Isolation of constitutive variants of a subfamily 10 histidine protein kinase (SppK) from Lactobacillus using random mutagenesis.
The histidine protein kinase SppK is a peptide pheromone-activated kinase that regulates the production of the bacteriocin sakacin P in Lactobacillus sakei. SppK belongs to subfamily 10 of histidine protein kinases (HPKs), which regulate important processes in Gram-positive bacteria, including virulence, competence and bacteriocin production. To obtain insight into the functional properties of this relatively unknown class of HPKs, we have subjected SppK to random mutagenesis by error-prone PCR, followed by selection for mutants displaying a constitutive phenotype. Most identified mutations were clustered in a predicted coiled coil-like region, which is an important part of the HPK dimer interface and which includes the autophosphorylated histidine. Other mutations were located in the junctions between the dimerization domain and the membrane receptor domain or the catalytic kinase domain. Interestingly, two previously identified constitutive variants of ComD, an SppK homologue involved in competence regulation in Streptococcus pneumoniae, contained single mutations in the same regions.
Mathiesen
Geir
G
Department of Chemistry, Biotechnology and Food Science, Norwegian University of Life Sciences, Chr. M. Falsensvei 1, PO Box 5003, 1432 As, Norway.
Axelsen
Gunnhild W
GW
Axelsson
Lars
L
Eijsink
Vincent G H
VG
D000595:Amino Acid Sequence; D001430:Bacteriocins / Q000096:biosynthesis; D019281:Dimerization; D015964:Gene Expression Regulation, Bacterial; D014644:Genetic Variation; D000071677:Histidine Kinase; D007778:Lactobacillus / Q000201:enzymology* / Q000235:genetics; D008969:Molecular Sequence Data; D016296:Mutagenesis; D009154:Mutation; D016133:Polymerase Chain Reaction / Q000379:methods; D011494:Protein Kinases / Q000737:chemistry / Q000235:genetics*; D015398:Signal Transduction
D001430:Bacteriocins; C087217:sakacin P protein, Lactobacillus; D011494:Protein Kinases; D000071677:Histidine Kinase