A wide range of microorganisms, the so-called acidophiles, inhabit acidic environments and grow optimally at pH values between 0 and 3. The intracellular pH of these organisms is, however, close to neutrality or slightly acidic. It is to be expected that enzymatic activities dedicated to extracellular functions would be adapted to the prevailing low pH of the environment (0-3), whereas intracellular enzymes would be optimally active at the near-neutral pH of the cytoplasm (4.6-7.0). The genes of several intracellular or cell-bound enzymes, a carboxylesterase and three alpha-glucosidases, from Ferroplasma acidiphilum, a cell wall-lacking acidophilic archaeon with a growth optimum at pH 1.7, were cloned and expressed in Escherichia coli, and their products purified and characterized. The Ferroplasmaalpha-glucosidases exhibited no sequence similarity to known glycosyl hydrolases. All enzymes functioned and were stable in vitro in the pH range 1.7-4.0, and had pH optima much lower than the mean intracellular pH of 5.6. This 'pH optimum anomaly' suggests the existence of yet-undetected cellular compartmentalization providing cytoplasmic pH patchiness and low pH environments for the enzymes we have analysed.
Alcanivorax borkumensis is a cosmopolitan marine bacterium that uses oil hydrocarbons as its exclusive source of carbon and energy. Although barely detectable in unpolluted environments, A. borkumensis becomes the dominant microbe in oil-polluted waters. A. borkumensis SK2 has a streamlined genome with a paucity of mobile genetic elements and energy generation-related genes, but with a plethora of genes accounting for its wide hydrocarbon substrate range and efficient oil-degradation capabilities. The genome further specifies systems for scavenging of nutrients, particularly organic and inorganic nitrogen and oligo-elements, biofilm formation at the oil-water interface, biosurfactant production and niche-specific stress responses. The unique combination of these features provides A. borkumensis SK2 with a competitive edge in oil-polluted environments. This genome sequence provides the basis for the future design of strategies to mitigate the ecological damage caused by oil spills.
We created a metagenome expression library from the brine:seawater interface of the Urania hypersaline basin, screened it for esterases, and characterized five of these. Two had no significant sequence homology to known esterases, hydrolyzed both carboxylesters and thioesters, and exhibited unusual, habitat-specific characteristics (preference for high hydrostatic pressure and salinity). One has an unusual structural signature incorporating three catalytic active centers mediating distinct hydrolytic activities and an adaptive tertiary-quaternary structure that alters between three molecular states, according to the prevailing physicochemical conditions. Some of the esterases have high activities, specificities, enantioselectivities, and exceptional stability in polar solvents, and they are therefore potentially useful for industrial biotransformations. One possesses the highest enantioselectivity toward an ester of the important chiral synthon solketal (E: 126[S]; 98%ee).
A metagenome expression library of bulk DNA extracted from the rumen content of a dairy cow was established in a phage lambda vector and activity-based screening employed to explore the functional diversity of the microbial flora. Twenty-two clones specifying distinct hydrolytic activities (12 esterases, nine endo-beta-1,4-glucanases and one cyclodextrinase) were identified in the library and characterized. Sequence analysis of the retrieved enzymes revealed that eight (36%) were entirely new and formed deep-branched phylogenetic lineages with no close relatives among known ester- and glycosyl-hydrolases. Bioinformatic analyses of the hydrolase gene sequences, and the sequences and contexts of neighbouring genes, suggested tentative phylogenetic assignments of the rumen organisms producing the retrieved enzymes. The phylogenetic novelty of the hydrolases suggests that some of them may have potential for new applications in biocatalysis.
Hydrocarbon-contaminated superficial sediments collected from the Harbor of Milazzo (Tirrenean Sea, northern Sicily), a zone strongly affected by anthropogenic activities, were examined for in situ biodegradative capacities. A culture-independent molecular phylogenetic approach was used to study the influence of hydrocarbon and nutrient addition on the activity and diversity of the indigenous microbiota during a microcosm evaluation. The autochthonous microbial community in non-polluted sediments was represented by eubacterial phylotypes grouped within Proteobacteria, CFB and Firmicutes. The archaeal domain was represented by members of Marine Group I of Crenarchaeota. The majority of recovered sequences was affiliated with heterotrophic genera Clostridium and Vibrio, typical members of eutrophic coastal environments. Amendments of hydrocarbons and mineral nutrients to microcosms dramatically changed the initial diversity of the microbial community. Only bacterial phylotypes affiliated with Proteobacteria and CFB division were detected. The decrease in diversity observed in several microcosms could be explained by the strong selection for microorganisms belonging to group of marine hydrocarbonoclastic gamma-Proteobacteria, namely Alcanivorax, Cycloclasticus, Marinobacter, Marinobacterium/Neptunomonas and Thalassolituus. This study demonstrated that nutrient amendment to hydrocarbon-contaminated superficial sediments enhanced the indigenous microbial biodegradation activity and that highly specialized marine hydrocarbonoclastic bacteria, representing a minor fraction in the natural microbial community, play an important role in the biodegradation of petroleum hydrocarbons accidentally entering the coastal environment.
