Role of plasmids in mercury transformation by bacteria isolated from the aquatic environment.

Eight mercury-resistant bacterial strains isolated from the Chesapeake Bay and one strain isolated from the Cayman Trench were examined for ability to volatilize mercury. Mercury volatilization was found to be variable in the strains tested. In addition, plasmids were detected in all strains. After curing, two of the bacterial strains lost mercury resistance, indicating that volatilization is plasmid mediated in these strains. Only two cultures demonstrated ability to methylate mercuric chloride under either aerobic or anaerobic conditions. Methylation of mercury, compared with volatilization, appears to be mediated by a separate genetic system in these bacteria. It is concluded that mercury volatilization in the estuarine environment can be mediated by genes carried on plasmids.     

Integrons of bacteria isolated from aquatic environment

Bacteria of the genus Pseudomonas, isolated from the water of the lakes Shira and Itkul (Republic of Khakassia, Russia) were shown to contain integrons of class 1 with gene cassettes, contained in the variable segment (sized 1 and 1.3 kb), were shown. Out of three detected integrons only one integron (in P. aeruginosa) included the sulfanilamide resistance gene contained in the 3'-conservative segment. The resistance of bacteria to kanamycin and ceftazidime was not seemingly linked with the presence of integrons. On the whole, the study revealed the presence of a significant proportion (27%) of integron-positive strains among aquatic bacteria with pronounced resistance to antibiotics.     

Retrotransfer of IncP1-like plasmids from aquatic bacteria

The first observation of plasmid retrotransfer by plasmids isolated from environmental sources is reported. A high incidence of retrotransferring ability amongst plasmids isolated from epilithic bacteria was found; some of these plasmids retrotransferred an IncQ plasmid at very high frequencies. Despite the broad host-range of the majority of the plasmids, only five out of 12 could be assigned to an incompatibility group by DNA hybridization. All five were designated IncP1; this revealed a limitation of probes derived from clinical sources for use with environmental isolates. Incompatibility testing by plate mating suggested that four additional plasmids displayed varying, albeit lower, degrees of incompatibility to the IncP1 plasmid RP1.     

Nucleotide sequences of mercury resistance determinants in bacteria isolated from mercury mines: detection of a family of recombinant mercury transposons in plasmids from Acinetobacter species

Partial nucleotide sequences were determined for mer operons located on large and small plasmids previously described in Acinetobacter spp. isolated from different mercury mines of the USSR. Inspection of the sequences shows that: 1. All Acinetobacter mer operons studied belong to a family of transposons homologous to transposons found in clinical isolates. 2. The transposons located on the small plasmids originated by recombinations between the transposons from the large plasmids and Tn501, a transposon found in a Pseudomonas hospital strain isolated in Australia. The left arm of each hybrid transposon was donated by a transposon of a large Acinetobacter plasmid and the right arm - by the Tn501.     

Plasmid-borne mercury resistance in aquatic bacteria

Abstract Bacteria isolated from the River Mersey were analysed for their tolerance to mercury (HgCl₂). About 40% of the population was tolerant to mercury and in 13 of 52 mercury-tolerant isolates tested the mercury resistance (Hg^®) was transferred to Escherichia coli in conjugal matings. These 13 isolates represented a range of gram-negative genera and in each case mercury resistance was coded by a conjugative plasmid. These plasmids (75 kb to > 250 kb in size) all expressed mercury resistance of the narrow spectrum variety, volatilised HgCl₂ to elemental Hg° vapour and showed some degree of temperature sensitivity of transfer. None expressed resistance to nine different antibiotics. These 13 Hg^R plasmids were classified by restriction mapping into three distinct groups typified by pMER11, pMER327 and pMER610. The eight pMER610 group plasmids are identical and belong to the IncHI-2 group. Two of the four pMER327 group plasmids are closely related while the other two contain some common restriction fragments. pMER11 is quite distinct from the other groups. These results imply that within this aquatic environment plasmids play an important role in the response of bacteria to contaminating mercury and that there is widespread plasmid transfer and considerable genetic rearrangement.     

