Biomedical subjects

K N Timmis

Publications and source records attributed to K N Timmis.

At least 55 records · Page 3Linked to original sources

PubMed1998

Multifunctional g3p-peptide tag for current phage display systems.

We have previously described a monoclonal antibody (mAb), 10C3, directed against the gene-3 protein (g3p) of filamentous phage M13, which was produced to study g3p fusion protein expression in Escherichia coli and its incorporation in the phage capsid [Tesar, M., Beckmann, C., Röttgen, P., Haase, B., Faude, U., Timmis, K., 1995. Monoclonal antibody against pIII of filamentous phage: an immunological tool to study pIII fusion protein expression in phage display systems. Immunology 1, 53-54]. In this study we report mapping of the antigenic epitope of the mAb 10C3, by means of short overlapping peptide-sequences [Frank, R., Overwin, H., 1996. Spot synthesis. In: Morris, G.E. (Ed.), Methods in Molecular Biology, Vol. 66: Epitope Mapping Protocols. Humana Press, Totowa, NJ, pp. 149-169.] comprising the C-terminal half of the g3-protein. A minimal recognizable peptide was found which is represented in the 11 amino acid sequence from positions 292 to 302 of g3p [Wezenbeek van, P.M.G.P., Hulsebos, T.J.M., Schoenmakers, J.G.G., 1980. Nucleotide sequence of the filamentous bacteriophage M13 DNA genome: comparison with phage fd. Gene 11, 129-148]. In order to use the antibody also for detection and purification of recombinant proteins, such as single chain antibodies, the epitope was introduced as a tag sequence into the phagemid pHEN1 [Hoogenboom, H.R., Griffith, A.D., Johnson, K., Chiswell, D.J., Hudson, P., Winter, G., 1991. Multi-subunit proteins on the surface of the filamentous phage: methodologies for displaying antibody (Fab) heavy and light chains. Nucleic Acid Res. 19, 4133-4137; Nissim, A., Hoogenboom, H.R., Tomlinson, I.M., Flynn, G., Midgley, C., Lane, D., Winter, G., 1994. Antibody fragments from a single pot phage display library as immunochemical reagents. EMBO J. 13 (3) 692-698]. Purified single chain antibodies containing this tag were detectable down to a concentration of 2 ng ml(-1) under non-denaturing conditions (ELISA) or 4 ng per lane on immunoblots. The high sensitivity of the antibody for the peptide tag was reflected in the antibody affinity constant K(D) of 6.80 x 10(-10) M, which was determined by real time biomolecular interaction analysis (BIA) based on surface plasmon resonance (SPR) [Karlsson, R., Fält, A., 1997. Experimental design for kinetic analysis of protein-protein interactions with surface plasmon resonance biosensors. J. Immunol. Methods 200, 121-133]. Finally, recombinant proteins in E. coli periplasmic extracts could be purified in a single step by affinity purification using immobilized mAb 10C3. These studies demonstrated that the new peptide-tag and its corresponding mAb represents a versatile tool for the detection of recombinant proteins selected by phage display technology.

Antibodies, Monoclonal↗

PubMed1998

Characterization of a gene cluster from Ralstonia eutropha JMP134 encoding metabolism of 4-methylmuconolactone.

