Purification and Characterization of a Major Extracellular Chitinase from a Biocontrol Bacterium, Paenibacillus elgii HOA73.

Chitinase-producing Paenibacillus elgii strain HOA73 has been used to control plant diseases. However, the antimicrobial activity of its extracellular chitinase has not been fully elucidated. The major extracellular chitinase gene (PeChi68) from strain HOA73 was cloned and expressed in Escherichia coli in this study. This gene had an open reading frame of 2,028 bp, encoding a protein of 675 amino acid residues containing a secretion signal peptide, a chitin-binding domain, two fibronectin type III domains, and a catalytic hydrolase domain. The chitinase (PeChi68) purified from recombinant E. coli exhibited a molecular mass of approximately 68 kDa on SDS-PAGE. Biochemical analysis indicated that optimum temperature for the actitvity of purified chitinase was 50ºC. However, it was inactivated with time when it was incubated at 40ºC and 50ºC. Its optimum activity was found at pH 7, although its activity was stable when incubated between pH 3 and pH 11. Heavy metals inhibited this chitinase. This purified chitinase completely inhibited spore germination of two Cladosporium isolates and partially inhibited germination of Botrytis cinerea spores. However, it had no effect on the spores of a Colletotricum isolate. These results indicate that the extracellular chitinase produced by P. elgii HOA73 might have function in limiting spore germination of certain fungal pathogens.

Quorum sensing is a key regulator for the antifungal and biocontrol activity of chitinase-producing Chromobacterium sp. C61.

Chromobacterium sp. strain C61 has strong biocontrol activity; however, the genetic and biochemical determinants of its plant disease suppression activity are not well understood. Here, we report the identification and characterization of two new determinants of its biocontrol activity. Transposon mutagenesis was used to identify mutants that were deficient in fungal suppression. One of these mutants had an insertion in a homologue of depD, a structural gene in the dep operon, that encodes a protein involved in non-ribosomal peptide synthesis. In the second mutant, the insertion was in a homologue of the luxI gene, which encodes a homoserine lactone synthase. The luxI- and depD- mutants had no antifungal activity in vitro and a dramatically reduced capacity to suppress various plant diseases in planta. Antifungal production and biocontrol were restored by complementation of the luxI- mutant. Other phenotypes associated with effective biological control, including motility and lytic enzyme secretion, were also affected by the luxI mutation. Biochemical analysis of ethyl acetate extracts of culture filtrates of the mutant and wild-type strains showed that a key antifungal compound, chromobactomycin, was produced by wild-type C61 and the complemented luxI- mutant, but not by the luxI- or depD- mutant. These data suggest that multiple biocontrol-related phenotypes are regulated by homoserine lactones in C61. Thus, quorum sensing plays an essential role in the biological control potential of diverse bacterial lineages.

Draft genome sequence of the biocontrol bacterium Chromobacterium sp. strain C-61.

Chromobacterium sp. strain C-61 is a plant-associated bacterium with proven capacities to suppress plant diseases. Here, we report the draft genome sequence and automatic annotation of strain C-61. A comparison of this sequence to the sequenced genome of Chromobacterium violaceum ATCC 12472 indicates the novelty of C-61 and a subset of gene functions that may be related to its biocontrol activities.

Inactivation of pqq genes of Enterobacter intermedium 60-2G reduces antifungal activity and induction of systemic resistance.

Enterobacter intermedium 60-2G, a phosphate solubilizing bacterium, has the ability to induce systemic resistance in plants against soft rot pathogen Erwinia carotovora. Glucose dehydrogenase, an enzyme that utilizes pyrroloquinoline quinone (PQQ) as a cofactor, is required for the synthesis of gluconic acid by E. intermedium 60-2G. Here, we report that the pqqA and pqqB genes are required for phosphate solubilization and induced systemic resistance against a soft rot pathogen in tobacco. Mutations in either the pqqA or pqqB gene abolished the production of 2-ketogluconic acid and eliminated the ability of E. intermedium to solubilize hydroxyapatite. Addition of gluconic acid to the growth media restored the ability of the pqqA mutant to produce 2-ketogluconic acid. Interestingly, both pqqA and pqqB mutants of E. intermedium lost their ability to inhibit the growth of the rice pathogen Magnaporthe grisea KI-409. Additionally, induced systemic resistance against the soft rot pathogen was attenuated in the pqq mutants. These functions were restored by complementation with the wild-type pqq gene cluster. Our findings suggest that PQQ plays an important function in beneficial traits including phosphate solubilization, antifungal activity, and induced systemic resistance of E. intermedium, possibly by acting as a cofactor for several enzymes including glucose dehydrogenase.

Cloning and high-level production of a chitinase from Chromobacterium sp. and the role of conserved or nonconserved residues on its catalytic activity.

A gene encoding an alkaline (pI of 8.67) chitinase was cloned and sequenced from Chromobacterium sp. strain C-61. The gene was composed of 1,611 nucleotides and encoded a signal sequence of 26 N-terminal amino acids and a mature protein of 510 amino acids. Two chitinases of 54 and 52 kDa from both recombinant Escherichia coli and C-61 were detected on SDS-PAGE. Maximum chitinase activity was obtained in the culture supernatant of recombinant E. coli when cultivated in TB medium for 6 days at 37 degrees C and was about fourfold higher than that from C-61. Chi54 from the culture supernatants could be purified by a single step based on isoelectric point. The purified Chi54 had about twofold higher binding affinity to chitin than to cellulose. The chi54 encoded a protein that included a type 3 chitin-binding domain belonging to group A and a family 18 catalytic domain belonging to subfamily A. In the catalytic domain, mutation of perfectly conserved residues and highly conserved residues resulted in loss of nearly all activity, while mutation of nonconserved residues resulted in enzymes that retained activity. In this process, a mutant (T218S) was obtained that had about 133% of the activity of the wild type, based on comparison of K (cat) values.

Influence of the transposition of the thermostabilizing domain of Clostridium thermocellum xylanase (XynX) on xylan binding and thermostabilization.

A xylanase gene, xynX, of Clostridium thermocellum had one thermostabilizing domain (TSD) between the signal peptide sequence and the catalytic domain (CD). The TSD of a truncated xylanase gene, xynX'(TSD-CD), was transpositioned from the N terminus to the C terminus of the CD by overlapping PCRs, and a modified product, xynX'(CD-TSD), was constructed. XynX'(TSD-CD) had a higher optimum temperature (70 degrees C versus 65 degrees C) and was more thermostable (residual activity of 68% versus 46% after a 20-min preincubation at 70 degrees C) than the one without the TSD, XynX'(CD). However, the domain-transpositioned enzyme, XynX'(CD-TSD), showed a lower optimum temperature (30 degrees C) and thermostability (20%) than XynX'(CD). Both XynX'(TSD-CD) and XynX'(CD-TSD) showed significantly higher binding capacity toward xylan than XynX'(CD), and the domain transposition did not cause any change in the binding ability. XynX'(TSD-CD) and XynX'(CD-TSD) also showed considerable binding to lichenan but not to carboxymethyl cellulose and laminarin. XynX'(TSD-CD) and XynX'(CD-TSD) had higher activities for insoluble xylan than XynX'(CD), while XynX'(CD) was more active against soluble xylan than XynX'(TSD-CD) and XynX'(CD-TSD). These results indicate that the TSD of XynX has dual functions, xylan binding and thermostabilization, and the domain should also be classified as a xylan-binding domain (XBD). The binding capacity of the XBD was not affected by domain transpositioning within the gene.