Mercury-resistant plasmids in bacteria from a mercury and antimony deposit area

Summary Most bacterial cells (Pseudomonas, Acinetobacter) obtained from the soil at the Khaidarkan mercury and antimony mine (Kirghiz USSR) contain R plasmids with mercury (HgCl₂) resistance determinants. The plasmids have a large molecular mass (about 100 MD, though smaller ones also occur), and at least some of them are transmissive. Many of the Hg^r bacteria also display an elevated antimony (SbCl₃) resistance, though this trait was not shown to be plasmid-dependent. There are practically no Hg^r plasmids in bacteria taken from the soil at different distances from the mine: the saturation of bacteria with Hg^r plasmids is maintained by selective pressure only in the area with a high enough toxin concentration.In the same mercury and antimony deposit area Hg^r plasmids were also found in Escherichia coli isolates from the gut of Mus musculus mice and Bufo viridis toads. At least some of the bacterial plasmids obtained from animals also carry antibiotic-resistance determinants (Tc^r, Cm^r, Sm^r). These plasmids are also transmissive. They display internal instability and lose their resistance determinants after a conjugation transfer to other E. coli trains.     

Frequency and characteristics of plasmids in bacteria isolated from deep-sea amphipods

Bacterial strains isolated from deep-sea amphipods were identified, classified, and screened for plasmid content. Plasmids were common, with 11 of 16 isolates carrying one or more plasmids; these ranged in size from 2.9 to 63 megadaltons. Several of the strains demonstrated distinctly different phenotypic traits yet contained plasmids of the same molecular weight. Results of agarose gel electrophoresis, DNA hybridization, and restriction analysis indicate that the plasmids detected in these deep-sea isolates are identical, suggesting that transmission may occur in the deep-sea environment and that plasmids are common in some deep-sea habitats.     

Diversity of mercury resistance plasmids obtained by exogenous isolation from the bacteria of sugar beet in three successive years

Abstract: Self-transmissible plasmids conferring mercury resistance were exogenously isolated from the bacterial populations of sugar beet roots (rhizoplane) and leaves (phyllosphere) into a Pseudomonas putida recipient. Fifty rhizoplane plasmids and 29 phyllosphere plasmids (60–383 kb) were purified. Numerical analysis of plasmid DNA restriction enzyme digest patterns identified five distinct groups. Three of these plasmid groups were isolated from sugar beet crops grown at the same site over three consecutive years, demonstrating their established presence. Each group of plasmids comprised individual isolates with structural additions or deletions. The frequency of exogenous isolation correlated with factors likely to influence plant growth, bacterial activity and the physiological state of donors prior to sampling. All plasmids investigated conferred narrow spectrum mercury resistance with a reductase detoxification mechanism. None of the plasmids conferred resistance to a range of antibiotics, other heavy metals, or to UV, and following transfer to recipient bacteria the range of carbon source utilisation was not altered. This is the first report of the persistence of Pseudomonas spp. plasmid structural types isolated over several years from a terrestrial habitat.     

Biotransformation of mercury by bacteria isolated from a river collecting cinnabar mine waters

One hundred six strains of aerobic bacteria were isolated from the Fiora River which drains an area of cinnabar deposits in southern Tuscany, Italy. Thirty-seven of the strains grew on an agar medium containing 10g/ml Hg (as HgCl₂) with all of these strains producing elemental mercury. Seven of the 37 strains also degraded methylmercury. None of 106 sensitive and resistant strains produced detectable monomethylmercury although 15 strains produced a benzene-soluble mercury species. Two strains of alkylmercury (methyl-, ethyl- and phenylmercury) degrading bacteria were tested for the ability to degrade several other analogous organometals and organic compounds, but no activity was detected toward these compounds. Mercury methylation is not a mechanism of Hg resistance in aerobic bacteria from this environment. Growth of bacteria on the agar medium containing 10g/ml HgCl₂ was diagnostic for Hg detoxification based on reduction.     

