Biomedical subjects

R T Hill

Publications and source records attributed to R T Hill.

At least 19 recordsLinked to original sources

PubMed2001

Chromium accumulation by two Streptomyces spp. isolated from riverine sediments.

Strains designated R22 and R25, isolated from Salí River sediments, Argentina, were highly resistant to chromium. These strains were shown by 16S rRNA sequencing studies to be Streptomyces spp.; this affiliation was consistent with morphological and chemical characteristics. Growth of strains R22 and R25 in medium containing 100 mg l(-1) chromate was reduced by only 23% and 34%, respectively, compared with growth in medium without added chromium. Streptomyces sp. strains R22 and R25 both accumulated chromium with yields of 10.0 and 5.6 mg Cr g(-1) of dry weight, respectively, and a chromate concentration of 50 mg ml(-1). Cell fractionation studies with strain R22 showed that the great majority of the chromium were associated with the cell wall fraction. Streptomyces strains R22 and R25 may have applications in bioremediation of chromium contamination.

Biodegradation, Environmental↗

PubMed2001

The effects of copper on the microbial community of a coral reef sponge.

Marine sponges often harbour communities of symbiotic microorganisms that fulfil necessary functions for the well-being of their hosts. Microbial communities associated with the sponge Rhopaloeides odorabile were used as bioindicators for sublethal cupric ion (Cu2+) stress. A combined strategy incorporating molecular, cultivation and electron microscopy techniques was adopted to monitor changes in microbial diversity. The total density of sponge-associated bacteria and counts of the predominant cultivated symbiont (alpha-proteobacterium strain NW001) were significantly reduced in response to Cu2+ concentrations of 1.7 microg l(-1) and above after 14 days of exposure. The number of operational taxonomic units (OTUs) detected by restriction fragment length polymorphism (RFLP) decreased by 64% in sponges exposed to 223 microg l(-1) Cu2+ for 48 h and by 46% in sponges exposed to 19.4 microg l(-1) Cu2+ for 14 days. Electron microscopy was used to identify 17 predominant bacterial morphotypes, composing 47% of the total observed cells in control sponges. A reduction in the proportion of these morphotypes to 25% of observed cells was evident in sponges exposed to a Cu2+ concentration of 19.4 microg l(-1). Although the abundance of most morphotypes decreased under Cu2+ stress, three morphotypes were not reduced in numbers and a single morpho-type actually increased in abundance. Bacterial numbers, as detected using fluorescence in situ hybridization (FISH), decreased significantly after 48 h exposure to 19.4 microg l(-1) Cu2+. Archaea, which are normally prolific in R. odorabile, were not detected after exposure to a Cu2+ concentration of 19.4 microg l(-1) for 14 days, indicating that many of the microorganisms associated with R. odorabile are sensitive to free copper. Sponges exposed to a Cu2+ concentration of 223 microg l(-1) became highly necrosed after 48 h and accumulated 142 +/- 18 mg kg(-1) copper, whereas sponges exposed to 19.4 microg l(-1) Cu2+ accumulated 306 +/- 15 mg kg(-1) copper after 14 days without apoptosis or mortality. Not only do sponges have potential for monitoring elevated concentrations of heavy metals but also examining changes in their microbial symbionts is a novel and sensitive bioindicator for the assessment of pollution on important microbial communities.

Alphaproteobacteria↗

PubMed2001

Phylogenetic diversity of bacteria associated with the marine sponge Rhopaloeides odorabile.

Molecular techniques were employed to document the microbial diversity associated with the marine sponge Rhopaloeides odorabile. The phylogenetic affiliation of sponge-associated bacteria was assessed by 16S rRNA sequencing of cloned DNA fragments. Fluorescence in situ hybridization (FISH) was used to confirm the presence of the predominant groups indicated by 16S rDNA analysis. The community structure was extremely diverse with representatives of the Actinobacteria, low-G+C gram-positive bacteria, the beta- and gamma-subdivisions of the Proteobacteria, Cytophaga/Flavobacterium, green sulfur bacteria, green nonsulfur bacteria, planctomycetes, and other sequence types with no known close relatives. FISH probes revealed the spatial location of these bacteria within the sponge tissue, in some cases suggesting possible symbiotic functions. The high proportion of 16S rRNA sequences derived from novel actinomycetes is good evidence for the presence of an indigenous marine actinomycete assemblage in R. odorabile. High microbial diversity was inferred from low duplication of clones in a library with 70 representatives. Determining the phylogenetic affiliation of sponge-associated microorganisms by 16S rRNA analysis facilitated the rational selection of culture media and isolation conditions to target specific groups of well-represented bacteria for laboratory culture. Novel media incorporating sponge extracts were used to isolate bacteria not previously recovered from this sponge.

