Table 1.
Homologous complementation of B. subtilis RNase P mutant strain SSB318 by B. subtilis rnpB wild type and mutant alleles
| rnpB variants in pHY300 | + IPTG | − IPTG | Aldose |
|---|---|---|---|
| B. subtilis PBs rnpB rnpBwt | + + + | + + + | None |
| B. subtilis PBs rnpB rnpBC258 | a | None | |
| B. subtilis PBs rnpB rnpBC259 | a | None | |
| B. subtilis Pxyl rnpBwt | + + + | + + +b | Xylose or glucose |
| B. subtilis Pxyl rnpBC258 | a | ||
| B. subtilis Pxyl rnpBC259 | + + + | − | Xylose |
| B. subtilis Pxyl rnpBwt + Pxyl rnpA | + + + | + + + | Xylose |
| B. subtilis Pxyl rnpBC258 + Pxyl rnpA | + + + | + + c | Xylose |
| B. subtilis Pxyl rnpBC259 + Pxyl rnpA | + + + | + + +d | Xylose |
| B. subtilis Pxyl rnpBwt + Pxyl rnpA-stop | + + + | + + + | Xylose |
| B. subtilis Pxyl rnpBC258 + Pxyl rnpA-stop | + + +e | − | Xylose |
| B. subtilis Pxyl rnpBC259 + Pxyl rnpA-stop | + + + | − | Xylose |
| pHY300 | + + + | − | Xylose |
| pHY300 + Pxyl rnpA | + + + | − | Xylose |
| pHY300 + Pxyl rnpA-stop | + + + | − | Xylose |
Growth of mutant strain SSB318 transformed with wild-type B. subtilis rnpB (rnpBwt) and mutant (rnpBC258, rnpBC259) alleles on plasmid pHY300; promoter types: PBs rnpB, native B. subtilis rnpB promoter; Pxyl, inducible xylose promoter; B. subtilis rnpA was overexpressed in parallel from the same plasmid; rnpA-stop designates the B. subtilis rnpA gene with two stop codons in the 5′-coding region. Cell growth was analyzed on LB plates (with appropriate antibiotics) in the presence (1 mM) or absence of IPTG; an aldose (xylose or glucose) at 2% (w/v) was added where indicated.
+ + +: growth with equal numbers of colonies on the corresponding + and − IPTG plates; ++: somewhat retarded growth, but equal numbers of colonies on + and − IPTG plates.; −: no growth;
aUnable to clone.
bBasal expression of xylose promoter (in the presence of 2% glucose) is sufficient for cell growth in the absence of IPTG.
cBasal expression of xylose promoter (in the presence of 2% glucose) is not sufficient for cell growth in the absence of IPTG.
dBasal expression of xylose promoter (in the presence of 2% glucose) is sufficient for cell growth in the absence of IPTG, but retarded cell growth.
eHere we succeeded to isolate a single colony with a stable rnpBC258 genotype; however, the majority of SSB318 cells transformed with rnpBC258 were still unstable and tended to form revertants to the wild type, most likely by recombination with the chromosomal rnpBwt gene.