Oxidation of arsenite to arsenate by a bacterium isolated from an aquatic environment

Arsenic is ubiquitous in the biosphere and frequently reported to be an environmental pollutant. Global cycling of arsenic is affected by microorganisms. This paper describes a new bacterial strain which is able to efficiently oxidize arsenite (As[III]) into arsenate (As[V]) in liquid medium. The rate of the transformation depends on the cell density. Arsenic species were separated by high performance liquid chromatography (HPLC) and quantified by inductively coupled plasma-atomic emission spectrometry (ICP-AES). The strain also exhibits high minimum inhibitory concentrations (MICs) for As[III] (6.65 mM (500 mg L^-1)) and other heavy metals, such as cadmium (1.42 mM (160 mg L^-1)) or lead (1.20 mM (250 mg L^-1)). Partial identification of the strain revealed a chemoorganotrophic, Gram-negative and motile rod. The results presented here demonstrate that this strain could represent a good candidate for arsenic remediation in heavily polluted sites.     

Incompatibility group K plasmids in bacteria isolated from a urinary tract infection

Citrobacter freundii and Klebsiella pneumoniae were concurrently isolated from a patient with a urinary tract infection. Transferable drug resistant plasmids were isolated from both strains, pMS434 and pMS435. These plasmids belonged to incompatibility group K and both carried genes governing resistance to various aminoglycoside antibiotics, i.e., kanamycin, gentamicin C complex, streptomycin, and 3',4'-dideoxykanamycin B, in addition to those governing resistance to sulfanilamide and ampicillin. They inactivated kanamycin, gentamicin C complex and 3',4'-dideoxykanamycin by adenylylation and kanamycin by phosphorylation. Electron microscopic observations disclosed that the molecular weights of the plasmids were about 67.8 megadaltons. These results indicated the similarity in genetic constitution of the two plasmids. This was the second isolation of incompatibility group K plasmids, following that reported by Hedges and Datta (Nature 234: 220-221, 1971).     

Distribution and characterization of kepone-resistant bacteria in the aquatic environment

Effects of the chlorinated insecticide Kepone on the ecology of Chesapeake Bay and James River bacteria were studied. Kepone-resistant bacteria present in a given environment were found to reflect the degree of fecal and/or high organic pollution of the sampling sites, based on total numbers and generic composition of the populations of Kepone-resistant bacteria. The presence of Kepone-resistant bacteria was found to be correlated (alpha = 0.01) with total coliforms, fecal coliforms, and total aerobic viable heterotrophic bacteria, but not with Kepone concentration, since Kepone-resistant bacteria were present in locations where Kepone could not be detected by the analytical methods used in this study. Only gram-negative bacteria, predominantly Pseudomonas, Vibrio, and Aeromonas spp., were found to be resistant to >/=10 mug of Kepone per ml. Gram-positive bacteria, i.e., Bacillus and Corynebacterium spp., were generally sensitive to >/=0.1 mug of Kepone per ml. From results of cluster analysis of taxonomic data, we determined that characteristics of Kepone-resistant bacteria included: resistance to pesticides and heavy metals; degradation of oil; positive oxidase and catalase reactions; and nitrate reduction. From results of the ecological and taxonomic analyses, we conclude that Kepone resistance in estuarine bacteria is due to the physicochemical composition of the gram-negative cell wall and not prior exposure to Kepone. Therefore, the presence of Kepone-resistant bacteria cannot serve as an indicator of Kepone contamination in the aquatic environment where gram-negative bacteria are predominant.     

Degradation of linear alkylbenzene sulfonate by a bacterial consortium isolated from the aquatic environment of Argentina

Aims:  To isolate and identify linear alkylbenzene sulfonate (LAS)-degrading bacteria from Río de la Plata and adjacent waters, and to assay their degradation capability as a consortium and as single organisms.
Methods and Results:  A consortium consisting of four bacterial strains: Aeromonas caviae (two strains), Pseudomonas alcaliphila and Vibrio sp. was identified by 16S rRNA analysis. Isolates grown as a consortium produced higher biomass from LAS and CO₂ release (mineralization) than individual cultures, and degraded 86% of LAS (20 mg l⁻¹), whereas pure strains degraded between 21% and 60%. Bacterial desulfonation from LAS was evidenced in the consortium and A. caviae strains. A complete disappearance of LAS (10 mg l⁻¹) was accomplished, and LAS levels of 50 and 100 mg l⁻¹ led to a pronounced decrease in the biodegradation extent and inhibition of culture growth.
Conclusions:  A bacterial consortium capable of complete LAS degradation was isolated from the Río de la Plata and adjacent waters. This consortium was more efficient for LAS degradation than individual cultures, and was sensitive to high LAS concentrations.
Significance and Impact of the Study:  The autochthonous consortium with high effectiveness on LAS biodegradation is a useful tool for LAS depletion from these polluted ecosystems.     

