Isolation and characterization of the Haemophilus influenzae uvrA gene

Morena, Maria L. de la; Hendrixson, David R.; Geme, Joseph W. St.

doi:10.1016/0378-1119(96)00264-8

Cited by 10 publications

(5 citation statements)

References 21 publications

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“…S. mutans cells were induced to genetic competence by addition of CSP, and the fused construct was then used to transform S. mutans WT strains, resulting in double-crossover recombination and replacement of the target gene with the Erm cassette. Prior to assessment of the uvrA mutant's ability to survive acid challenge, we first determined if a mutant defective in the putative uvrA gene was UV sensitive, since uvrA mutants in other bacteria were extremely sensitive to UV irradiation (8,10). It has been previously demonstrated that an adaptive response to one stress can often lead to cross-protection against other stresses (20).…”

Section: Resultsmentioning

confidence: 99%

“…In bacteria, the UvrABC complex is the principal component of the NER pathway (34), which is the main pathway for the removal of damage caused by UV light. Indeed, in E. coli and other organisms, gene knockouts in any of the NER constituents create mutants that are extremely UV sensitive (8,10,40), suggesting that any mutation made in the NER pathway completely obliterates the system. We have shown in S. mutans that uvrA mutants are also extremely sensitive to UV irradiation (Fig.…”

Section: Discussionmentioning

confidence: 99%

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uvrA Is an Acid-Inducible Gene Involved in the Adaptive Response to Low pH in Streptococcus mutans

et al. 2001

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The pH-inducible acid tolerance response (ATR) is believed to play a major role in acid adaptation and virulence of Streptococcus mutans. To study this phenomenon in S. mutans JH1005, differential display PCR was used to identify and clone 13 cDNA products that had increased expression in response to pH 5.0 compared to that of pH 7.5-grown cells. One of these products, confirmed to be pH inducible by RNA dot blot and reverse transcription-PCR analyses, had 67% identity to a uvrA-UV repair excinuclease gene in Bacillus subtilis. Further sequence analysis of the uvrA homologue using the S. mutans genome database revealed that the complete gene was encoded in an open reading frame (ORF) of 2,829 bp (944 amino acids; 104.67 kDa). Immediately 3 of uvrA was an ORF encoding a putative aminopeptidase gene (pepP). uvrA knockouts were constructed in S. mutans strains JH1005, NG8, and UA159 using allelic-exchange mutagenesis, replacing the entire gene with an erythromycin resistance cassette. As with uvrA mutants in other bacteria, the S. mutans uvrA mutants were extremely sensitive to UV irradiation. The uvrA mutant of S. mutans JH1005 was also more sensitive than the wild type to growth at pH 5.0, showing a 15% reduction in growth rate and a 14% reduction in final resting culture density. Acid-adapted S. mutans JH1005 uvrA mutants were shown to be more resistant to UV irradiation than was the parent but were unable to survive exposure to a killing pH of 3.0. Moreover, agarose gel electrophoretic analysis of chromosomal DNA isolated from uvrA-deficient cells exposed to low pH demonstrated more DNA damage than that for the wild-type strain. Here we suggest that uvrA and the nucleotide excision repair pathway are involved in the repair of acid-induced DNA damage and are associated with successful adaptation of S. mutans to low pH.The oral bacterium Streptococcus mutans is able to gain a selective advantage over other oral microbes by withstanding extreme fluctuations in plaque pH. In the plaque environment, resident bacteria metabolize dietary carbohydrate, which results in the production of organic acids and a decrease in plaque pH. Telemetric measurements of plaque pH indicate that the pH can drop from 7.0 to values ranging from 4.0 to 3.0 (23). The ability to adapt to moderate pH promotes the survival of S. mutans under lower-pH conditions that would otherwise be lethal (37). This adaptive response in S. mutans is called the acid tolerance response (ATR) (37, 42), and similar mechanisms have been identified in some enteric bacteria (11,12). Acid adaptation in S. mutans requires de novo protein synthesis (37) of up to 36 acid-regulated proteins (19) presumably encoded by acid-inducible genes.Aside from the general features of the cellular response to acid pH, relatively little is known about the function of the numerous proteins encoded by the pH-inducible genes that constitute the S. mutans ATR. The genes for the protein repair chaperone, DnaK (22), and the 54-kDa subunit homologue of the eukaryotic signal recognition pa...

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Section: Resultsmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

uvrA Is an Acid-Inducible Gene Involved in the Adaptive Response to Low pH in Streptococcus mutans

et al. 2001

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“…The first, ЈuvrA, located upstream of gusA beyond ORF-R, begins with an ATG start codon at nucleotide 515 and contains 171 codons. The deduced amino acid sequence from uvrAЈ shows similarities to several uvrA gene products (ABC excision nuclease subunit A) from organisms including Haemophilus influenzae (31% identity) (14) and Neisseria gonorrhoeae (37% identity) (9). The second, ЈbrnQ, contains 226 codons for the carboxy-terminal end of a protein whose amino acid sequence shows 40 and 31% identity to branched-chain amino acid transport system carrier proteins from Lactobacillus delbrueckii (46) and Pseudomonas aeruginosa (24), respectively.…”

