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

1999 ◽  
Vol 65 (3) ◽  
pp. 951-960 ◽  
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.

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

2000 ◽  
Vol 182 (21) ◽  
pp. 6123-6129 ◽  
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.

scholarly journals 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’

Acta Naturae ◽  
2016 ◽  
Vol 8 (1) ◽  
pp. 117-125 ◽  
Author(s):  
V. A. Chernukhin ◽  
D. A. Gonchar ◽  
M. A. Abdurashitov ◽  
O. A. Belichenko ◽  
V. S. Dedkov ◽  
...  
Keyword(s):  
Dna Sequence ◽  
Amino Acid Sequences ◽  
Open Reading Frames ◽  
Site Specific ◽  
Pcr Screening ◽  
Methylated Dna ◽  
Chromatographic Techniques ◽  
Dna Fragment ◽  
Reading Frames

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.

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

2019 ◽  
Vol 8 (1) ◽  
pp. 44 ◽  
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.

Sequence and characterization of a novel chromosomal aminoglycoside phosphotransferase gene, aph (3')-IIb, in Pseudomonas aeruginosa.

1996 ◽  
Vol 40 (5) ◽  
pp. 1254-1256 ◽  
Author(s):  
H Hächler ◽  
P Santanam ◽  
F H Kayser
Keyword(s):  
Amino Acids ◽  
Pseudomonas Aeruginosa ◽  
Amino Acid ◽  
Amino Acid Identity ◽  
Open Reading Frames ◽  
Acid Identity ◽  
Aminoglycoside Phosphotransferase ◽  
Psti Fragment ◽  
Reading Frames

A novel, probably chromosomally encoded, aminoglycoside phosphotransferase gene was cloned on a 2,996-bp PstI fragment from Pseudomonas aeruginosa and designated aph (3')-IIb. It coded for a protein of 268 amino acids that showed 51.7% amino acid identity with APH (3')-II [APH(3') is aminoglycoside-3' phosphotransferase] from Tn5. Two other open reading frames on the cloned fragment showed homology to a signal-transducing system in P. aeruginosa.

scholarly journals The Characterization of a Novel Phage, pPa_SNUABM_DT01, Infecting Pseudomonas aeruginosa

2021 ◽  
Vol 9 (10) ◽  
pp. 2040
Author(s):  
Jun Kwon ◽  
Sang Wha Kim ◽  
Sang Guen Kim ◽  
Jeong Woo Kang ◽  
Won Joon Jung ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Otitis Externa ◽  
Phage Genome ◽  
Novel Species ◽  
Major Complication ◽  
Open Reading Frames ◽  
Bacterial Cells ◽  
Genome Comparisons ◽  
Reading Frames

The bacterial genus Pseudomonas is a common causative agent of infections in veterinary medicine. In this study, we focused on Pseudomonas aeruginosa canine otitis externa isolates. Due to prolonged antibiotic treatment of otitis externa, antibiotic resistance is common and has become a major complication. Many alternatives to antibiotics have been studied, with bacteriophages emerging as the most promising alternatives. Here, we isolated and characterized a novel phage, pPa_SNUABM_DT01, by investigating its morphology, growth, lysis kinetics, and genomic characteristics. Phages have a vigorous capacity to eliminate bacterial cells through bacterial lysis. This capacity is dependent on the multiplicity of infection (MOI), but even at low MOIs, the phage successfully inhibited bacterial regrowth. The phage genome was 265,520 bp in size and comprised 312 putative open reading frames (ORFs). Comparative genome analysis demonstrated that the phage is a novel species in Myoviridae. The nucleotide similarity was moderately high compared with the Pseudomonas virus, Noxifer. However, a phylogenetic analysis and a dot plot indicated that pPa_SNUABM_DT01 is not closely related to the Phikzvirus or Noxifervirus genus but, instead, belongs to a novel one. The genome comparisons also indicate that the phage, pPa_SNUABM_DT01, could be a novel genus.

