A novel species of alkaliphilic Bacillus that produces an oxidatively stable alkaline serine protease

Saeki, Katsuhisa; Hitomi, Jun; Okuda, Mitsuyoshi; Hatada, Yuji; Kageyama, Yasushi; Takaiwa, Mikio; Kubota, Hiromi; Hagihara, Hiroshi; Kobayashi, Tohru; Kawai, Shuji; Ito, Susumu

doi:10.1007/s007920100224

Cited by 61 publications

(46 citation statements)

References 25 publications

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“…Intriguingly, RepB also has lower, but significant, similarity to putative replication proteins from plasmids of gram-positive bacteria, i.e., 47% and 50% similarity to Rep43 of Bacillus sp. strain KSM-KP43 plasmid (28) and to Rep of pIP404 from Clostridium perfringens (8), respectively. The conservation in the RepB homologs is sustained over the N-terminal 380 amino acids, whereas the C termini of the polypeptides are variable (Fig.…”

Section: Resultsmentioning

confidence: 99%

Genomic and Functional Characterization of the Modular Broad-Host-Range RA3 Plasmid, the Archetype of the IncU Group

Kulinska

Czeredys

Hayes

et al. 2008

Appl Environ Microbiol

117

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IncU plasmids are a distinctive group of mobile elements with highly conserved backbone functions and variable antibiotic resistance gene cassettes. The IncU archetype is conjugative plasmid RA3, whose sequence (45,909 bp) shows it to be a mosaic, modular replicon with a class I integron different from that of other IncU replicons. Functional analysis demonstrated that RA3 possesses a broad host range and can efficiently self-transfer, replicate, and be maintained stably in alpha-, beta-, and gammaproteobacteria. RA3 contains 50 open reading frames clustered in distinct functional modules. The replication module encompasses the repA and repB genes embedded in long repetitive sequences. RepA, which is homologous to antitoxin proteins from alpha-and gammaproteobacteria, contains a Cro/cI-type DNA-binding domain present in the XRE family of transcriptional regulators. The repA promoter is repressed by RepA and RepB. The minireplicon encompasses repB and the downstream repetitive sequence r1/r2. RepB shows up to 80% similarity to putative replication initiation proteins from environmental plasmids of beta-and gammaproteobacteria, as well as similarity to replication proteins from alphaproteobacteria and Firmicutes. Stable maintenance functions of RA3 are most like those of IncP-1 broad-host-range plasmids and comprise the active partitioning apparatus formed by IncC (ParA) and KorB (ParB), the antirestriction protein KlcA, and accessory stability components KfrA and KfrC. The RA3 origin of transfer was localized experimentally between the maintenance and conjugative-transfer operons. The putative conjugative-transfer module is highly similar in organization and in its products to transfer regions of certain broad-host-range environmental plasmids.Conjugative plasmids contribute greatly to the global spread of genetic information and gene exchange, as in some cases they can self-transfer even between distant bacterial species. Conjugative R factors assigned to the IncU incompatibility group have been isolated from a number of Aeromonas spp. and Escherichia coli strains from seawater fish hatcheries and diseased fish, as well as from clinical environments (2,4,6,15,26,30,31,42). Members of the IncU plasmid group are implicated particularly in the dissemination of antibiotic resistance in Aeromonas strains associated with aquatic environments (15).IncU representatives pAr-32 and pRAS1 contain resistance genes encoded within integrons, and on the basis of restriction enzyme analysis of both plasmid molecules, Sørum et al. (35) postulated a highly conserved backbone structure of IncU group members with variability in the region coding for antibiotic resistance. Similar observations were made with plasmids pASOT and pFBAOT (2,26,27). Plasmid pFBAOT6 (84,749 bp) has been sequenced recently and analyzed in silico (27). Plasmid Rms149 of the Pseudomonas IncP-6 group was assigned to the IncU group on the basis of incompatibility tests (12). Apart from homology between the replication genes of pFBAOT6 and Rms149, no conservati...

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Section: Resultsmentioning

confidence: 99%

Genomic and Functional Characterization of the Modular Broad-Host-Range RA3 Plasmid, the Archetype of the IncU Group

Kulinska

Czeredys

Hayes

et al. 2008

Appl Environ Microbiol

117

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“…The highest level of similarity at the amino acid level is the level of similarity with the putative replication protein (Rep43) from a cryptic plasmid found in Bacillus sp. strain KSM-KP43 (47). The Rep protein is also related to the replication protein encoded by plasmid pIP404 in Clostridium perfringens CPN50 (16) and to the nearly identical putative replication proteins (CTP1 and CTP45) encoded by Clostridium tetani plasmid pE88 (9).…”

Section: Resultsmentioning

confidence: 99%

Complete Nucleotide Sequence of the Conjugative Tetracycline Resistance Plasmid pFBAOT6, a Member of a Group of IncU Plasmids with Global Ubiquity

