Determination of biodegradation potential by two culture-independent methods in PAH-contaminated soils.

Biodegradation potentials of polycyclic aromatic hydrocarbons (PAHs) were determined with soil samples collected from various depths of a PAH-contaminated site and of a site nearby where PAHs were not found. Putative dioxygenase genes were amplified by a primer set specific for initial dioxygenases and identified by web-based database homology search. They were further categorized into several groups of which four dioxygenases were selected as probes for DNA hybridization. The hybridization signals according to the presence of putative dioxygenases were positively related to the extent of PAH contamination. However, the signal intensities varied depending on the probes hybridized and moreover were not consistent with PAH biodegradation activities determined by CO2 evolution. Despite widely accepted advantages of molecular biodegradation assessment, our data clearly present the variations of assessment results depending on the genetic information used and suggest that the methodology may tend to underestimate the real biodegradation capacity of a site probably due to the limited dioxygenase database available at the moment. Therefore, the molecular assessment of biodegradation potential should involve a very careful primer and probe design and an extensive microbiological examination of a site of interest to accurately delineate the biodegradation potential of the site.

Characterization of strain HY99, a novel microorganism capable of aerobic and anaerobic degradation of aniline.

We have characterized a novel microorganism, strain HY99, which is capable of aerobic and anaerobic degradation of aniline. Strain HY99 was found to aerobically metabolize aniline via catechol and 2-hydroxymuconic semialdehyde intermediates, and to transform aniline via p-aminobenzoate in anaerobic environments. Physiological and biochemical tests revealed that strain HY99 was most similar to Delftia acidovorans, but unlike D. acidovorans, strain HY99 was able to metabolize aniline under anaerobic conditions linked with nitrate reduction. Phylogenetic analysis based on 16S rDNA sequencing also revealed that strain HY99 was closely related to D. acidovorans, with 96% overall similarity.

Argon laser-assisted vasovasostomy.

Laser-assisted vasovasostomy recently has become a popular procedure in the United States. Only the microsurgical carbon dioxide laser is approved by the Food and Drug Administration for vasovasostomies. Using the HGM argon laser, the procedure was performed on 3 laboratory dogs with a patency rate of 100 percent (6/6) and anastomotic leak 17 percent (1/6). This procedure requires little microsurgical expertise, can be performed without the use of a microscope, and does not require a perfectly dry field. The argon laser is a superior technique to previously described laser-assisted vasovasostomies and further clinical correlation is recommended.

Cellular responses and proteomic analysis of Escherichia coli exposed to green tea polyphenols.

The purpose of this study was to characterize the cellular response and proteomic analysis of Escherichia coli exposed to tea polyphenols (TPP) extracted from Korean green tea (Camellia sinensis L). TPP showed a dose-dependent bactericidal effect on E. coli. Analysis of cell-membrane fatty acids of E. coli cultures treated with TPP identified unique changes in saturated and unsaturated fatty acids, whereas scanning electron microscopic analysis demonstrated the presence of perforations and irregular rod forms with wrinkled surfaces in cells treated with TPP. Two-dimensional polyacrylamide gel electrophoresis of soluble protein fractions from E. coli cultures exposed to TPP showed 17 protein spots increased or decreased by TPP. Nine upregulated proteins were identified (including GroEL and proteins involved in cellular defense, such as GyrA, RpoS, SodC, and EmrK), whereas the expression of eight proteins was downregulated by exposure to TPP (including proteins involved in carbon and energy metabolism, such as Eno, SdhA, and UgpQ, as well as those involved in amino-acid biosynthesis, such as GltK and TyrB). These results provide clues for understanding the mechanism of TPP-induced stress and cytotoxicity on E. coli.

Complete nucleotide sequence and overexpression of cat1 gene cluster, and roles of the putative transcriptional activator CatR1 in Acinetobacter lwoffii K24 capable of aniline degradation.

