Cloning, mapping, and characterization of the Escherichia coli prc gene, which is involved in C-terminal processing of penicillin-binding protein 3.

The prc gene, which is involved in cleavage of the C-terminal peptide from the precursor form of penicillin-binding protein 3 (PBP 3) of Escherichia coli, was cloned and mapped at 40.4 min on the chromosome. The gene product was identified as a protein of about 80 kDa in maxicell and in vitro systems. Fractionation of the maxicells producing the product suggested that the product was associated with the periplasmic side of the cytoplasmic membrane. This was consistent with the notion that the C-terminal processing of PBP 3 probably occurs outside the cytoplasmic membrane: the processing was found to be dependent on the secY and secA functions, indicating that the prc product or PBP 3 or both share the translocation machinery with other extracytoplasmic proteins. DNA sequencing analysis of the prc gene region identified an open reading frame, with two possible translational starts 6 bp apart from each other, that could code for a product with a calculated molecular weight of 76,667 or 76,432. The prc mutant was sensitive to thermal and osmotic stresses. Southern analysis of the chromosomal DNA of the mutant unexpectedly revealed that the mutation was a deletion of the entire prc gene and thus that the prc gene is conditionally dispensable. The mutation resulted in greatly reduced heat shock response at low osmolarity and in leakage of periplasmic proteins.     

High-level accumulation of a recombinant antibody fragment in the periplasm of Escherichia coli requires a triple-mutant (degP prc spr) host strain

During production of a humanized antibody fragment secreted into the periplasm of Escherichia coli, proteolytic degradation of the light chain was observed. In order to determine which protease(s) were responsible for this degradation, we compared expression of the F(ab')(2) antibody fragment in several E. coli strains carrying mutations in genes encoding periplasmic proteases. Analysis of strains cultured in high cell density fermentations showed that the combination of mutations in degP prc spr was necessary for the cells to produce high levels of the desired recombinant antibody fragment. In order to eliminate the possible effects of mutations in other genes, we constructed E. coli strains with protease mutations in isogenic backgrounds and repeated the studies in high cell density fermentations. Extensive light chain proteolysis persisted in degP strains. However, light chain proteolysis was substantially decreased in prc and prc spr strains, and was further decreased with the introduction of a degP mutation in prc and prc spr mutant strains. These results show that the periplasmic protease Prc (Tsp) is primarily responsible for proteolytic degradation of the light chain during expression of a recombinant antibody fragment in E. coli, and that DegP (HtrA) makes a minor contribution to this degradation as well. The results also show that spr, a suppressor of growth defects in prc strains, is required for a prc mutant to survive throughout high cell density fermentations.     

Genetic variation in the flanking regions of Shiga toxin 2 gene in Shiga toxin-producing Escherichia coli O157:H7 isolated in Japan

PM61 is a chain-forming envC strain of Escherichia coli with a leaky outer membrane. It was found to have an oversized penicillin-binding protein 3, which was the result of an IS4 insertion in the prc gene. The other properties of PM61 were caused by the envC mutation. We cloned the envC (yibP) gene and identified the mutation site, causing a single residue substitution, H366Y, in the PM61 envC allele. The gene product was predicted to be a periplasmic protein having coiled-coil structure in the N-terminal region and homology to lysostaphin in the C-terminal region. Overexpression of envC inhibited cell growth, and overexpression of the PM61 mutant allele caused cell lysis. Disruption of the chromosomal envC caused the same defects as the envC point mutation, indicating the gene is dispensable for growth but important for normal septation/separation and cell envelope integrity.     

Prevalence and chemosusceptibility of Neisseria meningitidis and Haemophilus influenzae in a population of central Italy

The purpose of this study was to determine the prevalence of nasopharyngeal carriers of Neisseria meningitidis and Haemophilus influenzae, and to evaluate their chemoresistance. N. meningitidis was more frequently isolated in adolescents (10-15 years). All of meningococci were susceptible to ceftriaxone and rifampin, only one isolate showed reduced susceptibility to chloramphenicol and four strains showed reduced penicillin susceptibility. The results show that these drugs are still effective for prophylaxis and treatment in our area. All strains of H. influenzae were susceptible to penicillin, ceftriaxone, chloramphenicol, rifampin, azithromicin and gentamicin. 6 nontypeable strains were resistant to sulfamethoxazole-trimethoprim, 7 strains of type a and c-f, and 3 non-typeable strains showed reduced susceptibility to tetracycline. In contrast with the current trend in the world, in our area the susceptibility of H. influenzae to betalactams was 100%, therefore these antibiotics are still the drugs of choice for treatment of invasive diseases.     