A 2,585 bp chromosomal DNA segment of Ralstonia eutropha JMP134 (formerly: Alcaligenes eutrophus JMP134) which contains a gene cluster encoding part of the modified ortho-cleavage pathway encodes a putative transport protein for 4-methylmuconolactone, a novel 4-methylmuconolactone methylisomerase and methylmuconolactone isomerase. The putative 4-methylmuconolactone transporter, a protein with a calculated molecular mass of 45.8 kDa, exhibits sequence homology to other members of the major superfamily of transmembrane facilitators and shows the common structural motif of 12 transmembrane-spanning alpha-helical segments and the hallmark amino acid motif characteristic of the superfamily. Consistent with the novelty of the reaction catalyzed by 4-methylmuconolactone methylisomerase, no primary sequence homologies were found between this enzyme or its gene and other proteins or genes in the data banks, suggesting that this enzyme represents a new type of isomerase. The molecular mass of the native 4-methylmuconolactone methylisomerase was determined by gel filtration analysis to be 25 +/- 2 kDa. From the polynucleotide sequence of the gene, a molecular mass of 12.9 kDa was calculated and hence we predict a homodimeric quaternary structure. The high sensitivity of 4-methylmuconolactone methylisomerase to heavy metals and thiol-modifying reagents implicates the involvement of sulfhydryl groups in the catalytic reaction. The methylmuconolactone isomerase - calculated molecular mass 10.3 kDa - has a primary structure related to the classical muconolactone isomerases (EC 5.3.3.4) of Acinetobacter calcoaceticus, of two Pseudomonas putida strains and of Ralstonia eutropha JMP134, suggesting that these are all isoenzymes. Consistent with this proposal is the finding that the purified protein exhibits muconolactone-isomerizing activity.

Alcaligenes↗

PubMed1998

Crystal structure of cis-biphenyl-2,3-dihydrodiol-2,3-dehydrogenase from a PCB degrader at 2.0 A resolution.

cis-Biphenyl-2,3-dihydrodiol-2,3-dehydrogenase (BphB) is involved in the aerobic biodegradation of polychlorinated biphenyls (PCBs). The crystal structure of the NAD+-enzyme complex was determined by molecular replacement and refined to an R-value of 17.9% at 2.0 A. As a member of the short-chain alcohol dehydrogenase/reductase (SDR) family, the overall protein fold and positioning of the catalytic triad in BphB are very similar to those observed in other SDR enzymes, although small differences occur in the cofactor binding site. Modeling studies indicate that the substrate is bound in a deep hydrophobic cleft close to the nicotinamide moiety of the NAD+ cofactor. These studies further suggest that Asn143 is a key determinant of substrate specificity. A two-step reaction mechanism is proposed for cis-dihydrodiol dehydrogenases.

Asparagine↗

PubMed1998

Alcanivorax borkumensis gen. nov., sp. nov., a new, hydrocarbon-degrading and surfactant-producing marine bacterium.

During screening for biosurfactant-producing, n-alkane-degrading marine bacteria, six heterotrophic bacterial strains were isolated from enriched mixed cultures, obtained from sea water/sediment samples collected near the isle of Borkum (North Sea), using Mihagol-S (C14,15-n-alkanes) as principal carbon source. These Gram-negative, aerobic, rod-shaped bacteria use a limited number of organic compounds, including aliphatic hydrocarbons, volatile fatty acids, and pyruvate and its methyl ether. During cultivation on n-alkanes as sole source of carbon and energy, all strains produced both extracellular and cell-bound surface-active glucose lipids which reduced the surface tension of water from 72 to 29 mN m-1 (16). This novel class of glycolipids was found to be produced only by these strains. The 16S rRNA gene sequence analysis showed that these strains are all members of the gamma-subclass of the Proteobacteria. Their phospholipids ester-linked fatty acid composition was shown to be similar to that of members of the genus Halmonas, although they did not demonstrate a close phylogenetic relationship to any previously described species. On the basis of the information summarized above, a new genus and species, Alcanivorax borkumensis, is described to include these bacteria. Strain SK2T is the type strain of A. borkumensis.

Base Composition↗

PubMed1998

Use of subtractive hybridization to design habitat-based oligonucleotide probes for investigation of natural bacterial communities.

