John Paul - Academia.edu (original) (raw)
Papers by John Paul
Microbiology (Reading, England), 1999
The population structure of Streptococcus pneumoniae in a sample of 134 carried antibiotic-suscep... more The population structure of Streptococcus pneumoniae in a sample of 134 carried antibiotic-susceptible isolates, and 53 resistant and susceptible invasive isolates, was examined using a DNA-based version of multilocus enzyme electrophoresis: multilocus restriction typing (MLRT). This involved RFLP analysis of PCR products generated from nine loci of housekeeping genes located around the pneumococcal chromosome. The combination of alleles at each of the nine loci gave an allelic profile or restriction type (RT). All carried (throat or nasopharyngeal) isolates from children or adults in Oxford and Manchester, UK, and from an HIV-seropositive cohort in Nairobi, Kenya, showed an epidemic population structure. Twelve carried clonal groups, each with different serotypes, were identified at both locations within the UK. Almost all of the carried clones examined (16/17) were found to possess identical RTs or sequence types (STs) to invasive isolates, indicating that frequently carried clone...
Applied and environmental microbiology, 1997
A total of 68 marine samples from various sites impacted by sewage and storm waters were analyzed... more A total of 68 marine samples from various sites impacted by sewage and storm waters were analyzed by both the plaque assay and a reverse transcriptase (RT) PCR technique for F(sup+)-specific coliphage. The coliphage levels detected by the plaque assay averaged 1.90 x 10(sup4) PFU/100.0 ml. Using a most probable number (MPN) PCR approach, the levels averaged 2.40 x 10(sup6) MPN-PCR units/100.0 ml. Two samples were positive by RT-PCR but negative by plaque assay, and 12 samples were positive by plaque assay but negative by RT-PCR (levels lower than 11.00 PFU/100.0 ml). The host system used for the plaque assay may detect somatic coliphage in addition to the F(sup+)-specific coliphage. When it is used as an indicator of pollution, contamination may be missed with more restrictive systems. The difference in results may be due to the sensitivity, specificity, or inhibition of RT-PCR in marine samples. This study provides information on quantifying PCR results by an MPN method and insight...
Applied and environmental microbiology, 1998
To determine the potential for bacteriophage-mediated gene transfer in the marine environment, we... more To determine the potential for bacteriophage-mediated gene transfer in the marine environment, we established transduction systems by using marine phage host isolates. Plasmid pQSR50, which contains transposon Tn5 and encodes kanamycin and streptomycin resistance, was used in plasmid transduction assays. Both marine bacterial isolates and concentrated natural bacterial communities were used as recipients in transduction studies. Transductants were detected by a gene probe complementary to the neomycin phosphotransferase (nptII) gene in Tn5. The transduction frequencies ranged from 1.33 x 10(-7) to 5.13 x 10(-9) transductants/PFU in studies performed with the bacterial isolates. With the mixed bacterial communities, putative transductants were detected in two of the six experiments performed. These putative transductants were confirmed and separated from indigenous antibiotic-resistant bacteria by colony hybridization probed with the nptII probe and by PCR amplification performed wit...
Applied and environmental microbiology, 1998
To understand the ecological and genetic role of viruses in the marine environment, it is critica... more To understand the ecological and genetic role of viruses in the marine environment, it is critical to know the infectivity of viruses and the types of interactions that occur between marine viruses and their hosts. We isolated four marine phages from turbid plaques by using four indigenous bacterial hosts obtained from concentrated water samples from Mamala Bay, Oahu, Hawaii. Two of the rod-shaped bacterial hosts were identified as Sphingomonas paucimobilis and Flavobacterium sp. All of the phage isolates were tailed phages and contained double-stranded DNA. Two of the phage isolates had morphologies typical of the family Siphoviridae, while the other two belonged to the families Myoviridae and Podoviridae. The head diameters of these viruses ranged from 47 to 70.7 nm, and the tail lengths ranged from 12 to 146 nm. The burst sizes ranged from 7.8 to 240 phage/bacterial cell, and the genome sizes, as determined by restriction digestion, ranged from 36 to 112 kb. The members of the Si...
