Petrotoga Mobilis Research Articles

The Synergistic Effect of GH13 and GH57 GBEs of Petrotoga mobilis Results in α-Glucan Molecules with a Higher Branch Density.

Glycogen is a biopolymer consisting of glycosyl units, with a linear backbone connected by α-1,4-linkages and branches attached via α-1,6-linkages. In microorganisms, glycogen synthesis involves multiple enzymes, with glycogen branching enzymes (GBEs) being vital for creating α-1,6-linkages. GBEs exist in two families: glycoside hydrolase (GH) 13 and GH57. Some organisms possess either a single GH13 or GH57 GBE, while others, such as Petrotoga mobilis, have both types of GBEs. In this study, the simultaneous use of a GH13 and GH57 GBE each from Petrotoga mobilis for α-glucan modification was investigated using a linear maltodextrin substrate with a degree of polymerization of 18 (DP18). The products from modifications by one or both GBEs in various combinations were analyzed and demonstrated a synergistic effect when both enzymes were combined, leading to a higher branch density in the glycogen structure. In this cooperative process, PmGBE13 was responsible for creating longer branches, whereas PmGBE57 hydrolyzed these branches, resulting in shorter lengths. The combined action of the two enzymes significantly increased the number of branched chains compared to when they acted individually. The results of this study therefore give insight into the role of PmGBE13 and PmGBE57 in glycogen synthesis, and show the potential use of both enzymes in a two-step modification to create an α-glucan structure with short branches at a high branch density.

Biochemical, structural and dynamical studies reveal strong differences in the thermal-dependent allosteric behavior of two extremophilic lactate dehydrogenases

In this work, we combined biochemical and structural investigations with molecular dynamics (MD) simulations to analyze the very different thermal-dependent allosteric behavior of two lactate dehydrogenases (LDH) from thermophilic bacteria. We found that the enzyme from Petrotoga mobilis (P. mob) necessitates an absolute requirement of the allosteric effector (fructose 1, 6-bisphosphate) to ensure functionality. In contrast, even without allosteric effector, the LDH from Thermus thermophilus (T. the) is functional when the temperature is raised. We report the crystal structure of P. mob LDH in the Apo state solved at 1.9 Å resolution. We used this structure and the one from T. the, obtained previously, as a starting point for MD simulations at various temperatures. We found clear differences between the thermal dynamics, which accounts for the behavior of the two enzymes. Our work demonstrates that, within an allosteric enzyme, some areas act as local gatekeepers of signal transmission, allowing the enzyme to populate either the T-inactive or the R-active states with different degrees of stringency.

Carbon steel corrosion by bacteria from failed seal rings at an offshore facility

Corrosion of carbon steel by microorganisms recovered from corroded seal rings at an offshore floating production facility was investigated. Microbial diversity profiling revealed that communities in all sampled seal rings were dominated by Pseudomonas genus. Nine bacterial species, Pseudomonas aeruginosa CCC-IOB1, Pseudomonas balearica CCC-IOB3, Pseudomonas stutzeri CCC-IOB10, Citrobacter youngae CCC-IOB9, Petrotoga mobilis CCC-SPP15, Enterobacter roggenkampii CCC-SPP14, Enterobacter cloacae CCC-APB1, Cronobacter sakazakii CCC-APB3, and Shewanella chilikensis CCC-APB5 were isolated from corrosion products and identified based on 16S rRNA gene sequence. Corrosion rates induced by the individual isolates were evaluated in artificial seawater using short term immersion experiments at 40 °C under anaerobic conditions. P. balearica, E. roggenkampii, and S. chilikensis, which have not been associated with microbiologically influenced corrosion before, were further investigated at longer exposure times to better understand their effects on corrosion of carbon steel, using a combination of microbiological and surface analysis techniques. The results demonstrated that all bacterial isolates triggered general and localised corrosion of carbon steel. Differences observed in the surface deterioration pattern by the different bacterial isolates indicated variations in the corrosion reactions and mechanisms promoted by each isolate.

Characterization of the GH13 and GH57 glycogen branching enzymes from Petrotoga mobilis SJ95 and potential role in glycogen biosynthesis.