The aniline-assimilating bacterium Acinetobacter lwoffii K24 has two cat gene clusters (cat1 and cat2). In this study, we completely sequenced 10-kb DNA fragment of cat1 genes of A. lwoffii K24, which had been cloned in plasmid pCD1-1. Sequence analysis revealed that the order of genes in the cat1 operon-containing gene cluster was ORF porin, catR1, catB1C1A1D, ORF1, and ORF2. Two ORFs located immediately downstream catD were most similar with two ORFs in cat gene cluster of Acinetobacter calcoaceticus ADP1 but the gene structure of catR1B1C1A1 was closest to that found in Frateurua sp. ANA-18 or Pseudomonas putida PRS2000. CatA1 gene product was significantly overexpressed and detected in SDS-PAGE when four cat1 genes (catB1C1A1D) were placed under the control of a lac promoter in pUC118 while overexpressions of other cat genes were accomplished under the control of a lac promoter in pET vector system. All gene products were verified by N-terminal amino acid sequencing. Gel retardation assay revealed that the putative regulatory gene activator CatR1 for the catB1C1A1 operon could bind the promoter region of catB2 as well as catB1, suggesting that transcription of catB1 or catB2 might be controlled by the putative gene activator CatR1. However, the promoter regions of catA1 and catA2 were found to have no affinity with catR1.

Characterization of cDNAs encoding small and large subunits of ADP-glucose pyrophosphorylases from watermelon (Citrullus vulgaris S.).

Three cDNA clones encoding ADP-glucose pyrophosphorylases were isolated from a full red fruit cDNA library of watermelon (Citrullus vulgaris S.). Sequence analyses indicated that one clone, wms1, corresponds to the small subunit, and two clones, wml1 and wml2 (a partial gene), are the large subunits of AGPase. The presumed AGPase proteins encoded by wms1, wml1, and wml2 have 526, 526, and 481 amino acids, respectively. The protein sequences have the conserved amino acids important for the substrate or regulator binding site, with some variation. Developmental changes in the amounts of wms1, wml1, and wml2 transcripts in fruits were measured by northern blot analysis. Their expression levels decreased from the small green to medium green stages, then increased in accordance with fruit ripening, which was different from those of tomato and oriental melon.

Genetic and functional analysis of the tbc operons for catabolism of alkyl- and chloroaromatic compounds in Burkholderia sp. strain JS150.

Burkholderia sp. strain JS150 is able to metabolize a wide range of alkyl-and chloroaromatic hydrocarbons through multiple, apparently redundant catabolic pathways. Previous research has shown that strain JS150 is able to synthesize enzymes for multiple upper pathways as well as multiple lower pathways to accommodate variously substituted catechols that result from degradation of complex mixtures of monoaromatic compounds. We report here the genetic organization and functional characterization of a gene cluster, designated tbc (for toluene, benzene, and chlorobenzene utilization), which has been cloned as a 14.3-kb DNA fragment from strain JS150 into vector pRO1727. The cloned DNA fragment expressed in Pseudomonas aeruginosa PAO1c allowed the recombinant to grow on toluene or benzene and to transform chlorobenzene, trichloroethylene, phenol, and cresols. The tbc genes are organized into two divergently transcribed operons, tbc1 and tbc2, each comprised of six open reading frames. Similarity searches of databases revealed that the tbc1 and tbc2 genes showed significant homology to multicomponent cresol and phenol hydroxylases and to toluene and benzene monooxygenases, respectively. Deletion mutagenesis and product analysis were used to demonstrate that tbc2 plays a role in the initial catabolism of the unactivated alkyl- or chloroaromatic substrate and that the tbc1 gene products play a role in the catabolism of the first metabolite that results from transformation of the initial substrate. Phylogenetic analysis was used to compare individual components of these tbc monooxygenases with similar sequences in the databases. These results provide further evidence for the existence of multiple, functionally redundant alkyl- and chloroaromatic monooxygenases in strain JS150.

Characterization and role of tbuX in utilization of toluene by Ralstonia pickettii PKO1.

