From accurate genome sequence to biotechnological application: The thermophile Mycolicibacterium hassiacum as experimental model.

Mycobacteria constitute a large group of microorganisms belonging to the phylum Actinobacteria encompassing some of the most relevant pathogenic bacteria and many saprophytic isolates that share a unique and complex cell envelope. Also unique to this group is the extensive capability to use and synthesize sterols, a class of molecules that include active signalling compounds of pharmaceutical use. However, few mycobacterial species and strains have been established as laboratory models to date, Mycolicibacterium smegmatis mc2 155 being the most common one. In this work, we focus on the use of a thermophilic mycobacterium, Mycolicibacterium hassiacum, which grows optimally above 50°C, as an emerging experimental model valid to extend our general knowledge of mycobacterial biology as well as for application purposes. To that end, accurate genomic sequences are key for gene mining, the study of pathogenicity or lack thereof and the potential for gene transfer. The combination of long- and short-massive sequencing technologies is strictly necessary to remove biases caused by errors specific to long-reads technology. By doing so in M. hassiacum, we obtained from the curated genome clues regarding the genetic manipulation potential of this microorganism from the presence of insertion sequences, CRISPR-Cas, type VII ESX secretion systems, as well as lack of plasmids. Finally, as a proof of concept of the applicability of M. hassiacum as a laboratory and industrial model, we used this high-quality genome of M. hassiacum to successfully knockout a gene involved in the use of phytosterols as source of carbon and energy, using an improved gene cassette for thermostable selection and a transformation protocol at high temperature.

Stabilization of Enzymes by Using Thermophiles.

Manufactured steroid compounds have many applications in the pharmaceutical industry. Due to the chemical complexity and chirality of steroids, there is an increasing demand for enzyme-based bioconversion processes to replace those based on chemical synthesis. In this context, thermostability of the involved enzymes is a highly desirable property as both the increased half-life of the enzyme and the enhanced solubility of substrates and products will improve the yield of the reactions. Metagenomic libraries from thermal environments are potential sources of thermostable enzymes of prokaryotic origin, but the number of expected hits could be quite low for enzymes handling substrates such as steroids, rarely found in prokaryotes. An alternative to metagenome screening is the selection of thermostable variants of well-known steroid-processing enzymes. Here we review and detail a protocol for such selection, where error-prone PCR (epPCR) is used to introduce random mutations into a gene to create a variants library for further selection of thermostable variants in the thermophile Thermus thermophilus. The method involves the use of folding interference vectors where the proper folding of the enzyme of interest at high temperature is linked to the folding of a reporter encoding a selectable property such as thermostable resistance to kanamycin, leading to a life-or-death selection of variants of reinforced folding independently of the activity of the enzyme.

Deletion of the primase-polymerases encoding gene, located in a mobile element in Thermus thermophilus HB27, leads to loss of function mutation of addAB genes.

DNA primase-polymerases (Ppol) have been shown to play active roles in DNA repair and damage tolerance, both in prokaryotes and eukaryotes. The ancestral thermophilic bacterium Thermus thermophilus strain HB27 encodes a Ppol protein among the genes present in mobile element ICETh2, absent in other T. thermophilus strains. Using different strategies we ablated the function of Ppol in HB27 cells, either by knocking out the gene through insertional mutagenesis, markerless deletion or through abolition of its catalytic activity. Whole genome sequencing of this diverse collection of Ppol mutants showed spontaneous loss of function mutation in the helicase-nuclease AddAB in every ppol mutant isolated. Given that AddAB is a major player in recombinational repair in many prokaryotes, with similar activity to the proteobacterial RecBCD complex, we have performed a detailed characterization of the ppol mutants in combination with addAB mutants. The results show that knockout addAB mutants are more sensitive to DNA damage agents than the wild type, and present a dramatic three orders of magnitude increase in natural transformation efficiencies with both plasmid and lineal DNA, whereas ppol mutants show defects in plasmid stability. Interestingly, DNA-integrity comet assays showed that the genome of all the ppol and/or addAB mutants was severely affected by widespread fragmentation, however, this did not translate in neat loss of viability of the strains. All these data support that Ppol appears to keep in balance the activity of AddAB as a part of the DNA housekeeping maintenance in T. thermophilus HB27, thus, playing a key role in its genome stability.

