The development of variation-based rifampicin resistance in Staphylococcus aureus deciphered through genomic and transcriptomic study.

Rifampicin (RIF) resistance imposes a challenge on the antimicrobial treatment of pathogen infections. Figuring out the development mechanism of RIF resistance is critical to improving antimicrobial therapy strategy in clinics and biological treatment strategy of RIF polluted sewage in environmental engineering. The RIF resistance development of Staphylococcus aureus (S. aureus) with exposure to RIF at sub-inhibitory concentrations was comprehensively investigated via genomic and transcriptomic approaches in this study. RIF minimal inhibitory concentration (MIC) for S. aureus rapidly increased from 0.032 to 256 mg/L. Membrane permeability decrease, biofilm formation enhancement, and ROS production increase associated with RIF resistance were observed in RIF-induced strains. Through comparative genomic analysis, mutations in rpoB and rpoC were considered to be associated with RIF resistance in S. aureus mutants. Pan-genome-wide single-nucleotide variant analysis indicated that mutations at rpoB-1412, rpoB-1451, and rpoB-1457 were prevalent in 13849 public genomes of S. aureus, while mutations at rpoB-2256, and rpoC-3092 were first discovered in this study. The panorama of adaptative alteration of cellular physiological processes was observed via transcriptomic analysis. The oxidation pressure responses, metabolism, transporters, virulence factors, and multiple steps of DNA and RNA machinery were found to be perturbed by RIF in S. aureus.

Mechanical loading attenuated negative effects of nucleotide analogue reverse-transcriptase inhibitor TDF on bone repair via Wnt/ß-catenin pathway.

The nucleotide analog reverse-transcriptase inhibitor, tenofovir disoproxil fumarate (TDF), is widely used to treat hepatitis B virus (HBV) and human immunodeficiency virus infection (HIV). However, long-term TDF usage is associated with an increased incidence of bone loss, osteoporosis, fractures, and other adverse reactions. We investigated the effect of chronic TDF use on bone homeostasis and defect repair in mice. In vitro, TDF inhibited osteogenic differentiation and mineralization in MC3T3-E1 cells. In vivo, 8-week-old C57BL/6 female mice were treated with TDF for 38 days to simulate chronic medication. Four-point bending test and µCT showed reduced bone biomechanical properties and microarchitecture in long bones. To investigate the effects of TDF on bone defect repair, we utilized a bilateral tibial monocortical defect model. µCT showed that TDF reduced new bone mineral tissue and bone mineral density (BMD) in the defect. To verify whether mechanical stimulation may be a useful treatment to counteract the negative bone effects of TDF, controlled dynamic mechanical loading was applied to the whole tibia during the matrix deposition phase on post-surgery days (PSDs) 5 to 8. Second harmonic generation (SHG) of collagen fibers and µCT showed that the reduction of new bone volume and bone mineral density caused by TDF was reversed by mechanical loading in the defect. Immunofluorescent deep tissue imaging showed that chronic TDF treatment reduced the number of osteogenic cells and the volume of new vessels. In addition, chronic TDF treatment inhibited the expressions of periostin and ß-catenin, but increased the expression of sclerostin. Both negative effects were reversed by mechanical loading. Our study provides strong evidence that chronic use of TDF exerts direct and inhibitory impacts on bone repair, but appropriate mechanical loading could reverse these adverse effects.

rpoB gene mutations in rifampin-resistant Mycobacterium tuberculosis isolates from rural areas of Zhejiang, China.

