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Marine hydrocarbonoclastic bacteria, like Alcanivorax borkumensis, play a globally important role in bioremediation of petroleum oil contamination in marine ecosystems. Accumulation of storage lipids, serving as endogenous carbon and energy sources during starvation periods, might be a potential adaptation mechanism for coping with nutrient limitation, which is a frequent stress factor challenging those bacteria in their natural marine habitats. Here we report on the analysis of storage lipid biosynthesis in A. borkumensis strain SK2. Triacylglycerols (TAGs) and wax esters (WEs), but not poly(hydroxyalkanoic acids), are the principal storage lipids present in this and other hydrocarbonoelastie bacterial species. Although so far assumed to be a characteristic restricted to gram-positive actinomycetes, substantial accumulation of TAGs corresponding to a fatty acid content of more than 23% of the cellular dry weight is the first characteristic of large-scale de novo TAG biosynthesis in a gram-negative bacterium. The acyltransferase AtfA1 (ABO_2742) exhibiting wax ester synthase/acyl-coenzyme A'diacylglycerol acyltransferase (WS/DGAT) activity plays a key role in both TAG and WE biosynthesis, whereas AtfA2 (ABO_1804) was dispensable for storage lipid formation. However, reduced but still substantial residual TAG levels in atfA1 and atfA2 knockout mutants compellingly indicate the existence of a yet unknown WS/DGAT-independent alternative TAG biosynthesis route. Storage lipids of A. borkumensis were enriched in saturated fatty acids and accumulated as insoluble intracytoplasmic inclusions exhibiting great structural variety. Storage lipid accumulation provided only a slight growth advantage during short-term starvation periods but was not required for maintaining viability and long-term persistence during extended starvation phases.

A novel mutant of the marine oil-degrading bacterium Alcanivorax borkumensis SK2, containing a mini-Tn5 transposon disrupting a 'tesB-like' acyl-coenzyme A (CoA) thioesterase gene, was found to hyperproduce polyhydroxyalkanoates (PHA), resulting in the extracellular deposition of this biotechnologically important polymer when grown on alkanes. The tesB-like gene encodes a distinct novel enzyme activity, which acts exclusively on hydroxylated acyl-CoAs and thus represents a hydroxyacyl-CoA-specific thioesterase. Inactivation of this enzyme results in the rechanneling of CoA-activated hydroxylated fatty acids, the cellular intermediates of alkane degradation, towards PHA production. These findings may open up new avenues for the development of simplified biotechnological processes for the production of PHA as a raw material for the production of bioplastics.

Alcanivorax borkumensis is a ubiquitous marine petroleum oil-degrading bacterium with an unusual physiology specialized for alkane metabolism. This 'hydrocarbonoclastic' bacterium degrades an exceptionally broad range of alkane hydrocarbons but few other substrates. The proteomic analysis presented here reveals metabolic features of the hydrocarbonoclastic lifestyle. Specifically, hexadecane-grown and pyruvate-grown cells differed in the expression of 97 cytoplasmic and membrane-associated proteins whose genes appeared to be components of 46 putative operon structures. Membrane proteins up-regulated in alkane-grown cells included three enzyme systems able to convert alkanes via terminal oxidation to fatty acids, namely, enzymes encoded by the well-known alkB1 gene cluster and two new alkane hydroxylating systems, a P450 cytochrome monooxygenase and a putative flavin-binding monooxygenase, and enzymes mediating [beta]-oxidation of fatty acids. Cytoplasmic proteins up-regulated in hexadecane-grown cells reflect a central metabolism based on a fatty acid diet, namely, enzymes of the glyoxylate bypass and of the gluconeogenesis pathway, able to provide key metabolic intermediates, like phosphoenolpyruvate, from fatty acids. They also include enzymes for synthesis of riboflavin and of unsaturated fatty acids and cardiolipin, which presumably reflect membrane restructuring required for membranes to adapt to perturbations induced by the massive influx of alkane oxidation enzymes. Ancillary functions up-regulated included the lipoprotein releasing system (Lol), presumably associated with biosurfactant release, and polyhydroxyalkanoate synthesis enzymes associated with carbon storage under conditions of carbon surfeit. The existence of three different alkane-oxidizing systems is consistent with the broad range of oil hydrocarbons degraded by A. borkumensis and its ecological success in oil-contaminated marine habitats.

Results demonstrate that ferredoxin Fdx3 identified in Sphingomonas sp. strain RW1 can serve as an electron donor for the dioxin dioxygenase. Data are also presented comparing Fdx3 with Fdx1 ferredoxin.

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.