We describe a rapid oligonucleotide probe design strategy based on subtractive hybridization which yields probes for 16S rRNA or rRNA genes of individual members of microbial communities that are specific within the context of those communities. This strategy circumvents the need to sequence many similar or identical clones of dominant members of a community. Radioactively labeled subfragments of a cloned 16S rRNA gene sequence for which a probe is required (target) were hybridized with biotinylated total 16S ribosomal DNA (rDNA) amplified from the microbial community, and the hybrids formed were subsequently discarded. The remaining enriched fragments were used to screen a library consisting of cloned subfragments of the target sequence by colony hybridization in order to identify the variable regions of the 16S rRNA gene with the required specificity. The sequencing of random clones in one 16S rDNA library demonstrated that only those clones with 100% sequence identity with the probe fragment were detected by it. Moreover, sequencing of other, randomly selected, probe-positive clones revealed 100% sequence identity with the probe. Probes developed in this way tended to correspond to more variable regions of the 16S rRNA if the target sequences were similar to the sequences of other clones in the library and to less variable regions if the target sequences were phylogenetically isolated within the clone library. Although the absolute specificity of the latter probes, as assessed by comparison with available database sequences, was lower than the absolute specificity of the probes from the more variable regions, they were specific within the context of the environmental samples from which they were derived.

Bacteria↗

PubMed1998

Activation and repression of transcription at the double tandem divergent promoters for the xylR and xylS genes of the TOL plasmid of Pseudomonas putida.

The xylR and xylS genes are divergent and control transcription of the TOL plasmid catabolic pathways for toluene metabolism. Four promoters are found in the 300-bp intergenic region: Pr1 and Pr2 are constitutive sigma70-dependent tandem promoters that drive expression of xylR, while expression of the xylS gene is driven from Ps2, a constitutive sigma70-dependent promoter, and by the regulatable sigma54 class Ps1 promoter. In Ps1 the XylR targets (upstream activator sequences [UASs]) overlap the Pr promoters, and two sites for integration host factor (IHF) binding are located at the region from positions -2 to -30 (-2/-30 region) and the -137/-156 region, the latter overlapping the Pr promoters. When the XylR protein binds to the UASs in the absence of effector, it represses expression from Pr promoters. In the XylR-plus background and in the absence of an effector, the level of expression from Ps1 is low, although detectable, whereas Ps2 is active. In this background and in the presence of an effector, XylR increases autorepression. In a sigma54-deficient Pseudomonas putida background, no expression occurred from Ps1 regardless of the presence of an effector. However, in the presence of an effector, the amount of RNA produced from Pr promoters was almost undetectable. This finding suggests that when no transcription occurred at the Ps1 promoter, clearance of XylR from the UASs was almost negligible. In this background, expression from Ps2 was very high regardless of the presence of an effector; this finding suggests that RNA polymerase containing sigma54 modulates expression from the downstream Ps2 sigma70-dependent promoter. In a P. putida IHF-minus background and in the presence of effector, Ps1 expression was the highest found; in contrast, the basal levels of this promoter were the lowest observed. This finding suggests that IHF acts in vivo as a repressor of the sigma54-dependent Ps1 promoter. In an IHF-deficient host background, expression from Ps2 in the presence of effector was negligible. Thus, binding of RNA polymerase containing sigma54 at the upstream promoter may modulate expression from the Ps2 promoter.

Bacterial Proteins↗

PubMed1998

Genetic analysis of dioxin dioxygenase of Sphingomonas sp. Strain RW1: catabolic genes dispersed on the genome.

