Rnc Gene Research Articles

Impact of a random TN5 mutation on endoglucanase secretion in ruminal cellulolytic Escherichia coli

ObjectiveMost protein secretion systems are found in gram-negative bacteria, but the mechanism of endoglucanase (BcsZ) secretion in Escherichia coli (E. coli) remains unclear. MethodsIn this study, we used JBZ-DH5α (which overexpresses BcsZ on the E. coli DH5α genome) as the initial strain. A mutant library was created by randomly inserting the TN5 transposon into the genome, and mutants with reduced transparent circles were identified on Congo red plates. The insertion sites of transposons in the genome were determined through whole-genome sequencing. ResultsThe results revealed that the genes rnc, lon, and suhB, which encode RNC-ribonuclease III (RNC), LON-protease (LON), and SuhB-inositol phosphatase (SuhB), respectively, were disrupted. BcsZ secretion decreased in E. coli DH5α when the lon, rnc, or suhB genes were deleted, but the overexpression of these genes restored their secretion levels. ConclusionThese findings suggest that the lon, rnc, and suhB genes play a role in BcsZ secretion in E. coli, potentially enhancing our knowledge of BcsZ secretion and offering a strategy to increase protein secretion in E. coli as a cell factory.

Comparative Transcriptomic Analysis of Flagellar-Associated Genes in Salmonella Typhimurium and Its rnc Mutant.

Salmonella enterica serovar Typhimurium (S. Typhimurium) is a globally recognized foodborne pathogen that affects both animals and humans. Endoribonucleases mediate RNA processing and degradation in the adaptation of bacteria to environmental changes and have been linked to the pathogenicity of S. Typhimurium. Not much is known about the specific regulatory mechanisms of these enzymes in S. Typhimurium, particularly in the context of environmental adaptation. Thus, this study carried out a comparative transcriptomic analysis of wild-type S. Typhimurium SL1344 and its mutant (∆rnc), which lacks the rnc gene encoding RNase III, thereby elucidating the detailed regulatory characteristics that can be attributed to the rnc gene. Global gene expression analysis revealed that the ∆rnc strain exhibited 410 upregulated and 301 downregulated genes (fold-change > 1.5 and p < 0.05), as compared to the wild-type strain. Subsequent bioinformatics analysis indicated that these differentially expressed genes are involved in various physiological functions, in both the wild-type and ∆rnc strains. This study provides evidence for the critical role of RNase III as a general positive regulator of flagellar-associated genes and its involvement in the pathogenicity of S. Typhimurium.

Fitness and Functional Landscapes of the E. coli RNase III Gene rnc.

How protein properties such as protein activity and protein essentiality affect the distribution of fitness effects (DFE) of mutations are important questions in protein evolution. Deep mutational scanning studies typically measure the effects of a comprehensive set of mutations on either protein activity or fitness. Our understanding of the underpinnings of the DFE would be enhanced by a comprehensive study of both for the same gene. Here, we compared the fitness effects and in vivo protein activity effects of ∼4,500 missense mutations in the E. coli rnc gene. This gene encodes RNase III, a global regulator enzyme that cleaves diverse RNA substrates including precursor ribosomal RNA and various mRNAs including its own 5' untranslated region (5'UTR). We find that RNase III's ability to cleave dsRNA is the most important determinant of the fitness effects of rnc mutations. The DFE of RNase III was bimodal, with mutations centered around neutral and deleterious effects, consistent with previously reported DFE's of enzymes with a singular physiological role. Fitness was buffered to small effects on RNase III activity. The enzyme's RNase III domain, which contains the RNase III signature motif and all active site residues, was more sensitive to mutation than its dsRNA binding domain, which is responsible for recognition and binding to dsRNA. Differential effects on fitness and functional scores for mutations at highly conserved residues G97, G99, and F188 suggest that these positions may be important for RNase III cleavage specificity.

RNase III coding genes modulate the cross-kingdom biofilm of Streptococcus mutans and Candida albicans.

