Modeling the environmental fate of manganese from methylcyclopentadienyl manganese tricarbonyl in urban landscapes.

The environmental impacts of gasoline additives such as lead (Pb) and Methyl Tertiary Butyl Ether (MTBE) are well documented, leading to the phasing out of these additives. In contrast, little is known about the health and environmental impacts of potential replacement chemicals such as Methylcyclopentadienyl Manganese Tricarbonyl (MMT). The combustion of MMT in gasoline leads to the formation of MnPO4 and MnSO4 and MMT is considered a recent source of inorganic Mn in urban landscapes particularly in high traffic areas. The main objective of this study is to estimate the automotive deposition of Mn from MMT relative to the traffic volume at sites near a major highway in the Greater Toronto Area of Canada, where MMT is currently being used. Manganese emission levels were estimated for two sites that varied according to Annual Average Daily Traffic (AADT) density, fuel consumption, distance traveled by automobiles, and Mn concentration (mg l(-1)) in gasoline. Multiple regression analysis was used to predict the AADT volume from year 2002-2010. Comparison of the mass balance between the ANOVA means of 15% Mn emitted from the automobile tailpipes at 10, and 18 mg of Mn l(-1) in gasoline was conducted for both study sites. The percentage difference between the Mn input at the selected concentrations of Mn in gasoline and output into surface soil were found to be 99% significant for both sites. Thus the predicted 15% tailpipe emission levels for 10 mg of Mn l(-1) of gasoline used in automobiles, which represented 1290.03 g/year for site 1 and 555.94 g/year for site 2, will add 5.73 and 2.47 mg/kg of Mn annually, respectively. These input levels are considered negligible when compared to the natural abundance of Mn in soil. Based on these data, it could take more than 95-256 years of continuous MMT usage in the region to double the content of Mn in surface soils at the respective sites.

Tetranucleotide frequencies in microbial genomes.

A computational strategy for determining the variability of long DNA sequences in microbial genomes is described. Composite portraits of bacterial genomes were obtained by computing tetranucleotide frequencies of sections of genomic DNA, converting the frequencies to color images and arranging the images according to their genetic position. The resulting images revealed that the tetranucleotide frequencies of genomic DNA sequences are highly conserved. Sections that were visibly different from those of the rest of the genome contained ribosomal RNA, bacteriophage, or undefined coding regions and had corresponding differences in the variances of tetranucleotide frequencies and GC content. Comparison of nine completely sequenced bacterial genomes showed that there was a nonlinear relationship between variances of the tetranucleotide frequencies and GC content, with the highest variances occurring in DNA sequences with low GC contents (less than 0.30 mol). High variances were also observed in DNA sequences having high GC contents (greater than 0.60 mol), but to a much lesser extent than DNA sequences having low GC contents. Differences in the tetranucleotide frequencies may be due to the mechanisms of intercellular genetic exchange and/or processes involved in maintaining intracellular genetic stability. Identification of sections that were different from those of the rest of the genome may provide information on the evolution and plasticity of bacterial genomes.

Protein method for investigating mercuric reductase gene expression in aquatic environments.

A colorimetric assay for NADPH-dependent, mercuric ion-specific oxidoreductase activity was developed to facilitate the investigation of mercuric reductase gene expression in polluted aquatic ecosystems. Protein molecules extracted directly from unseeded freshwater and samples seeded with Pseudomonas aeruginosa PU21 (Rip64) were quantitatively assayed for mercuric reductase activity in microtiter plates by stoichiometric coupling of mercuric ion reduction to a colorimetric redox chain through NADPH oxidation. Residual NADPH was determined by titration with phenazine methosulfate-catalyzed reduction of methyl thiazolyl tetrazolium to produce visible formazan. Spectrophotometric determination of formazan concentration showed a positive correlation with the amount of NADPH remaining in the reaction mixture (r2 = 0.99). Mercuric reductase activity in the protein extracts was inversely related to the amount of NADPH remaining and to the amount of formazan produced. A qualitative nitrocellulose membrane-based version of the method was also developed, where regions of mercuric reductase activity remained colorless against a stained-membrane background. The assay detected induced mercuric reductase activity from 10(2) CFU, and up to threefold signal intensity was detected in seeded freshwater samples amended with mercury compared to that in mercury-free samples. The efficiency of extraction of bacterial proteins from the freshwater samples was (97 +/- 2)% over the range of population densities investigated (10(2) to 10(8) CFU/ml). The method was validated by detection of enzyme activity in protein extracts of water samples from a polluted site harboring naturally occurring mercury-resistant bacteria. The new method is proposed as a supplement to the repertoire of molecular techniques available for assessing specific gene expression in heterogeneous microbial communities impacted by mercury pollution.

