Olga Zafra - Academia.edu (original) (raw)

Papers by Olga Zafra

FEBS Letters, Jun 18, 2002

A cytochrome c (NarC) is encoded as the ¢rst gene of the operon for nitrate respiration in Thermu... more A cytochrome c (NarC) is encoded as the ¢rst gene of the operon for nitrate respiration in Thermus thermophilus. NarC is required for anaerobic growth and for the synthesis of active nitrate reductase (NR). The K K and N N subunits (NarG, NarJ) of the NR were constitutively expressed in narC: :kat mutants, but NarG appeared in the soluble fraction instead of associated with the membranes. Our data demonstrate for NarC an essential role in the synthesis of active enzyme and for the attachment to the membrane of the respiratory NR from T. thermophilus.

Applied and Environmental Microbiology, Jul 1, 2004

Thermostable Mn-dependent catalases are promising enzymes in biotechnological applications as H 2... more Thermostable Mn-dependent catalases are promising enzymes in biotechnological applications as H 2 O 2-detoxifying systems. We cloned the genes encoding Mn-dependent catalases from Thermus thermophilus HB27 and HB8 and a less thermostable mutant carrying two amino acid replacements (M129V and E293G). When the wild-type and mutant genes were overexpressed in Escherichia coli, unmodified or six-His-tagged proteins of the expected size were overproduced as inactive proteins. Several attempts to obtain active forms or to activate the overproduced proteins were unsuccessful, even when soluble and thermostable proteins were used. Therefore, a requirement for a Thermus-specific activation factor was suggested. To overcome this problem, the Mn-dependent catalase genes were overexpressed directly in T. thermophilus under the control of the Pnar promoter. This promoter belongs to a respiratory nitrate reductase from of T. thermophilus HB8, whose transcription is activated by the combined action of nitrate and anoxia. Upon induction in T. thermophilus HB8, a 20-to 30-fold increase in catalase specific activity was observed, whereas a 90-to 110-fold increase was detected when the laboratory strain T. thermophilus HB27::nar was used as the host. The thermostability of the overproduced wild-type catalase was identical to that previously reported for the native enzyme, whereas decreased stability was detected for the mutant derivative. Therefore, our results validate the use of T. thermophilus as an alternative cell factory for the overproduction of thermophilic proteins that fail to be expressed in well-known mesophilic hosts.

SOLICITUD DE PATENTE A1 54 Título: Procedimiento para la obtención de anticuerpos monoclonales a ... more SOLICITUD DE PATENTE A1 54 Título: Procedimiento para la obtención de anticuerpos monoclonales a partir de muestras complejas de antígenos.

