Transformation of Coxiella burnetii to ampicillin resistance.

A 5.8-kb chromosomal fragment isolated from Coxiella burnetii initiates plasmid replication in Escherichia coli and was characterized as an autonomous replication sequence, ars (M. Suhan, S.-Y. Chen, H.A. Thompson, T.A. Hoover, A. Hill, and J.C. Williams, J. Bacteriol. 176:5233-5243, 1994). In the present study, an ars replicon was used to transform C. burnetii to ampicillin resistance. Plasmid pSKO(+)1000 contained the C. burnetii ars sequence cloned into a ColE1-type replicon encoding beta-lactamase. pSKO(+)1000 was introduced into C. burnetii by electroporation. Ampicillin-resistant cells were selected, and survivors were examined for the transformed genotype by Southern hybridization. Transformants stably maintained the pSKO(+)1000 bla DNA sequence in the chromosome as a result of homologous recombination. The recombination event resulted in the duplication of the 5.8-kb ars sequence in the C. burnetii chromosome. The bla gene was also located in an episome. However, an ampicillin resistance plasmid lacking the C. burnetii ars sequence did not stably transform C. burnetii. A biological assay analyzing beta-lactamase activity of C. burnetii transformants during acid activation in vitro provided evidence for expression of the bla (beta-lactamase) gene.     

Iodometric method for detection of beta-lactamase activity in yeast cells carrying ampicillin resistance gene in chimeric plasmids

In order to determine whether an ampicillin resistance gene in a chimeric plasmid is active in transformed yeast cells, it is necessary to have a simple and quick assay procedure. We describe here a procedure for achieving this goal using an iodometric color reaction. This method is based on the fact that the ampicillin resistance gene product, beta-lactamase, can hydrolyze penicillin G and release a reducing product, which can be visualized by the discoloration of a dark blue iodine-starch complex. We have improved this method so that the assay can be carried out on agar plate and in liquid culture. It permits the detection of the beta-lactamase enzyme activity in yeast liquid culture at a concentration as low as 1 X 10(5) cells/ml within 12 h. This method is especially useful for certain yeast transformation systems, such as industrial yeast cultures, where the transformants can be selected only by drug resistance.     

A simple method for maximizing the yields of membrane and exported proteins expressed in Escherichia coli

The feasibility of using a beta-lactamase fusion approach for maximizing the levels of periplasmic or membrane-bound proteins expressed in Escherichia coli was investigated. The coding region for mature TEM beta-lactamase was fused after the signal peptide and aminoterminal portion of the coding region of a weakly expressed periplasmic protein, PBP3*. The resultant plasmid was mutagenized and transformants expressing increased levels of ampicillin resistance were selected. The PBP3* gene of the unmutagenized beta-lactamase fusion plasmid, and of two mutant derivatives encoding increased ampicillin resistance, were then reassembled and the latter constructs were found to express increased levels of PBP3*. The applications of a beta-lactamase fusion approach in monitoring and optimizing levels of extracytoplasmic gene products expressed in E. coli are considered.     

Escherichia coli K-12 mutants hyperproducing chromosomal beta-lactamase by gene repetitions.

Escherichia coli K-12 ampicillin-resistant mutants hyperproducing chromosomal beta-lactamase arose spontaneously from strains carrying ampA1 ampC(+). Such mutants were found even in a recA background. Two Amp(r)-100 strains were analyzed genetically. The Amp(r)-100 resistance level of both strains could be transduced by direct selection for ampicillin resistance. Several classes of ampicillin-resistant transductants were found that differed from one another in the beta-lactamase activity and the ampicillin resistance mediated by an ampA1 ampC(+)-carrying strain. The data suggested that beta-lactamase hyperproduction was due to repetitions of the chromosomal amp genes. The size of the repeated region was calculated from cotransduction estimates, using the formula of Wu (Genetics 54:405-410, 1966), and was found to be about 1 min in one strain and 1.5 min in the other. Second-step Amp(r)-400 mutants were isolated from an Amp(r)-100 strain. The resistance of these mutants was apparently also due to repetitions, each mediating a resistance to about 10 mug/ml. Mutants of wild-type strains that were moderately resistant to ampicillin also gave rise to intermediate-resistance classes, suggesting repetitions of the wild-type amp alleles. F' factors hyperproducing chromosomal beta-lactamase by gene repetitions were constructed. They mediated levels of ampicillin resistance comparable to that of naturally occurring resistance plasmids. The expression of beta-lactamase hyperproduction was not affected by the presence of ampA and ampC alleles in trans and did not act in trans on the other alleles.     

