RNase III Is Required for Actinomycin Production in Streptomyces antibioticus

Using insertional mutagenesis, we have disrupted the RNase III gene, rnc, of the actinomycin-producing streptomycete, Streptomyces antibioticus. Disruption was verified by Southern blotting. The resulting strain grows more vigorously than its parent on actinomycin production medium but produces significantly lower levels of actinomycin. Complementation of the rnc disruption with the wild-type rnc gene from S. antibioticus restored actinomycin production to nearly wild-type levels. Western blotting experiments demonstrated that the disruptant did not produce full-length or truncated forms of RNase III. Thus, as is the case in Streptomyces coelicolor, RNase III is required for antibiotic production in S. antibioticus. No differences in the chemical half-lives of bulk mRNA were observed in a comparison of the S. antibioticus rnc mutant and its parental strain.     

Structure and regulation of the Salmonella typhimurium rnc-era-recO operon

The Escherichia coli rnc-era-recO operon encodes ribonuclease III (RNase III; a dsRNA endonuclease involved in rRNA and mRNA processing and decay), Era (an essential G-protein of unknown function) and RecO (involved in the RecF homologous recombination pathway). Expression of the rnc and era genes is negatively autoregulated: RNase III cleaves the rncO ‘operator’ in the untranslated leader, destabilizing the operon mRNA. As part of a larger effort to understand RNase III and Era structure and function, we characterized rnc operon structure, function and regulation in the closely related bacterium Salmonella typhimurium. Construction of a S typhimurium strain conditionally defective for RNase III and Era expression showed that Era is essential for cell growth. This mutant strain also enabled selection of recombinant clones containing the intact S typhimurium rnc-era-recO operon, whose nucleotide sequence, predicted protein sequence, and predicted rncO RNA secondary structure were all highly conserved with those of E coli. Furthermore, genetic and biochemical analysis revealed that S typhimurium rnc gene expression is negatively autoregulated by a mechanism very similar or identical to that in E coli, and that the cleavage specificities of RNase III_S.t. and RNase III_E.c. are indistinguishable with regard to rncO cleavage and S typhimurium 23S rRNA fragmentation in vivo.     

Development,Antibiotic Production,and Ribosome Assembly in Streptomyces venezuelae Are Impacted by RNase J and RNase III Deletion

RNA metabolism is a critical but frequently overlooked control element affecting virtually every cellular process in bacteria. RNA processing and degradation is mediated by a suite of ribonucleases having distinct cleavage and substrate specificity. Here, we probe the role of two ribonucleases (RNase III and RNase J) in the emerging model system Streptomyces venezuelae. We show that each enzyme makes a unique contribution to the growth and development of S. venezuelae and further affects the secondary metabolism and antibiotic production of this bacterium. We demonstrate a connection between the action of these ribonucleases and translation, with both enzymes being required for the formation of functional ribosomes. RNase III mutants in particular fail to properly process 23S rRNA, form fewer 70S ribosomes, and show reduced translational processivity. The loss of either RNase III or RNase J additionally led to the appearance of a new ribosomal species (the 100S ribosome dimer) during exponential growth and dramatically sensitized these mutants to a range of antibiotics.     

DNA sequencing of the Escherichia coli ribonuclease III gene and its mutations

Summary A 0.7 kb DNA fragment of the Escherichia coli K12 chromosome was shown to contain the structural gene for RNAse III (rnc). The DNA sequence of the gene was determined and its alteration in an RNAse III defective mutant, AB301-105, was identified. DNA sequence analysis also showed that a secondary-site suppressor of a temperature-sensitive mutation in the E. coli ribosomal protein gene, rpsL, occurred within the rnc gene, providing genetic evidence for the interaction of ribosomal proteins with RNAse III, which in turn acts on the nascent ribosomal RNA during assembly of ribosomes in E. coli.     

