Phylogeny and Evolution of RNA 3'-Nucleotidyltransferases in Bacteria.

The tRNA nucleotidyltransferases and poly(A) polymerases belong to a superfamily of nucleotidyltransferases. The amino acid sequences of a number of bacterial tRNA nucleotidyltransferases and poly(A) polymerases have been used to construct a rooted, neighbor-joining phylogenetic tree. Using information gleaned from that analysis, along with data from the rRNA-based phylogenetic tree, structural data available on a number of members of the superfamily and other biochemical information on the superfamily, it is possible to suggest a scheme for the evolution of the bacterial tRNA nucleotidyltransferases and poly(A) polymerases from ancestral species. Elements of that scheme are discussed along with questions arising from the scheme which can be explored experimentally.

Proteome analysis of biofilm produced by a Fusarium falciforme keratitis infectious agent.

Biofilms are structures that confer adaptive ability to and facilitate the virulence of fungal pathogens. Certain multi-functional proteins have been shown to be involved in fungal pathogenesis and these proteins may also be implicated in biofilm formation. The aim of this study was to identify a fungal agent isolated from the human cornea, to analyze the ability of this organism to form biofilms in vitro and to investigate protein expression in this condition. The fungus was identified by phylogenetic inference analysis. Biofilm formation and structure were evaluated by colorimetric methods and by optical and electron microscopy. We also resolved proteins obtained from biofilms and planktonic cultures by two-dimensional gel electrophoresis and identified those proteins by mass spectrometry. The fungus was identified as Fusarium falciforme. Colorimetric analysis and microscopy revealed that the highest level of biofilm formation was obtained at a concentration of 1 × 106 conidia/mL with 96 h of incubation at 28 °C. The biofilm architecture consisted of an extracellular matrix that embedded fungal filaments. We found nineteen proteins that were over-expressed in biofilms, as compared with planktonic cultures, and six proteins with unique expression in biofilms. Among the more abundant proteins identified were: transketolase, a putative antigen 1, enolase, phosphoglycerate kinase and ATP-citrate synthase. Some of these proteins are involved in basal metabolism, function as multi-functional proteins or have been described as potential virulence factors. We focused on the expression in biofilm of the enzyme, enolase, which was determined by real-time PCR. Our findings provide a perspective on the proteins associated with the formation of biofilms in vitro by an F. falciforme keratitis isolate.

Identification and biofilm development by a new fungal keratitis aetiologic agent.

BACKGROUND: In recent years, human keratitis caused by fungal plant pathogens has become more common. Biofilm is a structure that confers adaptations and virulence to fungi in keratitis. Neoscytalidium spp. are phytopathogenic and recently have been recognised as a human pathogen, using biofilm formation as a virulence factor. OBJECTIVES: The aim of this study was isolation, identification (at the species level) and characterisation of a new fungal keratitis agent. PATIENTS/METHODS: The fungus was isolated from a 67-year-old male patient with a corneal ulcer. Biofilm formation and structure were evaluated by colorimetric methods and microscopy. To identify the fungus, morphological characteristics were examined and a phylogenetic analysis was performed. RESULTS AND CONCLUSIONS: We report the identification of a fungus, a member of the genus Neoscytalidium which is associated with human keratitis. Phylogenetic analysis and morphological observations on conidiogenous cells, which occur only in arthric chains in aerial mycelium and the coelomycetous synasexual morph is absent, identified a new species, Neoscytalidium oculus sp. nov. The fungus formed biofilm at a concentration of 1 × 106  conidia/mL, during 96 hours of incubation at 37°C, and also manifested haemolysis and melanin production. This is the first report in Latin America of a new species of Neoscytalidium from a clinical isolate has been identified.

Linkage of catalysis and 5' end recognition in ribonuclease RNase J.

In diverse bacterial species, the turnover and processing of many RNAs is mediated by the ribonuclease RNase J, a member of the widely occurring metallo-ß-lactamase enzyme family. We present crystal structures of Streptomyces coelicolor RNase J with bound RNA in pre- and post-cleavage states, at 2.27 Å and 2.80 Å resolution, respectively. These structures reveal snapshots of the enzyme cleaving substrate directionally and sequentially from the 5' terminus. In the pre-cleavage state, a water molecule is coordinated to a zinc ion pair in the active site but is imperfectly oriented to launch a nucleophilic attack on the phosphate backbone. A conformational switch is envisaged that enables the in-line positioning of the attacking water and may be facilitated by magnesium ions. Adjacent to the scissile bond, four bases are stacked in a tightly sandwiching pocket, and mutagenesis results indicate that this organization helps to drive processive exo-ribonucleolytic cleavage. Like its numerous homologues, S. coelicolor RNase J can also cleave some RNA internally, and the structural data suggest how the preference for exo- versus endo-cleavage mode is linked with recognition of the chemical status of the substrate's 5' end.