Intrinsic penicillin resistance in penicillinase-producing Neisseria gonorrhoeae strains

The minimal inhibitory concentrations (MICs) of ampicillin for fifty strains of beta-lactamase-producing Neisseria gonorrhoeae (PPNG) isolated in Japan ranged from 1.56 to 200 micrograms/ml, and all the strains harbored a 4.5 megadalton plasmid. These strains were classified into two groups: dicloxacillin-susceptible (28%) and -resistant group (72%). A linear correlation was found in the dicloxacillin-susceptible strains between their beta-lactamase activity and the susceptibility to ampicillin, but not in the dicloxacillin-resistant strains. This suggests that the high ampicillin resistance in PPNG is due not only to acquiring the beta-lactamase producing plasmid, but also to some intrinsic resistance of the strains. To investigate a cause of the high ampicillin resistance, the beta-lactamase-producing plasmid, pTMS1, was transferred by conjugation to a penicillin-susceptible gonococcal strain as well as to its isogenic multiply antibiotic-resistant transformants, and the susceptibility of the transconjugants to ampicillin was determined. Acquisition of pTMS1 by a penicillin-susceptible strain resulted in a 32-fold increase in resistance to ampicillin, whereas the increase was 128-fold for its isogenic strains which contain some chromosomal mutations. These results suggest that reduced permeability of the outer membrane to ampicillin underlies the high ampicillin resistance of PPNG.     

低温菌启动子探针质粒的构建

为了在宿主菌Acinetobacter sp.DWC6中构建低温菌蛋白表达载体,以pBR322质粒为基础,去除质粒上β-内酰胺酶基因的启动子片段,取而代之为来源于质粒pJRD215的卡那霉素抗性基因片段,并在pBR322中插入Acinetobacter菌属特异性ori的DNA片段,构建了能在Acinetobacter sp.DWC6和E.coli中正常复制的启动子探针质粒pBAP1。通过在质粒pBAP1中的β-内酰胺酶基因上游随机导入Acinetobacter sp.DWC6基因组片段,通过检测宿主细胞的氨苄青霉素抗性和β-内酰胺酶活性,来筛选强启动子片段,并分析了启动子探针质粒载体的功能及启动子的强度。     

A staphylococcal plasmid that replicates and expresses ampicillin, gentamicin and amikacin resistance in Escherichia coli

Plasmid pPG1 from Staphylococcus aureus coding for ampicillin (Apr), gentamicin (Gmr) and amikacin (Akr) resistance was transformed into Escherichia coli. Transformation efficiency was about 2 x 10(3) transformants/micrograms of plasmid DNA. The plasmids present in the E. coli transformants were identical to pPG1 according to their restriction patterns. The copy number of pPG1 was estimated to be at least 20-times less in E. coli than in S. aureus. The minimal inhibitory concentrations (MICs) for Ap and Gm were lower in E. coli than in S. aureus. However, the MIC for Ak was higher in E. coli transformants than in S. aureus. pPG1 was maintained in the E. coli transformants for at least 80 generations at 37 degrees C without antibiotic selection pressure.     