Title: Expression of a temperature-sensitive esterase in a novel chaperone-based Escherichia coli strain Ferrer M, Chernikova TN, Timmis KN, Golyshin PN Ref: Applied Environmental Microbiology, 70:4499, 2004 : PubMed
A new principle for expression of heat-sensitive recombinant proteins in Escherichia coli at temperatures close to 4 degrees C was experimentally evaluated. This principle was based on simultaneous expression of the target protein with chaperones (Cpn60 and Cpn10) from a psychrophilic bacterium, Oleispira antarctica RB8(T), that allow E. coli to grow at high rates at 4 degrees C (maximum growth rate, 0.28 h(-1)). The expression of a temperature-sensitive esterase in this host at 4 to 10 degrees C yielded enzyme specific activity that was 180-fold higher than the activity purified from the non-chaperonin-producing E. coli strain grown at 37 degrees C (32,380 versus 190 micromol min(-1) g(-1)). We present evidence that the increased specific activity was not due to the low growth temperature per se but was due to the fact that low temperature was beneficial to folding, with or without chaperones. This is the first report of 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).
Most naturally occurring biofilms contain a vast majority of microorganisms which have not yet been cultured, and therefore we have little information on the genetic information content of these communities. Therefore, we initiated work to characterize the complex metagenome of model drinking water biofilms grown on rubber-coated valves by employing three different strategies. First, a sequence analysis of 650 16S rRNA clones indicated a high diversity within the biofilm communities, with the majority of the microbes being closely related to the Proteobacteria: Only a small fraction of the 16S rRNA sequences were highly similar to rRNA sequences from Actinobacteria, low-G+C gram-positives and the Cytophaga-Flavobacterium-Bacteroides group. Our second strategy included a snapshot genome sequencing approach. Homology searches in public databases with 5,000 random sequence clones from a small insert library resulted in the identification of 2,200 putative protein-coding sequences, of which 1,026 could be classified into functional groups. Similarity analyses indicated that significant fractions of the genes and proteins identified were highly similar to known proteins observed in the genera Rhizobium, Pseudomonas, and Escherichia: Finally, we report 144 kb of DNA sequence information from four selected cosmid clones, of which two formed a 75-kb overlapping contig. The majority of the proteins identified by whole-cosmid sequencing probably originated from microbes closely related to the alpha-, beta-, and gamma-Proteobacteria: The sequence information was used to set up a database containing the phylogenetic and genomic information on this model microbial community. Concerning the potential health risk of the microbial community studied, no DNA or protein sequences directly linked to pathogenic traits were identified.
Pseudomonas putida is a metabolically versatile saprophytic soil bacterium that has been certified as a biosafety host for the cloning of foreign genes. The bacterium also has considerable potential for biotechnological applications. Sequence analysis of the 6.18 Mb genome of strain KT2440 reveals diverse transport and metabolic systems. Although there is a high level of genome conservation with the pathogenic Pseudomonad Pseudomonas aeruginosa (85% of the predicted coding regions are shared), key virulence factors including exotoxin A and type III secretion systems are absent. Analysis of the genome gives insight into the non-pathogenic nature of P. putida and points to potential new applications in agriculture, biocatalysis, bioremediation and bioplastic production.
Title: A functional 4-hydroxysalicylate/hydroxyquinol degradative pathway gene cluster is linked to the initial dibenzo-p-dioxin pathway genes in Sphingomonas sp. strain RW1 Armengaud J, Timmis KN, Wittich RM Ref: Journal of Bacteriology, 181:3452, 1999 : PubMed
The bacterium Sphingomonas sp. strain RW1 is able to use dibenzo-p-dioxin, dibenzofuran, and several hydroxylated derivatives as sole sources of carbon and energy. We have determined and analyzed the nucleic acid sequence of a 9,997-bp HindIII fragment downstream of cistrons dxnA1A2, which encode the dioxygenase component of the initial dioxygenase system of the corresponding catabolic pathways. This fragment contains 10 colinear open reading frames (ORFs), apparently organized in one compact operon. The enzymatic activities of some proteins encoded by these genes were analyzed in the strain RW1 and, after hyperexpression, in Escherichia coli. The first three ORFs of the locus, designated dxnC, ORF2, and fdx3, specify a protein with a low homology to bacterial siderophore receptors, a polypeptide representing no significant homology to known proteins, and a putative ferredoxin, respectively. dxnD encodes a 69-kDa phenol monooxygenase-like protein with activity for the turnover of 4-hydroxysalicylate, and dxnE codes for a 37-kDa protein whose sequence and activity are similar to those of known maleylacetate reductases. The following gene, dxnF, encodes a 33-kDa intradiol dioxygenase which efficiently cleaves hydroxyquinol, yielding maleylacetate, the ketoform of 3-hydroxy-cis,cis-muconate. The heteromeric protein encoded by dxnGH is a 3-oxoadipate succinyl coenzyme A (succinyl-CoA) transferase, whereas dxnI specifies a protein exhibiting marked homology to acetyl-CoA acetyltransferases (thiolases). The last ORF of the sequenced fragment codes for a putative transposase. DxnD, DxnF, DxnE, DxnGH, and DxnI (the activities of most of them have also been detected in strain RW1) thus form a complete 4-hydroxysalicylate/hydroxyquinol degradative pathway. A route for the mineralization of the growth substrates 3-hydroxydibenzofuran and 2-hydroxydibenzo-p-dioxin in Sphingomonas sp. strain RW1 thus suggests itself.