SCO5745, a bifunctional RNase J ortholog, affects antibiotic production in Streptomyces coelicolor.

The bacterial RNases J are considered bifunctional RNases possessing both endo- and exonucleolytic activities. We have isolated an RNase J ortholog from Streptomyces coelicolor encoded by the gene sco5745. We overexpressed a decahistidine-tagged version of SCO5745 and purified the overexpressed protein by immobilized metal ion affinity chromatography. We demonstrated the presence of both 5'-to-3' exonucleolytic and endonucleolytic activities on the Bacillus subtilis thrS transcript. Exonucleoytic activity predominated with 5' monophosphorylated thrS, while endonucleolytic activity predominated with 5' triphosphorylated thrS. While sco5745 is the only RNase J allele in S. coelicolor, the gene is not essential. Its disruption resulted in delayed production of the antibiotic actinorhodin, overproduction of undecylprodigiosin, and diminished production of the calcium-dependent antibiotic, in comparison with the parental strain.

Streptomyces coelicolor polynucleotide phosphorylase can polymerize nucleoside diphosphates under phosphorolysis conditions, with implications for the degradation of structured RNAs.

We have examined the ability of wild-type polynucleotide phosphorylase (PNPase) from Streptomyces coelicolor and two mutant forms of the enzyme, N459D and C468A, to function in the polymerization of ADP and in the phosphorolysis of RNA substrates derived from the S. coelicolor rpsO-pnp operon. The wild-type enzyme was twice as active in polymerization as N459D and four times as active as C468A. The kcat/Km value for phosphorolysis of a structured RNA substrate by N459D was essentially the same as that observed for the wild-type enzyme, while C468A was 50% as active with this substrate. A mixture of all four common nucleoside diphosphates increased the kcat/Km for phosphorolysis of the structured substrate by the wild-type enzyme by a factor of 1.7 but did not affect phosphorolysis catalyzed by N459D or C468A. We conducted phosphorolysis of the structured substrate in the presence of nucleoside diphosphates and labeled the 3' ends of the products of those reactions using [(32)P]pCp. Digestion of the end-labeled RNAs and display of the products on a sequencing gel revealed that wild-type S. coelicolor PNPase was able to synthesize RNA 3' tails under phosphorolysis conditions while the N459D and C468A mutants could not. The wild-type enzyme did not add 3' tails to a substrate that already possessed an unstructured 3' tail. We propose a model in which the transient synthesis of 3' tails facilitates the phosphorolysis of structured substrates by Streptomyces PNPase.

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.

Streptomyces RNases - Function and impact on antibiotic synthesis.

Streptomyces are soil dwelling bacteria that are notable for their ability to sporulate and to produce antibiotics and other secondary metabolites. Antibiotic biosynthesis is controlled by a variety of complex regulatory networks, involving activators, repressors, signaling molecules and other regulatory elements. One group of enzymes that affects antibiotic synthesis in Streptomyces is the ribonucleases. In this review, the function of five ribonucleases, RNase E, RNase J, polynucleotide phosphorylase, RNase III and oligoribonuclease, and their impact on antibiotic production will be discussed. Mechanisms for the effects of RNase action on antibiotic synthesis are proposed.

RNA-Seq and RNA immunoprecipitation analyses of the transcriptome of Streptomyces coelicolor identify substrates for RNase III.

RNase III is a key enzyme in the pathways of RNA degradation and processing in bacteria and has been suggested as a global regulator of antibiotic production in Streptomyces coelicolor. Using RNA-Seq, we have examined the transcriptomes of S. coelicolor M145 and an RNase III (rnc)-null mutant of that strain. RNA preparations with reduced levels of structural RNAs were prepared by subtractive hybridization prior to RNA-Seq analysis. We initially identified 7,800 transcripts of known and putative protein-coding genes in M145 and the null mutant, JSE1880, along with transcripts of 21 rRNA genes and 65 tRNA genes. Approximately 3,100 of the protein-coding transcripts were categorized as low-abundance transcripts. For further analysis, we selected those transcripts of known and putative protein-coding genes whose levels changed by ≥ 2-fold between the two S. coelicolor strains and organized those transcripts into 16 functional categories. We refined our analysis by performing RNA immunoprecipitation of the mRNA preparation from JSE1880 using a mutant RNase III protein that binds to transcripts but does not cleave them. This analysis identified ca. 800 transcripts that were enriched in the RNA immunoprecipitates, including 28 transcripts whose levels also changed by ≥ 2-fold in the RNA-Seq analysis. We compare our results with those obtained by microarray analysis of the S. coelicolor transcriptome and with studies describing the characterization of small noncoding RNAs. We have also used the RNA immunoprecipitation results to identify new substrates for RNase III cleavage.

