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1.  Characterization of a Feedback-Resistant Mevalonate Kinase from the Archaeon Methanosarcina mazei▿ 
Applied and Environmental Microbiology  2011;77(21):7772-7778.
The mevalonate pathway is utilized for the biosynthesis of isoprenoids in many bacterial, eukaryotic, and archaeal organisms. Based on previous reports of its feedback inhibition, mevalonate kinase (MVK) may play an important regulatory role in the biosynthesis of mevalonate pathway-derived compounds. Here we report the purification, kinetic characterization, and inhibition analysis of the MVK from the archaeon Methanosarcina mazei. The inhibition of the M. mazei MVK by the following metabolites derived from the mevalonate pathway was explored: dimethylallyl diphosphate (DMAPP), geranyl pyrophosphate (GPP), farnesyl pyrophosphate (FPP), isopentenyl monophosphate (IP), and diphosphomevalonate. M. mazei MVK was not inhibited by DMAPP, GPP, FPP, diphosphomevalonate, or IP, a proposed intermediate in an alternative isoprenoid pathway present in archaea. Our findings suggest that the M. mazei MVK represents a distinct class of mevalonate kinases that can be differentiated from previously characterized MVKs based on its inhibition profile.
doi:10.1128/AEM.05761-11
PMCID: PMC3209144  PMID: 21908638
2.  p300 (Histone Acetyltransferase) biomarker predicts prostate cancer biochemical recurrence and correlates with changes in epithelia nuclear size and shape 
The Prostate  2008;68(10):1097-1104.
Background
p300 impacts the transcription of several genes involved in key pathways critical to PCa progression. Therefore, we evaluated the prognostic value of p300 expression and its correlation with nuclear alterations seen in tumor cells in men with long term follow-up after radical prostatectomy (RP).
Methods
NCI Cooperative Prostate Cancer Tissue Resource tissue microarray cores of 92 RP cases (56 non-recurrences and 36 PSA recurrences) were utilized for the study. p300 expression was assessed by quantitative immunohistochemistry and nuclear alterations in Feulgen-stained nuclei were evaluated by digital image analysis using the AutoCyte™ Pathology Workstation. Cox proportional hazards regression, Spearman’s rank correlation, and Kaplan-Meier plots were employed to analyze the data.
Results
p300 expression significantly correlated with nuclear alterations seen in tumor cells; specifically with circular form factor (p=0.012) and minimum feret (p=0.048). p300 expression in high grade tumors (Gleason score ≥7) was significantly higher compared to low grade tumors (Gleason score <7) [17.7% vs. 13.7%, respectively, p=0.03]. TNM stage, Gleason score, and p300 expression were univariately significant in the prediction of PCa biochemical recurrence free survival (p≤0.05). p300 expression remained significant in the multivariate model (p=0.03) while Gleason score showed a trend toward significance (p=0.06). Patients with a Gleason score ≥7 and p300 expression >24% showed the highest risk for PCa biochemical recurrence (p=0.002).
Conclusions
p300 expression correlates with nuclear alterations seen in tumor cells and has prognostic value in predicting long-term PCa biochemical recurrence free survival.
doi:10.1002/pros.20772
PMCID: PMC3099408  PMID: 18459105
3.  A Bistable Switch and Anatomical Site Control Vibrio cholerae Virulence Gene Expression in the Intestine 
PLoS Pathogens  2010;6(9):e1001102.
A fundamental, but unanswered question in host-pathogen interactions is the timing, localization and population distribution of virulence gene expression during infection. Here, microarray and in situ single cell expression methods were used to study Vibrio cholerae growth and virulence gene expression during infection of the rabbit ligated ileal loop model of cholera. Genes encoding the toxin-coregulated pilus (TCP) and cholera toxin (CT) were powerfully expressed early in the infectious process in bacteria adjacent to epithelial surfaces. Increased growth was found to co-localize with virulence gene expression. Significant heterogeneity in the expression of tcpA, the repeating subunit of TCP, was observed late in the infectious process. The expression of tcpA, studied in single cells in a homogeneous medium, demonstrated unimodal induction of tcpA after addition of bicarbonate, a chemical inducer of virulence gene expression. Striking bifurcation of the population occurred during entry into stationary phase: one subpopulation continued to express tcpA, whereas the expression declined in the other subpopulation. ctxA, encoding the A subunit of CT, and toxT, encoding the proximal master regulator of virulence gene expression also exhibited the bifurcation phenotype. The bifurcation phenotype was found to be reversible, epigenetic and to persist after removal of bicarbonate, features consistent with bistable switches. The bistable switch requires the positive-feedback circuit controlling ToxT expression and formation of the CRP-cAMP complex during entry into stationary phase. Key features of this bistable switch also were demonstrated in vivo, where striking heterogeneity in tcpA expression was observed in luminal fluid in later stages of the infection. When this fluid was diluted into artificial seawater, bacterial aggregates continued to express tcpA for prolonged periods of time. The bistable control of virulence gene expression points to a mechanism that could generate a subpopulation of V. cholerae that continues to produce TCP and CT in the rice water stools of cholera patients.