The dioxin dioxygenase of Sphingomonas sp. strain RW1 activates dibenzo-p-dioxin and dibenzofuran for further metabolism by introducing two atoms of oxygen at a pair of vicinal carbon atoms, one of which is involved in one of the bridges between the two aromatic rings, i.e., an angular dioxygenation. The dxnA1 and dxnA2 cistrons encoding this dioxygenase have been cloned and shown to be located just upstream of a hydrolase gene which specifies an enzyme involved in the subsequent step of the dibenzofuran biodegradative pathway. Genes encoding the electron supply system of the dioxygenase are not clustered with the dioxygenase gene but rather are located on two other distinct and separate genome segments. Moreover, whereas expression of dxnA1A2 is modulated according to the available carbon source, expression of the dbfB gene encoding the ring cleavage enzyme of the dibenzofuran pathway, which is located in the neighborhood of dxnA1A2 but oriented in the opposite direction, is constitutive. The scattering of genes for the component proteins of dioxin dioxygenase system around the genome of Sphingomonas sp. strain RW1, and the differential expression of dioxin pathway genes, is unusual and contrasts with the typical genetic organization of catabolic pathways where component cistrons tend to be clustered in multicistronic transcriptional units. The sequences of the alpha and beta subunits of the dioxin dioxygenase exhibit only weak similarity to other three component dioxygenases, but some motifs such as the Fe(II) binding site and the [2Fe-2S] cluster ligands are conserved. Dioxin dioxygenase activity in Escherichia coli cells containing the cloned dxnA1A2 gene was achieved only through coexpression of the cognate electron supply system from RW1. Under these conditions, exclusively angular dioxygenation of dibenzofuran and dibenzo-p-dioxin was obtained. The dioxin dioxygenase was not active in E. coli cells coexpressing a class IIB electron supply system. In the course of the isolation of the dxnA1 and dxnA2 cistrons, a number of other catabolic genes dispersed over different genome segments were identified, which may indicate greater catabolic potential than was previously suspected. This finding is consistent with the catabolic versatility of members of the genus Sphingomonas, which is becoming increasingly evident, and may indicate a less well evolved and regulated but more dynamic genetic organization in this organism than is the case for better-studied pathways in organisms such as Pseudomonas species.

Amino Acid Sequence↗

PubMed1998

Biochemical and genetic characterization of a gentisate 1, 2-dioxygenase from Sphingomonas sp. strain RW5.

A 4,103-bp long DNA fragment containing the structural gene of a gentisate 1,2-dioxygenase (EC 1.13.11.4), gtdA, from Sphingomonas sp. strain RW5 was cloned and sequenced. The gtdA gene encodes a 350-amino-acid polypeptide with a predicted size of 38.85 kDa. Comparison of the gtdA gene product with protein sequences in databases, including those of intradiol or extradiol ring-cleaving dioxygenases, revealed no significant homology except for a low similarity (27%) to the 1-hydroxy-2-naphthoate dioxygenase (phdI) of the phenanthrene degradation in Nocardioides sp. strain KP7 (T. Iwabuchi and S. Harayama, J. Bacteriol. 179:6488-6494, 1997). This gentisate 1,2-dioxygenase is thus a member of a new class of ring-cleaving dioxygenases. The gene was subcloned and hyperexpressed in E. coli. The resulting product was purified to homogeneity and partially characterized. Under denaturing conditions, the polypeptide exhibited an approximate size of 38.5 kDa and migrated on gel filtration as a species with a molecular mass of 177 kDa. The enzyme thus appears to be a homotetrameric protein. The purified enzyme stoichiometrically converted gentisate to maleylpyruvate, which was identified by gas chromatography-mass spectrometry analysis as its methyl ester. Values of affinity constants (Km) and specificity constants (Kcat/Km) of the enzyme were determined to be 15 microM and 511 s-1 M-1 x 10(4) for gentisate and 754 microM and 20 s-1 M-1 x 10(4) for 3, 6-dichlorogentisate. Three further open reading frames (ORFs) were found downstream of gtdA. The deduced amino acid sequence of ORF 2 showed homology to several isomerases and carboxylases, and those of ORFs 3 and 4 exhibited significant homology to enzymes of the glutathione isomerase superfamily and glutathione reductase superfamily, respectively.

Amino Acid Sequence↗

PubMed1998

Detoxification of protoanemonin by dienelactone hydrolase.

