Cloning and Characterization of a New Site-Specific Methyl-Directed ElmI Endonuclease Recognizing and Cleaving C5-methylated DNA Sequence 5’-G(5mC)^NG(5mC)-3’

Putative open reading frames of MD-endonucleases have been identified in Enterobacteria genomes as a result of the search for amino acid sequences homologous to MD-endonuclease BisI. A highly conserved DNA primary structure of these open reading frames in different genera of Enterobacteria (Escherichia, Klebsiella and Cronobacter) has allowed researchers to create primers for PCR screening, which was carried out on Enterobacteria DNA collected from natural sources. The DNA fragment, about 440 bp in length, was amplified by use of the genomic DNA of a wild E.coli LM N17 strain as a template and was inserted into the pMTL22 vector. Endonuclease activity was detected in an E.coli ER 2267 strain transformed with the obtained construction. A new enzyme named ElmI was purified by chromatographic techniques from the recombinant strain biomass. It was discovered that similarly to BisI this enzyme specifically cleaves the methylated DNA sequence 5-GCNGC- 3 before the central nucleotide N if this sequence contains two 5-methylcytosines. However, unlike BisI, ElmI more efficiently cleaves this sequence if more than two cytosine residues are methylated.

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Characterization of the Genes for Two Protocatechuate 3,4-Dioxygenases from the 4-Sulfocatechol-Degrading Bacterium Agrobacterium radiobacter Strain S2

Journal of Bacteriology ◽

10.1128/jb.182.21.6123-6129.2000 ◽

2000 ◽

Vol 182 (21) ◽

pp. 6123-6129 ◽

Cited By ~ 21

Author(s):

Matthias Contzen ◽

Andreas Stolz

Keyword(s):

Amino Acid Sequences ◽

Open Reading Frames ◽

Agrobacterium Radiobacter ◽

Sequence Alignments ◽

Degrading Bacterium ◽

Regulator Protein ◽

Putative Operon ◽

Genes Encoding ◽

Reading Frames

ABSTRACT The genes for two different protocatechuate 3,4-dioxygenases (P34Os) were cloned from the 4-sulfocatechol-degrading bacteriumAgrobacterium radiobacter strain S2 (DSMZ 5681). ThepcaH1G1 genes encoded a P34O (P34O-I) which oxidized protocatechuate but not 4-sulfocatechol. These genes were part of a protocatechuate-degradative operon which strongly resembled the isofunctional operon from the protocatechuate-degrading strainAgrobacterium tumefaciens A348 described previously by D. Parke (FEMS Microbiol. Lett. 146:3–12, 1997). The second P34O (P34O-II), encoded by the pcaH2G2 genes, was functionally expressed and shown to convert protocatechuate and 4-sulfocatechol. A comparison of the deduced amino acid sequences of PcaH-I and PcaH-II, and of PcaG-I and PcaG-II, with each other and with the corresponding sequences from the P34Os, from other bacterial genera suggested that the genes for the P34O-II were obtained by strain S2 by lateral gene transfer. The genes encoding the P34O-II were found in a putative operon together with two genes which, according to sequence alignments, encoded transport proteins. Further downstream from this putative operon, two open reading frames which code for a putative regulator protein of the IclR family and a putative 3-carboxymuconate cycloisomerase were identified.

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Characterization of ssfR andssgA, Two Genes Involved in Sporulation ofStreptomyces griseus

Journal of Bacteriology ◽

10.1128/jb.182.19.5521-5529.2000 ◽

2000 ◽

Vol 182 (19) ◽

pp. 5521-5529 ◽

Cited By ~ 33

Author(s):

Hao Jiang ◽

Kathleen E. Kendrick

Keyword(s):

Stop Codon ◽

Streptomyces Griseus ◽

Large Family ◽

Resistant Mutant ◽

Open Reading Frames ◽

Dna Fragment ◽

Morphological Differences ◽

White Mutant ◽

Reading Frames

ABSTRACT In the presence of cefoxitin, which inhibits septum formation during sporulation, Streptomyces griseus is unable to sporulate, retaining the sonication sensitivity of nonsporulating hyphae. Cefoxitin- and sonication-resistant mutant SKK2600 was isolated and showed many morphological differences from its parental strain. A 3.6-kb DNA fragment that complemented the mutations of SKK2600 contained two open reading frames (ORFs), either of which could complement SKK2600. One ORF, designated ssfR, encoded a protein containing a potential DNA-binding helix-turn-helix motif close to its N terminus. SsfR is similar to members of a large family of transcriptional regulators, particularly IclR of Escherichia coli. The second ORF was identified as ssgA, a previously described sporulation gene from S. griseus (S. Kawamoto and J. C. Ensign, Actinomycetology 9:136–151, 1995). A point mutation of C to T seven nucleotides upstream of the UGA stop codon of ssfR was responsible for the phenotype of isolated mutant strain SKK2600. Surprisingly, this mutation should not change the primary structure of SsfR. The ssfR andssgA disruption mutants were constructed and showed the “white” mutant phenotype, with some growth medium dependence. In addition, the ssfR null mutant sporulated ectopically in phosphate starvation medium.