Streptococcus mutans constantly coexists with Candida albicans in plaque biofilms of early childhood caries (ECC). The progression of ECC can be influenced by the interactions between S. mutans and C. albicans through exopolysaccharides (EPS). Our previous studies have shown that rnc, the gene encoding ribonuclease III (RNase III), is implicated in the cariogenicity of S. mutans by regulating EPS metabolism. The DCR1 gene in C. albicans encodes the sole functional RNase III and is capable of producing non-coding RNAs. However, whether rnc or DCR1 can regulate the structure or cariogenic virulence of the cross-kingdom biofilm of S. mutans and C. albicans is not yet well understood. By using gene disruption or overexpression assays, this study aims to investigate the roles of rnc and DCR1 in modulating the biological characteristics of dual-species biofilms of S. mutans and C. albicans and to reveal the molecular mechanism of regulation. The morphology, biomass, EPS content, and lactic acid production of the dual-species biofilm were assessed. Quantitative real-time polymerase chain reaction (qRT-PCR) and transcriptomic profiling were performed to unravel the alteration of C. albicans virulence. We found that both rnc and DCR1 could regulate the biological traits of cross-kingdom biofilms. The rnc gene prominently contributed to the formation of dual-species biofilms by positively modulating the extracellular polysaccharide synthesis, leading to increased biomass, biofilm roughness, and acid production. Changes in the microecological system probably impacted the virulence as well as polysaccharide or pyruvate metabolism pathways of C. albicans, which facilitated the assembly of a cariogenic cross-kingdom biofilm and the generation of an augmented acidic milieu. These results may provide an avenue for exploring new targets for the effective prevention and treatment of ECC.

Enterococcus faecalis rnc gene modulates its susceptibility to disinfection agents: a novel approach against biofilm

BackgroundEnterococcus faecalis (E. faecalis) plays an important role in the failure of root canal treatment and refractory periapical periodontitis. As an important virulence factor of E. faecalis, extracellular polysaccharide (EPS) serves as a matrix to wrap bacteria and form biofilms. The homologous rnc gene, encoding Ribonuclease III, has been reported as a regulator of EPS synthesis. In order to develop novel anti-biofilm targets, we investigated the effects of the rnc gene on the biological characteristics of E. faecalis, and compared the biofilm tolerance towards the typical root canal irrigation agents and traditional Chinese medicine fluid Pudilan.MethodsE. faecalis rnc gene overexpression (rnc+) and low-expression (rnc−) strains were constructed. The growth curves of E. faecalis ATCC29212, rnc+, and rnc− strains were obtained to study the regulatory effect of the rnc gene on E. faecalis. Scanning electron microscopy (SEM), confocal laser scanning microscopy (CLSM), and crystal violet staining assays were performed to evaluate the morphology and composition of E. faecalis biofilms. Furthermore, the wild-type and mutant biofilms were treated with 5% sodium hypochlorite (NaOCl), 2% chlorhexidine (CHX), and Pudilan. The residual viabilities of E. faecalis biofilms were evaluated using crystal violet staining and colony counting assays.ResultsThe results demonstrated that the rnc gene could promote bacterial growth and EPS synthesis, causing the EPS-barren biofilm morphology and low EPS/bacteria ratio. Both the rnc+ and rnc− biofilms showed increased susceptibility to the root canal irrigation agents. The 5% NaOCl group showed the highest biofilm removing effect followed by Pudilan and 2% CHX. The colony counting results showed almost complete removal of bacteria in the 5% NaOCl, 2% CHX, and Chinese medicine agents’ groups.ConclusionsThis study concluded that the rnc gene could positively regulate bacterial proliferation, EPS synthesis, and biofilm formation in E. faecalis. The rnc mutation caused an increase in the disinfectant sensitivity of biofilm, indicating a potential anti-biofilm target. In addition, Pudilan exhibited an excellent ability to remove E. faecalis biofilm.

