genomes
Nan Zhang* and Weilong HaoDepartment of Biological Sciences, Wayne State University, Detroit, Michigan, 48202
Mitochondrial DNA (mtDNA) encodes core subunits of enzymes involved in cell respiration. Mutations of mtDNA encoded genes would interrupt oxidative stress resistance and respiratory growth in yeast. It has been previously reported that mtDNA-encoded genes in Kazachstania servazzii contain single nucleotide insertions, which could presumably cause +1 translational frameshift. Unfortunately, the origin of +1 translational frameshift and its underlying mechanisms still remain largely elusive. In this study, we analyzed mitochondrial genomes in the Saccharomyces family, to which Kazachstania belongs. Among ten species we analyzed, +1 frameshift mutation is sporadically distributed in three mtDNA-encoded genes in seven species. The +1 frameshift mutations can potentially result in new amino residues, protein length elongation and contraction. Compared to yeast species without +1 frameshift mutation, the ones with +1 frameshift mutations produce comparable amount of protein products. Surprisingly, the yeast species with frameshift insertions show better respiratory growth than ones without frameshift insertion. This suggests +1 frameshift insertion is a rapid evolutionary force that can have significant consequences on cellular function. Our findings set the stage for developing more sophisticated analyses on the evolutionary and genetic mechanisms of frameshift insertions.
Phosphorylation of the Crp/Fnr Homolog, MrpC, by Eukaryotic-like Ser/Thr Kinases, Regulates Sporulation in Myxococcus xanthus
Brooke Feeley*, Vidhi Bhardwaj, Penelope I. Higgs
Myxococcus xanthus are Gram negative, soil dwelling bacteria that, upon nutrient depletion, enter a multicellular developmental program in which cells first aggregate into multicellular fruiting bodies and then differentiate into environmentally resistant spores. MrpC, a Crp/Fnr family transcriptional regulator, plays a key role in directing both aggregation and sporulation aspects of the developmental program. No ligand has been identified for MrpC, but it has been previously proposed that MrpC can be deactivated by phosphorylation from the Pkn8/Pkn14 eukaryotic-like Ser/Thr kinase cascade. We have determined that MrpC has a unique N-terminal extension bearing a putative TTSS phosphorylation motif which is essential for MrpC activity. An mrpC mutant bearing a substitution of the TTSS motif to AAAA (mrpCAAAA) fails to either aggregate or sporulate suggesting that phosphorylation of MrpC leads to its activation. We have shown that in vitro, Pkn14 directly phosphorylates wildtype MrpC, but not MrpCAAAA. While kinase-dead pkn14 (pkn14K48N) or pkn8 (pknK116N) single mutants develop normally, the double kinase-dead mutant (pkn14K48N pknK116N) aggregates normally, but only sporulates at approximately 35% of wildtype. Together, these data suggest that Pkn14-P and Pkn8-P act redundantly specifically on the sporulation pathway, and that other unidentified kinases influence MrpC to activate aggregation. We are currently investigating the effect of Pkn14 and Pkn8 on the phosphorylation status of MrpC in vivo, as well as how phosphorylated MrpC induces sporulation and aggregation. Finally, our data suggest that M. xanthus has adapted regulation of a widely utilized bacterial transcription factor to the large network of eukaryotic-like kinases, in order to regulate its multicellular behavior—a novel method of regulation of a Crp/Fnr homolog.