Expression of a polycistronic messenger RNA involved in antibiotic production in an rnc mutant of Streptomyces coelicolor.

RNase III is a double strand specific endoribonuclease that is involved in the regulation of gene expression in bacteria. In Streptomyces coelicolor, an RNase III (rnc) null mutant manifests decreased ability to synthesize antibiotics, suggesting that RNase III globally regulates antibiotic production in that species. As RNase III is involved in the processing of ribosomal RNAs in S. coelicolor and other bacteria, an alternative explanation for the effects of the rnc mutation on antibiotic production would involve the formation of defective ribosomes in the absence of RNase III. Those ribosomes might be unable to translate the long polycistronic messenger RNAs known to be produced by operons containing genes for antibiotic production. To examine this possibility, we have constructed a reporter plasmid whose insert encodes an operon derived from the actinorhodin cluster of S. coelicolor. We show that an rnc null mutant of S. coelicolor is capable of translating the polycistronic message transcribed from the operon. We show further that RNA species with the mobilities expected for mature 16S and 23S ribosomal RNAs are produced in the rnc mutant even though the mutant contains higher levels of the 30S rRNA precursor than the wild-type strain.

Insights into the Bacterial Diversity and Detection of Opportunistic Pathogens in Mexican Chili Powder.

Chili powder is the most frequently consumed spice in Mexican diets. Thus, the dissemination of microorganisms associated with chili powder derived from Capsicum annuum L. is significant during microbial quality analysis, with special attention on detection of potential pathogens. The results presented here describe the initial characterization of bacterial community structure in commercial chili powder samples. Our results demonstrate that, within the domain Bacteria, the most abundant family was Bacillaceae, with a relative abundance of 99% in 71.4% of chili powder samples, while 28.6% of samples showed an average relative abundance of 60% for the Enterobacteriaceae family. Bacterial load for aerobic mesophilic bacteria (AMB) ranged from 104 to 106 cfu/g, while for sporulated mesophilic bacteria (SMB), the count ranged from 102 to 105 cfu/g. Bacillus cereus sensu lato (s.l.) was observed at ca. ˂600 cfu/g, while the count for Enterobacteriaceae ranged from 103 to 106 cfu/g, Escherichia coli and Salmonella were not detected. Fungal and yeast counts ranged from 102 to 105 cfu/g. Further analysis of the opportunistic pathogens isolated, such as B. cereus s.l. and Kosakonia cowanii, using antibiotic-resistance profiles and toxinogenic characteristics, revealed the presence of extended-spectrum ß-lactamases (ESBLs) and Metallo-ß-lactamases (MBLs) in these organisms. These results extend our knowledge of bacterial diversity and the presence of opportunistic pathogens associated with Mexican chili powder and highlight the potential health risks posed by its use through the spread of antibiotic-resistance and the production of various toxins. Our findings may be useful in developing procedures for microbial control during chili powder production.

RNase III-dependent expression of the rpsO-pnp operon of Streptomyces coelicolor.

We have examined the expression of the rpsO-pnp operon in an RNase III (rnc) mutant of Streptomyces coelicolor. Western blotting demonstrated that polynucleotide phosphorylase (PNPase) levels increased in the rnc mutant, JSE1880, compared with the parental strain, M145, and this observation was confirmed by polymerization assays. It was observed that rpsO-pnp mRNA levels increased in the rnc mutant by 1.6- to 4-fold compared with M145. This increase was observed in exponential, transition, and stationary phases, and the levels of the readthrough transcript, initiated upstream of rpsO in the rpsO-pnp operon; the pnp transcript, initiated in the rpsO-pnp intergenic region; and the rpsO transcript all increased. The increased levels of these transcripts in JSE1880 reflected increased chemical half-lives for each of the three. We demonstrated further that overexpression of the rpsO-pnp operon led to significantly higher levels of PNPase activity in JSE1880 compared to M145, reflecting the likelihood that PNPase expression is autoregulated in an RNase III-dependent manner in S. coelicolor. To explore further the increase in the level of the pnp transcript initiated in the intergenic region in JSE1880, we utilized that transcript as a substrate in assays employing purified S. coelicolor RNase III. These assays revealed the presence of hitherto-undiscovered sites of RNase III cleavage of the pnp transcript. The position of those sites was determined by primer extension, and they were shown to be situated in the loops of a stem-loop structure.

