Search

Pseudomonas sp. strain B13 is a bacterium known to degrade chloroaromatic compounds. The properties to use 3- and 4-chlorocatechol are determined by a self-transferable DNA element, the clc element, which normally resides at two locations in the cell's chromosome. Here we report the complete nucleotide sequence of the clc element, demonstrating the unique catabolic properties while showing its relatedness to genomic islands and integrative and conjugative elements rather than to other known catabolic plasmids. As far as catabolic functions, the clc element harbored, in addition to the genes for chlorocatechol degradation, a complete functional operon for 2-aminophenol degradation and genes for a putative aromatic compound transport protein and for a muiticomponent aromatic ring dioxygenase similar to anthranilate hydroxylase. The genes for catabolic functions were inducible under various conditions, suggesting a network of catabolic pathway induction. For about half of the open reading frames (ORFs) on the clc element, no clear functional prediction could be given, although some indications were found for functions that were similar to plasmid conjugation. The region in which these ORFs were situated displayed a high overall conservation of nucleotide sequence and gene order to genomic regions in other recently completed bacterial genomes or to other genomic islands. Most notably, except for two discrete regions, the clc element was almost 100% identical over the whole length to a chromosomal region in Burkholderia xenovorans LB400. This indicates the dynamic evolution of this type of element and the continued transition between elements with a more pathogenic character and those with catabolic properties.

The chlorobenzene dioxygenase from Pseudomonas sp. strain P51 converts substituted aromatic compounds to the S configuration at the carbon atom. The enantiomeric excess was higher than the same reaction catalyzed by toluene dioxygenases. This is of interest to the biotechnology industry because cis-dihydrodiols are building blocks for molecules with multiple chiral centers.

Two operons of the hbpCAD gene cluster are independently regulated and their expression is mediated by HbpR protein, which requires RNA polymerase-sigma(sup)54 and an integration host factor for complete activation. The transcriptional organization of the gene cluster facilitates orderly expression of the hbp genes and 2-hydroxybiphenyl degradation pathway.

Research reveals a set of five genes localized to 5.9-kb tfdR-tfdK intergenic region of plasmid pJP4 forming a gene cluster. Data point out that the gene cluster encodes enzymes involved in the conversion of 2,4-dichlorophenol to beta-ketoadipate in Ralstonia eutropha JMP134.

Research has been conducted on the Pseudomonas strains that utilize m-toluate. The results of the isolation of these strains from the oil-contaminated soil samples in Belarus are discussed.

Research has been conducted on the 2-hydroxybiphenyl and 2,2'-dihydroxybiphenyl degradationin Pseudomonas azelaica. The regulation of this degradation by the regulatory gene hbpR is discussed.

Pseudomonas sp. strain P51 cis-chlorobenzene dihydrodiol dehydrogenase (CDD)(TcbB) expressed in Escherichia coli DH5alpha (pTCB149) has been found to catalyze enantioselective dehydrogenase reactions. The study was carried out with the object of investigating the potential of CDD for enantiomeric resolution of chiral dihydrodiols. CDD preferentially oxidized the para-halogenated cis-toluene dihydrodiol enantiomers preferentially formed by chlorobenzene dioxygenase (CDO).

Various microbial isolation and genetic techniques were used to determine whether chlorobenzene (CB) metabolism is a natural microbial response to contamination with synthetic chemicals. Results indicate that the ability to degrade CB is not an inherent property, but one which has evolved. The evolution is a result of simple horizontal gene transfer and gene recombination between different ancestral strains.

A study was undertaken to determine the unknown characteristics of the transmissible chlorocatechol degradation (clc) gene from the Pseudomonas specie strain B13 utilizing pulse-field gel eletrophoresis, DNA-DNA hybridizations and cosmid mapping. The B13 strain was considered a pseudomonad using the 3-chlorobenzoate as the main source of carbon and energy. Results revealed that clc genes were discovered on a transmissible element and the physical appearance of this element was detected by its tandem amplification in the host strain, P. putida F1.

A study was conducted to examine low-frequency horizontal transfers of chlorocatechol degradative genes from Pseudomonas sp. strain B13 in activated-sludge microcosms. Matings were carried out with cultures pre-grown on Luria-Bertani medium that was washed and resuspended in Z3 mineral medium. The microcosms were characterized by glass reactors and operated at a volume of about 05. liter of culture medium.

Ralstonia eutropha is able to grow on 2,4-dichlorophenoxyacetate (2,4-D) as the only source of carbon. The mechanism involved in the uptake of 2,4-D in Ralstonia is investigated by genetic analysis of the pJP4 plasmid. The tfdK gene on this plasmid codes for a product resembling the transport proteins of the major facilitator superfamily. The disruption of this gene reduced 2,4-D assimilation substantially, indicating that it has a role in the uptake of 2,4-D.

An Escherichia coli whole-cell biosensor was developed for linear alkane detection through the transcriptional fusion of Vibrio harveji promotorless luxAB genes and Pseudomonas oleovorans alkB promoter. Standardized and optimized assays were then employed in evaluating the biosensor strain's ability to detect octane concentrations. The biosensor was also found to be useful in estimating linear akane concentrations in groundwater samples contaminated with oil.