Rhizobia: contributions to diversity in prairie restorations
Allen, Jamie*; McBride, Justin*; Price, Paul; Grman, Emily; and Bauer, Jonathan
Biology Department, Eastern Michigan University, Ypsilanti, MI
In a world where climates and physical environments are rapidly changing due to human influence, scientists are trying to prevent the extinction of key species in various environments. In particular, Michigan prairies house many different plant and animal species that are at risk of extinction. To help reverse this trend, researchers have been actively attempting to transform former agricultural fields back into prairies by sowing seed for native prairie plant species, but many of these species fail to thrive in these prairie restorations. For example, many species of prairie legumes fail to properly establish during restorations. If we are able to more fully understand why these species fail to establish, we will be able to more effectively restore agricultural fields back to host the same biodiversity and provide the same ecosystem functions/services as the remnant (native) prairies that have never been disturbed. One of the leading hypotheses as to why some of these species fail to establish focuses on the effects that farming has on the soil microbial composition. To restore prairie ecosystems, it is important to understand how rhizobia affect the diversity of different species of legumes. To determine if the growth and nodulation of legumes depends on differences in rhizobia from restored versus remnant prairies, we placed three different species of legumes in 16 different soils: ten restored sites (age range 2-11 years) and six remnant sites. We found that legumes grown in soil from remnant prairies have greater biomass and more nodules than legumes grown in soil from restored prairies. However, there was no significant difference in legume biomass among restored prairies of different ages, indicating that older restored prairies might still lack the microbial communities needed to become as biodiverse as remnant prairies.
In Search of New Antimicrobials: New Methods For Mining Antibiotic-Producing Bacteria Kamal, Asad,* Steltz, Megan,* Shoukat, Maira,* McAlister-Raeburn, Madelaine, Price, Paul
Eastern Michigan University, Ypsilanti, MI
Abstract: Antibiotic resistance is quickly becoming one of the biggest healthcare issues facing humanity, as many of our existing antibiotics are less effective against a wide range of clinically relevant pathogens. There have been recent reports of bacterial pathogens that are completely resistant to all known antibiotics. Therefore there is an urgent need to develop or discover new classes of antibiotics. Many of our current classes of antibiotics were originally derived from soil microorganisms. However, soil mining for natural products that have antimicrobial activity, commonly referred to as antibiotics, has largely decreased over the past thirty years because the frequency of novel discoveries decreased substantially. Yet, new classes of antibiotics are still being discovered from soil dwelling bacteria. This project aimed to screen Michigan soil samples for bacteria that could potentially produce new/novel antibiotics. We used a modified Waksman method (competing soil microorganisms directly with bacterial pathogens on agar plates) to identify potential antibiotic-producing bacteria. Briefly, a soil sample was retrieved and serially diluted to obtain 200-300 bacteria per plate. Alternatively, diluted soil samples were combined with D-alanine auxotrophic pathogenic bacteria and plated on media containing D-alanine. Individual bacteria were and tested against a series of pathogenic bacteria and/or closely related pathogenic relatives. Bacteria that produced zones of clearing around the pathogenic bacteria were further characterized, noting the size of the zone of inhibition, and identified using 16S rRNA sequencing and BLAST searches against the 16S rRNA database. In total, we identified 59 unique bacterial isolates that produce antimicrobial compounds including 39 that were effective against methicillin-resistant Staphylococcus aureus. We plan to purify and study these antimicrobial compounds to determine their potential usefulness as clinically relevant antibiotics.
John S. Lee*1, Han-Shin Kim2, Jeffrey P. Nader1 and Yann S. Dufour1
1Michigan State University, East Lansing, MI 48824; 2Korea University, Seoul, 02841 South Korea
Background: Vibrio cholerae coordinates its motile and biofilm-forming lifestyles to establish infection in the human host. The second messenger, c-di-GMP, is central to the regulation of this phenotypic switching. However, previous studies were conducted on average population responses, masking the effect of single-cell phenotypes. A clonal population exhibits the stochastic generation of different phenotypes, termed phenotypic diversity, which can be beneficial as a bet-hedging or division of labor strategy. Phenotypic diversity in V. cholerae may play an important role during infection, but the control of phenotypic diversity remains largely uncharacterized. Method: We used video-microscopy to characterize single-cell motile behaviors under different growth conditions. Intracellular concentration of c-di-GMP was controlled using inducible di-guanylate cyclase (DGC) and phosphodiesterase (PDE). Phenotypic distribution in response to direct manipulation of c-di-GMP levels and to perturbation of signaling pathways that control phenotypic switching was measured as diffusion coefficients calculated from individual trajectories. Results: Dysregulation of transcriptional factors known to bind c-di-GMP, VpsR, VpsT, and FlrA, had little effect on population phenotypic distribution. On the other hand, TfoY, whose function remains unknown, promotes motility. The Plz protein family showed a greater effect on phenotypic distribution, suggesting the control of phenotypic diversity is also achieved at the post-transcriptional level. Conclusion: Clonal populations of V. cholerae are phenotypically diverse, and the c-di-GMP levels mediate changes in the proportion of motile and non-motile cells. No single pathway is directed to the regulation of phenotypic diversity, but instead, multiple regulatory pathways in the c-di-GMP network appear to be involved. Figure: Characterizing phenotypic diversity in V. cholerae isogenic populations. (A) Distributions of single-cell motile phenotypes for different growth phases. (B) Probability distribution changes upon PDE induction during exponential phase. (C) Probability distribution changes upon DGC induction during stationary phase.