Survival and detection of bacteria in an aquatic environment

A genetically engineered plasmid, pPSA131, was used as a DNA probe to detect homologous DNA in Escherichia coli HB101(pPSA131) after it was mixed with aquatic microorganisms from Lake Mead, Nevada, water samples. An isolate from the pLAFR1 chromosomal library of Pseudomonas syringae Cit 7 was used to detect parent P. syringae Cit 7 that had been mixed with Lake Mead water. E. coli(pPSA131) was kept in variously treated samples of lake water or buffer, and its survival was measured by viable cell counting on modified Luria-Bertani (LB) agar. Full-strength LB agar proved better than 0.1 x LB agar at recovering E. coli(pPSA131) after survival in low-nutrient environments. Survival of E. coli(pPSA131) remained high in filtered (0.22-micron pore size) lake water and salts buffer on both selective and nonselective agars but was lower in untreated lake water or lake water filtered with a 0.8-micron-pore-size membrane. Total recoverable colonies grown on LB agar were higher when lake water was filter treated (0.8-micron pore size) than when lake water was untreated. Microorganisms recovered from lake water alone grew rapidly on nonselective media, probably because of the "bottle effect." After being mixed with Lake Mead water, E. coli(pPSA131) and P. syringae were detected by colony blotting with non-radioactively labeled DNA probes. E. coli(pPSA131) were recovered at three times during 48 h from variously treated samples of lake water and from a mixture with Lake Mead water organisms. Colonies were supported on either nonselective or selective agar for comparison.(ABSTRACT TRUNCATED AT 250 WORDS)     

Comparative typing of Pseudomonas species isolated from the aquatic environment in Greece by SDS-PAGE and RAPD analysis

AIMS: Three broadly used typing methods were employed in order to assess and compare the identification and classification of environmental Pseudomonas strains. The reproducibility, typeability and discriminatory power of the methods were also compared to evaluate their application. Finally, the potential impact on public health of the isolates is to be discussed. METHODS AND RESULTS: Pseudomonas strains (160) isolated from the aquatic environment in Greece and identified by a rapid identification commercially available system (API20NE), were subjected to whole-cell protein electrophoresis (Sodium dodecyl sulfate-polyacrylamide gel electrophoresis) and Randomly Amplified Polymorphic DNAs (RAPD) using two 10-mer primers. In general, the obtained results were in agreement. Twenty isolates that could not be identified by the API20NE system were classified by the other methods. CONCLUSIONS: Rapid identification systems may serve only for a first rough identification of environmental Pseudomonads. In order to acquire further information, so that conclusions about their role in the ecosystem and human health could be drawn, other phenotypic or genotypic methods have to be applied. SIGNIFICANCE AND IMPACT OF STUDY: It is important, from a public health point of view, to monitor the identities of environmental Pseudomonas isolates using specific methods due to their ubiquity, heterogeneity and their pathogenicity, either established or potential.     

Biosequestration, transformation, and volatilization of mercury by Lysinibacillus fusiformis isolated from industrial effluent

In the present study, an efficient mercury-tolerant bacterial strain (RS-5) was isolated from heavy-metalcontaminated industrial effluent. Under shake flask conditions, 97% of the supplemented mercuric chloride was sequestered by the biomass of RS-5 grown in a tryptone soy broth. The sequestered mercuric ions were transformed inside the bacterial cells, as an XRD analysis of the biomass confirmed the formation of mercurous chloride, which is only feasible following the reaction of the elemental mercury and the residual mercuric chloride present within the cells. Besides the sequestration and intracellular transformation, a significant fraction of the mercury (63%) was also volatilized. The 16S rRNA gene sequence of RS-5 revealed its phylogenetic relationship with the family Bacillaceae, and a 98% homology with Lysinibacillus fusiformis, a Gram-positive bacterium with swollen sporangia. This is the first observation of the sequestration and volatilization of mercuric ions by Lysinibacillus sp.     