OBJECTIVE: The aim was to analyze genetic mutations in the rpoB gene of rifampin-resistant Mycobacterium tuberculosis isolates (RIFR-MTB) from Zhejiang, China. METHODS: We prospectively analyzed RIFR-associated mutations in 13 rural areas of Zhejiang. Isolates were subjected to species identification, phenotype drug susceptibility testing (DST), DNA extraction, and rpoB gene sequencing. RESULTS: A total of 103 RIFR isolates were identified by DST (22 RIFR only, 14 poly-drug resistant, 49 multidrug resistant, 13 pre-extensively drug resistant [pre-XDR], and 5 extensively drug resistant [XDR]) from 2152 culture-positive sputum specimens. Gene sequencing of rpoB showed that the most frequent mutation was S450L (37.86%, 39/103); mutations P280L, E521K, and D595Y were outside the rifampicin resistance-determining region (RRDR) but may be associated with RIFR. Mutations associated with poly-drug resistant, pre-XDR, and XDR TB were mainly located at codon 445 or 450 in the RRDR. CONCLUSIONS: The frequency of rpoB RRDR mutation in Zhejiang is high. Further studies are needed to clarify the relationships between RIFR and the TTC insertion at codon 433 in the RRDR and the P280L and D595Y mutations outside the RRDR.

Toehold-Mediated Strand Displacement in a Triplex Forming Nucleic Acid Clamp for Reversible Regulation of Polymerase Activity and Protein Expression.

Triplex forming oligonucleotides are used as a tool for gene regulation and in DNA nanotechnology. By incorporating artificial nucleic acids, target affinity and biological stability superior to that of natural DNA may be obtained. This work demonstrates how a chimeric clamp consisting of acyclic (L)-threoninol nucleic acid (aTNA) and DNA can bind DNA and RNA by the formation of a highly stable triplex structure. The (L)-aTNA clamp is released from the target again by the addition of a releasing strand in a strand displacement type of reaction. It is shown that the clamp efficiently inhibits Bsu and T7 RNA polymerase activity and that polymerase activity is reactivated by displacing the clamp. The clamp was successfully applied to the regulation of luciferase expression by reversible binding to the mRNA. When targeting a sequence in the double stranded plasmid, 40 % downregulation of protein expression is achieved.

A Self-Activated Mechanism for Nucleic Acid Polymerization Catalyzed by DNA/RNA Polymerases.

The enzymatic polymerization of DNA and RNA is the basis for genetic inheritance for all living organisms. It is catalyzed by the DNA/RNA polymerase (Pol) superfamily. Here, bioinformatics analysis reveals that the incoming nucleotide substrate always forms an H-bond between its 3'-OH and ß-phosphate moieties upon formation of the Michaelis complex. This previously unrecognized H-bond implies a novel self-activated mechanism (SAM), which synergistically connects the in situ nucleophile formation with subsequent nucleotide addition and, importantly, nucleic acid translocation. Thus, SAM allows an elegant and efficient closed-loop sequence of chemical and physical steps for Pol catalysis. This is markedly different from previous mechanistic hypotheses. Our proposed mechanism is corroborated via ab initio QM/MM simulations on a specific Pol, the human DNA polymerase-η, an enzyme involved in repairing damaged DNA. The structural conservation of DNA and RNA Pols supports the possible extension of SAM to Pol enzymes from the three domains of life.

Disruption of essential plastid gene expression caused by T7 RNA polymerase-mediated transcription of plastid transgenes during early seedling development.

Transcription of plastid transgenes by plastid-targeted T7 RNA polymerase (ptT7RNAP) during early seedling development in tobacco was associated with a pale-green leaf phenotype, depletion of plastid rRNAs and arrest of shoot development. Extensive analysis of mutant seedlings at the transcript level using DNA microarrays and RNA gel blotting revealed severe disruption of plastid rRNA accumulation at 4-days post-germination and reduced transcript accumulation for the essential gene clpP. Several nuclear genes encoding plastid proteins were differentially regulated in mutant seedlings over time. Ef-Tu was upregulated at 4-days post-germination and then subsequently downregulated, while RbcS was already downregulated at this early time point. The downregulation of nuclear genes encoding plastid proteins suggests disruption of plastid-to-nucleus signalling. In contrast, transcripts of three plastid genes showed increased accumulation in mutant seedlings. Transcripts of ndhC and ndhK accumulated at high levels possibly due to T7RNAP-mediated enhancement of transcription, while ptT7RNAP-mediated transcription through the phage T7 Tphi terminator into the adjacent plastome increased the level of accD transcripts. The leakiness of the Tphi terminator has implications for the use of T7RNAP-based expression systems in plastid biotechnology.

