Identification and characterization of two <Emphasis Type="Italic">uvrA</Emphasis> genes of <Emphasis Type="Italic">Xanthomonas axonopodis</Emphasis> pathovar citri

Two uvrA-like genes, designated uvrA1 and uvrA2, that may be involved in nucleotide excision repair in Xanthomonas axonopodis pv. citri (X. a. pv. citri) strain XW47 were characterized. The uvrA1 gene was found to be 2,964 bp in length capable of encoding a protein of 987 amino acids. The uvrA2 gene was determined to be 2,529 bp with a coding potential of 842 amino acids. These two proteins share 71 and 39% identity, respectively, in amino acid sequence with the UvrA protein of Escherichia coli. Analyses of the deduced amino acid sequence revealed that UvrA1 and UvrA2 have structures characteristic of UvrA proteins, including the Walker A and Walker B motifs, zinc finger DNA binding domains, and helix-turn-helix motif with a polyglycine hinge region. The uvrA1 or uvrA2 mutant, constructed by gene replacement, was more sensitive to DNA-damaging agents methylmethane sulfonate (MMS), mitomycin C (MMC), or ultraviolet (UV) than the wild type. The uvrA1 mutant was four orders of magnitude more sensitive to UV irradiation and two orders of magnitude more sensitive to MMS than the uvrA2 mutant. The uvrA1uvrA2 double mutant was one order of magnitude more sensitive to MMS, MMC, or UV than the uvrA1 single mutant. These results suggest that UvrA1 plays a more important role than UvrA2 in DNA repair in X. a. pv. citri. Both uvrA1 and uvrA2 genes were found to be constitutively expressed in the wild type and lexA1 or lexA2 mutant of X. a. pv. citri, and treatment of these cells with sublethal dose of MMC did not alter the expression of these two genes. Results of electrophoresis mobility shift assays revealed that LexA1 or LexA2 does not bind to either the uvrA1 or the uvrA2 promoter. These results suggest that uvrA expression in X. a. pv. citri is not regulated by the SOS response system.     

Cyanobacteria toxins in the Salton Sea

Background

Sequenced archaeal genomes contain a variety of bacterial and eukaryotic DNA repair gene homologs, but relatively little is known about how these microorganisms actually perform DNA repair. At least some archaea, including the extreme halophile Halobacterium sp. NRC-1, are able to repair ultraviolet light (UV) induced DNA damage in the absence of light-dependent photoreactivation but this 'dark' repair capacity remains largely uncharacterized. Halobacterium sp. NRC-1 possesses homologs of the bacterial uvrA, uvrB, and uvrC nucleotide excision repair genes as well as several eukaryotic repair genes and it has been thought that multiple DNA repair pathways may account for the high UV resistance and dark repair capacity of this model halophilic archaeon. We have carried out a functional analysis, measuring repair capability in uvrA, uvrB and uvrC deletion mutants.

Results

Deletion mutants lacking functional uvrA, uvrB or uvrC genes, including a uvrA uvrC double mutant, are hypersensitive to UV and are unable to remove cyclobutane pyrimidine dimers or 6–4 photoproducts from their DNA after irradiation with 150 J/m² of 254 nm UV-C. The UV sensitivity of the uvr mutants is greatly attenuated following incubation under visible light, emphasizing that photoreactivation is highly efficient in this organism. Phylogenetic analysis of the Halobacterium uvr genes indicates a complex ancestry.

Conclusion

Our results demonstrate that homologs of the bacterial nucleotide excision repair genes uvrA, uvrB, and uvrC are required for the removal of UV damage in the absence of photoreactivating light in Halobacterium sp. NRC-1. Deletion of these genes renders cells hypersensitive to UV and abolishes their ability to remove cyclobutane pyrimidine dimers and 6–4 photoproducts in the absence of photoreactivating light. In spite of this inability to repair UV damaged DNA, uvrA, uvrB and uvrC deletion mutants are substantially less UV sensitive than excision repair mutants of E. coli or yeast. This may be due to efficient damage tolerance mechanisms such as recombinational lesion bypass, bypass DNA polymerase(s) and the existence of multiple genomes in Halobacterium. Phylogenetic analysis provides no clear evidence for lateral transfer of these genes from bacteria to archaea.     