Biochimica et Biophysica Acta (BBA) - Nucleic Acids and Protein Synthesis, 1974
Soluble chromatin can be banded isopycnically in metrizamide gradients without prior fixation. Th... more Soluble chromatin can be banded isopycnically in metrizamide gradients without prior fixation. The chromatin--DNA of minimally sheared chromatin (750--1000 base-pairs) bands as a single, sharp peak, almost completely separated from the chromatin ribonucleoprotein particles. More extensive shearing (to about 360 base-pairs) leads to a bimodal distribution of the chromatin--DNA in the gradients; all of the DNA is complexed with proteins.
Clinical infectious diseases : an official publication of the Infectious Diseases Society of America, 2014
The ISME Journal, 2007
River plumes deliver large quantities of nutrients to oligotrophic oceans, often resulting in sig... more River plumes deliver large quantities of nutrients to oligotrophic oceans, often resulting in significant CO 2 drawdown. To determine the relationship between expression of the major gene in carbon fixation (large subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase, RuBisCO) and CO 2 dynamics, we evaluated rbcL mRNA abundance using novel quantitative PCR assays, phytoplankton cell analyses, photophysiological parameters, and pCO 2 in and around the Mississippi River plume (MRP) in the Gulf of Mexico. Lower salinity (30-32) stations were dominated by rbcL mRNA concentrations from heterokonts, such as diatoms and pelagophytes, which were at least an order of magnitude greater than haptophytes, a-Synechococcus or high-light Prochlorococcus. However, rbcL transcript abundances were similar among these groups at oligotrophic stations (salinity 34-36). Diatom cell counts and heterokont rbcL RNA showed a strong negative correlation to seawater pCO 2 . While Prochlorococcus cells did not exhibit a large difference between low and high pCO 2 water, Prochlorococcus rbcL RNA concentrations had a strong positive correlation to pCO 2 , suggesting a very low level of RuBisCO RNA transcription among Prochlorococcus in the plume waters, possibly due to their relatively poor carbon concentrating mechanisms (CCMs). These results provide molecular evidence that diatom/pelagophyte productivity is largely responsible for the large CO 2 drawdown occurring in the MRP, based on the cooccurrence of elevated RuBisCO gene transcript concentrations from this group and reduced seawater pCO 2 levels. This may partly be due to efficient CCMs that enable heterokont eukaryotes such as diatoms to continue fixing CO 2 in the face of strong CO 2 drawdown. Our work represents the first attempt to relate in situ microbial gene expression to contemporaneous CO 2 flux measurements in the ocean.
PLoS ONE, 2012
Microbial genomic sequence analyses have indicated widespread horizontal gene transfer (HGT). How... more Microbial genomic sequence analyses have indicated widespread horizontal gene transfer (HGT). However, an adequate mechanism accounting for the ubiquity of HGT has been lacking. Recently, high frequencies of interspecific gene transfer have been documented, catalyzed by Gene Transfer Agents (GTAs) of marine a-Proteobacteria. It has been proposed that the presence of bacterial genes in highly purified viral metagenomes may be due to GTAs. However, factors influencing GTA-mediated gene transfer in the environment have not yet been determined. Several genomically sequenced strains containing complete GTA sequences similar to Rhodobacter capsulatus (RcGTA, type strain) were screened to ascertain if they produced putative GTAs, and at what abundance. Five of nine marine strains screened to date spontaneously produced virus-like particles (VLP's) in stationary phase. Three of these strains have demonstrated gene transfer activity, two of which were documented by this lab. These two strains Roseovarius nubinhibens ISM and Nitratireductor 44B9s, were utilized to produce GTAs designated RnGTA and NrGTA and gene transfer activity was verified in culture. Cell-free preparations of purified RnGTA and NrGTA particles from marked donor strains were incubated with natural microbial assemblages to determine the level of GTA-mediated gene transfer. In conjunction, several ambient environmental parameters were measured including lysogeny indicated by prophage induction. GTA production in culture systems indicated that approximately half of the strains produced GTA-like particles and maximal GTA counts ranged from 10-30% of host abundance. Modeling of GTA-mediated gene transfer frequencies in natural samples, along with other measured environmental variables, indicated a strong relationship between GTA mediated gene transfer and the combined factors of salinity, multiplicity of infection (MOI) and ambient bacterial abundance. These results indicate that GTA-mediated HGT in the marine environment with the strains examined is favored during times of elevated bacterial and GTA abundance as well as in areas of higher salinity.
