Sequence analysis of the region downstream from a peptidoglycan hydrolase-encoding gene from Staphylococcus aureus NCTC8325

Borchardt, Scott A.; Babwah, Andy V.; Jayaswal, Radheshyam K.

doi:10.1016/0378-1119(93)90016-v

Cited by 15 publications

(8 citation statements)

References 19 publications

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“…Also, as indicated in Fig. 3, the downstream region of the lytA gene (nucleotides 3629-3779) displayed perfect homology with the attachment site of phage 4 1 1 (attP), nucleotides 254 1 -2704 showed significant homology to the downstream region of the staphylokinase (sak) gene, and nucleotides 3551-3672 showed significant homology to exfoliative toxin A (eta) gene of S. aureus as reported earlier (Borchardt et al 1993). A zymographic method was used to further confirm the activity of the product of ORF2.…”

supporting

confidence: 58%

Molecular analysis of lytic genes of bacteriophage 80α ofStaphylococcus aureus

Bon

Mani²,

Jayaswal³

1997

Can. J. Microbiol.

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Nucleotide sequencing of a 3779-bp fragment of the Staphylococcus aureus bacteriophage 80 alpha revealed two open reading frames: ORF1, designated as lytA, which encodes a polypeptide of 481 amino acids with an apparent M(r) of 53.81 kDa; and ORF2, designated as holin, which encodes for a hydrophobic polypeptide of 145 amino acids with an apparent M(r) of 15.58 kDa and exhibits two putative transmembrane helices. Both genes showed 100% sequence homology to that of the peptidoglycan hydrolase and holin genes of the S. aureus phage phi 11 reported earlier. In addition, the downstream sequences of the lytA gene were homologous to the phage attachment site (attP) of the phage phi 11. Based on our data we propose that the lytic system of the phage 80 alpha evolved from that of phage phi 11.

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supporting

confidence: 58%

Molecular analysis of lytic genes of bacteriophage 80α ofStaphylococcus aureus

Bon

Mani²,

Jayaswal³

1997

Can. J. Microbiol.

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“…The S. aureus gene codes for a 171-aminoacid polypeptide with a size of 18.6 kDa. Borchardt et al (5) were unable to detect expression of this gene in E. coli maxicell or in vitro transcription-translation experiments. The PfpI subunit that was 47% identical to an open reading frame in E. coli was also detected (Fig.…”

Section: Vol 178 1996 P Furiosus Gene Encoding a Novel Intracellulmentioning

confidence: 97%

Sequence, expression in Escherichia coli, and analysis of the gene encoding a novel intracellular protease (PfpI) from the hyperthermophilic archaeon Pyrococcus furiosus

et al. 1996

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A previously identified intracellular proteolytic activity in the hyperthermophilic archaeon Pyrococcus furiosus (I. I. Blumentals, A. S. Robinson, and R. M. Kelly, Appl. Environ. Microbiol. 56:1992Microbiol. 56: -1998Microbiol. 56: , 1990) was found to be a homomultimer consisting of 18.8-kDa subunits. Dissociation of this native P. furiosus protease I (PfpI) into a single subunit was seen by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) but only after trichloroacetic acid precipitation; heating to 95؇C in the presence of 2% SDS and 80 mM dithiothreitol did not dissociate the protein. The gene (pfpI) coding for this protease was located in genomic digests by Southern blotting with probes derived from the N-terminal amino acid sequence. pfpI was cloned, sequenced, and expressed in active form in Escherichia coli as a fusion protein with a histidine tag. The recombinant protease from E. coli showed maximum proteolytic activity at 95؇C, and its half-life was 19 min at this temperature. This level of stability was significantly below that previously reported for the enzyme purified by electroelution of a 66-kDa band from SDS-PAGE after extended incubation of cell extracts at 98؇C in 1% SDS (>30 h). The pfpI gene codes for a polypeptide of 166 amino acid residues lacking any conserved protease motifs; no protease activity was detected for the 18.8-kDa PfpI subunit (native or recombinant) by substrate gel assay. Although an immunological relationship of this protease to the eukaryotic proteasome has been seen previously, searches of the available databases identified only two similar amino acid sequences: an open reading frame of unknown function from Staphylococcus aureus NCTC 8325 (171 amino acid residues, 18.6 kDa, 41% identity) and an open reading frame also of unknown function in E. coli (172 amino acid residues, 18.8 kDa, 47% identity). Primer extension experiments with P. furiosus total RNA defined the 5 end of the transcript. There are only 10 nucleotides upstream of the start of translation; therefore, it is unlikely that there are any pre-or pro-regions associated with PfpI which could have been used for targeting or assembly of this protease. Although PfpI activity appears to be the dominant proteolytic activity in P. furiosus cell extracts, the physiological function of PfpI is unclear.Hyperthermophilic microorganisms (those that grow above 90ЊC with an optimum temperature of at least 80ЊC), which are mostly archaea (53), have been investigated for clues to evolutionary processes as well as to uncover biological strategies underlying life at elevated temperatures. Most of the focus has been on characterization of new isolates (3, 45) and on the mechanisms responsible for heightened levels of biomolecular thermostability (1). Although just as important, the physiology of this novel group of organisms has been less studied (20). This is not surprising since hyperthermophiles are generally difficult to culture, in addition to the fact that no genetic systems are available for directed ...

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“…From the sak + MRSA isolates, however, induction of the sak ‐converting phages by mitomycin C or UV radiation was unsuccessful (data not shown). Borchardt et al [19] reported that a part of the sak gene (only 221‐bp sequence) was present in the downstream region from the peptidoglycan hydrolase‐encoding gene ( lytA ). Based on these findings and our results, it is likely that the recombinational events near or in the region of the sak gene occurred repeatedly in S. aureus and/or bacteriophages.…”

Section: Resultsmentioning

confidence: 99%

The staphylokinase gene is located in the structural gene encodingN-acetylmuramyl-L-alanine amidase in methicillin-resistantStaphylococcus aureus

Horii¹,

Yokoyama²,

Barua³

et al. 2000

FEMS Microbiology Letters

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The nucleotide sequence of a 15600-bp DNA fragment containing the staphylokinase gene (sakNU3-1) of methicillin-resistant Staphylococcus aureus (MRSA) NU3-1 was determined. The sak gene was found within the ply gene encoding N-acetylmuramyl-L-alanine amidase and thus the ply gene should be inactivated. In the flanking region of the sak gene, the tandem repeat sequences (GAAGTGTT and GAATGGTT) were present as possible junction points between the sak and ply genes. No sequences characteristic of the presence of an IS-like element were found. Upstream from the ply gene, the kdpA, kdpB and kdpC homologues were present. Downstream from the ply gene, the tagA, tagH and tagG homologues were present. The sak gene was inserted into the same position of ply in 5/6 of sak(+) MRSA isolates with different genotypes. In all of these sak(+) isolates, Sak was detected in the culture supernatant.

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Sequence analysis of the region downstream from a peptidoglycan hydrolase-encoding gene from Staphylococcus aureus NCTC8325

Cited by 15 publications

References 19 publications

Molecular analysis of lytic genes of bacteriophage 80α ofStaphylococcus aureus

Molecular analysis of lytic genes of bacteriophage 80α ofStaphylococcus aureus

Sequence, expression in Escherichia coli, and analysis of the gene encoding a novel intracellular protease (PfpI) from the hyperthermophilic archaeon Pyrococcus furiosus

The staphylokinase gene is located in the structural gene encodingN-acetylmuramyl-L-alanine amidase in methicillin-resistantStaphylococcus aureus

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