UvrA and UvrB enhance mutations induced by oxidized deoxyribonucleotides

Oxidatively damaged DNA precursors (deoxyribonucleotides) are formed by reactive oxygen species. After the damaged DNA precursors are incorporated into DNA, they might be removed by DNA repair enzymes. In this study, to examine whether a nucleotide excision repair enzyme, Escherichia coli UvrABC, could suppress the mutations induced by oxidized deoxyribonucleotides in vivo, oxidized DNA precursors, 8-hydroxy-2′-deoxyguanosine 5′-triphosphate and 2-hydroxy-2′-deoxyadenosine 5′-triphosphate, were introduced into uvrA, uvrB, and uvrC E. coli strains, and mutations in the chromosomal rpoB gene were analyzed. Unexpectedly, these oxidized DNA precursors induced mutations only slightly in the uvrA and uvrB strains. In contrast, effect of the uvrC-deficiency was not observed. Next, mutT, mutT/uvrA, and mutT/uvrB E. coli strains were treated with H₂O₂, and the rpoB mutant frequencies were calculated. The frequency of the H₂O₂-induced mutations was increased in all of the strains tested; however, the increase was three- to four-fold lower in the mutT/uvrA and mutT/uvrB strains than in the mutT strain. Thus, UvrA and UvrB are involved in the enhancement, but not in the suppression, of the mutations induced by these oxidized deoxyribonucleotides. These results suggest a novel role for UvrA and UvrB in the processing of oxidative damage.     

Molecular cloning and sequencing of the gene encoding an exopolygalacturonase of a Bacillus isolate and properties of its recombinant enzyme

An exopolygalacturonase (exo-PGase; EC 3.2.1.82) was found in the culture broth of a Bacillus isolate. The gene encoding the exo-PGase, pehK, was cloned by polymerase chain reaction using mixed primers designed from N-terminal and internal amino acid (aa) sequences of the enzyme (PehK). The determined nucleotide (nt) sequence of pehK revealed a 2940 bp open reading frame (980 aa) that encoded a putative signal sequence (27 aa) and a mature protein (953 aa; 103 810 Da). The recombinant enzyme was purified to homogeneity from a culture broth of Bacillus subtilis harboring a pehK-containing plasmid. It had a molecular mass of 105 kDa and a pI value of 5.0. The maximum activity was observed at pH 8 and 55°C in Tris–HCl buffer. The degradation products from polygalacturonic or oligogalacturonic acids were digalacturonic acid, like the exo-PGases, PehX of Erwinia chrysanthemi and PehB of Ralstonia solanacearum. The deduced aa sequence of PehK exhibited moderate homology to those of PehX and PehB with approx. 30% identity for both. High homology was observed in a suitably aligned internal region of the three enzymes (65% identity), and some of the conserved aa residues appeared to form the catalytic core of the enzymes.     

N-Acetylglucosaminidase (chitobiase) from Serratia marcescens: gene sequence,and protein production and purification in Escherichia coli

The chitobiase (Chb) encoding gene (chb) from Serratia marcescens (Sm) has been cloned, sequenced and expressed in Escherichia coli (Ec). Sequencing has revealed an open reading frame encoding a protein of 885 amino acids (aa). Ec cells harbouring plasmids containing chb can produce enzymatically active Sm Chb protein which is secreted into the periplasm. An efficient purification scheme using cation-exchange chromatography is presented. This yields about 3 mg of >95% pure Sm Chb per litre of Ec culture. The deduced aa sequence is 27-aa longer at the N terminus than that determined by sequencing of the purified protein, suggesting that a leader sequence is removed during transport of the enzyme across the cell membrane. Comparison with the other members of the family 20 of glycosyl hydrolases revealed that Chb has a conserved central region which aligns with almost all members of this family. According to the crystal structure of Sm Chb, this region comprises the catalytic domain of Chb which has an α/β barrel fold     

A Structural Model for the Damage-sensing Complex in Bacterial Nucleotide Excision Repair