Microbial Ecology, 1989
A method for the concentration and detection of gene sequences in the dissolved DNA from freshwat... more A method for the concentration and detection of gene sequences in the dissolved DNA from freshwater and marine environments has been developed. The limit of detection in the dot blot format was 167 fg/ml (100 ml sample) for exogenous herpes simplex thymidine kinase (TK) gene that was added to artificial seawater or river water. This procedure has been used to determine the longevity and monitor progressive changes in molecular weight of a plasmid containing the TK gene added to eutrophic estuarine water. The onset of plasmid degradation as determined by change in molecular weight was rapid (within 5 min). Intact plasmid was detected for at least 4 hours and sequences hybridizable to the TK gene probe were present for up to 24 hours.
Marine Biotechnology, 2007
The gene for the large subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase (rbcL) has been... more The gene for the large subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase (rbcL) has been shown to be a useful target for molecular assays that quantify form- or clade-specific RNA transcript concentrations as a proxy for the carbon fixation activity of marine phytoplankton. To improve the phylogenetic specificity and sensitivity of RNA probe hybridization methods, a quantitative reverse transcription-polymerase chain reaction (RT-PCR) assay has been reported for diatom and pelagophyte rbcL RNA. Here we detail enhancements made to this PCR method and development of additional assays to specifically quantify rbcL expression from haptophytes, Synechococcus and high-light Prochlorococcus. In vitro RNA transcripts were tested to demonstrate specificity and quantitative accuracy. Application of these methods on seawater samples from two depth profiles in the northern Gulf of Mexico showed a fair degree of agreement between PCR and hybridization results, with results for the chromophytic or form ID rbcL-containing organisms having better agreement between the two methods. Diatoms and other heterokonts were shown to be the primary carbon fixers at these locations by PCR, in agreement with greater form ID rbcL RNA measured by hybridization.
Journal of Infectious Diseases, 2014
Journal of Infection, 1996
Although significant bacteriuria and urinary tract infection are more common in immunocompetent w... more Although significant bacteriuria and urinary tract infection are more common in immunocompetent women than men, studies linking HIV immunosuppression with an increased risk of developing urinary infection have so far only been carried out in men. We therefore examined the relationship between bacteriuria and HIV status and CD4+cell count in a relatively homogeneous cohort of female commercial sex workers (CSW) attending a community clinic in Nairobi. Two hundred and twenty-two women were enrolled, and grouped according to HIV status and CD4 count. Group 1 were HIV seronegative (n = 52); Group 2 were HIV seropositive with CD4 + counts above 500 x 10(6)/l (n = 51); Group 3 were HIV seropositive with CD4 + counts between 201 and 500 x 10(6)/l (n = 67); Group 4 were HIV seropositive with CD4+counts below 200 x 10(6)/l (n = 52). Clinical signs and symptoms were noted and mid-stream specimens of urine obtained for culture and sensitivity. Overall 23% (50/222) had significant bacteriuria. The rates in each group respectively were 25%, 29%, 19% and 23% and there was no significant association between bacteriuria and HIV status; or between bacteriuria and level of immuno-suppression as indicated by CD4 + count. Overall 19% (30/222) of women had symptoms (frequency; dysuria; loin pain; smelly urine) or signs (fever; loin tenderness) compatible with urinary tract infection. However there was no significant association between symptoms or signs of infection and bacteriuria or HIV status. A typical range of pathogens, predominantly Enterobacteriaceae, were isolated and there were high rates of resistance to commonly used antimicrobials as well as 10% resistance to ciprofloxacin. Although high rates of significant bacteriuria can occur in highly sexually-active women, this appears unrelated to HIV infection or the level of HIV-related immunosuppression and is generally asymptomatic or clinically indistinct.