Research was conducted to isolate and characterize a gram-positive bacterium, characterized as a new Rhodococcus strain based on its 16S rRNA gene sequence, with a high biodegradative potential. The biodegradative potential of Rhodococcus sp. strain RB1 was examined on gradient-agar plates and in liquid cultures. Results indicate a pathway for the mineralization of 2,4-DNP with 3-nitroadipate as a key metabolic intermediate.

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.

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 genes coding for the two subunits of integration host factor (IHF) from Pseudomonas putida were cloned, sequenced and functionally analyzed. Both the ihfA and ihfB genes of P. putida code for 100-amino acid-residue polypeptides that are 1 to 6 residues longer than the Escherichia coli IHF subunits, respectively. The P. putida ihfA and ihfB genes can effectively complement E. coli ihf mutants, indicating that the P. putida IHF subunits can form functional heterodimers with the IHF subunits of E. coli. In the evolution of IHF, amino acid changes were only in the N-terminal domains and in the extreme C terminal.

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.

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.

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.

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.

The dbfB gene of the enzyme 2,2', 3-trihydroxybiphenyl dioxygenase, a monomeric protein, is cloned from a cosmid library of the Sphingomonas strain which degrades dibenzofuran and dibenzo-p-dioxin. Kinetic evaluation of the enzyme yields similar K(m) values for 2,3-dihydroxybiphenyl and 2,2',-3-trihydroxybiphenyl.

Transcriptional lacZ fusions to the Pu and Pm promoters of toluene degradation plasmid introduced in monocopy in the Pseudomonas putida chromosome respond differently to their respective aromatic effectors, in terms of growth phase. Xyls-dependent activation of Pm-lacZ is observed soon after m-toluate addition. XylR and xylene-mediated induction of the sigma-54 promoter Pu is observed only after the cells show a decrease in exponential growth and enter the stationary stage.

The polychlorinated biphenyls (PCBs) are synthesized compounds which reveal exceptional stability, and as a result are used as dielectric fluids, plasticizers, flame retardants and hydraulic fluids. The extensive use of PCBs led to environmental contamination, and the Rhodococcus globerulus P6 was employed to degrade the PCB congeners. The PCB catabolic pathway was genetically and biochemically analyzed to reveal the presence of a PCB gene cluster that encodes three narrow-substrate specificity enzymes that meta cleave the first aromatic ring. A wide difference exists among PCB meta cleavage enzymes.

The Aeromonas hydrophila gene coding for an RNase I-like RNase was isolated and characterized. Sequence analysis showed the presence of a typical leader sequence at the beginning of the protein, whose sequence was highly similar to Escherichia coli RNase I. DNA hybridization analysis further showed strong homology to the corresponding E. coli K-12 and Shigella sp. sequences. However, the A. hydrophila RNase gene did not hybridize to the genomes of Vibrio or Plesiomonas, considered as more related genera.

A study was conducted on protoanemonin, a toxic metabolite of the degradation of some chloroaromatic compounds. The findings indicate that transformation of protoanemonin by dienelactone hydrolase of Pseudomonas sp strain B13 to cis-acetylacrylate is possible. It was also observed that the turnover rate of protoanemonin was only 1% that of cis-dienelactone, which suggests that at least this percentage of the enzyme is in the active state.

The frequency of cotransformation of alleles in Acinetobacter calcoaceticus may increase as the distance between alleles decreases. Researchers measured the frequency with which A. cacoaceticus cells were cotransformed with pca and pob alleles separated by different lengths of DNA. When donor alleles were separated by 9.2, 7.4, 6.3, and 5.1 kilobases, the cotransformation frequencies were 16%, 39%, 51%, and 60%. Ligating unlinked DNA fragments may allow them to be genetically linked via natural transformation.

Three genes, bphC1, bphC2, and bphC3, code for 2,3-dihydroxybiphenyl 1,2-dioxygenase isoenzymes in Rhodococcus globerulus. The catabolic activity of these isoenzymes, in terms of degradation of polychlorinated biphenyls, indicates that BphC2 and BphC3 overall show similar activities and higher activities than BphC1. Furthermore, the expression of the three genes differ and the phenotype of the bacterium is influenced by the substrate specificity of BphC1 and BphC2.

Research was conducted to examine the structure of two class IIB enzymes with complementary substrate specificities through an exchange of equivalent polypeptide sequences. The objective was to determine the region, amino acids and subunits involved in the dechlorination of tetrachlorobenzene. The exchange between the tecA and tod CBA dioxygenase systems of Bukholderia sp. strain produced chimeric dioxygenases. Results identified a single amino acid and interacting regions that were responsible for the dehalogenation of tetrachlorobenzene.