Evaluation of whole cells of Bacillus subtilis as substrate for measurement of autolysin activity

Iyer, Jyoti L.; Shetty, Prashanth; Pai, J. S.

doi:10.1016/j.procbio.2004.06.022

Cited by 6 publications

(4 citation statements)

References 12 publications

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“…Lysis of B. subtilis strains used for industrial purposes significantly decreases product yield [ 7 , 37 ]. We addressed this issue here by systematically deleting genes involved in autolysis from the wild-type strain B. subtilis 168 and assessed the effects of single and multiple deletions on cell lysis and biomass production.…”

Section: Discussionmentioning

confidence: 99%

Deleting multiple lytic genes enhances biomass yield and production of recombinant proteins by Bacillus subtilis

et al. 2014

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BackgroundBacillus subtilis is widely used in agriculture and industrial biotechnology; however, cell autolysis significantly decreases its yield in liquid cultures. Numerous factors mediate the lysis of B. subtilis, such as cannibalism factors, prophages, and peptidoglycan (PG) hydrolases. The aim of this work was to use molecular genetic techniques to develop a new strategy to prevent cell lysis and enhance biomass as well as the production of recombinant proteins.ResultsFive genes or genetic elements representing three different functional categories were studied as follows: lytC encoding PG hydrolases, the prophage genes xpf and yqxG-yqxH-cwlA (yGlA), and skfA and sdpC that encode cannibalism factors. Cell lysis was reduced and biomass was enhanced by deleting individually skfA, sdpC, xpf, and lytC. We constructed the multiple deletion mutant LM2531 (skfA sdpC lytC xpf) and found that after 4 h of culture, its biomass yield was significantly increased compared with that of prototypical B. subtilis 168 (wild-type) strain and that 15% and 92% of the cells were lysed in cultures of LM2531 and wild-type, respectively. Moreover, two expression vectors were constructed for producing recombinant proteins (β-galactosidase and nattokinase) under the control of the P43 promoter. Cultures of LM2531 and wild-type transformants produced 13741 U/ml and 7991 U/ml of intracellular β-galactosidase, respectively (1.72-fold increase). Further, the level of secreted nattokinase produced by strain LM2531 increased by 2.6-fold compared with wild-type (5226 IU/ml vs. 2028 IU/ml, respectively).ConclusionsOur novel, systematic multigene deletion approach designed to inhibit cell lysis significantly increased the biomass yield and the production of recombinant proteins by B. subtilis. These findings show promise for guiding efforts to manipulate the genomes of other B. subtilis strains that are used for industrial purposes.Electronic supplementary materialThe online version of this article (doi:10.1186/s12934-014-0129-9) contains supplementary material, which is available to authorized users.

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Section: Discussionmentioning

confidence: 99%

Deleting multiple lytic genes enhances biomass yield and production of recombinant proteins by Bacillus subtilis

et al. 2014

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“…Activity was tested both before and after extraction with 5% trichloroacetic acid (TCA), which removes teichoic acids, and no differences were noted between the effects of the different preparations. For peptidoglycan hydrolysis assays, crude cell wall preparations of 1-liter B. cereus cultures (grown in halfstrength TSB) were extracted with 10 M LiCl according to a method described previously (14) and were concentrated to a final volume of 10 ml in sterile deionized water.…”

Section: Methodsmentioning

confidence: 99%

Peptidoglycan from Bacillus cereus Mediates Commensalism with Rhizosphere Bacteria from the Cytophaga-Flavobacterium Group

