Incidence of Extended-Spectrum β-Lactamases and Characterization of Integrons in Extended-Spectrum β-Lactamase-producing Klebsiella pneumoniae Isolated in Shantou, China

This study is concerned with the level of antibiotic resistance of extended-spectrum β-lactamase （ESBL）-producing Klebsiella pneumoniae, isolated in Shantou, China, and its mechanism. Seventy- four non-repetitive clinical isolates of K. pneumoniae producing ESBLs were isolated over a period of 2 years. Antibiotic susceptibility, carried out by Epsilometer test, showed that most of the isolates were multiresistant. Polymerase chain reaction showed that, among the several types of β-lactamases, SHV was the most prevalent, TEM was the second most prevalent, and CTX-M was the least prevalent. Sixty-nine isolates were positive for integrase gene IntI1, but no IntI2 or IntI3 genes were found. The variable region of class 1 integrons were amplified and further identified by sequencing. Thirteen different gene cassettes and 11 different cassette combinations were detected. Dfr and aadA cassettes were predominant and cassette combinations dfrA12, orfF and aadA2 were most frequently found. No gene cassettes encoding ESBLs were found. Integrons were prevalent and played an important role in multidrug resistance in ESBL-producing K. pneumoniae.     

Occurrence of clinical isolates of Klebsiella pneumoniae harboring chromosomally mediated and plasmid-mediated CTX-M-15 β-lactamase in a Tunisian hospital

The spread of multidrug-resistant strains of Klebsiella pneumoniae in hospitals is of concern to clinical microbiologists, health care professionals, and physicians because of the impact infections caused by these bacteria have in causing morbidity and mortality. Clinical isolates of K.?pneumoniae have been found to show resistance to third-generation cephalosporins as a result of acquiring extended-spectrum β-lactamase-producing genes, such as bla(CTX-M). Since little is known about the mechanisms of antibiotic resistance observed in Kasserine hospital, Tunisia, this study was undertaken to investigate the mechanisms by which clinical isolates of K.?pneumoniae resist β-lactam antibiotics. Twelve strains of K.?pneumoniae were collected from patients admitted to Kasserine hospital; these isolates showed multiresistance phenotypes. Molecular genetics investigations using polymerase chain reaction, S1 digestion, and pulsed-field gel electrophoresisshowed that bla(CTX-M-15) in association with ISEcp1 is responsible for the resistance of these strains to third-generation cephalosporins. It has been determined that bla(CTX-M-15) is chromosomally mediated and plasmid mediated, which alarming need for infection control to prevent the outbreak of such a resistance mechanism.     

Characterization of Fosfomycin Resistant Extended-Spectrum β-Lactamase-Producing Escherichia coli Isolates from Human and Pig in Taiwan

To investigate the efficacy of fosfomycin against extended-spectrum β-lactamases (ESBL) producing Escherichia coli in Taiwan and the resistance mechanisms and characterization of human and pig isolates, we analyzed 145 ESBL-producing isolates collected from two hospitals (n = 123) and five farms (n = 22) in Taiwan from February to May, 2013. Antimicrobial susceptibilities were determined. Clonal relatedness was determined by PFGE and multi-locus sequence typing. ESBLs, ampC, and fosfomycin resistant genes were detected by PCR, and their flanking regions were determined by PCR mapping and sequencing. The fosfomycin resistant mechanisms, including modification of the antibiotic target (MurA), functionless transporters (GlpT and UhpT) and their regulating genes such as uhpA, cyaA, and ptsI, and antibiotic inactivation by enzymes (FosA and FosC), were examined. The size and replicon type of plasmids carrying fosfomycin resistant genes were analyzed. Our results revealed the susceptibility rates of fosfomycin were 94% for human ESBL-producing E. coli isolates and 77% for pig isolates. The PFGE analysis revealed 79 pulsotypes. No pulsotype was found existing in both human and pig isolates. Three pulsotypes were distributed among isolates from two hospitals. ISEcp1 carrying bla_{CTX-M-group 9} was the predominant transposable elements of the ESBL genes. Among the thirteen fosfomycin resistant isolates, functionless transporters were identified in 9 isolates. Three isolates contained novel amino acid substitutions (Asn67Ile, Phe151Ser and Trp164Ser, Val146Ala and His159Tyr, respectively) in MurA (the target of fosfomycin). Four isolates had fosfomycin modified enzyme (fosA3) in their plasmids. The fosA3 gene was harboured in an IncN-type plasmid (101 kbp) in the three pig isolates and an IncB/O-type plasmid (113 kbp) in the human isolate. In conclusion, we identified that 6% and 23% of the ESBL-producing E. coli from human and pigs were resistant to fosfomycin, respectively, in Taiwan. No clonal spread was found between human and pig isolates. Functionless transporters were the major cause of fosfomycin resistance, and the fosA3-transferring plasmid between isolates warrants further monitoring.     