The tbu regulon of Ralstonia pickettii PKO1 encodes enzymes involved in the catabolism of toluene, benzene, and related alkylaromatic hydrocarbons. The first operon in this regulon contains genes that encode the tbu pathway's initial catabolic enzyme, toluene-3-monooxygenase, as well as TbuT, the NtrC-like transcriptional activator for the entire regulon. It has been previously shown that the organization of tbuT, which is located immediately downstream of tbuA1UBVA2C, and the associated promoter (PtbuA1) is unique in that it results in a cascade type of up-regulation of tbuT in response to a variety of effector compounds. In our efforts to further characterize this unusual mode of gene regulation, we discovered another open reading frame, encoded on the strand opposite that of tbuT, 63 bp downstream of the tbuT stop codon. The 1,374-bp open reading frame, encoding a 458-amino-acid peptide, was designated tbuX. The predicted amino acid sequence of TbuX exhibited significant similarity to several putative outer membrane proteins from aromatic hydrocarbon-degrading bacteria, as well as to FadL, an outer membrane protein needed for uptake of long-chain fatty acids in Escherichia coli. Based on sequence analysis, transcriptional and expression studies, and deletion analysis, TbuX seems to play an important role in the catabolism of toluene in R. pickettii PKO1. In addition, the expression of tbuX appears to be regulated in a manner such that low levels of TbuX are always present within the cell, whereas upon toluene exposure these levels dramatically increase, even more than those of toluene-3-monooxygenase. This expression pattern may relate to the possible role of TbuX as a facilitator of toluene entry into the cell.

Characterization of Pseudomonas sp. HK-6 cells responding to explosive RDX (hexahydro-1,3,5-trinitro-1,3,5-triazine).

The cellular responses of Pseudomonas sp. HK-6 to explosive hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) have been extensively analyzed in this study. The stress shock proteins, which might contribute to enhancing the cellular resistance to the cytotoxic effect of RDX, were induced at different concentrations of RDX used as a substrate for cell culture of Pseudomonas sp. HK-6. The proteins were identified as 70-kDa DnaK and 60-kDa GroEL by SDS-PAGE and Western blot using the anti-DnaK and anti-GroEL monoclonal antibodies. The stress shock proteins induced by RDX were found to increase in proportion to the RDX concentration used for this work. Analysis of membrane fatty acids of strain HK-6 following exposure to RDX showed that the amounts of dominant lipids 16:1 omega7c/15:0 iso 2OH, 16:0 and 18:1 omega7c/omega9t/omega12t decreased substantially or were not detected in the cells exposed to RDX, while amounts of lipids 10:0 iso, 14:1 omega5c/omega5t and 16:10 methyl increased dramatically. Scanning electron microcopy analyses revealed the presence of perforations and irregular rod shapes with wrinkled surfaces for cells treated with 0.135 mM RDX for 12 h, suggesting that RDX has a substantial cytotoxic impact on cells of strain HK-6.

PAH utilization by Pseudomonas rhodesiae KK1 isolated from a former manufactured-gas plant site.

Pseudomonas rhodesiae KK1 was isolated from a former manufactured-gas plant site, due to its ability to grow rapidly in a mixture of polycyclic aromatic hydrocarbons (PAHs). Radiorespirometric analysis revealed that strain KK1 was found to be able to mineralize anthracene, naphthalene and phenanthrene. Notably, phenanthrene-grown cells were able to mineralize anthracene much more rapidly than naphthalene-grown cells. Comparative analysis of amino acid sequences from 17 randomly selected dioxygenases capable of hydroxylating unactivated aromatic nuclei indicated that the enzymes for catabolism of PAHs, such as naphthalene and phenanthrene, might exist redundantly in strain KK1. Northern hybridization for cells grown on naphthalene or phenanthrene, using the putative naphthalene or phenanthrene dioxygenase gene fragment as a probe, suggested that the enzyme for naphthalene catabolism might share some homology in deduced amino acid sequences with phenanthrene dioxygenases. Also, it was found that three lipids (17:0 cyclo, 18:1 omega7c, 19:0 cyclo) increased in response to both naphthalene and phenanthrene, while the shift of other lipids varied from substrate to substrate.