The rnc Gene Regulates the Microstructure of Exopolysaccharide in the Biofilm of Streptococcus mutans through the β-Monosaccharides

Streptococcus mutans is known as the crucial pathogen of human dental caries, owing to its contribution to the biofilm development via the capacity of synthesizing exopolysaccharide (EPS), which mainly compose of α-glycosidic bond and β-glycosidic bond. β-glycosidic bond is less flexible than α-glycosidic bond because of differences between their configurational properties. Previous studies have shown that the rnc gene is implicated in the EPS formation and the cariogenicity of S. mutans. However, the effects of rnc on the microstructure of EPS have been not well-understood yet. Here, we further investigated how the rnc gene worked to modulate microstructural properties of the extracellular polysaccharide of S. mutans using glycomics methods. The gas chromatography-mass spectrometer showed that the proportion of glucose was decreased in water-soluble EPS and galactose was absent in water-insoluble EPS from the S. mutans rnc-deficient strain (Smurnc), compared with the isogenic wild-type strain (UA159). The composition of functional groups and the displacement of hydrogen bond were analyzed by infrared radiation and 1H nuclear magnetic resonance, respectively. In addition, phenotypic modulation of the biofilm matrix was assessed by microscopy. We found that the EPS of UA159 and the rnc overexpression strain (Smurnc+) mainly consisted of β-glycosidic bonds. Conversely, the EPS of Smurnc were made up of mostly α-glycosidic bonds, leading to the attenuation of biofilm biomass and bacterial adhesion. Furthermore, the existence of β-glycosidic bond was verified by enzyme digestion. Collectively, the rnc gene modulates the conversion of β-glycosidic bonds, which may play important roles in regulating the micromolecule structure of the EPS matrix, thus affecting the characteristics of S. mutans biofilm. These data illustrate that β-glycosidic bonds mediated by rnc may be potential targets for the prevention and treatment of dental caries.

A novel plasmid-Escherichia coli system produces large batch dsRNAs for insect gene silencing.

RNA interference (RNAi)-based pest management requires efficient delivery and large-batch production of double-stranded (ds)RNA. We previously developed a nanocarrier-mediated dsRNA delivery system that could penetrate an insect's body and efficiently silence gene expression. However, there is a great need to improve the plasmid-Escherichia coli system for the mass production of dsRNA. Here, for efficient dsRNA production, we removed the rnc gene encoding endoribonuclease RNase III in E. coli BL21(DE3) and matched with the RNAi expression vector containing a single T7 promoter. The novel pET28-BL21(DE3) RNase III-system was successfully constructed to express vestigial (vg)-dsRNA against Harmonia axyridis. dsRNA was extracted and purified from cell cultures in four E. coil systems, and the yields of dsRNA in pET28-BL21(DE3) RNase III-, pET28-HT115(DE3), L4440-BL21(DE3) RNase III- and L4440-HT115(DE3) were 4.23, 2.75, 0.88 and 1.30 μg mL-1 respectively. The dsRNA expression efficiency of our novel E. coil system was three times that of L4440-HT115(DE3), a widely used dsRNA production system. The RNAi efficiency of dsRNA produced by our system and by biochemical synthesis was comparable when injected into Harmonia axyridis. Our system expressed dsRNA more efficiently than the widely used L4440-HT115(DE3) system, and the produced dsRNA showed a high gene-silencing effect. Notably, our pET28-BL21(DE3) RNase III-system provides a novel method for the mass production of dsRNA at low cost and high efficiency, which may promote gene function analysis and RNAi-based pest management. © 2020 Society of Chemical Industry.

Molecular characterization of RNase III protein of Asaia sp. for developing a robust RNAi-based paratransgensis tool to affect the sexual life-cycle of Plasmodium or Anopheles fitness