Hydrobiologia, 1991
... John H. Paul, Lisa H. Cazares, Andrew W. David, Mary F. DeFlaun' & Wade H. Jeffrey2 ... more ... John H. Paul, Lisa H. Cazares, Andrew W. David, Mary F. DeFlaun' & Wade H. Jeffrey2 Department of Marine Science, University of South ... July during the wet season, when seasonal flooding of area of leaf litter yielded high levels of dissolved organic carbon (DOC) which were ...
Harmful Algae, 2007
Blooms of Karenia brevis, the red tide forming dinoflagellate in the Gulf of Mexico, cause a myri... more Blooms of Karenia brevis, the red tide forming dinoflagellate in the Gulf of Mexico, cause a myriad of ecological and economic problems for coastal communities, including massive fish and mammal mortalities, and damage to tourism and fisheries/shellfish harvesting industries. There is a need for accurate detection and prediction of K. brevis blooms, including rapid and inexpensive monitoring of both water and shellfish meats to ensure the safety of shellfish harvested for human consumption. To address this issue, we have developed a protocol for easy field extraction of cellular RNA from water samples and coupled it with a handheld nucleic acid sequence-based amplification (NASBA) sensor that amplifies and detects target mRNA specific to the rbcL gene of K. brevis. This extraction protocol is a modified version of the Qiagen RNeasy Mini Kit spin protocol and requires no specialized equipment or training. Once extracted, the RNA is amplified and detected by NASBA in an in-house designed and produced handheld sensor that provides a real-time fluorescence plotting of the amplification. Both the field RNA extraction protocol and the handheld NASBA analyzer compared favorably to laboratory-based technologies. In duplicate reactions, the amplification curves generated with the handheld detector closely mirrored the curves generated with the bench top Nuclisens EasyQ NASBA analyzer and there was no difference in the sensitivity obtained using the handheld device versus the bench top models. This extraction protocol and detection sensor will be a valuable tool for rapidly monitoring K. brevis in field environments. #
Estuaries, 2005
The prophage induction assay provides a biologically based carcinogen-screening tool for environm... more The prophage induction assay provides a biologically based carcinogen-screening tool for environmental samples grounded in the parallel mechanisms of carcinogenesis and prophage induction. We developed an assay using a previously characterized marine bacterial Pseudomonas aeruginosa isolate designated as P94-4S3 for the detection of potentially genotoxic contamination in marine and estuarine environments. To perform the assay, the lysogenic isolate was exposed to either a known genotoxic compound or an environmental sample of interest. The response was considered positive when a statistically significant amount of prophage induction occurred in comparison to negative controls. Initial development of the assay for environmental samples included testing under a range of salinities and optimizing the method for the processing of water column and sediment samples. The assay has been field-tested over 2 yr in the Rookery Bay National Estuarine Research Reserve, Florida. The Marine Prophage Induction Assay (MPIA) was performed concurrently with laboratory toxicological analysis. There was good correspondence between positive MPIA results and detection of potentially toxic compounds by laboratory analysis. Five positive laboratory detections of known toxic compounds in natural samples occurred in conjunction with positive MPIA results. Two laboratory detections of compounds that are not genotoxic were accompanied by a negative MPIA response. Eight of the sediment samples contained detectable levels of arsenic. Four of these samples demonstrated a positive MPIA response, which may be due to the oxidation state of the arsenic within the sediment. One detection of a known toxic compound by the analytical laboratory was not accompanied by a positive induction response. Nine positive induction responses occurred without concurrent laboratory detection. This was possibly due to the limited range of compounds included in the laboratory testing performed, although false positive assay results cannot be ruled out.
Current Opinion in Biotechnology, 2005
Marine phages are the most abundant and diverse form of life on the planet, and their genomes hav... more 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.