HasF, a TolC-homolog of Serratia marcescens, is involved in energy-dependent efflux

A tolC-like gene (hasF) was identified upon scanning the incomplete database of the S. marcescens genome. This gene was amplified using PCR and cloned in the pUC18 vector to yield pUCHF. Sequencing of the S. marcescens tolC-like hasF gene and subsequent amino acid sequence prediction revealed approximately 80% amino acid homology with the Escherichia coli TolC. A tolC-deficient strain of E. coli (BL923) containing pUCHF/hasF was analyzed for susceptibility to fluoroquinolones (ciprofloxacin, norfloxacin, and ofloxacin), chloramphenicol, sodium dodecyl sulfate (SDS), and ethidium bromide. Antibiotic susceptibility assays of the E. coli tolC-deficient mutant BL923 demonstrated a 64-fold increase in resistance to SDS and ethidium bromide upon introduction of the S. marcescens tolC-like hasF gene. No change was observed for susceptibility to fluoroquinolones and chloramphenicol. Ethidium bromide accumulation assays performed using E. coli BL923:pUCHF established the role of the S. marcescens hasF gene product in proton gradient-dependent efflux.     

一种亨廷顿舞蹈症体外药物筛选细胞模型的建立

为建立以多聚谷氨酰胺（polyQ）为靶点的亨廷顿舞蹈症体外药物筛选细胞模型,以随机引物PCR法克隆了不同长度的 CAG片段,经序列测定正确后,分别融合到已经建立的氯霉素抗性融合蛋白表达系统pCAR中CAT的N端,重组质粒转化大肠杆菌,并在其中诱导表达,SDS-PAGE 和氯霉素抗性平板试验对目的蛋白可溶性与氯霉素活性进行测定。结果显示长度在40以上的polyQ为不溶性包涵体表达,表现为低水平的氯霉素抗性,40以下的polyQ则以可溶形式表达,表现为高水平氯霉素抗性,从而建立起能够模拟亨廷顿舞蹈症病理过程的体外细胞模型。通过检测模型细胞的氯霉素抗性,可以定性、定量地反映polyQ在体内的折叠状态和可溶性,故可以借助该模型对促溶药物或生物活性物质进行高通量筛选,为亨廷顿舞蹈症预防、诊断、治疗提供了新思路。     

Synergistic activity of coriander oil and conventional antibiotics against Acinetobacter baumannii

In this study we investigated the existence of synergistic antibacterial effect between coriander (Coriandrum sativum L.) essential oil and six different antibacterial drugs (cefoperazone, chloramphenicol, ciprofloxacin, gentamicin, tetracycline and piperacillin). The antibacterial activity of coriander oil was assessed using microdilution susceptibility testing and synergistic interaction by checkerboard assays. The association of coriander essential oil with chloramphenicol, ciprofloxacin, gentamicin and tetracycline against Acinetobacter baumannii showed in vitro effectiveness, which is an indicator of a possible synergistic interaction against two reference strains of A. baumannii (LMG 1025 and LMG 1041) (FIC index from 0.047 to 0.375). However, when tested the involvement between coriander essential oil and piperacillin or cefoperazone, the isobolograms and FIC index showed an additive interaction. The in vitro interaction could improve the antimicrobial effectiveness of ciprofloxacin, gentamicin and tetracycline and may contribute to resensitize A. baumannii to the action of chloramphenicol.     