Section: Resultsmentioning

confidence: 99%

Identification and Cloning of gusA , Encoding a New β-Glucuronidase from Lactobacillus gasseri ADH

Russell¹,

Klaenhammer

2001

Appl Environ Microbiol

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The gusA gene, encoding a new ␤-glucuronidase enzyme, has been cloned from Lactobacillus gasseri ADH. This is the first report of a ␤-glucuronidase gene cloned from a bacterial source other than Escherichia coli. A plasmid library of L. gasseri chromosomal DNA was screened for complementation of an E. coli gus mutant. Two overlapping clones that restored ␤-glucuronidase activity in the mutant strain were sequenced and revealed three complete and two partial open reading frames. The largest open reading frame, spanning 1,797 bp, encodes a 597-amino-acid protein that shows 39% identity to ␤-glucuronidase (GusA) of E. coli K-12 (EC 3.2.1.31). The other two complete open reading frames, which are arranged to be separately transcribed, encode a putative bile salt hydrolase and a putative protein of unknown function with similarities to MerR-type regulatory proteins. Overexpression of GusA was achieved in a ␤-glucuronidase-negative L. gasseri strain by expressing the gusA gene, subcloned onto a low-copy-number shuttle vector, from the strong Lactobacillus P6 promoter. GusA was also expressed in E. coli from a pET expression system. Preliminary characterization of the GusA protein from crude cell extracts revealed that the enzyme was active across an acidic pH range and a broad temperature range. An analysis of other lactobacilli identified ␤-glucuronidase activity and gusA homologs in other L. gasseri isolates but not in other Lactobacillus species tested.

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“…One of these paralogues is associated with the NER system [25], [26] while the other is a part of the uvrA-PG1037-pcrA operon. Similar to other organisms such as Heamophilus influenza [70], Streptococcus mutans [71], Bacillus subtilis [65] and Plasmodium falciparum [72], our P. gingivalis uvrA (FLL145) and P. gingivalis pcrA (FLL146) defective isogenic mutants showed extreme sensitivity to UV irradiation. The functions of these genes appear to be conserved as defects in their homologues in other organisms showed a similar phenotype [65], [70]–[72].…”

Section: Discussionmentioning

confidence: 64%

“…Similar to other organisms such as Heamophilus influenza [70], Streptococcus mutans [71], Bacillus subtilis [65] and Plasmodium falciparum [72], our P. gingivalis uvrA (FLL145) and P. gingivalis pcrA (FLL146) defective isogenic mutants showed extreme sensitivity to UV irradiation. The functions of these genes appear to be conserved as defects in their homologues in other organisms showed a similar phenotype [65], [70]–[72]. Additionally, when an essential component of the NER system in P. gingivalis was interrupted, creating the uvrB -defective mutant (FLL144), this isogenic strain was more sensitive to UV irradiation than the wild-type [28].…”

Section: Discussionmentioning

confidence: 64%

Protective Role of the PG1036-PG1037-PG1038 Operon in Oxidative Stress in Porphyromonas gingivalis W83

et al. 2013

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As an anaerobe, Porphyromonas gingivalis is significantly affected by the harsh inflammatory environment of the periodontal pocket during initial colonization and active periodontal disease. We reported previously that the repair of oxidative stress-induced DNA damage involving 8-oxo-7,8-dihydroguanine (8-oxoG) may occur by an undescribed mechanism in P. gingivalis. DNA affinity fractionation identified PG1037, a conserved hypothetical protein, among other proteins, that were bound to the 8-oxoG lesion. PG1037 is part of the uvrA-PG1037-pcrA operon in P. gingivalis which is known to be upregulated under H2O2 induced stress. A PCR-based linear transformation method was used to inactivate the uvrA and pcrA genes by allelic exchange mutagenesis. Several attempts to inactivate PG1037 were unsuccessful. Similar to the wild-type when plated on Brucella blood agar, the uvrA and pcrA-defective mutants were black-pigmented and beta-hemolytic. These isogenic mutants also had reduced gingipain activities and were more sensitive to H2O2 and UV irradiation compared to the parent strain. Additionally, glycosylase assays revealed that 8-oxoG repair activities were similar in both wild-type and mutant P. gingivalis strains. Several proteins, some of which are known to have oxidoreducatse activity, were shown to interact with PG1037. The purified recombinant PG1037 protein could protect DNA from H2O2-induced damage. Collectively, these findings suggest that the uvrA-PG1037-pcrA operon may play an important role in hydrogen peroxide stress-induced resistance in P. gingivalis.

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Isolation and characterization of the Haemophilus influenzae uvrA gene

Cited by 10 publications

References 21 publications

uvrA Is an Acid-Inducible Gene Involved in the Adaptive Response to Low pH in Streptococcus mutans

uvrA Is an Acid-Inducible Gene Involved in the Adaptive Response to Low pH in Streptococcus mutans

Identification and Cloning of gusA , Encoding a New β-Glucuronidase from Lactobacillus gasseri ADH

Protective Role of the PG1036-PG1037-PG1038 Operon in Oxidative Stress in Porphyromonas gingivalis W83

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