Co-expression with RadA and the characterization of stRad55B, a RadA paralog from the hyperthermophilic crenarchaea Sulfolobus tokodaii

ST0838 (designed stRad55B) is one of the four RadA paralogs (or Rad55 homologues) in the genome of the hyperthermophilic crenarchaeon Sulfolobus tokodaii. The gene is induced by UV irradiation, suggesting that it is involved in DNA recombinational repair in this organism. However, this protein could not be expressed normally in vitro. In this study, thermostable and soluble stRad55B was obtained by co-expression with S. tokodaii RadA (stRadA) in E. coli, and the enzymatic properties were examined. It was found that stRad55B bound ssDNA preferentially and had a very weak ATPase activity that was not stimulated by DNA. The recombinant protein inhibited the strand exchange activity promoted by stRadA, indicating that stRad55B might be an inhibitor to the homologous recombination in this archaeon. The results will be helpful for further functional and interaction analysis of RadA paralogs and for the understanding of the mechanism of recombinational repair in archaea. Supported by the National Basic Research Program of China (Grant No. 2004CB719604) and National Natural Science Foundation of China (Grant Nos. 30470386 and 30700011)     

Co-expression with RadA and the characterization of stRad55B, a RadA paralog from the hyperthermophilic crenarchaea Sulfolobus tokodaii

ST0838 (designed stRad55B) is one of the four RadA paralogs (or Rad55 homologues) in the genome of the hyperthermophilic crenarchaeon Sulfolobus tokodaii. The gene is induced by UV irradiation, suggesting that it is involved in DNA recombinational repair in this organism. However, this protein could not be expressed normally in vitro. In this study, thermostable and soluble stRad55B was obtained by co-expression with S. tokodaii RadA (stRadA) in E. coli, and the enzymatic properties were examined. It was found that stRad55B bound ssDNA preferentially and had a very weak ATPase activity that was not stimulated by DNA. The recombinant protein inhibited the strand exchange activity promoted by stRadA, indicating that stRad55B might be an inhibitor to the homologous recombination in this archaeon. The results will be helpful for further functional and interaction analysis of RadA paralogs and for the understanding of the mechanism of recombinational repair in archaea.     

The DNA binding and pairing preferences of the archaeal RadA protein demonstrate a universal characteristic of DNA strand exchange proteins

The archaeal RadA protein is a homologue of the Escherichia coli RecA and Saccharomyces cerevisiae Rad51 proteins and possesses the same biochemical activities. Here, using in vitro selection, we show that the Sulfolobus solfataricus RadA protein displays the same preference as its homologues for binding to DNA sequences that are rich in G residues, and under-represented in A and C residues. The RadA protein also displays enhanced pairing activity with these in vitro-selected sequences. These parallels between the archaeal, eukaryal and bacterial proteins further extend the universal characteristics of DNA strand exchange proteins.     

Genetic analysis of Escherichia coli RadA: functional motifs and genetic interactions

The RadA/Sms protein is a RecA‐related protein found universally in eubacteria and plants, implicated in processing of recombination intermediates. Here we show that the putative Zn finger, Walker A motif, KNRXG motif and Lon protease homology domain of the Escherichia coli RadA protein are required for DNA damage survival. RadA is unlikely to possess protease activity as the putative active site serine is not required. Mutants in RadA have strong synergistic phenotypes with those in the branch migration protein RecG. Sensitivity of radA recG mutants to azidothymidine (AZT) can be rescued by blocking recombination with recA or recF mutations or by overexpression of RuvAB, suggesting that lethal recombination intermediates accumulate in the absence of RadA and RecG. Synthetic genetic interactions for survival to AZT or ciprofloxacin exposure were observed between RadA and known or putative helicases including DinG, Lhr, PriA, Rep, RuvAB, UvrD, YejH and YoaA. These represent the first affected phenotypes reported for Lhr, YejH and YoaA. The specificity of these effects sheds new light on the role of these proteins in DNA damage avoidance and repair and implicates a role in replication gap processing for DinG and YoaA and a role in double‐strand break repair for YejH.     