T7 RNA polymerase-induced bending of promoter DNA is coupled to DNA opening.

To initiate transcription, T7 RNA polymerase (RNAP) forms a specific complex with its promoter DNA and melts several base pairs near the initiation site to form an open complex. Previous gel electrophoresis studies have indicated that the promoter DNA in the initiation complex is bent [Ujvari, A., and Martin, C. T. (2000) J. Mol. Biol. 295, 1173-1184]. Here we use fluorescence resonance energy transfer (FRET) to investigate the conformation of promoter DNA in the closed and open complexes of T7 RNAP. We have used steady state and time-resolved fluorescence approaches to measure the FRET efficiency in a doubly dye-labeled duplex promoter and in a premelted bubble promoter. Changes in the FRET efficiency and hence the DNA end-to-end distance changes are small when the duplex promoter forms a complex with T7 RNAP. On the other hand, FRET changes are relatively larger when the bubble promoter binds T7 RNAP or when initiating nucleotides are added to the duplex promoter-T7 RNAP complex. The shortening of DNA end-to-end distances is indicative of DNA bending in the bubble DNA complex and in the duplex promoter complex with the initiating nucleotides. Our results are consistent with the model in which in the absence of initiating nucleotides there is a distribution of closed and open complexes, and the promoter DNA is bent slightly by <40 degrees in the closed complex but bent more sharply by 86 degrees in the open complex. The energetics of DNA bending suggests that a significant part of the available free energy from promoter and polymerase interactions is utilized in DNA bending and/or untwisting. We propose that promoter opening occurs spontaneously upon DNA bending and/or untwisting as free energy is gained through interactions of the melted promoter with the T7 RNAP active site.

Two distinct curved DNAs upstream of the light-responsive psbA gene in a cyanobacterium.

A functional intrinsic DNA curvature, CIT, and potential DNA-binding factors for the basal transcription of psbA2 have been reported in a cyanobacterium, Microcystis aeruginosa K-81 (Asayama et al., Nucleic Acids Res., 30, 4658-4666 (2002)). In this article, we found another novel curved DNA, which was induced by RNA polymerases binding to the promoter region. Circular permutation analyses showed that the curved center of RNA polymerase-induced DNA bending (RIB) lies at approximately the +10 site, referring to the transcription start point as +1, in the RNA polymerase-DNA complex. Regions containing the curved center of RIB and CIT contributed to the basal transcription in vivo and in vitro. These results indicate that the region upstream of K-81 psbA2 has two distinct curved DNAs, CIT (sequence-directed type) and RIB (protein-induced type).

Natural minus-strand RNAs of alfalfa mosaic virus as in vitro templates for viral RNA polymerase. 3'-terminal non-coded guanosine and coat protein are insufficient factors for full-size plus-strand synthesis.

Replication complexes of alfalfa mosaic virus produce in vivo large quantities of plus-strand RNAs, but this production is fully dependent on the presence of coat protein. In order to study this process of RNA-dependent and coat protein-regulated RNA synthesis we have isolated the three natural minus-strand RNAs (containing any posttranscriptional modification that might have occurred) and have tested them for coat protein binding sites and template activity in an in vitro system with the viral RNA polymerase. The enzyme was prepared by an advanced isolation procedure. All three minus strands had a single non-coded G at their 3' terminus. They were not able to withdraw coat protein subunits from virions as free virion RNAs do. No sites protected by coat protein against ribonuclease T1 degradation were found. Two large T1 oligonucleotides from minus RNA 1 and one from minus RNA 3 were bound by coat protein to Millipore filters. Except for minus RNA 3 which caused a minute amount of full-size plus strand to be synthesized, the minus strands did not function as templates for full-size complementary strands. On the other hand, they gave rise to a number of well-defined shorter products, the synthesis of which was stimulated by the addition of coat protein. These products could not be elongated by a chase treatment and were probably the result of internal initiations. It is concluded that, although posttranscriptional modifications of the template and the presence of coat protein may be necessary factors for plus-strand RNA synthesis, they are certainly not sufficient. Our purified in vitro system needs further sophistication.