Actinobacteria↗

PubMed2000

A proposal for the reclassification of Bdellovibrio stolpii and Bdellovibrio starrii into a new genus, Bacteriovorax gen. nov. as Bacteriovorax stolpii comb. nov. and Bacteriovorax starrii comb. nov., respectively.

Bdellovibrios are unique bacteria with the ability to prey upon a wide variety of susceptible Gram-negative bacteria. Micro-organisms exhibiting this trait have been included in the genus Bdellovibrio despite their isolation from diverse habitats and relatively unstudied taxonomic relatedness. In this study, 16S rDNA sequences were compared from known terrestrial Bdellovibrio species, Bdellovibrio bacteriovorus 100T, Bdellovibrio stolpii Uki2T and Bdellovibrio starrii A3.12T in order to study their phylogenetic relationship. The two sequences from B. stolpii Uki2T and B. starrii A3.12T were 90.0% similar to each other but exhibited only 81.7% and 81.2% similarity, respectively to B. bacteriovorus 100T. Phylogenetic analysis indicated that B. bacteriovorus 100T clustered in a separate clade from B. starrii A3.12T and B. stolpii Uki2T, demonstrating only a distant relationship between B. bacteriovorus 100T and the other two recognized type species. DNA-DNA hybridization experiments also demonstrated <4% hybridization between these three species. On the basis of the results obtained from the phylogenetic analysis and DNA-DNA hybridization studies, it is proposed that B. stolpii Uki2T and B. starrii A3.12T should be transferred to a new genus, Bacteriovorax gen. nov. as Bacteriovorax stolpii comb. nov. and Bacteriovorax starrii comb. nov., respectively. It is also proposed that the type species for the new genus Bacteriovorax should be Bacteriovorax stolpii comb. nov.

Base Composition↗

PubMed2000

Interspecific transfer of Streptomyces giant linear plasmids in sterile amended soil microcosms.

The interspecific transfer of two giant linear plasmids was investigated in sterile soil microcosms. Plasmids pRJ3L (322 kb) and pRJ28 (330 kb), both encoding mercury resistance, were successfully transferred in amended soil microcosms from their streptomycete hosts, the isolates CHR3 and CHR28, respectively, to a plasmidless and mercury-sensitive strain, Streptomyces lividans TK24. Transconjugants of S. lividans TK24 were first observed after 2 to 3 days of incubation at 30 degrees C, which corresponded to the time taken for the formation of mycelia in soil. Transfer frequencies were 4.8 x 10(-4) and 3.6 x 10(-5) CFU/donor genome for pRJ3L and pRJ28, respectively. Transconjugants were analyzed by pulsed-field gel electrophoresis for the presence of plasmids, and plasmid identity was confirmed by restriction digests. Total genomic DNA digests confirmed that transconjugants were S. lividans TK24. The mercury resistance genes were shown to be on the plasmid in the transconjugants by hybridization analysis and were still functional. This is the first demonstration of transfer of giant linear plasmids in sterile soil microcosms. Giant linear plasmids were detected in many Streptomyces spp. isolated from mercury-contaminated sediments from Boston Harbor (United States), Townsville Harbor (Australia), and the Sali River (Tucuman, Argentina). Mercury resistance genes were shown to be present on some of these plasmids. Our findings that giant linear plasmids can be transferred between Streptomyces spp. and are common in environmental Streptomyces isolates suggest that these plasmids are important in gene transfer between streptomycetes in the environment.

Blotting, Southern↗

PubMed2000

Cloning and sequence analysis of the mercury resistance operon of Streptomyces sp. Strain CHR28 reveals a novel putative second regulatory gene.

A DNA library of pRJ28, a large linear plasmid encoding mercury resistance, was constructed, and the mercury resistance genes were cloned. The 5,921-bp sequence was analyzed and showed a high degree of similarity to the Streptomyces lividans 1326 mercury resistance operon. Genes merR, merT, merP, and orfIV were found in a similar order and in a single transcription unit. merA and merB were found to be transcribed in the opposite direction to genes merR, merT, merP, and orfIV, as in S. lividans 1326. A novel putative regulatory gene, orfX, was found 22 bp downstream of merA. orfX encodes a 137-amino acid protein with a potential helix-turn-helix motif in the N-terminal domain, characteristic of the MerR family of transcriptional regulators. Transcriptional studies showed that orfX is cotranscribed with merA and merB. It is hypothesized that orfX plays a role in the regulation of the mercury resistance operon, probably by binding at the MerR operator site.