Title: Genetic analysis of dioxin dioxygenase of Sphingomonas sp. Strain RW1: catabolic genes dispersed on the genome Armengaud J, Happe B, Timmis KN Ref: Journal of Bacteriology, 180:3954, 1998 : PubMed
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.
Title: Genetic and biochemical characterization of the broad spectrum chlorobenzene dioxygenase from Burkholderia sp. strain PS12--dechlorination of 1,2,4,5-tetrachlorobenzene Beil S, Happe B, Timmis KN, Pieper DH Ref: European Journal of Biochemistry, 247:190, 1997 : PubMed
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).
Title: 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 Diaz E, Timmis KN Ref: Journal of Biological Chemistry, 270:6403, 1995 : PubMed
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.
Title: Characterization of 2,2',3-trihydroxybiphenyl dioxygenase, an extradiol dioxygenase from the dibenzofuran- and dibenzo-p-dioxin-degrading bacterium Sphingomonas sp. strain RW1 Happe B, Eltis LD, Poth H, Hedderich R, Timmis KN Ref: Journal of Bacteriology, 175:7313, 1993 : PubMed
A key enzyme in the degradation pathways of dibenzo-p-dioxin and dibenzofuran, namely, 2,2',3-trihydroxybiphenyl dioxygenase, which is responsible for meta cleavage of the first aromatic ring, has been genetically and biochemically analyzed. The dbfB gene of this enzyme has been cloned from a cosmid library of the dibenzo-p-dioxin- and dibenzofuran-degrading bacterium Sphingomonas sp. strain RW1 (R. M. Wittich, H. Wilkes, V. Sinnwell, W. Francke, and P. Fortnagel, Appl. Environ. Microbiol. 58:1005-1010, 1992) and sequenced. The amino acid sequence of this enzyme is typical of those of extradiol dioxygenases. This enzyme, which is extremely oxygen labile, was purified anaerobically to apparent homogeneity from an Escherichia coli strain that had been engineered to hyperexpress dbfB. Unlike most extradiol dioxygenases, which have an oligomeric quaternary structure, the 2,2',3-trihydroxybiphenyl dioxygenase is a monomeric protein. Kinetic measurements with the purified enzyme produced similar Km values for 2,2',3-trihydroxybiphenyl and 2,3-dihydroxybiphenyl, and both of these compounds exhibited strong substrate inhibition. 2,2',3-Trihydroxydiphenyl ether, catechol, 3-methylcatechol, and 4-methylcatechol were oxidized less efficiently and 3,4-dihydroxybiphenyl was oxidized considerably less efficiently.
Title: Genetic analysis of a Pseudomonas locus encoding a pathway for biphenyl/polychlorinated biphenyl degradation Hofer B, Eltis LD, Dowling DN, Timmis KN Ref: Gene, 130:47, 1993 : PubMed
The cistronic organization of the bph locus, encoding a biphenyl/polychlorinated biphenyl (PCB) degradation pathway in Pseudomonas sp. LB400, has been elucidated. Seven structural genes, encoding biphenyl dioxygenase (bphA1A2A3A4), biphenyl-2,3-dihydrodiol-2,3-dehydrogenase (bphB), biphenyl-2,3-diol-1,2-dioxygenase (bphC) and 2-hydroxy-6-oxo-6-phenylhexa-2,4-dienoate hydrolase (bphD), have been located. The complete sequences of bphB, bphC and bphD are reported. Taken together with the data of Erickson and Mondello [J. Bacteriol. 174 (1992) 2903-2912], Pseudomonas sp. LB400 is now the first strain for which the sequences of all genes encoding the catabolism from biphenyls to benzoates have been determined. Comparisons of the deduced amino acid (aa) sequences of BphB, BphC and BphD with those of related proteins led to predictions about catalytically important aa residues. Six Bph have been detected and identified. Five of them could be obtained as the most abundant proteins when their genes were expressed in Escherichia coli.
