Kiyotaka Miyashita

Family 19 chitinases of Streptomyces species: characterization and distribution.

Chitinase C from Streptomyces griseus HUT6037, described in 1997, is the first family 19 chitinase found in an organism other than higher plants. In this study, some properties of chitinase C were compared with those of family 18 bacterial chitinases, and the distribution of family 19 chitinases in Streptomyces species was investigated. The specific hydrolysing activity of chitinase C against soluble and insoluble chitinous substrates was markedly higher than those of bacterial family 18 chitinases. Chitinase C exhibited marked antifungal activity, whereas the other bacterial chitinases examined had no antifungal activity. Chitinase C was insensitive to allosamidin, whereas the family 18 bacterial chitinases were sensitive. Taking advantage of this insensitivity to allosamidin, a search was made for family 19 chitinases in various Streptomyces species. Chitinases insensitive to allosamidin were detected in the culture supernatants of all tested Streptomyces species. Southern hybridization analysis using a labelled DNA fragment corresponding to the catalytic domain of chitinase C strongly suggested that these species have genes similar to the chiC gene of S. griseus HUT6037. DNA fragments corresponding to the major part of the catalytic domains were amplified by PCR. The amplified fragments encoded amino acid sequences very similar to that of the corresponding region of chitinase C. Therefore, it was concluded that Streptomyces species generally possess family 19 chitinases which are very similar to chitinase C. Comparison of their amino acid sequences with those of plant family 19 chitinases revealed that Streptomyces family 19 chitinases are class IV type in terms of the presence and positions of deletions of amino acid sequences which are characteristic of plant class IV chitinases.

Comparison of Enzymatic and Antifungal Properties between Family 18 and 19 Chitinases from S. coelicolor A3(2)

Streptomyces coelicolor A3(2) has 13 chitinase genes encoding 11 family 18 and two family 19 chitinases. To compare enzymatic properties of family 19 chitinase and family 18 chitinases produced by the same organism, the four chitinases (Chi18bA, Chi18aC, Chi18aD, and Chi19F), whose genes are expressed at high levels in the presence of chitin, were produced in Eschericha coli and purified. The effect of pH on the hydrolytic activity was very different not only among the four chitinases but also among the substrates. The hydrolytic activity of Chi19F, family 19 chitinase, against soluble substrates was remarkably high as compared with three family 18 chitinases, but was the lowest against crystalline substrates among the four chitinases. On the contrary, Chi18aC, a family 18-subfamily A chitinase, showed highest activity against crystalline substrates. Only Chi19F exhibited significant antifungal activity. Based on these observations, the roles of family 19 chitinases are discussed.

Molecular cloning and characterization of chitinase genes from Streptomyces lividans 66

Summary: In order to study the genetic control of chitinolytic activity in Streptomyces, chitinase genes were cloned from S. lividans TK64 into the multicopy plasmid pIJ702 and their expression monitored in their natural host by measuring increases in chitinase productivity. Four independent clones were obtained, and the plasmids named pEMJ1, pEMJ5, pEMJ7 and pEMJ8. Restriction endonuclease digestion showed that although two of the plasmids (pEMJ7 and pEMJ8) shared a common DNA fragment, there were no substantial similarities between the inserts of plasmids pEMJ1, pEMJ5 and pEMJ7. This was confirmed by DNA-DNA hybridization studies. Four chitinases (A, B, C, and D) were identified, with molecular masses of 36, 46, 65, and 41 kDa, respectively. Production of chitinases A and B was specified by the plasmids pEMJ1 and pEMJ5, respectively. Genes for the other two chitinases (C and D) were carried by plasmid pEMJ7. Although significant differences were observed between chitinases A, B, and C in terms of optimum pH for activity and mode of digestion of substrates, chitinases C and D were very similar in these respects. Cloned genes were also expressed in S. coelicolor M130 and in Escherichia coli.

