Biomedical subjects

John H Paul

Publications and source records attributed to John H Paul.

12 recordsLinked to original sources

PubMed2006

Development and applications of microbial ecogenomic indicators for monitoring water quality: report of a workshop assessing the state of the science, research needs and future directions.

This article brings forth recommendations from a workshop sponsored by the U.S. Environmental Protection Agency's Science to Achieve Results (STAR) and Environmental Monitoring and Assessment (EMAP) Programs and by the Council of State Governments, held during May 2002 in Kansas City, Kansas. The workshop assembled microbial ecologists and environmental scientists to determine what research and science is needed to bring existing molecular biological approaches and newer technologies arising from microbial genomic research into environmental monitoring and water quality assessments. Development of genomics and proteomics technologies for environmental science is a very new area having potential to improve environmental water quality assessments. The workshop participants noted that microbial ecologists are already using molecular biological methods well suited for monitoring and water quality assessments and anticipate that genomics-enabled technologies could be made available for monitoring within a decade. Recommendations arising from the workshop include needs for (i) identification of informative microbial gene sequences, (ii) improved understandings of linkages between indicator taxa, gene expression and environmental condition, (iii) technological advancements towards field application, and (iv) development of the appropriate databases.

Animals↗

PubMed2006

The genome of deep-sea vent chemolithoautotroph Thiomicrospira crunogena XCL-2.

Presented here is the complete genome sequence of Thiomicrospira crunogena XCL-2, representative of ubiquitous chemolithoautotrophic sulfur-oxidizing bacteria isolated from deep-sea hydrothermal vents. This gammaproteobacterium has a single chromosome (2,427,734 base pairs), and its genome illustrates many of the adaptations that have enabled it to thrive at vents globally. It has 14 methyl-accepting chemotaxis protein genes, including four that may assist in positioning it in the redoxcline. A relative abundance of coding sequences (CDSs) encoding regulatory proteins likely control the expression of genes encoding carboxysomes, multiple dissolved inorganic nitrogen and phosphate transporters, as well as a phosphonate operon, which provide this species with a variety of options for acquiring these substrates from the environment. Thiom. crunogena XCL-2 is unusual among obligate sulfur-oxidizing bacteria in relying on the Sox system for the oxidation of reduced sulfur compounds. The genome has characteristics consistent with an obligately chemolithoautotrophic lifestyle, including few transporters predicted to have organic allocrits, and Calvin-Benson-Bassham cycle CDSs scattered throughout the genome.

Bacterial Adhesion↗

PubMed2005

Marine phage genomics: what have we learned?

Marine phages are the most abundant and diverse form of life on the planet, and their genomes have been described as the largest untapped reservoir of genomic information. To date, however, the complete genome sequences of only 17 marine phage are known. Nevertheless, these genomes have revealed some interesting features, including the presence of photosynthetic genes in cyanophage and common patterns of genomic organization. Intriguing findings are also being made from studies of the uncultivated marine viral community genome ('metavirome'). The greatest challenge in interpreting the biology of these phages, and for making comparisons with their terrestrial counterparts, is the high proportion of unidentifiable open reading frames (approximately 60%). Future studies are likely to focus on sequencing more marine phage genomes from disparate hosts and diverse environments and on further basic studies of the biology of existing marine phages.

Bacteriophages↗

PubMed2005

Increased precision of microbial RNA quantification using NASBA with an internal control.

Detection and quantification of low abundance target RNA has wide utility in the fields of clinical diagnostics, environmental monitoring, gene expression analysis, and biodefense. Nucleic acid based sequence amplification (NASBA) is an isothermal amplification method that provides the sensitivity needed for these applications. However, the requirement for three separate enzymes in NASBA often results in a greater variability between replicate samples than that seen in PCR-based assays. To overcome this problem, we have adapted the bioMérieux Nuclisens Basic Kit and Nuclisens EasyQ Analyzer along with the introduction of a synthetic internal control RNA (IC-RNA) for quantification of potentially any RNA sequence. Using the rbcL gene from the Florida red tide organism Karenia brevis as our target, we describe a simple method to accurately quantify the native target by computing the ratio of the time to positivity (TTP) values for both the wild-type and IC-RNA, and plotting this ratio against the starting number of target molecules or cells. By utilizing this simple method, we have significantly increased our accuracy and precision of prediction over the standard TTP calculations.

Animals↗

PubMed2005

Complete genome sequence of phiHSIC, a pseudotemperate marine phage of Listonella pelagia.