The TodK histidine kinase and its role in cell fate segregation during the M. xanthus developmental program.
Maike Glaser1,2, Chris Mataczynski1, and Penelope I. Higgs1
1Dept. of Biological Sciences, Wayne State University, Detroit, MI, USA2
Quantifying the Genome-Wide Protein Synthesis Economy of C. crescentusAretakis, James R.* Schrader, Jared M.
Department of Biological Sciences, Wayne State University, Detroit, MI, 48202
Protein synthesis is one of the most energetically costly processes for a bacterial cell, yet energy waste can be minimized by careful control of protein production from each gene for optimal cell growth. In C. crescentus, a model for the bacterial cell cycle, over 900 mRNAs are cell cycle-regulated and our lab recently found that 484 of these are additionally controlled at the translational level. However, we do not have a systematic understanding of the relative importance of transcription, mRNA decay, and translation on the gene regulatory network controlling the cell cycle. Here we present a genome-wide approach to quantify the relative impact of the sub-steps of gene expression (including transcription, mRNA turnover, and translation) that control protein production in C. crescentus. While genome-wide transcription rates cannot be measured directly, we can measure steady state mRNA levels and mRNA degradation using RNA-seq, allowing us to calculate the rate of transcription. Translation rates were also measured by using ribosome profiling, a genome wide method that is based on the sequencing of ribosomes protected mRNA fragments. We have performed these three global measurements in log-phase cultures of C. crescentus and are now focused on analyzing these measurements and incorporating them into a model representing the kinetic parameters of protein synthesis rates across the genome. Interestingly, we noted that a vitamin B12 receptor is one of the most highly synthesized proteins in C. cresentus, suggesting that the cells may be starving for this cofactor. To test if cells were starved for vitamin B12 we supplemented the medium with vitamin B12 and observed increases in growth rates. Genetic disruption of the vitamin B12 receptor gene no longer showed enhanced growth, suggesting that the B12 receptor is required for B12 enhanced growth. Therefore, the kinetic analysis of protein synthesis will likely yield important insights into C. crescentus physiology and cell cycle regulation.
Response of microbial communities to grazing by mosquito larvae
B. C. Norman* and E. D. WalkerDepartment of Microbiology and Molecular Genetics, Michigan State University, East Lansing MI 48824
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.
Effects of Commensal Clostridium sporogenesis derived Indole-3-propionic acid on Campylobacter jejuni
Authors: Ritam Sinha*, Victor J. DiRita
Michigan State University, East Lansing, MI 48824.
Gut metabolites like indole and its derivatives are considered as a important signaling molecule in the gut which produce by the commensal gut microbiota. Several recent studies have shown that indole and its derivatives regulate bacterial bacterial motility, chemotaxis, antibiotic resistance and secretion of virulence factors. In this study, we are focusing to study the effects of indole and its derivatives on Campylobacter jejuni, a main causative agent of gastroenteritis in developed and developing countries. While being a pathogen in humans, C. jejuni is a normal, benign inhabitant of the chicken gastrointestinal tract, and we are investigating mechanisms that differentially affect its behavior in pathogenic vs. commensal outcomes. Our initial studies showed that high concentration of indole significantly reduce bacterial growth. Inhibition of C. jejuni growth could be overcome in the presence of formate, an intestinal short chain fatty acid. At sub-inhibitory concentrations, indole, and its derivatives such as indole-3-propionic acid, reduces motility of C.jejuni in semisolid agar. Co-culturing of C. jejuni with the gut commensal Clostridium sporogenesis, which produces indole propionic acid, also reduces its motility in semisolid agar. From this study we conclude indole (and its derivatives) from gut commensal microbes may play a regulatory role on C.jejuni motility. We are extending these in vitro experiments to examine if production of indole and derivatives in vivo by the gut commensals modulates C. jejuni colonization and pathogenicity in a host-dependent manner.