BackgroundAccording to scientific recommendations, paratransgenesis is one of the solutions for improving the effectiveness of the Global Malaria Eradication Programme. In paratransgenesis, symbiont microorganisms are used for distorting or blocking the parasite life-cycle, affecting the fitness and longevity of vectors or reducing the vectorial competence. It has been revealed recently that bacteria could be used as potent tools for double stranded RNA production and delivery to insects. Moreover, findings showed that RNase III mutant bacteria are more competent for this aim. Asaia spp. have been introduced as potent paratransgenesis candidates for combating malaria and, based on their specific features for this goal, could be considered as effective dsRNA production and delivery tools to Anopheles spp. Therefore, we decided to characterize the rnc gene and its related protein to provide the basic required information for creating an RNase III mutant Asaia bacterium.MethodsAsaia bacteria were isolated from field-collected Anopheles stephensi mosquitoes. The rnc gene and its surrounding sequences were characterized by rapid amplification of genomic ends. RNase III recombinant protein was expressed in E. coli BL21 and biological activity of the purified recombinant protein was assayed. Furthermore, Asaia RNaseIII amino acid sequence was analyzed by in silico approaches such as homology modeling and docking to determine its structural properties.ResultsIn this study, the structure of rnc gene and its related operon from Asaia sp. was determined. In addition, by performing superimposition and docking with specific substrate, the structural features of Asaia RNaseIII protein such as critical residues which are involved and essential for proper folding of active site, binding of magnesium ions and double stranded RNA molecule to protein and cleaving of dsRNA molecules, were determined.ConclusionsIn this study, the basic and essential data for creating an RNase III mutant Asaia sp. strain, which is the first step of developing an efficient RNAi-based paratransgenesis tool, were acquired. Asaia sp. have been found in different medically-important vectors and these data are potentially very helpful for researchers studying paratransgenesis and vector-borne diseases and are interested in applying the RNAi technology in the field.

Effects of S. mutans gene-modification and antibacterial monomer dimethylaminohexadecyl methacrylate on biofilm growth and acid production

ObjectivesAntibacterial quaternary ammonium monomers (QAMs) are used in resins. The rnc gene in Streptococcus mutans (S. mutans) plays a key role in resisting antibiotics. The objectives of this study were to investigate for the first time: (1) the effects of rnc deletion on S. mutans biofilms and acid production; (2) the combined effects of rnc deletion with dimethylaminohexadecyl methacrylate (DMAHDM) on biofilm-inhibition efficacy. MethodsParent S. mutans strain UA159 (ATCC 700610) and the rnc-deleted S. mutans were used. Bacterial growth, minimum inhibitory concentration (MIC), and minimal bactericidal concentration (MBC) were measured to analyze the bacterial susceptibility of the parent and rnc-deleted S. mutans against DMAHDM, with the gold-standard chlorhexidine (CHX) as control. Biofilm biomass, polysaccharide and lactic acid production were measured. ResultsThe drug-susceptibility of the rnc-deleted S. mutans to DMAHDM or CHX was 2-fold higher than parent S. mutans. The drug-susceptibility did not increase after 10 passages (p < 0.05). Deleting the rnc gene increased the biofilm susceptibility to DMAHDM or CHX by 2-fold. The rnc-deletion in S. mutans reduced biofilm biomass, polysaccharide and lactic acid production, even at no drugs. DMAHDM was nearly 40 % more potent than the gold-standard CHX. The combination of rnc deletion+DMAHDM treatment achieved the greatest reduction in biofilm biomass, polysaccharide synthesis, and lactic acid production. SignificanceGene modification by deleting the rnc in S. mutans reduced the biofilm growth and acid production, and the rnc deletion+DMAHDM method showed the greatest biofilm-inhibition efficacy, for the first time. The dual strategy of antibacterial monomer+bacterial gene modification shows great potential to control biofilms and inhibit caries.

Carbohydrate Metabolism Regulated by Antisense vicR RNA in Cariogenicity

Streptococcus mutans is a major cariogenic pathogen that resides in multispecies oral microbial biofilms. The VicRK 2-component system is crucial for bacterial adaptation, virulence, and biofilm organization and contains a global and vital response regulator, VicR. Notably, we identified an antisense vicR RNA (ASvicR) associated with an adjacent RNase III–encoding (rnc) gene that was relevant to microRNA-size small RNAs (msRNAs). Here, we report that ASvicR overexpression significantly impeded bacterial growth, biofilm exopolysaccharide synthesis, and cariogenicity in vivo. Transcriptome analysis revealed that the ASvicR RNA mainly regulated carbohydrate metabolism. In particular, overproducing ASvicR demonstrated a reduction in galactose and glucose metabolism by monosaccharide composition analysis. The results of high-performance gel permeation chromatography revealed that the water-insoluble glucans isolated from ASvicR presented much lower molecular weights. Furthermore, direct evidence showed that total RNAs were disrupted by rnc-encoded RNase III. With the coexpression of T4 RNA ligase, putative msRNA1657, which is an rnc-related messenger RNA, was verified to bind to the 5′-UTR regions of the vicR gene. Furthermore, ASvicR regulation revealed a sponge regulatory-mediated network for msRNA associated with adjacent RNase III–encoding genes. There was an increase in ASvicR transcript levels in clinical S. mutans strains from caries-free children, while the expression of ASvicR was decreased in early childhood caries patients; this outcome may be explored as a potential strategy contributing to the management of dental caries. Taken together, our findings suggest an important role of ASvicR-mediated sponge regulation in S. mutans, indicating the characterization of lactose metabolism by a vital response regulator in cariogenicity. These findings have a number of implications and have reshaped our understanding of bacterial gene regulation from its transcriptional conception to the key roles of regulatory RNAs.