Optimal conditions for genetic transformation of the cyanobacterium Anacystis nidulans R2

Under optimal conditions, the cyanobacterium Anacystis nidulans R2 was transformed to ampicillin resistance at frequencies of greater than 10(7) transformants per microgram of plasmid (pCH1) donor DNA. No stringent period of competency was detected, and high frequencies of transformation were achieved with cultures at various growth stages. Transformation increased with time after addition of donor DNA up to 15 to 18 h. The peak of transformation efficiency (transformants/donor molecule) occurred at plasmid concentrations of 125 to 325 ng/ml with an ampicillin resistance donor plasmid (pCH1) and 300 to 625 ng/ml for chloramphenicol resistance conferred by plasmid pSG111. The efficiency of transformation was enhanced by excluding light during the incubation or by blocking photosynthesis with the electron transport inhibitor 3-(3, 4-dichlorophenyl)-1, 1-dimethylurea (DCMU) or the uncoupler carbonyl cyanide-m-chlorophenyl hydrazone. Preincubation of cells in darkness for 15 to 18 h before addition of donor DNA significantly decreased transformation efficiency. Growth of cells in iron-deficient medium before transformation enhanced efficiency fourfold. These results were obtained with selection for ampicillin (pCH1 donor plasmid)- or chloramphenicol (pSG111 donor plasmid)-resistant transformants. Approximately 1,000 transformants per microgram were obtained when chromosomal DNA from an herbicide (DCMU)-resistant mutant was used as donor DNA. DCMU resistance was also transferred to recipient cells by using restriction fragments of chromosomal DNA from DCMU-resistant mutants. This procedure allowed size classes of fragments to be assayed for the presence of the DCMU resistance gene.     

Selection for signal sequence mutations that enhance production of secreted human proinsulin by Escherichia coli

This paper describes a method for the positive selection of signal sequence mutations that result in enhanced production of secreted human proinsulin by Escherichia coli. Coding sequences for the structural portion of beta-lactamase (EC 3.5.2.6) were substituted for those of the C terminus of proinsulin in a plasmid that normally directs the synthesis and secretion of proinsulin. The resulting plasmid directed the synthesis of a proinsulin/beta-lactamase fusion protein that was secreted into the periplasmic space and conferred resistance to low levels of ampicillin (Ap). Beneficial changes to the signal sequence were selected by the host's ability to grow on high levels of Ap. The beta-lactamase coding sequences were then replaced with those of human proinsulin, resulting in plasmids which directed enhanced production of secreted proinsulin.     

Mediation, by Saccharomyces cerevisiae translocation signals, of beta-lactamase transport through the Escherichia coli inner membrane and sensitive method for detection of signal sequences.

Signal sequences of Saccharomyces cerevisiae invertase and alpha-factor pheromone were tested for the ability to mediate protein transport through the inner membrane of Escherichia coli by fusion to bacterial beta-lactamase lacking the signal sequence (blaS0). Both types of transformants exhibited ampicillin resistance in accordance with the transport of the fused protein to the periplasmic compartment. This compartment contained most of the beta-lactamase activity present in the cell. Therefore, the tested yeast signal sequences, which conferred translocation of their proteins across the membrane of the endoplasmic reticulum in S. cerevisiae, can provide the same function in E. coli. The screening for ampicillin resistance among blaS0 fusions provides a convenient method for the isolation of functional yeast and possibly higher eucaryotic signal sequences.     

