Ken-ji Yokoi

Genetic and biochemical characterization of glutamyl endopeptidase of Staphylococcus warneri M

A Staphylococcus warneri strain M, newly isolated from processed seafood (smoked Watasenia scintillans), produced an extracellular protease. The protease, designated to as m-PROM (the mature form of PROM), selectively cleaved the carbonyl side of glutamic acid residues in beta-casein. Sequence of N-terminal 27 amino acids of m-PROM, RANVILPNNDRHQINDTTLGHYAPVTF, was found to be similar to those of other glutamyl endopeptidases, V8 protease (Staphylococcus aureus strain V8) and SPase (S. aureus ATCC 12600). To determine the complete primary structure and precursor of PROM, its gene (proM) was cloned and sequenced. The gene proM was found to encode for a protein of 316 amino acids. The amino acid residues from 64 to 90 completely coincided with the N-terminal 27 amino acids of the m-PROM, suggesting that the N-terminal 63 amino acids region of p-PROM (the precursor form of PROM) might be processed posttranslationally. Moreover, the whole amino acid sequence deduced from the primary structure of proM shows significant similarity to those of other glutamyl endopeptidases, V8 protease and SPase. These results suggested that PROM belongs to the glutamyl endopeptidase class. PROM, however, differs from V8 and SPase proteases in the processing site and the C-terminal region.

Molecular properties of the putative autolysin AtlWM encoded by Staphylococcus warneri M: Mutational and biochemical analyses of the amidase and glucosaminidase domains

The putative autolysin Atl(WM) of Staphylococcus warneri M is a modular protein exhibiting two enzyme activities, an N-terminal side amidase (ami(atlwm)-R1-R2) and a C-terminal side glucosaminidase (R3-glu(atlwm)). Zymographic analysis of the protein overproduced in Escherichia coli showed that both enzymes were active toward 17 Gram-positive bacteria, including staphylococci, lactobacilli, lactococci, enterococci, and micrococci. The purified enzyme core ami(atlwm) (or glu(atlwm)) had the pH and temperature optima of about 7.0 (5.5) and 41 (50) degrees C, respectively. ami(atlwm) was inactivated by EDTA, and was stimulated by such salts as CoCl(2), MnCl(2), CaCl(2), or ZnCl(2). Six mutations within ami(atlwm), (H362A, E421A, H467A, H479, D481A, and Y491D) drastically reduced cell-lytic activity. Comparative analysis with other related amidases suggested that the three residues H362, H467, and D481 likely act as ligands (and/or active sites). The lytic activity of glu(atlwm) markedly declined in four mutants (E1238A, E1238Q, T1239A, and Y1332A). For determination of the putative cell-recognition regions, four domains (R1-R2, R1, R2, and R3) were purified; all the proteins substantially bound to S. warneri M cells from exponential to stationary growth phases, and R1-R2 aggregated the cells. Protein sequencing and immunoblot analysis suggested that the extacellular Atl(WM) might be primarily processed at two specific sites (one between pro and ami(atlwm), and the other between R2 and R3) to yield the mature amidase and glucosaminidase.

Properties of the inulinase gene levH1 of Lactobacillus casei IAM 1045; cloning, mutational and biochemical characterization.

Though some genetic features of lactobacillar fructan hydrolases were elucidated, information about their enzymology or mutational analyses were scarce. Lactobacillus casei IAM1045 exhibits extracellular activity degrading inulin. After partial purification of the inulin-degrading protein from the spent culture medium, several fragments were obtained by protease digestion. Based on their partial amino-acid sequences, oligonucleotide primers were designed, and its structural gene (levH1) was determined using the gene library constructed in the E. coli system. The levH1 gene encoded a protein (designated as LevH1), of which calculated molecular mass and pI were 138.8-kDa and 4.66, respectively. LevH1 (1296 amino-acids long) was predicted to have a four-domain structure, containing (i) an N-terminal secretion signal of 40 amino-acids, (ii) variable domain of about 140 residues whose function is unclear, (iii) a catalytic domain of about 630 residues with glycoside-hydrolase activity consisting of two modules, a five-blade β-propeller module linked to a β-sandwich module, (iv) a C-terminal domain of about 490 residues comprising five nearly perfect repeat sequences of 80 residues homologous to equivalents of other hypothetical cell surface proteins, followed by 37-residues rich in Ser/Thr/Pro/Gly, a pentad LPQAG (the LPXTG homologue). When overproduced in E. coli, the putative variable-catalytic domain region of about 770 residues exhibited exo-inulinase activity. Deletion analyses demonstrated that the variable-catalytic domain region containing two modules is important for enzymatic activity. Presence of eight conserved motifs (I-VIII) was suggested in the catalytic domain by comparative analysis, among which motif VIII was newly identified in the β-sandwich module in this study. Site-directed mutagenesis of conserved amino-acids in these motifs revealed that D198, R388, D389 and E440, were crucial for inulinase activity. Moreover, mutations of D502A and D683A in motif VI and VIII respectively caused significant decrease in the activity. These results suggested that the variable domain and β-sandwich module, besides the β-propeller module, are important for inulin-degrading activity of LevH1.

