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K N Timmis

Publications and source records attributed to K N Timmis.

At least 91 records · Page 5Linked to original sources
PubMed1996

Degradation of Chlorinated Dibenzofurans and Dibenzo-p-Dioxins by Sphingomonas sp. Strain RW1.

The ability of the dibenzofuran- and dibenzo-p-dioxin-mineralizing bacterium Sphingomonas sp. strain RW1 (R.-M. Wittich, H. Wilkes, V. Sinnwell, W. Francke, and P. Fortnagel, Appl. Environ. Microbiol. 58:1005-1010, 1992) to oxidize chlorinated derivatives of dibenzofuran and dibenzo-p-dioxin was analyzed. Strain RW1 degraded several mono- and dichlorinated dibenzofurans and dibenzo-p-dioxins, but it did not degrade more highly chlorinated congeners. Most mono- and dichlorinated dibenzofurans and dibenzo-p-dioxins investigated in this study were degraded to the corresponding mono- and dichlorinated salicylates and catechols, respectively, together with salicylate and catechol. This indicates an initial dioxygenolytic attack on the substituted as well as on the nonsubstituted aromatic nucleus of most of the target compounds. Strain RW1 could not grow at the expense of monochlorinated dibenzo-p-dioxins and dibenzofurans as carbon sources, with the exception of 4-chlorodibenzofuran, which was stoichiometrically converted to 3-chlorosalicylate.

Journal Article↗
PubMed1996

Use of colicin e3 for biological containment of microorganisms.

The genetic determinant of the lethal antibiotic colicin E3 was cloned under the control of a tightly regulated promoter in the absence of the gene for its cognate inhibitor. Combination of this killing cassette with a stringent regulatory element provided a substrate-dependent conditional suicide system that was exploited for the biological containment of a Pseudomonas putida strain. The lethality of a single gene copy and the distinct and universal cellular target of the antibiotic suggest colicin E3 as an ideal candidate for combination with other lethal functions to design highly efficient containment systems for microorganisms.

Journal Article↗
PubMed1996

Identification of an immunoglobulin A binding motif located in the beta-antigen of the c protein complex of group B streptococci.

The beta-antigen of the c protein complex of group B streptococci contains two immunoglobulin A (IgA)-binding domains called A and B. A 73-amino-acid segment in domain A is responsible for most of the IgA-binding activity. To identify the IgA binding motif, the 73-amino-acid domain was divided into 60 14-amino-acid overlapping peptides spot synthesized onto a cellulose membrane. A 20-residue putative antigenic epitope was identified and expressed as a fusion protein. The fusion protein was purified by fast protein liquid chromatography and used to raise rabbit antiserum. By use of a membrane with spot-synthesized peptide amino acids of decreasing length (from 14 to 6 amino acids), the major antigenic epitope recognized by the anti-fusion protein antibodies was mapped to motif MLKKIE. Anti-fusion protein antibodies inhibited the binding of IgA to group B streptococci. This inhibition could be blocked by the peptide containing the motif MLKKIE. These results indicate that the motif MLKKIE is located in the IgA-binding site. The IgA-binding domain of beta-antigen from three group B streptococcal strains reacted with the anti-fusion protein antibodies, and their coding sequences gave positive signals in Southern hybridization. The sequences of beta-antigen from these strains were amplified by PCR, and sequence analysis showed them to be identical. The results indicate that the motif MLKKIE is required for IgA binding and is present in different group B streptococcal strains.

Amino Acid Sequence↗
PubMed1996

Effect of heterogeneity of hydrophobic moieties on surface activity of lichenysin A, a lipopeptide biosurfactant from Bacillus licheniformis BAS50.

Lichenysin A, a surface-active lipopeptide produced by Bacillus licheniformis, strain BAS50, contains longchain beta-hydroxy fatty acids. Regulation of the synthesis of fatty acids and beta-hydroxy fatty acids was studied by modifying the culture medium. Addition of branched-chain alpha-amino acids to the medium caused similar changes to both cellular fatty acid and to beta-hydroxy fatty acid composition in the lipophilic part of lichenysin A. Production of lichenysin A was enhanced about two- and four-fold by addition of L-glutamic acid and L-asparagine respectively. It is suggested that these amino acids may be involved in the control of lipopeptide formation. Elucidation of the structure-function relationship of surface-active lipopeptides by analysis of the activities of structurally characterized compounds is discussed. Fractions of lichenysin A with branched beta-hydroxy acids in the lipid tail demonstrated lower surface-tension activity than the fractions of lichenysin A having straight beta-hydroxy acids. The presence of a lichenysin A fraction with beta-hydroxymyristic [(C14)n] acid residues appears to have an important influence on the surface activity of a mixture of lichenysins A.