A RecA / RAD51 homologue from a hyperthermophilic archaeon retains the major RecA domain only

 A gene encoding a RecA/RAD51 homologue from a hyperthermophilic archaeon, Pyrococcus sp. KOD1 (Pk), was cloned, sequenced and expressed in Escherichia coli. The deduced 210-amino acid sequence was compared to homologues from bacteria (RecA), eukaryotes (RAD51, DMC1) and archaea (RadA). The entire protein from Pk (Pk-REC) basically corresponds to the essential central domain of its counterparts and lacks the two smaller RecA subdomains at the N- and C-termini. The sequence comparison suggests that Pk-REC represents a common prototype of RecA, RAD51, DMC1 and RadA, with higher enzymatic activity. Recombinant Pk-REC was fully active and complemented the ultraviolet light sensitivity of an E. coli recA mutant strain. Received: 11 June 1996 / Accepted: August 31 1996     

Identification of sn-Glycerol-1-phosphate Dehydrogenase Activity from Genomic Information on a Hyperthermophilic Archaeon,Sulfolobus tokodaii Strain 7

sn-Glycerol-1-phosphate dehydrogenase is responsible for the formation of sn-glycerol-1-phosphate, the backbone of membrane phospholipids of Archaea. This activity had never been detected in cell-free extract of Sulfolobus sp. Here we report the detection of this activity on the thermostable ST0344 protein of Sulfolobus tokodaii expressed in Escherichia coli, which was predicted from genomic information on S. tokodaii. This is another line of evidence for the general mechanism of sn-glycerol-1-phosphate formation by the enzyme.     

Inactivation of Dipeptidyl Peptidase IV Attenuates the Virulence of <Emphasis Type="Italic">Streptococcus suis</Emphasis> Serotype 2 that Causes Streptococcal Toxic Shock Syndrome

Di-peptidyl peptidase IV (DPP IV), originally recognized as CD26 in eukaryotic cells, is distributed widely in microbial pathogens, including Streptococcus suis (S. suis), an emerging zoonotic agent. However, the role of DPP IV in S. suis virulence remains unclear. Here, we identified a dpp IV homologue from highly invasive isolate of S. suis 2 (SS2) causing streptococcal toxic shock syndrome (STSS). Enzymatic assays reproduced its enzymatic activity of dpp IV protein product as a functional DPP IV, and ELISA analysis demonstrated that SS2 DPP IV can interact with human fibronectin. An isogenic SS2 mutant of dpp IV, Δdpp IV, was obtained by homologous recombination. Experimental animal infection suggested that an inactivation of dpp IV attenuates greatly its high virulence of Chinese virulent strains of SS2. Functional complementation can restore this defect in SS2 pathogenicity. To our knowledge, it may confirm, for the first time, that DPP IV contributes to SS2 virulence. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users. Authors Junchao Ge, Youjun Feng and Hongfeng Ji contributed equally.     

Both RadA and RadB are involved in homologous recombination in Pyrococcus furiosus

RecA and Rad51 proteins are essential for homologous recombination in Bacteria and Eukarya, respectively. Homologous proteins, called RadA, have been described for Archaea. Here we present the characterization of two RecA/Rad51 family proteins, RadA and RadB, from Pyrococcus furiosus. The radA and radB genes were not induced by DNA damage resulting from exposure of the cells to gamma and UV irradiation and heat shock, suggesting that they might be constitutively expressed in this hyperthermophile. RadA had DNA-dependent ATPase, D-loop formation, and strand exchange activities. In contrast, RadB had a very weak ATPase activity that is not stimulated by DNA. This protein had a strong binding affinity for DNA, but little strand exchange activity could be detected. A direct interaction between RadA and RadB was detected by an immunoprecipitation assay. Moreover, RadB, but not RadA, coprecipitated with Hjc, a Holliday junction resolvase found in P. furiosus, in the absence of ATP. This interaction was suppressed in the presence of ATP. The Holliday junction cleavage activity of Hjc was inhibited by RadB in the absence, but not in the presence, of ATP. These results suggest that RadB has important roles in homologous recombination in Archaea and may regulate the cleavage reactions of the branch-structured DNA.     