Sinorhizobium meliloti RNase III: Catalytic Features and Impact on Symbiosis.

Members of the ribonuclease (RNase) III family of enzymes are metal-dependent double-strand specific endoribonucleases. They are ubiquitously found and eukaryotic RNase III-like enzymes include Dicer and Drosha, involved in RNA processing and RNA interference. In this work, we have addressed the primary characterization of RNase III from the symbiotic nitrogen-fixing α-proteobacterium Sinorhizobium meliloti. The S. meliloti rnc gene does encode an RNase III-like protein (SmRNase III), with recognizable catalytic and double-stranded RNA (dsRNA)-binding domains that clusters in a branch with its α–proteobacterial counterparts. Purified SmRNase III dimerizes, is active at neutral to alkaline pH and behaves as a strict metal cofactor-dependent double-strand endoribonuclease, with catalytic features distinguishable from those of the prototypical member of the family, the Escherichia coli ortholog (EcRNase III). SmRNase III prefers Mn2+ rather than Mg2+ as metal cofactor, cleaves the generic structured R1.1 substrate at a site atypical for RNase III cleavage, and requires higher cofactor concentrations and longer dsRNA substrates than EcRNase III for optimal activity. Furthermore, the ultraconserved E125 amino acid was shown to play a major role in the metal-dependent catalysis of SmRNase III. SmRNase III degrades endogenous RNA substrates of diverse biogenesis with different efficiency, and is involved in the maturation of the 23S rRNA. SmRNase III loss-of-function neither compromises viability nor alters morphology of S. meliloti cells, but influences growth, nodulation kinetics, the onset of nitrogen fixation and the overall symbiotic efficiency of this bacterium on the roots of its legume host, alfalfa, which ultimately affects plant growth. Our results support an impact of SmRNase III on nodulation and symbiotic nitrogen fixation in plants.

Regulation mechanism of rnc gene on Streptococcus mutans environmental tolerance and its mechanism

Regulation mechanism of rnc gene on Streptococcus mutans environmental tolerance and its mechanism

Activity of Streptococcus mutans VicR Is Modulated by Antisense RNA

The VicRK 2-component system of Streptococcus mutans regulates genes associated with cell wall biogenesis and biofilm formation. A putative RNase III–encoding gene (rnc) is located downstream from the vicRKX operon. The goals of this study were to investigate the potential role of VicR in the regulation of adjacent downstream genes and evaluate transcription levels of vicR during planktonic and biofilm growth. Quantitative real-time polymerase chain reaction (qRT-PCR) was used to investigate whether vicRKX and adjacent downstream genes were cotranscribed. Binding of purified recombinant VicR protein to promoter regions of vicR, rnc, and syfA genes was confirmed by electrophoretic mobility shift assay and by chromatin immunoprecipitation analyses. VicR antisense (ASvicR) RNA was detected by Northern blotting and qRT-PCR assays. ASvicR overexpression mutants were constructed, and the biofilm biomass was determined by crystal violet microtiter assay. Adjacent downstream genes rnc, smc, syfA, smu.1511, and syfB were cotranscribed with vicRKX. The predicted promoter regions of vicR, rnc, and syfA genes were directly regulated by VicR. An ASvicR RNA transcript was detected upstream of the rnc gene. Expression of the ASvicR RNA transcript was elevated in planktonic cultures and repressed during biofilm growth. In addition, Western blot data showed that expression of the VicR protein decreased by 35% in planktonic as compared with biofilm cultures. Furthermore, we show that overexpression of ASvicR led to a reduction in biofilm formation. The downstream genes rnc, smc, syfA, smu.1511, and syfB are cotranscribed with vicRKX. VicR is autophosphorylated, and rnc and syfA are directly regulated by VicR. Expression of VicR protein correlated inversely with different levels of ASvicR RNA transcript and growth conditions. The biofilm biomass decreased in the ASvicR overexpression mutant. These data suggest a role for the ASvicR RNA transcript in posttranscriptional regulation of VicR protein production in S. mutans.

