Length and structural effect of signal peptides derived from Bacillus subtilis alpha-amylase on secretion of Escherichia coli beta-lactamase in B. subtilis cells.

The precursor of Bacillus subtilis alpha-amylase contains an NH2-terminal extension of 41 amino acid residues as the signal sequence. The E. coli beta-lactamase structural gene was fused with the DNA for the promoter and signal sequence regions. Activity of beta-lactamase was expressed and more than 95% of the activity was secreted into the culture medium. DNA fragments coding for short signal sequences 28, 31, and 33 amino acids from the initiator Met were prepared and fused with the beta-lactamase structural gene. The sequences of 31 and 33 amino acid residues with Ala COOH-terminal amino acid were able to secrete active beta-lactamase from B. subtilis cells. However beta-lactamase was not secreted into the culture medium by the shorter signal sequence of 28 amino acid residues, which was not cleaved. Molecular weight analysis of the extracellular and cell-bound beta-lactamase suggested that the signal peptide of B. subtilis alpha-amylase was the first 31 amino acids from the initiator Met. The significance of these results was discussed in relation to the predicted secondary structure of the signal sequences.     

Effect of the potential triplex DNA region on the in vitro expression of bacterial beta-lactamase gene in superhelical recombinant plasmids.

The effect of a pyrimidine/purine-biased stretch which has the potential to form an unusual triplex DNA structure on gene expression has been analyzed by measuring the activity of beta-lactamase as a reporter gene in recombinant plasmids. The Escherichia coli transformant carrying the plasmid p7ERS which has a potential triplex DNA region expressed about twofold more beta-lactamase activity than that carrying the plasmid pUC19. Since the expression of beta-lactamase has been shown to be affected by template topology in vitro, this in vivo observation suggests that the inserted pyrimidine/purine-biased stretch modulates the topology of flanking regions by forming unusual DNA structure to keep the template at the superhelicity favorable for the expression of beta-lactamase.     

Specific processing of the bacterial beta-lactamase precursor in Saccharomyces cerevisiae

Synthesis and processing of the bacterial enzyme beta-lactamase (E.C. 3.5. 2.6) were studied in Saccharomyces cerevisiae. The 2-micron DNA vector pADH040-2 containing the yeast ADH1 promoter fused to the bacterial gene was used in order to obtain enhanced synthesis of the bacterial protein in yeast transformants. Both precursor and mature beta-lactamase were shown to be present in yeast cells, the precursor being the major product. The mature enzyme was purified about 500-fold over crude extracts to apparent homogeneity and thus represents nearly 0.2% of the total yeast protein. No difference in specific activity and molecular weight could be observed when compared with the authentic beta-lactamase from Escherichia coli. Specificity of the processing of beta-lactamase in yeast cells was verified by partial amino acid sequence analysis demonstrating the removal of the signal peptide at the correct position.     

Beta-lactamase genes of Streptomyces badius, Streptomyces cacaoi and Streptomyces fradiae: cloning and expression in Streptomyces lividans

Genes encoding extracellular beta-lactamases (EC 3.5.2.6) of Gram-positive Streptomyces badius, Streptomyces cacaoi and Streptomyces fradiae have been cloned into Streptomyces lividans. The beta-lactamase gene of S. badius was initially isolated on a 7 kb BamHI fragment and further located on a 1300 bp DNA segment. An 11 kb BamHI fragment was isolated encompassing the S. cacaoi beta-lactamase gene, which was subcloned to a 1250 bp DNA fragment. The beta-lactamase gene of S. fradiae was cloned on an 8 kb BamHI fragment and mapped to a 4 kb DNA segment. Each of the three BamHI fragments encompassing the beta-lactamase genes hybridized to a BamHI fragment of the corresponding size in chromosomal DNA from the respective strain used for cloning. The activities of the three beta-lactamases were predominantly found to be extracellular in the S. lividans recombinants. The S. badius and S. cacaoi beta-lactamases exhibited a 10-100-times lower activity in S. lividans, whereas the S. fradiae beta-lactamase showed an approximately 10-fold higher activity in the cloned state, compared with the activities found in the original strains.     