Author Summary
Most pathogenic microorganisms infect in a stepwise manner: colonization of host surfaces is followed by invasion and injury of host tissues and, late in the infectious process, dissemination to other hosts occurs. During its residence in the host, the pathogen produces essential virulence determinants and often replicates rapidly, leading to a vast expansion of its biomass. Although this scenario is well established also for Vibrio cholerae, the cause of a potentially fatal diarrheal illness, it has not previously been possible to identify precisely when or where virulence determinants are produced in the intestine. We addressed this question by investigating the expression of virulence genes by individual V. cholerae during infection of the small intestine. Virulence genes were found to be powerfully expressed early in the infectious process by bacteria in close proximity to epithelial surfaces. Increased replication rates were also localized to epithelial surfaces. During later stages of the infection, the population of V. cholerae bifurcates into two fractions: one subpopulation continues to express virulence genes, whereas these genes are silenced in the other subpopulation. The genetic program controlling the continued production of virulence genes may mediate the persistence of a hyper-infectious subpopulation of bacteria in the stools of cholera patients.
doi:10.1371/journal.ppat.1001102
PMCID: PMC2940755  PMID: 20862321
4.  Roles for RNA in Telomerase Nucleotide and Repeat Addition Processivity 
Molecular cell  2003;11(6):1673-1683.
Summary
Telomerase is a ribonucleoprotein reverse transcriptase with two subunits critical for catalytic activity, the protein telomerase reverse transcriptase (TERT) and telomerase RNA. In this study, we establish additional roles of the telomerase RNA subunit by demonstrating that RNA motifs stimulate the processivity of nucleotide and repeat addition. These functions are both functionally and physically separable from the roles of other RNA motifs in establishing a properly defined template. Binding of Tetrahymena telomerase RNA stem IV to TERT enhances nucleotide addition processivity, while a cooperation of the RNA pseudoknot and stem IV promotes repeat addition processivity. The low processivity of DNA synthesis by telomerase ribonucleoproteins lacking the pseudoknot and/or stem IV can be rescued by addition of the deleted region in trans. These findings demonstrate RNA elements with roles in telomerase elongation processivity that are distinct from RNA elements that specify the internal template.
PMCID: PMC2917597  PMID: 12820978
5.  Genomic and Phenotypic Diversity of Coastal Vibrio cholerae Strains Is Linked to Environmental Factors▿ †  
Applied and Environmental Microbiology  2007;73(11):3705-3714.
Studies of Vibrio cholerae diversity have focused primarily on pathogenic isolates of the O1 and O139 serotypes. However, autochthonous environmental isolates of this species routinely display more extensive genetic diversity than the primarily clonal pathogenic strains. In this study, genomic and metabolic profiles of 41 non-O1/O139 environmental isolates from central California coastal waters and four clinical strains are used to characterize the core genome and metabolome of V. cholerae. Comparative genome hybridization using microarrays constructed from the fully sequenced V. cholerae O1 El Tor N16961 genome identified 2,787 core genes that approximated the projected species core genome within 1.6%. Core genes are almost universally present in strains with widely different niches, suggesting that these genes are essential for persistence in diverse aquatic environments. In contrast, the dispensable genes and phenotypic traits identified in this study should provide increased fitness for certain niche environments. Environmental parameters, measured in situ during sample collection, are correlated to the presence of specific dispensable genes and metabolic capabilities, including utilization of mannose, sialic acid, citrate, and chitosan oligosaccharides. These results identify gene content and metabolic pathways that are likely selected for in certain coastal environments and may influence V. cholerae population structure in aquatic environments.
doi:10.1128/AEM.02736-06
PMCID: PMC1932678  PMID: 17449702
6.  Detection and Transformation of Genome Segments That Differ within a Coastal Population of Vibrio cholerae Strains▿ †  
Applied and Environmental Microbiology  2007;73(11):3695-3704.