Revertants from RNase III negative strains of Escherichia coli

Summary E. coli strains carrying the rnc-105 allele do not show any level of RNase III in extracts, grow slower than rnc ⁺ strains at temperatures up to 45°C and fail to grow at 45°C. Revertants which can grow at 45°C were isolated. The vast majority of them still do not grow as fast as rnc ⁺ strains and did not regain RNase III activity. The mutation(s) which caused them are suppressor mutations (physiological suppressors) which do not map in the immediate vicinity of the rnc gene. A few of the revertants regain normal growth, and contain normal levels of RNase III. They do not harbor the rnc-105 allele and therefore are considered to be true revertants. By using purines other than adenine it was possible to isolate rnc ⁺ pur ^- revertants from an rnc ^- pur ^- strain with relative ease. They behaved exactly like the true rnc ⁺ revertants isolated from rnc ^- strains at 45°C.A merodiploid strain which contains the rnc ⁺ gene on an episome behaves exactly like an rnc ⁺ strain with respect to growth and RNA metabolism, eventhough its specific RNase III activity is about 60% of that of an rnc ⁺ strain; thus the level of RNase III is not limiting in the cell.The rnc ^- strains show a characteristic pattern of transitory molecules, related to rRNA, 30S, 25S, p23 and 18S, which are not observed in rnc ⁺ strains. This pattern is unchanged in rnc ^- strains and in the revertants which are still lacking RNase III, regardless of the temperature in which RNA synthesis was examined (30° to 45°C). On the other hand, in the rnc ⁺ strains as well as in the true revertants and the rnc ⁺/rnc ^- merodiploid, the normal pattern of p16 and p23 is observed at all temperatures. These findings suggest that all the effects observed in RNase III^- strains are due to pleiotropic effects of the rnc-105 allele, and that the enzyme RNase III is not essential for the viability of the E. coli cell.     

Regulation of the AbrA1/A2 Two-Component System in Streptomyces coelicolor and the Potential of Its Deletion Strain as a Heterologous Host for Antibiotic Production

The Two-Component System (TCS) AbrA1/A2 from Streptomyces coelicolor M145 is a negative regulator of antibiotic production and morphological differentiation. In this work we show that it is able to auto-regulate its expression, exerting a positive induction of its own operon promoter, and that its activation is dependent on the presence of iron. The overexpression of the abrA2 response regulator (RR) gene in the mutant ΔabrA1/A2 results in a toxic phenotype. The reason is an excess of phosphorylated AbrA2, as shown by phosphoablative and phosphomimetic AbrA2 mutants. Therefore, non-cognate histidine kinases (HKs) or small phospho-donors may be responsible for AbrA2 phosphorylation in vivo. The results suggest that in the parent strain S. coelicolor M145 the correct amount of phosphorylated AbrA2 is adjusted through the phosphorylation-dephosphorylation activity rate of the HK AbrA1. Furthermore, the ABC transporter system, which is part of the four-gene operon comprising AbrA1/A2, is necessary to de-repress antibiotic production in the TCS null mutant. Finally, in order to test the possible biotechnological applications of the ΔabrA1/A2 strain, we demonstrate that the production of the antitumoral antibiotic oviedomycin is duplicated in this strain as compared with the production obtained in the wild type, showing that this strain is a good host for heterologous antibiotic production. Thus, this genetically modified strain could be interesting for the biotechnology industry.     

Molecular and functional analysis of the ribosomal L11 and S12 protein genes ( rplK and rpsL ) of Streptomyces coelicolor A3(2)

A RelC deletion mutant, KO-100, of Streptomyces coelicolor A3(2) has been isolated from a collection of spontaneous thiostrepton-resistant mutants. KO-100 grows as vigorously as the parent strain and possesses a 6-bp deletion within the rplK, previously termed relC. When the wild-type rplK gene was propagated on a low-copy-number vector in mutant KO-100, the ability to produce ppGpp, actinorhodin and undecylprodigiosin, which had been lost in the RelC mutant, was completely restored. Allele replacement by gene homogenotization demonstrated that the RelC mutation is responsible for the resistance to thiostrepton and the inactivation of ppGpp, actinorhodin and undecylprodigiosin production. Western blotting showed that ribosomes from the RelC mutant KO-100 contain only one-eighth the amount of L11 protein found in ribosomes of the parent strain. The impairment of antibiotic production in KO-100 could be rescued by the introduction of mutations that confer resistance to streptomycin (str), which result in alteration of Lys-88 in ribosomal protein S12 to Glu or Arg. No accompanying restoration of ppGpp synthesis was detected in these RelC str double mutants. Received: 12 May 1997 / Accepted: 22 July 1997     