Evaluation of a double synergy differential test (DSDT) for differential detection of ESBL and AmpC-type β-lactamases in Escherichia coli, Klebsiella pneumoniae and Proteus mirabilis

This work describes a simple and practical double synergy differential test (DSDT) that couples the detection of ESBLs and AmpC-type enzymes by means of a combo-disk approach using cefotaxime and ceftazidime as indicator substrates, and clavulanate and boronic acid as enzyme inhibitors. The DSDT was tested with a collection of 118 Escherichia coli, Klebsiella pneumoniae and Proteus mirabilis strains with different beta-lactamase profiles, and proved to be highly sensitive and specific for the detection of ESBL and AmpC-producing isolates.     

Phylogenetic Distribution and Prevalence of Genes Encoding Class I Integrons and CTX-M-15 Extended-Spectrum β-Lactamases in Escherichia coli Isolates from Healthy Humans in Chandigarh,India

Escherichia coli is generally considered as a commensal inhabitant of gastrointestinal tract of humans and animals. The aim of this study was to gain insight on the distribution of phylotypes and presence of genes encoding integrons, extended β-lactamases and resistance to other antimicrobials in the commensal E. coli isolates from healthy adults in Chandigarh, India. PCR and DNA sequencing were used for phylogenetic classification, detections of integrase genes, gene cassettes within the integron and extended β-lactamases. The genetic structure of E. coli revealed a non-uniform distribution of isolates among the seven phylogenetic groups with significant representation of group A. Integron-encoded integrases were detected in 25 isolates with class 1 integron-encoded intI1 integrase being in the majority (22 isolates). The gene cassettes identified were those for trimethoprim, streptomycin, spectinomycin and streptothricin. The dfrA12-orfF-aadA2 was the most commonly found gene cassette in intI1 positive isolates. Phenotypic assay for screening the potential ESBL producers suggested 16 isolates to be ESBL producers. PCR detection using gene-specific primers showed that 15 out of these 16 ESBL-producing E. coli harboured the bla _CTX-M-15 gene. Furthermore, molecular studies helped in characterizing the genes responsible for tetracycline, chloramphenicol and sulphonamides resistance. Collectively, our study outlines the intra-species phylogenetic structure and highlights the prevalence of class 1 integron and bla _CTX-M-15 in commensal E. coli isolates of healthy adults in Chandigarh, India. Our findings further reinforce the relevance of commensal E. coli strains on the growing burden of antimicrobial resistance.     

Coexpression of β-mannanase and xylanase genes in Escherichia coli

[目的]β-甘露聚糖酶和木聚糖酶都属于半纤维素酶,它们已经同时运用于工农业生产的许多领域.构建β-甘露聚糖酶和木聚糖酶共表达菌株并进行相关评价.[方法]通过设计一个共同的酶切位点,将菌株Bacillus subtilis BE-91中的β-甘露聚糖酶和木聚糖酶基因串联到表达载体pET28a(+)上,转化大肠杆菌构建了一株能够共表达β-甘露聚糖酶和木聚糖酶的菌株B.pET28a-man-xyl.[结果]菌株诱导21h后,发酵液中β-甘露聚糖酶和木聚糖酶的酶活分别为713.34 U/mL和1455.83 U/mL,是胞内酶活的11.8倍和2.53倍.[结论]SDS-PAGE分析、水解圈活性检测和胞外酶与胞内酶酶活检测表明:两个酶均以功能蛋白独立分泌到胞外.此外,与β-甘露聚糖酶和木聚糖酶单独酶解半纤维素相比,复合酶的酶解效果更好.菌株的成功构建为复合酶制剂(半纤维素酶制剂)的研究和生产奠定基础.     