Vibrio cholerae is an autochthonous member of diverse aquatic ecosystems around the globe. Collectively, the genomes of environmental V. cholerae strains comprise a large repository of encoded functions which can be acquired by individual V. cholerae lineages through uptake and recombination. To characterize the genomic diversity of environmental V. cholerae, we used comparative genome hybridization to study 41 environmental strains isolated from diverse habitats along the central California coast, a region free of endemic cholera. These data were used to classify genes of the epidemic V. cholerae O1 sequenced strain N16961 as conserved, variably present, or absent from the isolates. For the most part, absent genes were restricted to large mobile elements and have known functions in pathogenesis. Conversely, genes present in some, but not all, California isolates were in smaller contiguous clusters and were less likely to be near genes with functions in DNA mobility. Two such clusters of variable genes encoding different selectable metabolic phenotypes (mannose and diglucosamine utilization) were transformed into the genomes of environmental isolates by chitin-dependent competence, indicating that this mechanism of general genetic exchange is conserved among V. cholerae. The transformed DNA had an average size of 22.7 kbp, demonstrating that natural competence can mediate the movement of large chromosome fragments. Thus, whether variable genes arise through the acquisition of new sequences by horizontal gene transfer or by the loss of preexisting DNA though deletion, natural transformation provides a mechanism by which V. cholerae clones can gain access to the V. cholerae pan-genome.
doi:10.1128/AEM.02735-06
PMCID: PMC1932674  PMID: 17449699
7.  RpoS Controls the Vibrio cholerae Mucosal Escape Response 
PLoS Pathogens  2006;2(10):e109.
Vibrio cholerae causes a severe diarrhoeal disease by secreting a toxin during colonization of the epithelium in the small intestine. Whereas the initial steps of the infectious process have been intensively studied, the last phases have received little attention. Confocal microscopy of V. cholerae O1-infected rabbit ileal loops captured a distinctive stage in the infectious process: 12 h post-inoculation, bacteria detach from the epithelial surface and move into the fluid-filled lumen. Designated the “mucosal escape response,” this phenomenon requires RpoS, the stationary phase alternative sigma factor. Quantitative in vivo localization assays corroborated the rpoS phenotype and showed that it also requires HapR. Expression profiling of bacteria isolated from ileal loop fluid and mucus demonstrated a significant RpoS-dependent upregulation of many chemotaxis and motility genes coincident with the emigration of bacteria from the epithelial surface. In stationary phase cultures, RpoS was also required for upregulation of chemotaxis and motility genes, for production of flagella, and for movement of bacteria across low nutrient swarm plates. The hapR mutant produced near-normal numbers of flagellated cells, but was significantly less motile than the wild-type parent. During in vitro growth under virulence-inducing conditions, the rpoS mutant produced 10- to 100-fold more cholera toxin than the wild-type parent. Although the rpoS mutant caused only a small over-expression of the genes encoding cholera toxin in the ileal loop, it resulted in a 30% increase in fluid accumulation compared to the wild-type. Together, these results show that the mucosal escape response is orchestrated by an RpoS-dependent genetic program that activates chemotaxis and motility functions. This may furthermore coincide with reduced virulence gene expression, thus preparing the organism for the next stage in its life cycle.
Synopsis
Vibrio cholerae, a pathogenic microbe, causes a severe diarrhoeal disease mainly in Third World countries. Although the pathogenicity of this organism has been intensively studied for more than a century, most research has focused on the initial stages of the infection, especially colonization of the intestine and virulence gene expression. However, the last stages of the infectious process have received very little attention. In the present manuscript, the authors use the rabbit ileal loop model of cholera to show how this organism, late in the infection, detaches from the epithelial surface and migrates into the luminal fluid, a process the authors termed the “mucosal escape response.” This study identifies, for the first time, how the alternative starvation sigma factor RpoS regulates this process. Features of this genetic program include the dramatic induction of genes involved in motility and chemotaxis functions. This study furthermore identifies RpoS as an important regulator of virulence gene expression and shows that the mucosal escape response may coincide with diminished virulence gene expression. This work is essential for understanding a key and under-appreciated step in the life cycle of this important human pathogen: its exit from the intestine and how this serves to prepare it for transmission into environmental reservoirs or to new human hosts.
doi:10.1371/journal.ppat.0020109
PMCID: PMC1617127  PMID: 17054394

Results 1-7 (7)