A 6.2-kb DNA fragment with genes for the nitrilotriacetate (NTA) monooxygenase of Chelatobacter heintzii ATCC 29600 was cloned and characterized by DNA sequencing and expression experiments. The nucleotide sequence has three main open reading frames (ORFs). Two of the ORFs could be assigned to the NTA monooxygenase components A and B. The predicted N-terminal amino acid sequences of these ORFs were similar to those determined for the purified components. The deduced gene product of the third ORF had homology in the N-terminal region with the GntR class of bacterial regulator proteins and therefore may encode a regulator protein.

An analysis of induction and repression of denitrification activity in Paracoccus denitrificans shows that denitrification starts during the transition from aerobic to anaerobic conditions and stops in the reverse process. The transition from aerobic to anaerobic respiration causes an increase in the mRNA levels for the individual denitrification enzymes. The delay nitrite reductase formation, as compared to the formation of nitrate and nitrous oxide reductase, indicates that these three genes have different modes of regulation.

LysR-type transcriptional activator, TcbR, causes a bend in the binding site of tcbC and tcbR promoter sequences in the DNA strands. The TcbR gene encodes the tcbR activator protein in Pseudomonas sp. strain P51. The TcbR binding sites occupy the regions between -85 and -40 and consist of a LysR-type recognition sequence motif. TcbR treatment of certain DNA regions causes DNaseI digestion to occur, resulting in the bend in the DNA site.

Microorganisms from various areas of the planet had been found to have developed evolutionary changes in the catabolism of manmade compounds. The influence of the amount of xenobiotic compounds introduced by man into the environment on the evolutionary process remains unanswered. However, it was known that gene transfer, mutational drift, and genetic recombination and transposition were the mechanisms that spurred the enviromental adaption.

The different regulatory systems involved in biodegradation pathways of aromatic compounds linking them to another known protein families are detailed. The information on regulatory systems is used to construct whole-cell living bioreporters that are used to measure the quality of the aqueous, soil, and air environment.

Research has been conducted on partial nitrification of ammonium to nitrite under oxic conditions. The authors have investigated the role of pH, low temperature and dilution rate in the formation, composition and activity of ammonia-oxidizing biomass in bioreactors from sewage sludge.

Research was conducted to examine the synthesis of a new recombinase gene (Int-B13) of the phage P4 integrase family. A further objective was to demonstrate its function in site-specific integration of the clc element. Results provide evidence that Int-B13 caused the site-specific integrative recombination between the clc element's attachment site and chromosomal attachment sites. It is predicted that the Int-B13 gene causes site-specific chromosomal integration of the complete clc element.

Summary Green fluorescent proteins (GFPs) are frequently used as marker and reporter systems to assess the fate and activity of microbial strains with the ability to degrade xenobiotic compounds. To evaluate the potential of this tool for tracking herbicide-degrading microorganisms in the environment a promoterless gfp was linked to the tfd C promoter, which is activated during degradation of the herbicide 2,4-dichlorophenoxyacetic acid (2,4-D), and integrated into the chromosome of the 2,4-D–degrading strain Ralstonia eutropha JMP 134. The effects of the inserted gfp gene on the kinetics of 2,4-D degradation by R. eutropha in batch and chemostat culture were compared to those of the wild-type strain. In batch culture with 2,4-D as the only carbon and energy source the maximum specific growth rate of the gfp -marked strain did not differ significantly from the wild type. However, compared to the wild type, the 2,4-D steady-state concentration in 2,4-D-limited chemostat cultures of the gfp -marked strain was higher at all dilution rates tested. The reduced competitiveness of the gfp -marked strain at low substrate concentrations was confirmed in a competition experiment for 2,4-D in continuous culture at a dilution rate of 0.075 h[sup -1] . Reproducibly, the gfp -marked strain was displaced by the wild-type strain. The study clearly demonstrates that fitness of constructs cannot be assessed by measuring µ[sub max] with selected substrates in batch cultures only but that a thorough kinetic analysis is needed, which also considers slow, carbon-limited growth conditions as they occur in the environment. [ABSTRACT FROM AUTHOR]

Genetically engineered bioreporters are an excellent complement to traditional methods of chemical analysis. The application of fluorescence flow cytometry to detection of bioreporter response enables rapid and efficient characterization of bacterial bioreporter population response on a single-cell basis. In the present study, intrapopulation response variability was used to obtain higher analytical sensitivity and precision. We have analyzed flow cytometric data for an arsenic-sensitive bacterial bioreporter using an artificial neural network-based adaptive clustering approach (a single-layer perceptron model). Results for this approach are far superior to other methods that we have applied to this fluorescent bioreporter (e.g., the arsenic detection limit is 0.01 microM, substantially lower than for other detection methods/algorithms). The approach is highly efficient computationally and can be implemented on a real-time basis, thus having potential for future development of high-throughput screening applications.