The rnc Gene Promotes Exopolysaccharide Synthesis and Represses the vicRKX Gene Expressions via MicroRNA-Size Small RNAs in Streptococcus mutans.

Dental caries is a biofilm-dependent disease that largely relies on the ability of Streptococcus mutans to synthesize exopolysaccharides. Although the rnc gene is suggested to be involved in virulence mechanisms in many other bacteria, the information regarding it in S. mutans is very limited. Here, using deletion or overexpression mutant assay, we demonstrated that rnc in S. mutans significantly positively regulated exopolysaccharide synthesis and further altered biofilm formation. Meanwhile, the cariogenecity of S. mutans was decreased by deletion of rnc in a specific pathogen-free (SPF) rat model. Interestingly, analyzing the expression at mRNA level, we found the downstream vic locus was repressed by rnc in S. mutans. Using deep sequencing and bioinformatics analysis, for the first time, three putative microRNA-size small RNAs (msRNAs) targeting vicRKX were predicted in S. mutans. The expression levels of these msRNAs were negatively correlated with vicRKX but positively correlated with rnc, indicating rnc probably repressed vicRKX expression through msRNAs at the post-transcriptional level. In all, the results present that rnc has a potential role in the regulation of exopolysaccharide synthesis and can affect vicRKX expressions via post-transcriptional repression in S. mutans. This study provides an alternative avenue for further research aimed at preventing caries.

Production of double-stranded RNA for interference with TMV infection utilizing a bacterial prokaryotic expression system

In many species, the introduction of double-stranded RNA (dsRNA) induces potent and specific gene silencing, a phenomenon called RNA interference (RNAi). RNAi is the process of sequence-specific, posttranscriptional gene silencing (PTGS) in animals and plants, mediated by dsRNA homologous to the silenced genes. In plants, PTGS is part of a defense mechanism against virus infection, and dsRNA is the pivotal factor that induces gene silencing. Here, we report an efficient method that can produce dsRNA using a bacterial prokaryotic expression system. Using the bacteriophage lambda-dependent Red recombination system, we knocked out the rnc genes of two different Escherichia coli strains and constructed three different vectors that could produce dsRNAs. This work explores the best vector/host combinations for high output of dsRNA. In the end, we found that strain M-JM109 or the M-JM109lacY mutant strain and the vector pGEM-CP480 are the best choices for producing great quantities of dsRNA. Resistance analyses and Northern blot showed that Tobacco mosaic virus infection could be inhibited by dsRNA, and the resistance was an RNA-mediated virus resistance. Our findings indicate that exogenous dsRNA could form the basis for an effective and environmentally friendly biotechnological tool that protects plants from virus infections.

Homologous and heterologous expression of RNase III from Lactococcus lactis

The endoribonuclease III (RNase III), encoded by the rnc gene, is an important enzyme for RNA metabolism. In this report a chromosomal fragment containing the rnc gene from Lactococcus lactis was cloned and its expression was analyzed. Complementation assays performed in Escherichia coli demonstrate that the lactococcal RNase III (Lac-RNase III) is able to process rRNAs and to regulate the levels of polynucleotide phosphorylase (PNPase). These results demonstrate that the lactococcal enzyme is able to substitute the Ec-RNase III not only in the rRNA processing, but also in the processing of mRNAs. The amount of lactococcal rnc transcript in an E. coli Δrnc strain was 3.3-fold higher than in the wild type strain, suggesting that the E. coli RNase III triggers the degradation of the heterologous rnc mRNA. Lac-RNase III is able to cleave an in vitro synthesized mRNA substrate specific for the Bacillus subtilis homolog. Using this substrate, we standardized an enzymatic assay which allows the specific detection of the endonucleolytic activity of Lac-RNase III in L. lactis and E. coli crude extracts.