Retinal Innate Response to Candida albicans in Exogenous Fungal Endophthalmitis
Bruce G. Rottmann* 1, Pawan Kumar Singh1,2, and Ashok Kumar1,2,31
Department of Anatomy and Cell Biology, Wayne State University School of Medicine, Detroit, MI, USA 482012
Deparmtnet of Ophthalmology, Kresge Eye Institute, Wayne State University School of Medicine, Detroit, MI, USA 482013
Deparmtnet of Immunology and Microbiology, Wayne State University School of Medicine, Detroit, MI, USA 48201
Purpose: Among the fungal pathogens, Candida (C.) albicans is a major cause of fungal endophthalmitis. The purpose of this study is to develop a mouse model of C. albicans endophthalmitis and examine the innate immune response in this disease.Methods: Exogenous C. albicans endophthalmitis was experimentally induced the eyes of C57BL/6 and BALB/c mice by intravitreal injection of a standard laboratory strain of C. albicans (ATCC SC5317). Disease progression was monitored by daily slit-lamp exam and assigning clinical scores. Following euthanasia, fungal burden was estimated via a serial dilution method. The level of pro-inflammatory cytokines and chemokines was determined by qRT-PCR and ELISA. Neutrophil infiltration was determined by flow cytometry and immunostaining. The extent of retinal tissue damage was assessed by H&E and TUNEL staining. Retinal function was measured by electroretinography (ERG).Results: Our dose response study revealed that 6,500 CFU of C. albicans induced reproducible endophthalmitis in both mouse strains. Our time-course experiments revealed that fungal burden is increased by 24 and 48h post-infection, followed by a decline at 72 and 96h. This trend was more pronounced for the BALB/c mice than the C57BL/B6 mice. The levels of pro-inflammatory cytokines followed a similar trend, peaking at 24/48h and declining by 72/96h in both mouse strains. Unexpectedly, PMN infiltration remained high in C57BL/6 mice, even at 96h post-infection, while decreased in BALB/c mice following a peak at 48h. ERG analysis revealed a time-dependent decline in retinal function, which coincided with increased retinal tissue damage, as evidenced by increased retinal detachment/folding and more TUNEL positive cells.Conclusions: Our results indicate that C. albicans causes endophthalmitis and elicits a significant inflammatory response in the eyes of both C57BL/6 and BALB/c mice. Furthermore, there appears to be no discernible difference in disease outcome between these two models apart from the degree of PMN infiltration. The establishment of this new mouse model will enable us to evaluate therapeutic efficacy of antifungal drugs.Note: I presented this poster at the American Society of Microbiology (ASM) Microbe 2017.
Mutations in ALIX-binding Domains of HCMV pUL103, a Multifunctional, Herpesviridae-Conserved Tegument Protein, Greatly Impair Virus Replication
Anderson, Ashley N.*, Ortiz, Daniel A., Glassbrook, James E., and Pellett, Philip E.