The genome for the marine pseudotemperate member of the Siphoviridae phiHSIC has been sequenced using a combination of linker amplification library construction, restriction digest library construction, and primer walking. phiHSIC enters into a pseudolysogenic relationship with its host, Listonella pelagia, characterized by sigmoidal growth curves producing >10(9) cells/ml and >10(11) phage/ml. The genome (37,966 bp; G+C content, 44%) contained 47 putative open reading frames (ORFs), 17 of which had significant BLASTP hits in GenBank, including a beta subunit of DNA polymerase III, a helicase, a helicase-like subunit of a resolvasome complex, a terminase, a tail tape measure protein, several phage-like structural proteins, and 1 ORF that may assist in host pathogenicity (an ADP ribosyltransferase). The genome was circularly permuted, with no physical ends detected by sequencing or restriction enzyme digestion analysis, and lacked a cos site. This evidence is consistent with a headful packaging mechanism similar to that of Salmonella phage P22 and Shigella phage Sf6. Because none of the phage-like ORFs were closely related to any existing phage sequences in GenBank (i.e., none more than 62% identical and most <25% identical at the amino acid level), phiHSIC is unique among phages that have been sequenced to date. These results further emphasize the need to sequence phages from the marine environment, perhaps the largest reservoir of untapped genetic information.

Electrophoresis, Gel, Pulsed-Field↗

PubMed2004

Development and evaluation of a method to detect and quantify enteroviruses using NASBA and internal control RNA (IC-NASBA).

We have developed a rapid, sensitive, and specific assay for the detection and quantification of enteroviruses using nucleic acid sequence-based amplification (NASBA). The inclusion of an internal control (IC) increased the precision and accuracy of the method over a standard NASBA assay and provided a way to detect assay inhibition. The assay was sensitive to 10 viral particles with amplification and detection occurring in as little as 18 min. The assay detected a variety of different enteroviruses to the exclusion of non-target viruses. The standard NASBA method resulted in predictions of viral load to within an order of magnitude of the expected number, as compared with prediction to within less than a half order of magnitude using the IC-NASBA method. Rapid and sensitive detection of enteroviruses is important in both clinical samples to diagnose illness and in environmental samples to assess risk of wastewater contamination and potential health hazards.

Enterovirus↗

PubMed2004

Detection and quantification of the red tide dinoflagellate Karenia brevis by real-time nucleic acid sequence-based amplification.

Nucleic acid sequence-based amplification (NASBA) is an isothermal method of RNA amplification that has been previously used in clinical diagnostic testing. A real-time NASBA assay has been developed for the detection of rbcL mRNA from the red tide dinoflagellate Karenia brevis. This assay is sensitive to one K. brevis cell and 1.0 fg of in vitro transcript, with occasional detection of lower concentrations of transcript. The assay did not detect rbcL mRNA from a wide range of nontarget organisms and environmental clones, while 10 strains (all tested) of K. brevis were detected. By the use of standard curves based on time to positivity, concentrations of K. brevis in environmental samples were predicted by NASBA and classified into different levels of blooms per the Florida Fish and Wildlife Conservation Commission (FWC) system. NASBA classification matched FWC classification (based on cell counts) 72% of the time. Those samples that did not match were off by only one class. NASBA is sensitive, rapid, and effective and may be used as an additional or alternative method to detect and quantify K. brevis in the marine environment.

Animals↗

PubMed2004

Geochemical rate-RNA integration study: ribulose-1,5-bisphosphate carboxylase/oxygenase gene transcription and photosynthetic capacity of planktonic photoautotrophs.

A pilot field experiment to assess the relationship between traditional biogeochemical rate measurements and transcriptional activity of microbial populations was carried out at the LEO 15 site off Tuckerton, N.J. Here, we report the relationship between photosynthetic capacity of autotrophic plankton and transcriptional activity of the large subunit gene (rbcL) for ribulose-1,5-bisphosphate carboxylase/oxygenase (RubisCO), the enzyme responsible for primary carbon fixation during photosynthesis. Similar diel patterns of carbon fixation and rbcL gene expression were observed in three of four time series, with maxima for photosynthetic capacity (P(max)) and rbcL mRNA occurring between 10 a.m. and 1 p.m. The lowest P(max) and rbcL levels were detected between 6 p.m. and 10:30 p.m. A significant correlation was found between P(max) and form ID rbcL mRNA (R(2) = 0.56) and forms IA and IB (R(2) = 0.41 and 0.47, respectively). The correlation between the abundance of "diatom" rbcL and P(max) mRNA was modest (R(2) = 0.49; n = 12) but improved dramatically (R(2) = 0.97; n = 10) upon removal of two outliers which represented afternoon samples with high P(max) but lower mRNA levels. Clone libraries from reverse transcription-PCR-amplified rbcL mRNA indicated the presence of several chromophytic algae (diatoms, prymnesiophytes, and chrysophytes) and some eukaryotic green flagellates. Analogous results were obtained from amplified small rRNA sequences and secondary pigment analysis. These results suggest that diatoms were a major contributor to carbon fixation at LEO 15 at the time of sampling and that photosynthetic carbon fixation was partially controlled by transcriptional regulation of the RubisCO gene.

