Phosphorylation and functionality of CdtR in Clostridium difficile.

The production of TcdA, TcdB and CDT in Clostridium difficile PCR ribotype 027, is regulated by the two-component system response regulator CdtR. Despite this, little is known about the signal transduction pathway leading to the activation of CdtR. In this study, we generated R20291ΔPalocΔcdtR model strains expressing CdtR phospho-variants in which our predicted phospho-accepting Asp, Asp61 was mutated for Ala or Glu. The constructs were assessed for their ability to restore CDT production. Dephospho-CdtR-Asp61Ala was completely non-functional and mirrored the cdtR-deletion mutant, whilst phospho-CdtR-Asp61Glu was functional, possessing 38-52% of wild-type activity. Taken together, these data suggest that CdtR is activated by phosphorylation of Asp61. The same principles were applied to assess the function of PCR ribotype 078-derived CdtR, which was shown to be non-functional owing to polymorphisms present within its coding gene. Conversely, polymorphisms present within its promoter region, provide significantly enhanced promoter activity compared with its PCR ribotype 027 counterpart. To ensure our data were representative for each ribotype, we determined that the cdtR nucleotide sequence was conserved in a small library of eight PCR ribotype 027 clinical isolates and nineteen PCR ribotype 078 isolates from clinical and animal origin.

Effect of antibiotic treatment on the formation of non-spore Clostridium difficile persister-like cells.

Background: The spore is the virulence factor identified to be involved in the recurrence of the disease caused by Clostridium difficile. Objectives: To demonstrate that lethal antibiotic concentrations induce the appearance of C. difficile persister-like non-spore cells. Methods: C. difficile and derivative spo0A mutant strains were tested for their susceptibility to antibiotics, as determined using an agar dilution method. Persister-cell generation was determined for all strains using up to 10â€Š× the MIC of every antibiotic for up to 6 days. Results: Using up to 10â€Š× the MIC of every antibiotic, we were able to induce the appearance of persister-like behaviour since biphasic killing curves could be observed in response to treatment antibiotics. Conclusions: To the best of our knowledge, this work provides, for the first time, experimental evidence of the appearance of C. difficile persister-like cells, opening a new research avenue in the pathogenesis of this nosocomial pathogen.

Development of Clostridium difficile R20291ΔPaLoc model strains and in vitro methodologies reveals CdtR is required for the production of CDT to cytotoxic levels.

Assessing the regulation of Clostridium difficile transferase (CDT), is complicated by the presence of a Pathogenicity locus (PaLoc) which encodes Toxins A and B. Here we developed R20291ΔPaLoc model strains and cell-based assays to quantify CDT-mediated virulence. Their application demonstrated that the transcriptional regulator, CdtR, was required for CDT-mediated cytotoxicity.

A Novel Bacteriophage with Broad Host Range against Clostridioides difficile Ribotype 078 Supports SlpA as the Likely Phage Receptor.

Bacteriophages represent a promising option for the treatment of Clostridioides difficile (formerly Clostridium difficile) infection (CDI), which at present relies on conventional antibiotic therapy. The specificity of bacteriophages should prevent dysbiosis of the colonic microbiota associated with antibiotic treatment of CDI. While numerous phages have been isolated, none have been characterized with broad host range activity toward PCR ribotype (RT) 078 strains, despite their relevance to medicine and agriculture. In this study, we isolated four novel C. difficile myoviruses: ΦCD08011, ΦCD418, ΦCD1801, and ΦCD2301. Their characterization revealed that each was comparable with other C. difficile phages described in the literature, with the exception of ΦCD1801, which exhibited broad host range activity toward RT 078, infecting 15/16 (93.8%) of the isolates tested. In order for wild-type phages to be exploited in the effective treatment of CDI, an optimal phage cocktail must be assembled that provides broad coverage against all C. difficile RTs. We conducted experiments to support previous findings suggesting that SlpA, a constituent of the C. difficile surface layer (S-layer) is the likely phage receptor. Through interpretation of phage-binding assays, our data suggested that ΦCD1801 could bind to an RT 012 strain only in the presence of a plasmid-borne S-layer cassette corresponding to the slpA allele found in RT 078. Armed with this information, efforts should be directed toward the isolation of phages with broad host range activity toward defined S-layer cassette types, which could form the basis of an effective phage cocktail for the treatment of CDI. IMPORTANCE Research into phage therapy has seen a resurgence in recent years owing to growing concerns regarding antimicrobial resistance. Phage research for potential therapy against Clostridioides difficile infection (CDI) is in its infancy, where an optimal "one size fits all" phage cocktail is yet to be derived. The pursuit thus far has aimed to find phages with the broadest possible host range. However, for C. difficile strains belonging to certain PCR ribotypes (RTs), in particular RT 078, phages with broad host range activity are yet to be discovered. In this study, we isolate four novel myoviruses, including ΦCD1801, which exerts the broadest host range activity toward RT 078 reported in the literature. Through the application of ΦCD1801 to phage-binding assays, we provide data to support the prior notion that SlpA represents the likely phage receptor on the bacterial cell surface. Our finding directs research attention toward the isolation of phages with activity toward strains possessing defined S-layer cassette types.