Glycogen is a highly branched α-glucan polymer widely used as energy and carbon reserve by many microorganisms. The branches are introduced by glycogen branching enzymes (EC 2.4.1.18), that are classified into glycoside hydrolase families 13 (GH13) and 57 (GH57). Most microorganisms have typically only a single glycogen branching enzyme (gbe) gene. Only a few microorganisms carry both GH13 and GH57 gbe genes, such as Petrotoga mobilis and Mycobacterium tuberculosis. Here we report the basic characteristics of the GH13 and GH57 GBE of P. mobilis, both heterologously expressed in E. coli. The GH13 GBE has a considerably higher branching activity towards the linear α-glucan amylose, and produces a highly branched α-glucan with a high molecular weight which is very similar to glycogen. The GH57 GBE, on the contrary, makes a much smaller branched α-glucan. While the GH13 GBE acts as a classical glycogen branching enzyme involved in glycogen synthesis, the role of GH57 GBE remains unclear.

Synthesis of highly branched α-glucans with different structures using GH13 and GH57 glycogen branching enzymes

Glycogen branching enzymes (GBEs) convert starch into branched α-glucan polymers. To explore if the amylose content of substrates effects the structure of the branched α-glucans, mixtures of amylose and amylopectin were converted by four thermophilic GBEs. The degree of branching and molecular weight of the products increased with an increasing percentage of amylose with the GH57 GBEs of Thermus thermophilus and Thermococcus kodakarensis, and the GH13 GBEs of Rhodothermus marinus and Petrotoga mobilis. The only exception is that the degree of branching of the Petrotoga mobilis GBE products is not influenced by the amylose content. A second difference is the relatively high hydrolytic activity of two GH57 GBEs, while the two GH13 GBEs have almost no hydrolytic activity. Moreover, the two GH13 GBEs synthesize branched α-glucans with a narrow molecular weight distribution, while the two GH57 GBEs products consist of two or three molecular weight fractions.

Environmental Factors Modulating the Stability and Enzymatic Activity of the Petrotoga mobilis Esterase (PmEst).

Enzymes isolated from thermophilic organisms found in oil reservoirs can find applications in many fields, including the oleochemical, pharmaceutical, bioenergy, and food/dairy industries. In this study, in silico identification and recombinant production of an esterase from the extremophile bacteria Petrotoga mobilis (designated PmEst) were performed. Then biochemical, bioinformatics and structural characterizations were undertaken using a combination of synchrotron radiation circular dichroism (SRCD) and fluorescence spectroscopies to correlate PmEst stability and hydrolytic activity on different substrates. The enzyme presented a high Michaelis-Menten constant (KM 0.16 mM) and optimum activity at ~55°C for p-nitrophenyl butyrate. The secondary structure of PmEst was preserved at acid pH, but not under alkaline conditions. PmEst was unfolded at high concentrations of urea or guanidine through apparently different mechanisms. The esterase activity of PmEst was preserved in the presence of ethanol or propanol and its melting temperature increased ~8°C in the presence of these organic solvents. PmEst is a mesophilic esterase with substrate preference towards short-to medium-length acyl chains. The SRCD data of PmEst is in agreement with the prediction of an α/β protein, which leads us to assume that it displays a typical fold of esterases from this family. The increased enzyme stability in organic solvents may enable novel applications for its use in synthetic biology. Taken together, our results demonstrate features of the PmEst enzyme that indicate it may be suitable for applications in industrial processes, particularly, when the use of polar organic solvents is required.

Genomic characterization of Defluviitoga tunisiensis L3, a key hydrolytic bacterium in a thermophilic biogas plant and its abundance as determined by metagenome fragment recruitment