Kinetics↗

PubMed2003

Pathogenic human viruses in coastal waters.

This review addresses both historical and recent investigations into viral contamination of marine waters. With the relatively recent emergence of molecular biology-based assays, a number of investigations have shown that pathogenic viruses are prevalent in marine waters being impacted by sewage. Research has shown that this group of fecal-oral viral pathogens (enteroviruses, hepatitis A viruses, Norwalk viruses, reoviruses, adenoviruses, rotaviruses, etc.) can cause a broad range of asymptomatic to severe gastrointestinal, respiratory, and eye, nose, ear, and skin infections in people exposed through recreational use of the water. The viruses and the nucleic acid signature survive for an extended period in the marine environment. One of the primary concerns of public health officials is the relationship between the presence of pathogens and the recreational risk to human health in polluted marine environments. While a number of studies have attempted to address this issue, the relationship is still poorly understood. A contributing factor to our lack of progress in the field has been the lack of sensitive methods to detect the broad range of both bacterial and viral pathogens. The application of new and advanced molecular methods will continue to contribute to our current state of knowledge in this emerging and important field.

Humans↗

PubMed2002

Marine phage genomics.

Marine phages are the most abundant biological entities in the oceans. They play important roles in carbon cycling through marine food webs, gene transfer by transduction and conversion of hosts by lysogeny. The handful of marine phage genomes that have been sequenced to date, along with prophages in marine bacterial genomes, and partial sequencing of uncultivated phages are yielding glimpses of the tremendous diversity and physiological potential of the marine phage community. Common gene modules in diverse phages are providing the information necessary to make evolutionary comparisons. Finally, deciphering phage genomes is providing clues about the adaptive response of phages and their hosts to environmental cues.

Bacteriophages↗

PubMed1991

Detection of Gene Expression in Genetically Engineered Microorganisms and Natural Phytoplankton Populations in the Marine Environment by mRNA Analysis.

A simple method that combines guanidinium isothiocyanate RNA extraction and probing with antisense and sense RNA probes is described for analysis of microbial gene expression in planktonic populations. Probing of RNA sample extracts with sense-strand RNA probes was used as a control for nonspecific hybridization or contamination of mRNA with target DNA. This method enabled detection of expression of a plasmid-encoded neomycin phosphotransferase gene (nptII) in as few as 10Vibrio cells per ml in 100 ml of seawater. We have used this method to detect expression of the ribulose-1,5-bisphosphate carboxylase large-subunit gene (rbcL) in Synechococcus cultures and natural phytoplankton populations in the Dry Tortugas, Florida. During a 36-h diel study, rbcL expression of the indigenous phytoplankton was greatest in the day, least at night (1100, 0300, and 0100 h), and variable at dawn or dusk (0700 and 1900 h). These results are the first report of gene expression in natural populations by mRNA isolation and probing. This methodology should be useful for the study of gene expression in microorganisms released into the environment for agricultural or bioremediation purposes and indigenous populations containing highly conserved target gene sequences.

Journal Article↗

PubMed1988

Seasonal and Diel Variability in Dissolved DNA and in Microbial Biomass and Activity in a Subtropical Estuary.

Dissolved DNA and microbial biomass and activity parameters were measured over a 15-month period at three stations along a salinity gradient in Tampa Bay, Fla. Dissolved DNA showed seasonal variation, with minimal values in December and January and maximal values in summer months (July and August). This pattern of seasonal variation followed that of particulate DNA and water temperature and did not correlate with bacterioplankton (direct counts and [H]thymidine incorporation) or phytoplankton (chlorophyll a and CO(2) fixation) biomass and activity. Microautotrophic populations showed maxima in the spring and fall, whereas microheterotrophic activity was greatest in late summer (September). Both autotrophic and heterotrophic microbial activity was greatest at the high estuarine (low salinity) station and lowest at the mouth of the bay (high salinity station), irrespective of season. Dissolved DNA carbon and phosphorus constituted 0.11 +/- 0.05% of the dissolved organic carbon and 6.6 +/- 6.5% of the dissolved organic phosphorus, respectively. Strong diel periodicity was noted in dissolved DNA and in microbial activity in Bayboro Harbor during the dry season. A noon maximum in primary productivity was followed by an 8 p.m. maximum in heterotrophic activity and a midnight maximum in dissolved DNA. This diel periodicity was less pronounced in the wet season, when microbial parameters were strongly influenced by episodic inputs of freshwater. These results suggest that seasonal and diel production of dissolved DNA is driven by primary production, either through direct DNA release by phytoplankton, or more likely, through growth of bacterioplankton on phytoplankton exudates, followed by excretion and lysis.

Journal Article↗