Thermostability Engineering of a Class II Pyruvate Aldolase from Escherichia coli by in Vivo Folding Interference.

The use of enzymes in industrial processes is often limited by the unavailability of biocatalysts with prolonged stability. Thermostable enzymes allow increased process temperature and thus higher substrate and product solubility, reuse of expensive biocatalysts, resistance against organic solvents, and better "evolvability" of enzymes. In this work, we have used an activity-independent method for the selection of thermostable variants of any protein in Thermus thermophilus through folding interference at high temperature of a thermostable antibiotic reporter protein at the C-terminus of a fusion protein. To generate a monomeric folding reporter, we have increased the thermostability of the moderately thermostable Hph5 variant of the hygromycin B phosphotransferase from Escherichia coli to meet the method requirements. The final Hph17 variant showed 1.5 °C higher melting temperature (T m) and 3-fold longer half-life at 65 °C compared to parental Hph5, with no changes in the steady-state kinetic parameters. Additionally, we demonstrate the validity of the reporter by stabilizing the 2-keto-3-deoxy-l-rhamnonate aldolase from E. coli (YfaU). The most thermostable multiple-mutated variants thus obtained, YfaU99 and YfaU103, showed increases of 2 and 2.9 °C in T m compared to the wild-type enzyme but severely lower retro-aldol activities (150- and 120-fold, respectively). After segregation of the mutations, the most thermostable single variant, Q107R, showed a T m 8.9 °C higher, a 16-fold improvement in half-life at 60 °C and higher operational stability than the wild-type, without substantial modification of the kinetic parameters.

Self-sufficient asymmetric reduction of ß-ketoesters catalysed by a novel and robust thermophilic alcohol dehydrogenase co-immobilised with NADH.

ß-Hydroxyesters are essential building blocks utilised by the pharmaceutical and food industries in the synthesis of functional products. Beyond the conventional production methods based on chemical catalysis or whole-cell synthesis, the asymmetric reduction of ß-ketoesters with cell-free enzymes is gaining relevance. To this end, a novel thermophilic (S)-3-hydroxybutyryl-CoA dehydrogenase from Thermus thermophilus HB27 (Tt27-HBDH) has been expressed, purified and biochemically characterised, determining its substrate specificity towards ß-ketoesters and its dependence on NADH as a cofactor. The immobilization of Tt27-HBDH on agarose macroporous beads and its subsequent coating with polyethyleneimine has been found the best strategy to increase the stability and workability of the heterogeneous biocatalyst. Furthermore, we have embedded NADH in the cationic layer attached to the porous surface of the carrier. Since Tt27-HBDH catalyses cofactor recycling through 2-propanol oxidation, we achieve a self-sufficient heterogeneous biocatalyst where NADH is available for the immobilised enzymes but its lixiviation to the reaction bulk is avoided. Taking advantage of the autofluorescence of NADH, we demonstrate the activity of the enzyme towards the immobilised cofactor through single-particle analysis. Finally, we tested the operational stability in the asymmetric reduction of ß-ketoesters in batch, succeeding in the reuse of both the enzyme and the co-immobilised cofactor up to 10 reaction cycles.

Biochemical and Structural Characterization of a novel thermophilic esterase EstD11 provide catalytic insights for the HSL family.