Protoanemonin is a toxic metabolite which may be formed during the degradation of some chloroaromatic compounds, such as polychlorinated biphenyls, by natural microbial consortia. We show here that protoanemonin can be transformed by dienelactone hydrolase of Pseudomonas sp. strain B13 to cis-acetylacrylate. Although similar Km values were observed for cis-dienelactone and protoanemonin, the turnover rate of protoanemonin was only 1% that of cis-dienelactone. This indicates that at least this percentage of the enzyme is in the active state, even in the absence of activation. The trans-dienelactone hydrolase of Pseudomonas sp. strain RW10 did not detectably transform protoanemonin. Obviously, Pseudomonas sp. strain B13 possesses at least two mechanisms to avoid protoanemonin toxicity, namely a highly active chloromuconate cycloisomerase, which routes most of the 3-chloro-cis,cis-muconate to the cis-dienelactone, thereby largely preventing protoanemonin formation, and dienelactone hydrolase, which detoxifies any small amount of protoanemonin that might nevertheless be formed.

Biodegradation, Environmental↗

PubMed1998

Identification of chlorobenzene dioxygenase sequence elements involved in dechlorination of 1,2,4,5-tetrachlorobenzene.

The TecA chlorobenzene dioxygenase and the TodCBA toluene dioxygenase exhibit substantial sequence similarity yet have different substrate specificities. Escherichia coli cells producing recombinant TecA enzyme dioxygenate and simultaneously eliminate a halogen substituent from 1,2,4,5-tetrachlorobenzene but show no activity toward benzene, whereas those producing TodCBA dioxygenate benzene but not tetrachlorobenzene. A hybrid TecA dioxygenase variant containing the large alpha-subunit of the TodCBA dioxygenase exhibited a TodCBA dioxygenase specificity. Acquisition of dehalogenase activity was achieved by replacement of specific todC1 alpha-subunit subsequences by equivalent sequences of the tecA1 alpha-subunit. Substrate transformation specificities and rates by E. coli resting cells expressing hybrid systems were analyzed by high-performance liquid chromatography. This allowed the identification of both a single amino acid and potentially interacting regions required for dechlorination of tetrachlorobenzene. Hybrids with extended substrate ranges were generated that exhibited activity toward both benzene and tetrachlorobenzene. The regions determining substrate specificity in (chloro)benzene dioxygenases appear to be different from those previously identified in biphenyl dioxygenases.

Amino Acid Sequence↗

PubMed1997

Oral somatic transgene vaccination using attenuated S. typhimurium.

An attenuated strain of S. typhimurium has been used as a vehicle for oral genetic immunization. Eukaryotic expression vectors containing truncated genes of ActA and listeriolysin--two virulence factors of Listeria monocytogenes--have been used to transform S. typhimurium aroA. Multiple or even single oral immunizations with such transformants induced excellent cellular and humoral responses. In addition, protective immunity was induced with listeriolysin transformants. The quality of the responses suggested a transfer of plasmid DNA from the bacterial carrier to the host. Such transfer was unequivocally shown in vitro with primary peritoneal macrophages. We describe a highly versatile system for antigen delivery, identification of protective antigens for vaccination, and efficient generation of antibodies against the product of open reading frames present on virtually any DNA segment.

3-Phosphoshikimate 1-Carboxyvinyltransferase↗

PubMed1997

Green fluorescent protein-based reporter systems for genetic analysis of bacteria including monocopy applications.

The green fluorescent protein (GFP) gene, gfp, was used to develop versatile reporter systems for genetic analysis in, and monitoring of bacteria. This reporter system is available on a plasmid and on a mini-transposon located in a suicide delivery plasmid for generation of chromosomal fusions. To achieve sensitivity levels necessary for use in monocopy applications and for detection of single cells, the 3'-end of gfp was replaced by that of a modified gfp gene characterized by a 45-fold stronger fluorescence signal than that exhibited by the natural GFP. This modified gfp gene was also equipped with the strong translation signals of the atpE gene. Transfer of the mini-transposon into two different Pseudomonas spp. and Alcaligenes eutrophus produced random chromosomal fusions, some 5% of which exhibited fluorescence detectable by eye. Individual GFP+ cells were readily observed by fluorescence microscopy.

Bacteria↗

PubMed1997

Molecular characterization of Fdx1, a putidaredoxin-type [2Fe-2S] ferredoxin able to transfer electrons to the dioxin dioxygenase of Sphingomonas sp. RW1.

