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.

Overexpression, purification, and characterization of Paenibacillus cell surface-expressed chitinase ChiW with two catalytic domains.

Paenibacillus sp. strain FPU-7 produces several different chitinases and effectively hydrolyzes robust chitin. Among the P. FPU-7 chitinases, ChiW, a novel monomeric chitinase with a molecular mass of 150 kDa, is expressed as a cell surface molecule. Here, we report that active ChiW lacking the anchoring domains in the N-terminus was successfully overproduced in Escherichia coli and purified to homogeneity. The two catalytic domains at the C-terminal region were classified as typical glycoside hydrolase family 18 chitinases, whereas the N-terminal region showed no sequence similarity to other known proteins. The vacuum-ultraviolet circular dichroism spectrum of the enzyme strongly suggested the presence of a ß-stranded-rich structure in the N-terminus. Its biochemical properties were also characterized. Various insoluble chitins were hydrolyzed to N,N'-diacetyl-D-chitobiose as the final product. Based on amino acid sequence similarities and site-directed mutagenesis, Glu691 and Glu1177 in the two GH-18 domains were identified as catalytic residues.

Cooperative degradation of chitin by extracellular and cell surface-expressed chitinases from Paenibacillus sp. strain FPU-7.

Chitin, a major component of fungal cell walls and invertebrate cuticles, is an exceedingly abundant polysaccharide, ranking next to cellulose. Industrial demand for chitin and its degradation products as raw materials for fine chemical products is increasing. A bacterium with high chitin-decomposing activity, Paenibacillus sp. strain FPU-7, was isolated from soil by using a screening medium containing α-chitin powder. Although FPU-7 secreted several extracellular chitinases and thoroughly digested the powder, the extracellular fluid alone broke them down incompletely. Based on expression cloning and phylogenetic analysis, at least seven family 18 chitinase genes were found in the FPU-7 genome. Interestingly, the product of only one gene (chiW) was identified as possessing three S-layer homology (SLH) domains and two glycosyl hydrolase family 18 catalytic domains. Since SLH domains are known to function as anchors to the Gram-positive bacterial cell surface, ChiW was suggested to be a novel multimodular surface-expressed enzyme and to play an important role in the complete degradation of chitin. Indeed, the ChiW protein was localized on the cell surface. Each of the seven chitinase genes (chiA to chiF and chiW) was cloned and expressed in Escherichia coli cells for biochemical characterization of their products. In particular, ChiE and ChiW showed high activity for insoluble chitin. The high chitinolytic activity of strain FPU-7 and the chitinases may be useful for environmentally friendly processing of chitin in the manufacture of food and/or medicine.

Cloning of a novel dehalogenase from environmental DNA.

Cloning of pceA, the gene of tetrachloroethene (PCE)-reductive dehalogenase, was undertaken from environmental DNA. Two genes were amplified using PCR primer deduced from pceA. Functional expression of these genes was unsuccessful in Escherichia coli, but PceA1 synthesized in vitro was enzymatically active. In recombinant E. coli, PceA1 formed a complex with host DnaK and caused filamentous growth.

Molecular properties of the putative autolysin Atl(WM) 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.

Mutational and biochemical analyses of the endolysin Lys(gaY) encoded by the Lactobacillus gasseri JCM 1131T phage phi gaY.

The Lys(gaY) of Lactobacillus gasseri JCM 1131(T) phage phigaY endolysin was purified to homogeneity using the Escherichia coli/His.Tag system. Zymographic and spectrophotometric assays showed that Lys(gaY) lysed over 20 heated Gram-positive bacterial species as the substrates, including lactobacilli, lactococci, enterococci, micrococci, and staphylococci. The enzymatic activity had the pH and temperature optima of about 6.5 and 37 degrees C, respectively. Amino-acid substitution analysis revealed that 13 residues of Lys(gaY) were involved in cell-lytic activity: in the beta/alpha(gaY) domain, G10, D12, E33, D36, H60, Y61, D96, E98, V124, L132, and D198; in the SH3b(gaY) domain, Y272 and W284. In addition, deletion analysis demonstrated that the beta/alpha(gaY) domain of N-terminal 216 residues is the core enzyme portion, although the cell-lytic ability is lower than that of Lys(gaY). These mutational experiments suggested that beta/alpha(gaY) (in which two acidic residues of D12 and E98 likely act as catalytic residues) is responsible for cell-lytic activity, and SH3b(gaY) promotes beta/alpha(gaY) possibly through cell-wall binding function. The purified His-tagged SH3b(gaY) domain containing 94 residues from S217 to K310 (i) bound to Gram-positive bacteria susceptible to Lys(gaY), (ii) induced aggregation of exponentially growing cells of L. gasseri JCM 1131(T), L. casei IAM 1045, Lactococcus lactis C2, L. lactis MG 1363, and Enterococcus hirae IAM 1262 by forming thread-like chained cells, (iii) inhibited lytic activity of Lys(gaY), and (iv) impeded autolysis of L. gasseri JCM 1131(T) in buffer systems. A truncated protein HDeltaSH3b(gaY) lacking in N-terminal 21 residues (from S217 to E237) of SH3b(gaY) and an amino-acid substituted protein HSH3b(gaY)G (W284G) lost the activities of HSH3b(gaY), showing that the N-terminal region and W284 probably play important roles in the SH3b(gaY) function(s).