A vector for the construction of translational fusions to TEM beta-lactamase and the analysis of protein export signals and membrane protein topology

A plasmid vector, pJBS633, that facilitates the construction of translational fusions of genes of interest to the coding region of the mature form of TEM beta-lactamase has been developed. Transformants containing in-frame fusions can be identified by their ability to grow when plated at high inocula on agar containing ampicillin (Ap). The cellular location of the beta-lactamase moiety of the fusion proteins can then be determined since only those that direct the translocation of the beta-lactamase across the cytoplasmic membrane to the periplasm result in the ability of individual cells of Escherichia coli to form isolated colonies in the presence of Ap. Conversely, those fusion proteins in which the beta-lactamase moiety remains cytoplasmic do not protect individual cells against Ap. Transformants expressing the latter class of fusion proteins can, however, be identified when plated at high inocula since, as cells start to lyse, the cytoplasmic beta-lactamase activity is released and provides Ap resistance to the surrounding cells. The vector contains the origin of replication of f1 phage so that single-stranded plasmid DNA can be obtained in the appropriate orientation to allow sequencing across the fusion junction using a universal primer complementary to the start of the coding region of mature TEM beta-lactamase. pJBS633 should be useful as a general vector for the construction of beta-lactamase fusions and, in particular, for the analysis of protein export signals and the determination of the organisation of proteins in the E. coli cytoplasmic membrane.     

Interaction between the helicases genetically linked to Fanconi anemia group J and Bloom's syndrome

Bloom's syndrome (BS) and Fanconi anemia (FA) are autosomal recessive disorders characterized by cancer and chromosomal instability. BS and FA group J arise from mutations in the BLM and FANCJ genes, respectively, which encode DNA helicases. In this work, FANCJ and BLM were found to interact physically and functionally in human cells and co-localize to nuclear foci in response to replication stress. The cellular level of BLM is strongly dependent upon FANCJ, and BLM is degraded by a proteasome-mediated pathway when FANCJ is depleted. FANCJ-deficient cells display increased sister chromatid exchange and sensitivity to replication stress. Expression of a FANCJ C-terminal fragment that interacts with BLM exerted a dominant negative effect on hydroxyurea resistance by interfering with the FANCJ-BLM interaction. FANCJ and BLM synergistically unwound a DNA duplex substrate with sugar phosphate backbone discontinuity, but not an 'undamaged' duplex. Collectively, the results suggest that FANCJ catalytic activity and its effect on BLM protein stability contribute to preservation of genomic stability and a normal response to replication stress.     

Preparation and characterization of a beta-lactamase-Fab' conjugate for the site-specific activation of oncolytic agents.

Antibody-directed catalysis (ADC) is a two-step method for the targeted delivery of chemotherapeutic agents in which enzyme-antibody conjugates, prelocalized to antigen-bearing cells, activate prodrugs designed to be substrates for the enzyme. An enzyme-Fab' conjugate exhibiting both native beta-lactamase activity and immunoreactivity toward carcinoembryonic antigen (CEA) was constructed. Treatment of CEA-expressing LS174T cells with this conjugate imparted beta-lactamase activity to the cells; beta-lactamase activity was not imparted by treatment with unconjugated beta-lactamase and not to CEA negative cells treated with conjugate. Cephalosporin-based prodrugs, and other substrates synthesized as model compounds, were found to have wide variations in their kinetic parameters toward the conjugate, with kcat values ranging from 16 to 3300 s-1 and KM values ranging from 5 to 160 microM. The prodrug derived from desacetylvinblastine-3-carboxylic acid hydrazide (DAVLBHYD) was studied in vitro and found to be 5-fold less cytotoxic to LS174T cells than the parent DAVLBHYD. For antigen-positive cells preincubated with conjugate, however, the prodrug showed the same potency as the parent drug. Thus, the combination of conjugate and prodrug appears to provide antigen-dependent toxicity to tumor cells.     

Identification of amino acid substitutions that alter the substrate specificity of TEM-1 beta-lactamase.