Antisense therapy of influenza.

The liposomally encapsulated and the free antisense phosphorothioate oligonucleotides (S-ODNs) with four target sites (PB1, PB2, PA, and NP) were tested for their abilities to inhibit virus-induced cytopathogenic effects by a MTT assay using MDCK cells. The liposomally encapsulated S-ODN complementary to the sites of the PB2-AUG initiation codon showed highly inhibitory effects. On the other hand, the inhibitory effect of the liposomally encapsulated S-ODN targeted to PB1 was considerably decreased in comparison with those directed to the PB2 target sites. The liposomally encapsulated antisense phosphorothioate oligonucleotides exhibited higher inhibitory activities than the free oligonucleotides, and showed sequence-specific inhibition, whereas the free antisense phosphorothioate oligonucleotides were observed to inhibit viral absorption to MDCK cells. Therefore, the antiviral effects of S-ODN-PB2-AUG and PA-AUG were examined in a mouse model of influenza virus A infection. Balb/c mice exposed to the influenza virus A (A/PR/8/34) strain at dose of 100 LD(50)s were treated i.v. with various doses (5-40 mg/kg) of liposomally (Tfx-10) encapsulated PB2-AUG or PA-AUG before virus infection and 1 and 3 days postinfection. PB2-AUG oligomer treated i.v. significantly prolonged the mean survival time in days (MDS) and increased the survival rates with a dose-dependent manner. We demonstrate the first successful in vivo antiviral activity of antisense administered i.v. in experimental respiratory tract infections induced with influenza virus A.

The effects of transcription and RNA processing on the initiation of chloroplast DNA replication in Chlamydomonas reinhardtii.

In Chlamydomonas reinhardtii, the origin for chloroplast DNA replication, Ori A, overlaps the coding region for the chloroplast ribosomal protein Rpl16. In an in vitro DNA replication system that uses cloned Ori A as template, alteration of transcription across rpl16 affects replication activity. S1 nuclease protection mapping of cellular RNA derived from this region revealed multiple 5' and 3' ends, and several 3' ends were mapped within mini Ori A (224 bp), the core region for replication initiation. We also demonstrated that the protein fraction used in the in vitro DNA replication system contained an RNA processing activity responsible for the generation of multiple 3' ends. The 3' ends of some of the processed RNA species coincided with those of the cellular transcripts. Initiation of DNA replication in the in vitro system changed the abundance of some of the processed RNA species, and the S1 nuclease protection pattern generated by the 3' ends now mimicked that of the in vivo transcripts. We also monitored the pattern of 3' ends in cellular transcripts from the rpl16 region during gametogenesis--when the chloroplast DNA is under-replicated--and detected a change in transcript abundance that correlated with that seen in the in vitro study. Measurements of the template activity of mutants with targeted sequences change near the sites of processing also supported the notion that the processed transcripts play an important role in DNA replication.

Activation of the alfalfa mosaic virus genome by viral coat protein in non-transgenic plants and protoplasts. The protection model biochemically tested.

In non-transgenic host plants and protoplasts alfalfa mosaic virus displays a strong need for coat protein when starting an infection cycle. The "protection model" states that the three viral RNAs must have a few coat protein subunits at their 3' termini in order to protect them in the host cell against degradation by 3'- to- 5' exoribonucleases [Neeleman L, Van der Vossen EAG, Bol JF (1993) Virology 196: 883-887]. We demonstrated that the naked genome RNAs are slightly infectious, if the inoculation is done at very high concentrations, or if it is preceded by an additional inoculation with the RNAs 1 and 2 (encoding subunits for the viral RNA polymerase). This could mean that the necessity for protection by coat protein is lost if the RNAs in large quantities can overcome the activity of the degrading enzymes, or are protected by association with the RNA polymerase, respectively. However, after having tested in protoplasts the survival of separately preinoculated naked RNA 1 during several hours before RNA 2 was inoculated, on the one hand, or of simultaneously inoculated RNAs 1 and 2, with cycloheximide in the medium during the first hours after inoculation, on the other hand, we had to conclude that the viral genome RNAs are quite stable in the cell in the absence of coat protein or RNA polymerase, respectively. This invalidates the protection model. Accommodation of the above findings by our published "messenger release model" for genome activation [Houwing CJ, Jaspars EMJ (1993) Biochimie 75: 617-621] is discussed.