Microbiology (Reading, England), 1999
The population structure of Streptococcus pneumoniae in a sample of 134 carried antibiotic-suscep... more The population structure of Streptococcus pneumoniae in a sample of 134 carried antibiotic-susceptible isolates, and 53 resistant and susceptible invasive isolates, was examined using a DNA-based version of multilocus enzyme electrophoresis: multilocus restriction typing (MLRT). This involved RFLP analysis of PCR products generated from nine loci of housekeeping genes located around the pneumococcal chromosome. The combination of alleles at each of the nine loci gave an allelic profile or restriction type (RT). All carried (throat or nasopharyngeal) isolates from children or adults in Oxford and Manchester, UK, and from an HIV-seropositive cohort in Nairobi, Kenya, showed an epidemic population structure. Twelve carried clonal groups, each with different serotypes, were identified at both locations within the UK. Almost all of the carried clones examined (16/17) were found to possess identical RTs or sequence types (STs) to invasive isolates, indicating that frequently carried clone...
Applied and environmental microbiology, 1997
A total of 68 marine samples from various sites impacted by sewage and storm waters were analyzed... more A total of 68 marine samples from various sites impacted by sewage and storm waters were analyzed by both the plaque assay and a reverse transcriptase (RT) PCR technique for F(sup+)-specific coliphage. The coliphage levels detected by the plaque assay averaged 1.90 x 10(sup4) PFU/100.0 ml. Using a most probable number (MPN) PCR approach, the levels averaged 2.40 x 10(sup6) MPN-PCR units/100.0 ml. Two samples were positive by RT-PCR but negative by plaque assay, and 12 samples were positive by plaque assay but negative by RT-PCR (levels lower than 11.00 PFU/100.0 ml). The host system used for the plaque assay may detect somatic coliphage in addition to the F(sup+)-specific coliphage. When it is used as an indicator of pollution, contamination may be missed with more restrictive systems. The difference in results may be due to the sensitivity, specificity, or inhibition of RT-PCR in marine samples. This study provides information on quantifying PCR results by an MPN method and insight...
Applied and environmental microbiology, 1998
To determine the potential for bacteriophage-mediated gene transfer in the marine environment, we... more To determine the potential for bacteriophage-mediated gene transfer in the marine environment, we established transduction systems by using marine phage host isolates. Plasmid pQSR50, which contains transposon Tn5 and encodes kanamycin and streptomycin resistance, was used in plasmid transduction assays. Both marine bacterial isolates and concentrated natural bacterial communities were used as recipients in transduction studies. Transductants were detected by a gene probe complementary to the neomycin phosphotransferase (nptII) gene in Tn5. The transduction frequencies ranged from 1.33 x 10(-7) to 5.13 x 10(-9) transductants/PFU in studies performed with the bacterial isolates. With the mixed bacterial communities, putative transductants were detected in two of the six experiments performed. These putative transductants were confirmed and separated from indigenous antibiotic-resistant bacteria by colony hybridization probed with the nptII probe and by PCR amplification performed wit...
Applied and environmental microbiology, 1998
To understand the ecological and genetic role of viruses in the marine environment, it is critica... more To understand the ecological and genetic role of viruses in the marine environment, it is critical to know the infectivity of viruses and the types of interactions that occur between marine viruses and their hosts. We isolated four marine phages from turbid plaques by using four indigenous bacterial hosts obtained from concentrated water samples from Mamala Bay, Oahu, Hawaii. Two of the rod-shaped bacterial hosts were identified as Sphingomonas paucimobilis and Flavobacterium sp. All of the phage isolates were tailed phages and contained double-stranded DNA. Two of the phage isolates had morphologies typical of the family Siphoviridae, while the other two belonged to the families Myoviridae and Podoviridae. The head diameters of these viruses ranged from 47 to 70.7 nm, and the tail lengths ranged from 12 to 146 nm. The burst sizes ranged from 7.8 to 240 phage/bacterial cell, and the genome sizes, as determined by restriction digestion, ranged from 36 to 112 kb. The members of the Si...
