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The biphenyl dioxygenase of Burkholderia sp. strain LB400 dioxygenates polychlorobiphenyls at different positions on the ring depending on the placement of substituents. The 2,4-disubstituted ring is dioxygenated at positions 2 and 3, whereas the 2,5-dichlorinated ring is dioxygenated at positions 3 and 4.

The utilization of 1,2,3,4-tetrachloro-, 1,2,4-trichloro-, the three isomeric dichlorobenzenes and fructose as the sole carbon and energy sources at nanomolar concentrations was studied in batch experiments with Burkholderia sp. strain PS14. In liquid culture, all chlorobenzenes were metabolized within one hour from their initial concentration of 500 nM. In soil microcosms, Burkholderia sp. strain PS14 metabolized tetrachlorobenzene over a 72-hour incubation period. These indicate that strain PS14 exhibits a very high affinity for chlorobenzenes at nanomolar concentrations.

Research was conducted to examine the hypothesis that the genetic and biochemical analyses of the tec operon suggest a route for the evolution of chlorobenzene degradation genes. Southern blot analysis of BamHI-, Bg/II-, and double-digested PS12 total DNA revealed a single band which indicate that the tecA gene is present at only one locus. Biochemical characterization of TodF-like protein T1pF and a genetically refunctionalized TodE-like protein produced in Escherichia coli provided data consistent with the proposed relationships.

Changes in the microbial community found in a highly-complex activates sludge ecosystem after the addition of phenols have been studied through thethermal gradient gel electrophoresis analysis of ribosomal sequences from community DNA. The introduction of phenols decreases the Shannon index of diversity from 1.13 to 0.22 indicating that the microbial community structure has been compromised. The addition of genetically-engineered Pseudomonas sp. strain B13 SN45RE protected the microbial community from further degradation while the nonengineered parental strain B13 did not offer any protection.

Previous experiments resulted in the cloning of the ferredoxin gene fdx1 and the reductase A2 gene redA2 of the electron transport system of the dioxin dioxygenase of Sphingomonas sp. strain RW1 and characterized the corresponding proteins. Fdx1 was found to be clustered with genes that appeared to encode two atypical decarboxylases and a glutathione-S transferase. A study was conducted to identify the open reading frames encoding the dioxin diogenase itself and derived functional expression of this enzyme system.

The cloning, sequencing and characterization of the gene encoding a gentisate 1,2-dioxygenase (gtdA) from Sphingomonas sp. strain RW5 were described. The gtdA gene encoded a 350-amino acid polypeptide. Results showed that the gentisate 1,2-dioxygenase is a member of a new class of ring-cleaving dioxygenases. Studies indicated that the cloned gtdA tolerates unchlorinated gentisate as well as dichlorinated gentisate as substrates. It was found that cloned gtdA plays an essential role in the degradation of 3,6-dichloro-2-methoxybenzoate.

The xylR autoregulation of Pr promoter and expression from Ps promoters from a wide-array of Pseudomonas putida genetic backgrounds, with promoters in their TOL plasmid, was studied. The findings revealed that the autoregulation of XylR increases when exposed to pathway substrates. The results indicated that integration host factor functions in vivo as a repressor of the sigma-54-dependent Ps1 promoter and that binding of RNA polymerase having sigma-54 at the upstream region can control expression from the Ps2 promoter.

A rapid oligonucleotide probe design strategy based on subtractive hybridization was devised to eliminate the need to sequence many similar clones of dominant members of a community. The method yields probes for 16S rRNA or rRNA genes of individual members of microbial communities specific within the context of those communities. Results revealed that only those clones with 100% sequence identity with the probe fragment were detected. Probes also were inclined to correspond to more variable regions of the 16S rRNA if the target sequences of other clones in the library.