Nucleotide excision repair is distinguished from other DNA repair pathways by its ability to process a wide range of structurally unrelated DNA lesions. In bacteria, damage recognition is achieved by the UvrA·UvrB ensemble. Here, we report the structure of the complex between the interaction domains of UvrA and UvrB. These domains are necessary and sufficient for full-length UvrA and UvrB to associate and thereby form the DNA damage-sensing complex of bacterial nucleotide excision repair. The crystal structure and accompanying biochemical analyses suggest a model for the complete damage-sensing complex.Nucleotide excision repair is distinguished from other DNA repair pathways by its ability to process a diverse set of lesions. In bacteria, the initial steps are carried out by three proteins: UvrA, UvrB, and UvrC. The UvrA·UvrB complex conducts surveillance of DNA and recognizes damage. Having located a lesion, UvrA “loads” UvrB onto the DNA at the damaged sites and then dissociates. Damage searching, formation of the UvrB·DNA “preincision” complex, and dissociation of UvrA are regulated by ATP (1). UvrB subsequently recruits the endonuclease UvrC, which catalyzes incisions on either side of the lesion (2, 3). Following incision, UvrC and the damage-containing oligonucleotide are removed by UvrD (helicase II), whereas UvrB remains bound to the gapped DNA and recruits DNA polymerase I for repair synthesis. Sealing of the single-stranded nick completes the repair process and restores the original DNA sequence (4).Since its discovery more than 40 years ago, bacterial nucleotide excision repair has been extensively studied, resulting in a large body of work that describes the protein components and the details of how they operate. Notwithstanding the trove of genetic and biochemical data, several key questions remain unanswered. For example, how does the same set of proteins handle a diverse set of lesions while maintaining specificity? How do UvrA and UvrB cooperate during damage recognition, and what is the precise role of ATP? Ongoing studies in the field, including those described below, aim to address these issues.Recently, we reported the structure of Geobacillus stearothermophilus UvrA and the identification of binding sites for DNA and UvrB (5). We also established that the identified UvrB-binding domain is necessary and sufficient to mediate the UvrA-UvrB interaction and that the isolated interaction domains of UvrA (5) and UvrB (6) bind to each other in solution.To understand the interaction between UvrA and UvrB, we have determined the crystal structure of the complex between the two isolated interaction domains. The structure revealed that UvrA-UvrB interaction interface is largely polar, mediated by several highly conserved charged residues. Site-directed mutagenesis and biochemical characterization of the mutant proteins confirmed the importance of the observed interactions. Based on the interaction domain complex structure, we have constructed a structural model for the full-length UvrA·UvrB ensemble and propose two models for lesion recognition that will serve as a basis for future experiments.     

A 14-kDa Arabidopsis thaliana RNA polymerase III subunit contains two α-motifs flanked by a highly charged C terminus

We have sequenced a cDNA and a gene, AtRPC14, from Arabidopsis thaliana (At) (ecotype Columbia) that encode a protein related to the yeast RNA polymerases (Pol) I and III subunits, yAC19. Polyclonal antibodies raised against the recombinant At polypeptide (AtC14) bind to the Pol I and/or III subunits of about 13–15 kDa, but do not bind to any Pol II subunit in Pol purified from cauliflower, wheat or At. The amino acid (aa) sequence derived from the AtRPC14 cDNA and genomic clones consists of 122 aa, as compared to the 142 aa in the yeast yAC19 subunit and 143 aa in a putative Caenorhabditis elegans CeAC16 subunit. AtC14, yAC19 and CeAC16 contain a conserved sequence of about 85 aa which is related to two motifs in the α subunit of Escherichia coli (Ec) Pol. AtC14 lacks a highly charged N terminus of about 50 aa found in both yAC19 and CeAC16, but has a highly charged C terminus of about 30 aa not found in yAC19 and CeAC16.     

Nucleotide sequence of the gene coding for SEC14p in Candida (torulopsis) glabrata

A gene coding for SEC14p from Candida glabrata has been cloned and characterized. Nucleotide (nt) sequence analysis reveals an open reading frame of 909 bp and predicts the synthesis of a polypeptide of 302 amino acid (aa) residues. Comparison of nt and aa sequences shows that the gene exhibits a much higher homology to the Saccharomyces cerevisiae (72% and 87%, respectively) than to the Candida albicans (55% and 65%, respectively) SEC14 gene.     