Stability of wild-type and mutant RTEM-1 beta-lactamases: effect of the disulfide bond

Uniquely among class A beta-lactamases, the RTEM-1 and RTEM-2 enzymes contain a single disulfide bond between Cys 77 and Cys 123. To study the possible role of this naturally occurring disulfide in stabilizing RTEM-1 beta-lactamase and its mutants at residue 71, this bond was removed by introducing a Cys 77----Ser mutation. Both the wild-type enzyme and the single mutant Cys 77----Ser confer the same high levels of resistance to ampicillin in vivo to Escherichia coli; at 30 degrees C the specific activity of purified Cys 77----Ser mutant is also the same as that of the wild-type enzyme. Also, neither wild-type enzyme nor the Cys 77----Ser mutant is inactivated by brief exposure to p-hydroxymercuribenzoate. However, above 40 degrees C the mutant enzyme is less stable than wild-type enzyme. After introduction of the Cys 77----Ser mutation, none of the double mutants (containing the second mutations at residue 71) confer resistance to ampicillin in vivo at 37 degrees C; proteins with Ala, Val, Leu, Ile, Met, Pro, His, Cys, and Ser at residue 71 confer low levels of resistance to ampicillin in vivo at 30 degrees C. The use of electrophoretic blots stained with antibodies against beta-lactamase to analyze the relative quantities of mutant proteins in whole-cell extracts of E. coli suggests that all 19 of the doubly mutant enzymes are proteolyzed much more readily than their singly mutant analogues (at Thr 71) that contain a disulfide bond.(ABSTRACT TRUNCATED AT 250 WORDS)     

Cloning of methylated transforming DNA from Neurospora crassa in Escherichia coli.

An arg-2 mutant of Neurospora crassa was transformed to prototrophy with a pBR322-N. crassa genomic DNA library. Repeated attempts to recover the integrated transforming DNA or segments thereof by digestion, ligation, and transformation of Escherichia coli, with selection for the plasmid marker ampicillin resistance, were unsuccessful. Analyses of a N. crassa transformant demonstrated that the introduced DNA was heavily methylated at cytosine residues. This methylation was shown to be responsible for our inability to recover transformants in standard strains of E. coli; transformants were readily obtained in a strain which is deficient in the two methylcytosine restriction systems. Restriction of methylated DNA in E. coli may explain the general failure to recover vector or transforming sequences from N. crassa transformants.     

Integration of exogenous DNA into the genome of Azotobacter vinelandii

The soil bacterium Azotobacter vinelandii was genetically transformed by chromosomal integration to ampicillin and/or tetracycline resistance using restriction endonuclease-linearized plasmids. Polyacrylamide gel electrophoresis of protein extracts from three independently isolated ampicillin resistant transformants showed the presence of a 28 Kd band which is the approximate size of the ampicillin resistance gene product (i.e., -lactamase). Moreover, with nitrocefin, a chromogenic cephalosporin, as a substrate, it was shown that all of the ampicillin resistant transformants produced functional -lactamase. DNA hybridization showed that the chromosomal DNA from transformed cells contained plasmid DNA sequences at discrete sites. Growth experiments indicated that stable A. vinelandii transformants that carry functional integrated DNA were physiologically impaired.     

Overcoming resistance to beta-lactamase inhibitors: comparing sulbactam to novel inhibitors against clavulanate resistant SHV enzymes with substitutions at Ambler position 244

Amino acid changes at Ambler position R244 in class A TEM and SHV beta-lactamases confer resistance to ampicillin/clavulanate, a beta-lactam/beta-lactamase inhibitor combination used to treat serious infections. To gain a deeper understanding of this resistance phenotype, we investigated the activities of sulbactam and two novel penem beta-lactamase inhibitors with sp2 hybridized C3 carboxylates and bicyclic R1 side chains against a library of SHV beta-lactamase variants at the 244 position. Compared to SHV-1 expressed in Escherichia coli, all 19 R244 variants exhibited increased susceptibility to ampicillin/sulbactam, an important difference compared to ampicillin/clavulanate. Kinetic analyses of SHV-1 and three SHV R244 (-S, -Q, and -L) variants revealed the Ki for sulbactam was significantly elevated for the R244 variants, but the partition ratios, kcat/kinact, were markedly reduced (13 000 --> 相似文献   

Surprisingly fast disappearance of beta-lactam selection pressure in cultivation as detected with novel biosensing approaches