A strain of Salmonella typhimurium was transfected with the truncated genes for ActA and listeriolysin, virulence factors from Listeria monocytogenes, and used in oral vaccination. The transformed Salmonella elicited cellular and the humoral responses from the host mice. The response rate indicates that there has been a transfer of genetic material from the bacteria to the host which was substantiated by in vitro studies using peritoneal macrophages.

Microbiological research shows the mineralization of the polyhalogenated dibenzofuran 4-chlorodibenzofuran using a coculture of dibenzofuran-degrading bacterium Sphingomonas sp. strain RW1 and the chlorosalicy-late-degrading bacterium Burkholderia sp. strain JWS (13,15). The complete degradation of 4-chlorodibenzofuran with concomitant release od Cl- and formation of biomass is observed.

The presentation of heterologous O antigens on the surface of a well-characterized live vaccine strain was utilized for the development of vaccine candidates against Shigella flexneri. The introduction of the rfb-rfb gene cassette into the attenuated Shigella flexneri vaccine strain SFL124 resulted in the expression of homologous and heterologous O antigens. Furthermore, the expression of longer O-antigen chain length was induced by providing heterologous rol genes.

The functional expression of Rhodococcus globerulus ring-hydroxylating biphenyl dioxygenase (BDO) gene in Pseudomonas putida was analyzed to characterize the biochemical activity of its protein product. Analysis of Pseudomonas putida expressing the Rhodococccus globerulus bphA1A2A3A4 gene cluster indicated the presence of BDO activity. However, the expression of the BDO genes in Escherichia coli did not induce BDO activity due to the absence of the T7 promoter which mediated the transcription of the BDO genes in the gram-negative host.

The metabolism of 4-chlorocatechol via the 3-oxoadipate pathway produces the antibiotic protoanemonin that is toxic to indigenous and supplemented 4-chlorobiphenyl-cometabolizing bacteria in soil. The survival of soil bacteria and fungi is adversely affected by the direct addition of protoanemonin, but is unaffected by its degradation. The addition of organisms that contain the meta-cleavage pathway for 4-chlorocatechol metabolism increases the survival of the soil bacteria. This suggests that protoanemonin is formed via the ortho-cleavage pathway.

A minitransposon containing the tox operon, controled by a promoter responsive to an aromatic inducer, enhances the production of pertussis toxin (PT) by Bordetella bronchiseptica. Characterization of recombinant clones also reveals the tox operon under the control of the Psal promoter. The growth pattern of PT is independent of noninducing conditions and low inducer concentration. Thus, the strain performance is unaffected in bioreactors after uncoupling from gene expression. The biological activity of the recombinant PT is similar to the PT produced by B. pertussis.

INTRODUCTION Metabolism of Aromatic Hydrocarbons Specified by TOL Plasmids Plasmids that specify the degradation of toluene and xylenes have been found in bacteria isolated in different locations around the world, [...]

The bacterium Sphingomonas sp. strain RW1 degrades many mono- and dichlorinated dibenzofurans, and dibenzo-p-dioxins, to the mono- and dichlorinated salicylates and catechols. The degradation is apparently a dioxygenolytic attack on the substituted and nonsubstituted aromatic nucleus of the target compounds. The strain RW1 uses monochlorinated dibenzo-p-dioxins and dibenzofurans as the carbon source.

The usefulness of the Shigella flexneri aroD strain as a live vaccine is suggested by its production of properly linked heterologous O antigen polysaccharide. After transfection with plasmids carrying the rfp and rfb genetic determinants of S. dysenteriae type 1 O antigen, the S. flexneri strains SFL124 and SFL1070 were analyzed for the quality and quantity of heterologous O antigen expression. Linkage of O antigen to the S. flexneri R3 core oligosaccharide is discussed.

Most of the mono- and dichlorobiphenyls and 2 of 6 trichlorobiphenyls are metabolized to benzoates, by the encoding enzymes of the upper biphenyl pathway. The bph locus of Pseudomonas sp. strain LB400 are used for the encoding. The enzymes used for the polychlorobiphenyl degradation attack the substituted aromatic ring in the order of non- > ortho- > meta- > para. The rate of chlorobenzoates formation depends on the initial attack rate. The presence or absence of a chlorine substituent at an ortho position, yield a corresponding absorption spectra of phenylhexadienoates.