Removal of caffeine in sewage by Pseudomonas putida: Implications for water pollution index.

A strain of Pseudomonas putida (biotype A) capable of growing on caffeine (1,3,7-trimethylxanthine) was isolated from a domestic wastewater processing operation. It used caffeine as the sole carbon source with a mean growth rate constant (k) of 0.049 h(-1) (approximately 20 h per generation), whereas k for glucose utilization under similar incubation conditions was 0.31 (3.3 h per generation). The isolate contained at least two plasmids, and the increased expression of a 40 kDa protein was attributable to growth on caffeine. Degradation byproducts of caffeine metabolism by the bacterial isolate included other xanthine derivatives. The slow bacterial catabolism of caffeine in sewage has implications for the effectiveness of wastewater purification, re-use and disposal.

Bacterial genetic exchange in nature.

Most bacteria are haploid organisms containing only one copy of each gene per cell for most of the growth cycle. This means that the chance for correcting random mutations in bacterial genes would depend entirely on the complementarity inherent in DNA structures, unless homologous DNA sequences can be imported from outside the cell. Bacteria, like all living organisms have evolved at least one autonomous mechanism, conjugation, for exchanging portions of genetic materials between two related cells. The ecological benefits of conjugation include the expansion of metabolic versatility and resistance to hazardous environmental conditions. Natural bacterial genetic exchange also occurs through virus infections (transduction) and through the uptake of extracellular DNA (transformation). The origin and ecological benefits of transduction and transformation are difficult to assess because they are driven by factors external to the affected cell. Bacterial genetic exchange has implications for the evolution of phenotypes that are either beneficial to humans, such as biodegradation of toxic xenobiotic chemicals, or that are detrimental, such as the evolution of pathogenesis and the spread of antibiotic resistance. Understanding natural bacterial genetic exchange mechanisms is also relevant to the assessment of dispersal risks associated with genetically engineered bacteria and recombinant genes in the environment.

Transduction of a freshwater microbial community by a new Pseudomonas aeruginosa generalized transducing phage, UT1.

A pseudolysogenic, generalized transducing bacteriophage, UT1, isolated from a natural freshwater habitat, is capable of mediating the transfer of both chromosomal and plasmid DNA between strains of Pseudomonas aeruginosa. Several chromosomal alleles from three different P. aeruginosa strains were found to transduce at frequencies from 10(-8) to 10(-10) transductants per PFU at multiplicities of infection (MOI) between 0.1 and 1. Transduction frequencies of certain alleles increased up to 1000-fold as MOIs were decreased to 0.01. UT1 is also capable of transducing plasmid DNA to indigenous populations of microorganisms in natural lake-water environments. Data obtained in this study suggest that environmentally endemic bacteriophages such as UT1 are formidable transducers of naturally occurring microbial communities. It should be possible to develop model systems to test transduction in freshwater environments using components derived exclusively from these environments.

Application of DNA probes to analysis of bacteriophage distribution patterns in the environment.

Radiolabeled bacteriophage DNA probes have been used in this study to determine the distribution of Pseudomonas aeruginosa-infecting bacteriophages in natural samples of lake water, sediment, soil, and sewage. The sensitivity of detection of bacteriophage with the DNA probes was between 10(3) and 10(4) PFU and 10(6) to 10(7) CFU of lysogenized bacteria detectable with a homologous phage DNA probe. Analyses of environmental samples suggest that up to 40% of P. aeruginosa in natural ecosystems contain DNA sequences homologous to phage genomes. By using different bacteriophage DNA probes, the diversity of the bacteriophage population in sewage was estimated to be higher than that in other natural samples. The indication that transducing phages and prophages are widely distributed in the Pseudomonas populations investigated has considerable implications for the frequency of natural gene transfer by transduction and of lysogenic conversion of host bacteria in natural ecosystems.