Characterization of the histidine decarboxylase gene of Staphylococcus epidermidis TYH1 coded on the staphylococcal cassette chromosome.

Histamine production from histidine in fermented food results in food spoilage, and is harmful to consumers. From fish-miso, we have isolated a new bacterial strain Staphylococcus epidermidis TYH1, which produced histamine under acidic condition in the medium supplemented with glucose. Using oligonucleotides deduced from the histidine decarboxylase gene (hdcA) of Lactobacillus hilgardii, about 14-kbp DNA region of the TYH1 genome was cloned and sequenced. This region contained two putative genes hdcA(TYH1) and hdcP(TYH1) encoding proteins HdcA(TYH1) (310 amino acid residues) and HdcP(TYH1) (495 residues), respectively. Nucleotide sequence around this hdc cluster showed similarity to SCCpbp4 region of S. epidermidis ATCC 12228. Downstream of the cluster, ccrA, ccrB (Type II, respectively) and pbp4 were located. The CcrA and CcrB proteins catalyzed excision of the hdc cluster from the TYH1 chromosome, upon introduction into the TYH1 strain via multicopy plasmid. When hdcA(TYH1) was expressed in Staphylococcus warneri M, histamine was extracellularly accumulated in dependence on exogenous histidine. These results indicate that the gene encoding a histidine decarboxylase resides in a movable genetic element, SCC. This new element is designated as SCChdc.

Molecular properties of the glucosaminidase AcmA from Lactococcus lactis MG1363: mutational and biochemical analyses.

The major autolysin AcmA of Lactococcus lactis ssp. cremoris MG1363 is a modular protein consisting of an N-terminal signal sequence, a central enzymatic region (glu(acma) as a glucosaminidase), and a C-terminal cell-recognition domain (LysM123). glu(acma) (about 160 amino acids) belongs to the glycoside hydrolase (GH) 73 family, and the two acidic residues E128 and D153 have been thought to be catalytically important. In this study, amino-acid substitution analysis of AcmA was first carried out in the Escherichia coli system. Point mutations E94A, E94Q, E128A, D153A, and Y191A markedly reduced cell-lytic activity (3.8%, 1.1%, 4.2%, 4.8%, and 2.4%, respectively), whereas E128Q and D153N retained significant residual activities (32.1% and 44.0%, respectively). On the other hand, Y191F and Y191W mutations retained high activities (66.2% and 46.0%, respectively). These results showed that E94 (rather than E128 and D153) and the aromatic residue Y191 probably play important roles in catalysis of AcmA. Together with mutational analysis of another GH73 glucoaminidase Glu(atlwm) from the Staphylococcus warneri M autolysin Atl(WM), these results suggested that the GH73 members cleave a glycosidic bond via a substrate-assisted mechanism, as postulated in the GH20 members. AcmA and Glu(atlwm) were purified from E. coli recombinant cells, and their enzymatic properties were studied.