The genome sequence of Defluviitoga tunisiensis L3 originating from a thermophilic biogas-production plant was established and recently published as Genome Announcement by our group. The circular chromosome of D. tunisiensis L3 has a size of 2,053,097bp and a mean GC content of 31.38%. To analyze the D. tunisiensis L3 genome sequence in more detail, a phylogenetic analysis of completely sequenced Thermotogae strains based on shared core genes was performed. It appeared that Petrotoga mobilis DSM 10674T, originally isolated from a North Sea oil-production well, is the closest relative of D. tunisiensis L3. Comparative genome analyses of P. mobilis DSM 10674T and D. tunisiensis L3 showed moderate similarities regarding occurrence of orthologous genes. Both genomes share a common set of 1351 core genes. Reconstruction of metabolic pathways important for the biogas production process revealed that the D. tunisiensis L3 genome encodes a large set of genes predicted to facilitate utilization of a variety of complex polysaccharides including cellulose, chitin and xylan. Ethanol, acetate, hydrogen (H2) and carbon dioxide (CO2) were found as possible end-products of the fermentation process. The latter three metabolites are considered to represent substrates for methanogenic Archaea, the key organisms in the final step of the anaerobic digestion process. To determine the degree of relatedness between D. tunisiensis L3 and dominant biogas community members within the thermophilic biogas-production plant, metagenome sequences obtained from the corresponding microbial community were mapped onto the L3 genome sequence. This fragment recruitment revealed that the D. tunisiensis L3 genome is almost completely covered with metagenome sequences featuring high matching accuracy. This result indicates that strains highly related or even identical to the reference strain D. tunisiensis L3 play a dominant role within the community of the thermophilic biogas-production plant.

Asymmetric reductive amination by a wild-type amine dehydrogenase from the thermophilic bacteria Petrotoga mobilis

Biocatalytic potential of a new wild-type amine dehydrogenase used in an enzyme-catalyzed synthesis of an enantiomerically pure primary amine.

Knowledge-based discovery for designing CRISPR-CAS systems against invading mobilomes in thermophiles.

Clustered regularly interspaced short palindromic repeats (CRISPRs) are direct features of the prokaryotic genomes involved in resistance to their bacterial viruses and phages. Herein, we have identified CRISPR loci together with CRISPR-associated sequences (CAS) genes to reveal their immunity against genome invaders in the thermophilic archaea and bacteria. Genomic survey of this study implied that genomic distribution of CRISPR-CAS systems was varied from strain to strain, which was determined by the degree of invading mobiloms. Direct repeats found to be equal in some extent in many thermopiles, but their spacers were differed in each strain. Phylogenetic analyses of CAS superfamily revealed that genes cmr, csh, csx11, HD domain, devR were belonged to the subtypes of cas gene family. The members in cas gene family of thermophiles were functionally diverged within closely related genomes and may contribute to develop several defense strategies. Nevertheless, genome dynamics, geological variation and host defense mechanism were contributed to share their molecular functions across the thermophiles. A thermophilic archaean, Thermococcus gammotolerans and thermophilic bacteria, Petrotoga mobilis and Thermotoga lettingae have shown superoperons-like appearance to cluster cas genes, which were typically evolved for their defense pathways. A cmr operon was identified with a specific promoter in a thermophilic archaean, Caldivirga maquilingensis. Overall, we concluded that knowledge-based genomic survey and phylogeny-based functional assignment have suggested for designing a reliable genetic regulatory circuit naturally from CRISPR-CAS systems, acquired defense pathways, to thermophiles in future synthetic biology.

Mannosylglucosylglycerate biosynthesis in the deep-branching phylum Planctomycetes: characterization of the uncommon enzymes from Rhodopirellula baltica

The biosynthetic pathway for the rare compatible solute mannosylglucosylglycerate (MGG) accumulated by Rhodopirellula baltica, a marine member of the phylum Planctomycetes, has been elucidated. Like one of the pathways used in the thermophilic bacterium Petrotoga mobilis, it has genes coding for glucosyl-3-phosphoglycerate synthase (GpgS) and mannosylglucosyl-3-phosphoglycerate (MGPG) synthase (MggA). However, unlike Ptg. mobilis, the mesophilic R. baltica uses a novel and very specific MGPG phosphatase (MggB). It also lacks a key enzyme of the alternative pathway in Ptg. mobilis – the mannosylglucosylglycerate synthase (MggS) that catalyses the condensation of glucosylglycerate with GDP-mannose to produce MGG. The R. baltica enzymes GpgS, MggA, and MggB were expressed in E. coli and characterized in terms of kinetic parameters, substrate specificity, temperature and pH dependence. This is the first characterization of genes and enzymes for the synthesis of compatible solutes in the phylum Planctomycetes and for the synthesis of MGG in a mesophile.

