Characterization of new l,d-endopeptidase gene product CwlK (previous YcdD) that hydrolyzes peptidoglycan in Bacillus subtilis

We generated a conditional CCase mutant of Bacillus subtilis to explore the participation in vivo of the tRNA nucleotidyltransferase (CCA transferase or CCase) in the maturation of the single-copy tRNACys, which lacks an encoded CCA 3′ end. We observed that shorter tRNACys species, presumably lacking CCA, only accumulated when the inducible Pspac : cca was introduced into an rnr mutant strain, but not in combination with pnp. We sequenced the tRNA 3′ ends produced in the various mutant tRNACys species to detect maturation and decay intermediates and observed that decay of the tRNACys occurs through the addition of poly(A) or heteropolymeric tails. A few clones corresponding to full-size tRNAs contained either CCA or other C and/or A sequences, suggesting that these are substrates for repair and/or decay. We also observed editing of tRNACys at position 21, which seems to occur preferentially in mature tRNAs. Altogether, our results provide in vivo evidence for the participation of the B. subtilis cca gene product in the maturation of tRNAs lacking CCA. We also suggest that RNase R exoRNase in B. subtilis participates in the quality control of tRNA.

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A highly thermostable neutral protease from Bacillus caldolyticus: cloning and expression of the gene in Bacillus subtilis and characterization of the gene product.

Journal of Bacteriology ◽

10.1128/jb.173.13.4107-4115.1991 ◽

1991 ◽

Vol 173 (13) ◽

pp. 4107-4115 ◽

Cited By ~ 35

Author(s):

B van den Burg ◽

H G Enequist ◽

M E van der Haar ◽

V G Eijsink ◽

B K Stulp ◽

...

Keyword(s):

Bacillus Subtilis ◽

Gene Product ◽

Cloning And Expression ◽

Neutral Protease

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Characterization of the Depletion of 2-C-Methyl-d-Erythritol-2,4-Cyclodiphosphate Synthase in Escherichia coli and Bacillus subtilis

Journal of Bacteriology ◽

10.1128/jb.184.20.5609-5618.2002 ◽

2002 ◽

Vol 184 (20) ◽

pp. 5609-5618 ◽

Cited By ~ 35

Author(s):

Tracey L. Campbell ◽

Eric D. Brown

Keyword(s):

Escherichia Coli ◽

Cell Wall ◽

Bacillus Subtilis ◽

Gene Product ◽

Cell Walls ◽

Isoprenoid Biosynthesis ◽

Synthetic Lethal ◽

E Coli

ABSTRACT The ispF gene product in Escherichia coli has been shown to catalyze the formation of 2-C-methyl-d-erythritol 2,4-cyclodiphosphate (MEC) in the deoxyxylulose (DOXP) pathway for isoprenoid biosynthesis. In this work, the E. coli gene ispF and its Bacillus subtilis orthologue, yacN, were deleted and conditionally complemented by expression of these genes from distant loci in the respective organisms. In E. coli, complementation was achieved through integration of ispF at the araBAD locus with control from the arabinose-inducible araBAD promoter, while in B. subtilis, yacN was placed at amyE under control of the xylose-inducible xylA promoter. In both cases, growth was severely retarded in the absence of inducer, consistent with these genes being essential for survival. E. coli cells depleted of MEC synthase revealed a filamentous phenotype. This was in contrast to the depletion of MEC synthase in B. subtilis, which resulted in a loss of rod shape, irregular septation, multicompartmentalized cells, and thickened cell walls. To probe the nature of the predominant deficiency of MEC synthase-depleted cells, we investigated the sensitivity of these conditionally complemented mutants, grown with various concentrations of inducer, to a wide variety antibiotics. Synthetic lethal behavior in MEC synthase-depleted cells was prevalent for cell wall-active antibiotics.

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