Bacterium Sphingomonas sp. strain RW1 is, under aerobic conditions, able to degrade dibenzofuran and dibenzo-p-dioxin. The first step of the pathway is performed by a ring-dihydroxylating enzyme. Bunz and Cook have reported the purification and characterization of this dioxin dioxygenase and a ferredoxin able to transfer electrons to the dioxygenase [Bunz, P. V. & Cook, A. M. (1993) J. Bacteriol. 175, 6467-6475]. The gene encoding this [2Fe-2S] ferredoxin was identified by screening a genomic library constructed in pLAFR3 with a probe generated by a nested-PCR amplification. Primers for the amplification were designed based on the N-terminus sequence of the purified ferredoxin and on sequence comparisons with related proteins. Several cosmids were obtained and the ferredoxin gene, fdx1, was subcloned from one of them. The nucleotide sequence of a 4.6-kb DNA fragment encompassing the ferredoxin gene was determined. While in the case of all known multi-component dioxygenases, genes encoding the alpha and beta subunits are found to be contiguous with the gene of the specific electron carrier, the fdx1 gene in Sphingomonas sp. RW1 does not appear to be directly linked with the dioxin dioxygenase genes. Rather, it is clustered with genes apparently encoding two atypical decarboxylases/isomerases and a glutathione S-transferase. The ferredoxin gene was hyperexpressed and the recombinant ferredoxin was purified. Spectroscopic characterization of Fdx1 demonstrated the presence of a putidaredoxin-type [2Fe-2S] cluster in this protein. Its redox potential was determined to be -245 (+/- 5) mV versus the normal hydrogen electrode at 25 degrees C, pH 8.0. Therefore, the protein is closely related to [2Fe-2S] ferredoxins known to be electron donors to monooxygenases involved in hydroxylation of aromatic compounds. Thus, this report provides clear evidence that a putidaredoxin-type [2Fe-2S] ferredoxin, namely Fdx1, is able to transfer electrons to the dioxin dioxygenase of Sphingomonas sp. RW1.

Amino Acid Sequence↗

PubMed1997

Genetic and biochemical characterization of the broad spectrum chlorobenzene dioxygenase from Burkholderia sp. strain PS12--dechlorination of 1,2,4,5-tetrachlorobenzene.

The bacterium, Burkholderia (previously Pseudomonas) sp. strain PS12, reported earlier to degrade 1,2,4-trichlorobenzene is shown here to utilize also 1,2,4,5-tetrachlorobenzene (Cl4-benzene) as a growth substrate. To investigate the possibility that this organism attacks Cl4-benzene with a chlorobenzene dioxygenase which concomitantly causes dehalogenation, and to analyze the substrate range of the initial enzyme, a 5503-bp DNA fragment from PS12, exhibiting high similarity to genes coding for class IIB dioxygenases, was cloned and expressed in Escherichia coli. The sequence includes the tec genes coding for the alpha-subunit and beta-subunit of a terminal dioxygenase, a ferredoxin and a reductase. E. coli cells producing these proteins were able to dioxygenolytically attack a range of aromatic compounds including chlorinated benzenes and toluene, and also dinuclear aromatics such as biphenyl and dibenzo-p-dioxin. The enzyme was shown by (18)O2 incorporation experiments to dioxygenolytically attack a chlorosubstituted carbon atom of Cl4-benzene, thereby forming an unstable diol intermediate which spontaneously rearomatizes with concomitant chloride elimination to the corresponding 3,4,6-trichlorocatechol (Cl3-catechol).

Amino Acid Sequence↗

PubMed1997

16S rRNA gene sequence analyses and inter- and intrageneric relationships of Xanthomonas species and Stenotrophomonas maltophilia.

