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Genomic analysis and initial characterization of the chitinolytic system of Microbulbifer degradans strain 2-40.

ABSTRACT: The marine bacterium Microbulbifer degradans strain 2-40 produces at least 10 enzyme systems for degrading insoluble complex polysaccharides (ICP). The draft sequence of the 2-40 genome allowed a genome-wide analysis of the chitinolytic system of strain 2-40. The chitinolytic system includes three secreted chitin depolymerases (ChiA, ChiB, and ChiC), a secreted chitin-binding protein (CbpA), periplasmic chitooligosaccharide-modifying enzymes, putative sugar transporters, and a cluster of genes encoding cytoplasmic proteins involved in N-acetyl-D-glucosamine (GlcNAc) metabolism. Each chitin depolymerase was detected in culture supernatants of chitin-grown strain 2-40 and was active against chitin and glycol chitin. The chitin depolymerases also had a specific pattern of activity toward the chitin analogs 4-methylumbelliferyl-beta-D-N,N'-diacetylchitobioside (MUF-diNAG) and 4-methylumbelliferyl-beta-D-N,N',N"-triacetylchitotrioside (MUF-triNAG). The depolymerases were modular in nature and contained glycosyl hydrolase family 18 domains, chitin-binding domains, and polycystic kidney disease domains. ChiA and ChiB each possessed polyserine linkers of up to 32 consecutive serine residues. In addition, ChiB and CbpA contained glutamic acid-rich domains. At 1,271 amino acids, ChiB is the largest bacterial chitinase reported to date. A chitodextrinase (CdxA) with activity against chitooligosaccharides (degree of polymerization of 5 to 7) was identified. The activities of two apparent periplasmic (HexA and HexB) N-acetyl-beta-D-glucosaminidases and one cytoplasmic (HexC) N-acetyl-beta-D-glucosaminidase were demonstrated. Genes involved in GlcNAc metabolism, similar to those of the Escherichia coli K-12 NAG utilization operon, were identified. NagA from strain 2-40, a GlcNAc deacetylase, was shown to complement a nagA mutation in E. coli K-12. Except for the GlcNAc utilization cluster, genes for all other components of the chitinolytic system were dispersed throughout the genome. Further examination of this system may provide additional insight into the mechanisms by which marine bacteria degrade chitin and provide a basis for future research on the ICP-degrading systems of strain 2-40.

SUBMITTER: Howard MB

PROVIDER: S-EPMC155392 | biostudies-literature | 2003 Jun

REPOSITORIES: biostudies-literature

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Genomic analysis and initial characterization of the chitinolytic system of Microbulbifer degradans strain 2-40.

Howard Michael B MB Ekborg Nathan A NA Taylor Larry E LE Weiner Ronald M RM Hutcheson Steven W SW

Journal of bacteriology 20030601 11

The marine bacterium Microbulbifer degradans strain 2-40 produces at least 10 enzyme systems for degrading insoluble complex polysaccharides (ICP). The draft sequence of the 2-40 genome allowed a genome-wide analysis of the chitinolytic system of strain 2-40. The chitinolytic system includes three secreted chitin depolymerases (ChiA, ChiB, and ChiC), a secreted chitin-binding protein (CbpA), periplasmic chitooligosaccharide-modifying enzymes, putative sugar transporters, and a cluster of genes e ...[more]

PMID: 12754233

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Project description:The Gram-negative rod-shaped bacterium Serratia marcescens is an opportunistic pathogen of many organisms, including insects. We report the identification and optimal in vitro chitinase production conditions of a novel chitinolytic S. marcescens strain TC-1 isolated from a naturally infected white grub (Anomala corpulenta) collected from a peanut field at Nanyang city, Henan province, China. Strain identification was conducted by morphological, physiological, biochemical and molecular analyses. The amplified 16S rRNA gene of TC-1 showed a similarity greater than 99% with multiple strains of S. marcescens. Based on Neighbor-joining phylogenetic tree analysis of bacterial 16S rRNA gene sequences, TC-1 formed a clade with S. marcescens, clearly separated from other Serratia spp. The strain TC-1 showed larvicidal activities against five insect species (A. corpulenta, Plutella xylostella, Spodoptera exigua, Helicoverpa armigera, Bombyx mori) and the nematode Caenorhabditis elegans, but not against S. litura. The operating parameters of chitinase production by TC-1 were optimized by response surface methodology using a three-factor, three-level Box-Behnken experimental design. The effects of three independent variables i.e. colloidal chitin concentration (7-13 g l-1), incubation time (24-72 h) and incubation temperature (24-32 °C) on chitinase production by TC-1 were investigated. A regression model was proposed to correlate the independent variables for an optimal chitinase activity predicted as 20.946 U ml-1, using a combination of colloidal chitin concentration, incubation time and incubation temperature of 9.06 g l-1, 63.83 h and 28.12 °C, respectively. The latter agreed well with a mean chitinase activity of 20.761 ± 0.102 U ml-1 measured in the culture supernatants of TC-1 grown under similar conditions with a colloidal chitin concentration, incubation time and incubation temperature of 9 g l-1, 64 h and 28 °C, respectively. Our study revealed the S. marcescens strain TC-1 with potential as a biocontrol agent of insect pests and nematodes and demonstrated the proposed regression model's potential to guide chitinase production by this strain.

Dataset Information

Genomic analysis and initial characterization of the chitinolytic system of Microbulbifer degradans strain 2-40.

Publications

Genomic analysis and initial characterization of the chitinolytic system of Microbulbifer degradans strain 2-40.

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