Recent advances in the genetics of the clostridia.

Several laboratories around the world have started work on genetic analysis of clostridia. Interest in this diverse group of anaerobic organisms has grown with increasing awareness of the benefits that may accrue from their biotechnological exploitation. Research to date has focussed on construction of shuttle vectors containing replicons from clostridial and streptococcal plasmids, development of methods for transferring genes, and molecular cloning of genes--especially those involved in toxigenicity, fermentative metabolism and polysaccharide utilization. In selected species gene transfer by protoplast transformation, electroporation and conjugation has been accomplished and transposable elements have been introduced. It can be anticipated that our understanding of the molecular biology of these interesting organisms will grow rapidly in the future, bringing with it improved prospects for rational biotechnological exploitation.

Chemotherapeutic tumour targeting using clostridial spores.

The toxicity associated with conventional cancer chemotherapy is primarily due to a lack of specificity for tumour cells. In contrast, intravenously injected clostridial spores exhibit a remarkable specificity for tumours. This is because, following their administration, clostridial spores become exclusively localised to, and germinate in, the hypoxic/necrotic tissue of tumours. This unique property could be exploited to deliver therapeutic agents to tumours. In particular, genetic engineering could be used to endow a suitable clostridial host with the capacity to produce an enzyme within the tumour which can metabolise a systemically introduced, non-toxic prodrug into a toxic metabolite. The feasibility of this strategy (clostridial-directed enzyme prodrug therapy, CDEPT) has been demonstrated by cloning the Escherichia coli B gene encoding nitroreductase (an enzyme which converts the prodrug CB1954 to a highly toxic bifunctional alkylating agent) into a clostridial expression vector and introducing the resultant plasmid into Clostridium beijerinckii (formerly C. acetobutylicum) NCIMB 8052. The gene was efficiently expressed, with recombinant nitroreductase representing 8% of the cell soluble protein. Following the intravenous injection of the recombinant spores into mice, tumour lysates have been shown, by Western blots, to contain the E. coli-derived enzyme.

Improved plasmid vectors for the isolation of translational lac gene fusions.

The beta-galactosidase fusion vector pMC1403 has been modified to include the unique cloning sites EcoRI, SmaI, BamHI, SalI, AccI, PstI and HindIII. The new vectors (pNM480, pNM481 and pNM482) allow the fusion of genes to beta-galactosidase in all three translational reading frames, and exhibit an increased sensitivity of promoter detection due to a higher copy number.

The complete nucleotide sequence of the Pseudomonas gene coding for carboxypeptidase G2.

The complete nucleotide sequence of the Pseudomonas chromosomal gene coding for the enzyme carboxypeptidase G2 (CPG2) has been determined. The nucleotide sequence obtained has been confirmed by comparing the predicted amino acid sequence with that of randomly derived peptide fragments and by N-terminal sequencing of the purified protein. The gene has been shown to code for a 22 amino acid signal peptide at its N-terminus which closely resembles the signal peptides of other secreted proteins. An alternative 36 amino acid signal peptide which may function in Pseudomonas has also been identified. The codon utilisation of the gene is influenced by the high G + C (67.2%) content of the DNA and exhibits a 92.8% preference for codons ending in G or C. This unusual codon preference may contribute to the generally observed weak expression of Pseudomonas genes in Escherichia coli. A region of DNA upstream of the structural gene has also been sequenced and a ribosome binding site and two putative promoter sequences identified.

Cloning and complete nucleotide sequence of the Bacillus stearothermophilus tryptophanyl tRNA synthetase gene.