Rhodes

Parkhill

Bird

et al. 2004

Appl Environ Microbiol

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This study presents the first complete sequence of an IncU plasmid, pFBAOT6. This plasmid was originally isolated from a strain of Aeromonas caviae from hospital effluent (Westmorland General Hospital, Kendal, United Kingdom) in September 1997 (G. Rhodes, G. Huys, J. Swings, P. McGann, M. Hiney, P. Smith, and R. W. Pickup, Appl. Environ. Microbiol. 66:3883-3890, 2000) and belongs to a group of related plasmids with global ubiquity. pFBAOT6 is 84,748 bp long and has 94 predicted coding sequences, only 12 of which do not have a possible function that has been attributed. Putative replication, maintenance, and transfer functions have been identified and are located in a region in the first 31 kb of the plasmid. The replication region is poorly understood but exhibits some identity at the protein level with replication proteins from the gram-positive bacteria Bacillus and Clostridium. The mating pair formation system is a virB homologue, type IV secretory pathway that is similar in its structural organization to the mating pair formation systems of the related broad-host-range (BHR) environmental plasmids pIPO2, pXF51, and pSB102 from plant-associated bacteria. Partitioning and maintenance genes are homologues of genes in IncP plasmids. The DNA transfer genes and the putative oriT site also exhibit high levels of similarity with those of plasmids pIPO2, pXF51, and pSB102. The genetic load region encompasses 54 kb, comprises the resistance genes, and includes a class I integron, an IS630 relative, and other transposable elements in a 43-kb region that may be a novel Tn1721-flanked composite transposon. This region also contains 24 genes that exhibit the highest levels of identity to chromosomal genes of several plant-associated bacteria. The features of the backbone of pFBAOT6 that are shared with this newly defined group of environmental BHR plasmids suggest that pFBAOT6 may be a relative of this group, but a relative that was isolated from a clinical bacterial environment rather than a plant-associated bacterial environment.

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“…strain KSM-KP43 (10). This protease also possesses the conserved methionine, but it resists oxidizing agents (10). Multiple amino acid sequence comparisons between KP-43 and related enzymes in the subtilisin family have indicated that KP-43 does not belong to any of the subfamilies of the subtilisin family (11).…”

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confidence: 99%

“…strain KSM-KP43 (10). This protease also possesses the conserved methionine, but it resists oxidizing agents (10).…”

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confidence: 99%

The Crystal Structure of an Oxidatively Stable Subtilisin-like Alkaline Serine Protease, KP-43, with a C-terminal β-Barrel Domain

Nonaka

Fujihashi

Kita

et al. 2004

Journal of Biological Chemistry

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The crystal structure of an oxidatively stable subtilisinlike alkaline serine protease, KP-43 from Bacillus sp. KSM-KP43, with a C-terminal extension domain, was determined by the multiple isomorphous replacements method with anomalous scattering. The native form was refined to a crystallographic R factor of 0.134 (R free of 0.169) at 1.30-Å resolution. KP-43 consists of two domains, a subtilisin-like ␣/␤ domain and a C-terminal jelly roll ␤-barrel domain. The topological architecture of the molecule is similar to that of kexin and furin, which belong to the subtilisin-like proprotein convertases, whereas the amino acid sequence and the binding orientation of the C-terminal ␤-barrel domain both differ in each case. Since the C-terminal domains of subtilisin-like proprotein convertases are essential for folding themselves, the domain of KP-43 is also thought to play such a role. KP-43 is known to be an oxidation-resistant protease among the general subtilisin-like proteases. To investigate how KP-43 resists oxidizing reagents, the structure of oxidized KP-43 was also determined and refined to a crystallographic R factor of 0.142 (R free of 0.212) at 1.73-Å resolution. The structure analysis revealed that Met-256, adjacent to catalytic Ser-255, was oxidized similarly to an equivalent residue in subtilisin BPN. Although KP-43, as well as proteinase K and subtilisin Carlsberg, lose their hydrolyzing activity against synthetic peptides after oxidation treatment, all of them retain 70 -80% activity against proteinaceous substrates. These results, as well as the ␤-casein digestion pattern analysis, have indicated that the oxidation of the methionine adjacent to the catalytic serine is not a dominant modification but might alter the substrate specificities.

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A novel species of alkaliphilic Bacillus that produces an oxidatively stable alkaline serine protease

Cited by 61 publications

References 25 publications

Genomic and Functional Characterization of the Modular Broad-Host-Range RA3 Plasmid, the Archetype of the IncU Group

Genomic and Functional Characterization of the Modular Broad-Host-Range RA3 Plasmid, the Archetype of the IncU Group

Complete Nucleotide Sequence of the Conjugative Tetracycline Resistance Plasmid pFBAOT6, a Member of a Group of IncU Plasmids with Global Ubiquity

The Crystal Structure of an Oxidatively Stable Subtilisin-like Alkaline Serine Protease, KP-43, with a C-terminal β-Barrel Domain

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