Cloning and sequencing of a new holin-encoding gene of Bacillus licheniformis

A Bacillus licheniformis DNA fragment which exhibits homology with the upstream region of the cell-wall hydrolase-encoding gene, cwlL, was cloned into Escherichia coli (Ec). Nucleotide sequencing indicated that there are two open reading frames (tentatively designated as xpaG1 and xpaG2) which encode polypeptide of 89 and 88 amino acids (aa) (10044 and 9764 Da, respectively). Ec cells harboring two compatible plasmids (pMWB1 and pHSGKH) containing the Bacillus subtilis cell-wall hydrolase-encoding gene, cwlA, and xpaG1–G2, respectively, exhibited higher extracellular cell-wall hydrolase activity than did cells harboring pMWB1 and a control plasmid, pHSG398. The aa sequence homology of XpaG2 with other polypeptides indicated that xpaG2 is a holin-encoding gene. Moreover, Ec C600 harboring a plasmid containing xpaG1-xpaG2 led to leakage of β-galactosidase into the extracellular fraction.     

The DrrC protein of Streptomyces peucetius, a UvrA-like protein, is a DNA-binding protein whose gene is induced by daunorubicin

DrrC, a daunorubicin resistance protein with a strong sequence similarity to the UvrA protein involved in excision repair of DNA, is induced by daunorubicin in Streptomyces peucetius and behaves like an ATP-dependent, DNA binding protein in vitro. The refolded protein obtained from expression of the drrC gene in Escherichia coli was used to conduct gel retardation assays. DrrC bound a DNA segment containing the promoter region of a daunorubicin production gene only in the presence of ATP and daunorubicin. This result suggests that DrrC is a novel type of drug self-resistance protein with DNA binding properties like those of UvrA. Western blotting analysis with a polyclonal antiserum generated against His-tagged DrrC showed that the appearance of DrrC in S. peucetius is coincident with the onset of daunorubicin production and that the drrC gene is induced by daunorubicin. These data also showed that the DnrN and DnrI regulatory proteins are required for drrC expression. The level of DrrA, another daunorubicin resistance protein that resembles ATP-dependent bacterial antiporters, was regulated in the same way as that of DrrC.     

Ribonucleotides as nucleotide excision repair substrates

The incorporation of ribonucleotides in DNA has attracted considerable notice in recent years, since the pool of ribonucleotides can exceed that of the deoxyribonucleotides by at least 10–20-fold, and single ribonucleotide incorporation by DNA polymerases appears to be a common event. Moreover ribonucleotides are potentially mutagenic and lead to genome instability. As a consequence, errantly incorporated ribonucleotides are rapidly repaired in a process dependent upon RNase H enzymes. On the other hand, global genomic nucleotide excision repair (NER) in prokaryotes and eukaryotes removes damage caused by covalent modifications that typically distort and destabilize DNA through the production of lesions derived from bulky chemical carcinogens, such as polycyclic aromatic hydrocarbon metabolites, or via crosslinking. However, a recent study challenges this lesion-recognition paradigm. The work of Vaisman et al. (2013) [34] reveals that even a single ribonucleotide embedded in a deoxyribonucleotide duplex is recognized by the bacterial NER machinery in vitro. In their report, the authors show that spontaneous mutagenesis promoted by a steric-gate pol V mutant increases in uvrA, uvrB, or uvrC strains lacking rnhB (encoding RNase HII) and to a greater extent in an NER-deficient strain lacking both RNase HI and RNase HII. Using purified UvrA, UvrB, and UvrC proteins in in vitro assays they show that despite causing little distortion, a single ribonucleotide embedded in a DNA duplex is recognized and doubly-incised by the NER complex. We present the hypothesis to explain the recognition and/or verification of this small lesion, that the critical 2′-OH of the ribonucleotide – with its unique electrostatic and hydrogen bonding properties – may act as a signal through interactions with amino acid residues of the prokaryotic NER complex that are not possible with DNA. Such a mechanism might also be relevant if it were demonstrated that the eukaryotic NER machinery likewise incises an embedded ribonucleotide in DNA.     

Identification of a novel protein, PriB, in Klebsiella pneumoniae

PriB is a primosomal protein required for the reinitiation of replication in bacteria. Here, we report the identification and characterization of a novel PriB protein in Klebsiella pneumoniae (KPN_04595; KpPriB). Unlike the well-studied Escherichia coli PriB protein (EcPriB), which exists as a homodimer comprising 104-aa polypeptides, KpPriB forms a monomer of only 55 aa, due to the absence of the 49 aa N-terminus in KpPriB. Although this N-terminal region (1–49 aa) in EcPriB contains several important residues, such as K18, R34, and W47, which are crucial for ssDNA binding, we found that KpPriB binds ssDNA, but not ssRNA, with comparable affinity as that for EcPriB. Results from filter-binding assays demonstrate that the KpPriB–ssDNA interaction is cooperative and salt-sensitive. Substituting the residue K33 in KpPriB with alanine, the position corresponding to the classic ssDNA-binding residue K82 of EcPriB located in loop L₄₅, significantly reduced ssDNA-binding activity and cooperativity. These results reveal that the 1–49 aa region of the classical PriB protein is unnecessary for ssDNA binding. On the basis of these findings, the structure–function relationships of KpPriB are discussed.     