A novel esterase, EstD11, has been discovered in a hot spring metagenomic library. It is a thermophilic and thermostable esterase with an optimum temperature of 60°C. A detailed substrate preference analysis of EstD11 was done using a library of chromogenic ester substrate that revealed the broad substrate specificity of EstD11 with significant measurable activity against 16 substrates with varied chain length, steric hindrance, aromaticity and flexibility of the linker between the carboxyl and the alcohol moiety of the ester. The tridimensional structures of EstD11 and the inactive mutant have been determined at atomic resolutions. Structural and bioinformatic analysis, confirm that EstD11 belongs to the family IV, the hormone-sensitive lipase (HSL) family, from the α/ß-hydrolase superfamily. The canonical α/ß-hydrolase domain is completed by a cap domain, composed by two subdomains that can unmask of the active site to allow the substrate to enter. Eight crystallographic complexes were solved with different substrates and reaction products that allowed identification of the hot-spots in the active site underlying the specificity of the protein. Crystallization and/or incubation of EstD11 at high temperature provided unique information on cap dynamics and a first glimpse of enzymatic activity in vivo. Very interestingly, we have discovered a unique Met zipper lining the active site and the cap domains that could be essential in pivotal aspects as thermo-stability and substrate promiscuity in EstD11.

Integrative and Conjugative Element ICETh1 Functions as a Pangenomic DNA Capture Module in Thermus thermophilus.

Transjugation is an unconventional conjugation mechanism in Thermus thermophilus (Tth) that involves the active participation of both mating partners, encompassing a DNA secretion system (DSS) in the donor and an active natural competence apparatus (NCA) in the recipient cells. DSS is encoded within an integrative and conjugative element (ICETh1) in the strain Tth HB27, whereas the NCA is constitutively expressed in both mates. Previous experiments suggested the presence of multiple origins of transfer along the genome, which could generate genomic mosaicity among the progeny. Here, we designed transjugation experiments between two closely related strains of Tth with highly syntenic genomes, containing enough single nucleotide polymorphisms to allow precise parenthood analysis. Individual clones from the progeny were sequenced, revealing their origin as derivatives of our ICETh1-containing intended "donor" strain (HB27), which had acquired separate fragments from the genome of the ICETh1-free HB8 cells, which are our intended recipient. Due to the bidirectional nature of transjugation, only assays employing competence-defective HB27 derivatives as donors allowed the recovery of HB8-derived progeny. These results show a preference for a retrotransfer mechanism in transjugation in ICETh1-bearing strains, supporting an inter-strain gene-capture function for ICETh1. This function could benefit the donor-capable host by facilitating the acquisition of adaptive traits from external sources, ultimately increasing the open pangenome of Thermus, maximizing the potential repertoire of physiological and phenotypical traits related to adaptation and speciation.

Nitrate Respiration in Thermus thermophilus NAR1: from Horizontal Gene Transfer to Internal Evolution.

Genes coding for enzymes of the denitrification pathway appear randomly distributed among isolates of the ancestral genus Thermus, but only in few strains of the species Thermus thermophilus has the pathway been studied to a certain detail. Here, we review the enzymes involved in this pathway present in T. thermophilus NAR1, a strain extensively employed as a model for nitrate respiration, in the light of its full sequence recently assembled through a combination of PacBio and Illumina technologies in order to counteract the systematic errors introduced by the former technique. The genome of this strain is divided in four replicons, a chromosome of 2,021,843 bp, two megaplasmids of 370,865 and 77,135 bp and a small plasmid of 9799 pb. Nitrate respiration is encoded in the largest megaplasmid, pTTHNP4, within a region that includes operons for O2 and nitrate sensory systems, a nitrate reductase, nitrate and nitrite transporters and a nitrate specific NADH dehydrogenase, in addition to multiple insertion sequences (IS), suggesting its mobility-prone nature. Despite nitrite is the final product of nitrate respiration in this strain, the megaplasmid encodes two putative nitrite reductases of the cd1 and Cu-containing types, apparently inactivated by IS. No nitric oxide reductase genes have been found within this region, although the NorR sensory gene, needed for its expression, is found near the inactive nitrite respiration system. These data clearly support that partial denitrification in this strain is the consequence of recent deletions and IS insertions in genes involved in nitrite respiration. Based on these data, the capability of this strain to transfer or acquire denitrification clusters by horizontal gene transfer is discussed.