Molecular characterization of NADase-streptolysin O operon of hemolytic streptococci.

Whether slo, the gene encoding streptolysin O (SLO), a streptococcal cytolysin, has its own promoter or not is unsettled as yet. Present analyses demonstrate that slo is a member of an operon covering the upper-stream nusG and nga (NADase) genes, from which transcription of slo proceeds polycistronically, and major transcript is produced by readthrough from nga promoter. Mutational conversion of the sixth nucleotide T at the putative -10 region of chromosomal nga gene into C caused a drastic decrease in both NADase and SLO activities and the disappearance of the two corresponding mRNA bands from the Northern blot profile. The initiation site of the transcription was determined at 56 bp upstream (NusG gene) and 25 bp upstream (NADase gene) of each initiation codon. Although the promoter region of slo gene is highly conserved between group A and C streptococci, the proper slo promoter is nonfunctional in group C strain H46A. Moreover, commonly conserved arrangement was limited to the nusG-nga-orf1-slo region. These results indicate an intimate relationship between NADase and SLO in the regulation of their biosynthesis. Additional results suggest that NADase, synthesized as precursor with feeble activity, is activated by removing the carboxyl terminal region during or after secretion into culture medium.

Crystal Structure of Chitinase ChiW from Paenibacillus sp. str. FPU-7 Reveals a Novel Type of Bacterial Cell-Surface-Expressed Multi-Modular Enzyme Machinery.

The Gram-positive bacterium Paenibacillus sp. str. FPU-7 effectively hydrolyzes chitin by using a number of chitinases. A unique chitinase with two catalytic domains, ChiW, is expressed on the cell surface of this bacterium and has high activity towards various chitins, even crystalline chitin. Here, the crystal structure of ChiW at 2.1 Å resolution is presented and describes how the enzyme degrades chitin on the bacterial cell surface. The crystal structure revealed a unique multi-modular architecture composed of six domains to function efficiently on the cell surface: a right-handed ß-helix domain (carbohydrate-binding module family 54, CBM-54), a Gly-Ser-rich loop, 1st immunoglobulin-like (Ig-like) fold domain, 1st ß/α-barrel catalytic domain (glycoside hydrolase family 18, GH-18), 2nd Ig-like fold domain and 2nd ß/α-barrel catalytic domain (GH-18). The structure of the CBM-54, flexibly linked to the catalytic region of ChiW, is described here for the first time. It is similar to those of carbohydrate lyases but displayed no detectable carbohydrate degradation activities. The CBM-54 of ChiW bound to cell wall polysaccharides, such as chin, chitosan, ß-1,3-glucan, xylan and cellulose. The structural and biochemical data obtained here also indicated that the enzyme has deep and short active site clefts with endo-acting character. The affinity of CBM-54 towards cell wall polysaccharides and the degradation pattern of the catalytic domains may help to efficiently decompose the cell wall chitin through the contact surface. Furthermore, we clarify that other Gram-positive bacteria possess similar cell-surface-expressed multi-modular enzymes for cell wall polysaccharide degradation.

The two-component cell lysis genes holWMY and lysWMY of the Staphylococcus warneri M phage varphiWMY: cloning, sequencing, expression, and mutational analysis in Escherichia coli.

From the genome library of Staphylococcus warneri M, the two successive cell-lysis genes (holWMY and lytWMY) were cloned and characterized. The lytWMY gene encoded a protein (LysWMY), whose calculated molecular mass and pI were 54 kDa and 8.95, respectively. When overproduced in Escherichia coli, lysWMY directed a protein of 45 kDa (smaller than the predicted molecular mass), having N-terminal 13 residues identical with those predicted from DNA. Comparative analysis revealed that LysWMY significantly resembles the putative N-acetylmuramoyl-L-alanine amidases encoded by the staphylococcal phages phi11, 80 alpha, and Twort. Examination of modular organization of LysWMY identified three putative domains CHAP (for D-alanyl-glycyl endopeptidase), amidase (L-muramoyl-L-alanine amidase), and SH3 (cell wall recognition). Gene knockout analysis revealed that each of the two domains of CHAP and amidase was responsible for cell-lytic activity on a zymogram gel. Site-directed mutation of Cys29Ala, His92Ala, or Asn114Ala in the CHAP domain substantially reduced cell-lytic activity, suggesting that this Cys-His-Asn triad is crucial for the enzymatic function. On the other hand, the holWMY gene encoded a protein (HolWMY) with molecular mass and pI of 16 kDa and 4.36; this protein contained two potential transmembrane helices, resembling other predicted holins (a cytoplasmic membrane-disrupting protein) encoded by the S. aureus phage, phi11, 80 alpha, and Twort. Upon mitomycin C exposure of S. warneri M, a prophage (phiWMY) was induced and the virion was examined under electron microscopy. PCR amplification and sequencing revealed the presence of the holWMY-lysWMY genes in the phage genome.