Amino Acid Sequence↗
PubMed1995

From xenobiotic to antibiotic, formation of protoanemonin from 4-chlorocatechol by enzymes of the 3-oxoadipate pathway.

Chloroaromatics, a major class of industrial pollutants, may be oxidatively metabolized to chlorocatechols by soil and water microorganisms that have evolved catabolic activities toward these xenobiotics. We show here that 4-chlorocatechol can be further transformed by enzymes of the ubiquitous 3-oxoadipate pathway. However, whereas chloromuconate cycloisomerases catalyze the dechlorination of 3-chloro-cis,cis-muconate to form cis-dienelactone, muconate cycloisomerases catalyze a novel reaction, i.e. the dechlorination and concomitant decarboxylation to form 4-methylenebut-2-en-4-olide (protoanemonin), an ordinarily plant-derived antibiotic that is toxic to microorganisms.

Adipates↗
PubMed1995

Degradation of chlorobiphenyls catalyzed by the bph-encoded biphenyl-2,3-dioxygenase and biphenyl-2,3-dihydrodiol-2,3-dehydrogenase of Pseudomonas sp. LB400.

In order to characterize the metabolites produced in vivo by biphenyl-2,3-dioxygenase and biphenyl-2,3-dihydrodiol-2,3-dehydrogenase, the first two enzymes of the (polychloro)biphenyl catabolic pathway encoded by the bph locus of Pseudomonas sp. LB400, recombinant E. coli strains expressing the respective genes were constructed. Biphenyl-2,3-dioxygenase attack on 2,2'- or 2,4'-dichlorobiphenyl was shown to give rise to virtually quantitative ortho-dechlorination of these congeners by hydroxylation at the chlorinated carbon 2 and its unsubstituted neighbour. Elimination of hydrochloric acid directly leads to 2,3-dihydroxy-chlorobiphenyls and obviates the need for biphenyl-2,3-dihydrodiol-2,3-dehydrogenase for the catabolism of such congeners.

Biphenyl Compounds↗
PubMed1995

Crystal structure of the biphenyl-cleaving extradiol dioxygenase from a PCB-degrading pseudomonad.

Polychlorinated biphenyls (PCBs) typify a class of stable aromatic pollutants that are targeted by bioremediation strategies. In the aerobic degradation of biphenyl by bacteria, the key step of ring cleavage is catalyzed by an Fe(II)-dependent extradiol dioxygenase. The crystal structure of 2,3-dihydroxybiphenyl 1,2-dioxygenase from a PCB-degrading strain of Pseudomonas cepacia has been determined at 1.9 angstrom resolution. The monomer comprises amino- and carboxyl-terminal domains. Structural homology between and within the domains reveals evolutionary relationships within the extradiol dioxygenase family. The iron atom has five ligands in square pyramidal geometry: one glutamate and two histidine side chains, and two water molecules.

Amino Acid Sequence↗
PubMed1995

Evidence of histidine coordination to the catalytic ferrous ion in the ring-cleaving 2,2',3-trihydroxybiphenyl dioxygenase from the dibenzofuran-degrading bacterium Sphingomonas sp. strain RW1.

The 1H NMR spectra of an aromatic ring-cleaving extradiol dioxygenase, 2,2',3-trihydroxybiphenyl dioxygenase of the dibenzofuran-degrading bacterium Sphingomonas sp. strain RW1, are reported. In the catalytically active reduced form of the monomeric enzyme (MW = 32 kDa), three broad strongly downfield shifted signals were observed, two of which disappeared in D2O solution. Their shifts and linewidths are consistent with ring NH and meta-like protons of coordinated histidines. These signals show strong sensitivity to the presence of the substrate. The oxidized form of the enzyme shows no hyperfine shifted signals. It is suggested that the high spin Fe(II) ion present in the active form of the enzyme is coordinated by at least two histidines. This is the first report of hyperfine shifted NMR signals being detected for an extradiol dioxygenase.