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An endogenous retrovirus presumed to have been endogenized or relocated recently in a marsupial, the red-necked wallaby

Genome ◽

10.1139/gen-2021-0047 ◽

2022 ◽

Author(s):

Sakura Hayashi ◽

Konami Shimizu ◽

Yusuke Honda ◽

Yukako Katsura ◽

Akihiko Koga

Keyword(s):

Endogenous Retrovirus ◽

Amino Acid Sequences ◽

Open Reading Frames ◽

Length Variation ◽

Wild Type ◽

Long Terminal Repeats ◽

Recent Event ◽

Melanin Biosynthesis ◽

Dna Fragment ◽

Reading Frames

An albino infant wallaby was born to a mother with the wild-type body color. PCR and sequencing analyses of TYR (encoding tyrosinase, which is essential for melanin biosynthesis) of this albino wallaby revealed a 7.1-kb-long DNA fragment inserted in the first exon. Because the fragment carried long terminal repeats, we assumed it to be a copy of an endogenous retrovirus, which we named walb. We cloned other walb copies residing in the genomes of this species and another wallaby species. The copies exhibited length variation, and the longest copy (>8.0 kb) contained open reading frames whose deduced amino acid sequences were well aligned with those of gag, pol, and env of retroviruses. It is not known through which of the following likely processes the walb copy was inserted into TYR: endogenization (infection of a germline cell by an exogenous virus), reinfection (infection by a virus produced from a previously endogenized provirus), or retrotransposition (intracellular relocation of a provirus). In any case, the insertion into TYR is considered to have been a recent event on an evolutionary timescale because albino mutant alleles generally do not persist for long because of their deleterious effects in wild circumstances.

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Isolation and Characterization of Genes Responsible for Naphthalene Degradation from Thermophilic Naphthalene Degrader, Geobacillus sp. JF8

Microorganisms ◽

10.3390/microorganisms8010044 ◽

2019 ◽

Vol 8 (1) ◽

pp. 44 ◽

Cited By ~ 1

Author(s):

Daisuke Miyazawa ◽

Le Thi Ha Thanh ◽

Akio Tani ◽

Masaki Shintani ◽

Nguyen Hoang Loc ◽

...

Keyword(s):

Amino Acid ◽

Thermophilic Bacterium ◽

Amino Acid Sequences ◽

Open Reading Frames ◽

Pcr Analysis ◽

Naphthalene Degradation ◽

Isolation And Characterization ◽

Dihydrodiol Dehydrogenase ◽

Reading Frames

Geobacillus sp. JF8 is a thermophilic biphenyl and naphthalene degrader. To identify the naphthalene degradation genes, cis-naphthalene dihydrodiol dehydrogenase was purified from naphthalene-grown cells, and its N-terminal amino acid sequence was determined. Using a DNA probe encoding the N-terminal region of the dehydrogenase, a 10-kb DNA fragment was isolated. Upstream of nahB, a gene for dehydrogenase, there were two open reading frames which were designated as nahAc and nahAd, respectively. The products of nahAc and nahAd were predicted to be alpha and beta subunit of ring-hydroxylating dioxygenases, respectively. Phylogenetic analysis of amino acid sequences of NahB indicated that it did not belong to the cis-dihydrodiol dehydrogenase group that includes those of classical naphthalene degradation pathways. Downstream of nahB, four open reading frames were found, and their products were predicted as meta-cleavage product hydrolase, monooxygenase, dehydrogenase, and gentisate 1,2-dioxygenase, respectively. A reverse transcriptase-PCR analysis showed that transcription of nahAcAd was induced by naphthalene. These findings indicate that we successfully identified genes involved in the upper pathway of naphthalene degradation from a thermophilic bacterium.