Molecular properties of the two-component cell lysis system encoded by prophage phigaY of Lactobacillus gasseri JCM 1131T: cloning, sequencing, and expression in Escherichia coli.

Shotgun cloning of the Lactobacillus gasseri JCM 1131T whole DNA yielded two recombinant plasmids, p118gaY1 and p118gaY2, which directed cell lysis activity. Sequencing analysis revealed that the two plasmids carried almost identical inserted genes in following orders (truncated genes, in parentheses): in p118gaY1, (orf149)-orf92-holgaY-lysgaY-orf35-attL-(mnaAgaY1); in p118gaY2, (orfXgaY1)-orf169-orf149-orf92-holgaY-lysgaY-orf35-attP-(intgaY). The lysgaY-encoded protein (designated as LysgaY, 33.7 kDa) showed significant homology with putative muramidases (peptidoglycan-degrading enzyme) of the Lactobacillus phage phiadh, Lj965, Lj928, LL-H, mv4, and mv1. By zymogram analysis, LysgaY overproduced in Escherichia coli exhibited lytic activity towards 17 Gram-positive bacterial strains, including lactobacilli, lactococci, and staphylococci. The holgaY-encoded protein (15.7 kDa) contained three potential transmembrane helices, resembling putative holins (cytoplasmic membrane-disrupting protein) of Lj928 and Lj965. On the other hand, another clone p118gaYR obtained by EcoRI-shotgun cloning carried the (ptsCgaY1)-attR-(intgaY) genes. Three sequences, attL, attP, and attR, had a 47-bp common (core) sequence, and the core of attR was located in 3' region of a potential tRNA(Arg) gene. These results suggested that (i) attL and attR are phage-host junctions, left- and right-arms, respectively, (ii) attP is a phage attachment site, and (iii) intgaY is an integrase gene for phage integration and/or excision. After mitomycin C-induction, phage particles were demonstrated by electron microscopy. The prophage (phigaY) is somewhat leaky in the host, and has the two-component lysis system (HolgaY-LysgaY), closely resembling that of Lj928 as well as Lj965.

Molecular analysis of the lysis protein Lys encoded by Lactobacillus plantarum phage phig1e.

The putative cell-lysis gene lys of Lactobacillus plantarum G1e phage phig1e encodes for a 442 amino-acids protein Lys. The N-terminal region (about 80 amino acids) of Lys consists of two discrete regions (the signal-peptide-like domain and the DE domain containing putative active sites of endolysin). To elucidate functions of the regions of Lys, mutational (random, site-directed, and/or fusion) analysis was performed. The plasmid pNdEHL, expressing the wild type Lys protein under promoter of lacZ' gene in Escherichia coli, was constructed. Two molecular species (44 kDa; referred to as pre-Lys, and 42 kDa; mature-Lys) from the protein extract of XL1-Blue/pNdEHL were detected on a sodium dodecyl sulfate gel and zymogram with L. plantarum G1e cells. Based on the N-terminal amino acid sequences, the two molecules were determined as; pre-Lys (the amino acid position deduced from lys gene, 1-7) MKLKNKL, mature-Lys (27-33) QTLSSQS. The mature Lys was hardly detected in the cells treated with sodium azide. These results suggested that the N-terminal 26 amino acids region of Lys precursor form is possibly processed posttranslationally, by a SecA-dependent manner at least in E. coli. Analysis of the point mutants (pLD36A, pLE39A, pLE55A, pLE67A and pLD71A), indicated that the acidic residues (aspartic acids at position 36, 71 and glutamic acids at position 39, 55) of N-terminal region and the serine at the position 48 of phig1e Lys are essential for the lytic activity.

Characterization of lytic enzyme activities of Lactobacillus gasseri with special reference to autolysis.