DNAse footprint analysis of the xyl gene operons of Pseudomonas putida TOL plasmid showed that its binding site for XylR activator and the region containing the Ps promoter are protected by integration host factor. Genetic transformation of the Ps promoter produced a negative reaction from IHF. However, Escherichia coli mutants lacking IHF exhibited an elevated level of transcriptional activity, while E. coli IHF-deficient mutant exhibited no alteration in the synthesis of XylR protein.

Bacillus licheniformis produces lichenysin A, a lipopeptide biosurfactant which has seven amino acids in each molecule of the protein moiety. The bacteria grows on salty substrates and produces lichenysin A both aerobically and anaerobically. Lichenysin A reduces the interfacial tension between oil and salts and can be used for enhanced oil recovery at oil sites where salinity and temperature is very high. Lichenysin A is more effective at low concentrations because its critical micelle concentration is less.

Two broad-host-range cell lysis systems consisting of lysis genes S, R and Rz from bacteriophage lambda and the lysis gene E from bacteriophage PhiX174 enable the analysis of bacterial transformation and the environmental fate of cell death-generated DNA levels. Production of high levels of nucleic acids and activation of cellular autolysins accompany the induction of cell lysis by the two systems. The second cell lysis system contains a bacterial plasmid pDKLO2 that enhances the efficiency of the system for analyzing the fate of the DNA released due to cell death.

Determination of the partition ratio and Km of four mutants, 4ECR1, 4ECR6, 3MCS and 3MCR, of the catecholamine 2, 3-dioxygenase that is coded by TOL plasmid pWWO of Pseudomonas putida, help characterize them. Sequencing the structural genes helps determine the amino acid sequences of all four mutants. Catalysis of 4-ethylcatechol nullifies the wild-type catechol 2,3,-dioxygenase, indicating a low value of partition ratio for this substrate while 4ECR1 and 4ECR6 have higher partition ratios. The partition ratio of 3MCS is lower than that of 3MCR for 3-methylcatechol.

Primer extension mapping analysis of Xyls-dependent and Xyls-independent initiation of Pm transcription by benzoate in the Pseudomonas putida plasmid pWWo strain shows that the same PM core promoter structure with two cis-acting regulatory sequences mediates the transcription activation. Mutations and deletions in the promoter region indicate that the Pm transcription responds homologously to benzoate in the presence and absence of Xyls. The two regulators are Xyls and a chromosome encoded benzoate degradation pathway-related regulator.

The TOL plasmid pWW0 of Pseudomonas putida, involved in the metabolism of toluene and xylenes for carbon utilization, is regulated by XylS protein which activates its Pm promoter region. The XylS protein regulator is activated by substituted benzoates or an over production of XylS and the XylS-Pm interaction involves Om sequence within the promoter.

Genetically engineered microorganisms (GEMs) may be utilized for in situ bioremediation. GEMs do not possess much physiological differences from parental organisms. This implies that estimates on the likely behavior and survival of a GEM in its intended environment may be obtained by introducing a GEM and its parental strain into a microcosm and comparing the results to field experiments with the parental strain.

The transcriptional induction kinetics of mRNA synthesis from plasmid pWW0 of Pseudomonas putida are studied under various growth conditions. The XylR protein is the regulatory element which controls the transcriptional circuit of TOL plasmid pWWO catabolic pathways. The plasmid-encoded pathway provides a global understanding of cell metabolism.

Operon expression of xyl of Pseudomonas putida TOL plasmid is repressed on addition of glucose and related compounds. This repression produces two primary targets, xyl promoters Pu and Ps. The promoter function of xyl is inhibited by the presence of glucose. The promoter activation of xyl is not the same as the classical system of enteric catabolic repression.