Biochimica et Biophysica Acta (BBA) - Nucleic Acids and Protein Synthesis, 1974
Soluble chromatin can be banded isopycnically in metrizamide gradients without prior fixation. Th... more Soluble chromatin can be banded isopycnically in metrizamide gradients without prior fixation. The chromatin--DNA of minimally sheared chromatin (750--1000 base-pairs) bands as a single, sharp peak, almost completely separated from the chromatin ribonucleoprotein particles. More extensive shearing (to about 360 base-pairs) leads to a bimodal distribution of the chromatin--DNA in the gradients; all of the DNA is complexed with proteins.
Clinical infectious diseases : an official publication of the Infectious Diseases Society of America, 2014
The ISME Journal, 2007
River plumes deliver large quantities of nutrients to oligotrophic oceans, often resulting in sig... more River plumes deliver large quantities of nutrients to oligotrophic oceans, often resulting in significant CO 2 drawdown. To determine the relationship between expression of the major gene in carbon fixation (large subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase, RuBisCO) and CO 2 dynamics, we evaluated rbcL mRNA abundance using novel quantitative PCR assays, phytoplankton cell analyses, photophysiological parameters, and pCO 2 in and around the Mississippi River plume (MRP) in the Gulf of Mexico. Lower salinity (30-32) stations were dominated by rbcL mRNA concentrations from heterokonts, such as diatoms and pelagophytes, which were at least an order of magnitude greater than haptophytes, a-Synechococcus or high-light Prochlorococcus. However, rbcL transcript abundances were similar among these groups at oligotrophic stations (salinity 34-36). Diatom cell counts and heterokont rbcL RNA showed a strong negative correlation to seawater pCO 2 . While Prochlorococcus cells did not exhibit a large difference between low and high pCO 2 water, Prochlorococcus rbcL RNA concentrations had a strong positive correlation to pCO 2 , suggesting a very low level of RuBisCO RNA transcription among Prochlorococcus in the plume waters, possibly due to their relatively poor carbon concentrating mechanisms (CCMs). These results provide molecular evidence that diatom/pelagophyte productivity is largely responsible for the large CO 2 drawdown occurring in the MRP, based on the cooccurrence of elevated RuBisCO gene transcript concentrations from this group and reduced seawater pCO 2 levels. This may partly be due to efficient CCMs that enable heterokont eukaryotes such as diatoms to continue fixing CO 2 in the face of strong CO 2 drawdown. Our work represents the first attempt to relate in situ microbial gene expression to contemporaneous CO 2 flux measurements in the ocean.
PLoS ONE, 2012
Microbial genomic sequence analyses have indicated widespread horizontal gene transfer (HGT). How... more Microbial genomic sequence analyses have indicated widespread horizontal gene transfer (HGT). However, an adequate mechanism accounting for the ubiquity of HGT has been lacking. Recently, high frequencies of interspecific gene transfer have been documented, catalyzed by Gene Transfer Agents (GTAs) of marine a-Proteobacteria. It has been proposed that the presence of bacterial genes in highly purified viral metagenomes may be due to GTAs. However, factors influencing GTA-mediated gene transfer in the environment have not yet been determined. Several genomically sequenced strains containing complete GTA sequences similar to Rhodobacter capsulatus (RcGTA, type strain) were screened to ascertain if they produced putative GTAs, and at what abundance. Five of nine marine strains screened to date spontaneously produced virus-like particles (VLP's) in stationary phase. Three of these strains have demonstrated gene transfer activity, two of which were documented by this lab. These two strains Roseovarius nubinhibens ISM and Nitratireductor 44B9s, were utilized to produce GTAs designated RnGTA and NrGTA and gene transfer activity was verified in culture. Cell-free preparations of purified RnGTA and NrGTA particles from marked donor strains were incubated with natural microbial assemblages to determine the level of GTA-mediated gene transfer. In conjunction, several ambient environmental parameters were measured including lysogeny indicated by prophage induction. GTA production in culture systems indicated that approximately half of the strains produced GTA-like particles and maximal GTA counts ranged from 10-30% of host abundance. Modeling of GTA-mediated gene transfer frequencies in natural samples, along with other measured environmental variables, indicated a strong relationship between GTA mediated gene transfer and the combined factors of salinity, multiplicity of infection (MOI) and ambient bacterial abundance. These results indicate that GTA-mediated HGT in the marine environment with the strains examined is favored during times of elevated bacterial and GTA abundance as well as in areas of higher salinity.