Benzofurans↗
PubMed1995

The evolutionary relationship of biphenyl dioxygenase from gram-positive Rhodococcus globerulus P6 to multicomponent dioxygenases from gram-negative bacteria.

The Gram+ bacterium Rhodococcus globerulus P6 (RgP6) catabolizes a range of polychlorinated biphenyl (PCB) congeners, thus being of interest in bioelimination processes for PCB. The first step in the pathway, a dioxygenase attack of one of the biphenyl (BP) rings, is catalyzed by biphenyl dioxygenase (BDO). In this study, the nucleotide (nt) sequences of the four clustered cistrons, bphA1A2A3A4, encoding the subunits of BDO and forming part of the bph operon of RgP6 for BP degradation, were determined. A conserved motif proposed to bind a Rieske-type [2Fe-2S] cluster was identified in the deduced amino acid (aa) sequence of both the a subunit of the terminal oxygenase (BphA1) and ferredoxin (BphA3). The ferredoxin reductase subunit (BphA4) contains conserved sites for FAD and NADH binding. Deduced aa sequences of the BDO subunits shared homologies to multicomponent aromatic ring-hydroxylating dioxygenases from Gram- microorganisms. Stronger identity was found to toluene dioxygenase (TDO) of Pseudomonas putida F1 than to other BDO. Aa sequence comparisons suggest that BP degradation genes of RgP6 may have originated in Gram- microorganisms, probably Pseudomonas, and subsequently transferred to this Gram+ bacterium.

Amino Acid Sequence↗
PubMed1995

Identification of functional residues in a 2-hydroxymuconic semialdehyde hydrolase. A new member of the alpha/beta hydrolase-fold family of enzymes which cleaves carbon-carbon bonds.

The 2-hydroxymuconic semialdehyde hydrolase, XylF, of the Pseudomonas putida TOL plasmid-encoded pathway for the catabolism of toluene and xylenes, catalyzes one of the rarest types of enzyme reaction (EC 3.7.1.9), the hydrolysis of a carbon-carbon bond in its substrate, the ring-fission product of 3-alkyl-substituted catechols. In this study, amino acid sequence comparisons between XylF and other hydrolases, and analysis of the similarity between the predicted secondary structure of XylF and the known secondary structure of the haloalkane dehalogenase from Xanthobacter autotrophicus strain GJ10, led us to identify several conserved residues likely to have a functional role in the catalytic center of XylF. Three amino acids, Ser107, Asp228, and His256, were found to be arranged in a sequential order similar to that in alpha/beta hydrolase-fold enzymes. Investigations of the potential functional role of these and other residues through amino acid modification and in vitro site-directed mutagenesis experiments provided evidence in support of the hypothesis that XylF is a serine hydrolase of the alpha/beta hydrolase-fold family of enzymes, and pointed to the residues identified above as the catalytic triad of XylF. These studies also provided information on other conserved residues in XylF-related enzymes. Interestingly, the substitution of Phe by Met in position 108 of XylF created an enzyme with increased thermostability and altered substrate specificity.

Amino Acid Sequence↗
PubMed1995

Cloning, nucleotide sequence, and expression of the gene encoding a novel dioxygenase involved in metabolism of carboxydiphenyl ethers in Pseudomonas pseudoalcaligenes POB310.

Pseudomonas pseudoalcaligenes strain POB310 degrades 3- and 4-carboxydiphenyl ether. The initial reaction involves an angular dioxygenation yielding an unstable hemiacetal that spontaneously decays to phenol and protocatechuate. We cloned a DNA fragment containing the gene encoding the initial dioxygenase from an unstable, self-transmissible plasmid. Sequence analysis revealed two open reading frames encoding proteins with putative molecular masses of 46.3 and 33.6 kDa. The deduced amino acid sequences showed homologies to oxygenase and reductase subunits of aromatic ring-activating dioxygenases, and contained regions identical to consensus sequences that bind chloroplast-like and Rieske-type [2Fe2S] clusters, suggesting that the initial dioxygenase is a class IA aromatic ring-activating dioxygenase system. Initial dioxygenase activity was induced in bacteria grown in M9 minimal medium containing 3- or 4-carboxydiphenyl ether or phenol as carbon source, indicating that the regulation is dependent on the phenol pathway. The maximal specific activity was measured at the beginning of the exponential growth phase.