The Bacillus stearothermophilus NCA 1503 tryptophanyl tRNA synthetase (WTS; EC 6.1.1.2) gene has been cloned in Escherichia coli and the amino acid (aa) sequence of the enzyme deduced unequivocally from the DNA sequence of the cloned gene. The predicted aa sequence of the WTS enzyme agrees with the previously determined aa sequence except that the DNA sequence indicates a third Arg residue at the C terminus of the enzyme over the two Arg residues indicated by sequencing the protein itself. The trpS gene consists of a 984-bp open reading frame commencing with an ATG start codon and ending with a TAA stop codon. Putative transcriptional promoters, a Shine-Dalgarno sequence and a transcription terminator have been identified. Thus the trpS gene probably constitutes a single transcriptional unit.

The pMTL nic- cloning vectors. I. Improved pUC polylinker regions to facilitate the use of sonicated DNA for nucleotide sequencing.

A series of nic- cloning vectors have been constructed analogous to the pUC plasmids but which are smaller in size and carry more extensive polylinker regions within the lacZ' gene. The vectors pMTL20 and pMTL21 carry six additional sites (AatII, MluI, NcoI, BglII, XhoI and StuI) to those present in pUC18 and pUC19, while pMTL22 and -23 possess eleven new cloning sites (ActII, MluI, NcoI, BglII, XhoI, StuI, NaeI, EcoRV, ClaI, NdeI and NruI). More importantly, the relative order of the restriction sites within the polylinker of these latter vectors has been totally rearranged, relative to pUC18 and pUC19, to facilitate the conversion of DNA fragments with incompatible ends to fragments with compatible termini. The availability of such DNA fragments is a crucial requirement when M13 templates are generated for dideoxy sequencing by the sonication procedure. Derivatives of these vectors have also been constructed which demonstrate improved segregational stability by incorporation of the pSC101 par locus. During the construction of these new vectors data were obtained which demonstrated that the pUC and pMTL plasmids contain a previously unreported single base pair difference within the RNA I/RNA II region (compared to pBR322) responsible for a three-fold increase in plasmid copy number. The pUC and pMTL plasmids were also shown to be functionally nic-, thus affording the lowest categorisation in genetic manipulation experiments.

Complete nucleotide sequence of the Rhodosporidium toruloides gene coding for phenylalanine ammonia-lyase.

The complete nucleotide sequence of the Rhodosporidium toruloides gene coding for the enzyme phenylalanine ammonia-lyase (PAL) has been determined. The primary structure of PAL was deduced from the nucleotide sequence of the two cDNA clones, pPAL1 and pPAL2, which covered the entire amino acid-coding sequence. Comparison of cDNA and genomic sequences of pal revealed the presence of six introns. The nucleotide sequences of these introns were compared to those from other fungi. The primary amino acid sequence of the enzyme exhibits only 30.8% identity with the determined primary sequence of PAL from Phaseolus vulgaris. Upstream from the structural gene there is a stretch of C + T-rich DNA similar to that found upstream from a number of Neurospora and Saccharomyces cerevisiae genes. In the case of S. cerevisiae, these C + T-rich sequences are thought to be involved in the transcription of highly expressed genes.

Nucleotide sequence analysis of the gene for protein A from Staphylococcus aureus Cowan 1 (NCTC8530) and its enhanced expression in Escherichia coli.

Nucleotide sequence analysis of the gene (spa) for staphylococcal protein A (SPA) from Staphylococcus aureus strain Cowan 1 (NCTC8530) shows that the sequence differs from previously reported SPA nucleotide sequences, especially in the number of repeat units in the cell-wall-binding region of the gene. Dot matrix comparison with streptococcal protein G and the macrophage receptor for the constant fragment (Fc) of immunoglobulins shows a limited but significant homology. The homology to the latter probably identifies the Fc-binding region in the immunoglobulin-binding domains of SPA. Enhanced production of SPA in Escherichia coli was achieved using the lac promoter immediately upstream from the spa gene.

High-level expression of the phenylalanine ammonia lyase-encoding gene from Rhodosporidium toruloides in Saccharomyces cerevisiae and Escherichia coli using a bifunctional expression system.