Critical Reviews in Oncology Hematology, Sep 1, 2023

Revista de la Sociedad Española de Microbiología

Frontiers in Microbiology

Regulation of eDNA release Function of eDNA References GRAM-NEGATIVE BACTERIA Acinetobacter calco... more Regulation of eDNA release Function of eDNA References GRAM-NEGATIVE BACTERIA Acinetobacter calcoaceticus Lysis Unknown Unknown Palmen and Hellingwerf, 1995 Campylobacter jejuni Autolysis Unknown Biofilm matrix Svensson et al., 2014 Caulobacter crescentus Lysis Unknown Biofilm dispersal Berne et al., 2010 Haemophilus influenzae Unknown Unknown Biofilm matrix Izano et al., 2009 Helicobacter pylori Vesicles Unknown Biofilm matrix Grande et al., 2011, 2015 Neisseria gonorrhoeae Type IV secretion system Unknown HGT and biofilm Hamilton et al., 2005; Steichen et al., 2011 Neisseria meningitidis Lysis Unknown Biofilm matrix Lappann et al., 2010 Pseudomonas aeruginosa Vesicles/prophage QS Biofilm matrix and nutrient source Kadurugamuwa and Beveridge, 1996; Allesen-Holm et al., 2006; Mulcahy et al., 2010 Pseudomonas chlororaphis Autolysis Unknown Biofilm matrix Wang et al., 2016 Pseudomonas fluorescens Unknown Unknown Unknown Catlin and Cunningham, 1958 Pseudomonas stuzeri Lysis Unknown HGT Stewart et al., 1983 Rhodovulum sulfidophilum Unknown QS Biofilm matrix (flocculation) Watanabe et al., 1998 Suzuki et al., 2009 Shewanella oneidensis Autolysis Phage-induced upon iron-mediated oxidative stress Biofilm matrix and nutrient source Gödeke et al., 2011a,b; Binnenkade et al., 2014 Vibrio costicolus Unknown Unknown Unknown Smithies and Gibbons, 1955 Xanthomonas citri Unknown Unknown Biofilm matrix Sena-Vélez et al., 2016 GRAM-POSITIVE BACTERIA Bacillus cereus Unknown Unknown Biofilm matrix Vilain et al., 2009 Bacillus subtilis Lytic-independent mechanism QS (early competence) HGT, nutrient source? Takahashi, 1962 Zafra et al., 2012 Deinococcus radiodurans Unknown Unknown DNA repair Boling and Setlow, 1966 Enterococcus faecalis Fratricidal-mechanism of autolysis Unknown Biofilm matrix Thomas et al., 2008, 2009 Lysteria monocytogenes Unknown Unknown Biofilm matrix Harmsen et al., 2010 Micrococcus halodenitrificans Unknown Unknown Unknown Smithies and Gibbons, 1955 Micrococcus sodonensis Unknown Unknown Unknown Campbell et al., 1961 Mycobacterium avium Unknown Unknown Biofilm matrix Rose et al., 2015 Staphylococcus aureus Autolysis QS Biofilm matrix Rice et al., 2007 Brackman et al., 2016 Staphylococcus epidermidis Autolysis Unknown Biofilm matrix Qin et al., 2007 Staphylococcus lugdunensis Lytic-independent mechanism Competence (comEB) Biofilm matrix Rajendran et al., 2015 Streptococcus gordonii Lytic-independent mechanism QS (competence) Biofilm matrix Kreth et al., 2009; Jack et al., 2015 Streptococcus intermedius Unknown Unknown Biofilm matrix Petersen et al., 2004; Nur et al., 2013 Streptococcus mutans Vesicles Unknown Biofilm matrix Liao et al., 2014 Streptococcus pneumoniae Competence-induced lysis QS (CSP) (competence) Biofilm matrix, HGT, nutrient source? Steinmoen et al., 2002; Moscoso et al., 2006 Streptococcus sanguinis Lytic-independent mechanism Unknown Biofilm matrix (aggregation) Kreth et al., 2009 ARCHAEA Halorubrum lacusprofundi Unknown Unknown Biofilm matrix Fröls et al., 2012 Haloferax volcanii Unknown Unknown Biofilm matrix, HGT, nutrient source Chimileski et al., 2014a,b Thermococcus spp. Vesicles/unknown Unknown Unknown Soler et al., 2008 Thermococcus onnurineus Vesicles Unknown Unknown Choi et al., 2015 EUKARYOTES Aspergillus fumigatus Autolysis Unknown Biofilm matrix (antifungal resistance) Rajendran et al., 2013 Candida albicans Unknown Unknown Biofilm matrix (antifungal resistance)

DNA repair, Apr 1, 2017

8-oxo-7,8-dihydro-2'-deoxyguanosine (8oxodG) is a major lesion resulting from oxidative stres... more 8-oxo-7,8-dihydro-2'-deoxyguanosine (8oxodG) is a major lesion resulting from oxidative stress and found in both DNA and dNTP pools. Such a lesion is usually removed from DNA by the Base Excision Repair (BER), a universally conserved DNA repair pathway. 8oxodG usually adopts the favored and promutagenic syn-conformation at the active site of DNA polymerases, allowing the base to hydrogen bonding with adenine during DNA synthesis. Here, we study the structural determinants that affect the glycosidic torsion-angle of 8oxodGTP at the catalytic active site of the family X DNA polymerase from Bacillus subtilis (PolXBs). We show that, unlike most DNA polymerases, PolXBs exhibits a similar efficiency to stabilize the anti and syn conformation of 8oxodGTP at the catalytic site. Kinetic analyses indicate that at least two conserved residues of the nucleotide binding pocket play opposite roles in the anti/syn conformation selectivity, Asn263 and His255 that favor incorporation of 8oxodGMP...