Tetracycline and beta-lactam resistances among others are used as selection markers in the production of recombinant proteins. The beta-lactam resistance is based on degradation, i.e. the selection pressure gradually disappears from the culture, whereas tetracycline resistance is based on active efflux. We have studied the kinetics of the stability of antibiotic selection pressure in culture using a simple model system (pBR322 in Escherichia coli). Concentrations of ampicillin, carbenicillin and tetracycline were measured with novel sensor cells developed in our lab. These cells are specifically induced to produce light in the presence of the drugs and here their performance was shown to be excellent in monitoring antibiotic concentrations in cell culture. The sensor cells are cheap to produce and use and a high number of samples can be analysed simultaneously. To our surprise, ampicillin and carbenicillin were completely degraded after 2.5-3.0 h of culture, although it has been widely claimed that especially carbenicillin is a good selective agent, whereas tetracycline was stable in culture. beta-lactamase activity in culture was found to correlate with the kinetics of ampicillin degradation.     

Resistance plasmids in Pseudomonas cepacia 4G9.

Pseudomonas cepacia 4G9 utilizes 2-tridecanone as its sole carbon source and has been shown to be resistant to a variety of antibiotics. To ascertain whether any of these characteristics were plasmid mediated, Escherichia coli HB101 was transformed with plasmid DNA isolated from Pseudomonas cepacia 4G9. No 2-tridecanone-utilizing transformants were obtained. Tetracycline (Tc)- and ampicillin (Ap)- resistant transformants were obtained at a low frequency. Plasmid deoxyribonucleic acid from antibiotic-resistant E. coli HB101 transformants had molecular weights of 2.9 x 10(6) for pJW2 Tcr and 5.4 x 10(6) for pJW3 Apr as determined by electron microscopy. Electron microscopy of plasmid deoxyribonucleic acid from P. cepacia 4G9 revealed a single plasmid species, pJW1 of 1.78 x 10(6). Tetracycline resistance in both P. cepacia 4G9 and E. coli HB101(pJW2) was inducible, whereas ampicillin resistance in P. cepacia 4G9 was constitutive. The level of ampicillin resistance coded by pJW3 was lower in P. cepacia 4G9 than in the transformant E. coli HB101(pJW3).     

Beta-lactamase activity of bacteria of the genus Proteus]

beta-Lactamases of Proteus and their role in the mechanism of the microbe resistance to penicillins and ceporin were studied. It was found that the beta-lactamase of Proteus had low activity and were produced by both beta-lactamide resistant and sensitive clinical strains of Proteus. The resistant cultures of Proteus produced enzymes more frequently (3.4--5 times) than the sensitive ones. The synthesis of beta-lactamase in the clinical Proteus strains was inducable. The high induction coefficient was achieved only in the presence of high concentrations of the inductor. No significant dependence of the culture sensitivity level of ampicillin and ceporin on the induction level was observed. The most significant part of the constitutive enzyme in Proteus was intracellular, while that of the inducable enzyme was extracellular. No correlative dependence between the culture resistance levels to penicillins and ceporin and the enzyme activity was noted. The beta-lactamase activity was not found in the transconjugants with the in vitro acquired R-factor controlling the ampicillin and ceporin resistance, as well as in the resistant mutants selected on the media with increasing concentrations of the above antibiotics. Induction of beta-lactamase synthesis was not found in these strains either. The ability of Proteus to synthesize beta-lactamase can be lost on the strain storage under laboratory conditions which was not always accompanied by reduction of the culture sensitivity to ampicillin and ceporin. The enzymatic destruction of beta-lactamides was not the main mechanism of Proteus resistance to the above antibiotics.     