Title: DNA sequence determination of the TOL plasmid (pWWO) xylGFJ genes of Pseudomonas putida: implications for the evolution of aromatic catabolism Horn JM, Harayama S, Timmis KN Ref: Molecular Microbiology, 5:2459, 1991 : PubMed
The meta operon of the Pseudomonas putida TOL plasmid (pWWO) encodes all enzymes of a meta-cleavage pathway for the metabolism of benzoic acids to Krebs-cycle intermediates. We have determined and analysed the nucleic acid sequence of a 3442 bp region of the meta operon containing the xyl-GFJ genes whose products are involved in the post meta-ring fission transformation of catechols. Homology analysis of the xylGFJ gene products revealed evidence of biochemical relatedness, suggested enzymatic mechanisms, and permitted us to propose evolutionary events which may have generated the current variety of aromatic degradative pathways. The xylG gene, which specifies 2-hydroxymuconic semialdehyde dehydrogenase (HMSD), was found to encode a protein of 51.7 kDa. The predicted protein sequence exhibits significant homology to eukaryotic aldehyde dehydrogenases (ADHs) and to the products of two other Pseudomonas catabolic genes, i.e. xylC and alkH. Expansion of the ADH superfamily to include these prokaryotic enzymes permitted a broader analysis of functionally critical ADH residues and phylogenetic relationships among superfamily members. The importance of three regions of these enzymes previously thought to be critical to ADH activity was reinforced by this analysis. However glutamine-487, also thought to be critical, is less well conserved. The revised ADH phylogeny proposed here suggests early catabolic ADH divergence with subsequent interkingdom gene exchange. The xylF gene, which specifies 2-hydroxymuconic semialdehyde hydrolase (HMSH), was delineated by N-terminal sequence analysis of the purified gene product and is shown to encode a protein of 30.6 kDa. Homology analysis revealed sequence similarity to a chromosomally encoded serine hydrolase, especially in the region of the previously identified active-site serine residue, suggesting that HMSH may also possess a serine hydrolytic enzymatic mechanism. Likewise, the xylJ gene, which specifies 2-hydroxy-pent-2,4-dienoate hydratase (HPH), was delineated by N-terminal sequence analysis of purified HPH, and was found to encode a 23.9 kDa protein. Sequence comparisons revealed that both HMSH and HPH have analogues in the tod gene cluster, which specifies a toluene/benzene degradative pathway. Although the newly identified todF and todJ genes had been at least partially sequenced (Zylstra and Gibson, 1989), the open reading frames had not been positively identified. The presence of todJ provides strong evidence that the reactions following ring fission in the tod pathway are identical to those of the TOL pathway.
Title: Characterization of a plasmid-specified pathway for catabolism of isopropylbenzene in Pseudomonas putida RE204 Eaton RW, Timmis KN Ref: Journal of Bacteriology, 168:123, 1986 : PubMed
A Pseudomonas putida strain designated RE204, able to utilize isopropylbenzene as the sole carbon and energy source, was isolated. Tn5 transposon mutagenesis by means of the suicide transposon donor plasmid pLG221 yielded mutant derivatives defective in isopropylbenzene metabolism. These were characterized by the identification of the products which they accumulated when grown in the presence of isopropylbenzene and by the assay of enzyme activities in cell extracts. Based on the results obtained, the following metabolic pathway is proposed: isopropylbenzene----2,3-dihydro -2,3-dihydroxyisopropylbenzene----3-isopropylcatechol----2 -hydroxy-6-oxo-7-methylocta-2,4-dienoate----isobutyrate + 2-oxopent-4-enoate----amphibolic intermediates. Plasmid DNA was isolated from strain RE204 and mutant derivatives and characterized by restriction enzyme cleavage analysis. Isopropylbenzene-negative isolates carried a Tn5 insert within a 15-kilobase region of a 105-kilobase plasmid designated pRE4. DNA fragments of pRE4 carrying genes encoding isopropylbenzene catabolic enzymes were cloned in Escherichia coli with various plasmid vectors; clones were identified by (i) selection for Tn5-encoded kanamycin resistance in the case of Tn5 mutant plasmids, (ii) screening for isopropylbenzene dioxygenase-catalyzed oxidation of indole to indigo, and (iii) use of a Tn5-carrying restriction fragment, derived from a pRE4::Tn5 mutant plasmid, as a probe for clones carrying wild-type restriction fragments. These clones were subsequently used to generate a transposon insertion and restriction enzyme cleavage map of the isopropylbenzene metabolic region of pRE4.