Urinary Tract Infections in Kidney Transplant Patients Due to Escherichia coli and Klebsiella pneumoniae-Producing Extended-Spectrum β-Lactamases: Risk Factors and Molecular Epidemiology

Urinary tract infection (UTI) is a common complication after kidney transplantation, often associated to graft loss and increased healthcare costs. Kidney transplant patients (KTPs) are particularly susceptible to infection by Enterobacteriaceae-producing extended-spectrum β-lactamases (ESBLs). A retrospective case-control study was conducted to identify independent risk factors for ESBL-producing Escherichia coli and Klebsiella pneumoniae in non-hospitalized KTPs with UTI. Forty-nine patients suffering from UTI by ESBL-producing bacteria (ESBL-P) as case group and the same number of patients with UTI by ESBL negative (ESBL-N) as control-group were compared. Clinical data, renal function parameters during UTI episodes, UTI recurrence and relapsing rate, as well as risk factors for recurrence, molecular characterization of isolates and the respective antimicrobial susceptibility profile were evaluated. Diabetes mellitus (p <0.007), previous antibiotic prophylaxis (p=0.017) or therapy (p<0.001), previous UTI (p=0.01), relapsing infection (p=0.019) and patients with delayed graft function after transplant (p=0.001) represented risk factors for infection by ESBL positive Enterobacteriaceae in KTPs. Interestingly, the period of time between data of transplantation and data of UTI was shorter in case of ESBL-P case-group (28.8 months) compared with ESBL-N control-group (50.9 months). ESBL-producing bacteria exhibited higher resistance to fluoroquinolones (p=0.002), trimethoprim-sulfamethoxazole (p<0.001) and gentamicin (p<0.001). Molecular analysis showed that bla _CTX-M was the most common ESBL encoding gene (65.3%), although in 55.1% of the cases more than one ESBL gene was found. In 29.4% of K. pneumoniae isolates, three bla-genes (bla _CTX-M-bla _TEM-bla _SHV) were simultaneously detected. Low estimated glomerular filtration rate (p=0.009) was found to be risk factor for UTI recurrence. Over 60% of recurrent UTI episodes were caused by genetically similar strains. UTI by ESBL-producing Enterobacteriaceae in KTPs represent an important clinical challenge regarding not only hospitalized patients but also concerning outpatients.     

PCR-Based Detection of Extended-Spectrum β-Lactamases (bla CTX-M-1 and bla TEM ) in Escherichia coli, Salmonella spp. and Klebsiella pneumoniae Isolated from Pigs in North Eastern India (Mizoram)

Cephalosporins are major antimicrobials used to treat serious infections. However, their effectiveness is being compromised by the emergence of extended-spectrum β-lactamases (ESBLs). A total of 138 enteric bacteria were isolated from 53 faecal samples of pigs collected from different districts of Mizoram, of which 102 (73.91 %) were Escherichia coli, 26 (18.84 %) were Salmonella spp. and 10 (7.25 %) were Klebsiella pneumoniae. Phenotypic confirmatory test (Double Discs Synergy Test) showed that 8 (5.80 %) E. coli isolates were ESBLs producer. PCR analysis confirmed that out of the eight isolate, 7 (5.07 %) harboured bla _CTX-M-1 gene and/or bla _TEM gene. Of the eight positive isolates, 7 (5.07 %) and 3 (2.17 %) were found to be positive for bla _CTX-M-1 gene and bla _TEM gene, respectively, of which 3 (2.17 %) isolates were positive for both the genes. Only 4 (2.90 %) E. coli isolates carried bla _CTX-M-1 gene alone. Agarose gel electrophoresis showed that all the isolates were carrying plasmids ranging between 0.9 and ~30 kb. Out of the seven isolates positive for bla _CTX-M-1 and/or bla _TEM , 2 (1.84 %) isolates were confirmed for bla _CTX-M-1 gene in their plasmid. Only one E. coli isolate was found to be positive for both the genes in its plasmid. The resistance plasmid could not be transferred to a recipient by in vitro horizontal gene transfer method.     

Characterization of Plasmid-Mediated Quinolone Resistance (PMQR) Genes in Extended-Spectrum β-Lactamase-Producing Enterobacteriaceae Pediatric Clinical Isolates in Mexico