Microbial Ecology, 1989
A method for the concentration and detection of gene sequences in the dissolved DNA from freshwat... more A method for the concentration and detection of gene sequences in the dissolved DNA from freshwater and marine environments has been developed. The limit of detection in the dot blot format was 167 fg/ml (100 ml sample) for exogenous herpes simplex thymidine kinase (TK) gene that was added to artificial seawater or river water. This procedure has been used to determine the longevity and monitor progressive changes in molecular weight of a plasmid containing the TK gene added to eutrophic estuarine water. The onset of plasmid degradation as determined by change in molecular weight was rapid (within 5 min). Intact plasmid was detected for at least 4 hours and sequences hybridizable to the TK gene probe were present for up to 24 hours.
Marine Biotechnology, 2007
The gene for the large subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase (rbcL) has been... more The gene for the large subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase (rbcL) has been shown to be a useful target for molecular assays that quantify form- or clade-specific RNA transcript concentrations as a proxy for the carbon fixation activity of marine phytoplankton. To improve the phylogenetic specificity and sensitivity of RNA probe hybridization methods, a quantitative reverse transcription-polymerase chain reaction (RT-PCR) assay has been reported for diatom and pelagophyte rbcL RNA. Here we detail enhancements made to this PCR method and development of additional assays to specifically quantify rbcL expression from haptophytes, Synechococcus and high-light Prochlorococcus. In vitro RNA transcripts were tested to demonstrate specificity and quantitative accuracy. Application of these methods on seawater samples from two depth profiles in the northern Gulf of Mexico showed a fair degree of agreement between PCR and hybridization results, with results for the chromophytic or form ID rbcL-containing organisms having better agreement between the two methods. Diatoms and other heterokonts were shown to be the primary carbon fixers at these locations by PCR, in agreement with greater form ID rbcL RNA measured by hybridization.
Journal of Infectious Diseases, 2014
Journal of Infection, 1996
Although significant bacteriuria and urinary tract infection are more common in immunocompetent w... more Although significant bacteriuria and urinary tract infection are more common in immunocompetent women than men, studies linking HIV immunosuppression with an increased risk of developing urinary infection have so far only been carried out in men. We therefore examined the relationship between bacteriuria and HIV status and CD4+cell count in a relatively homogeneous cohort of female commercial sex workers (CSW) attending a community clinic in Nairobi. Two hundred and twenty-two women were enrolled, and grouped according to HIV status and CD4 count. Group 1 were HIV seronegative (n = 52); Group 2 were HIV seropositive with CD4 + counts above 500 x 10(6)/l (n = 51); Group 3 were HIV seropositive with CD4 + counts between 201 and 500 x 10(6)/l (n = 67); Group 4 were HIV seropositive with CD4+counts below 200 x 10(6)/l (n = 52). Clinical signs and symptoms were noted and mid-stream specimens of urine obtained for culture and sensitivity. Overall 23% (50/222) had significant bacteriuria. The rates in each group respectively were 25%, 29%, 19% and 23% and there was no significant association between bacteriuria and HIV status; or between bacteriuria and level of immuno-suppression as indicated by CD4 + count. Overall 19% (30/222) of women had symptoms (frequency; dysuria; loin pain; smelly urine) or signs (fever; loin tenderness) compatible with urinary tract infection. However there was no significant association between symptoms or signs of infection and bacteriuria or HIV status. A typical range of pathogens, predominantly Enterobacteriaceae, were isolated and there were high rates of resistance to commonly used antimicrobials as well as 10% resistance to ciprofloxacin. Although high rates of significant bacteriuria can occur in highly sexually-active women, this appears unrelated to HIV infection or the level of HIV-related immunosuppression and is generally asymptomatic or clinically indistinct.
