RNase II levels change according to the growth conditions: characterization of gmr, a new Escherichia coli gene involved in the modulation of RNase II.

In Escherichia coli, ribonucleases are effectors that rapidly modulate the levels of mRNAs for adaptation to a changing environment. Factors involved in the regulation of these ribonucleases can be relevant for mRNA stability. RNase II is one of the main ribonucleases responsible for exonucleolytic activity in E. coli extracts. We have identified and characterized a new E. coli gene, which was named gmr (gene modulating RNase II). The results demonstrate that a deletion of gmr can be associated with changes in RNase II levels and activity. Western analysis and exoribonuclease activity assays showed a threefold increase in RNase II in the gmr deletion strain. Gmr does not affect RNase II mRNA, but modulates RNase II at the level of protein stability. RNase II protein turnover is slower in the gmr deletion strain. We also show that RNase II levels change in different media, and that this regulation is abolished in a strain lacking gmr. The data presented here show that the regulation of ribonucleolytic activity can depend on growth conditions, and this regulation can be mediated by factors that are not RNases.

A Bacillus subtilis secreted protein with a role in endospore coat assembly and function.

Bacterial endospores are encased in a complex protein coat, which confers protection against noxious chemicals and influences the germination response. In Bacillus subtilis, over 20 polypeptides are organized into an amorphous undercoat, a lamellar lightly staining inner structure, and an electron-dense outer coat. Here we report on the identification of a polypeptide of about 30 kDa required for proper coat assembly, which was extracted from spores of a gerE mutant. The N-terminal sequence of this polypeptide matched the deduced product of the tasA gene, after removal of a putative 27-residue signal peptide, and TasA was immunologically detected in material extracted from purified spores. Remarkably, deletion of tasA results in the production of asymmetric spores that accumulate misassembled material in one pole and have a greatly expanded undercoat and an altered outer coat structure. Moreover, we found that tasA and gerE mutations act synergistically to decrease the efficiency of spore germination. We show that tasA is the most distal member of a three-gene operon, which also encodes the type I signal peptidase SipW. Expression of the tasA operon is enhanced 2 h after the onset of sporulation, under the control of sigmaH. When tasA transcription is uncoupled from sipW expression, a presumptive TasA precursor accumulates, suggesting that its maturation depends on SipW. Mature TasA is found in supernatants of sporulating cultures and intracellularly from 2 h of sporulation onward. We suggest that, at an early stage of sporulation, TasA is secreted to the septal compartment. Later, after engulfment of the prespore by the mother cell, TasA acts from the septal-proximal pole of the spore membranes to nucleate the organization of the undercoat region. TasA is the first example of a polypeptide involved in coat assembly whose production is not mother cell specific but rather precedes its formation. Our results implicate secretion as a mechanism to target individual proteins to specific cellular locations during the assembly of the bacterial endospore coat.

Assembly requirements and role of CotH during spore coat formation in Bacillus subtilis.

We report Western blot data showing that the 42.8-kDa product of the previously characterized cotH locus (8) is a structural component of the Bacillus subtilis spore coat. We show that the assembly of CotH requires both CotE and GerE. In agreement with these observations, the ultrastructural analysis of purified spores suggests that CotH is needed for proper formation of both inner and outer layers of the coat.

Bacillus subtilis spore coat assembly requires cotH gene expression.

Endospores of Bacillus subtilis are encased in a protein shell, known as the spore coat, composed of a lamella-like inner layer and an electron-dense outer layer. We report the identification and characterization of a gene, herein called cotH, located at 300 degrees on the B. subtilis genetic map between two divergent cot genes, cotB and cotG. The cotH open reading frame extended for 1,086 bp and corresponded to a polypeptide of 42.8 kDa. Spores of a cotH null mutant were normally heat, lysozyme, and chloroform resistant but were impaired in germination. The mutant spores were also pleiotropically deficient in several coat proteins, including the products of the previously cloned cotB, -C, and -G genes. On the basis of the analysis of a cotE cotH double mutant, we infer that CotH is probably localized in the inner coat and is involved in the assembly of several proteins in the outer layer of the coat.

PNPase modulates RNase II expression in Escherichia coli: implications for mRNA decay and cell metabolism.

PNPase and RNase II are the key regulatory exonucleases controlling mRNA decay in Escherichia coli. The rnb transcripts were found to proceed through the terminator and PNPase was found to be involved in the 3' to 5' degradation of rnb mRNA. Analysis of these longer 3' termini revealed that they are located in UA-rich regions. Comparison of single and double mutants suggested that PNPase and RNase II could have different roles in the degradation of these unstructured regions. We have shown that RNase II levels can vary over a fivefold range in haploid cells and that its expression depends on PNPase levels. PNPase-deficient strains were found to have a 2-2.5-fold increase in RNase II activity, while PNPase-overproducing strains reduced the rnb message and RNase II levels. Conversely, the amount of PNPase in the rnb deletion strain was approximately twofold higher than that in the wild-type strain. These observations suggest that the two main exonucleases are inter-regulated through a fine tuning mechanism. We discuss the implications of these results with regard to mRNA degradation and cell metabolism.

The role of endonucleases in the expression of ribonuclease II in Escherichia coli.