Amino Acid Sequence↗

PubMed1998

Mercury-resistant actinomycetes from the Chesapeake Bay.

Two actinomycete strains, CHR3 and CHR28, were isolated from metal-contaminated sediments from Baltimore Inner Harbor. The isolates were classified as Streptomyces spp. Agar diffusion assays showed both isolates to be resistant to mercuric chloride and phenylmercuric acetate. Hybridization experiments indicated that genes homologous to the mercuric reductase and organomercurial lyase of Streptomyces lividans 1326 were present in strains CHR3 and CHR28. Strain CHR28 grew at both low and high salt concentrations; however, strain CHR3 showed enhanced growth in the presence of salt, evidence of its habitat being marine or estuarine sediment.

Baltimore↗

PubMed1998

Mercury resistance is encoded by transferable giant linear plasmids in two chesapeake bay Streptomyces strains.

The Streptomyces strains CHR3 and CHR28, isolated from the Baltimore Inner Harbor, contained two and one, respectively, giant linear plasmids which carry terminally bound proteins. The plasmids pRJ3L (322 kb), from CHR3, and pRJ28 (330 kb), from CHR28, carry genes homologous to the previously characterized chromosomal Streptomyces lividans 66 operon encoding resistance against mercuric compounds. Both plasmids are transmissible (without any detectable rearrangement) to the chloramphenicol-resistant S. lividans TK24 strain lacking plasmids and carrying a chromosomal deletion of the mer operon. S. lividans TK24 conjugants harboring pRJ3L or pRJ28 exhibited profiles of mercury resistance to mercuric compounds similar to those of Streptomyces strains CHR3 and CHR28.

Baltimore↗

PubMed1996

Distribution of Vibrio vulnificus in the Chesapeake Bay.

Vibrio vulnificus is a potentially lethal human pathogen capable of producing septicemia in susceptible persons. Disease is almost always associated with consumption of seafood, particularly raw oysters, or with exposure of wounds to seawater. An oligonucleotide DNA probe (V. vulnificus alkaline phosphatase-labeled DNA probe [VVAP]), previously shown to be highly specific for V. vulnificus, was used to enumerate this species in environmental samples collected from the Chesapeake Bay between April 1991 and December 1992. Total aerobic, heterotrophic, culturable bacteria were enumerated by plate counts on nonselective medium. The number of V. vulnificus organisms was determined by colony lifts of spread plates for subsequent hybridization with VVAP. V. vulnificus was not detected in any samples collected during February and March (water temperature of < 8 degrees C) but was found in 80% of the water samples collected during May, July, September, and December (water temperature of > 8 degrees C), with concentrations ranging from 3.0 x 10(1) to 2.1 x 10(2)/ml (ca. 8% of the total culturable heterotrophic bacteria). In a multiple regression analysis, increased V. vulnificus concentrations were correlated with lower salinities and with isolation from samples collected closer to the bottom. Isolation from oysters was demonstrable when water temperatures were 7.6 degrees C, with concentrations ranging from 1.0 x 10(3) to 4.7 x 10(4)/g (ca. 12% of total culturable bacteria). In samples collected in May and July, V. vulnificus was identified in seven of seven plankton samples and four of nine sediment samples. Our data demonstrate that V. vulnificus is a widespread and important component of the bacterial population of the Chesapeake Bay, with counts that are comparable to those reported from the Gulf of Mexico.

Animals↗

PubMed1996

Effects of sunlight on bacteriophage viability and structure.

Current estimates of viral abundance in natural waters rely on direct counts of virus-like particles (VLPs), using either transmission or epifluorescence microscopy. Direct counts of VLPs, while useful in studies of viral ecology, do not indicate whether the observed VLPs are capable of infection and/or replication. Rapid decay in bacteriophage viability under environmental conditions has been observed. However, it has not been firmly established whether there is a corresponding degradation of the virus particles. To address this question, viable and direct counts were carried out employing two Chesapeake Bay bacteriophages in experimental microcosms incubated for 56 h at two depths in the York River estuary. Viruses incubated in situ in microcosms at the surface yielded decay rates in full sunlight of 0.11 and 0.06 h-1 for CB 38 phi and CB 7 phi, respectively. The number of infective particles in microcosms in the dark and at a depth of 1 m was not significantly different from laboratory controls, with decay rates averaging 0.052 h-1 for CB 38 phi and 0.037 h-1 for CB 7 phi. Direct counts of bacteriophages decreased in teh estuarine microcosms, albeit only at a rate of 0.028 h-1, and were independent of treatment. Destruction of virus particles is concluded to be a process separate from loss of infectivity. It is also concluded that strong sunlight affects the viability of bacteriophages in surface waters, with the result that direct counts of VLPs overestimate the number of bacteriophage capable of both infection and replication. However, in deeper waters, where solar radiation is not a significant factor, direct counts should more accurately estimate numbers of viable bacteriophage.