Amino Acid Sequence↗
PubMed1995

Monoclonal antibody against pIII of filamentous phage: an immunological tool to study pIII fusion protein expression in phage display systems.

UNLABELLED: A monoclonal antibody directed against the gene 3 product (pIII) of filamentous phage M13 was produced to study pIII-fusion protein expression in E. coli and its incorporation in the phage capsid. The protein was gel-purified from E. coli expression cultures harboring the genetic information of pIII under the control of an inducible lac promoter. To study pIII-fusion protein expression, phage display systems were applied in which either the whole pIII or the C-terminal half was used (McCafferty et al. (1990) Nature (London) 348, 552-554; Szardenings and Collins (1990) Gene 94, 1-7; Barbas and Lerner (1991) In: METHODS: Companion to METHODS in Enzymology, Combinatorial Immunoglobulin Libraries on the Surface of Phage (Phabs): Rapid Selection of Antigen-Specific Fabs, Vol. 2, Academic Press, Orlando, pp. 119-124). In all cases, the monoclonal antibody was able to detect the native and the recombinant protein in E. coli and on the phage tip using non-denaturing (ELISA) and denaturing (SDS-PAGE, immunoblot analysis) conditions. All selected pIII-specific monoclonal antibodies were found to be directed against epitopes within amino acids 198 to 406 of pIII, which is necessary for capsid incorporation and therefore included in all pIII-mediated phage display designs.

Animals↗
PubMed1995

Identification of Salmonella typhi promoters activated by invasion of eukaryotic cells.

The interaction of pathogenic microorganisms with host tissues, and the underlying genetic events which regulate these interactions, are difficult to analyse where no suitable animal model exists. The approach described here, for obtaining information on the genes involved in these interactions, employs an infection system based on the invasion of Henle cells by Salmonella typhi to select promoter-containing DNA sequences able to activate gene expression inside eukaryotic cells. Several DNA fragments exhibiting different promoter strengths and extent of selective activation within eukaryotic cells were identified. Three were selected and characterized according to the expression level of the reporter gene, the polynucleotide sequence, the transcription start, and the dependence upon regulatory proteins. All fragments gave much stronger expression of the reporter gene when the recombinant S. typhi carrier strains invaded cells compared with the expression measured in growth medium. One promoter-containing region exhibited sequence homology to sigma 54-dependent promoters, whereas another appears to be dependent on the stationary-phase RNA polymerase subunit sigma s. S. typhi containing the S1 subunit gene of pertussis toxin cloned under the control of these promoters, selectively expressed the S1 subunit following infection of different phagocytic and non-phagocytic cell lines of human or murine origin. Deletion and point mutant derivatives of two promoters enabled the identification of the main motif required for promoter activity. This method may be helpful for the analysis of pathogenesis in organisms previously difficult to study because of the lack of a convenient animal model, and could provide insights into the chronology and topology of gene expression during infection, including a possible genetic basis for tissue tropism.

Amino Acid Sequence↗
PubMed1995

Tracking the Response of Burkholderia cepacia G4 5223-PR1 in Aquifer Microcosms.

The introduction of bacteria into the environment for bioremediation purposes (bioaugmentation) requires analysis and monitoring of microbial population dynamics to define persistence and activity from both efficacy and risk assessment perspectives. Burkholderia cepacia G4 5223-PR1 is a Tn5 insertion mutant which constitutively expresses a toluene ortho-monooxygenase that degrades trichloroethylene (TCE). This ability of G4 5223-PR1 to degrade TCE without aromatic induction may be useful for bioremediation of TCE-containing aquifers and groundwater. Thus, a simulated aquifer sediment system and groundwater microcosms were used to monitor the survival of G4 5223-PR1. The fate of G4 5223-PR1 in sediment was monitored by indirect immunofluorescence microscopy, a colony blot assay, and growth on selective medium. G4 5223-PR1 was detected immunologically by using a highly specific monoclonal antibody which reacted against the O-specific polysaccharide chain of the lipopolysaccharides of this organism. G4 5223-PR1 survived well in sterilized groundwater, although in nonsterile groundwater microcosms rapid decreases in the G4 5223-PR1 cell population were observed. Ten days after inoculation no G4 5223-PR1 cells could be detected by selective plating or immunofluorescence. G4 5223-PR1 survival was greater in a nonsterile aquifer sediment microcosm, although after 22 days of elution the number of G4 5223-PR1 cells was low. Our results demonstrate the utility of monoclonal antibody tracking methods and the importance of biotic interactions in determining the persistence of introduced microorganisms.