RNase III deficient Salmonella typhimurium LT2 contains intervening sequences (IVSs) in its 23S rRNA

Salmonella typhimurium LT2 contains intervening sequences (IVSs) of 90–110 nt within all its 23S rRNA that are cleaved out by RNase III, resulting in rRNA fragmentation. In order to determine the functionality of 23S rRNA that contains unexcised IVSs, we constructed an S. typhimurium RNase III (rnc) deficient strain by transducing a mini-Tn10 (rnc-14::Tn10) from Escherichia coli K-12. The resulting strain of S. typhimurium was viable, contained IVSs within all of its 23S rRNA, and showed a growth reduction similar to that observed for the RNase III deficient strain of E. coli. These results indicate that ribosomes containing 23S rRNA in which IVSs are not excised are functional in translation, and make it unlikely that RNase III excision of IVSs from strain LT2 23S rRNA is dictated by a selective pressure to uphold the functional integrity of ribosomes.     

Translational fidelity in Escherichia coli: Antagonistic effects of neaA and ramC gene products on the ribosome function

Summary A double mutant carrying the ramC and neaA mutations has been constructed by Plvir transductions. This mutant, which carries alterations in ribosomal proteins S5 and S17, behaves like to wildtype bacteria in the following respects: it no longer exhibits the restriction of informational suppressors normally associated with the neaA mutation (altered protein S17); ribosomes from the double mutant show increased intrinsic and neamine-induced misreading in vitro in contrast to ribosomes from the neaA strain, although still less than the misreading level of ribosomes from the ramC (altered protein S5) strain.These properties suggest that ribosomal proteins S5 and S17 act cooperatively to balance translational fidelity.     

Molecular and functional analysis of the ribosomal L11 and S12 protein genes (rplK and rpsL) of Streptomyces coelicolor A3(2)

A RelC deletion mutant, KO-100, of Streptomyces coelicolor A3(2) has been isolated from a collection of spontaneous thiostrepton-resistant mutants. KO-100 grows as vigorously as the parent strain and possesses a 6-bp deletion within the rplK, previously termed relC. When the wild-type rplK gene was propagated on a low-copy-number vector in mutant KO-100, the ability to produce ppGpp, actinorhodin and undecylprodigiosin, which had been lost in the RelC mutant, was completely restored. Allele replacement by gene homogenotization demonstrated that the RelC mutation is responsible for the resistance to thiostrepton and the inactivation of ppGpp, actinorhodin and undecylprodigiosin production. Western blotting showed that ribosomes from the RelC mutant KO-100 contain only one-eighth the amount of L11 protein found in ribosomes of the parent strain. The impairment of antibiotic production in KO-100 could be rescued by the introduction of mutations that confer resistance to streptomycin (str), which result in alteration of Lys-88 in ribosomal protein S12 to Glu or Arg. No accompanying restoration of ppGpp synthesis was detected in these RelC str double mutants.     

Impairment of ribosomal subunit synthesis in aminoglycoside-treated ribonuclease mutants of Escherichia coli

The bacterial ribosome is an important target for many antimicrobial agents. Aminoglycoside antibiotics bind to both 30S and 50S ribosomal subunits, inhibiting translation and subunit formation. During ribosomal subunit biogenesis, ribonucleases (RNases) play an important role in rRNA processing. E. coli cells deficient for specific processing RNases are predicted to have an increased sensitivity to neomycin and paromomycin. Four RNase mutant strains showed an increased growth sensitivity to both aminoglycoside antibiotics. E. coli strains deficient for the rRNA processing enzymes RNase III, RNase E, RNase G or RNase PH showed significantly reduced subunit amounts after antibiotic treatment. A substantial increase in a 16S RNA precursor molecule was observed as well. Ribosomal RNA turnover was stimulated, and an enhancement of 16S and 23S rRNA fragmentation was detected in E. coli cells deficient for these enzymes. This work indicates that bacterial RNases may be novel antimicrobial targets.     