Hydrobiologia, 1991
... John H. Paul, Lisa H. Cazares, Andrew W. David, Mary F. DeFlaun' & Wade H. Jeffrey2 ... more ... John H. Paul, Lisa H. Cazares, Andrew W. David, Mary F. DeFlaun' & Wade H. Jeffrey2 Department of Marine Science, University of South ... July during the wet season, when seasonal flooding of area of leaf litter yielded high levels of dissolved organic carbon (DOC) which were ...
Harmful Algae, 2007
Blooms of Karenia brevis, the red tide forming dinoflagellate in the Gulf of Mexico, cause a myri... more Blooms of Karenia brevis, the red tide forming dinoflagellate in the Gulf of Mexico, cause a myriad of ecological and economic problems for coastal communities, including massive fish and mammal mortalities, and damage to tourism and fisheries/shellfish harvesting industries. There is a need for accurate detection and prediction of K. brevis blooms, including rapid and inexpensive monitoring of both water and shellfish meats to ensure the safety of shellfish harvested for human consumption. To address this issue, we have developed a protocol for easy field extraction of cellular RNA from water samples and coupled it with a handheld nucleic acid sequence-based amplification (NASBA) sensor that amplifies and detects target mRNA specific to the rbcL gene of K. brevis. This extraction protocol is a modified version of the Qiagen RNeasy Mini Kit spin protocol and requires no specialized equipment or training. Once extracted, the RNA is amplified and detected by NASBA in an in-house designed and produced handheld sensor that provides a real-time fluorescence plotting of the amplification. Both the field RNA extraction protocol and the handheld NASBA analyzer compared favorably to laboratory-based technologies. In duplicate reactions, the amplification curves generated with the handheld detector closely mirrored the curves generated with the bench top Nuclisens EasyQ NASBA analyzer and there was no difference in the sensitivity obtained using the handheld device versus the bench top models. This extraction protocol and detection sensor will be a valuable tool for rapidly monitoring K. brevis in field environments. #
Estuaries, 2005
The prophage induction assay provides a biologically based carcinogen-screening tool for environm... more The prophage induction assay provides a biologically based carcinogen-screening tool for environmental samples grounded in the parallel mechanisms of carcinogenesis and prophage induction. We developed an assay using a previously characterized marine bacterial Pseudomonas aeruginosa isolate designated as P94-4S3 for the detection of potentially genotoxic contamination in marine and estuarine environments. To perform the assay, the lysogenic isolate was exposed to either a known genotoxic compound or an environmental sample of interest. The response was considered positive when a statistically significant amount of prophage induction occurred in comparison to negative controls. Initial development of the assay for environmental samples included testing under a range of salinities and optimizing the method for the processing of water column and sediment samples. The assay has been field-tested over 2 yr in the Rookery Bay National Estuarine Research Reserve, Florida. The Marine Prophage Induction Assay (MPIA) was performed concurrently with laboratory toxicological analysis. There was good correspondence between positive MPIA results and detection of potentially toxic compounds by laboratory analysis. Five positive laboratory detections of known toxic compounds in natural samples occurred in conjunction with positive MPIA results. Two laboratory detections of compounds that are not genotoxic were accompanied by a negative MPIA response. Eight of the sediment samples contained detectable levels of arsenic. Four of these samples demonstrated a positive MPIA response, which may be due to the oxidation state of the arsenic within the sediment. One detection of a known toxic compound by the analytical laboratory was not accompanied by a positive induction response. Nine positive induction responses occurred without concurrent laboratory detection. This was possibly due to the limited range of compounds included in the laboratory testing performed, although false positive assay results cannot be ruled out.
Current Opinion in Biotechnology, 2005
Marine phages are the most abundant and diverse form of life on the planet, and their genomes hav... more 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.