A thermostable DNA primase-polymerase from a mobile genetic element involved in defence against environmental DNA.

Primase-polymerases (Ppol) are one of the few enzymes able to start DNA synthesis on ssDNA templates. The role of Thermus thermophilus HB27 Ppol, encoded along a putative helicase (Hel) within a mobile genetic element (ICETh2), has been studied. A mutant lacking Ppol showed no effects on the replication of the element. Also, no apparent differences in the sensitivity to DNA damaging agents and other stressors or morphological changes in the mutant cells were detected. However, the mutants lacking Ppol showed an increase in two to three orders of magnitude in their transformation efficiency with plasmids and genomic DNA acquired from the environment (eDNA), independently of its origin and G + C content. In contrast, no significant differences with the wild type were detected when the cells received the DNA from other T. thermophilus partners in conjugation-like mating experiments. The similarities of this behaviour with that shown by mutants lacking the Argonaute (ThAgo) protein suggests a putative partnership Ppol-ThAgo in the DNA-DNA interference mechanism of defence, although other eDNA defence mechanisms independent of ThAgo cannot be discarded.

Hypoxanthine-Guanine Phosphoribosyltransferase/adenylate Kinase From Zobellia galactanivorans: A Bifunctional Catalyst for the Synthesis of Nucleoside-5'-Mono-, Di- and Triphosphates.

In our search for novel biocatalysts for the synthesis of nucleic acid derivatives, we found a good candidate in a putative dual-domain hypoxanthine-guanine phosphoribosyltransferase (HGPRT)/adenylate kinase (AMPK) from Zobellia galactanivorans (ZgHGPRT/AMPK). In this respect, we report for the first time the recombinant expression, production, and characterization of a bifunctional HGPRT/AMPK. Biochemical characterization of the recombinant protein indicates that the enzyme is a homodimer, with high activity in the pH range 6-7 and in a temperature interval from 30 to 80°C. Thermal denaturation experiments revealed that ZgHGPRT/AMPK exhibits an apparent unfolding temperature (Tm) of 45°C and a retained activity of around 80% when incubated at 40°C for 240 min. This bifunctional enzyme shows a dependence on divalent cations, with a remarkable preference for Mg2+ and Co2+ as cofactors. More interestingly, substrate specificity studies revealed ZgHGPRT/AMPK as a bifunctional enzyme, which acts as phosphoribosyltransferase or adenylate kinase depending upon the nature of the substrate. Finally, to assess the potential of ZgHGPRT/AMPK as biocatalyst for the synthesis of nucleoside-5'-mono, di- and triphosphates, the kinetic analysis of both activities (phosphoribosyltransferase and adenylate kinase) and the effect of water-miscible solvents on enzyme activity were studied.

The Thermus thermophilus DEAD-box protein Hera is a general RNA binding protein and plays a key role in tRNA metabolism.

RNA helicases catalyze the ATP-dependent destabilization of RNA duplexes. DEAD-box helicases share a helicase core that mediates ATP binding and hydrolysis, RNA binding and unwinding. Most members of this family contain domains flanking the core that can confer RNA substrate specificity and guide the helicase to a specific RNA. However, the in vivo RNA substrates of most helicases are currently not defined. The DEAD-box helicase Hera from Thermus thermophilus contains a helicase core, followed by a dimerization domain and an RNA binding domain that folds into an RNA recognition motif (RRM). The RRM mediates high affinity binding to an RNA hairpin, and an adjacent duplex is then unwound by the helicase core. Hera is a cold-shock protein, and has been suggested to act as an RNA chaperone under cold-shock conditions. Using crosslinking immunoprecipitation of Hera/RNA complexes and sequencing, we show that Hera binds to a large fraction of T. thermophilus RNAs under normal-growth and cold-shock conditions without a strong sequence preference, in agreement with a structure-specific recognition of RNAs and a general function in RNA metabolism. Under cold-shock conditions, Hera is recruited to RNAs with high propensities to form stable secondary structures. We show that selected RNAs identified, including a set of tRNAs, bind to Hera in vitro, and activate the Hera helicase core. Gene ontology analysis reveals an enrichment of genes related to translation, including mRNAs of ribosomal proteins, tRNAs, tRNA ligases, and tRNA-modifying enzymes, consistent with a key role of Hera in ribosome and tRNA metabolism.