RraAS1 inhibits the ribonucleolytic activity of RNase ES by interacting with its catalytic domain in <Emphasis Type="Italic">Streptomyces coelicolor</Emphasis>

RraA is a protein inhibitor of RNase E, which degrades and processes numerous RNAs in Escherichia coli. Streptomyces coelicolor also contains homologs of RNase E and RraA, RNase ES and RraAS1/RraAS2, respectively. Here, we report that, unlike other RraA homologs, RraAS1 directly interacts with the catalytic domain of RNase ES to exert its inhibitory effect. We further show that rraAS1 gene deletion in S. coelicolor results in a higher growth rate and increased production of actinorhodin and undecylprodigiosin, compared with the wild-type strain, suggesting that RraAS1-mediated regulation of RNase ES activity contributes to modulating the cellular physiology of S. coelicolor.     

Accumulation of Barley Stripe Mosaic Virus is Significantly Reduced in Transgenic Wheat Plants Expressing a Bacterial Ribonuclease

An rnc70 gene encoding a mutant bacterial ribonuclease III (RNase III) was introduced into wheat (Triticum aestivum cv. Bobwhite) by microprojectile bombardment. T1, T2, and T3 plants regenerated from three transgenic callus lines were challenged with barley stripe mosaic virus. Plants expressing RNase III exhibited a high level of resistance to the virus infection. This resistance was evidenced by the absence of virus symptoms and reduced accumulation of virions in these plants. The result demonstrates that this pathogen-targeted resistance strategy can be effectively employed in conferring resistance to viral diseases of cereal crops.     

An Arabidopsis RNase III-like protein,AtRTL2, cleaves double-stranded RNA in vitro

Class 1 ribonuclease III (RNase III), found in bacteria and yeast, is involved in processing functional RNA molecules such as ribosomal RNAs (rRNAs). However, in Arabidopsis thaliana, the lack of an obvious phenotype or quantitative change in mature rRNAs in class 1 RNase III (AtRTL2) mutants and overexpressing plants suggests that AtRTL2 is not involved in rRNA maturation. We characterized the in vitro activity of AtRTL2 to consider its in vivo function. AtRTL2 cleaved double-stranded RNA (dsRNA) specifically in vitro, yielding products of approximately 25 nt or longer in length, in contrast to 10–20 nt long products in bacteria and yeasts. Although dsRNA-binding activity was not detected, the dsRNA-binding domains in AtRTL2 were essential for its dsRNA-cleaving activity. Accumulation of small RNAs derived from transgene dsRNAs was increased when AtRTL2 was transiently expressed in Nicotiana benthamiana leaves by agroinfiltration. These results raise the possibility that AtRTL2 has functions distinct from those of other class 1 RNase IIIs in vivo.     

The effect of both homologous and heterologous DNA on integration efficiencies in pneumococcal transformation

Summary A mutant of the yeast Saccharomyces cerevisiae has been isolated that is resistant to narciclasine, an inhibitor of peptide bond formation on 80S ribsomes. The mutant shows cross-resistance to a number of inhibitors of peptidyl transferase including anthelmycin, a 4-aminohexosyl cytosine antibiotic, which does not compete with narciclasine for its ribosomal binding site. The mutation is within the gene tcm1 or a closely linked gene on chromosome XV; it is expressed in the 60S ribosomal subunit. The parameters of the binding of (³H)narciclasine to ribosomes and ribosomal subunits from both wild-type and mutant strains have been calculated by ultracentrifugation. One molecule of narciclasine is bound per ribosome or per 60S ribosomal subunit, the values of the dissociation constants being 0.054 and 0.13 m respectively, for 80S and 60S particles from the wild-type cells. Ribosomes of the mutant strain have a lower affinity for narciclasine and trichodermin than ribosomes from wild-type cells. The mutation is semidominant in heterozygous diploid cells.