CRP interacts with promoter-bound sigma54 RNA polymerase and blocks transcriptional activation of the dctA promoter.

The cAMP receptor protein (CRP) is an activator of sigma70-dependent transcription. Analysis of the sigma54-dependent dctA promoter reveals a novel negative regulatory function for CRP. CRP can bind to two distant sites of the dctA promoter, sites which overlap the upstream activator sequences for the DctD activator. CRP interacts with Esigma54 bound at the dctA promoter via DNA loop formation. When the CRP-binding sites are deleted, CRP still interacts in a cAMP-dependent manner with the stable Esigma54 closed complex via protein-protein contacts. CRP is able to repress activation of the dctA promoter, even in the absence of specific CRP-binding sites. CRP affects both the final level and the kinetics of activation. The establishment of the repression and its release by the NtrC activator proceed via slow processes. The kinetics suggest that CRP favours a new form of closed complex which interconverts slowly with the classical closed intermediate. Only the latter is capable of interacting with an activator to form an open promoter complex. Thus, Esigma54 promoters are responsive to CRP, a protein unrelated to sigma54 activators, and the repression exerted is the direct result of an interaction between Esigma54 and the CRP-cAMP complex.

Targets for inhibition of hepatitis C virus replication.

Considerable progress has been made in characterizing the proteins involved in hepatitis C virus (HCV) replication, despite the lack of a cell culture system. A number of systems have been developed to examine the processes involved in viral replication, including the initiation and processing of the viral proteins required for RNA replication, the unwinding activities of the RNA helicase and the synthesis of RNA by the viral polymerase. These processes have been examined using individually cloned proteins expressed in various in vitro systems, which may be suitable targets for antiviral agents. The viral helicase and protease domains have now been crystallized, which may enable the rational design of specific inhibitors. The recent developments in HCV research in understanding the function of the viral non-structural proteins and the establishment of in vitro screening assays may aid in the development of new antiviral agents.

redD and actII-ORF4, pathway-specific regulatory genes for antibiotic production in Streptomyces coelicolor A3(2), are transcribed in vitro by an RNA polymerase holoenzyme containing sigma hrdD.

redD and actII-ORF4, regulatory genes required for synthesis of the antibiotics undecylprodigiosin and actinorhodin by Streptomyces coelicolor A3(2), were transcribed in vitro by an RNA polymerase holoenzyme containing sigma hrdD. Disruption of hrdD had no effect on antibiotic production, indicating that redD and actII-ORF4 are transcribed in vivo by at least one other RNA polymerase holoenzyme. These data provide the first experimental evidence that HrdD can function as a sigma factor.

Transcription by T7 RNA polymerase using benzo[a]pyrene-modified templates.

Synthesis of T7 RNA polymerase is inhibited by the presence of bulky adducts in the DNA template. Of the types of adducts tested, those formed by the potent carcinogen benzo[a]pyrene (B[a]P) caused the greatest inhibition. M13 DNA molecules containing a single late T7 RNA polymerase promoter have been prepared containing B[a]P adducts in either the displaced or template strand and these have been used as templates for in vitro RNA transcription by the T7 RNA polymerase. We find that the level of inhibition of RNA synthesis is substantially greater (greater than or equal to 10-fold) when adducts are positioned specifically in the template strand. Polyacrylamide gel analysis of the products synthesized off these strand-specifically modified templates showed that adducts situated in the template strand gave rise to discrete bands which presumably represent the termination of synthesis at the adduct site while the product derived from a template containing adducts in the displaced resembled that obtained using a native template.

Preparative in vitro mRNA synthesis using SP6 and T7 RNA polymerases.