Ribonuclease II (RNase II), encoded by the rnb gene, is one of the two major Escherichia coli exonucleases involved in mRNA degradation. Some of the ribonucleases implicated in this process have recently been shown to be inter-regulated. In this paper we studied the effects of the endonucleases RNase E and RNase III in rnb expression. We have shown that RNase E cleaves the rnb message internally: when this ribonuclease is inactivated rnb mRNA accumulates with a concomitant increase in RNase II activity. RNase III also affects RNase II expression but in an indirect way. We discuss these implications for the regulation of mRNA degradation.

Precise physical mapping of the Escherichia coli rnb gene, encoding ribonuclease II.

Ribonuclease II (encoded by rnb) is one of the two main exonucleases involved in mRNA degradation in Escherichia coli. We report the precise physical mapping of rnb to 29 min on the chromosomal map in the vicinity of pyrF, and clarify the genetic and physical maps of this E. coli chromosomal region. The results were confirmed by the construction of a strain partially deleted for rnb.

Construction and characterization of an absolute deletion mutant of Escherichia coli ribonuclease II.

The degradation of mRNA plays a central role in the control of protein synthesis. In Escherichia coli, the rnb gene encodes ribonuclease II (RNase II), one of the two main exonucleases involved in mRNA decay. We have constructed strain CMA201, in which the rnb promoter region and the gene were deleted from the chromosome and replaced by a tetr cassette. This is the first rnb absolute deletion mutant that shows the complete absence of rnb-specific mRNA. This strain has growth characteristics similar to the wild-type, even though it has no RNase II activity, and it should be useful in studies of mRNA metabolism.

DNA sequencing and expression of the gene rnb encoding Escherichia coli ribonuclease II.

The Escherichia coli ribonuclease II (RNase II) is an exonuclease involved in mRNA degradation that hydrolyses single-stranded polyribonucleotides processively in the 3' to 5' direction. Sequencing of a 2.2 kb MseI-RsaI fragment containing the rnb gene revealed an open reading frame of 1794 nucleotides that encodes a protein of 598 amino acid residues, whose calculated molecular mass is 67,583 Da. This value is in good agreement with that obtained by sodium dodecyl sulphate/polyacrylamide gel electrophoresis of polypeptides synthesized by expression with the T7 RNA polymerase/promoter system. This system was also used to confirm the correct orientation of rnb. Translation initiation was confirmed by rnb-lacZ fusions. The mRNA start site was determined by S1 nuclease mapping. Two E. coli mutants harbouring different rnb alleles deficient in RNase II activity were complemented with the expressed fragment carrying the rnb gene.

Gene heterogeneity for tetracycline resistance in Staphylococcus spp.

Nucleotide sequences related to four tet genes were studied by hybridization in 183 clinical Staphylococcus isolates. tet(K) predominated in strains resistant only to tetracycline, while tet(M) was responsible for combined tetracycline and minocycline resistance. In strains harboring both genes, they contributed additively. tet(L) was detected in only five strains, and no hybridization was observed with tet(O).

Nucleotide sequence of the fosB gene conferring fosfomycin resistance in Staphylococcus epidermidis.

We have determined the nucleotide sequence of gene fosB of plasmid pIP1842, which confers resistant to fosfomycin in Staphylococcus epidermidis BM2641. The resistance gene was identified as a coding sequence of 417 bases pairs corresponding to a protein with a calculated Mw of 16,345 daltons. This value is in good agreement with that of 15,000, estimated by SDS-polyacrylamide gel electrophoresis of a bacterial cell-free coupled transcription-translation system. The fosB gene product exhibits 48% sequence homology with the fosfomycin resistance protein FOSA of Enterobacteriaceae. The substitutions are scattered throughout indicating that the two corresponding genes have diverged from a common ancestor. Downstream from fosB is located an open reading frame that was partially sequenced. The deduced amino-acid sequence was 50% homologous to REP alpha 1, a protein involved in the replication of the Enterococcus faecalis plasmid pAM alpha 1.

Occurrence of the Campylobacter resistance gene tetO in Enterococcus and Streptococcus spp.

The distribution of nucleotide sequences related to tetK, tetL, tetM, and tetO was studied by dot blot hybridization in 178 strains of Streptococcus and Enterococcus spp. that were resistant to tetracycline. The tetO gene, which is responsible for tetracycline resistance in Campylobacter spp., was detected in six Streptococcus strains and two Enterococcus strains, in which it was borne by similar plasmids. This observation confirms our previous proposal that tetO originated in gram-positive cocci. tetM, the most prevalent resistance gene, was present alone in 109 strains and associated with tetL in 33 strains in which the two genes contributed cooperatively to high-level tetracycline resistance. tetL was present alone in five Enterococcus strains, and tetK was detected in a single Streptococcus strain. The existence of 22 strains that did not hybridize to the probes suggest that tetracycline resistance in streptococci and enterococci involves additional gene classes as well.

Non-radioactive gene probes for the detection of tetracycline and/or minocycline resistance in staphylococci.

This report describes the development of a non-radioactive gene probe for the characterization of tetracycline and/or minocycline resistant staphylococci. Both radio-labelled and non-radioactive gene probes yielded a better characterization of the strains than the determination of minimal inhibitory concentration (MIC). A 100% correlation was found between radioactive and non-radioactive hybridization tests. The latter technique detected genes present in a single copy per bacterial genome, could be performed in 8 h, avoided radioactive hazards and autoradiography delay, and appeared therefore to be a useful tool for clinical and epidemiological studies.