Identification and molecular characterisation of CmeB, a Campylobacter jejuni multidrug efflux pump

A multidrug efflux pump gene (cmeB) was identified from the published Campylobacter jejuni genome sequence. Secondary structural analysis showed that the gene encoded a protein belonging to the resistance nodulation cell division (RND) family of efflux transporters. The gene was inactivated by insertional mutagenesis. Compared with the wild-type strain (NCTC 11168), the resultant knockout strain (NCTC 11168-cmeB::kan(r)) displayed increased susceptibility to a range of antibiotics including beta-lactams, fluoroquinolones, macrolides, chloramphenicol, tetracycline, ethidium bromide, the dye acridine orange and the detergent sodium dodecyl sulfate. Accumulation of ciprofloxacin was increased in the knockout mutant, but carbonyl cyanide m-chlorophenyl hydrazone, a proton motive force inhibitor, had less effect upon ciprofloxacin accumulation in the knockout mutant compared with NCTC 11168. These data show that the identified gene encodes an RND-type multi-substrate efflux transporter, which contributes to intrinsic resistance to a range of structurally unrelated compounds in C. jejuni. This efflux pump has been named CmeB (for Campylobacter multidrug efflux).     

Versatile cloning vectors derived from the runaway-replication plasmid pKN402

Two cloning vectors have been constructed employing runaway-replication mutants of plasmid R1. One of these, pMOB45, carries tetracycline and chloramphenicol resistance. The other, pMOB48, carries chloramphenicol resistance, lacOP, and an assayable part of the lacPOZ operon. Both of these plasmids can be amplified to high levels by heat induction, which condition does not lead to inhibition of protein synthesis; thus the plasmid can produce large amounts of DNA and protein. In pMOB48, a unique BamHI site is present near the amino-terminus of the beta-galactosidase gene. Chimeras formed by the insertion of restriction fragments at this site can be detected on X-gal plates, and can be used for the lacIq-controlled expression of proteins which are fused to the amino-terminus of beta-galactosidase. Induction with IPTG at 40 degrees C leads to the synthesis of extremely high levels of proteins whose gene have been cloned into this site.     

Heterologous gene expression in Bacteroides fragilis.

Bacteroides fragilis and other gastrointestinal tract Bacteroides are unusual gram-negative eubacteria in that genes from other gram-negative eubacteria are not expressed when introduced into these organisms. To analyze gene expression in Bacteroides, expression vector and promoter probe (detection) vector systems were developed. The essential feature of the expression vector was the incorporation of a Bacteroides insertion sequence element, IS4351, which possesses promoter activity directed outward from its ends. Genes inserted into the multiple cloning site downstream from an IS4351 DNA fragment were readily expressed in B. fragilis. The chloramphenicol acetyltransferase (cat) structural gene from Tn9 was tested and conferred chloramphenicol resistance on B. fragilis. Both chloramphenicol resistance and CAT activity were shown to be dependent on the IS4351 promoters. Similar results were obtained with the Escherichia coli beta-glucuronidase gene (uidA) but activity was just 30% of the levels seen with cat. Two tetracycline resistance determinants, tetM from Streptococcus agalactiae and tetC from E. coli, also were examined. tetC did not result in detectable tetracycline resistance but the gram-positive tetM gene conferred high-level resistance to tetracycline and minocycline in Bacteroides hosts. Based on the cat results, promoter probe vectors containing the promoterless cat gene were constructed. These vectors were used to clone random B. fragilis promoters from partial genomic libraries and the recombinants displayed a range of CAT activities and chloramphenicol MICs in B. fragilis hosts. In addition, known E. coli promoters (Ptet, Ptac, Ptrc, Psyn, and P1P2rrnB) were tested for activity in B. fragilis. No chloramphenicol resistance or CAT activity was observed in B. fragilis with these promoters.     

Occurrence of Chloramphenicol-acetylating Enzymes in Various Gram-negative Bacilli