Peterson

Dunn

Klimowicz

et al. 2006

Appl Environ Microbiol

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Previous research in our laboratory revealed that the introduction of Bacillus cereus UW85 can increase the populations of bacteria from the Cytophaga-Flavobacterium (CF) group of the Bacteroidetes phylum in the soybean rhizosphere, suggesting that these rhizosphere microorganisms have a beneficial relationship (G. S. Gilbert, J. L. Parke, M. K. Clayton, and J. Handelsman, Ecology 74:840-854, 1993). In the present study, we determined the frequency at which CF bacteria coisolated with B. cereus strains from the soybean rhizosphere and the mechanism by which B. cereus stimulates the growth of CF rhizosphere strains in root exudate media. In three consecutive years of sampling, CF strains predominated among coisolates obtained with B. cereus isolates from field-grown soybean roots. In root exudate media, the presence of B. cereus was required for CF coisolate strains to reach high population density. However, rhizosphere isolates from the phylum Proteobacteria grew equally well in the presence and absence of B. cereus, and the presence of CF coisolates did not affect the growth of B. cereus. Peptidoglycan isolated from B. cereus cultures stimulated growth of the CF rhizosphere bacterium Flavobacterium johnsoniae, although culture supernatant from B. cereus grown in root exudate media did not. These results suggest B. cereus and CF rhizosphere bacteria have a commensal relationship in which peptidoglycan produced by B. cereus stimulates the growth of CF bacteria.

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“…The cell of B. subtilis might autolysed during fermentation, so resulting in a large number of cell deaths and reducing bacterial fermentation concentration [32]. There were many factors that led to autolysis of the bacterium [33][34][35][36].…”

Section: Discussionmentioning

confidence: 99%

Arabinose Plays an Important Role in Regulating the Growth and Sporulation of <i>Bacillus subtilis</i> NCD-2

Fu,

Liu,

et al. 2023

Preprint

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Bacillus subtilis NCD-2 showed a promising biocontrol effect against plant soil-borne diseases, and was developed as a commercial microbial fungicide against cotton verticillium wilt in China. Spores were main ingredient of the fungicide and played the crucial role in biological control of plant diseases. Therefore, the key to reducing the cost of fungicide was to find ways to increase the number of spores of strain NCD-2 during fermentation. In this study, 755 substances were evaluated by Phenotype Microarray technology, and 5 kinds of carbon sources and 1 kind of nitrogen source were found to promote the metabolism of strain NCD-2. Among carbon sources L-arabinose showed strongest ability to promote the bacteria growth and sporulation of strain NCD-2. When L-arabinose was used as a single carbon source, it could increase the bacteria concentration and the sporulation efficiency of strain NCD-2 by 2.04 times and 1.99 times, respectively, compared with D-glucose. Moreover, L-arabinose could significantly decrease the autolysis of strain NCD-2 revealed by microscopy observation. The mechanism for promoting the sporulation by L-arabinose was analysed by RNA-seq sequencing. Results showed that total of 790, 923 and 1270 genes were up-regulated and 639, 850 and 1001 genes were down-regulated under L-arabinose supplies when cultured at 8 h, 12 h and 16 h, respectively. Fourteen differentially expressed genes associated with arabinose transportation and sporulation were selected for qRT-PCR analysis, and the result showed basically consistent expression trend with transcriptome. Notably, genes associated with arabinose metabolism, sporulation, spore resistance to heat and spore coat formation were significantly up-regulated, and genes associated with sporulation-delaying protein were significantly down-regulated under L-arabinose supplies. msmX gene which was involved in arabinose transport in Bacillus genus was deleted, and the mutant decreased the growth and sporulation by 53.71% and 86.46%, respectively, when compared to strain NCD-2 wild type. Complementary of the mutant by importing intact msmX gene could restore the growth and sporulation of the mutant strain. In conclusion, arabinose played an important role in regulating the growth and sporulation of strain NCD-2.

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Evaluation of whole cells of Bacillus subtilis as substrate for measurement of autolysin activity

Cited by 6 publications

References 12 publications

Deleting multiple lytic genes enhances biomass yield and production of recombinant proteins by Bacillus subtilis

Deleting multiple lytic genes enhances biomass yield and production of recombinant proteins by Bacillus subtilis

Peptidoglycan from Bacillus cereus Mediates Commensalism with Rhizosphere Bacteria from the Cytophaga-Flavobacterium Group

Arabinose Plays an Important Role in Regulating the Growth and Sporulation of <i>Bacillus subtilis</i> NCD-2

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