This work describes the characterization of plasmid-mediated quinolone-resistance (PMQR) genes from a multicenter study of ESBL-producing Enterobacteriaceae pediatric clinical isolates in Mexico. The PMQR gene-positive isolates were characterized with respect to ESBLs, and mutations in the GyrA and ParC proteins were determined. The phylogenetic relationship was established by PFGE and the transfer of PMQR genes was determined by mating assays. The prevalence of the PMQR genes was 32.1%, and the rate of qnr-positive isolates was 15.1%; 93.3% of the latter were qnrB and 6.4% were qnrA1. The distribution of isolates in terms of bacterial species was as follows: 23.5% (4/17) corresponded to E. cloacae, 13.7% (7/51) to K. pneumoniae, and 13.6% (6/44) to E. coli. In addition, the prevalence of aac(6’)-Ib-cr and qepA was 15.1% and 1.7%, respectively. The molecular characteristics of qnr- and qepA-positive isolates pointed to extended-spectrum β-lactamase (ESBL) CTX-M-15 as the most prevalent one (70.5%), and to SHV-12 in the case of aac(6’)-Ib-cr-positive isolates. GyrA mutations at codons Ser-83 and Asp-87, and ParC mutations at codons Ser-80 were observed in 41.1% and 35.2% of the qnr-positive isolates, respectively. The analysis of the transconjugants revealed a co-transmission of bla_CTX-M-15 with the qnrB alleles. In general, the prevalence of PMQR genes (qnr and aac(6’)-Ib-cr) presented in this work was much lower in the pediatric isolates, in comparison to the adult isolates in Mexico. Also, ESBL CTX-M-15 was the main ESBL identified in the pediatric isolates, whereas in the adult ones, ESBLs corresponded to the CTX-M and the SHV families. In comparison with other studies, among the PMQR-genes identified in this study, the qnrB-alleles and the aac(6’)-Ib-cr gene were the most prevalent, whereas the qnrS1, qnrA1 and qnrB-like alleles were the most prevalent in China and Uruguay.     

Expression and characterization of Kluyveromyces lactis β-galactosidase in Escherichia coli

Kluyveromyces lactis -galactosidase gene, LAC4, was expressed in Escherichia coli as a soluble His-tagged recombinant enzyme under the optimized culture conditions. The expressed protein was multimeric with a subunit molecular mass of 118 kDa. The dimeric form of the -galactosidase was the major fraction but had a lower activity than those of the multimeric forms. The purified enzyme required Mn²⁺ for activity and was inactivated irreversibly by imidazole above 50 mM. The activity was optimal at 37 and 40 °C for o-nitrophenyl--d-galactopyranoside (oNPG) and lactose, respectively. The optimum pH value is 7. The K _m and V _max values of the purified enzyme for oNPG were 1.5 mM and 560 mol min^–1 mg^–1, and for lactose 20 mM and 570 mol min^–1 mg^–1, respectively.     

Activity and stability of Escherichia coli β-galactosidase in cosolvent systems

The kinetic parameters of E.coli -galactosidase were not altered by the addition of 2-propanol or ethyl acetate (1.6% v/v). While ethylene glycol (1.6% v/v) doubled the values of both KM (0.29 mM) and kcat (1393 s–), tetraethyleneglycol-dimethylether (Tetraglyme,1.6% v/v) preserved KM, but decreased kcat. At 50°C all the cosolvents dramatically shortened the enzymatic half life, and so did Tetraglyme and 2-propanol at 28°C. At 28°C, both ethyl acetate and ethylene glycol stabilised the enzyme 9- and 6-fold respectively. This fact, together with the activation effect of ethylene glycol may lead to practical applications. © Rapid Science Ltd. 1998     

Prevalence and Characteristics of Extended-Spectrum β-Lactamase and Plasmid-Mediated Fluoroquinolone Resistance Genes in Escherichia coli Isolated from Chickens in Anhui Province,China

The aim of this study was to characterize the prevalence of extended-spectrum β-lactamase (ESBL) genes and plasmid-mediated fluoroquinolone resistance (PMQR) determinants in 202 Escherichia coli isolates from chickens in Anhui Province, China, and to determine whether ESBL and PMQR genes co-localized in the isolates. Antimicrobial susceptibility for 12 antimicrobials was determined by broth microdilution. Polymerase chain reactions (PCRs), DNA sequencing, and pulsed field gel electrophoresis (PFGE) were employed to characterize the molecular basis for β-lactam and fluoroquinolone resistance. High rates of antimicrobial resistance were observed, 147 out of the 202 (72.8%) isolates were resistant to at least 6 antimicrobial agents and 28 (13.9%) of the isolates were resistant to at least 10 antimicrobials. The prevalence of bla _CTX-M, bla _TEM-1 and bla _TEM-206 genes was 19.8%, 24.3% and 11.9%, respectively. Seventy-five out of the 202 (37.1%) isolates possessed a plasmid-mediated quinolone resistance determinant in the form of qnrS (n = 21); this determinant occurred occasionally in combination with aac(6′)-1b-cr (n = 65). Coexistence of ESBL and/or PMQR genes was identified in 31 of the isolates. Two E. coli isolates carried bla _TEM-1, bla _CTX-M and qnrS, while two others carried bla _CTX-M, qnrS and aac(6′)-1b-cr. In addition, bla _TEM-1, qnrS and aac(6′)-1b-cr were co-located in two other E. coli isolates. PFGE analysis showed that these isolates were not clonally related and were genetically diverse. To the best of our knowledge, this study is the first to describe detection of TEM-206-producing E. coli in farmed chickens, and the presence of bla _TEM-206, qnrS and aac(6′)-1b-cr in one of the isolates.     