Ultrastructure of two oil-degrading bacteria isolated from the tropical soil environment

Two oil-degrading bacteria identified as Pseudomonas aeruginosa and Micrococcus luteus were isolated from crude-oil-polluted soils in Nigeria. The organisms were grown on n-hexadecane and sodium succinate and then examined for the presence of hydrocarbon inclusions. Inclusion bodies were found in n-hexadecane-grown cells and were absent in succinate-grown cells. Formation of hydrocarbon inclusion bodies appears to be a general phenomenon among hydrocarbon utilizers.     

Naphthalene-utilizing and mercury-resistant bacteria isolated from an acidic environment

Soil samples were taken from areas of low pH (2.5–3.5) surrounding an outdoor coal storage pile. These samples were added to medium with naphthalene as the sole carbon source to enrich for organisms capable of degrading polycyclic aromatic hydrocarbons (PAH) at low pH. Five such bacterial strains were isolated. Sequencing of the 16S rDNA showed them to be members of the genera Clavibacter, Arthrobacter and Acidocella. These organisms were all capable of growth with naphthalene as a sole carbon source at low pH. The genes nahAc, nahAd, phnAc, nahH, xylE or GST, which are known to be associated with PAH degradation were not detected. Isolate 10, the Acidocella strain, tolerated high levels of mercury. PCR amplification and sequencing of genes from the mer operon from isolate 10 DNA suggested that mercury is transported into the bacterial cell and subsequently detoxified since the enzymes encoded by genes in this operon are involved in these processes.     

Survival and detection of bacteria in an aquatic environment.

A genetically engineered plasmid, pPSA131, was used as a DNA probe to detect homologous DNA in Escherichia coli HB101(pPSA131) after it was mixed with aquatic microorganisms from Lake Mead, Nevada, water samples. An isolate from the pLAFR1 chromosomal library of Pseudomonas syringae Cit 7 was used to detect parent P. syringae Cit 7 that had been mixed with Lake Mead water. E. coli(pPSA131) was kept in variously treated samples of lake water or buffer, and its survival was measured by viable cell counting on modified Luria-Bertani (LB) agar. Full-strength LB agar proved better than 0.1 x LB agar at recovering E. coli(pPSA131) after survival in low-nutrient environments. Survival of E. coli(pPSA131) remained high in filtered (0.22-micron pore size) lake water and salts buffer on both selective and nonselective agars but was lower in untreated lake water or lake water filtered with a 0.8-micron-pore-size membrane. Total recoverable colonies grown on LB agar were higher when lake water was filter treated (0.8-micron pore size) than when lake water was untreated. Microorganisms recovered from lake water alone grew rapidly on nonselective media, probably because of the "bottle effect." After being mixed with Lake Mead water, E. coli(pPSA131) and P. syringae were detected by colony blotting with non-radioactively labeled DNA probes. E. coli(pPSA131) were recovered at three times during 48 h from variously treated samples of lake water and from a mixture with Lake Mead water organisms. Colonies were supported on either nonselective or selective agar for comparison.(ABSTRACT TRUNCATED AT 250 WORDS)     

Ability of bacteria isolated from the hospital environment to proliferate in infusion fluids

The ability of some bacteria isolated from environmental sources to grow in commonly administered infusion fluids was investigated. Infusion fluids may become contaminated with bacteria present in the environment in which they are being administered and the ability of such bacteria to proliferate within commonly used infusion fluids is therefore clinically significant. Bacteria present in the air and sink drains in wards at the Ife State Hospital were isolated, identified, and comprised fifteen Gram-positive and five Gram-negative organisms. Fourteen of the organisms obtained were introduced into sterile distilled water and five different infusion fluid preparations and were monitored over a 48 h period. It was found that, in general, Gram-positive organisms did not grow in the fluids used, while Gram-negative organisms were able to proliferate in the same fluids. Some Gram-positive organisms showed remarkable ability to remain viable in infusion fluids for more than 48 h.