NdgR,an IclR-like regulator involved in amino-acid-dependent growth,quorum sensing,and antibiotic production in <Emphasis Type="Italic">Streptomyces coelicolor</Emphasis>

NdgR (regulator for nitrogen source-dependent growth and antibiotic production), an IclR-like regulator, has been initially identified as a binding protein to the promoters of doxorubicin biosynthetic genes in Streptomcyes peucetius by DNA affinity capture assay method. NdgR is well conserved throughout the Streptomcyes species and many other bacteria such as Mycobacteria and Corynebacteria. In Streptomcyes coelicolor, ndgR deletion mutant showed slow cell growth and defects in differentiation and enhances the production of actinorhodin (ACT) in minimal media containing certain amino acids where wild-type strain could not produce ACT. Although deletion mutant of ndgR showed different antibiotic production in minimal media containing Leu or Gln, it only showed reduced mRNA expression levels of the genes involved in leucine metabolism. Neither NdgR-dependent expression of glnA nor direct binding of NdgR protein to glnA, glnII, and glnR promoters was observed. However, ScbR, which is governed by NdgR shown by gel mobility shift assay, binds to promoter of glnR, suggesting indirect regulation of glutamine metabolism by NdgR. NdgR protein binds to intergenic region of ndgR–leuC, and scbR–scbA involved in γ-butyrolactone. Two-dimensional gel analysis has shown a global effect of ndgR deletion in protein expression, including up-regulated proteins involved in ACT synthesis and down-regulation of chaperones such as GroEL, GroES, and DnaK. These results suggest a global regulatory role for NdgR in amino acid metabolisms, quorum sensing, morphological changes, antibiotic production, and expression of chaperonines in S. coelicolor. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Distorted octahedral coordination of tungstate in a subfamily of specific binding proteins

Bacteria and archaea import molybdenum and tungsten from the environment in the form of the oxyanions molybdate (MoO₄ ²⁻) and tungstate (WO₄ ²⁻). These substrates are captured by an external, high-affinity binding protein, and delivered to ATP binding cassette transporters, which move them across the cell membrane. We have recently reported a crystal structure of the molybdate/tungstate binding protein ModA/WtpA from Archaeoglobus fulgidus, which revealed an octahedrally coordinated central metal atom. By contrast, the previously determined structures of three bacterial homologs showed tetracoordinate molybdenum and tungsten atoms in their binding pockets. Until then, coordination numbers above four had only been found for molybdenum/tungsten in metalloenzymes where these metal atoms are part of the catalytic cofactors and coordinated by mostly non-oxygen ligands. We now report a high-resolution structure of A. fulgidus ModA/WtpA, as well as crystal structures of four additional homologs, all bound to tungstate. These crystal structures match X-ray absorption spectroscopy measurements from soluble, tungstate-bound protein, and reveal the details of the distorted octahedral coordination. Our results demonstrate that the distorted octahedral geometry is not an exclusive feature of the A. fulgidus protein, and suggest distinct binding modes of the binding proteins from archaea and bacteria. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users. K. Hollenstein and M. Comellas-Bigler contributed equally to this work.     

Characterization of a new RNase HII and its essential amino acid residues in the archaeon <Emphasis Type="Italic">Sulfolobus tokodaii</Emphasis> reveals a regulatory C-terminus

The archaea possess RNase H proteins that share features of both prokaryotic and eukaryotic forms. Although the Sulfolobus RNase HI has been reported to have unique structural and biochemical properties, its RNase HII has not yet been investigated and its biochemical properties remain unknown. In the present study, we have characterized the ST0519 RNase HII from S. tokodaii as a new form. The enzyme utilized hybrid RNA/DNA as a substrate and had an optimal temperature between 37 and 50°C. The activity of wild-type protein was stimulated by Mn²⁺, whereas this cation significantly inhibited the activity of C-terminal truncated mutant proteins. A series of mutation assays revealed a regulatory C-terminal tail in the S. tokodaii RNase HII. One mutant, ST0519 (residues 1–195), retained only partial activity, while ST0519 (residues 1–196) completely lost its activity. Based on the presumed structure, the C-terminus might form a short α-helix in which two residues, I195 and L196, are essential for the cleavage activity. Our data suggest that the C-terminal α-helix is likely involved in the Mn²⁺-dependent substrate cleavage activity through stabilization of a flexible loop structure. Our findings offer important clues for further understanding the structure and function of both archaeal and eukaryotic RNase HII.     