International journal of food microbiology, Jan 30, 2016

Ribera de Duero Spanish wines are appreciated worldwide for their organoleptic characteristics; h... more Ribera de Duero Spanish wines are appreciated worldwide for their organoleptic characteristics; however, the wine market is very competitive, and the demand for high quality natural wines has been increasing in recent years. The microbiology of the process, specifically the yeasts involved in the alcoholic fermentation, constitutes an essential element directly related to the complexity and quality of the wine. Our work has focused on the development of a procedure to identify the indigenous wine yeasts present in complex samples of must and wine, without requiring colony isolation or a microbiological culture. The procedure is based on the use of AFLP molecular markers. The AFLP allele profiles obtained from complex samples are compared with the species-specific ones previously determined and included in a database using a sorting algorithm. The system allows a fast and efficient identification of yeast species and strains present in complex must and wine samples. This information ...

Nucleic Acids Research, 2016

Bacillus subtilis is one of the bacterial members provided with a nonhomologous end joining (NHEJ... more Bacillus subtilis is one of the bacterial members provided with a nonhomologous end joining (NHEJ) system constituted by the DNA-binding Ku homodimer that recruits the ATP-dependent DNA Ligase D (BsuLigD) to the double-stranded DNA breaks (DSBs) ends. BsuLigD has inherent polymerization and ligase activities that allow it to fill the short gaps that can arise after realignment of the broken ends and to seal the resulting nicks, contributing to genome stability during the stationary phase and germination of spores. Here we show that BsuLigD also has an intrinsic 5-2-deoxyribose-5-phosphate (dRP) lyase activity located at the N-terminal ligase domain that in coordination with the polymerization and ligase activities allows efficient repairing of 2deoxyuridine-containing DNA in an in vitro reconstituted Base Excision Repair (BER) reaction. The requirement of a polymerization, a dRP removal and a final sealing step in BER, together with the joint participation of BsuLigD with the spore specific AP endonuclease in conferring spore resistance to ultrahigh vacuum desiccation suggest that BsuLigD could actively participate in this pathway. We demonstrate the presence of the dRP lyase activity also in the homolog protein from the distantly related bacterium Pseudomonas aeruginosa, allowing us to expand our results to other bacterial LigDs.

Metabolic Engineering, 2015

Surveying the dynamics of metabolic networks of Gram-negative bacteria often requires the conditi... more Surveying the dynamics of metabolic networks of Gram-negative bacteria often requires the conditional shutdown of enzymatic activities once the corresponding proteins have been produced. We show that given biochemical functions can be entirely suppressed in vivo with camel antibodies (VHHs, nanobodies) that target active sites of cognate enzymes expressed in the cytoplasm. As a proof of principle, we raised VHHs against 2,5-dihydroxypyridine dioxygenase (NicX) of Pseudomonas putida, involved in nicotinic acid metabolism. Once fused to a thioredoxin domain, the corresponding nanobodies inhibited the enzyme both in E. coli and in P. putida cells, which then accumulated the metabolic substrate of NicX. VHHs were further engineered to track the antigen in vivo by C-terminal fusion to a fluorescent protein. Conditional expression of the resulting VHHs allows simultaneously to track and target proteins of interest and enables the design of transient phenotypes without mutating the genetic complement of the bacteria under study.

