Browsing by Author "Stewart, Charles R."

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    A host-shutoff early gene of Bacillus subtilis bacteriophage SPO1
    (1994) Wei, Ping; Stewart, Charles R.
    Shutoff of host biosynthesis is one of the earliest and most dramatic events occurring in viral infection and requires the expression of viral early genes. To understand the mechanisms of bacteriophage SPO1 induced host-shutoff, two SPO1 early genes, e3 and e22, were cloned and sequenced, and the roles of e3 in host-shutoff and in phage growth were studied. Both e3 and e22 are novel genes, and are actively expressed during the first few minutes of infection before being promptly shut off. Expression of a plasmid-borne e3 gene, in either B. subtilis or E. coli, caused the inhibition of host DNA, RNA and protein synthesis, and ultimately led to cell death. To identify the primary target of e3-induced shutoff, an e3-resistant E. coli mutant was isolated and analyzed. Plasmid libraries of this mutant's genomic DNA, when screened for genes that could protect wild-type E. coli against e3, yielded the rpoB and dksA genes, which specify the RNA polymerase $\beta$ subunit and a suppressor for DnaK, respectively. The wild-type dksA gene, but not the wild-type rpoB gene, was able to protect against e3, suggesting that the primary e3-resistant mutation was in the rpoB gene and that protection by the dksA gene depended upon overexpression from the plasmid. I suggest that e3 acts by distorting the structure of the host RNA polymerase, thus preventing host transcription, and that this distortion can be prevented or reversed by a chaperonin-like activity specified by dksA. The host-shutoff still occurred normally during infection by an SPO1 mutant which lacked e3 activity, and it occurred much more rapidly than that caused by expression of e3 in uninfected cells. Thus, the e3 product must be only one component of the host-shutoff machinery, which must include elements whose function is redundant to that of e3. At high multiplicities of infection, the mutant SPO1 produced more phage progeny than the wild-type SPO1, suggesting that high concentrations of e3 can be inhibitory to phage growth as well as to host function. Perhaps for that reason, expression of both e3 and e22 is shut off after a brief period of high activity.
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    Anticipation of Nitric Oxide Stress in the Human Commensal Fungus Candida albicans
    (2013-07-24) Lynn, Jed; Bennett, George N.; Braam, Janet; Segatori, Laura; Stewart, Charles R.
    Candida albicans is the most common human commensal fungus, able to colonize host niches such as skin, mouth and gastrointestinal tract. Colonization of diverse microenvironments requires the ability to evade or overcome innate host protection and adapt to rapid transitions between environments with different stresses and nutrient availability. Colonization of the gastrointestinal tract requires passage through the stomach containing toxic levels of nitric oxide, generated from acidification of nitrite in the low pH of the stomach. Although resistance of C. albicans to nitric oxide is mediated by the flavohemoglobin Yhb1, little is known about the physiologically relevant ligands that regulate YHB1 expression. Here I propose the hypothesis that nontoxic saliva chemicals induce YHB1 expression and promote resistance to nitric oxide generated in the stomach. Supporting this hypothesis is the observation that two ions actively concentrated in the saliva – nitrate and thiocyanate – induce YHB1 expression. Indeed, whole-genome transcriptional analysis of C. albicans treated with nitrate or thiocyanate produce gene expression profiles nearly identical to cells treated with nitrite or nitric oxide. Pretreatment of C. albicans with either of these two nontoxic compounds increases resistance of the yeast to nitric oxide. I propose that this is an evolved response in which C. albicans anticipates nitric oxide stress generated in the stomach. C. albicans thus upregulates nitric oxide stress response genes in response to saliva signals that precede nitric oxide formation further on in the gut. Only a few examples of anticipatory signaling have so far been identified and it is not known how common this type of regulation is among microbes. Expression of the YHB1 gene in response to nitric oxide is regulated by the transcription factor Cta4. I show that Cta4 binds to the YHB1 promoter in vivo as a homodimer and is necessary, but not sufficient, for nitric oxide, nitrate and thiocyanate induced expression of YHB1. Based on these data I propose a model in which Cta4 transcriptional activation is inhibited under non-inducing conditions by a negative regulator. Understanding the mechanism by which C. albicans senses and responds to nitric oxide, nitrate and thiocyanate remains a question for future research.
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    Antimicrobial proteins from the SPO1 bacteriophage
    (2007-01-02) Stewart, Charles R.; Shamoo, Yousif A.; Rice University; United States Patent and Trademark Office
    Anti-bacterial peptides are provided which are derived from the bacteriophage SPO1.
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    Bacteriophage SP01 Gene Product 56 Inhibits Bacillus subtilis Cell Division by Interacting with FtsL and Disrupting Pbp2B and FtsW Recruitment
    (American Society for Microbiology, 2020) Bhambhani, Amit; Iadicicco, Isabella; Lee, Jules; Ahmed, Syed; Belfatto, Max; Held, David; Marconi, Alexia; Parks, Aaron; Stewart, Charles R.; Margolin, William; Levin, Petra Anne; Haeusser, Daniel P.