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Characterization of the Bacillus subtilis ywsC Gene, Involved in γ-Polyglutamic Acid Production

Journal of Bacteriology ◽

10.1128/jb.184.2.337-343.2002 ◽

2002 ◽

Vol 184 (2) ◽

pp. 337-343 ◽

Cited By ~ 62

Author(s):

Yuji Urushibata ◽

Shinji Tokuyama ◽

Yasutaka Tahara

Keyword(s):

Bacillus Subtilis ◽

Western Blot ◽

Blot Analysis ◽

Northern Blot Analysis ◽

Nitrilotriacetic Acid ◽

Open Reading Frames ◽

Polyglutamic Acid ◽

Dna Fragment ◽

Reading Frames

ABSTRACT The genes required for γ-polyglutamic acid (PGA) production were cloned from Bacillus subtilis IFO16449, a strain isolated from fermented soybeans. There were four open reading frames in the cloned 4.2-kb DNA fragment, and they were almost identical to those in the ywsC and ywtABC genes of B. subtlis 168. Northern blot analysis showed that the four genes constitute an operon. Three genes, ywsC, ywtA, and ywtB, were disrupted to determine which gene plays a central role in PGA biosynthesis. No PGA was produced in ΔywsC and ΔywtA strains, indicating that both of these genes are essential for PGA production. To clarify the function of the YwsC protein, histidine-tagged YwsC (YwsC-His) was produced in the ΔywsC strain and purified from the lysozyme-treated lysate of the transformant by Ni-nitrilotriacetic acid affinity chromatography. Western blot analysis revealed that the YwsC-His protein consists of two subunits, the 44-kDa and 33-kDa proteins, which are encoded by in-phase overlapping in the ywsC gene. 14C-labeled PGA was synthesized by the purified proteins from l-[14C]-glutamate in the presence of ATP and MnCl2, through an acylphosphate intermediate, indicating that the ywsC gene encodes PGA synthetase (EC 6.3.2), a crucial enzyme in PGA biosynthesis.

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Characterization of the Type 8 Capsular Gene Cluster of Streptococcus pneumoniae

Journal of Bacteriology ◽

10.1128/jb.181.19.6214-6219.1999 ◽

1999 ◽

Vol 181 (19) ◽

pp. 6214-6219 ◽

Cited By ~ 23

Author(s):

Rosario Muñoz ◽

Marta Mollerach ◽

Rubens López ◽

Ernesto García

Keyword(s):

Streptococcus Pneumoniae ◽

Nucleotide Sequence ◽

Gene Cluster ◽

Complete Nucleotide Sequence ◽

Capsular Polysaccharide ◽

Open Reading Frames ◽

Dna Fragment ◽

Type 3 ◽

Reading Frames

ABSTRACT The complete nucleotide sequence of the capsular gene cluster (cap8) responsible for the biosynthesis of the capsular polysaccharide of Streptococcus pneumoniae type 8 has been determined. The cap8 gene cluster, located between the genes dexB and aliA, is composed of 12 open reading frames. A 14.7-kb DNA fragment embracing the cap8genes was sufficient to transform an unencapsulated type 3 S. pneumoniae strain to a strain with the type 8 capsule. A possible scenario for the evolution of pneumococcal types 2 and 8 is outlined.

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Molecular Characterization of the Complete 23F Capsular Polysaccharide Locus of Streptococcus pneumoniae

Journal of Bacteriology ◽

10.1128/jb.180.19.5273-5278.1998 ◽

1998 ◽

Vol 180 (19) ◽

pp. 5273-5278 ◽

Cited By ~ 28

Author(s):

Mario Ramirez ◽

Alexander Tomasz

Keyword(s):

Streptococcus Pneumoniae ◽

Molecular Characterization ◽

Dna Sequence ◽

Capsular Polysaccharide ◽

Open Reading Frames ◽

The Other ◽

Reading Frames

ABSTRACT The complete DNA sequence of the capsular locus 23F ofStreptococcus pneumoniae is presented. The 18.6-kbcps23f locus is composed of 18 open reading frames flanked at the 5′ and 3′ ends by the genes dexB andaliA, an arrangement similar to those of some of the other identified cps loci.

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Molecular Characterization of the GenespcaG and pcaH, Encoding Protocatechuate 3,4-Dioxygenase, Which Are Essential for Vanillin Catabolism inPseudomonas sp. Strain HR199

Applied and Environmental Microbiology ◽

10.1128/aem.65.3.951-960.1999 ◽

1999 ◽

Vol 65 (3) ◽

pp. 951-960 ◽

Cited By ~ 35

Author(s):

Jörg Overhage ◽

Andreas U. Kresse ◽

Horst Priefert ◽

Horst Sommer ◽

Gerhard Krammer ◽

...