Nucleic acids research, 2014

Intracellular reactive oxygen species as well as the exposure to harsh environmental conditions c... more Intracellular reactive oxygen species as well as the exposure to harsh environmental conditions can cause, in the single chromosome of Bacillus subtilis spores, the formation of apurinic/apyrimidinic (AP) sites and strand breaks whose repair during outgrowth is crucial to guarantee cell viability. Whereas double-stranded breaks are mended by the nonhomologous end joining (NHEJ) system composed of an ATP-dependent DNA Ligase D (LigD) and the DNA-end-binding protein Ku, repair of AP sites would rely on an AP endonuclease or an AP-lyase, a polymerase and a ligase. Here we show that B. subtilis Ku (BsuKu), along with its pivotal role in allowing joining of two broken ends by B. subtilis LigD (BsuLigD), is endowed with an AP/deoxyribose 5'-phosphate (5'-dRP)-lyase activity that can act on ssDNA, nicked molecules and DNA molecules without ends, suggesting a potential role in BER during spore outgrowth. Coordination with BsuLigD makes possible the efficient joining of DNA ends with...

PLoS ONE, 2012

Extracellular DNA (eDNA) release is a widespread capacity described in many microorganisms. We id... more Extracellular DNA (eDNA) release is a widespread capacity described in many microorganisms. We identified and characterized lysis-independent eDNA production in an undomesticated strain of Bacillus subtilis. DNA fragments are released during a short time in late-exponential phase. The released eDNA corresponds to whole genome DNA, and does not harbour mutations suggesting that is not the result of error prone DNA synthesis. The absence of eDNA was linked to a spread colony morphology, which allowed a visual screening of a transposon library to search for genes involved in its production. Transposon insertions in genes related to quorum sensing and competence (oppA, oppF and comXP) and to DNA metabolism (mfd and topA) were impaired in eDNA release. Mutants in early competence genes such as comA and srfAA were also defective in eDNA while in contrast mutations in late competence genes as those for the DNA uptake machinery had no effect. A subpopulation of cells containing more DNA is present in the eDNA producing strains but absent from the eDNA defective strain. Finally, competent B. subtilis cells can be transformed by eDNA suggesting it could be used in horizontal gene transfer and providing a rationale for the molecular link between eDNA release and earlycompetence in B. subtilis that we report.

Plasmid, 2003

A specific expression system for Thermus spp. is described. Plasmid pMKE1 contains replicative or... more A specific expression system for Thermus spp. is described. Plasmid pMKE1 contains replicative origins for Escherichia coli and Thermus spp., a selection gene encoding a thermostable resistance to kanamycin, and a 720 bp DNA region containing the promoter (Pnar), and the regulatory sequences of the respiratory nitrate reductase operon of Thermus thermophilus HB8. Two genes, encoding a thermophilic beta-galactosidase and an alkaline phosphatase were cloned in pMKE1 as cytoplasmic and periplasmic reporters, respectively. The expression of the reporters was specifically induced by the combined action of nitrate and anoxia in facultative anaerobic derivatives of T. thermophilus HB27 to which the gene cluster for nitrate respiration was transferred by conjugation. Overexpressions in the range of approximately 200-fold were obtained for the cytoplasmic reporter, whereas that of the periplasmic reporter was limited to approximately 20-fold, with respect to their intrinsic respective activities.

Molecular Microbiology, 2008

The bc1 respiratory complex III constitutes a key energy-conserving respiratory electron transpor... more The bc1 respiratory complex III constitutes a key energy-conserving respiratory electron transporter between complex I (type I NADH dehydrogenase) and II (succinate dehydrogenase) and the final nitrogen oxide reductases (Nir, Nor and Nos) in most denitrifying bacteria. However, we show that the expression of complex III from Thermus thermophilus is repressed under denitrification, and that its role as electron transporter is replaced by an unusual nitrate reductase (Nar) that contains a periplasmic cytochrome c (NarC). Several lines of evidence support this conclusion: (i) nitrite and NO are as effective signals as nitrate for the induction of Nar; (ii) narC mutants are defective in anaerobic growth with nitrite, NO and N 2O; (iii) such mutants present decreased NADH oxidation coupled to these electron acceptors; and (iv) complementation assays of the mutants reveal that the membranedistal heme c of NarC was necessary for anaerobic growth with nitrite, whereas the membrane-proximal heme c was not. Finally, we show evidence to support that Nrc, the main NADH oxidative activity in denitrification, interacts with Nar through their respective membrane subunits. Thus, we propose the existence of a Nrc-Nar respiratory supercomplex that is required for the development of the whole denitrification pathway in T. thermophilus.