The occurrence of a chloramphenicol-acetylating enzyme, similar to that found in Escherichia coli, carrying an R factor was investigated in various gram-negative bacilli. The acetylated products of chloramphenicol were identified by chromatography and quantitatively assayed after benzene extraction. The investigated strains were of the Salmonella-Arizona group, the Klebsiella-Aerobacter group, Serratia marcescens, the Proteus group, and Pseudomonas aeruginosa, most of which were isolated from 1947 to 1957. Both chloramphenicol-sensitive and -resistant strains were included, but none of them was able to transfer chloramphenicol resistance by conjugation. In the Proteus group, a significant level of a chloramphenicol-acetylating enzyme was found in most strains, whether they were sensitive or resistant to chloramphenicol; the resistant strains showed higher levels of the enzyme. Some chloramphenicol-sensitive strains lacked this enzyme. Only the sensitive strains containing the enzyme could easily produce chloramphenicol-resistant mutants with higher enzyme activity. Thus, the chloramphenicol resistance of this group can be reasonably explained on the basis of the chloramphenicol-acetylating enzyme. All of the Pseudomonas aeruginosa strains were resistant to chloramphenicol, and most strains showed low levels of the enzyme (which, however, did not appear sufficient to explain their resistance). All of the strains of the other groups (except one strain of Enterobacter cloacae) lacked the enzyme, although most strains of the Klebsiella-Aerobacter group and of S. marcescens were resistant to chloramphenicol. With respect to the origin of the resistance gene of the R factor, it is noteworthy that the strains of Proteus mirabilis isolated in 1947 possessed this enzyme before the discovery of chloramphenicol.     

Spirochaeta aurantia has diacetyl chloramphenicol esterase activity

The free-living spirochete Spirochaeta aurantia was nearly as susceptible to diacetyl chloramphenicol, the product of chloramphenicol acetyltransferase, as it was to chloramphenicol itself. This unexpected susceptibility to diacetyl chloramphenicol was wholly or partly the consequence of intrinsic carboxylesterase activity, as indicated by high-performance liquid chromatography, thin-layer chromatography, and microbiological assays. The esterase converted the diacetate to chloramphenicol, thus inhibiting spirochete growth. The esterase activity was cell associated, reduced by proteinase K, eliminated by boiling, and independent of the presence of either chloramphenicol or diacetyl chloramphenicol. S. aurantia extracts also hydrolyzed other esterase substrates, and two of these, alpha-napthyl acetate and 4-methylumbelliferyl acetate, identified an esterase of approximately 75 kDa in a nondenaturing gel. Carboxylesterases occur in Streptomyces species, but in this study their activity was weaker than that of S. aurantia. The S. aurantia esterase could reduce the effectiveness of cat as either a selectable marker or a reporter gene in this species.     

Mobilization and location of the genetic determinant of chloramphenicol resistance from Lactobacillus plantarum caTC2R.

The mobilization of a nonconjugative plasmid (pCaT) that mediates chloramphenicol resistance in Lactobacillus plantarum caTC2R was achieved by comobilization with the conjugative plasmid pAM beta 1. The conjugation studies confirmed that the 8.5-kb pCaT in L. plantarum caTC2R contains the gene responsible for chloramphenicol resistance and that the plasmid has several unique restriction sites which make it useful for genetic studies in Carnobacterium spp. Cloning studies showed that the gene responsible for chloramphenicol resistance is located in the 2.6-kb EcoRV-SalI region of pCaT. This was confirmed by probing the 3.0-kb BglII fragment of pCaT with a biotin-labeled 1.6-kb BstEII-HpaII fragment from the streptococcal-derived plasmid pVA797(Cmr). Expression of chloramphenicol resistance in Carnobacterium as well as in other Lactobacillus species was achieved by electrotransformation using donor DNA from pCaT.     

RNT4 3'-UTR insertion/deletion polymorphisms are not associated with atrial septal defect in Chinese Han population: a brief communication

Atrial septal defect (ASD) is a common type of congenital heart disease, which is defined as any communication through atrial septum. Several studies have revealed that genetic factors may influence the susceptibility of ASD. Recent studies have shown that reticulon 4 (RTN4) gene might be involved in some processes relevant to heart development, such as regulation of cell migration and vascular remodeling. This study aimed to evaluate RTN4 gene polymorphisms of CAA and TATC insertion/deletion in relation to the risk of ASD in Chinese Han population. A total of 175 ASD patients and 308 unrelated healthy controls were successfully investigated. The polymorphisms of patients were determined by polymerase chain reaction-polyacrylamide gel electrophoresis. There was no significant difference in the allele frequencies of CAA and TATC insertion/deletion in RNT4 gene between ASD patients and controls. The same results were seen in their genotypes. The present study suggests that CAA and TATC insertion/deletion polymorphisms of RNT4 gene may not be a useful marker to predict the susceptibility of ASD in Chinese Han population.