A chimeric yeast promoter (pPGK::REP2), capable of directing high-level gene expression in both Saccharomyces cerevisiae and Escherichia coli, has been constructed. It was derived by fusing the promoter of the yeast PGK gene (encoding phosphoglycerate kinase) to a region residing immediately 5' to the yeast 2 mu plasmid REP2 gene (encoding a trans-acting plasmid maintenance protein). In S. cerevisiae, transcripts initiated within the REP2-derived moiety of the promoter, but the transcription start point was dictated by the PGK determinator sequence. Promoter function in E. coli was due to the presence of consensus prokaryotic -35 and -10 motifs in the REP2 moiety. To facilitate expression studies, the promoter was incorporated into a versatile series of S. cerevisiae/E. coli shuttle vectors which provided a choice of selectable marker and copy number in S. cerevisiae. To maximise translational efficiency, a novel cloning strategy was devised which allows the juxtaposition of genes to the promoter such that the heterologous AUG replaces that of the REP2 AUG, without any alteration in the surrounding nucleotide (nt) context. This strategy was used to place both the Tn903 neo gene and the Rhodosporidium toruloides phenylalanine ammonia lyase (PAL)-encoding gene under the transcriptional control of pPGK::REP2. In the former case, cells became resistant to extremely high levels of Geneticin (> 3 mg/ml in the case of S. cerevisiae). In the case of the latter, PAL was shown to accumulate to approx. 9 and 10% of total soluble protein in S. cerevisiae and E. coli, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

Nucleotide sequence of the Erwinia chrysanthemi NCPPB 1066 L-asparaginase gene.

The complete nucleotide sequence of the Erwinia chrysanthemi NCPPB 1066 gene coding for the chemotherapeutic enzyme L-asparaginase has been determined. The structural gene consists of an open reading frame commencing with an ATG start codon of 1044 bp followed by a TGA stop codon. Confirmation of the nucleotide sequence was obtained by comparing the predicted amino acid (aa) sequence with that derived by N-terminal aa sequencing of the purified protein. The gene has been shown to code for a 21-aa signal peptide at its N terminus which closely resembles the signal peptides of other secreted proteins. In common with highly expressed Escherichia coli genes, little use is made of modulator codons. The predicted aa sequence of the enzyme exhibits 46% identity with the determined primary sequence of the E. coli L-asparaginase, although the predicted secondary structure of both proteins indicates more extensive homology. Downstream of the TGA stop codon is a G + C-rich region of dyad symmetry (delta G = -25.4 kcal) characteristic of E. coli Rho-independent transcription terminators. Upstream of the structural gene there are no sequences which bear a strong resemblance to the consensus -35 and -10 regions of E. coli promoters. A sequence is present (CTGGCTCTCCTCTTGAT), however, which exhibits strong homology to the nif promoter consensus sequence (CTGGCACN5TTGCA). Upstream of this region is a sequence which strongly resembles the consensus sequence for promoter regions which are subject to catabolite repression.

Cloning and sequence analysis of the genes encoding phosphotransbutyrylase and butyrate kinase from Clostridium acetobutylicum NCIMB 8052.

An 8.1-kb fragment of chromosomal DNA from Clostridium acetobutylicum NCIMB 8052 (formerly NCIB 8052) has been cloned into plasmid pAT153 and shown to allow the growth of Escherichia coli LJ32 (F+ atoC2c atoD32 fadR) on butyrate as the sole source of carbon and energy. Deletion analysis delineated a 3.9-kb subfragment capable of complementation. The nucleotide sequence of this fragment was determined and it was shown to encode three complete, and two incomplete open reading frames (ORFs). Based on enzymic studies of recombinant clones, two of these ORFs were shown to encode phosphotransbutyrylase and butyrate kinase. The above enzymes are involved in the acidogenic phase of fermentation in C. acetobutylicum. The fragment also carries an incomplete ORF encoding a polypeptide exhibiting substantial similarity to dihydropteroate synthase.

Physical characterisation of the replication region of the Streptococcus faecalis plasmid pAM beta 1.

The complete nucleotide (nt) sequence of a 5.1-kb EcoRI DNA restriction fragment carrying the replication region of the Streptococcus faecalis plasmid pAM beta 1 has been determined. Of the seven major open reading frames (ORF A-G) identified within this fragment, two (C and E) were shown to be encoding by in vitro transcription/translation assays. Evidence was obtained that synthesis of the polypeptide (Mr 57,380) encoded by the largest ORF (E) was essential for replication. Deletion analysis indicated that the minimum unit of DNA required for replication resided on a 2.59-kb AccI-HpaI subfragment. ORF C resided outside of this fragment and encompassed an extensive region of directly repeated nt sequence. The encoded polypeptide (Mr 30,471) was therefore composed of large tracts of reiterated amino acid sequence (11 x VDP and 35 x TEP tripeptides) which probably caused the observed anomalous electrophoretic mobility of the synthesised protein (equivalent to 61 kDa). Deletion of a 416-bp segment of DNA between unique KpnI and StyI sites caused an increase in copy number, which correlated with the in vitro production of higher levels of ORF E polypeptide. Although homology was detected between the sequenced DNA, and the replicon of a closely related streptococcal plasmid (pSM19035), none was evident to any other characterised Gram+ plasmid.