Wayne State University School of Medicine, Detroit, MI, 48201
Human cytomegalovirus (HCMV) is an enveloped, single segment, double-stranded DNA betaherpesvirus, and a global threat to human health. HCMV is present in over 50% of the adult population and causes disease in immunocompromised and naïve populations, consisting of transplant recipients, HIV patients, fetuses, and infants. During infection, HCMV remodels the cellular secretory machinery (Golgi, endosomes) to form the cytoplasmic virion assembly compartment (cVAC). New virus particles are assembled within the cVAC and are then trafficked out of the cell. The cVAC is essential for efficient HCMV viral replication. We previously identified pUL103 as necessary for cVAC biogenesis, as well as cell-to-cell spread and virion maturation. pUL103 is an HCMV tegument (layer of proteins and mRNA between the envelope and capsid) protein that is conserved across the Herpesviridae (homolog of HSV pUL7). Knockdown of pUL103 leads to decreased numbers of properly formed cVACs, smaller plaques, and increased numbers of irregularly-formed virions. We detected two potential ALIX-binding late domains in pUL103 (1: GWPVGLGLL and 2: YPNL). ALIX, a cellular protein, is a hub of protein interactions involved in Endosomal Sorting Complex Required for Transport (ESCRT)-mediated membrane scission events and other processes. Using proteomic methods, we found that pUL103 interacts with ALIX. In transfected cells, mutating either domain resulted in decreased ALIX binding by pUL103. In infected cells, mutating the first late domain decreased virus titers over one hundred-fold. When both late domains were mutated, virus titers decreased over a thousand-fold, suggesting a synergistic defect. Further studies are required to determine whether the growth defect associated with mutations in pUL103’s ALIX-binding domains is due to loss of ALIX binding or is due to the mutations causing a stability defect in pUL103.
Effectiveness of Alkali Treatment of Ballast Water Against Cyanobacteria
AuthorsSeth Benson1*, Jeffery Ram2, and Greg Colores1
1. Central Michigan University, Mount Pleasant, Michigan 48859 USA;
2. State University, Detroit, Michigan 482013.
AbstractCyanobacteria are a diverse phylum with impacts on almost all ecosystems with access to sunlight. These impacts can be both beneficial and detrimental to ecological diversity. Cyanobacteria contain species responsible for potent cyanotoxins, which are a major health concern in the public water supply following aquatic blooms. Ballast water from industrial shipping vessels serves as the putative vector for invasive marine organisms such as cyanotoxin-producing cyanobacteria. Treatment methods are in the process of being implemented but remain largely understudied. The focus of this project is on alkali treatment of ballast water. For this study we are using DNA samples from Great Lakes shipping vessel ballast water left untreated and ballast water treated with a strong base. PCR products were generated for cyanobacteria-specific 16s rRNA genes. Early results reveal cyanobacterial PCR products in both treated and untreated water. Future work will involve PCR amplification of the microcystin producing gene, mcyD, and sequencing of PCR products to determine the cyanobacterial species present pre- and post treatment.
Role of mRNA Folding in non-Shine-Dalgarno Translation Initiation
Bharmal, Mohammed-Husain M., Schrader, Jared M.
Department of Biological Sciences, Wayne State University, Detroit, MI, 48202
Translation initiation is an essential process in which ribosomes engage the mRNA at the start codon. In E. coli, translation initiation at the start codon is facilitated by base pairing of the ribosome and a Shine-Dalgarno (SD) site in the mRNA. Surprisingly, recent genome surveys revealed that only half of bacterial genes contain SD sequences, with some bacterial species having as few as 8% of their genes encoded with upstream SD sequences. To understand the mechanism(s) of non-SD translation initiation, we utilize Caulobacter crescentus, an ?-proteobacterium that is highly adapted to non-SD initiation. We hypothesize that mRNA folding plays a major role in non-SD mRNA translation initiation. To test this hypothesis, we used computational analysis of the mRNA folding stability in C. crescentus translation initiation regions which revealed that the mRNA structures are typically less stable around the start codon than at AUG codons within the coding sequence. To test if the start codon region’s structural stability is functionally relevant to initiation we made different mutations in the 5’ UTR’s of SD and non-SD mRNAs that alter the mRNA’s structural stability and assayed their translation using YFP. Mutations destabilizing secondary structures surrounding the start codon increase translation, while mutations stabilizing the secondary structure surrounding the start codon lower translation. These data support a model in which the C. crescentus ribosome initiates preferentially on single stranded AUG codons. Interestingly, the leaderless mRNAs which completely lack a 5’ UTR, show a high amount of YFP production as compared to SD or non-SD mRNAs, suggesting that C. crescentus is also highly adapted for leaderless mRNA translation.