Journal of Biological Chemistry, 2004

A four-gene operon (nrcDEFN) was identified within a conjugative element that allows Thermus ther... more A four-gene operon (nrcDEFN) was identified within a conjugative element that allows Thermus thermophilus to use nitrate as an electron acceptor. Three of them encode homologues to components of bacterial respiratory chains: NrcD to ferredoxins; NrcF to iron-sulfurcontaining subunits of succinate-quinone oxidoreductase (SQR); and NrcN to type-II NADH dehydrogenases (NDHs). The fourth gene, nrcE, encodes a membrane protein with no homologues in the protein data bank. Nitrate reduction with NADH was catalyzed by membrane fractions of the wild type strain, but was severely impaired in nrc::kat insertion mutants. A fusion to a thermophilic reporter gene was used for the first time in Thermus spp. to show that expression of nrc required the presence of nitrate and anoxic conditions. Therefore, a role for the nrc products as a new type of membrane NDH specific for nitrate respiration was deduced. Consistent with this, nrc::kat mutants grew more slowly than the wild type strain under anaerobic conditions, but not in the presence of oxygen. The oligomeric structure of this Nrc-NDH was deduced from the analysis of insertion mutants and a two-hybrid bacterial system. Attachment to the membrane of NrcD, NrcF, and NrcN was dependent on NrcE, whose cytoplasmic C terminus interacts with the three proteins. Interactions were also detected between NrcN and NrcF. Inactivation of nrcF produced solubilization of NrcN, but not of NrcD. These data lead us to conclude that the Nrc proteins form a distinct third type of bacterial respiratory NDH.

Journal of Bacteriology, 2005

The nar operon, coding for the respiratory nitrate reductase of Thermus thermophilus (NRT), encod... more The nar operon, coding for the respiratory nitrate reductase of Thermus thermophilus (NRT), encodes a di-heme b-type (NarJ) and a di-heme c-type (NarC) cytochrome. The role of both cytochromes and that of a putative chaperone (NarJ) in the synthesis and maturation of NRT was studied. Mutants of T. thermophilus lacking either NarI or NarC synthesized a soluble form of NarG, suggesting that a putative NarCI complex constitutes the attachment site for the enzyme. Interestingly, the NarG protein synthesized by both mutants was inactive in nitrate reduction and misfolded, showing that membrane attachment was required for enzyme maturation. Consistent with its putative role as a specific chaperone, inactive and misfolded NarG was synthesized by narJ mutants, but in contrast to its Escherichia coli homologue, NarJ was also required for the attachment of the thermophilic enzyme to the membrane. A bacterial two-hybrid system was used to demonstrate the putative interactions between the NRT proteins suggested by the analysis of the mutants. Strong interactions were detected between NarC and NarI and between NarG and NarJ. Weaker interaction signals were detected between NarI, but not NarC, and both NarG and NarH. These results lead us to conclude that the NRT is a heterotetrameric (NarC/NarI/NarG/NarH) enzyme, and we propose a model for its synthesis and maturation that is distinct from that of E. coli. In the synthesis of NRT, a NarCI membrane complex and a soluble NarGJH complex are synthesized in a first step. In a second step, both complexes interact at the cytoplasmic face of the membrane, where the enzyme is subsequently activated with the concomitant conformational change and release of the NarJ chaperone from the mature enzyme.