TEM-1 beta-lactamase is the most prevalent plasmid-mediated beta-lactamase in gram-negative bacteria. Recently, TEM beta-lactamase variants with amino acid substitutions in the active-site pocket of the enzyme have been identified in natural isolates with increased resistance to extended-spectrum cephalosporins. To identify other amino acid substitutions that alter the activity of TEM-1 towards extended-spectrum cephalosporins, we probed regions around the active-site pocket by random-replacement mutagenesis. This mutagenesis technique involves randomizing the DNA sequence of three to six codons in the blaTEM-1 gene to form a library containing all or nearly all of the possible substitutions for the region randomized. In total, 20 different residue positions that had been randomized were screened for amino acid substitutions that increased enzyme activity towards the extended-spectrum cephalosporin cefotaxime. Substitutions at positions 104, 168, and 238 in the TEM-1 beta-lactamase that resulted in increased enzyme activity towards extended-spectrum cephalosporins were found. In addition, small deletions in the loop containing residues 166 to 170 drastically altered the substrate specificity of the enzyme by increasing activity towards extended-spectrum cephalosporins while virtually eliminating activity towards ampicillin.     

Mutagenic effect of acridine orange on the expression of penicillin G acylase and beta-lactamase in Escherichia coli

AIMS: The present work aimed to improve the production of penicillin G acylase (PGA) and reduce the beta-lactamase activity through acridine orange (AO) induced mutation in Escherichia coli. METHODS AND RESULTS: Three wild E. coli strains BDCS-N-FMu10, BDCS-N-S21 and BDCS-N-W50, producing both the enzymes PGA and beta-lactamase were treated by AO. Minimum inhibitory concentration of AO was 10 microg ml(-1) and it was noted that bacterial growth was gradually suppressed by increasing the concentration of AO from 10 to 100 microg ml(-1). The highest concentration that gave permissible growth rate was 50 microg ml(-1). The isolated survivals were screened on the bases of PGA and beta-lactamase activities. Among the retained mutants, the occurrence of beta-lactamase deficient ones (91%) was significantly higher than penicillin acylase deficient ones (27%). CONCLUSIONS: In seven of the mutants, PGA activity was enhanced with considerable decrease in beta-lactamase activity. One of the mutant strains (BDCS-N-M36) exhibited very negligible expression of beta-lactamase activity and twofold increase in PGA activity [12.7 mg 6-amino-penicillanic acid (6-APA) h(-1) mg(-1) wet cells] compared with that in the wild-type strain (6.3 mg 6-APA h(-1) mg(-1) wet cells). SIGNIFICANCE AND IMPACT OF THE STUDY: The treatment of E. coli cells with AO resulted in mutants with enhanced production of PGA and inactivation of beta-lactamase. These mutants could be used for industrial production of PGA.     

Polyamine depletion inhibits the differentiation of L6 myoblast cells

Exposure to alpha-difluoromethylornithine, an enzyme-activated irreversible inhibitor of ornithine decarboxylase, inhibited the insulin induced differentiation of L6 myoblast cells. Differentiation was assessed by measuring creatine kinase activity and by determining the percentage of nuclei in myotubes. The levels of putrescine and spermidine increased in stimulated cultures prior to their differentiation and these increases were blocked by alpha-difluoromethylornithine. Provision of exogenous putrescine was able to reverse the inhibitory effect of the drug. The anti-differentiative effect is observed only if alpha-difluoromethylornithine is added within twenty-four hours of insulin stimulation. In the experimental protocol used, alpha-difluoromethylornithine was added as the cultures approached confluence and had no effect on their ultimate DNA content. Therefore, the effect of alpha-difluoromethylornithine on myoblast differentiation is not secondary to an effect on cellular proliferation. These results indicate that polyamines may be involved in the mediation of muscle cell differentiation.     

Secretion activities of Bacillus subtilis alpha-amylase signal peptides of different lengths in Escherichia coli cells

The B. subtilis alpha-amylase promoter and signal peptide are functional in E. coli cells. DNA fragments coding for signal peptides with different lengths (28, 31, 33 and 41 amino acids from the translation initiator Met) were prepared and fused with the E. coli beta-lactamase structural gene. In B. subtilis cells, the sequences of 31, 33 and 41 amino acids were able to secrete beta-lactamase into the surrounding media, but the 28 amino acid sequence was not. In contrast, all of the four sequences were able to export beta-lactamase into the periplasmic space of E. coli cells. Thus, the recognition of the B. subtilis alpha-amylase signal peptide in E. coli cells seems to be different from that in B. subtilis cells.     