Molecular Specificities of R Factor-Determined Beta-Lactamases: Correlation with Plasmid Compatibility

Beta (beta)-lactamases determined by 29 ampicillin resistance plasmids could be divided into two types. One, TEM-type, was very uniform with respect to substrate specificity but heterogeneous in absolute levels of beta-lactamase activity. The TEM-type beta-lactamase was determined by R factors of compatibility groups FII, Ialpha, Iepsilon, N, C, A, T, W, P, L, and X, and by prophage phi Amp. The other type, characterized by the ability to hydrolyze oxacillin, was less common, showed lower absolute levels of activity, and was heterogeneous as regards substrate specificities. Oxacillin-hydrolyzing beta-lactamases were determined by R factors of compatibility groups FI, Ialpha, N, C, and O.     

Precursor of beta-lactamase is enzymatically inactive. Accumulation of the preprotein in Saccharomyces cerevisiae

Synthesis and properties of the bacterial precursor of beta-lactamase (E.C.3.5.2.6) were studied in Saccharomyces cerevisiae transformants. A protease-deficient yeast mutant was transformed with the plasmid pADH040-2 conferring high expression of the bla gene. Besides precisely processed beta-lactamase, transformed yeast cells contained mainly bla precursor up to the amount of 2% of total cellular protein. The precursor was shown to be synthesized on free polysomes in vivo but could be processed with rough microsomal membranes in a cell-free translation system. By applying an isolation procedure using high-salt conditions, the labile precursor could be separated in a native form from the mature beta-lactamase. Thereby it could be shown that the pre-beta-lactamase had virtually no enzymatic activity in contrast to the mature enzyme, which was indistinguishable from bacterial beta-lactamase. Furthermore, the precursor was highly susceptible to proteolytic degradation by trypsin under conditions which did not affect the mature enzyme. Accordingly, the protein conformation of the precursor must be substantially different from that of the authentic beta-lactamase, demonstrating that specific processing and transport of beta-lactamase is associated with directing the protein to a distinct conformation.     

Construction of an Escherichia coli-Rhodococcus shuttle vector and plasmid transformation in Rhodococcus spp.

A plasmid transformation system for Rhodococcus sp. strain H13-A was developed by using an Escherichia coli-Rhodococcus shuttle plasmid constructed in this study. Rhodococcus sp. strain H13-A contains three cryptic indigenous plasmids, designated pMVS100, pMVS200, and pMVS300, of 75, 19.5, and 13.4 kilobases (kb), respectively. A 3.8-kb restriction fragment of pMVS300 was cloned into pIJ30, a 6.3-kb pBR322 derivative, containing the E. coli origin of replication (ori) and ampicillin resistance determinant (bla), as well as a Streptomyces gene for thiostrepton resistance, tsr. The resulting 10.1-kb recombinant plasmid, designated pMVS301, was isolated from E. coli DH1(pMVS301) and transformed into Rhodococcus sp. strain AS-50, a derivative of strain H13-A, by polyethylene glycol-assisted transformation of Rhodococcus protoplasts and selection for thiostrepton-resistant transformants. Thiostrepton-resistant transformants were also ampicillin resistant and were shown to contain pMVS301, which was subsequently isolated and transformed back into E. coli. The cloned 3.8-kb fragment of Rhodococcus DNA in pMVS301 contains a Rhodococcus origin of replication, since the hybrid plasmid was capable of replication in both genera. The plasmid was identical in E. coli and Rhodococcus transformants as determined by restriction analysis and was maintained as a stable, independent replicon in both organisms. Optimization of the transformation procedure resulted in transformation frequencies in the range of 10(5) transformants per micrograms of pMVS301 DNA in Rhodococcus sp. strain H13-A and derivative strains. The plasmid host range extends to strains of Rhodococcus erythropolis, R. globulerus, and R. equi, whereas stable transformants were not obtained with R. rhodochrous or with several coryneform bacteria tested as recipients. A restriction map demonstrated 14 unique restriction sites in pMVS301, some of which are potentially useful for molecular cloning in Rhodococcus spp. and other actinomycetes. This is the first report of plasmid transformation and of heterologous gene expression in a Rhodococcus sp.