Keyword(s):

Pseudomonas Aeruginosa ◽

Sole Carbon Source ◽

Genomic Library ◽

Amino Acid Sequences ◽

Open Reading Frames ◽

Regulatory Proteins ◽

Β Subunit ◽

Catabolic Pathway ◽

Reading Frames

ABSTRACT Pseudomonas sp. strain HR199 is able to utilize eugenol (4-allyl-2-methoxyphenol), vanillin (4-hydroxy-3-methoxybenzaldehyde), or protocatechuate as the sole carbon source for growth. Mutants of this strain which were impaired in the catabolism of vanillin but retained the ability to utilize eugenol or protocatechuate were obtained after nitrosoguanidine mutagenesis. One mutant (SK6169) was used as recipient of a Pseudomonas sp. strain HR199 genomic library in cosmid pVK100, and phenotypic complementation was achieved with a 5.8-kbp EcoRI fragment (E58). The amino acid sequences deduced from two corresponding open reading frames (ORF) identified on E58 revealed high degrees of homology to pcaGand pcaH, encoding the two subunits of protocatechuate 3,4-dioxygenase. Three additional ORF most probably encoded a 4-hydroxybenzoate 3-hydroxylase (PobA) and two putative regulatory proteins, which exhibited homology to PcaQ of Agrobacterium tumefaciens and PobR of Pseudomonas aeruginosa, respectively. Since mutant SK6169 was also complemented by a subfragment of E58 that harbored only pcaH, this mutant was most probably lacking a functional β subunit of the protocatechuate 3,4-dioxygenase. Since this mutant was still able to grow on protocatechuate and lacked protocatechuate 4,5-dioxygenase and protocatechuate 2,3-dioxygenase, the degradation had to be catalyzed by different enzymes. Two other mutants (SK6184 and SK6190), which were also impaired in the catabolism of vanillin, were not complemented by fragment E58. Since these mutants accumulated 3-carboxy muconolactone during cultivation on eugenol, they most probably exhibited a defect in a step of the catabolic pathway following the orthocleavage. Moreover, in these mutants cyclization of 3-carboxymuconic acid seems to occur by a syn absolute stereochemical course, which is normally only observed forcis,cis-muconate lactonization in pseudomonads. In conclusion, vanillin is degraded through theortho-cleavage pathway in Pseudomonas sp. strain HR199 whereas protocatechuate could also be metabolized via a different pathway in the mutants.

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DNA Sequence Analysis of a Bioluminescent Marine Bacterium

10.28971/142008rb71 ◽

2008 ◽

Author(s):

Benjamin Ryder

Keyword(s):

Sequence Analysis ◽

Dna Sequence ◽

Marine Bacterium ◽

Dna Sequence Analysis ◽

Open Reading Frames ◽

Pha Synthase ◽

Putative Gene ◽

Dna Fragment ◽

Reading Frames

Studies the sequencing of the DNA fragment containing the gene phaC (PHA synthase) and undertakes the search for open reading frames and putative gene matches in a bioluminescent marine bacterium.

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Characterization of the Serogroup O11 O-Antigen Locus of Pseudomonas aeruginosa PA103

Journal of Bacteriology ◽

10.1128/jb.181.14.4275-4284.1999 ◽

1999 ◽

Vol 181 (14) ◽

pp. 4275-4284 ◽

Cited By ~ 56

Author(s):

C. R. Dean ◽

C. V. Franklund ◽

J. D. Retief ◽

M. J. Coyne ◽

K. Hatano ◽

...

Keyword(s):

Pseudomonas Aeruginosa ◽

Open Reading Frames ◽

Striking Similarity ◽

Insertion Mutation ◽

Significant Similarity ◽

O Antigen ◽

Dna Fragment ◽

Similarity Searches ◽

Reading Frames

ABSTRACT We previously cloned a genomic DNA fragment from the serogroup O11Pseudomonas aeruginosa strain PA103 that contained all genes necessary for O-antigen synthesis and directed the expression of serogroup O11 antigen on recombinant Escherichia coli andSalmonella. To elucidate the pathway of serogroup O11 antigen synthesis, the nucleotide sequence of the biosynthetic genes was determined. Eleven open reading frames likely to be involved in serogroup O11 O-antigen biosynthesis were identified and are designated in order as wzz PaO111 (wzz fromP. aeruginosa serogroup O11),wzx PaO11, wbjA,wzy PaO11, wbjB to wbjF,wbpL O11 and wbpM O11(wbpL and wbpM from serogroup O11). Consistent with previous descriptions of O-antigen biosynthetic gene loci, the entire region with the exception of wbpM O11 has a markedly reduced G+C content relative to the chromosomal average. WzyPaO11 shows no significant similarity at the protein or DNA sequence level to any database sequence and is very hydrophobic, with 10 to 12 putative transmembrane domains, both typical characteristics of O-antigen polymerases. A nonpolar chromosomal insertion mutation in wzy PaO11 in P. aeruginosa PA103 confirmed the identity of this gene. There is striking similarity between WbjBCDE and Cap(5/8)EFGL, involved in type 5 and type 8 capsule biosynthesis in Staphylococcus aureus. There is nearly total identity between wbpM O11and wbpM O5, previously shown by others to be present in all 20 P. aeruginosa serogroups. Using similarity searches, we have assigned functions to the proteins encoded by the PA103 O-antigen locus and present the potential steps in the pathway for the biosynthesis of P. aeruginosa serogroup O11 O antigen.

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