Natural genetic competence in Bacillus subtilis natto OK2

Abstract

We isolated a Bacillus subtilis natto strain, designated OK2, from a lot of commercial fermented soybean natto and studied its ability to undergo natural competence development using a comG-lacZ fusion at the amyE locus. Although transcription of the late competence genes was not detected in the B. subtilis natto strain OK2 during competence development, these genes were constitutively transcribed in the OK2 strain carrying either the mecA or the clpC mutation derived from B. subtilis 168. In addition, both OK2 mutants exhibited high transformation frequencies, comparable with that observed for B. subtilis 168. Moreover, as expected from these results, overproduction of ComK derived from strain 168 in strain OK2 resulted in a high transformation frequency as well as in induction of the late competence genes. These results clearly indicated that ComK produced in both the mecA and clpC mutants of strain OK2 (ComK(OK2)) could activate the transcription of the whole set of late competence genes and suggested that ComK(OK2) was not activated in strain OK2 during competence development. We therefore sequenced the comS gene of OK2 and compared it with that of 168. The comS(OK2) had a single-base change, resulting in the replacement of Ser (strain 168) by Cys (strain OK2) at position 11.

FIG. 1

Construction of pULI7KS27 carrying the Pspac-controlled comK gene. The 601-bp fragment containing the Shine-Dalgarno (SD) and coding sequences of the B. subtilis 168 comK gene was amplified by PCR and was inserted at the XbaI site of pULI7 as described in Materials and Methods.

FIG. 2

Expression of comG-lacZ in B. subtilis 168 and the B. subtilis natto strain OK2. Cells were grown in CI medium, and samples (200 to 500 μl) were collected at the indicated times. β-Galactosidase activities were determined as described in Materials and Methods. Time zero is defined as the end of exponential growth. Symbols: ○, RIK1001 (168 amyE::comG-lacZ); ●, RIK7101 (OK2 amyE::comG-lacZ).

FIG. 3

The effect of mecA or clpC mutation on comG-lacZ expression in the B. subtilis natto strain OK2. Growth conditions and measurement of β-galactosidase activities were as specified in the legend to Fig. 2. Time zero is defined as the end of exponential growth. Symbols: ○, RIK7101 (OK2 amyE::comG-lacZ); ●, RIK7102 (OK2 amyE::comG-lacZ mecA::spc); ▵, RIK7103 (OK2 amyE::comG-lacZ clpC::spc).

FIG. 4

Effect of comK induction on comG-lacZ expression in B. subtilis 168 during exponential growth. RIK1027 cells carrying pULI7KS27 (Fig. 1) were grown in LB medium containing 7.5 μg of kanamycin/ml. When the cell density reached an OD₆₆₀ of 0.1, IPTG was added to the culture at various concentrations. Symbols: ○, 0 mM IPTG; ●, 0.1 mM IPTG; ▵, 1 mM IPTG.

FIG. 5

Effect of comK induction on comG-lacZ expression in OK2 during competence development. RIK7127 cells carrying plasmid pULI7KS27 were grown in CI medium containing 7.5 μg of kanamycin/ml and 0.1 μg of biotin/ml as described in Materials and Methods. At the point shown by the arrow (T₀, defined as the end of exponential growth), IPTG was added to the culture at the indicated concentrations, and β-galactosidase activities were measured as specified in the legend to Fig. 2. Symbols: ○, 0 mM IPTG; ●, 0.1 mM IPTG; ▵, 1 mM IPTG.