Multiple domain structure in a chitinase gene (chiC) of Streptomyces lividans

One of the chitinases of Streptomyces lividans, chitinase C, was encoded by a 2 kb smaI-XhoI restriction fragment contained in the recombinant plasmid pEMJ7. DNA sequence analysis of this region revealed the presence of two open reading frames (ORF1 and ORF2) which had opposite orientations. Northern analysis showed that only the mRNA complementary to ORF1 was transcribed, and that this transcription was induced by chitin and repressed by glucose. ORF1 showed a codon distribution typical of Streptomyces. A sequence identical to that of the N-terminus of mature secreted chitinase C was found from amino acid residue 31 in the deduced amino acid sequence of ORF1 (619 amino acids), implying that ORF1 encodes a pre-protein of chitinase C. The pre-protein of chitinase C consisted of four discrete domains. The 30 amino acid N-terminal sequence, domain 1, was characteristic of a signal peptide. Domain 2 consisted of 105 N-terminal amino acids of mature chitinase C, and was similar to cellulose-binding domains of several cellulases. Domain 3 (94 amino acids) showed homology with type III homology units of fibronectin. Domain 4, a C-terminal 390 amino acid sequence, is probably the catalytic domain of the chitinase, since it exhibited identity with several other chitinolytic enzymes.

Distribution and Phylogenetic Analysis of Family 19 Chitinases in Actinobacteria

ABSTRACT In organisms other than higher plants, family 19 chitinase was first discovered in Streptomyces griseus HUT6037, and later, the general occurrence of this enzyme in Streptomyces species was demonstrated. In the present study, the distribution of family 19 chitinases in the class Actinobacteria and the phylogenetic relationship of Actinobacteria family 19 chitinases with family 19 chitinases of other organisms were investigated. Forty-nine strains were chosen to cover almost all the suborders of the class Actinobacteria, and chitinase production was examined. Of the 49 strains, 22 formed cleared zones on agar plates containing colloidal chitin and thus appeared to produce chitinases. These 22 chitinase-positive strains were subjected to Southern hybridization analysis by using a labeled DNA fragment corresponding to the catalytic domain of ChiC, and the presence of genes similar to chiC of S. griseus HUT6037 in at least 13 strains was suggested by the results. PCR amplification and sequencing of the DNA fragments corresponding to the major part of the catalytic domains of the family 19 chitinase genes confirmed the presence of family 19 chitinase genes in these 13 strains. The strains possessing family 19 chitinase genes belong to 6 of the 10 suborders in the order Actinomycetales, which account for the greatest part of the Actinobacteria. Phylogenetic analysis suggested that there is a close evolutionary relationship between family 19 chitinases found in Actinobacteria and plant class IV chitinases. The general occurrence of family 19 chitinase genes in Streptomycineae and the high sequence similarity among the genes found in Actinobacteria suggest that the family 19 chitinase gene was first acquired by an ancestor of the Streptomycineae and spread among the Actinobacteria through horizontal gene transfer.

Transcriptional co-regulation of five chitinase genes scattered on the Streptomyces coelicolor A3(2) chromosome

Streptomyces coelicolor A3(2) strain M145 has eight chitinase genes scattered on the chromosome: six genes for family 18 (chiA, B, C, D, E and H) and two for family 19 (chiF and G). In this study, the expression and regulation of these genes were investigated. The transcription of five of the genes (chiA, B, C, D and F) was induced in the presence of colloidal chitin while that of the other three genes (chiE, G and H) was not. The transcripts of the five induced chi genes increased and reached their maximum at 4 h after the addition of colloidal chitin, all showing the same temporal patterns. The induced levels of the transcripts of chiB were significantly lower than those of the other four genes. Dynamic analysis of the transcripts of the chi genes indicated that chiA and chiC were induced more strongly than chiD and chiF. Addition of chitobiose also induced transcription of the chi genes, but significantly earlier than did colloidal chitin. When cells were cultured in the presence of colloidal chitin, an exponential increase of chitobiose concentration in the culture supernatant was observed prior to the induced transcription of the chi genes. This result, together with the immediate effect of chitobiose on the induction, suggests that chitobiose produced from colloidal chitin is involved in the induction of transcription of the chi genes. The transcription of the five chi genes was repressed by glucose. This repression was apparently mediated by the glucose kinase gene glkA.

Nucleotide sequence and expression of a gene (chiB) for a chitinase from Streptomyces lividans