Cloning, expression and complete nucleotide sequence of the Bacillus stearothermophilus L-lactate dehydrogenase gene.

The structural gene for L-lactate dehydrogenase (LDH; EC 1.1.1.27) from Bacillus stearothermophilus NCA 1503 has been cloned in Escherichia coli and its complete nucleotide sequence determined. The predicted amino acid (aa) sequence of the LDH enzyme agrees with the previously determined aa sequence except to three positions: aa 125 and 126, Ser-Glu, are inverted whilst His at position 130 has been replaced by Ser in our sequence. The lct gene consists of an open reading frame (ORF) commencing from the ATG start codon of 951 bp followed by a TGA stop codon. Upstream from the start codon is a strong (delta G = -14.4 kcal) Shine-Dalgarno (SD) sequence, a feature typical of Gram-positive ribosome binding sites. Putative RNA polymerase recognition signals (-35 and -10 regions) have been identified upstream from the lct structural gene but there are no structures resembling Rho-independent transcription termination signals downstream from the TGA stop codon. Two further ORFs, preceded by SD sequences, are present downstream from the lct gene. Thus the lct gene may constitute the first gene of an operon. Subclones of the lct gene have been constructed in the expression plasmid pKK223-3 and the LDH enzyme produced in soluble form at levels of up to 36% of the E. coli soluble cell protein.

Expression of the bacterial nitroreductase enzyme in mammalian cells renders them selectively sensitive to killing by the prodrug CB1954.

A recombinant retrovirus encoding E. coli nitroreductase (NTR) was used to infect mammalian cells. NIH3T3 cells expressing NTR were killed by the prodrug CB1954, which NTR converts to a bifunctional alkylating agent. Admixed, unmodified NIH3T3 cells could also be killed. In contrast to the Herpes simplex virus (HSV) thymidine kinase (TK)/ganciclovir(GCV) enzyme/prodrug system, NTR/CB1954 cell killing was effective in non-cycling cells. Co-operative killing was observed when cells expressing both NTR and TK were treated with a combination of CB1954 and GCV. NTR expression in human melanoma, ovarian carcinoma or mesothelioma cells also rendered them sensitive to CB1954 killing. These data suggest that delivery of the NTR gene to human tumours, followed by treatment with CB1954, may provide a novel tumour gene therapy approach.

Cloning and nucleotide sequences of the mdh and sucD genes from Thermus aquaticus B.

A 3 kb DNA fragment containing the gene (mdh) encoding malate dehydrogenase (MDH) from the thermophile Thermus aquaticus B was cloned in Escherichia coli and its nucleotide sequence determined. Comparative analysis showed the nucleotide sequence to be very closely related to that determined for the Thermus flavus mdh gene and flanking regions, with no differences between the predicted amino acid sequences of the MDHs. A proximal open reading frame, identified as the sucD gene, and the mdh gene may be parts of the same operon in T. aquaticus B. Expression of the T. aquaticus B mdh gene in E. coli was found to be at a relatively low level. A simple method for purification of thermostable MDH from the E. coli clone containing the T. aquaticus B mdh gene is presented.

Molecular analysis of the malR gene of Clostridium butyricum NCIMB 7423, a member of the LacI-GalR family of repressor proteins.

Deletion of a region of DNA 5' to a previously characterised malQ gene of Clostridium butyricum resulted in increased production of the enzyme activity encoded by malQ, 4-alpha-glucanotransferase. Nucleotide sequence analysis revealed the presence of an open reading frame capable of encoding a protein of 335 amino acids. This protein was found to share 33% amino acid sequence identity with the Bacillus subtilis CcpA (catabolite control protein) repressor, 28% identity with the Streptomyces coelicolor MalR repressor, and 30%, 25%, and 21% amino acid identity with the Escherichia coli repressors GalR, LacI and MalI, respectively. The amino-terminal domain was predicted to be able to form a helix-turn-helix structure, and shared highest similarity with the equivalent functional domain from the E. coli LacI repressor. Interruption of malR by the generation of a frameshift mutation led to a 10-fold increase in MalQ activity. These data suggest that the identified open reading frame encodes a repressor of the C. butyricum malQ gene, and of the adjacent malP gene. The gene has, therefore, been designated malR, and its encoded gene product MalR.