A highly thermoactive and salt-tolerant α-amylase isolated from a pilot-plant biogas reactor

Aiming at the isolation of novel enzymes from previously uncultured thermophilic microorganisms, a metagenome library was constructed from DNA isolated from a pilot-plant biogas reactor operating at 55 °C. The library was screened for starch-degrading enzymes, and one active clone was found. An open reading frame of 1,461 bp encoding an α-amylase from an uncultured organism was identified. The amy13A gene was cloned in Escherichia coli, resulting in high-level expression of the recombinant amylase. The novel enzyme Amy13A showed the highest sequence identity (75 %) to α-amylases from Petrotoga mobilis and Halothermothrix orenii. Amy13A is highly thermoactive, exhibiting optimal activity at 80 °C, and it is also highly salt-tolerant, being active in 25 % (w/v) NaCl. Amy13A is one of the few enzymes that tolerate high concentrations of salt and elevated temperatures, making it a potential candidate for starch processing under extreme conditions.

Phylogeny and molecular signatures for the phylum Thermotogae and its subgroups

Thermotogae species are currently identified mainly on the basis of their unique toga and distinct branching in the rRNA and other phylogenetic trees. No biochemical or molecular markers are known that clearly distinguish the species from this phylum from all other bacteria. The taxonomic/evolutionary relationships within this phylum, which consists of a single family, are also unclear. We report detailed phylogenetic analyses on Thermotogae species based on concatenated sequences for many ribosomal as well as other conserved proteins that identify a number of distinct clades within this phylum. Additionally, comprehensive analyses of protein sequences from Thermotogae genomes have identified >60 Conserved Signature Indels (CSI) that are specific for the Thermotogae phylum or its different subgroups. Eighteen CSIs in important proteins such as PolI, RecA, TrpRS and ribosomal proteins L4, L7/L12, S8, S9, etc. are uniquely present in various Thermotogae species and provide molecular markers for the phylum. Many CSIs were specific for a number of Thermotogae subgroups. Twelve of these CSIs were specific for a clade consisting of various Thermotoga species except Tt. lettingae, which was separated from other Thermotoga species by a long branch in phylogenetic trees; Fourteen CSIs were specific for a clade consisting of the Fervidobacterium and Thermosipho genera and eight additional CSIs were specific for the genus Thermosipho. In addition, the existence of a clade consisting of the deep branching species Petrotoga mobilis, Kosmotoga olearia and Thermotogales bacterium mesG1 was supported by seven CSIs. The deep branching of this clade was also supported by a number of CSIs that were present in various Thermotogae species, but absent in this clade and all other bacteria. Most of these clades were strongly supported by phylogenetic analyses based on two datasets of protein sequences and they identify potential higher taxonomic grouping (viz. families) within this phylum. We also report 16 CSIs that are shared by either some or all Thermotogae species and some species from other taxa such as Archaea, Aquificae, Firmicutes, Proteobacteria, Deinococcus, Fusobacteria, Dictyoglomus, Chloroflexi and eukaryotes. The shared presence of some of these CSIs could be due to lateral gene transfers between these groups. However, no clear preference for any particular group was observed in this regard. The molecular probes based on different genes/proteins, which contain these Thermotogae-specific CSIs, provide novel and highly specific means for identification of both known as well as previously unknown Thermotogae species in different environments. Additionally, these CSIs also provide valuable tools for genetic and biochemical studies that could lead to discovery of novel properties that are unique to these bacteria.

Two Alternative Pathways for the Synthesis of the Rare Compatible Solute Mannosylglucosylglycerate inPetrotoga mobilis

