HtrA Protein Hyperproduction Increases the Viability of Bacillus subtilis Cells under the Stress Conditions and Stimulates Biofilm Formation
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HtrA Protein Hyperproduction Increases the Viability of Bacillus subtilis Cells under the Stress Conditions and Stimulates Biofilm Formation

L.S. Chernova*, I.S. Sharafutdinov**, A.R. Kayumov***

Kazan Federal University, Kazan, 420008 Russia

E-mail: *lsch-888@live.com, **irwad@yandex.ru, *** kairatr@yandex.ru

Received December 14, 2016

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Abstract

HtrA (high-temperature requirement A) are the heat shock-induced serine proteases widely spread in pro- and eukaryotic cells. These enzymes control the quality of intracellular proteins by hydrolysis of denatured proteins, thereby protecting cells from various stresses. In many pathogenic bacteria, they are factors of pathogenicity. Thus, in Streptococcus mutans, HtrA participates in quorum-dependent processes and biofilm formation via digestion of the pheromone ComX. In this work, Bacillus subtilis HtrA Hy with an overexpressionof the HtrA protein has been obtained and its physiological features have been characterized. The overexpression of HtrA in Bacillus subtilis cells increases cell viability at high temperature conditions, ethanol and salt stresses. In addition, B. subtilis HtrA Hy is characterized by the intensive biofilm formation with an increased synthesis of extracellular matrix. Congo red staining of the co lonyhas revealed that HtrA overexpression induces the increased production of amyloid-like proteins. Moreover, these cells exhibited intensive swarming in contrast to the wild-type strain. The overexpression of the HtrA protease results in an increased level of expression of the eps and yqxM genes encoding the components of the biofilm matrix. This fact is probably related to the effect of the enzyme on regulatory processes, possibly due to the hydrolysis of signal regulatory peptides.

Keywords: protease HtrA, biofilm formation, viability, temperature stress, protease hyperproduction

Acknowledgments. The study was supported by the Russian Science Foundation (project no. 15-14-00046).

Figure Captions

Fig. 1. The dynamics of growth of B. subtilis strains at 37 ?С (a) and at 49 ?С (b). Key: wt – B. subtilis wt, hy – B. subtilis HyHtrA.

Fig. 2. Assessment of the viability in B. subtilis cultivated under the conditions of temperature shock using differential fluorescent staining (a) and CFU counting (b). Key: wt – B. subtilis wt, hy – B. subtilis HyHtrA.

Fig. 3. The dynamics of growth of B. subtilis strains after 5% (a) and 7.5% (b) ethanol exposure and 1 M NaCl (c). Key: wt – B. subtilis wt, hy – B. subtilis HyHtrA.

Fig. 4. Quantification of biofilm formation by B. subtilis wt, B. subtilis HyHtrA: a – with the help of Congo red staining, b – a cross section of the colony, c – depending on the optical density OD550 by crystal violet staining intensity. Key: wt – B. subtilis wt, hy – B. subtilis HyHtrA.

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For citation: Chernova L.S., Sharafutdinov I.S., Kayumov A.R. HtrA protein hyperproduction increases the viability of Bacillus subtilis cells under the stress conditions and stimulates biofilm formation. Uchenye Zapiski Kazanskogo Universiteta. Seriya Estestvennye Nauki, 2017, vol. 159, no. 2, pp. 262–271. (In Russian)


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