Expression in Chinese hamster transformant TK+-cells of the bacterial gene for beta-lactamase

Using monospecific immune antiserum the expression of beta-lactamase bacterial gene (from the plasmid pBR322) in transformed TK+ Chinese hamster cells was studied. It is shown that in some TK+-clones the beta-lactamase gene is expressed and this expression is stimulated by an inhibitor of DNA methylation-5-azacytidine. Two physically linked genes (the Herpes simplex thymidine kinase gene, and the beta-lactamase gene) introduced into the Chinese hamster cells on the same plasmid are found to be expressed independently.     

Replication and expression of plasmid pBR 322 during discontinuous culture of a stringent and relaxed Escherichia coli strain

The E. coli strains CP78 and CP79 carrying the plasmid pBR 322 display similar growth kinetics in discontinuous culture. During limitation of amino acids the stringent strain CP78 is able to synthesize guanosine-5'diphosphate-3'-diphosphate (ppGpp) and guanosine-5'-triphosphate-3' diphosphate, but the relaxed strain can not produce highly phosphorylated guanosine nucleotides. During the logarithmic phase of growth both strains contain similar amounts of plasmid DNA. During amino acid starvation plasmid DNA is amplified in the relaxed strain only, whereas in the stringent strain the plasmid content per cell remains constant. In stationary phase cells of CP78 a higher activity of plasmid-coded beta-lactamase than in CP79 cells was detected. Furthermore, remarkable differences between both strains were observed in the composition of proteins derived from the periplasmic fraction and separated by polyacrylamide gel electrophoresis. Our results might indicate a negative control of pBR 322 DNA replication by ppGpp during amino acid starvation.     

Modification, processing, and subcellular localization in Escherichia coli of the pCloDF13-encoded bacteriocin release protein fused to the mature portion of beta-lactamase.

A fusion between the pCloDF13-derived bacteriocin release protein and beta-lactamase was constructed to investigate the subcellular localization and posttranslational modification of the bacteriocin release protein in Escherichia coli. The signal sequence and 25 of the 28 amino acid residues of the mature bacteriocin release protein were fused to the mature portion of beta-lactamase. The hybrid protein (Mr, 31,588) was expressed in minicells and whole cells and possessed full beta-lactamase activity. Immunoblotting of subcellular fractions revealed that the hybrid protein is present in both the cytoplasmic and outer membranes of E. coli. Radioactive labeling experiments in the presence or absence of globomycin showed that the hybrid protein is modified with a diglyceride and fatty acids and is processed by signal peptidase II, as is the murein lipoprotein. The results indicated that the pCloDF13-encoded bacteriocin release protein is a lipoprotein which is associated with both membranes of E. coli cells.     

Analysis of aclarubicin-induced cell death in human fibroblasts

In the present study we investigated the mode of cell death induced by aclarubicin (ACL) in trisomic (BB) and normal (S-2) human fibroblasts. Cells were incubated with ACL for 2h and then cultured in drug-free medium for up to 96h. Using fluorescence microscopy, agarose gel electrophoresis and comet assay we demonstrate that ACL induced time-dependent morphological and biochemical changes in both cell types. The population of apoptotic cells, analysed by acridine orange and ethidium bromide nuclear staining reached its maximum at 24-48h. Prolonged post-treatment time progressively increased the level of necrotic cells. At 24-48h time points we also observed a significant increase in caspase-3 activity, oligonucleosomal DNA fragmentation and DNA strand breaks. Cotreatment of cells with the specific caspase-3 inhibitor Ac-DEVD-CHO partly reduced the extent of apoptosis and necrosis and DNA degradation. In conclusion, trisomic and normal fibroblasts demonstrate similar response to aclarubicin treatment. Drug induced the apoptotic and necrotic pathway of cell death that was mediated by caspase-3.     

Induction of beta-lactamase influences the course of development in Myxococcus xanthus.