Intraparticle pH Sensing Within Immobilized Enzymes: Immobilized Yellow Fluorescent Protein as Optical Sensor for Spatiotemporal Mapping of pH Inside Porous Particles.

pH is a fundamental variable in enzyme catalysis and its measurement therefore is crucial for understanding and optimizing enzyme-catalyzed reactions. Whereas measurements within homogeneous bulk liquid solution are prominently used, enzymes immobilized inside porous particles often suffer from pH gradients due to partition effects and heterogeneously catalyzed biochemical reactions. Unfortunately, the measurements of intraparticle pH are not available due to the lack of useful suitable methodologies; as a consequence the biocatalyst characterization is hampered. Here, a fully biocompatible methodology for real-time optical sensing of pH within porous materials is described. A genetically encoded ratiometric pH indicator, the superfolder yellow fluorescent protein (sYFP), is used to functionalize the internal surface of enzyme carrier supports. By using controlled, tailor-made immobilization, sYFP is homogeneously distributed within these materials, and so enables, via self-referenced imaging analysis, pH measurements in high accuracy and with useful spatiotemporal resolution. The hydrolysis of penicillin by a penicillin acylase, taking place in solution or confined to the solid surface of the porous matrix is used to show the monitoring of evolution of internal pH. Thus, pH sensing based on immobilized sYFP represents a broadly applicable technique to the study of the internally heterogeneous environment of immobilized enzymes into solid particles.

Methods to Identify and Analyze Vesicle-Protected DNA Transfer.

Conjugation, transformation, and transduction constitute the three classical mechanisms involved in horizontal gene transfer (HGT) among prokaryotes. In addition, alternative HGT mechanisms exist in groups of organisms. Among them, the use of DNA-containing membrane vesicles as shuttle elements for HGT has been described for a number of microorganisms, including both thermophiles and mesophiles. Here we describe the methods followed to detect, purify, and analyze these vesicles.

Thermostability enhancement of the Pseudomonas fluorescens esterase I by in vivo folding selection in Thermus thermophilus.

Prolonged stability is a desired property for the biotechnological application of enzymes since it allows its reutilization, contributing to making biocatalytic processes more economically competitive with respect to chemical synthesis. In this study, we have applied selection by folding interference at high temperature in Thermus thermophilus to obtain thermostable variants of the esterase I from Pseudomonas fluorescens (PFEI). The most thermostable variant (Q11L/A191S) showed a melting temperature (Tm ) of 77.3 ± 0.1°C (4.6°C higher than the wild-type) and a half-life of over 13 hr at 65°C (7.9-fold better than the wild-type), with unchanged kinetic parameters. Stabilizing mutations Q11L and A191S were incorporated into PFEI variant L30P, previously described to be enantioselective in the hydrolysis of the (-)-enantiomer of the Vince lactam. The final variant Q11L/L30P/A191S showed a significant improvement in thermal stability (Tm of 80.8 ± 0.1°C and a half-life of 65 min at 75°C), while retaining enantioselectivity (E > 100). Structural studies revealed that A191S establishes a hydrogen bond network between a V-shaped hairpin and the α/ß hydrolase domain that leads to higher rigidity and thus would contribute to explaining the increase in stability.

Functional Characterization and Structural Analysis of NADH Oxidase Mutants from Thermus thermophilus HB27: Role of Residues 166, 174, and 194 in the Catalytic Properties and Thermostability.