Bacteriophages↗

PubMed1996

Distribution of sewage indicated by Clostridium perfringens at a deep-water disposal site after cessation of sewage disposal.

Clostridium perfringens, a marker of domestic sewage contamination, was enumerated in sediment samples obtained from the vicinity of the 106-Mile Site 1 month and 1 year after cessation of sewage disposal at this site. C. perfringens counts in sediments collected at the disposal site and from stations 26 nautical miles (ca. 48 km) and 50 nautical miles (ca. 92 km) to the southwest of the site were, in general, more than 10-fold higher than counts from an uncontaminated reference site. C. perfringens counts at the disposal site were not significantly different between 1992 and 1993, suggesting that sewage sludge had remained in the benthic environment at this site. At stations where C. perfringens counts were elevated (i.e., stations other than the reference station), counts were generally higher in the top 1 cm and decreased down to 5 cm. In some cases, C. perfringens counts in the bottom 4 or 5 cm showed a trend of higher counts in 1993 than in 1992, suggesting bioturbation. We conclude that widespread sludge contamination of the benthic environment has persisted for at least 1 year after cessation of ocean sewage disposal at the 106-Mile Site.

Clostridium perfringens↗

PubMed1995

Temperature-induced recovery of Vibrio cholerae from the viable but nonculturable state: growth or resuscitation?

Vibrio cholerae cells were incubated at 4 degrees C in nutrient-limited artificial seawater (ASW) microcosms. Plate counts declined from 8 x 10(5) to less than 2 c.f.u. ml-1 in about 23 d. When samples of microcosms were shifted to 30 degrees C, plate counts increased to 2.2 x 10(5) c.f.u. ml-1 in 72 h. An experiment was performed to determine whether culturable cells obtained after temperature upshifts were the result of 'resuscitation', or outgrowth, of nonculturable cells or of cell division and growth of the few culturable cells that remained in samples. Prior to temperature upshift, samples from the microcosms were diluted 10- and 100-fold in filter-sterilized (0.1 microns) ASW from the microcosms. Undiluted, 1/10, and 1/100 diluted samples recovered culturability to about 2.2 x 10(5) c.f.u. ml-1 within 72 h of temperature upshift. If resuscitation of nonculturable cells had occurred, the resultant number of culturable cells in diluted samples would have been 1/10 and 1/100 that of undiluted samples, respectively. In microcosms where plate counts had declined to less than 1 c.f.u. ml-1, 1/100 diluted samples did not regain culturability, i.e. no culturable cells remained from which growth could occur. Our conclusions are that in the experiments reported here, recovery of culturable cells on temperature upshifts resulted from growth and that there were no growth-inhibiting factors in the spent growth medium, supported by the finding that about 10(2) recovered V. cholerae cells ml-1 inoculated into filter-sterilized microcosm ASW grew to about 6.2 x 10(5) c.f.u. ml-1 in 24 h, confirming that V. cholerae is capable of significant growth in ASW.

Colony Count, Microbial↗

PubMed1995

Decrease in culturability of Vibrio cholerae caused by glucose.

The culturability of Vibrio cholerae O1 serotype Inaba strain 569B was decreased by the addition of glucose to cell suspensions in starvation media. A similar effect was observed with sucrose, maltose, and fructose. We term this inhibitory effect glucose shock. It was not observed with arabinose or xylose or with carboxylates, such as acetate and pyruvate. No acidification of the medium occurred in the presence of these carbohydrates. Glucose shock was prevented by the addition of nitrogen or phosphorus sources. In the presence of phosphate, the bacterium produced formic acid from glucose. The phenomenon of glucose shock was also observed in V. cholerae O1 serotype Inaba strain RIMD 2203082 but not in strain RIMD 2203088 (O1 Inaba), IID 936 (O1 Ogawa), or RIMD 2214034 (non-O1). The culturability of Escherichia coli, Enterobacter aerogenes, and Listonella anguillarum did not decrease in starvation media with added glucose. Hence, the phenomenon should have ecological significance in determining the distribution of bacteria in marine ecosystems in situations where carbohydrates are abundant, but nitrogen and phosphorus are limiting.

Glucose↗

PubMed1994

Isolation of a Vibrio cholerae transposon-mutant with an altered viable but nonculturable response.