Cloning and characterization of the Actinobacillus actinomycetemcomitans gene encoding a heat-shock protein 90 homologue

In a search for clones from a λgtl 1 expression library of Actinobacillus actinomycetemcomitans (Aa) genomic DNA that expressed epitopes from a 70-kDa iron-repressible membrane protein, we inadvertently identified clones that encoded a member of the 90-kDa heat-shock protein (HSP 90) family. The gene appears to encode a homologue of HtpG, as the nucleotide sequence has ∼70% identity with the Escherichia coli (Ec) and Vibrio fischeri htpG. Growth of an Aa htpG insertion mutant at 42°C was reduced to 50% of the parent strain, similar to an Ec htpG deletion mutant. These data suggest that Aa HtpG performs a function similar to Ec HtpG.     

Characterization of the Haemophilus influenzae topA locus: DNA topoisomerase I is required for genetic competence

A gene essential for the development of genetic competence in Haemophilus influenzae (Hi) was identified as a homolog of the Escherichia coli (Ec) topA gene, which encodes DNA topoisomerase I (TopI). The Hi topA locus was initially identified by mini-TnlOkan mutagenesis. Three independent insertion events within 500 bp of each other resulted in mutant strains that shared a similar phenotype. Each was deficient in competence-induced DNA binding, showed increased sensitivity to UV irradiation, and had an increased doubling time as compared to the wild-type (wt) strain. The nucleotide sequence of a 6.6-kb fragment containing the wt allele was determined. The sequence contained an open reading frame (ORF) of 868 amino acids (aa) that was interrupted by each of the mini-Tn10kan mutations. The deduced aa sequence had a molecular mass of 98 155 Da, a pI of 8.59 and showed strong similarity to Ec TopI. Examination of the topoisomer distribution of a test plasmid in an Hi mutant carrying an insertion in this ORF showed an increase in the level of supercoiling, indicating that TopI is necessary to relax supercoiled DNA in Hi. Complementation studies and insertional inactivation of genes downstream from topA indicated that TopI and not some downstream gene product was essential for competence. Four other ORFs were identified and two of these had homology to known genes. ORF1, which was truncated at one end of the sequenced region, shared strong sequence similarity to the C-terminal end of Ec pyridine nucleotide transhydrogenase β subunit. ORF4, which was also truncated, showed strong sequence similarity to the N-terminal end of Ec threonyl-tRNA synthetase.     

Cloning and analysis of a cDNA encoding a two-domain hemoglobin chain from the water flea Daphnia magna

A cDNA encoding a two-domain hemoglobin (Hb) chain of Daphnia magna was cloned and its nucleotide (nt) sequence of 1261 bp was determined. The nt sequence contained 74 bp of the leader sequence, 1047 bp of an open reading frame (ORF), and 119 bp of the 3′-untranslated region (UTR), excluding the polyadenylation tail. A sequence, AATACA, located 24 bp upstream from the polyA sequence was considered to be a polyadenylation signal. cDNA-derived amino acid (aa) sequence revealed that D. magna Hb chain is synthesized as a secretory precursor with a signal peptide of 18 aa. Mature D. magna Hb chain consists of 330-aa residues with a calculated molecular weight of 36 227, which is composed of two large repeated domains, domain 1 and 2. Several key aa that are invariant in all or most of other Hb and required for functional heme-binding are conserved in each of the two domains. The N-terminal extension (pre-A segment) of domain 1 was unusually long and contained an unusual threonine-rich sequence. The homology between the aa sequences of the two domains (24% identity) was much lower than that observed in other two-domain Hb chains from clams or nematode. Hb mRNA level in D. magna reared under low oxygen concentration was more than 12 times higher than that in D. magna reared with sufficient aeration, indicating that the expression of Hb gene is regulated by mRNA level.