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. 2006 Oct;188(19):6816-23.
doi: 10.1128/JB.00756-06.

Analysis of RNase P protein (rnpA) expression in Bacillus subtilis utilizing strains with suppressible rnpA expression

Markus Gössringer  1 , Rosel Kretschmer-Kazemi Far, Roland K Hartmann
Affiliations

Analysis of RNase P protein (rnpA) expression in Bacillus subtilis utilizing strains with suppressible rnpA expression

Markus Gössringer et al. J Bacteriol. 2006 Oct.

Abstract

Bacterial RNase P is composed of an RNA subunit and a single protein subunit (encoded by the rnpB and rnpA genes, respectively). We constructed Bacillus subtilis mutant strains that conditionally express the RNase P protein under control of the xylose promoter (P(xyl)). In one strain (d7), rnpA expression was efficiently repressed in the absence of the inducer xylose, leading to cell growth arrest. Growth could be restored by a second functional rnpA allele. This is the first RNase P protein knockdown strain, providing the first direct proof that the rnpA gene is essential in B. subtilis and, by inference, in other bacteria. We further show (i) that, in the wild-type context, rnpA expression is attenuated by transcriptional polarity and (ii) that translation of rnpA mRNA in B. subtilis can be initiated at two alternative start codons. His-tagged RNase P protein variants are functional in vivo and permit purification of in vivo-assembled holoenzymes by affinity chromatography. Simultaneous expression of plasmid-encoded RNase P RNA and His-tagged protein increased RNase P holoenzyme yields. Massive overproduction of RNase P protein in strain d7 is compatible with cell viability.

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Figures

FIG. 1.
FIG. 1.
Schematic of the WT rpmH-rnpA region in B. subtilis YB886 (wt, top) in comparison with those of integration vectors pS2.1 and pD2b (for construction details, see Fig. S1 in the supplemental material). In construct pS2.1, the Pxyl promoter was directly fused to the rnpA gene, whereas Pxyl was fused to the upstream rpmH gene in construct pD2b; here, rpmH has a 5-bp deletion in its 5′ region [rpmH(Oc)], preventing translation of a functional rpmH gene product. The chloramphenicol resistance gene (cat) was used as a selection marker. Primer pair dnaA-5 and jag-3 (horizontal arrows at the top) was used to amplify this chromosomal region for insertion into plasmid pSP64 to yield integration vectors pS2.1 and pD2b. The direction of transcription is indicated by the open arrowheads within boxes representing the coding regions of cat, dnaA, jag, rnpA, rpmH, and spoIIIJ. P2 indicates the natural tandem promoters preceding rpmH; the rpmH and rnpA genes are shaded in gray.
FIG. 2.
FIG. 2.
Sequence of the genomic rpmH-rnpA region in B. subtilis 168 derivative strain YB886. Tandem promoters (16) and the SD sequence preceding rpmH are indicated by horizontal dashed or solid lines above the sequence. Coding regions for rpmH and rnpA are depicted by gray boxes. The highlighted nucleotides at the beginning of rpmH indicate a 5-bp deletion (del.) in construct pD2b (Fig. 1). The rnpA cistron contains two potential start codons (boxed); a possible SD sequence is absent with respect to the GUG start codon; a potential SD sequence for the putative UUG start codon of rnpA is indicated by the dotted line; the third position of each potential start codon was mutated singly or simultaneously to C in plasmids d7(pB.s.[rpmH(Oc)-rnpA-GTc]), d7(pB.s.[rpmH(Oc)-rnpA-TTc]), and d7(pB.s.[rpmH(Oc)-rnpA-GTcTTc]) (Table 1). G (stop) and T (stop) above the rnpA sequence depict two point mutations, resulting in stop codons, which were introduced simultaneously into rnpA in plasmid pB.s.[rpmH(Oc)-rnpA(Am,Op)]. Arrowheads below the intergenic sequence depict regions of complementarity, the first one fulfilling the requirements of a transcription terminator.
FIG. 3.
FIG. 3.
RT-PCR analysis of strain d7. PCR products were analyzed on 2.5% agarose gels. (A) Time course of rnpA expression after transfer into medium supplemented with 2% xylose (X) or 2% glucose (G). Total RNA was isolated at the indicated time points from the respective d7 culture. Con, PCR control with genomic DNA as the template; M, DNA marker with fragment sizes indicated at the right. (B) The rpsR transcript encoding ribosomal protein S18 was used to control for fluctuations in total RNA amounts. (C) Growth curves of the cultures used for RT-PCR in panels A and B. OD600, optical density at 600 nm.
FIG. 4.
FIG. 4.
Genetic complementation of strain d7. (A) Growth curves of B. subtilis d7, d7(pDG148), and d7(pB.s.[rpmH(Oc)-rnpA]) in the presence of either 2% xylose or 2% glucose. Initially, cultures were grown in medium supplemented with 2% xylose. At time point zero, duplicate cultures were inoculated to an optical density at 600 nm (OD600) of ca. 0.2 into fresh LB medium supplemented with either xylose or glucose. (B) Corresponding growth curves for strain d7(pB.s.rnpA-NH) grown in the presence of 2% glucose, with or without induction by 1 mM IPTG.
FIG. 5.
FIG. 5.
Western blot analysis to determine levels of P protein expression. Total protein was prepared from strain d7(pDG148) (lane 1), d7(pB.s.[rpmH(Oc)-rnpA-CH]) (lanes 2), or d7(pB.s.rnpA-NH) (lane 3) 1.5 h after IPTG induction by treatment of cell suspensions with trichloroacetic acid. Recombinant N-terminally His-tagged B. subtilis P protein (60 ng) was loaded as a control in lane 4. Samples were subjected to 13% sodium dodecyl sulfate-PAGE and either blotted (panel A) or stained with Coomassie blue (panel B) to control for differences in the amount of loaded total protein.
FIG. 6.
FIG. 6.
Endonucleolytic processing of 5′-32P-labeled ptRNAGly by RNase P holoenzyme bound to Ni-NTA agarose via His-tagged B. subtilis P protein. The enrichment of RNase P activity was determined after incubation of Ni-NTA resin with cell lysate from strain d7(pB.s.[rpmH(Oc)-rnpA]), d7(pB.s.[rpmH(Oc)-rnpA-CH]), d7(pB.s.rnpA-NH), or d7(pB.s.[rnpA-NH+rnpB]). Relative processing rates (krel), given at the bottom, are normalized to activity with extract from strain d7(pB.s.[rpmH(Oc)-rnpA-CH]). Vector pB.s.[rnpA-NH+rnpB] additionally encoded the B. subtilis rnpB gene under control of the Pspac promoter, including the natural Rho-independent transcription terminator downstream of rnpB. The terminator is of the type that can function in both transcription directions (4). This was assumed to prevent potential antisense interference due to the opposite orientation of rnpA and rnpB in pB.s.[rnpA-NH+rnpB].
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