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. 2016 May 25;11(5):e0156247.
doi: 10.1371/journal.pone.0156247. eCollection 2016.

Loss of GltB Inhibits Biofilm Formation and Biocontrol Efficiency of Bacillus subtilis Bs916 by Altering the Production of γ-Polyglutamate and Three Lipopeptides

Huafei Zhou  1   2 , Chuping Luo  1   3 , Xianwen Fang  2 , Yaping Xiang  1   2 , Xiaoyu Wang  2 , Rongsheng Zhang  2 , Zhiyi Chen  1   2
Affiliations

Loss of GltB Inhibits Biofilm Formation and Biocontrol Efficiency of Bacillus subtilis Bs916 by Altering the Production of γ-Polyglutamate and Three Lipopeptides

Huafei Zhou et al. PLoS One. .

Abstract

Aims: This study examined the contribution of GltB on biofilm formation and biocontrol efficiency of B. subtilis Bs916.

Methods and results: The gltB gene was identified through a biofilm phenotype screen and a bioinformatics analysis of serious biofilm formation defects, and then a gltB single knockout mutant was constructed using homologous recombination. This mutant demonstrated severe deficits in biofilm formation and colonisation along with significantly altered production ofγ-polyglutamate (γ-PGA) and three lipopeptide antibiotics (LPs) as measured by a transcriptional analysis of both the wild type B. subtilis Bs916 and the gltB mutant. Consequently, the mutant strain retained almost no antifungal activity against Rhizoctonia solani and exhibited decreased biocontrol efficiency against rice sheath blight. Very few gltB mutant cells colonised the rice stem, and they exhibited no significant nutrient chemotaxis compared to the wild type B. subtilis Bs916. The mechanism underlying these deficits in the gltB mutant appears to be decreased significantly in production of γ-PGA and a reduction in the production of both bacillomycin L and fengycin. Biofilm restoration of gltB mutant by additionγ-PGA in the EM medium demonstrated that biofilm formation was able to restore significantly at 20 g/L.

Conclusions: GltB regulates biofilm formation by altering the production ofγ-PGA, the LPs bacillomycin L and fengcin and influences bacterial colonisation on the rice stem, which consequently leads to poor biocontrol efficiency against rice sheath blight.

Significance and impact of study: This is the first report of a key regulatory protein (GltB) that is involved in biofilm regulation and its regulation mechanism and biocontrol efficiency by B. subtilis.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Fig 1
Fig 1. Changes in biofilm formation due to the B. subtilis Bs916 and ΔgltB mutants in MSgg culture medium with 20 μg/mL Congo Red and 10 μg/mL Coomassie brilliant blue.
(1) B. subtilis Bs916 biofilm was dense and solid with clear lines. In contrast, the ΔgltB mutant formed an uneven biofilm with an irregular shape. (2) The net weight of the ΔgltB mutant biofilm was more than three times less than the net weight of the WT B. subtilis Bs916 biofilm.
Fig 2
Fig 2. Differences in antibacterial activity against R. solani between WT B. subtilis Bs916 and the ΔgltB mutant.
The ΔgltB mutant completely lost its antibacterial activity compared to the WT B. subtilis Bs916.
Fig 3
Fig 3. Colonisation of the rice plant against rice sheath blight.
Over time, the number of both WT B. subtilis Bs916 and ΔgltB mutant cells initially increased and then began to decrease. However, although WT B. subtilis Bs916 could produce normal clusters of cells, the ΔgltB mutant also demonstrated this same trend. By the last time point (i.e., 15 d), there were very few cells in either the WT B. subtilis Bs916 or the ΔgltB mutant, and the clustering of WT B. subtilis Bs916’s had disappeared.
Fig 4
Fig 4
Antibiotic secretion of bacillomycin L (a), fengycin (c), and surfactin (b). The ΔgltB mutant no longer secreted bacillomycin L or fengycin. In contrast, the ΔgltB mutant produced significantly higher (approximately five times higher) levels of surfactin compared to the WT B. subtilis Bs916.
Fig 5
Fig 5. γ-PGA and glutamate content of WT B. subtilis Bs916 and the ΔgltB mutant in the process of biofilm formation.
For glutamate detection, in addition to standard L-glutamate and double dilution to B. subtilis Bs916, all treatments were diluted tenfold for detection. For γ-PGA detection, all treatments were also diluted tenfold for detection.
Fig 6
Fig 6. Biofilm restoration in the ΔgltB mutant.
A final concentration of 10 g/L, 20 g/L, and 30 g/Lγ-PGA solution were added into the ΔgltB mutant in 4mL EM medium. The final concentration of 10 g/L and 20 g/Lγ-PGA were able to recover biofilm formation in the ΔgltB mutant, however, high final concentration of 30 g/Lγ-PGA inhibited biofilm formation in WT B. subtilis Bs916 and the ΔgltB mutant.
Fig 7
Fig 7. Biofilm restoration by B. subtilis Bs916’sγ-PGA in the ΔgltB mutant.
The ΔgltB mutant grew next to B. subtilis Bs916 on solid EM plate with 1.5% agar to observe their biofilm formation. Biofilm formation of ΔgltB mutant was no significant restoration before 36 h. But in 72h, its biofilm formation had been restored to most parts.
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References

    1. O'Toole GA, Pratt LA, Watnick PI, et al. Genetic approaches to study of biofilms. Methods in enzymology, 1999; 310: 91–109. ISBN 9780121822118 - PubMed
    1. O'Toole GA, Kaplan HB, Kolter R. Biofilm formation as microbial development. Annual Reviews in Microbiology, 2000; 54(1): 49–79. 10.1146/annurev.micro.54.1.49 - DOI - PubMed
    1. Branda SS, Vik S, Friedman L, Kolter R. Biofilms: the matrix revisited. Trends in microbiology. 2005; 13(1):20–6. 10.1016/j.tim.2004.11.006 . - DOI - PubMed
    1. Kolter R, Greenberg EP. Microbial sciences: the superficial life of microbes. Nature. 2006; 441(7091):300–2. 10.1038/441300a . - DOI - PubMed
    1. Haggag WM, Timmusk S. Colonization of peanut roots by biofilm-forming Paenibacillus polymyxa initiates biocontrol against crown rot disease. Journal of applied microbiology. 2008; 104(4):961–9. 10.1111/j.1365-2672.2007.03611.x . - DOI - PubMed

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Grants and funding

This work was supported by National Natural Science Foundation of China (grant 31570061); National High-tech R&D Program of China (2011AA10A201); Science Foundation of the Jiangsu Academy of Agricultural Sciences [grant CX(14)2128]; public welfare industry (agriculture) research special funds (201103002-3).

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