A molecular dynamics study of Cyclophilin A free and in complex with the Ala-Pro dipeptide

Six different molecular dynamics simulations of Cyclophilin A, three with the protein free in water and three with the Ala-Pro dipeptide bound to the protein, have been performed, and analysed with respect to structure and hydration of the active site. The water structure in the binding pocket of the free Cyclophilin A was found to mimic the experimentally obtained binding cis conformation of the dipeptide. Cyclophilin A is a peptidyl–prolyl cis–trans isomerase (PPIase), but the mechanism of the cis/trans isomerization is not exactly clear. This study was performed to understand better the binding between dipeptide and Cyclophilin A, but also two previously proposed isomerization mechanisms are discussed. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Structural and Functional Characterisation of a Conserved Archaeal RadA Paralog with Antirecombinase Activity

DNA recombinases (RecA in bacteria, Rad51 in eukarya and RadA in archaea) catalyse strand exchange between homologous DNA molecules, the central reaction of homologous recombination, and are among the most conserved DNA repair proteins known. RecA is the sole protein responsible for this reaction in bacteria, whereas there are several Rad51 paralogs that cooperate to catalyse strand exchange in eukaryotes. All archaea have at least one (and as many as four) RadA paralog, but their function remains unclear. Herein, we show that the three RadA paralogs encoded by the Sulfolobus solfataricus genome are expressed under normal growth conditions and are not UV inducible. We demonstrate that one of these proteins, Sso2452, which is representative of the large archaeal RadC subfamily of archaeal RadA paralogs, functions as an ATPase that binds tightly to single-stranded DNA. However, Sso2452 is not an active recombinase in vitro and inhibits D-loop formation by RadA. We present the high-resolution crystal structure of Sso2452, which reveals key structural differences from the canonical RecA family recombinases that may explain its functional properties. The possible roles of the archaeal RadA paralogs in vivo are discussed.     

Overproduction of soluble recombinant transglutaminase from <Emphasis Type="Italic">Streptomyces netropsis</Emphasis> in <Emphasis Type="Italic">Escherichia coli</Emphasis>

A novel microbial transglutaminase (TGase) from the cultural filtrate of Streptomyces netropsis BCRC 12429 (Sn) was purified. The specific activity of the purified TGase was 18.2 U/mg protein with an estimated molecular mass of 38 kDa by sodium dodecyl sulfate polyacrylamide gel electrophoresis analysis. The TGase gene of S. netropsis was cloned and an open reading frame of 1,242 bp encoding a protein of 413 amino acids was identified. The Sn TGase was synthesized as a precursor protein with a preproregion of 82 amino acid residues. The deduced amino acid sequence of the mature S. netropsis TGase shares 78.9–89.6% identities with TGases from Streptomyces spp. A high level of soluble Sn TGase with its N-terminal propeptide fused with thioredoxin was expressed in E. coli. A simple and efficient process was applied to convert the purified recombinant protein into an active enzyme and showed activity equivalent to the authentic mature TGase. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Cloning of milk-derived bioactive peptides in <Emphasis Type="Italic">Streptococcus thermophilus</Emphasis>

The enzymatic breakdown of milk proteins releases bioactive peptides. Two such peptides are the 11-residue antimicrobial peptide from bovine lactoferrin (BL-11) and the 12-residue hypotensive peptide from α_s1-casein (C-12). These two peptides have now been cloned in Streptococcus thermophilus to develop strains that enhance the functionality and nutritional value of dairy food products. Nucleic acid sequences encoding the peptides were generated by overlapping PCR and were subsequently cloned into a new expression vector under control of the ST₂₂₀₁ promoter. S. thermophilus transformants were successfully identified using GFP as a selectable marker. The presence of the synthetic gene constructs in S. thermophilus was confirmed by PCR. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

The SOSS1 single‐stranded DNA binding complex promotes DNA end resection in concert with Exo1

The human SSB homologue 1 (hSSB1) has been shown to facilitate homologous recombination and double‐strand break signalling in human cells. Here, we compare the DNA‐binding properties of the SOSS1 complex, containing SSB1, with Replication Protein A (RPA), the primary single‐strand DNA (ssDNA) binding complex in eukaryotes. Ensemble and single‐molecule approaches show that SOSS1 binds ssDNA with lower affinity compared to RPA, and exhibits less stable interactions with DNA substrates. Nevertheless, the SOSS1 complex is uniquely capable of promoting interaction of human Exo1 with double‐strand DNA ends and stimulates its activity independently of the MRN complex in vitro. Both MRN and SOSS1 also act to mitigate the inhibitory action of the Ku70/80 heterodimer on Exo1 activity in vitro. These results may explain why SOSS complexes do not localize with RPA to replication sites in human cells, yet have a strong effect on double‐strand break resection and homologous recombination.     