Expression and characterization of Pichia etchellsii β-glucosidase in Escherichia coli

The β-glucosidase enzyme is important as the terminal enzyme involved in hydrolysis of cellobiose and short-chain cellodextrins generated during enzymatic cellulose degradation. Under controlled reaction conditions the enzyme also displays cello-oligosaccharide synthesizing ability (based on either the thermodynamic or kinetic approach). We present here the purification of the enzyme β-glucosidase (BGL) of Pichia etchellsii from recombinant pBG55 Escherichia coli clone. The kinetic parameters, substrate specificity and oligosaccharide synthesizing ability of the purified enzyme are also reported. The purified 200-kDa protein (tetramer of 50 kDa) was identified as a broad-substrate-specificity enzyme exhibiting increased temperature and glucose tolerance compared to the native yeast enzyme. Temperature directed substrate specificity for aryl β,1–4 linkage, and β(1–2), β(1–4), β(1–6) and β(2-1) linkages in various natural disaccharides was observed. Glycosylation of the enzyme was found to be unimportant for enzyme activity. With both cellobiose and glucose, oligosaccharide synthesis was detected. The implications of this information with regard to cellulose hydrolysis and oligosaccharide synthesis are discussed.     

Expression of a β-galactosidase gene from Lactobacillus sake in Escherichia coli

Summary A -galactosidase gene from Lactobacillus sake coding for lactose hydrolysis was cloned and expressed in Escherichia coli. Chromosomal DNA from L. sake was partially digested with the restriction enzyme Sau3AI, and the 3–6 Kb fragment was ligated to the cloning vector pSP72 digested with BamHI. One E. coli transformant expressing -galactosidase was isolated on X-gal plates. It contained a plasmid with an insertion of approx. 4 Kb. The restriction map of the recombinant plasmid was constructed. The characteristics of the recombinant -galactosidase were compared with those of the wild type. The optima pH and temperature for both enzymes was 6.5 and 50°C, respectively. Stability of the enzymes at different temperatures and activity on lactose were determined.     

Molecular cloning and expression of a Micromonospora chalcae β-glucosidase encoding gene in Escherichia coli

A Sau3A I genomic library from the actinomycete Micromonospora chalae was constructed in Escherichia coli using the expression vector pUC18. Using the chromogenic substrate 5-bromo-4-chloro-3-indolyl--glucoside (X-glu), a number of positive recombinant colonies were identified. One of those exhibiting the strongest phenotype contained a recombinant plasmid, pANNA1 which harboured a 4.2kb DNA insert. Using restriction endonuclease site mapping and subcloning strategies a 2.3kb DNA fragment encoding the -glucosidase activity was identified. Characterization of the strongly expressed recombinant enzyme demonstrated that it had a dramatically increased thermal stability at 50 °C. The Km values obtained for the recombinant enzyme and that from M. chalcae using the substrate p-nitrophenyl--D-glucoside were 0.19mM and 0.25mM, respectively.     

Occurrence of extended-spectrum β-lactamases-producing Escherichia coli isolates over gradient pollution in an urban tropical estuary

Bacterial resistance to antimicrobials is a global public health problem that surpasses the human context and can be increased by pollution. However, the lack of systematic monitoring of resistance in some aquatic matrices, such as tropical estuaries, makes it unknown whether its occurrence is associated with anthropogenic pollution in these environments. Therefore, we investigated the occurrence of extended-spectrum beta-lactamases (ESBLs) producing Escherichia coli as a resistance indicator for 12 consecutive months at three representative points of a pollution gradient in Guanabara Bay (GB), Brazil. Sixty-six E. coli strains were selected from 72 samples of GB waters in the presence of ceftriaxone (8 μg mL⁻¹) and identified by MALDI-TOF MS. Of the 66, 55 (83.3%) strains were ESBL producers. They carried beta-lactamase/ESBL genes, with the predominance of bla_CTX-M (54, 98.2%), especially the bla_CTX-M-1,2 allele (49.1%). These strains were detected frequently (81.8%) from the point with the highest pollution levels. Furthermore, the marker for Class 1 integron, intI1 gene, was detected in 54.5% of ESBL producers. These data suggest an association between antimicrobial-resistant E. coli and sewage pollution in aquatic environments raising concerns about the possible risks of human exposure to these waters and fish consumption.     