The Thermus thermophilus strain HB27 NADH-oxidase (Tt27-NOX) catalyzes the oxidation of nicotinamide adenine dinucleotide (NAD(P)H) by reducing molecular oxygen to hydrogen peroxide in a two-electron transfer mechanism. Surprisingly, Tt27-NOX showed significant differences in catalytic properties compared to its counterpart from the strain HB8 (Tt8-NOX), despite a high degree of sequence homology between both variants. The sequence comparison between both enzymes revealed only three divergent amino acid residues at positions 166, 174, and 194. Motivated with these findings, in this work we performed mutagenesis experiments in the former three positions to study the specific role of these residues in the catalytic properties and thermostability of Tt27-NOX. We subjected five mutants, along with the wild-type enzyme, to biochemical characterization and thermal stability studies. As a result, we identified two more active and more thermostable variants than any Tt8-NOX variant reported in the literature. The most active and thermostable variant K166/H174/Y194 retained 90% of its initial activity after 5 h at pH 7 and 80 °C and an increase in melting temperature of 48.3 °C compared with the least active variant K166/R174/Y194 (inactivated after 15 min of incubation). These results, supported by structural analysis and molecular dynamics simulation studies, suggest that Lys at position 166 may stabilize the loop in which His174 is located, increasing thermal stability.

A Modular Vector Toolkit with a Tailored Set of Thermosensors To Regulate Gene Expression in Thermus thermophilus.

Modular plasmid architectures have shown to be a very useful resource to standardize, build, share, and compare biological parts and functional vectors, and are being applied in an increasing number of microorganisms. Here, we present a modular plasmid toolkit for Thermus thermophilus, a species considered as a workhorse for biotechnology and a model for high-temperature biology. Apart from integrating improved versions of already existing parts, we have characterized specific promoters and developed a thermosensor-based palette that restricts the expression to Thermus and, at the same time, controls protein expression in this organism in a temperature-dependent manner.

Efecto de la vacunación antigripal en trabajadores de atención primaria y población general de Gran Canaria: estudio transversal / Effect of influenza vaccination in Primary Healthcare workers and the general population in Gran Canaria: A cross-sectional study

Objetivo: Estimar el efecto de la vacunación antigripal en los trabajadores de atención primaria y en la población atendida durante la campaña de vacunación antigripal 2015/2016. Diseño: Estudio transversal. Emplazamiento: Todos los centros asistenciales de atención primaria del Área de Salud de Gran Canaria. Participantes: Un total de 1.868 profesionales (33,5% hombres; 66,5% mujeres) y 795.605 personas de la población general (49,4% hombres; 50,6% mujeres). Mediciones principales: En profesionales, las variables de resultado fueron: declaraciones de gripe al Sistema de Vigilancia Epidemiológica y días de incapacidad temporal por enfermedad; en población general: gripes declaradas y coberturas vacunales en función del estado vacunal del sanitario. Se estimó la magnitud de asociación entre la vacunación y la morbilidad mediante modelos de regresión logística. Resultados: Los profesionales sanitarios que no se vacunaron tuvieron un riesgo de tener gripe 1,7 veces superior al de los profesionales que se vacunaron, aunque la diferencia no fue estadísticamente significativa; en población general se encontró una asociación significativa solo en mujeres (OR: 1,3; IC95%: 1,1-1,5). La cobertura poblacional fue significativamente mayor cuando médico y enfermero estaban vacunados (OR: 1,3; IC 95%: 1,3-1,3), y el número de gripes declaradas disminuyó cuando el enfermero estaba vacunado (OR: 0,9; IC 95%: 0,9-0,9). Conclusiones: Se observó un posible efecto protector de la vacunación antigripal en la población general, así como una influencia del estado vacunal de los sanitarios en las coberturas de los pacientes. Aun así, las bajas coberturas registradas apuntan a una necesidad de implementar medidas que propicien una actitud más favorable frente a la vacunación antigripal

