Genetic and biochemical properties of an extracellular neutral metalloprotease from Staphylococcus hyicus subsp. hyicus

Ayora, Silvia; Götz, Friedrich

doi:10.1007/bf00281792

Cited by 61 publications

(47 citation statements)

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“…Strains were grown overnight in LB medium in the presence or absence of 5 µM AHLs or dichloromethane extracts of spent culture supernatants from different strains. Proteolytic activity was measured as described by Ayora & Go$ tz (1994 Fig. 1.…”

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confidence: 99%

N-Acylhomoserine-lactone-mediated communication between Pseudomonas aeruginosa and Burkholderia cepacia in mixed biofilms

Riedel¹,

Hentzer²,

et al. 2001

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Pseudomonas aeruginosa and Burkholderia cepacia are capable of forming mixed biofilms in the lungs of cystic fibrosis patients. Both bacteria employ quorum-sensing systems, which rely on N-acylhomoserine lactone (AHL) signal molecules, to co-ordinate expression of virulence factors with the formation of biofilms. As both bacteria utilize the same class of signal molecules the authors investigated whether communication between the species occurs. To address this issue, novel Gfp-based biosensors for non-destructive, in situ detection of AHLs were constructed and characterized. These sensors were used to visualize AHL-mediated communication in mixed biofilms, which were cultivated either in artificial flow chambers or in alginate beads in mouse lung tissue. In both model systems B. cepacia was capable of perceiving the AHL signals produced by P. aeruginosa, while the latter strain did not respond to the molecules produced by B. cepacia. Measurements of extracellular proteolytic activities of defined quorum-sensing mutants grown in media complemented with AHL extracts prepared from culture supernatants of various wild-type and mutant strains supported the view of unidirectional signalling between the two strains.

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Section: Introductionmentioning

confidence: 99%

N-Acylhomoserine-lactone-mediated communication between Pseudomonas aeruginosa and Burkholderia cepacia in mixed biofilms

Riedel¹,

Hentzer²,

et al. 2001

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“…The lipolytic activity was quantified by determination of the OD 410 value. Proteolytic activity was investigated as described previously (Ayora & Gotz, 1994). An aliquot (150 ml) of filter-sterilized culture supernatant was incubated with 250 ml substrate for 6 h at 37˚C.…”

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Quorum-sensing-directed protein expression in Serratia proteamaculans B5a

et al. 2003

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N-Acyl-L-homoserine-lactone-producing Serratia species are frequently encountered in spoiling foods of vegetable and protein origin. The role of quorum sensing in the food spoiling properties of these bacteria is currently being investigated. A set of luxR luxI homologous genes encoding a putative quorum sensor was identified in the N-(3-oxo-hexanoyl)-L-homoserine lactone (3-oxo-C6-HSL)-producing Serratia proteamaculans strain B5a. The 3-oxo-C6-HSL synthase SprI showed 79 % similarity with EsaI from Pantoea stewartii and the putative regulatory protein SprR was 86 % similar to the SpnR of Serratia marcescens. Proteome analysis suggested that the presence of at least 39 intracellular proteins was affected by the 3-oxo-C6-HSL-based quorum sensing system. The lipB-encoded secretion system was identified as one target gene of the quorum sensing system. LipB was required for the production of extracellular lipolytic and proteolytic activities, thus rendering the production of food-deterioration-relevant exoenzymes indirectly under the control of quorum sensing. Strain B5a caused quorum-sensing-controlled spoilage of milk. Furthermore, chitinolytic activity was controlled by quorum sensing. This control appeared to be direct and not mediated via LipB. The data presented here demonstrate that quorum-sensing-controlled exoenzymic activities affect food quality. INTRODUCTIONThe bacterial phenomenon of quorum sensing enables bacteria to communicate and thereby coordinate the expression of specific target genes in response to the population density. In a minimum of 30 Gram-negative bacterial species, quorum sensing is mediated by small diffusible signal molecules, N-acylated derivatives of L-homoserine lactone, which may differ in the length and substitution of their respective acyl side chains. N-Acyl-Lhomoserine lactone (AHL) molecules with N-acyl side chains ranging from 4 to 14 carbons and with an oxo-, hydroxy-or no substituent at the C3 position have been identified [for recent reviews see Whitehead et al. (2001), Fuqua et al. (2001) and Miller & Bassler (2001)]. One of the best characterized quorum sensing systems is present in the marine bacterium Vibrio fischeri, in which LuxI synthesizes N-(3-oxo-hexanoyl)-L-homoserine lactone (3-oxo-C6-HSL). It is believed that the signal molecule 3-oxo-C6-HSL at a certain threshold concentration binds to a regulatory protein, LuxR, and thereby renders it active. LuxR hereafter activates transcription of the luxICDABEG operon. As a consequence, the bacterial culture expresses a bioluminescent phenotype when it colonizes the light organs of certain fish and squids and a non-bioluminescent phenotype when the bacteria live in a planktonic state in sea water [(for reviews see Sitnikov et al. (1995) and Dunlap (1999)]. This is one of the few known examples of quorumsensing-regulated gene expression in relation to a symbiotic relationship. In most other cases, quorum sensing is related to pathogenicity traits such as conjugal transfer of the Ti plasmids from Agrobacterium tumefaciens ...