Effect of 2-hydroxybenzoate on the maintenance of naphthalene-degrading pseudomonads in seeded and unseeded soil.

The addition of specific nontoxic inducers of catabolic operons to contaminated sites is an approach that may enhance the efficiency of in situ biodegradation. We determined the genetic response of six pseudomonads to salicylate (also known as 2-hydroxybenzoate) added directly to 50 g of nonsterile soil samples. The strains, isolated from a polyaromatic hydrocarbon-contaminated soil, metabolized naphthalene as the sole source of available carbon, and their DNA sequences show significant homology to the nahAB genes of the degradative plasmid NAH7. Duplicate nonsterile soil cultures were incubated for up to 30 days. Experimental soil cultures were seeded with naphthalene-degrading strains (10(8) CFU g-1) originally isolated from the soil and amended with salicylate (16 or 160 micrograms g-1). Soil samples were analyzed periodically for the population density of heterotrophic bacteria and naphthalene degraders and for the abundance of the naphthalene-degradative genotype in the bacterial community. At 160 micrograms g-1, salicylate sustained the density of naphthalene degraders at the introduced density for 30 days in addition to producing a two- to sixfold increase in the occurrence in the bacterial community of DNA sequences homologous to the nah operon. No change in recoverable bacterial population densities was observed when soil samples were amended with 16 micrograms of salicylate g-1, but this concentration of salicylate induced a significant increase in the level of nah-related genes in the population.

Transduction of linked chromosomal genes between Pseudomonas aeruginosa strains during incubation in situ in a freshwater habitat.

Both transduction of single chromosomal loci and cotransduction of closely linked loci were observed between lysogenic and nonlysogenic strains of Pseudomonas aeruginosa in a freshwater habitat. Transductants were recovered at frequencies of 10(-6) to 10(-5) transductants per CFU. Transductants of lysogenized strains were recovered 10- to 100-fold more frequently than were transductants of nonlysogenic parents. Lysogens are thus capable of introducing phages which mediate generalized transduction into the natural microbial community and serving as recipients of transduced DNA. It would appear that lysogeny has the potential of increasing the size and flexibility of the gene pool available to natural populations of bacteria. The ability to generate and select new genetic combinations through phage-mediated exchange can be significant in the face of a continually changing environment and may contribute to the apparent fitness of the lysogenic state in natural ecosystems.

Dynamic interactions ofPseudomonas aeruginosa and bacteriophages in lake water.

The persistence and interaction between newly isolated strains ofPseudomonas aeruginosa and resident bacteriophages indigenous to a freshwater environment was monitored over 45 days in lake water microcosms. The interaction between susceptible and resistant bacteria with pure phage (UT1) particles or a mixed phage population (M1) was investigated by following temporal changes in host density, phage-to-bacteria ratio (PBR), and the appearance of apparent prophage carriers within the host population. Decay rates of the phage (UT1) ranged from 0.054 hour(-1) in natural water to 0.027 hour(-1) in filtered lake water. About 45% of sensitive bacteria incubated with phase UT1 were pseudolysogenic within 12 hours of incubation in natural lake water. This process was delayed until 72 hours in the steile lake water control, suggesting that host-phage interaction is promoted in the presence of a viable natural microbial community. Phage UT1 appeared to stabilize the density of host bacteria in lake water at a level of 10(4) colony-forming units (cfu) ml(-1). Bacterial coexistence with the mixed phage (M1) population resulted in an oscillating equilibrium with the PBR stabilizing at about 3. The presence of extraneous homoimmune phages appeared to be detrimental to the stability of the pseudolysogens, which were maintained at a lower population density than prophage-free cells in lake water containing the mixed phage (M1) population.