Objective: To estimate the effect of the influenza vaccination in Primary Healthcare workers and the general population vaccinated during the 2015/2016 campaign. Design: Cross-sectional study. Setting: All the Primary Healthcare centres within the Gran Canaria healthcare region. Participants: A total of 1,868 Primary Healthcare workers (33.5% men; 66.5% women) and 795,605 individuals from the general population (49.4% men; 50.6% women). Principal measurements: The outcome variables in Primary Healthcare workers were: influenza cases reported to the Epidemiological Surveillance System, and the sick leave days due to illness. In the general population: reported flu cases and vaccination coverage in connection with the vaccination status of the healthcare professional. The magnitude of association between vaccination and morbidity was estimated applying logistic regression models. Results: Although not statistically significant, healthcare professionals that were not vaccinated had 1.7-fold increase in the risk of having influenza than those vaccinated. In the general population the association was significant in the female population (OR: 1.3; 95% CI: 1.1-1.5). Population coverage was significantly higher when both the doctor and nurse were vaccinated (OR: 1.3; 95 %CI: 1.3-1.3), and reported flu cases decreased when the nurse was vaccinated (OR: 0.9; 95%CI: 0.9-0.9). Conclusion: A possible protective effect of influenza vaccination was observed in the general population, as well as an influence of Primary Healthcare workers on the patients regarding this. Even so, the low coverages registered point to a need to implement measures that may lead to a more favourable attitude towards influenza vaccination

A new family of nitrate/nitrite transporters involved in denitrification

Denitrifying bacteria carry out nitrate and nitrite respiration inside and outside the cell, respectively. In Thermus thermophilus, nitrate and nitrite transport processes are carried out by major facilitator superfamily (MFS) transporters. The sequence of the nar operon of nitrate-only respiring strains of T. thermophilus includes two tandemly organized MFS transporter genes (narK and narT) of the NarK1 and NarK2 families. Both can function as nitrate/nitrite antiporters, but NarK has been proposed as more specific for nitrate whereas NarT more specific for nitrite. In some nitrate- and nitrite-respiring strains of the same species, a single MFS transporter (NarO) belonging to a different MFS subfamily appears. To analyze the role of this single MFS in the same genetic context, we transferred the two types of nar operon to the aerobic strain HB27, and further included in both of them the ability to respire nitrite. The new denitrifying strains HB27dn, with two MFS, and HB27dp, with a single one, were used to isolate mutants devoid of transporters. Through in trans complementation experiments, we demonstrate that the NarO single MFS works efficiently in the transport of both nitrate and nitrite

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A new family of nitrate/nitrite transporters involved in denitrification.

Denitrifying bacteria carry out nitrate and nitrite respiration inside and outside the cell, respectively. In Thermus thermophilus, nitrate and nitrite transport processes are carried out by major facilitator superfamily (MFS) transporters. The sequence of the nar operon of nitrate-only respiring strains of T. thermophilus includes two tandemly organized MFS transporter genes (narK and narT) of the NarK1 and NarK2 families. Both can function as nitrate/nitrite antiporters, but NarK has been proposed as more specific for nitrate whereas NarT more specific for nitrite. In some nitrate- and nitrite-respiring strains of the same species, a single MFS transporter (NarO) belonging to a different MFS subfamily appears. To analyze the role of this single MFS in the same genetic context, we transferred the two types of nar operon to the aerobic strain HB27, and further included in both of them the ability to respire nitrite. The new denitrifying strains HB27dn, with two MFS, and HB27dp, with a single one, were used to isolate mutants devoid of transporters. Through in trans complementation experiments, we demonstrate that the NarO single MFS works efficiently in the transport of both nitrate and nitrite.

Complete Genome Sequence of Mycolicibacterium hassiacum DSM 44199.

Mycolicibacterium hassiacum is the most thermophilic of all the mycobacteria. A partial sequence based on Illumina technology of around 5 Mbp was published in 2012. Here, we report the 5,269,097-bp complete genome sequence assembled into a single circular chromosome.