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“…Previously, we characterized an extracellular neutral metalloprotease, named ShpI, from S. hyicus subsp. hyicus (3). A gene encoding a protein of 438 amino acids with a deduced molecular mass of 49.7 kDa was cloned and expressed in S. camosus TM300.…”

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Biochemical properties of a novel metalloprotease from Staphylococcus hyicus subsp. hyicus involved in extracellular lipase processing

1994

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Two extracellular proteases from Staphylococcus hyicus subsp. hyicus, ShpI and ShpII, have been characterized. ShpI is a neutral metalloprotease with broad substrate specificity; the gene has been cloned and sequenced. ShpII, characterized here, is mainly produced in the late logarithmic growth phase in contrast to ShpI, which is mainly produced in the late stationary growth phase. ShpII was purified from culture medium of S. hyicus by ammonium sulfate precipitation and DEAE-Sepharose chromatography. The molecular mass, estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, was 34 kDa. The temperature optimum of ShpH was 55°C, and the pH optimum was 7.4. ShpII, a neutral metalloprotease, was strongly inhibited by zinc and calcium chelators. The amino-terminal sequence of the active enzyme was similar to the corresponding region of a Staphylococcus epidermidis metalloprotease. The substrate specificity of ShpII was similar to that of thermolysin-like proteases, with the exception that ShpII also recognized aromatic amino acids. We demonstrated in vitro that ShpII, but not ShpI, cleaved the 86-kDa S. hyicus subsp. hyicus prolipase between Thr-245 and Val-246 to generate the mature 46-kDa lipase. Results of additional in vivo experiments supported the model that ShpII is necessary for the extracellular processing and maturation of S. hyicus subsp. hyicus lipase.Several extracellular proteolytic enzymes from various staphylococcal species have been studied. Three types of proteases from Staphylococcus aureus, a serine protease, a metalloprotease, and a thiol protease, have been characterized (2). The serine proteases only cleave peptide bonds on the COOH-terminal side of dicarboxylic amino acids and are inhibited by diisopropylphosphofluoridate. This inhibition indicates that the enzyme has a serine residue in the active site and is, therefore, probably related to subtilisin and trypsin. The second type of protease, the metalloprotease, normally has broad substrate specificity but has a preference for the NH2-terminal side of hydrophobic amino acid residues. The metalloprotease usually requires zinc for catalytic activity and calcium for stabilizing the tertiary structure and is required for processing other enzymes (e.g., the serine protease). The third type of protease, thiol protease, has broad substrate specificity and also exhibits esterase activity (1). Thiol protease is only active in the presence of reducing agents and is completely inhibited by Hg2+, Ag2+, and Zn2+. The characteristics of thiol protease are shared by papain and partly by streptococcal proteases. Proteases from other staphylococcal species usually fall into one of these groups.Strains of Staphylococcus hyicus subsp. hyicus have strong proteolytic activity (8), which generally can be detected by the use of casein as a substrate. A preliminary biochemical characterization of a protease produced by a strain of S. hyicus subsp. hyicus was carried out by Takeuchi et al. (20,21). We previously demonstrated that the lipase of S. hy...

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Genetic and biochemical properties of an extracellular neutral metalloprotease from Staphylococcus hyicus subsp. hyicus

Cited by 61 publications

References 56 publications

N-Acylhomoserine-lactone-mediated communication between Pseudomonas aeruginosa and Burkholderia cepacia in mixed biofilms

N-Acylhomoserine-lactone-mediated communication between Pseudomonas aeruginosa and Burkholderia cepacia in mixed biofilms

Quorum-sensing-directed protein expression in Serratia proteamaculans B5a

Biochemical properties of a novel metalloprotease from Staphylococcus hyicus subsp. hyicus involved in extracellular lipase processing

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