    Previous work identified gene product 56 (gp56), encoded by the lytic bacteriophage SP01, as being responsible for inhibition of Bacillus subtilis cell division during its infection. Assembly of the essential tubulin-like protein FtsZ into a ring-shaped structure at the nascent site of cytokinesis determines the timing and position of division in most bacteria. This FtsZ ring serves as a scaffold for recruitment of other proteins into a mature division-competent structure permitting membrane constriction and septal cell wall synthesis. Here, we show that expression of the predicted 9.3-kDa gp56 of SP01 inhibits later stages of B. subtilis cell division without altering FtsZ ring assembly. Green fluorescent protein-tagged gp56 localizes to the membrane at the site of division. While its localization does not interfere with recruitment of early division proteins, gp56 interferes with the recruitment of late division proteins, including Pbp2b and FtsW. Imaging of cells with specific division components deleted or depleted and two-hybrid analyses suggest that gp56 localization and activity depend on its interaction with FtsL. Together, these data support a model in which gp56 interacts with a central part of the division machinery to disrupt late recruitment of the division proteins involved in septal cell wall synthesis.IMPORTANCE Studies over the past decades have identified bacteriophage-encoded factors that interfere with host cell shape or cytokinesis during viral infection. The phage factors causing cell filamentation that have been investigated to date all act by targeting FtsZ, the conserved prokaryotic tubulin homolog that composes the cytokinetic ring in most bacteria and some groups of archaea. However, the mechanisms of several phage factors that inhibit cytokinesis, including gp56 of bacteriophage SP01 of Bacillus subtilis, remain unexplored. Here, we show that, unlike other published examples of phage inhibition of cytokinesis, gp56 blocks B. subtilis cell division without targeting FtsZ. Rather, it utilizes the assembled FtsZ cytokinetic ring to localize to the division machinery and to block recruitment of proteins needed for septal cell wall synthesis.
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    The BIophysical Basis for Adaptation: Predicting Evolutionary Outcomes from Physicochemical Properties
    (2013-05-13) Benitez Cardenas, Andres; Shamoo, Yousif; Stewart, Charles R.; Nikonowicz, Edward P.; Silberg, Jonathan J.; Segatori, Laura
    Experimental evolution can be used in conjunction with biophysical characterization of enzymes to determine the link between cellular fitness and physicochemical properties of enzymes. Sequencing of ancestral and evolved populations can be used to compare the outcomes of experimental evolution with measurements of fitness, using growth rate assays to correlate fitness outcomes to specific mutations. Combined with enzyme assays of kinetic properties that can provide a direct link between genotypic and phenotypic changes of adaptive mutants, we can model the complex relationship between genotypic changes and evolutionary outcomes. Two experimental evolution systems were used to explore the link between enzyme properties and fitness outcomes. In the first series of studies, a “weak link” evolution experiment was used to explore the effect of reducing selection strength on altering accessible pathways for adaptation. In the weak link method the essential gene for adenylate kinase (AK) was replaced in the chromosome of the thermophile Geobacillus stearothermophilus with a homolog from Bacillus subtilis. Replacement with the maladapted gene confers a high fitness cost, and therefore mutations that restore function of AK are strongly favored. Two triple mutants of AK containing a new combination of single point mutants identified under strong selection, AKQ199R/A193V/Q16L and AKQ199R/T179I/Q16L were discovered through an adaptation experiment using a weak temperature ramp; suggesting that the adaptive landscape for AK thermostability is highly constrained. A thermostable coupled assay was developed for measuring adenylate kinase activity using LDHTTHERMOPHILUS and PKGSTEAROTHERMOPHILUS at high temperatures. The triple mutants had increased function compared with the double mutant ancestors, but the triple mutants displayed diminishing returns epistasis on fitness. In the second experimental evolution system, a mathematical model was developed to investigate the role of adaptive mutations, in the tetracycline inactivation enzyme TetX2, on antibiotic resistance to minocycline (MCN). Growth rates measurements, enzyme kinetics, and flux balance equations were used to develop a model to predict the effect on growth rates of TetX2 and seven adaptive TetX2 variants at different MCN concentrations. Population histogram measurements for the experimental evolution study were measured using a high throughput Illumina sequencing method (FREQ-SEQ). We found that the model was able to accurately predict the fitness outcomes for the wild type and the seven single mutants of TetX2 that were originally isolated, as well as for a double mutant that was not used in the development of the original model. The mathematical model accurately predicts that the two mutants TetX2T280A and TetX2N371I provide the largest fitness benefits, in agreement with the results of in vitro experiments on adaptation to MCN. The model was also able to accurately predict enzyme parameters from growth rates values, with a specific emphasis on predicting the ratio of Vmax/KM(MCN). The model allows us to make predictions about the fitness benefits of physicochemical changes to enzymes, and can be used as a high throughput method for determining enzyme kinetic parameters without requiring protein purification. Understanding how physicochemical changes of enzymes relate to phenotypic changes, and ultimately to fitness, requires knowledge of both the molecular basis for determining enzyme properties, and how selection acts on fitness differences to determine evolutionary outcomes. This research provides direct links between physicochemical changes and adaptive phenotypes, as well providing observations of how adaptive landscapes and fitness changes affect evolutionary outcomes.
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    The Induction of Infectivity in Human Astrovirus in Response to Capsid Proteolysis
    (2013-06-28) Harper, Justin; Tao, Yizhi Jane; McNew, James A.; Stewart, Charles R.
    Astrovirus is a non-enveloped, T=3, positive-sense RNA virus that presents with self-limiting gastroenteritis; however, it has been additionally associated with serious presentations such as nephritis, hepatitis, and encephalitis, which is compounded by its propensity to engage in cross-species penetrations. Astrovirus undergoes a complex capsid maturation process mediated by host proteases in which an inert, immature capsid composed of VP90 is sequentially cleaved to yield a highly infectious particle composed of VP34 and VP27/VP25, which form the capsid shell and spikes, respectively. By overexpressing a VP9070-418 truncate in insect cells, we have demonstrated that the shell domain alone cannot support particle assembly, implying a crucial role for the dimeric contacts within the spike. Various monomeric, shell domain truncates (i.e. VP9071-252, VP9071-283, VP9071-313, and VP9071-415) have been successfully expressed and purified, but none yielded useful crystals, suggesting the structural context of the capsid lattice may be needed to stabilize their conformational flexibility.