Phosphorylation and functional analysis of the sporulation initiation factor Spo0A from Clostridium botulinum.
The initiation of sporulation in aerobic Bacillus species is regulated by the phosphorelay consisting of several sensor histidine kinases, the Spo0F response regulator, the Spo0B phosphotransferase and the Spo0A transcription factor that upon phosphorylation represses genes for growth and activates the developmental process. Clostridium species lack both Spo0F and Spo0B and the identities of the sensor histidine kinases are unknown. The amino acid sequence of Spo0A is highly conserved in Clostridium botulinum relative to Bacillus subtilis but the cloned C. botulinum Spo0A was unable to complement a spo0A mutant of B. subtilis for sporulation. However, it was able to repress the abrB gene of B. subtilis. Active site mutations in Spo0A still repressed, indicating this activity was independent of phosphorylation. An orphan sensor histidine kinase of C. botulinum appeared to normally phosphorylate C. botulinum Spo0A and expression of this kinase in combination with C. botulinum Spo0A in B. subtilis was lethal, suggesting phosphorylation of C. botulinum Spo0A repressed essential growth genes as a prerequisite to sporulation but could not compensate for this effect by inducing sporulation. A chimera Spo0A consisting of a B. subtilis Spo0A response regulator domain fused to a C. botulinum DNA-binding domain was capable of restoring sporulation to a spo0A mutant of B. subtilis albeit at less than wild-type levels. The data suggest that induction of sporulation requires interactions of both domains of Spo0A with other conserved proteins and despite the high conservation of the amino acid sequence of C. botulinum Spo0A, some of these interactions have been lost.
Wörner
Kristina
K
The Scripps Research Institute, 10550 N. Torrey Pines Road, La Jolla, CA 92037, USA.
Szurmant
Hendrik
H
Chiang
Christina
C
Hoch
James A
JA
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D003001:Cloning, Molecular; D003014:Clostridium botulinum / Q000378:metabolism / Q000502:physiology*; D008969:Molecular Sequence Data; D010766:Phosphorylation; D017434:Protein Structure, Tertiary; D012097:Repressor Proteins / Q000235:genetics / Q000378:metabolism*; D013171:Spores, Bacterial / Q000378:metabolism*; D020798:Two-Hybrid System Techniques
D001426:Bacterial Proteins; D012097:Repressor Proteins