Integrative, xylE-based promoter probe vectors for use in Streptomyces.

Two promoter probe plasmid vectors, designated pIPP1 and pIPP2, were constructed from the existing plasmids pXE4 and pSET152. pIPP1 and 2 use the xylE gene of Pseudomonas putida as a reporter and can be transferred to streptomycetes by conjugation from Escherichia coli. The function of these plasmids as promoter probes was demonstrated in Streptomyces antibioticus and Streptomyces coelicolor using the phenoxazinone synthase and polynucleotide phosphorylase promoters from S. antibioticus. xylE activity could be detected in colonies on agar plates or via the in vitro assay for catechol dioxygenase. The integration into the S. antibioticus chromosome of the constructs containing the phsA promoter was verified by Southern blotting. The presence of the bla locus in pIPP1 allows the recovery of putative promoters by marker rescue.

Acquisition of pcnB [poly(A) polymerase I] genes via horizontal transfer from the ß, γ-Proteobacteria.

Poly(A) polymerases (PAPs) and tRNA nucleotidyltransferases belong to a superfamily of nucleotidyltransferases and modify RNA 3'-ends. The product of the pcnB gene, PAP I, has been characterized in a few ß-, γ- and δ-Proteobacteria. Using the PAP I signature sequence, putative PAPs were identified in bacterial species from the α- and ε-Proteobacteria and from four other bacterial phyla (Firmicutes, Actinobacteria, Bacteroidetes and Aquificae). Phylogenetic analysis, alien index and G+C content calculations strongly suggest that the PAPs in the species identified in this study arose by horizontal gene transfer from the ß- and γ-Proteobacteria.

Detection of Bacillus cereus sensu lato Isolates Posing Potential Health Risks in Mexican Chili Powder.

The potential presence of spore-forming bacteria related to the Bacillus cereus group in Mexican chili powder elaborated from Capsicum annuum L. is of commercial and clinical interest, because chili powder is an essential spice in the Mexican diet and in diets around the globe. To facilitate detection and isolation of members of this group of spore-forming bacteria from Mexican chili powder samples, we identified colonies that grew on agar medium selective for Bacillus cereus sensu lato, supplemented with polymyxin B (10 µg/mL) and ampicillin (10 to 100 µg/mL). The presumptive B. cereus (s.l.) isolates were tested using a tRNACys-PCR-based approach and the results identified species related phylogenetically to B. cereus, B. thuringiensis, and B. toyonensis. Their toxigenic potential was assessed by serological tests to detect enterotoxins (Nhe and Hbl) and by PCR targeting the hemolysin BL (hbl) component C (hblC) and non-hemolytic enterotoxin component A (nheA). The antibiotic profiles of the isolates showed a high resistance to ß-lactams (100% of the isolates), trimethoprim-sulfamethoxazole (100%), tetracycline (90%), erythromycin (77%), clindamycin (74%), and chloramphenicol (42%). Our results indicate the presence of B. cereus s.l. with toxigenic characteristics in Mexican chili powder. Because of the potential for these organisms to cause disease through their production of various toxins, and resistance to antibiotics, we recommend that a microbiological risk assessment must be considered in the Mexican regulatory requirements.

(p)ppGpp inhibits polynucleotide phosphorylase from streptomyces but not from Escherichia coli and increases the stability of bulk mRNA in Streptomyces coelicolor.

ppGpp regulates gene expression in a variety of bacteria and in plants. We proposed previously that ppGpp or its precursor, pppGpp [referred to collectively as (p)ppGpp], or both might regulate the activity of the enzyme polynucleotide phosphorylase in Streptomyces species. We have examined the effects of (p)ppGpp on the polymerization and phosphorolysis activities of PNPase from Streptomyces coelicolor, Streptomyces antibioticus, and Escherichia coli. We have shown that (p)ppGpp inhibits the activities of both Streptomyces PNPases but not the E. coli enzyme. The inhibition kinetics for polymerization using the Streptomyces enzymes are of the mixed noncompetitive type, suggesting that (p)ppGpp binds to a region other than the active site of the enzyme. ppGpp also inhibited the phosphorolysis of a model RNA substrate derived from the rpsO-pnp operon of S. coelicolor. We have shown further that the chemical stability of mRNA increases during the stationary phase in S. coelicolor and that induction of a plasmid-borne copy of relA in a relA-null mutant increases the chemical stability of bulk mRNA as well. We speculate that the observed inhibition in vitro may reflect a role of ppGpp in the regulation of antibiotic production in vivo.