The compatible solute mannosylglucosylglycerate (MGG), recently identified in Petrotoga miotherma, also accumulates in Petrotoga mobilis in response to hyperosmotic conditions and supraoptimal growth temperatures. Two functionally connected genes encoding a glucosyl-3-phosphoglycerate synthase (GpgS) and an unknown glycosyltransferase (gene Pmob_1143), which we functionally characterized as a mannosylglucosyl-3-phosphoglycerate synthase and designated MggA, were identified in the genome of Ptg. mobilis. This enzyme used the product of GpgS, glucosyl-3-phosphoglycerate (GPG), as well as GDP-mannose to produce mannosylglucosyl-3-phosphoglycerate (MGPG), the phosphorylated precursor of MGG. The MGPG dephosphorylation was determined in cell extracts, and the native enzyme was partially purified and characterized. Surprisingly, a gene encoding a putative glucosylglycerate synthase (Ggs) was also identified in the genome of Ptg. mobilis, and an active Ggs capable of producing glucosylglycerate (GG) from ADP-glucose and d-glycerate was detected in cell extracts and the recombinant enzyme was characterized, as well. Since GG has never been identified in this organism nor was it a substrate for the MggA, we anticipated the existence of a nonphosphorylating pathway for MGG synthesis. We putatively identified the corresponding gene, whose product had some sequence homology with MggA, but it was not possible to recombinantly express a functional enzyme from Ptg. mobilis, which we named mannosylglucosylglycerate synthase (MggS). In turn, a homologous gene from Thermotoga maritima was successfully expressed, and the synthesis of MGG was confirmed from GDP-mannose and GG. Based on the measurements of the relevant enzyme activities in cell extracts and on the functional characterization of the key enzymes, we propose two alternative pathways for the synthesis of the rare compatible solute MGG in Ptg. mobilis.

Social bacteria and asocial eukaryotes

The end of 2007 brought us draft genome sequences of two eukaryotic microorganisms, Babesia bovis and Malassezia globosa, as well as complete genomes of the ammonia-oxidizing archaeal chemoautotroph Nitrosopumilus maritimus, and several environmental bacteria (Table 1). In terms of genome size, this list covers both sides of the spectrum: the 245 kb genome of an obligate insect symbiont Sulcia muelleri is the second smallest microbial genome sequenced so far, whereas the 13 033 kb chromosome of the social myxobacterium Sorangium cellulosum breaks the record as the largest bacterial genome – and the largest DNA molecule in the prokaryotic world.

Petrotoga mexicana sp. nov., a novel thermophilic, anaerobic and xylanolytic bacterium isolated from an oil-producing well in the Gulf of Mexico.

A novel anaerobic, thermophilic, xylanolytic, motile rod-shaped bacterium with a sheath-like outer structure (toga) was isolated from a Mexican oil well in the Gulf of Mexico. Strain MET12T was a Gram-negative bacterium, reducing elemental sulfur, thiosulfate and sulfite to hydrogen sulfide. Its optimum growth conditions were 55 degrees C, pH 6.6, 3% NaCl and 0.15% MgCl2.6H2O. The DNA G+C content was 36.1 mol%. Phylogenetically, strain MET12T was related to members of genus Petrotoga, with similarities to Petrotoga mobilis, Petrotoga sibirica, Petrotoga miotherma and Petrotoga olearia varying from 97.6 to 98.8%. However DNA-DNA relatedness values between these species and strain MET12T were lower than 70%. As strain MET12T (=DSM 14811T=CIP 107371T) was genomically and phenotypically different from existing Petrotoga species, it is proposed as the type strain of a novel species, Petrotoga mexicana sp. nov.

Petrotoga mobilis sp. nov., from a North Sea oil-production well.

Rod-shaped, thermophilic bacteria with a sheath-like outer structure (toga) were isolated from hot oilfield water of a North Sea oil reservoir. One of the isolates, designated SJ95(T), is an obligately anaerobic, sheathed, Gram-negative, fermentative bacterium capable of reducing elemental sulfur to hydrogen sulfide and tolerating high salt concentrations. The optimum growth conditions for this isolate are 58-60 degrees Celsius and pH 6.5-7.0 with 3-4% NaCl and 0.7% MgSO(4). 7H(2)O in the medium. Vitamins are required for growth. Growth is stimulated by yeast extract. Cells of strain SJ95(T) vary in size from 1-2 to 40-50 micron in length and are motile with a subpolar flagellation. Cels grown on xylan have xylanase activity, presumably associated with the toga, and glucose isomerase activity was detected in xylose-grown cells. The DNA G+C content is 31 and 34 mol%, determined by the thermal denaturation and HPLC methods, respectively. Phylogenetically, strain SJ95(T) is most closely related to Petrotoga miotherma with a 97.7% similarity level between their 165 rDNA sequences. The DNA-DNA reassociation value between the two DNAs was 35.6%. On the basis of differences in genotypic, phenotypic and immunological characteristics, strain SJ95t (=DSM 10674t) is proposed as the type strain of a new species, Petrotoga mobilis. It can be readily distinguished from P. miotherma by its motility.