Effect of Bacillus subtilis spo0A mutation on cell wall lytic enzymes and extracellular proteases, and prevention of cell lysis

Kodama, Takeko; Endo, Kiyoshi; Ara, Katsutoshi; Ozaki, Katsuya; Kakeshita, Hiroshi; Yamane, Kunio; Sekiguchi, Junichi

doi:10.1263/jbb.103.13

Cited by 43 publications

(47 citation statements)

References 55 publications

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“…Cell lysis has been intensively studied, and its mechanism has been correlated to autolysins (25). We examined a B. subtilis lytC lytD double mutant SH128 lacking two major autolysin genes and its parental strain 1A304.…”

Section: Resultsmentioning

confidence: 99%

Nonclassical Protein Secretion by Bacillus subtilis in the Stationary Phase Is Not Due to Cell Lysis

Yang¹,

Ewis²,

Zhang³

et al. 2011

Journal of Bacteriology

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The carboxylesterase Est55 has been cloned and expressed in Bacillus subtilis strains. Est55, which lacks a classical, cleavable N-terminal signal sequence, was found to be secreted during the stationary phase of growth such that there is more Est55 in the medium than inside the cells. Several cytoplasmic proteins were also secreted in large amounts during late stationary phase, indicating that secretion in B. subtilis is not unique to Est55. These proteins, which all have defined cytoplasmic functions, include GroEL, DnaK, enolase, pyruvate dehydrogenase subunits PdhB and PdhD, and SodA. The release of Est55 and those proteins into the growth medium is not due to gross cell lysis, a conclusion that is supported by several lines of evidence: constant cell density and secretion in the presence of chloramphenicol, constant viability count, the absence of EF-Tu and SecA in the culture medium, and the lack of effect of autolysin-deficient mutants. The shedding of these proteins by membrane vesicles into the medium is minimal. More importantly, we have identified a hydrophobic ␣-helical domain within enolase that contributes to its secretion. Thus, upon the genetic deletion or replacement of a potential membrane-embedding domain, the secretion of plasmid gene-encoded mutant enolase is totally blocked, while the wild-type chromosomal enolase is secreted normally in the same cultures during the stationary phase, indicating differential specificity. We conclude that the secretion of Est55 and several cytoplasmic proteins without signal peptides in B. subtilis is a general phenomenon and is not a consequence of cell lysis or membrane shedding; instead, their secretion is through a process(es) in which protein domain structure plays a contributing factor.Bacillus subtilis secretes large amounts of proteins into the growth medium (43). Of the known secretory pathways in B. subtilis, the majority of proteins are exported from the cytoplasm by the Sec-dependent pathway, through which secretory proteins are synthesized as precursors with typical cleavable N-terminal signal peptides (3, 36). Fewer proteins are released into the medium via the cleavable twin-arginine translocation (TAT) system (39). Still other proteins are exported into the medium via ATP-binding cassette transporters, a dedicated pseudopilin export pathway, a competence development system or an ESAT-6 (Mycobacterium tuberculosis early secreted antigenic target of 6 kDa)-like system (31).The genome of B. subtilis 168 is 4,215 kbp in length and contains about 4,100 genes that are predicted to include over 250 extracellular proteins; the majority of these proteins are secreted through the aforementioned pathways (3, 18). However, proteomic studies have revealed that genome-based predictions reflect only 50% of the actual composition of the extracellular proteome. This significant discrepancy is mainly due to the difficulties in the prediction of extracellular proteins lacking signal peptides (including cytoplasmic proteins) and lipoproteins (3, 18). These findin...

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Section: Resultsmentioning

confidence: 99%

Nonclassical Protein Secretion by Bacillus subtilis in the Stationary Phase Is Not Due to Cell Lysis

Yang¹,

Ewis²,

Zhang³

et al. 2011

Journal of Bacteriology

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“…The 5 0 and 3 0 regions of sscA (fragments 1 and 2) were amplified using B. subtilis 168 chromosomal DNA and two pairs of primers (61up+379F and 61up+379R for fragment 1, and 61dn+410F and 61dn+410R for fragment 2). The spectinomycin resistance gene was amplified using pBlueSPR 32) and primers PB-M13-20 and PB-M13Rev (fragment 3). The 5 0 -end sequences of primers 61up+379R and 61dn+410F are complementary to those of primers PB-M13-20 and PB-M13Rev respectively.…”

Section: Methodsmentioning

confidence: 99%

“…Preparation of total RNA was performed as described previously. 37,38) Aliquots (10 mg) of the total RNA were electrophoresed and blotted on a positively charged Hybond-N+membrane (GE Healthcare, Japan). Hybridizations were performed with digoxigenin-labeled RNA probes (10 ng) following the manufacturer's instructions, with minor modifications (Roche Diagnostics, Japan).…”

Section: Methodsmentioning

confidence: 99%

A Novel Small Protein ofBacillus subtilisInvolved in Spore Germination and Spore Coat Assembly

Kodama

Matsubayashi

Yanagihara

et al. 2011

Bioscience, Biotechnology, and Biochemistry

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“…Given the co-induction of lytE and phoA by heat stress (Helmann et al, 2001;Huang et al, 2013;Tseng et al, 2011) and the co-inhibition of the levels of LytE and PhoA by Spo0A in B. subtilis (Hulett et al, 1994;Jensen et al, 1993;Kodama et al, 2007), we wondered whether there would be other correlation(s) between expression of lytE and phoA. Firstly, effect of mutation of lytE on transcription of phoA was investigated by using PCR and the primer pair A64 plus A65 (Table S1) to construct the pDLbased integrative plasmid pGS2095 (Table 1), which carries a transcriptional fusion of the regulatory region of phoA with bgaB.…”

mentioning

confidence: 99%

Expressions of alkaline phosphatase genes during phosphate starvation are under positive influences of multiple cell wall hydrolase genes in <i>Bacillus subtilis</i>

Shen¹,

Hu²,

Shaw³

2016

J. Gen. Appl. Microbiol.

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Effect of Bacillus subtilis spo0A mutation on cell wall lytic enzymes and extracellular proteases, and prevention of cell lysis

Cited by 43 publications

References 55 publications

Nonclassical Protein Secretion by Bacillus subtilis in the Stationary Phase Is Not Due to Cell Lysis

Nonclassical Protein Secretion by Bacillus subtilis in the Stationary Phase Is Not Due to Cell Lysis

A Novel Small Protein ofBacillus subtilisInvolved in Spore Germination and Spore Coat Assembly

Expressions of alkaline phosphatase genes during phosphate starvation are under positive influences of multiple cell wall hydrolase genes in <i>Bacillus subtilis</i>

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