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

1999 ◽  
Vol 181 (14) ◽  
pp. 4275-4284 ◽  
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.

scholarly journals Identification and Characterization of a Novel Family of Pneumococcal Proteins That Are Protective against Sepsis

2001 ◽  
Vol 69 (2) ◽  
pp. 949-958 ◽  
Author(s):  
John E. Adamou ◽  
Jon H. Heinrichs ◽  
Alice L. Erwin ◽  
William Walsh ◽  
Tony Gayle ◽  
...  
Keyword(s):  
Genomic Sequence ◽  
Systemic Disease ◽  
Amino Acid Sequences ◽  
Open Reading Frames ◽  
Gene Products ◽  
Homologous Proteins ◽  
Potential Vaccine ◽  
Identification And Characterization ◽  
Reading Frames

ABSTRACT Four pneumococcal genes (phtA, phtB, phtD, andphtE) encoding a novel family of homologous proteins (32 to 87% identity) were identified from the Streptococcus pneumoniae genomic sequence. These open reading frames were selected as potential vaccine candidates based upon their possession of hydrophobic leader sequences which presumably target these proteins to the bacterial cell surface. Analysis of the deduced amino acid sequences of these gene products revealed the presence of a histidine triad motif (HxxHxH), termed Pht (pneumococcal histidine triad) that is conserved and repeated several times in each of the four proteins. The four pht genes (phtA, phtB, phtD, and a truncated version of phtE) were expressed inEscherichia coli. A flow cytometry-based assay confirmed that PhtA, PhtB, PhtD and, to a lesser extent, PhtE were detectable on the surface of intact bacteria. Recombinant PhtA, PhtB, and PhtD elicited protection against certain pneumococcal capsular types in a mouse model of systemic disease. These novel pneumococcal antigens may serve as effective vaccines against the most prevalent pneumococcal serotypes.

scholarly journals Sequence Analysis and Initial Characterization of Two Isozymes of Hydroxylaminobenzene Mutase from Pseudomonas pseudoalcaligenes JS45

2000 ◽  
Vol 66 (7) ◽  
pp. 2965-2971 ◽  
Author(s):  
John K. Davis ◽  
George C. Paoli ◽  
Zhongqi He ◽  
Lloyd J. Nadeau ◽  
Charles C. Somerville ◽  
...  
Keyword(s):  
Amino Acid ◽  
Amino Acid Sequence ◽  
Genomic Library ◽  
Amino Acid Sequences ◽  
Open Reading Frames ◽  
Pcr Analysis ◽  
Pseudomonas Pseudoalcaligenes ◽  
E Coli ◽  
Specific Activities ◽  
Reading Frames

ABSTRACT Pseudomonas pseudoalcaligenes JS45 grows on nitrobenzene by a partially reductive pathway in which the intermediate hydroxylaminobenzene is enzymatically rearranged to 2-aminophenol by hydroxylaminobenzene mutase (HAB mutase). The properties of the enzyme, the reaction mechanism, and the evolutionary origin of the gene(s) encoding the enzyme are unknown. In this study, two open reading frames (habA and habB), each encoding an HAB mutase enzyme, were cloned from a P. pseudoalcaligenes JS45 genomic library and sequenced. The open reading frames encoding HabA and HabB are separated by 2.5 kb and are divergently transcribed. The deduced amino acid sequences of HabA and HabB are 44% identical. The HAB mutase specific activities in crude extracts of Escherichia coli clones synthesizing either HabA or HabB were similar to the specific activities of extracts of strain JS45 grown on nitrobenzene. HAB mutase activity in E. coli extracts containing HabB withstood heating at 85�C for 10 min, but extracts containing HabA were inactivated when they were heated at temperatures above 60�C. HAB mutase activity in extracts of P. pseudoalcaligenesJS45 grown on nitrobenzene exhibited intermediate temperature stability. Although both the habA gene and thehabB gene conferred HAB mutase activity when they were separately cloned and expressed in E. coli, reverse transcriptase PCR analysis indicated that only habA is transcribed in P. pseudoalcaligenes JS45. A mutant strain derived from strain JS45 in which the habA gene was disrupted was unable to grow on nitrobenzene, which provided physiological evidence that HabA is involved in the degradation of nitrobenzene. A strain in which habB was disrupted grew on nitrobenzene. Gene Rv3078 of Mycobacterium tuberculosisH37Rv encodes a protein whose deduced amino acid sequence is 52% identical to the HabB amino acid sequence. E. colicontaining M. tuberculosis gene Rv3078 cloned into pUC18 exhibited low levels of HAB mutase activity. Sequences that exhibit similarity to transposable element sequences are present between habA and habB, as well as downstream ofhabB, which suggests that horizontal gene transfer resulted in acquisition of one or both of the hab genes.

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