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 Genetic and Functional Analysis of the tbc Operons for Catabolism of Alkyl- and Chloroaromatic Compounds inBurkholderia sp. Strain JS150

2001 ◽  
Vol 67 (10) ◽  
pp. 4805-4816 ◽  
Author(s):  
Hyung-Yeel Kahng ◽  
Juliana C. Malinverni ◽  
Michelle M. Majko ◽  
Jerome J. Kukor
Keyword(s):  
Functional Characterization ◽  
Open Reading Frames ◽  
Gene Products ◽  
Product Analysis ◽  
Initial Substrate ◽  
Wide Range ◽  
Dna Fragment ◽  
Similarity Searches ◽  
Reading Frames

ABSTRACT Burkholderia sp. strain JS150 is able to metabolize a wide range of alkyl-and chloroaromatic hydrocarbons through multiple, apparently redundant catabolic pathways. Previous research has shown that strain JS150 is able to synthesize enzymes for multiple upper pathways as well as multiple lower pathways to accommodate variously substituted catechols that result from degradation of complex mixtures of monoaromatic compounds. We report here the genetic organization and functional characterization of a gene cluster, designatedtbc (for toluene, benzene, and chlorobenzene utilization), which has been cloned as a 14.3-kb DNA fragment from strain JS150 into vector pRO1727. The cloned DNA fragment expressed in Pseudomonas aeruginosa PAO1c allowed the recombinant to grow on toluene or benzene and to transform chlorobenzene, trichloroethylene, phenol, and cresols. The tbc genes are organized into two divergently transcribed operons, tbc1 and tbc2, each comprised of six open reading frames. Similarity searches of databases revealed that the tbc1 and tbc2 genes showed significant homology to multicomponent cresol and phenol hydroxylases and to toluene and benzene monooxygenases, respectively. Deletion mutagenesis and product analysis were used to demonstrate thattbc2 plays a role in the initial catabolism of the unactivated alkyl- or chloroaromatic substrate and that thetbc1 gene products play a role in the catabolism of the first metabolite that results from transformation of the initial substrate. Phylogenetic analysis was used to compare individual components of these tbc monooxygenases with similar sequences in the databases. These results provide further evidence for the existence of multiple, functionally redundant alkyl- and chloroaromatic monooxygenases in strain JS150.

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

2000 ◽  
Vol 182 (19) ◽  
pp. 5521-5529 ◽  
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.

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 Characterization of the Major Control Region ofVibrio cholerae Bacteriophage K139: Immunity, Exclusion, and Integration

1999 ◽  
Vol 181 (9) ◽  
pp. 2902-2913 ◽  
Author(s):  
Jutta Nesper ◽  
Julia Blaß ◽  
Michael Fountoulakis ◽  
Joachim Reidl
Keyword(s):  
Control Region ◽  
Open Reading Frames ◽  
Periplasmic Protein ◽  
Early Step ◽  
Significant Similarity ◽  
Attenuation Effect ◽  
El Tor ◽  
Phage Exclusion ◽  
Reading Frames

ABSTRACT The temperate bacteriophage K139 is highly associated with pathogenic O1 Vibrio cholerae strains. The nucleotide sequence of the major control region of K139 was determined. The sequences of four (cox, cII, cI, and int) of the six deduced open reading frames and their gene order indicated that K139 is related to the P2 bacteriophage family. Two genes of the lysogenic transcript from the mapped promoter PL encode homologs to the proteins CI and Int, with deduced functions in prophage formation and maintenance. Between thecI and int genes, two additional genes were identified: orf2, which has no significant similarity to any other gene, and the formerly characterized gene glo. Further analysis revealed that Orf2 is involved in preventing superinfection. In a previous report, we described that mutations inglo cause an attenuation effect in the cholera mouse model (J. Reidl and J. J. Mekalanos, Mol. Microbiol. 18:685–701, 1995). In this report, we present strong evidence that Glo participates in phage exclusion. Glo was characterized to encode a 13.6-kDa periplasmic protein which inhibits phage infection at an early step, hence preventing reinfection of vibriophage K139 into K139 lysogenic cells. Immediately downstream of gene int, the attPsite was identified. Upon analysis of the correspondingattB site within the V. cholerae chromosome, it became evident that phage K139 is integrated between the flagellin genes flaA and flaC of O1 El Tor and O139V. cholerae lysogenic strains.

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

2002 ◽  
Vol 184 (2) ◽  
pp. 337-343 ◽  
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.

scholarly journals Characterization of the Type 8 Capsular Gene Cluster of Streptococcus pneumoniae

1999 ◽  
Vol 181 (19) ◽  
pp. 6214-6219 ◽  
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.

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.

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 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.

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