Molecular Properties and Extracellular Processing of the Lipase of Staphylococcus warneri M

Staphylococcus warneri M exhibited extracellular lipase activity. By zymogram analysis of extracellular proteins, multiple bands were detected and the profiles changed depending on the bacterial growth phase. N-terminal amino acid sequences of three bands (N1–N3) were determined. From the genome library of S. warneri M whole DNA, the gene-directing lipase activity (named gehCWM) was cloned and characterized. The gehCWM gene encoded a protein (GehCWM), whose calculated molecular mass was 83.4 kDa, and the sequence was similar to the other staphylococcal lipases. Though two lipases have been known from S. warneri 863, GehCWM differs from both of them, indicating that this enzyme is the third extracellular lipase of the S. warneri strain. The N-terminal sequences of the N1–N3 polypeptides completely coincided with the deduced amino acid sequences in GehCWM. GehCWM was predicted to be a prepro-protein. In vitro processing and protein sequencing suggested that pro-GehCWM is possibly processed by extracellular glutamyl endopeptidase, PROM. Inductively coupled plasma-atomic emission spectrometer analysis showed that purified his-tagged mature GehCWM possessed zinc ion. A gehCWM knockout mutant was constructed by insertion of an erythromycin resistance gene into the gehCWM. Zymogram and immunoblot analyses of the gehCWM mutant indicated that GehCWM was a major extracellular lipase of S. warneri M.

Inactivation of the serine proteinase operon (proMCD) of Staphylococcus warneri M: serine proteinase and cysteine proteases are involved in the autolysis.

Unlike other members of coagulase negative staphylococci (CNS), strain warneri has proMCD operon, a homologue of sspABC proteinase operon of S. aureus. The proM and proC encode serine glutamyl endopeptidase and cysteine protease respectively, whereas proD directs homologue of SspC, putative cytoplasmic inhibitor which protects the host bacterium from premature activation of SspB. We determined whole nucleotide sequence of proMCD operon of S. warneri M, succeeded in expression of these genes, and investigated their functions by gene inactivation and complementation experiments. In gelatin zymography of the culture supernatant, a 20-kDa band corresponding to PROC cysteine protease was detected. By Western blotting, PROD was also confirmed in the cytoplasmic protein fraction. PROC and PROD showed significant similarity to SspB and SspC of S. aureus (73% and 58%, respectively). Inactivation mutants of proMCD, proCD and proD genes were established, separately. In the proMCD mutant, degradation/processing of extracellular proteins was drastically reduced, suggesting that PROM was responsible for the cleavage of extracellular proteins. By the proD mutation, the growth profile was not affected, and secretion of PROC was retained. Extracellular protein profiles of the proCD and proD mutants were not so different each other, but autolysin profiles were slightly dissimilar, around 39-48 kDa and 20kDa bands in zymogram. Experiments in buffer systems showed that autolysis was significantly diminished in proMCD mutant, and was promoted by addition of purified PROM. The proC gene was cloned into a multicopy plasmid, and introduced into the proMCD mutant. Compared with the wild type, autolysis of the proC-complemented strain was definitely enhanced by addition of purified PROM. These results suggested that PROM and PROC affected the coccal autolysis, through processing of the autolysin.

Characterization of the major tail protein gpP encoded by Lactobacillus plantarum phage phi gle.

Lactobacillus plantarum temperate phage phi g1e encodes a major virion protein gpP. In the present study, the gpP protein was overproduced in Escherichia coli under plac, and purified. Like the native-gpP protein from phi gle particles (Kakikawa et al., 1996), the purified-gpP protein had an apparent molecular mass of 26.0 kDa on SDS polyacrylamide gel electrophoresis (PAGE), larger than that (18.8 kDa) predicted from the DNA sequence, and was deficient in the first methionine as revealed by the N-terminal protein sequencing. In addition, analysis by immunoelectron microscopy demonstrated that immunogold particles (associated with antigpP-sera) specifically bound to the tails of phi gle particles, indicating that gpP is a main tail component (putatively a tube protein).

Aureolysin of Staphylococcus warneri M accelerates its proteolytic cascade, and participates in biofilm formation

The aureolysin (Aur) gene of S. warneri M (aurWM) was cloned and sequenced. Analyses of the aurWM-inactivated mutant (S. warneri Mau) suggested that AurWM was probably associated with efficient processing of the PROM protease (homolog of V8/SspA serine protease), whereas considerable amount of mature-PROC protease (homolog of SspB cysteine protease) accumulated without AurWM. Additionally, AurWM appeared to affect biofilm formation in an uncertain suppressive way.