We have isolated more than 2500 mutants of Vibrio cholerae by using transposon mutagenesis. Mutants were screened under low nutrient conditions in artificial seawater for an altered viable but nonculturable response, compared to the wild-type. Mutant JR09H1 entered the viable but nonculturable state more rapidly than the wild-type at both 25 degrees C and 4 degrees C.

Bacteriological Techniques↗

PubMed1994

A gene for the enterotoxin zonula occludens toxin is present in Vibrio mimicus and Vibrio cholerae O139.

The presence of the zonula occludens toxin (ZOT) gene, which encodes an enterotoxin produced by serotype O1 strains of the pathogenic bacterium, Vibrio cholerae, in addition to cholera toxin, was investigated in selected strains of V. mimicus and the new pandemic V. cholerae non-O1 serotype O139. The zot gene was detected by polymerase chain reaction (PCR) amplification, using sets of primers based on the sequence of the V. cholerae O1 zot sequence. PCR amplification of genomic DNAs of both cholera toxin gene (ctx) positive and ctx- strains of V. mimicus detected the presence of zot gene. An AccI-EcoRV V. cholerae zot gene fragment designed to overlap PCR products was used as a probe. Southern hybridization studies confirmed that the PCR fragments from V. mimicus and V. cholerae O139 were strongly homologous to the V. cholerae O1 zot gene. The zot gene was found with 3 of 5 strains of V. mimicus of which only one strain harbored the ctx gene. The presence of a zot gene in ctx- toxigenic V. mimicus indicates a possible role of ZOT in the toxigenicity of this species. We conclude that, in addition to ctx, V. mimicus and V. cholerae O139 have the potential to produce ZOT.

Base Sequence↗

PubMed1992

Distribution of viruses in the Chesapeake Bay.

High virus counts were found in water samples collected from the Chesapeake Bay. Viruses were enumerated by ultracentrifugation of water samples onto grids which were visualized by transmission electron microscopy. Virus counts in September 1990, April 1991, June 1991, August 1991, and October 1991 ranged between 2.6 x 10(6) and 1.4 x 10(8) viruses ml-1 with a mean of 2.5 x 10(7) viruses ml-1. Virus counts were usually at least three times higher than direct bacterial counts in corresponding samples. Virus counts in August and October were significantly higher than at the other sampling times, whereas bacterial counts were significantly lower at that time, yielding mean virus-to-bacterium ratios of 12.6 and 25.6, respectively. From analysis of morphology of the virus particles, it is concluded that a large proportion of the viruses are bacteriophages. The high virus counts obtained in this study suggest that viruses may be an important factor affecting bacterial populations in the Chesapeake Bay, with implications for gene transfer in natural aquatic bacterial populations and release of genetically engineered microorganisms to estuarine and coastal environments.

Bacteria↗

PubMed1989

Development of pLR591, a Streptomyces-Escherichia coli positive selection shuttle vector.

The Escherichia coli positive selection vector pEcoR251 was ligated with the broad host range, high copy number Streptomyces plasmid pIJ702 to produce pLR591, a Streptomyces-E. coli positive selection shuttle vector. The EcoRI and thiostrepton resistance genes of pLR591 were expressed in E. coli and Streptomyces lividans respectively. The positive selection shuttle vector pLR591 facilitates the construction in E. coli of genomic libraries which can be screened in Streptomyces strains.

Cloning, Molecular↗

PubMed1989

Molecular analysis of a novel glutamine synthetase of the anaerobe Bacteroides fragilis.

The nucleotide sequence of a 2777 bp DNA segment containing the Bacteroides fragilis glnA gene was determined. The B. fragilis glnA open reading frame of 2187 bp encoded a glutamine synthetase (GS) subunit of 729 amino acid residues with a calculated Mr of 82,827. The apparent Mr of the GS subunit determined by SDS-PAGE was approximately 75,000. A single mRNA transcription start point was identified upstream of the B. fragilis glnA open reading frame. The B. fragilis GS subunit is approximately 270 and 400 amino acids longer than the GSI and GSII subunits, respectively, of other prokaryotes and eukaryotes. The GSI and GSII holoenzymes are dodecamers and octamers respectively, whereas the GS of B. fragilis is a hexamer. Although GSI and GSII subunits show amino acid similarity in five conserved regions, this similarity is not strongly conserved in the B. fragilis GS. The GS of B. fragilis is not regulated by adenylylation and lacks the adenylylation site. It also lacks the Trp residue associated with the active site in GSI and GSII enzymes from other prokaryotes and eukaryotes.

Amino Acid Sequence↗