Functional study of Lactococcus lactis RNase III in Escherichia coli

Endoribonuclease III (RNase III) is known to participate in a number of RNA maturation and decay pathways. Previous research carried out on citQRP mRNA processing had provided strong evidence that RNase III exists in Lactococcus lactis and acts as a major endoribonuclease in the control of citQRP mRNA stability. The DNA coding ( rnc) for the putative lactococcal RNase III was cloned by PCR technology. In addition, we provide the transcriptional analysis of rnc gene and compared the lactococcal RNase III sequence to those of Gram-positive and Gram-negative organisms. This computer-assisted comparison showed an identity ranking from 37% to 54%, and the highest score was obtained with Streptococcus pneumoniae RNase III. Moreover, heterologous complementation indicated that lactococcal RNase III is able to complement the loss of Escherichia coli RNase III for both 30S RNA processing and λ N posttranscriptional regulator.

Cloning and analysis of the rnc-era-recO operon from Pseudomonas aeruginosa.

The rnc operon from Pseudomonas aeruginosa has been cloned and characterized. The three genes comprising this operon, rnc, era, and recO, are arranged similarly to those in some other gram-negative bacteria. Multicopy plasmids carrying the rnc operon of P. aeruginosa functionally complement mutations of the rnc, era, and recO genes in Escherichia coli. In particular, the P. aeruginosa era homolog rescues the conditional lethality of era mutants in E. coli, and the presumptive protein has 60% identity with the Era of E. coli. We discuss these data and evidence suggesting that a GTPase previously purified from P. aeruginosa and designated Pra is not an Era homolog.

Bacillus subtilis RNase III gene: cloning, function of the gene in Escherichia coli, and construction of Bacillus subtilis strains with altered rnc loci.

The rnc gene of Bacillus subtilis, which has 36% amino acid identity with the gene that encodes Escherichia coli RNase III endonuclease, was cloned in E. coli and shown by functional assays to encode B. subtilis RNase III (Bs-RNase III). The cloned B. subtilis rnc gene could complement an E. coli rnc strain that is deficient in rRNA processing, suggesting that Bs-RNase III is involved in rRNA processing in B. subtilis. Attempts to construct a B. subtilis rnc null mutant were unsuccessful, but a strain was constructed in which only a carboxy-terminal truncated version of Bs-RNase III was expressed. The truncated Bs-RNase III showed virtually no activity in vitro but was active in vivo. Analysis of expression of a copy of the rnc gene integrated at the amy locus and transcribed from a p(spac) promoter suggested that expression of the B. subtilis rnc is under regulatory control.

Escherichia coli RNase III (rnc) autoregulation occurs independently of rnc gene translation.

Control of mRNA stability is an established means of regulating gene expression. However, the detailed mechanisms by which such control is achieved are only now emerging. In particular, there remains a question about the involvement of translation. Escherichia coli ribonuclease III (RNase III) negatively autoregulates expression of its own gene (rnc) approximately 10-fold, by cleaving the untranslated leader and initiating approximately 10-fold more rapid decay of the rnc mRNA, after which RNase III plays no further role. Here, we define the mechanism of this control further. Mutations that increase rnc gene translation abolish autoregulation by increasing the stability of the RNase III-cleaved transcript RNA approximately 10-fold, with no effect on the uncleaved species. Mutations that decrease translation destabilize the rnc mRNA in the presence or absence of RNase III. In so doing, they reveal a pathway of rnc transcript decay distinct from the RNase III-dependent pathway. Stability of a 'mini-rnc' transcript containing the rnc leader and only the first two codons of the rnc gene is unaffected by decreased translation, presumably because sequences required for this pathway were removed. Importantly, this mini-rnc transcript is regulated normally by RNase III. Moreover, rnc transcripts synthesized in vitro do not decay in cell-free extracts lacking ribosomes, unless they are first cleaved by RNase III. These two results show that RNase III cleavage can initiate rnc transcript decay independently of rnc gene translation, unambiguously establishing that control of mRNA decay need not involve changes in translation. How rnc gene translation is optimized for efficient autoregulation will also be discussed.