Abstract Streptomyces lividans 66 has three distinct chitinase genes. The nucleotide sequence of one gene for chitinase ( chiB ) from S. lividans was determined, and the structure and expression of the gene were analyzed. The structural gene consisted of 1,830 bp encoding 610 amino acid residues. Comparison of the deduced amino acid sequence of chitinase B with those of chitinases of other bacteria revealed a domain structure from N-terminal to C-terminal of the following order: signal peptide, substrate-binding domain, type III repeating unit, and catalytic domain. When the three chitinase genes from S. lividans ( chiA, chiB, chiC ) were compared with one another, the overall similarities between the nucleotide sequences of chiA and chiB and between the amino acid sequences deduced from them were found to be 49% and 59%, respectively, whereas chiC showed no relatedness to either chiA or chiB . It is, therefore, suggested that chiA and chiB diverged relatively recently. chiB of S. lividans was found to be more than 90% similar to chi01 of Streptomyces olivaceoviridis in both nucleotide and amino acid sequences, and chiC of S. lividans was found to be almost identical to chtA of S. plicatus . Southern hybridization studies conducted using chiA, chiB , and chiC of S. lividans as probes against genomic DNA from several Streptomyces strains revealed that these genes of S. lividans are distributed and highly conserved among the genus Streptomyces . Although a pair of direct repeat sequences similar to those found in the promoter region of other chitinase genes of Streptomyces are present in chiB , the motif is least conserved in chiB . The level of expression of chiB was shown to be lower than that of either chiA or chiC . It is assumed that chitinases A and C play a major role in chitin degradation in S. lividans .

The dasABC Gene Cluster, Adjacent to dasR, Encodes a Novel ABC Transporter for the Uptake of N,N′-Diacetylchitobiose in Streptomyces coelicolor A3(2)

ABSTRACT N,N′-Diacetylchitobiose [(GlcNAc)2] induces the transcription of chitinase (chi) genes in Streptomyces coelicolor A3(2). Physiological studies showed that (GlcNAc)2 addition triggered chi expression and increased the rate of (GlcNAc)2 concentration decline in culture supernatants of mycelia already cultivated with (GlcNAc)2, suggesting that (GlcNAc)2 induced the synthesis of its own uptake system. Four open reading frames (SCO0531, SCO0914, SCO2946, and SCO5232) encoding putative sugar-binding proteins of ABC transporters were found in the genome by probing the 12-bp repeat sequence required for regulation of chi transcription. SCO5232, named dasA, showed transcriptional induction by (GlcNAc)2 and N,N′,N‴-triacetylchitotriose [(GlcNAc)3]. Surface plasmon resonance analysis showed that recombinant DasA protein exhibited the highest affinity for (GlcNAc)2 (equilibrium dissociation constant [KD] = 3.22 × 10−8). In the dasA-null mutant, the rate of decline of the (GlcNAc)2 concentration in the culture supernatant was about 25% of that in strain M145. The in vitro and in vivo data clearly demonstrated that dasA is involved in (GlcNAc)2 uptake. Upstream and downstream of dasA, the transcriptional regulator gene (dasR) and two putative integral membrane protein genes (dasBC) are located in the opposite and same orientations, respectively. The expression of dasR and dasB, which seemed independent of dasA transcription, was also induced by (GlcNAc)2 and (GlcNAc)3.

Chitin-Induced Gene Expression in Secondary Metabolic Pathways of Streptomyces coelicolor A3(2) Grown in Soil

ABSTRACT Microarray analyses revealed that the expression of genes for secondary metabolism together with that of primary metabolic genes was induced by chitin in autoclaved soil cultures of Streptomyces coelicolor A3(2). The data also indicated that DasR was involved in the regulation of gene expression for chitin catabolism, secondary metabolism, and stress responses.

Expression of 2-halobenzoate dioxygenase genes (cbdSABC) involved in the degradation of benzoate and 2-halobenzoate in Burkholderia sp. TH2

Burkholderia sp. TH2, isolated from soil, utilizes 2-chlorobenzoate (2CB) and benzoate (BA) as its sole source of carbon and energy. The genes for 2-halobenzoate dioxygenase (cbdABC) from Burkholderia sp. TH2 were cloned and sequenced. The predicted amino acid sequences of all the gene products are highly similar to the cbd gene products of Pseudomonas sp. 2CBS. Disruption of the promoter region of cbdA resulted in loss of growth on 2CB and BA, indicating that these genes are involved in the growth of TH2 on these substrates. Expression of the cbd genes was analyzed by transcriptional fusion assay. The cbdS gene, a possible araC/xylS-type transcriptional regulatory gene, was shown to positively regulate the expression of cbdA. In addition, the effectors of CbdS were shown to be 2CB, 2-bromobenzoate, o-toluate (2-methylbenzoate), 2-iodobenzoate, and BA. Primer extension analysis showed that the cbdA mRNA started at two positions, 14 and 15 nucleotides upstream from the cbdA start codon, ATG. A pair of direct repeats, identical to that of the Pm promoter of the TOL plasmid, was found upstream of -35 hexamer of the cbdA promoter.