Journal Article↗
PubMed1995

Characterization of a new lipopeptide surfactant produced by thermotolerant and halotolerant subsurface Bacillus licheniformis BAS50.

Strain BAS50, isolated from a petroleum reservoir at a depth of 1,500 m and identified as Bacillus licheniformis, grew and produced a lipopeptide surfactant when cultured on a variety of substrates at salinities of up to 13% NaCl. Surfactant production occurred both aerobically and anaerobically and was optimal at 5% NaCl and temperatures between 35 and 45 degrees C. The biosurfactant, termed lichenysin A, was purified and chemically characterized. A tentative structure and composition for the surfactant are described. Lichenysin A is a mixture of lipopeptides, with the major components ranging in size from 1,006 to 1,034 Da. The lipid moiety contains a mixture of 14 linear and branched beta-hydroxy fatty acids ranging in size from C12 to C17. There are seven amino acids per molecule. The peptide moiety is composed of the following amino acids: glutamic acid as the N-terminal amino acid, asparagine, valine, leucine, and isoleucine as the C-terminal amino acid, at a ratio of 1.1:1.1:1.0:2.8:1.0, respectively. Purified lichenysin A decreases the surface tension of water from 72 mN/m to 28 mN/m and achieves the critical micelle concentration with as little as 12 mg/liter, characterizing the product as a powerful surface-active agent that compares favorably to others surfactants. The antibacterial activity of lichenysin A has been demonstrated.

Amino Acids↗
PubMed1995

Formation of Dimethylmuconolactones from Dimethylphenols by Alcaligenes eutrophus JMP 134.

2,3-, 2,4-, 2,5-, 3,4-, and 3,5-dimethylphenols were cometabolized by 2,4-dichlorophenoxyacetate-grown Alcaligenes eutrophus JMP 134 or the constitutive derivative JMP 134-1 via the ortho pathway into dimethylmuconolactones as dead-end products. Formation of two distinct lactones from 3,4-dimethylphenol is indicative of 2- as well as 6-hydroxylation. Induction of the meta-cleavage pathway by 2,3- and 3,4-dimethylphenols resulted in growth and no accumulation of products. In contrast, 3,5-dimethylphenol is not metabolized by the meta-cleavage pathway.

Journal Article↗
PubMed1995

Conversion of chlorobiphenyls into phenylhexadienoates and benzoates by the enzymes of the upper pathway for polychlorobiphenyl degradation encoded by the bph locus of Pseudomonas sp. strain LB400.

Metabolism of 21 chlorobiphenyls by the enzymes of the upper biphenyl catabolic pathway encoded by the bph locus of Pseudomonas sp. strain LB400 was investigated by using recombinant strains harboring gene cassettes containing bphABC or bphABCD. The enzymes of the upper pathway were generally able to metabolize mono- and dichlorinated biphenyls but only partially transform most trichlorinated congeners investigated: 14 of 15 mono- and dichlorinated and 2 of 6 trichlorinated congeners were converted into benzoates. All mono- and at least 8 of 12 dichlorinated congeners were attacked by the bphA-encoded biphenyl dioxygenase virtually exclusively at ortho and meta carbons. This enzyme exhibited a high degree of selectivity for the aromatic ring to be attacked, with the order of ring preference being non- > ortho- > meta- > para-substituted for mono- and dichlorinated congeners. The influence of the chlorine substitution pattern of the metabolized ring on benzoate formation resembled its influence on the reactivity of initial dioxygenation, suggesting that the rate of benzoate formation may frequently be determined by the rate of initial attack. The absorption spectra of phenylhexadienoates formed correlated with the presence or absence of a chlorine substituent at an ortho position.

Benzoates↗