Expression in Escherichia coli of cDNA Encoding Barley β-Amylase and Properties of Recombinant β-Amylase

To express the cloned β-amylase cDNA in Escherichia coli under control of the tac promoter, a plasmid pBETA92 was constructed. The plasmid consisted of 6312 bp. An extract of E. coli JM109 harboring pBETA92 had β-amylase activity that produced β-maltose from soluble starch. The enzyme production started in the logarithmic phase, increased linearly, and reached a maximum after 12 h. The recombinant barley β-amylase gave two major (pI 5.43 and 5.63) and four minor (pI 5.20, 5.36, 5.80, and 6.13) activity bands on isoelectric focusing, and their pIs didn’t change throughout the incubation. But Western blot analysis found that one β-amylase having a molecular weight of about 56,000 was synthesized. The recombinant β-amylase was purified from the cells by consecutive column chromatography. The purified enzyme gave a single band of protein on SDS–PAGE but showed heterogeneity on isoelectric focusing. The N-terminal amino acid sequence showed that the recombinant β-amylase lacked four amino acids at positions 2–5 (Glu-Val-Asn-Val) when compared with the presumed amino acid sequence of barley β-amylase. Therefore, the recombiant β-amylase consisted of 531 amino acids, and its molecular weight was calculated to be 59,169. The N-terminal amino acid sequence of the recombinant β-amylase and the nucleotide sequence of the junction position in plasmid pBETA92 indicated that GTG (Val-5 in the case of barley β-amylase) at positions 27–29 from the SD sequence (AGGA) was the translation initiation codon. The properties of the recombinant β-amylase were almost the same as those of barley β-amylase except for the pI and the K_m values for maltohexaose and maltoheptaose. The pI of recombiant barley β-amylase calculated by Genetyx Version 9 based on the presumed amino acid sequence was 5.60, but the real pIs were 5.20–6.13. Therefore, some post-translational reaction(s) might happen after protein synthesis in E. coli cells, and this modification might cause the differences in the pI and the K_m values for maltohexaose and maltoheptaose between the barley and the recombinant β-amylases.     

Expression in Escherichia coli of a cloned β-glucanase gene from Bacillus amyloliquefaciens

Summary The recombinant phage G1 has been identified by screening 700 plaques of a Charon 4A library, containing DNA of Bacillus amyloliquefaciens, for phage clones directing the hydrolysis of lichenan in Escherichia coli, as indicated by haloes surrounding plaques on lichenan agar. The gene coding for an endo--1.3–1.4-glucanase was recloned within a 3.6 kb EcoRI fragment into the EcoRI site of plasmid pBR322, in both orientations.The location and extent of the bgl gene on the 3.6 kb Bacillus DNA insert was estimated by insertion mutagenesis with transposon Tn5 and restriction mapping of Tn5 insertions within or near to the bgl gene.The -glucanase synthesized by E. coli harbouring plasmids pEG1 or pEG2 was shown to accumulate mainly in the periplasmic space but -glucanase activities were also detected extracellulary and in the cytoplasm. The molecular weight of the enzyme synthesized in E. coli harbouring pEG1 was estimated by SDS-polyacrylamide gel electrophoresis to be about 24000. It was shown that the level of bgl gene expression in E. coli varies about 10-fold, depending on the orientation of the 3.6 kb DNA-fragment cloned within the EcoRI site of pBR322. After insertion of HindIII-DNA fragments from phage into the HindIII site of the -glucanase-high-expression plasmid pEG1, we obtained clones also showing an approximately 10-fold reduction in -glucanase activites. It was thus concluded that on plasmid pEG1 the leftward acting Ap^r (PI) promotor of plasmid pBR322 strongly increases the expression in E. coli of the cloned B. amyloliquefaciens bgl gene.Abbreviations Ap ampicillin, Km, kanamycin - kd kilodalton - kb kilobase pairs - moi multiplicity of infection - pfu plaque forming units - SDS sodium dodecylsulphate - Tc tetracycline