A method for preparative in vitro mRNA synthesis is presented. The method makes it possible to generate several hundred RNA copies per molecule of linearized DNA template. The protocol is applicable to the synthesis of RNAs of differing lengths (from 200 to 3000 bases), and both SP6 and T7 RNA polymerases can be used. The transcription can be scaled up to at least 6-12 ml, yielding 4.5-18 mg of homogeneous biologically active mRNA.

Structure of transcriptionally active chromatin: radiological evidence for requirement of torsionally constrained DNA.

Synthesis of alpha- and beta-globin RNA in DMSO-induced Friend's erythroleukemia cells and synthesis of immunoglobulin gamma- and kappa-chain RNA, total RNA, 5S RNA, and tRNA in mouse myeloma cells (MPC-11) was inhibited by gamma-irradiation. For all RNA species, synthesis decreased nearly exponentially as a function of radiation dose, whereas RNA size distributions, turnover rates, and specific activities of radioactively labeled RNA were affected only insignificantly. D37 values for the loss of synthesis of various RNA species correspond to target sizes ranging from 21,000 to 53,000 kd, or 30-80 kbp of DNA. These target sizes are several-fold larger than the structural genes in question; however, they correspond well with the size of DNA loops, or "domains" constrained by the nuclear matrix. The data suggest that the eukaryotic transcription unit is the torsionally constrained chromatin loop, transcription of which may be inactivated, or significantly reduced by a DNA single-strand break.

Detection of polycistronic transcripts in Newcastle disease virus infected cells and identification of their sequence content.

The synthesis of six to seven polycistronic transcripts of Newcastle disease virus (NDV) in BHK cells was detected by Northern hybridization using cDNA clones generated by reverse transcription of five NDV mRNAs. Within the molecular weight range resolved by the gel electrophoresis system employed, four of the transcripts were suggested to be distronic, containing sequences of two genes, NP-P, P-M, M-F0 and F0-HN, respectively. In addition, tricistronic molecules of M-F0-HN and possibly of NP-P-M as well as P-M-F0 appeared to develop, although they were very low in amount. These data suggest a gene order of NP-P-M-F0-HN on the NDV genome. The polycistronic as well as monocistronic transcripts were generated with an almost constant proportion in amount throughout the virus replication. Further, at least several of them were also generated under the conditions where only the primary transcription was allowed by inhibiting de novo protein synthesis. Therefore, it appears likely that there is no distinct temporal control in NDV genome expression.

RNA polymerase specificity of mRNA production and enhancer action.

To examine the RNA polymerase (EC 2.7.7.6) specificity of RNA maturation/utilization and transcriptional enhancement, we constructed a chimeric plasmid (pPolI-CAT) in which a promoter for mouse rRNA gene transcription was placed adjacent the coding sequences for chloramphenicol acetyltransferase (CAT; EC 2.3.1.28). A number of other constructs, including plasmids also containing a murine sarcoma virus enhancer or lacking any natural eukaryotic promoter sequences, were also prepared. In apparent agreement with earlier conclusions that an RNA polymerase I transcript can act as a messenger RNA, transient transfection of mouse L cells with pPolI-CAT yielded both high levels of transcription from the RNA polymerase I promoter and enzymatically active CAT protein. However, further examination revealed that CAT protein is not translated from RNA that begins at the normal rRNA transcription initiation site. Polysomal RNA is devoid of such RNA and instead consists of CAT-encoding transcripts that begin elsewhere in the mouse ribosomal DNA (rDNA) region. Since transcription of these aberrant RNAs is stimulated by the addition of a murine sarcoma virus enhancer segment, they are probably transcribed by RNA polymerase II. Transcripts that map to the authentic rRNA start site are not similarly enhanced. Moreover, unlike the RNAs deriving from the rRNA initiation site, these aberrant RNAs are more stable and the level of translatable CAT transcripts is suppressed by inclusion of larger segments of the rDNA promoter regions. Fortuitously initiated mRNAs are also formed in the absence of any natural eukaryotic promoter sequence. From these data we conclude that there is no evidence that normal RNA polymerase I transcription yields functional mRNA and that transcriptional enhancement appears to be RNA polymerase specific.