Characterization of tRNA(Cys) processing in a conditional Bacillus subtilis CCase mutant reveals the participation of RNase R in its quality control.

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 tRNA(Cys), which lacks an encoded CCA 3' end. We observed that shorter tRNA(Cys) 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 tRNA(Cys) species to detect maturation and decay intermediates and observed that decay of the tRNA(Cys) 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 tRNA(Cys) 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.

Analysis of tRNA Cys processing under salt stress in Bacillus subtilis spore outgrowth using RNA sequencing data.

Background: In spore-forming bacteria, the molecular mechanisms of accumulation of transfer RNA (tRNA) during sporulation must be a priority as tRNAs play an essential role in protein synthesis during spore germination and outgrowth. However, tRNA processing has not been extensively studied in these conditions, and knowledge of these mechanisms is important to understand long-term stress survival.    Methods:To gain further insight into tRNA processing during spore germination and outgrowth, the expression of the single copy tRNA Cys gene was analyzed in the presence and absence of 1.2 M NaCl in Bacillus subtilis using RNA-Seq data obtained from the Gene Expression Omnibus (GEO) database. The CLC Genomics work bench 12.0.2 (CLC Bio, Aarhus, Denmark, https://www.qiagenbioinformatics.com/) was used to analyze reads from the tRNA Cys gene.  Results:The results show that spores store different populations of tRNA Cys-related molecules.  One such population, representing 60% of total tRNA Cys, was composed of tRNA Cys fragments.  Half of these fragments (3´-tRF) possessed CC, CCA or incorrect additions at the 3´end. tRNA Cys with correct CCA addition at the 3´end represented 23% of total tRNA Cys, while with CC addition represented 9% of the total and with incorrect addition represented 7%. While an accumulation of tRNA Cys precursors was induced by upregulation of the rrnD operon under the control of  σ A -dependent promoters under both conditions investigated, salt stress produced only a modest effect on tRNA Cys expression and the accumulation of tRNA Cys related species. Conclusions:The results demonstrate that tRNA Cys molecules resident in spores undergo dynamic processing to produce functional molecules that may play an essential role during protein synthesis.

Geobacter sulfurreducens contains separate C- and A-adding tRNA nucleotidyltransferases and a poly(A) polymerase.

The genome of Geobacter sulfurreducens contains three genes whose sequences are quite similar to sequences encoding known members of an RNA nucleotidyltransferase superfamily that includes tRNA nucleotidyltransferases and poly(A) polymerases. Reverse transcription-PCR using G. sulfurreducens total RNA demonstrated that the genes encoding these three proteins are transcribed. These genes, encoding proteins designated NTSFI, NTSFII, and NTSFIII, were cloned and overexpressed in Escherichia coli. The corresponding enzymes were purified and assayed biochemically, resulting in identification of NTSFI as a poly(A) polymerase, NTSFII as a C-adding tRNA nucleotidyltransferase, and NTSFIII as an A-adding tRNA nucleotidyltransferase. Analysis of G. sulfurreducens rRNAs and mRNAs revealed the presence of heteropolymeric RNA 3' tails. This is the first characterization of a bacterial system that expresses separate C- and A-adding tRNA nucleotidyltransferases and a poly(A) polymerase.

Running rings around RNA: a superfamily of phosphate-dependent RNases.

The exosome of Saccharomyces cerevisiae and the degradosome of Escherichia coli are multienzyme complexes involved in the degradation of mRNA. Both contain enzymes that are similar to the phosphate-dependent exoribonuclease RNase PH. These enzymes are phosphorylases that degrade RNA from the 3'-end. A recent X-ray crystallographic study of the polynucleotide phosphorylase (PNPase) from Streptomyces antibioticus reveals, for the first time, the atomic structure of a member of the RNase PH superfamily. Here, information from the structure of PNPase is used to address two related issues. First, the structure supports the idea that PNPase, which is a trimer of multidomain subunits, arose by duplication of a gene encoding an RNase PH-like enzyme. Second, the structure might explain how RNase PH-like enzymes associate into oligomeric rings that degrade RNA in a processive reaction.