Bacillus subtilis RadA/Sms contributes to chromosomal transformation and DNA repair in concert with RecA and circumvents replicative stress in concert with DisA

Bacillus subtilis radA is epistatic to disA and recA genes in response to methyl methane sulfonate- and 4-nitroquinoline-1-oxide-induced DNA damage. We show that ΔradA cells were sensitive to mitomycin C- and H₂O₂-induced damage and impaired in natural chromosomal transformation, whereas cells lacking DisA were not. RadA/Sms mutants in the conserved H1 (K104A and K104R) or KNRFG (K255A and K255R) motifs fail to rescue the sensitivity of ΔradA in response to the four different DNA damaging agents. A RadA/Sms H1 or KNRFG mutation impairs both chromosomal and plasmid transformation, but the latter defect was suppressed by inactivating RecA. RadA/Sms K255A, K255R and wild type RadA/Sms reduced the diadenylate cyclase activity of DisA, whereas RadA/Sms K104A and K104R blocked it. Single-stranded and Holliday junction DNA are preferentially bound over double-stranded DNA by RadA/Sms and its variants. Moreover, RadA/Sms ATPase activity was neither stimulated by a variety of DNA substrates nor by DisA. RadA/Sms possesses a 5´→3´ DNA helicase activity. The RadA/Sms mutants neither hydrolyze ATP nor unwind DNA. Thus, we propose that RadA/Sms has two activities: to modulate DisA and to promote RecA-mediated DNA strand exchange. Both activities are required to coordinate responses to replicative stress and genetic recombination.     

Phenotypic and genotypic diversity among strains of <Emphasis Type="Italic">Aureobasidium pullulans</Emphasis> in comparison with related species

Intra-specific diversity of 200 Aureobasidium pullulans strains isolated from different sources and their relatives Kabatiella lini CBS 125.21 T and Hormonema prunorum CBS 933.72 T were studied by assessment of macromorphological, and physiological tests, sodium dodecyl sulfate-polyacrylamide gel electrophoresis technique (SDS–PAGE) of whole-cell proteins as well as enterobacterial repetitive intergenic consensus (ERIC)-, repetitive extragenic palindromic (REP)- and BOX-PCR techniques (collectively known as rep-PCR). Rep-PCR is an efficient procedure for discrimination of A. pullulans in terms of simplicity and rapidity. RFLP-PCR technique was applied for the identification of A. pullulans isolates and distinction from related species. This technique was insufficient for investigation of intra-specific diversity. The tested strains of A. pullulans could be divided into two groups based on their macromorphological, protein patterns obtained after SDS-PAGE as well as rep-PCR patterns. The first group of strains shared similar characteristics and was very different from the second one, designated as “complex group”, consisting of strains with very little similarities within the group. Phenetic analysis of ERIC banding patterns failed to group the isolates on the basis of their substrate or geographical origin. Using 18S rDNA gene sequence analysis of selected isolates, three strains: HoHe3 km, A. pullulans DSM 62074 and H. prunorum CBS 933.72 T were distinguished from all other analysed members of genera Aureobasidium and Kabatiella. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

<Emphasis Type="Italic">Schizosaccharomyces pombe</Emphasis> replication protein Cdc45/Sna41 requires Hsk1/Cdc7 and Rad4/Cut5 for chromatin binding

Cdc45 is a conserved protein required for firing of replication origins and processive DNA replication. We used an in situ chromatin-binding assay to determine factors required for fission yeast Cdc45p chromatin binding. Assembly of the pre-replicative complex is essential for Cdc45p chromatin binding, but pre-replicative complex assembly occurs independently of Cdc45p. Fission yeast Cdc45p associates with MCM proteins in asynchronously growing cells and cells arrested in S phase by hydroxyurea, but not in cells arrested at the G2/M transition. Both hsk1⁺ (the fission yeast CDC7 homologue) and rad4⁺/cut5⁺ (the fission yeast DPB11 homologue) are required for Cdc45p chromatin binding. Cdc45p also remains chromatin-bound in mutants that fail to recover from replication arrest. In summary, Cdc45p chromatin binding requires an intact pre-replicative complex as well as signaling from both the Dbf4-dependent kinase and cyclin-dependent kinases.