FEBS Letters, 2002

Extremophiles, 2002

The presence of a periplasmic space within the cell envelope of Thermus thermophilus was analyzed... more The presence of a periplasmic space within the cell envelope of Thermus thermophilus was analyzed in a mutant (HB8 ∆ UTR1) defective in the regulation of its Slayer (surface crystalline layer). This mutant forms round multicellular bodies (MBs) surrounded by a common envelope as the culture approaches the stationary phase. Confocal microscopy revealed that the origin of the MBs is the progressive detachment of the external layers coupled with the accumulation of NH 2-containing material between the external envelopes and the peptidoglycan. A specific pattern of proteins was found as soluble components of the intercellular space of the MBs by a single fractionation procedure, suggesting that they are periplasmic-like components. To demonstrate this, we cloned a gene (phoA) from T. thermophilus HB8 encoding a signal peptide-wearing alkaline phosphatase (AP), and engineered it to be overexpressed in the mutant from a shuttle vector. Most of the AP activity (>80%) was found as a soluble component of the MBs' intercellular fraction. All these data indicate that Thermus thermophilus actually has a periplasmic space which is functionally similar to that of Proteobacteria. The potential application of the HB8 ∆ UTR1 mutant for the overexpression of periplasmic thermophilic proteins is discussed.

Environmental Microbiology, 2011

Functional studies of biodegradative activities in environmental microorganisms require molecular... more Functional studies of biodegradative activities in environmental microorganisms require molecular tools for monitoring catabolic enzymes in the members of the native microbiota. To this end, we have generated repertories of single-domain V(HH) fragments of camel immunoglobulins (nanobodies) able to interact with multiple proteins that are descriptors of environmentally relevant processes. For this, we immunized Camelus dromedarius with a cocktail of up to 12 purified enzymes that are representative of major types of detoxifying activities found in aerobic and anaerobic microorganisms. Following the capture of the antigen-binding modules from the mRNA of the camel lymphocytes and the selection of sub-libraries for each of the enzymes in a phage display system we found a large number of V(HH) modules that interacted with each of the antigens. Those associated to the enzyme 2,3 dihydroxybiphenyl dioxygenase of Burkholderia xenovorans LB400 (BphC) and the arsenate reductase of Staphylococcus aureus (ArsC) were examined in detail and found to hold different qualities that were optimal for distinct protein recognition procedures. The repertory of anti-BphC V(HH) s included variants with a strong affinity and specificity for linear epitopes of the enzyme. When the anti-BphC V(HH) library was recloned in a prokaryotic intracellular expression system, some nanobodies were found to inhibit the dioxygenase activity in vivo. Furthermore, anti-ArsC V(HH) s were able to discriminate between proteins stemming from different enzyme families. The easiness of generating large collections of binders with different properties widens considerably the molecular toolbox for analysis of biodegradative bacteria and opens fresh possibilities of monitoring protein markers and activities in the environment.

Environmental Microbiology, 2008

The nitrate conjugative element (NCE) encodes the ability to respire nitrate in the facultative T... more The nitrate conjugative element (NCE) encodes the ability to respire nitrate in the facultative Thermus thermophilus NAR1 strain. This process is carried out by two heterotetrameric enzymes that catalyse the oxidation of NADH (Nrc) and the reduction of nitrate (Nar), whose expression is activated by the NCE-encoded transcription factors DnrS and DnrT. We report the presence of NCE in other facultative strains of T. thermophilus and analyse its role in subsequent steps of the denitrification pathway. We encountered that nrc mutants of denitrifying strains show a decrease in anaerobic growth rates not only with nitrate, but also with nitrite, NO and N(2)O, which is concomitant to their lower NADH oxidation activities in vitro. We show that nitrate, nitrite and NO are activating signals for transcription of nrc in these strains. Finally, we demonstrate that DnrS and DnrT are required for anaerobic growth not only with nitrate, but also with nitrite, NO and N(2)O. These data allow us to conclude that: (i) Nrc constitutes the main electron donor for the four reductases of the denitrification pathway, and (ii) the NCE controls the expression of the whole denitrification pathway and the repression of the aerobic respiration through the transcription factors DnrS and DnrT.