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Constitutive high level expression of an endoxylanase gene from the newly isolated Bacillus subtilis AQ1 in Escherichia coli.

ABSTRACT: A xylanolytic bacterium was isolated from the sediment of an aquarium. Based on the 16S rDNA sequence as well as morphological and biochemical properties the isolate was identified and denoted as Bacillus subtilis (B. subtilis) AQ1 strain. An endoxylanase-encoding gene along with its indigenous promoter was PCR amplified and after cloning expressed in E. coli. In E. coli the recombinant enzyme was found in the extracellular, in the cytoplasmic, and in the periplasmic fraction. The specific activity of the extracellular AQ1 recombinant endoxylanase after 24-hour fermentation was very high, namely, 2173.6 ± 51.4 and 2745.3 ± 11?U/mg in LB and LB-xylan medium, respectively. This activity was clearly exceeding that of the native B. subtilis AQ1 endoxylanase and that of 95% homologous recombinant one from B. subtilis DB104. The result shows that the original AQ1 endoxylanase promoter and the signal peptide gave a very high constitutive extracellular expression in E. coli and hence made the production in E. coli feasible.

SUBMITTER: Helianti I

PROVIDER: S-EPMC2947863 | biostudies-literature | 2010

REPOSITORIES: biostudies-literature

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Constitutive high level expression of an endoxylanase gene from the newly isolated Bacillus subtilis AQ1 in Escherichia coli.

Helianti Is I Nurhayati Niknik N Ulfah Maria M Wahyuntari Budiasih B Setyahadi Siswa S

Journal of biomedicine & biotechnology 20100921

A xylanolytic bacterium was isolated from the sediment of an aquarium. Based on the 16S rDNA sequence as well as morphological and biochemical properties the isolate was identified and denoted as Bacillus subtilis (B. subtilis) AQ1 strain. An endoxylanase-encoding gene along with its indigenous promoter was PCR amplified and after cloning expressed in E. coli. In E. coli the recombinant enzyme was found in the extracellular, in the cytoplasmic, and in the periplasmic fraction. The specific activ ...[more]

PMID: 20936136

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Project description:Bacillus subtilis is recognized as a safe and reliable human and animal probiotic and is associated with bioactivities such as production of vitamin and immune stimulation. Additionally, it has great potential to be used as an alternative to antimicrobial drugs, which is significant in the context of antibiotic abuse in food animal production. In this study, we isolated one strain of B. subtilis, named WS-1, from apparently healthy pigs growing with sick cohorts on one Escherichia coli endemic commercial pig farm in Guangdong, China. WS-1 can strongly inhibit the growth of pathogenic E. coli in vitro. The B. subtilis strain WS-1 showed typical Bacillus characteristics by endospore staining, biochemical test, enzyme activity analysis, and 16S rRNA sequence analysis. Genomic analysis showed that the B. subtilis strain WS-1 shares 100% genomic synteny with B. subtilis with a size of 4,088,167 bp. Importantly, inoculation of newborn piglets with 1.5 × 1010 CFU of B. subtilis strain WS-1 by oral feeding was able to clearly inhibit diarrhea (p < 0.05) and death (p < 0.05) caused by pathogenic E. coli in piglets. Furthermore, histopathological results showed that the WS-1 strain could protect small intestine from lesions caused by E. coli infection. Collectively, these findings suggest that the probiotic B. subtilis strain WS-1 acts as a potential biocontrol agent protecting pigs from pathogenic E. coli infection. Importance: In this work, one B. subtilis strain (WS-1) was successfully isolated from apparently healthy pigs growing with sick cohorts on one E. coli endemic commercial pig farm in Guangdong, China. The B. subtilis strain WS-1 was identified to inhibit the growth of pathogenic E. coli both in vitro and in vivo, indicating its potential application in protecting newborn piglets from diarrhea caused by E. coli infections. The isolation and characterization will help better understand this bacterium, and the strain WS-1 can be further explored as an alternative to antimicrobial drugs to protect human and animal health.

Project description:RasP is a predicted intramembrane metalloprotease of Bacillus subtilis that has been proposed to cleave the stress response anti-sigma factors RsiW and RsiV, the cell division protein FtsL, and remnant signal peptides within their transmembrane segments. To provide evidence for direct effects of RasP on putative substrates, we developed a heterologous coexpression system. Since expression of catalytically inactive RasP E21A inhibited expression of other membrane proteins in Escherichia coli, we added extra transmembrane segments to RasP E21A, which allowed accumulation of most other membrane proteins. A corresponding active version of RasP appeared to promiscuously cleave coexpressed membrane proteins, except those with a large periplasmic domain. However, stable cleavage products were not observed, even in clpP mutant E. coli Fusions of transmembrane segment-containing parts of FtsL and RsiW to E. coli maltose-binding protein (MBP) also resulted in proteins that appeared to be RasP substrates upon coexpression in E. coli, including FtsL with a full-length C-terminal domain (suggesting that prior cleavage by a site 1 protease is unnecessary) and RsiW designed to mimic the PrsW site 1 cleavage product (suggesting that further trimming by extracytoplasmic protease is unnecessary). Purified RasP cleaved His6-MBP-RsiW(73-118) in vitro within the RsiW transmembrane segment based on mass spectrometry analysis, demonstrating that RasP is an intramembrane protease. Surprisingly, purified RasP failed to cleave His6-MBP-FtsL(23-117). We propose that the lack of α-helix-breaking residues in the FtsL transmembrane segment creates a requirement for the membrane environment and/or an additional protein(s) in order for RasP to cleave FtsL.IMPORTANCE Intramembrane proteases govern important signaling pathways in nearly all organisms. In bacteria, they function in stress responses, cell division, pathogenesis, and other processes. Their membrane-associated substrates are typically inferred from genetic studies in the native bacterium. Evidence for direct effects has come sometimes from coexpression of the enzyme and potential substrate in a heterologous host and rarely from biochemical reconstitution of cleavage in vitro We applied these two approaches to the B. subtilis enzyme RasP and its proposed substrates RsiW and FtsL. We discovered potential pitfalls and solutions in heterologous coexpression experiments in E. coli, providing evidence that both substrates are cleaved by RasP in vivo but, surprisingly, that only RsiW was cleaved in vitro, suggesting that FtsL has an additional requirement.

Dataset Information

Constitutive high level expression of an endoxylanase gene from the newly isolated Bacillus subtilis AQ1 in Escherichia coli.

Publications

Constitutive high level expression of an endoxylanase gene from the newly isolated Bacillus subtilis AQ1 in Escherichia coli.

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