Mutational analysis of Bacillus megaterium QM B1551 cortex-lytic enzymes.
Molecular-genetic and muropeptide analysis techniques have been applied to examine the function in vivo of the Bacillus megaterium QM B1551 SleB and SleL proteins. In common with Bacillus subtilis and Bacillus anthracis, the presence of anhydromuropeptides in B. megaterium germination exudates, which is indicative of lytic transglycosylase activity, is associated with an intact sleB structural gene. B. megaterium sleB cwlJ double mutant strains complemented with engineered SleB variants in which the predicted N- or C-terminal domain has been deleted (SleB-ΔN or SleB-ΔC) efficiently initiate and hydrolyze the cortex, generating anhydromuropeptides in the process. Additionally, sleB cwlJ strains complemented with SleB-ΔN or SleB-ΔC, in which glutamate and aspartate residues have individually been changed to alanine, all retain the ability to hydrolyze the cortex to various degrees during germination, with concomitant release of anhydromuropeptides to the surrounding medium. These data indicate that while the presence of either the N- or C-terminal domain of B. megaterium SleB is sufficient for initiation of cortex hydrolysis and the generation of anhydromuropeptides, the perceived lytic transglycosylase activity may be derived from an enzyme(s), perhaps exclusively or in addition to SleB, which has yet to be identified. B. megaterium SleL appears to be associated with the epimerase-type activity observed previously in B. subtilis, differing from the glucosaminidase function that is apparent in B. cereus/B. anthracis.
Christie
Graham
G
Department of Chemical Engineering and Biotechnology, Institute of Biotechnology, University of Cambridge, Cambridge, England CB2 1QT, United Kingdom. gc301@cam.ac.uk
Ustok
Fatma Isik
FI
Lu
Qiaozhi
Q
Packman
Len C
LC
Lowe
Christopher R
CR
D000581:Amidohydrolases / Q000235:genetics / Q000378:metabolism; D000595:Amino Acid Sequence; D001410:Bacillus megaterium / Q000201:enzymology / Q000235:genetics* / Q000378:metabolism; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D015964:Gene Expression Regulation, Bacterial / Q000502:physiology; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D011487:Protein Conformation; D013171:Spores, Bacterial / Q000502:physiology
D001426:Bacterial Proteins; C101656:SleB protein, Bacillus; D000581:Amidohydrolases