The nearly complete, PCR-amplified, 16S rRNA gene sequences have been determined from the representative type strains of eight xanthomonad phena, including six validly described species of the genus Xanthomonas and Stenotrophomonas maltophilia. Pairwise sequence comparisons and phylogenetic analysis demonstrated that the xanthomonads comprise a monophyletic lineage-within the gamma-subclass of the Proteobacteria. Although the genus Xanthomonas was observed to comprise a cluster of very closely related species, the observed species-specific primary sequence differences were confirmed through sequencing additional strains belonging to the respective species.

Base Sequence↗

PubMed1997

Temperature dependent expression of an acid phosphatase by Bordetella bronchiseptica: role in intracellular survival.

Bordetella bronchiseptica has the ability to invade and survive intracellularly. This potential to survive for extended periods within eukaryotic cells might play an important role in the pathogenesis of the infections caused by this microorganism. The bacterial factors involved in this process, however, have not yet been determined. In this study we have identified an acid phosphatase produced by B. bronchiseptica, but not by other Bordetella spp. The expression of this enzyme was demonstrated to be strictly regulated by temperature (optimal expression at 30 degrees C) and seems to be partially repressed by the product of the bvg locus. The enzyme was localized in the cytoplasmic fraction, the optimal activity was observed at pH 5.5, and the apparent molecular mass obtained by zymogram was 40 kDa. To further investigate the pathogenic role of this enzyme, mutants lacking acid phosphatase activity were obtained from both bvg-positive and bvg-negative parental strains using minitransposons. The growth pattern of these recombinant clones in vitro was similar to the parent strains, however, the tested clones exhibited a significant reduction (P < or = 0.05) in their intracellular survival ability. This newly described acid phosphatase from B. bronchiseptica seems to play a role in intracellular survival, and therefore represents a novel pathogenicity factor.

Acid Phosphatase↗

PubMed1997

Construction and characterization of genetically-marked bivalent anti-Shigella dysenteriae 1 and anti-Shigella flexneri Y live vaccine candidates.

Bivalent vaccine candidates were developed against Shigella dysenteriae 1 and Shigella flexneri, which are among the most frequent causative agents of shigellosis in developing countries. The rfp and rfb gene clusters, which code for S. dysenteriae serotype 1 O-antigen biosynthesis, were inserted into an arsenite resistance minitransposon and randomly integrated into the attenuated S. flexneri aroD serotype Y strain SFL124. Nine recombinant clones that efficiently expressed both homologous and heterologous O-antigens were obtained. Southern blot analysis showed that in one clone the S. dysenteriae 1 genes had integrated into the chromosome, whereas in all the others they had integrated into the virulence plasmid. All recombinant clones exhibited normal growth characteristics, were able to invade and survive within eukaryotic cells to the same extent as the parental strain, and expressed efficiently the recombinant lipopolysaccharide within invaded cells. Immunization of mice with two of the recombinant clones resulted in the production of antibodies specific for both homologous and heterologous O-antigens. The recombinant clones constitute promising vaccine candidates which can readily be distinguished from endemic shigellae by their non-antibiotic resistance marker.

Animals↗

PubMed1997

Bioprotection of microbial communities from toxic phenol mixtures by a genetically designed pseudomonad.

Pseudomonas sp. B13 SN45RE is a genetically engineered microorganism (GEM) that is able to simultaneously degrade mixtures of chloro- and methylaromatics ordinarily toxic for microbial communities via a designed novel ortho-cleavage pathway. The utility of the GEM was investigated in a laboratory scale sewage plant fed with mixtures of either 4-chlorophenol and 4-methyphenol or 3-chlorophenol and 4-methylphenol. In the model system the GEM significantly increased the rate and extent of degradation of the phenol mixtures. In the absence of the GEM, shock loads of the phenol mixtures (1 mM of each compound) reduced the numbers of culturable bacteria by three orders of magnitude, completely eliminated protozoa and metazoa, and caused a drastic decrease in oxygen consumption, whereas the presence of the GEM protected the indigenous microbial community and assured continued functioning of the sewage plant.

Biodegradation, Environmental↗