Myxococcus xanthus is a gram-negative bacterium that develops in response to starvation on a solid surface. The cells assemble into multicellular aggregates in which they differentiate from rod-shaped cells into spherical, environmentally resistant spores. Previously, we have shown that the induction of beta-lactamase is associated with starvation-independent sporulation in liquid culture (K. A. O'Connor and D. R. Zusman, Mol. Microbiol. 24:839-850, 1997). In this paper, we show that the chromosomally encoded beta-lactamase of M. xanthus is autogenously induced during development. The specific activity of the enzyme begins to increase during aggregation, before spores are detectable. The addition of inducers of beta-lactamase in M. xanthus, such as ampicillin, D-cycloserine, and phosphomycin, accelerates the onset of aggregation and sporulation in developing populations of cells. In addition, the exogenous induction of beta-lactamase allows M. xanthus to fruit on media containing concentrations of nutrients that are normally too high to support development. We propose that the induction of beta-lactamase is an integral step in the development of M. xanthus and that this induction is likely to play a role in aggregation and in the restructuring of peptidoglycan which occurs during the differentiation of spores. In support of this hypothesis, we show that exogenous induction of beta-lactamase can rescue aggregation and sporulation of certain mutants. Fruiting body spores from a rescued mutant are indistinguishable from wild-type fruiting body spores when examined by transmission electron microscopy. These results show that the signal transduction pathway leading to the induction of beta-lactamase plays an important role in aggregation and sporulation in M. xanthus.     

掺入大肠杆菌膜中的磷脂酰胆碱(PC)影响青霉素b-内酰胺酶的分泌

[目的]为了研究磷脂酰胆碱(PC)在原核生物细胞中的生物学作用,探讨PC对细菌膜系统的功能的影响.[方法]使用ptac 85质粒作载体,将螺旋菌pcs基因导入E.coli Top10细胞构建了E.coli Fop10 pcs 菌株,并在特定的条件下培养细菌,使细菌膜磷脂中合成30%左右的磷脂酰胆碱.然后再使用抗生素抗性分析、β-内酰胺酶的酶活测定以及Western blot杂交技术,分析质粒编码的β-内酰胺酶从细胞质到细胞问质的分泌情况.[结果]抗生素抗性分析发现,高浓度的氨苄青霉素抑制E.coliTop10 pcs 细菌的生长的氨苄青霉素剂量低于对照组,其半致死剂量IC50在700～800μg/mL之间.酶活检测显示E.coli Top10 pcs 细菌周质内β-内酰胺酶的酶活性只有对照菌株的1,5,Western blot进一步分析发现周质内β-内酰胺酶的含量也为对照菌株的1/5.由此可见,周质内低含量的β-内酰胺酶是导致E.coli Top10pcs 细菌氨苄青霉素抗性降低的原因.[结论]掺入细菌膜磷脂双分子层的PC影响p.内酰胺酶通过Sec转运途径从细胞质分泌到细菌周质空间内,提示细菌磷脂酰胆碱可能在调节蛋白转运和分泌方面起着重要的作用.     

Effect of sulbactam on anti-pseudomonal activity of beta-lactam antibiotics in cells producing various levels of the MexAB-OprM efflux pump and beta-lactamase

The beta-lactamase inhibitor, sulbactam, was tested for beta-lactamase inhibitory activity in Pseudomonas aeruginosa cells producing various levels of both the MexAB-OprM efflux pump and beta-lactamase. We found that sulbactam lowered the MICs of cefoperazone and piperacillin by inhibiting the beta-lactamase 8-fold in the cell producing a constitutively high level of AmpC-type beta-lactamase and a wild-type level of MexAB-OprM pump compared with that without sulbactam. The MICs of cefoperazone and piperacillin in the cell producing a constitutively high level of both the efflux pump and beta-lactamase under the presence of sulbactam were 8 and 4 times, respectively, lower than that without sulbactam. The MICs of sulbactam in the cell producing a constitutively high and a wild-type level of the efflux pump were 16- and 8-fold higher, respectively, than that in the mutant lacking the efflux pump. We concluded that sulbactam exerts potent beta-lactamase inhibitory activity in the cell producing a high level of efflux pump, in spite of the fact that sulbactam serves as a substrate of the MexAB-OprM pump. Increasing amounts of sulbactam over the weight of beta-lactams further strengthen the effect of beta-lactam antibiotics.