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A new principle for expression of heat-sensitive recombinant proteins in Escherichia coli at temperatures close to 4 degrees C. was experimentally evaluated. The successful use of a chaperone-based E. coli strain to express heat-labile recombinant proteins at temperatures below the theoretical minimum growth temperature of a common E. coli strain (7.5 degrees C.) is reported.

Most soil bacteria are adequately represented in clone libraries prepared from either rRNA or rDNA. These libraries are used to identify bacteria using PCR rather than traditional culture methods.

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.

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.

The aerobic mineralization of 1,2,3,4-tetrachlorobenzene by a pure culture of Pseudomonas chlororaphis RW71 was demonstrated. The reaction produced stoichiometric amounts of chloride. The transient excretion of tetrachlorocatechol in the early growth phase suggested an initial attack by a dioxygenase to form the corresponding dihydrodiol which rearomatizes to the catechol. Extraordinarily high activity was found in chlorocatechol 1,2-dioxygenase in crude cell extracts.

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.

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 metabolism of 2-chloro and 3-chlorotoluene by Burkholderia sp. strain PS12 and Pseudomonas putida F1 was analyzed to determine the biochemical characteristics of the doixygenolytic pathway. The toluene dioxygenase of Pseudomonas putida F1 and the chlorobenzene dioxygenase of Burkholderia sp. PS12 strain catalyzed the conversion of 2-, 3- and 4-chlorotoluene to dihydrodiols. The synthetic process was characterized by the formation of minor products such as methyl- and chloro-substituted 1,2-dihydroxycyclohexadienes from 2- and 3-chlorotoluene.

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.

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.

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.

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.

The production and selection of Pseudomonas putida strains with enhanced mercurial detoxification characteristics involved the genetical combination of a benzene degradative route, which can reduce Hg(II) at higher rates. A few derivative strains could dissociate chemically different parts of an organomercurial compound, phenyl mercuric acetate (PMA) into its metal and aromatic parts, and subsequently detoxify these parts.

Pseudomonas cepacia G4 causes the degradation of trichloroethylene (TCE) by a pathway for aromatic compounds induced by substrates such as phenol and tryptophan. Pseudomonas cepacia G4 5223 PR1(PR1) is a Tn5 insertion mutant which expresses the toulene ortho-monooxygenase responsible for TCF degradation. In ground water microcosms, phenol-induced strain G4 and noninduced strain PR1 caused degradation to TCE to nondetectable levels within 24 hours at densities of 108 cells per milliliter. At lower densities degradation of TCE was not noticed after 48 hours. Aquifer sediment microcosms, indicated that TCE diminished from 60 to < 0.1 micro mols within 24 hours at 5 x 108 PR1 organisms per gram (wet weight) of sediment and from 60 to 26 micro mols over a period of 10 weeks at 5 x 107 PR1 organisms per gram.

A genetically engineered microorganism (GEM), Pseudomonas sp. strain B13 FR1, was created via a constructed ortho cleavage pathway. This organism is capable of degrading chloro- and methylaromatics. The fate and function of of this GEM in aquatic sediments was investigated with the use of a microcosm system model. The genetically engineered Pseudomonad survived in the microcosm system for four weeks and increased the degradation rate of exogenously applied 3-chlorobenzoate and 4-methybenzoate. These results suggest that this GEM is potentially useful for bioremediation.

Aquatic sediment microcosms of 20 layered sediment cores were incubated and their ecosystem parameters were compared to those of the source ecosystem. Parameters for both the microcosms and the source ecosystem remained the same for two weeks but small differences were discerned by the third and fourth week. However, these differences were so small, that microcosms can serve as laboratory models for source ecosystems up to four weeks. This ability of microcosm systems to simulate conditions in a source ecosystem can be used to assess the effects of exogenous microorganisms on an aquatic environment.

The feasibility of using colicin E3 for the biological containment of microorganisms is evaluated by cloning the antibiotic in the absence of the gene for its cognate inhibitor. The process is carried out under the control of a tightly regulated promoter. Results show that colicin E3 has lethal single gene copy, making it an ideal material for highly efficient containment systems for microorganisms.

The spatial and compositional structure and complex dynamics of the developing biofilm and its polychlorinated biphenyls (PCB)-degrading activity, leading to the identification of three stages of biofilm development is reported. The biofilm developing from PCB-contaminated soil on PCB droplets displayed a pronounced pattern of maturation not reported before from pure-culture experiments.

The germination of conidia of Aspergillus nidulans, a sensitive indicator of water stress in the presence of ethanol is corelated with the intracellular concentration of the compatible solutes glycerol and erythritol, which protect against both osmotic and nonturgor forms of water stress is reported. The conclusion states that the osmoprotectants glycerol and erythritol protect against ethanol induced water stress in A. nidulans.