Lactobacillus gasseri JCM 1130 and JCM 1131(T) exhibited autolytic activity in agar containing autoclaved cells of each strain as substrate. By zymogram analysis of JCM 1131(T), two lytic bands with apparent molecular masses of 54.5 and 35 kDa, were detected. Similarly, JCM 1130 yielded two lytic bands with apparent molecular masses of 35 and 33.5 kDa. In simple buffers as well, JCM 1131(T) suffered a drastic decrease in cell turbidity, but JCM 1130 did not undergo the decrease. The optimal pH for autolysis of JCM 1131(T) was in the range of 6.0-7.0, and the lysis was completely inhibited at pH 4-5. The lysis of JCM 1131(T) was suppressed by NaCl, in a concentration-dependent way. When subjected to UV irradiation or mitomycin C (MMC) treatment, cultures of both strains elicited conspicuous turbidity decrease after 2-4 h of growth, suggesting the occurrence of prophage induction. The 35-kDa lytic band of JCM 1131(T) and the 33.5-kDa protein of JCM 1130 were considerably increased by UV irradiation.

Crystallization and preliminary X-ray analysis of the catalytic domains of Paenibacillus sp. strain FPU-7 cell-surface-expressed chitinase ChiW.

The polysaccharide chitin is effectively hydrolyzed and utilized as a carbon and nitrogen source by the Gram-positive bacterium Paenibacillus sp. strain FPU-7. ChiW is a unique cell-surface-expressed chitinase among the Paenibacillus sp. strain FPU-7-secreted chitinases. An N-terminally truncated ChiW protein, primarily comprised of the two catalytic domains of the full-length protein, was successfully overexpressed in Escherichia coli, purified as a functional recombinant protein with a molecular mass of approximately 98 kDa and crystallized. Preliminary X-ray analysis showed that the crystal diffracted to 1.93 Šresolution and belonged to the orthorhombic space group P212121, with unit-cell parameters a = 112.1, b = 128.2, c = 162.6 Å, suggesting the presence of two molecules in an asymmetric unit.

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.

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 ß-propeller module linked to a ß-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 ß-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 ß-sandwich module, besides the ß-propeller module, are important for inulin-degrading activity of LevH1.

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 gehC(WM)) was cloned and characterized. The gehC(WM )gene encoded a protein (GehC(WM)), 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, GehC(WM) 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 GehC(WM). GehC(WM) was predicted to be a prepro-protein. In vitro processing and protein sequencing suggested that pro-GehC(WM) is possibly processed by extracellular glutamyl endopeptidase, PROM. Inductively coupled plasma-atomic emission spectrometer analysis showed that purified his-tagged mature GehC(WM) possessed zinc ion. A gehC(WM) knockout mutant was constructed by insertion of an erythromycin resistance gene into the gehC(WM). Zymogram and immunoblot analyses of the gehC(WM )mutant indicated that GehC(WM) was a major extracellular lipase of S. warneri M.

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.

Discoidin domain of chitosanase is required for binding to the fungal cell wall.

Previously, we reported properties of a glycosylase belonging to GH-8 glycosyl hydrolase (GH) and having both chitosanase and glucanase activities. This enzyme (D2), whose molecular mass (86 kDa) was the largest among the GH-8 group, has its catalytic domain at the N-terminal region, and discoidin domain (DD) at the C-terminal region. Although various chitosanases, chitinases and glucanases have been known, DD is unique to the D2 enzyme. Glucanase and chitinase, but not chitosanase, are known to have functional domain such as carbohydrate-binding module, besides catalytic domain. Accordingly, function of the DD of D2 chitosanase was analyzed, using zygomycete cell wall containing chitosan, glucan and chitin as the basic constituents. The DD specifically and tightly bound to chitosan, but did not participate in affinity for glucan and chitin. Deletion of the DD caused marked reduction in absorbability to cell wall and in hydrolytic activity toward chitosan and glucan. These results suggest that the DD is concerned in binding of the enzyme to cell wall and in effective digestion of the insoluble substrate, through hydrolysis of not only chitosan but also coexisting glucan. Thus, this is the first example of chitosan-binding domain among various carbohydrate-binding modules reported thus far.

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.

Effect of synthetic hydroxy isothiocyanates on a bacterial virus and DNA.

The Effect of hydroxy isothiocyanates on a bacterial virus and M13 DNA was examined. Hydroxy-substituted phenyl and phenyl alkyl isothiocyanates, especially 2-(3,4-dihydroxyphenyl)ethyl isothiocyanate(IT-Dop) synthesized from dopamine, showed antiviral activity on psiK. In transfection experiments with M13 mp DNA species, IT-Dop inhibited the single-stranded (SS) molecule more effectively than the double stranded replicative form (RF) DNA. These effects were dependent on reaction time, and on IT-Dop concentration. An additional experiment indicated that treatment with IT-Dop suppressed annealing (reassociation) of denatured DNA. These results indicate that IT-Dop reacts mildly with virus and SS DNA.