Glucosaminidase of Bacillus subtilis: cloning, regulation, primary structure and biochemical characterization

Abstract

The 90 kDa glucosaminidase protein was purified to apparent homogeneity from vegetative cells of Bacillus subtilis AC327, and then the corresponding gene was cloned into Escherichia coli in two inactive forms by standard procedures. Nucleotide sequencing of the glucosaminidase region revealed a monocistronic operon, (designated lytD = w l G ) encoding a 95.6 kDa protein, comprising 880 amino acid residues, which has a typical signal peptide. Moreover, another monocistronic operon (designated pmi = orfX), encoding a 35.4 kDa protein, was found upstream of the glucosaminidase gene. Expression of a IytD-lacZ fusion gene, driven by lytD regulatory sequences, was observed during the exponential growth phase. The introduction of a sigD null mutation greatly reduced (by about 95%) the expression of the fusion. Amino acid sequence analysis of the glucosaminidase showed two types of direct repeats, each type being present twice, in the N-terminal-to-central region of the glucosaminidase: these repeats probably represent the cell-wall-binding domain. Zymographic analysis revealed that the 90 kDa glucosaminidase is partly processed to several smaller proteins (35-39 kDa), retaining lytic activity. Processing of these proteins occurred between the N-terminal cellwall-binding and C-terminal catalytic domains of the glucosaminidase, the site being located between the 569th and 606th codons of the glucosaminidase. Serial deletions from the N-terminus of the glucosaminidase revealed that the loss of more than one repeating unit drastically reduces its lytic activity toward cell walls. The IytD gene product, in either an intact or a truncated form, was found to be lethal for E. coli, and the N-terminally truncated glucosaminidase proteins, produced in E. coli, were very unstable. The partially purified glucosaminidase from B. subtilis was found to be very unstable at low ionic strength at 37 "C, but this instability was overcome by the addition of either SDS-purified cell wall or protease inhibitor (PMSF) to the enzyme or after purification of the glucosaminidase to apparent homogeneity.

Characterization of new l,d-endopeptidase gene product CwlK (previous YcdD) that hydrolyzes peptidoglycan in Bacillus subtilis

“…In particular, several autolysins such as lytC (cwlB), lytD (cwlG), and lytF (cwlE) are transcribed by Eσ D RNA polymerase (Kuroda and Sekiguchi 1993;Lazarevic et al 1992;Margot et al 1994;Ohnishi et al 1999;Rashid et al 1995). Thus, it is predicted that the transcription of cwlK may depend on Eσ A and/or Eσ D RNA polymerases because cwlK is transcribed during the vegetative growth phase (Fig.…”

“…Several hydrolases produced during the vegetative growth phase, such as LytC (CwlB), LytD (CwlG), LytG (YubE), LytE (CwlF), LytF (CwlE), CwlO (YvcE), and CwlS (YojL), have been characterized in B. subtilis (Blackman et al 1998;Foster and Popham 2002;Fukushima et al 2006;Horsburgh et al 2003b;Ishikawa et al 1998;Kuroda and Sekiguchi 1991;Lazarevic et al 1992;Margot et al 1994;Margot et al 1998;Margot et al 1999;Ohnishi et al 1999;Rashid et al 1995;Shida and Sekiguchi 2005;Yamaguchi et al 2004). LytD and LytG have been identified as N-acetylglucosaminidases (Horsburgh et al 2003b;Margot et al 1994;Rashid et al 1995), LytC as an N-acetylmuramoyl-L-alanine amidase (Kuroda and Sekiguchi 1991), and LytF, CwlO, and CwlS as D,L-endopeptidases hydrolyzing the D-γ-glutamyl-meso-diaminopimelic acid linkage in the peptidoglycan of B. subtilis (Fukushima et al 2006;Margot et al 1999;Ohnishi et al 1999;Yamaguchi et al 2004).…”

“…LytD and LytG have been identified as N-acetylglucosaminidases (Horsburgh et al 2003b;Margot et al 1994;Rashid et al 1995), LytC as an N-acetylmuramoyl-L-alanine amidase (Kuroda and Sekiguchi 1991), and LytF, CwlO, and CwlS as D,L-endopeptidases hydrolyzing the D-γ-glutamyl-meso-diaminopimelic acid linkage in the peptidoglycan of B. subtilis (Fukushima et al 2006;Margot et al 1999;Ohnishi et al 1999;Yamaguchi et al 2004). Moreover, PgdS (YwtD) is a polyglutamic acid-degrading enzyme produced during the vegetative growth phase and it belongs to the "D,L-endopeptidase" family (Suzuki and Tahara 2003).…”

Information decomposition method to analyze symbolical sequences

“…Fig. 5 (Bi) shows that the major part of the beta-N-acetylglucosaminidase gene [33,34] has latent periodicity with the period length equal to 120 bases. In Fig.…”

“…Bi -Bacillus subtilis gene [33,34] for beta-N-acetylglucosaminidase (1296-3938 b.p.) from sequence BACORFX.…”