Isolation of BamHI variants with reduced cleavage activities

Derivation of the bamhIR sequence (Brooks, J. E., Nathan, P.D., Landry, D., Sznyter, L.A., Waite-Rees, P., Ives, C. C., Mazzola, L. M., Slatko, B. E., and Benner, J. S. (1991) Nucleic Acids Res., in press), the gene coding for BamHI endonuclease, has facilitated construction of an Escherichia coli strain that overproduces BamHI endonuclease (W. E. Jack, L. Greenough, L. F. Dorner, S. Y. Xu, T. Strezelecka, A. K. Aggarwal, and I. Schildkraut, submitted for publication). As expected, low-level constitutive expression of the bamhIR gene in E. coli from the Ptac promotor construct is lethal to the host unless the bamHIM gene, which encodes the BamHI methylase, is also expressed within the cell. We identified four classes of BamHI endonuclease variants deficient in catalysis by selecting for survival of a host deficient for bamHIM gene, transformed with mutagenized copies of the bamhIR gene, and then screening the surviving cell extracts for DNA cleavage and binding activities. Class I variants (G56S, G91S/T153I, T114I, G130R, E135K, T153I, T157I, G194D) displayed 0.1-1% of the wild-type cleavage activity; class II variant (D94N) lacked cleavage activity but retained wild-type DNA binding specificity; class III variants (E77K, E113K) lacked cleavage activity but bound DNA more tightly; class IV variants (G56D, G90D, G91S, R122H, R155H) lacked both binding and cleavage activities. Variants with residual cleavage activities induced the E. coli SOS response and thus are presumed to cleave chromosomal DNA in vivo. We conclude that Glu77, Asp94, and Glu113 residues are essential for BamHI catalytic function.     

Localized mutagenesis of the tetracycline gene in the plasmid pBR322 induced by sodium bisulfite in vitro

The technique of localized in vitro mutagenesis in the cohesive ends of plasmid pBR322 DNA has been elaborated (separately for BamHI and HindIII sites). Plasmid DNA digested by restriction endonucleases has been treated with sodium bisulphite deaminating cytosine to form uracil in single stranded DNA (cohesive ends of the plasmid). The mutagenized plasmid DNA, free of mutagen, has been treated with bacteriophage T4 ligase. E. coli C600 cells were subsequently transformed by the ligated DNA preparation. The clones having tetracycline gene mutagenized represented 4.0-11.1% and 1.2-3.1% among HindIII and BamHI mutants, respectively, selected as TcR----TcS transformants. Selection of mutagenized DNA by the second endonuclease restriction has increased the mutant yields up to 55.6-78.0% and 10.0-75.4%, respectively. The yield of TcS mutations in the control DNA treated at all stages of experiment, except for mutagen treatment, has reached 0.06% and 0.2%, respectively.     

Identification and characterization of integron-mediated antibiotic resistance among Shiga toxin-producing Escherichia coli isolates

A total of 50 isolates of Shiga toxin-producing Escherichia coli (STEC), including 29 O157:H7 and 21 non-O157 STEC strains, were analyzed for antimicrobial susceptibilities and the presence of class 1 integrons. Seventy-eight (n = 39) percent of the isolates exhibited resistance to two or more antimicrobial classes. Multiple resistance to streptomycin, sulfamethoxazole, and tetracycline was most often observed. Class 1 integrons were identified among nine STEC isolates, including serotypes O157:H7, O111:H11, O111:H8, O111:NM, O103:H2, O45:H2, O26:H11, and O5:NM. The majority of the amplified integron fragments were 1 kb in size with the exception of one E. coli O111:H8 isolate which possessed a 2-kb amplicon. DNA sequence analysis revealed that the integrons identified within the O111:H11, O111:NM, O45:H2, and O26:H11 isolates contained the aadA gene encoding resistance to streptomycin and spectinomycin. Integrons identified among the O157:H7 and O103:H2 isolates also possessed a similar aadA gene. However, DNA sequencing revealed only 86 and 88% homology, respectively. The 2-kb integron of the E. coli O111:H8 isolate contained three genes, dfrXII, aadA2, and a gene of unknown function, orfF, which were 86, 100, and 100% homologous, respectively, to previously reported gene cassettes identified in integrons found in Citrobacter freundii and Klebsiella pneumoniae. Furthermore, integrons identified among the O157:H7 and O111:NM strains were transferable via conjugation to another strain of E. coli O157:H7 and to several strains of Hafnia alvei. To our knowledge, this is the first report of integrons and antibiotic resistance gene cassettes in STEC, in particular E. coli O157:H7.     

An integron of class 1 is present on the plasmid pCG4 from Gram-positive bacterium Corynebacterium glutamicum

The streptomycin/spectinomycin resistance determinant of the 29-kb plasmid pCG4 from Corynebacterium glutamicum was found to be a part of a typical class 1 integron. The sequence analysis revealed that the integron (designated InCg) identified in this Gram-positive bacterium is almost identical to the integron InC present on the plasmid pSA1700 from the Gram-negative bacterium Pseudomonas aeruginosa. Differences in only two base pairs were found in the 3.8-kb sequence. One base substitution (G→C) is present in the streptomycin/spectinomycin resistance determinant which is thus identical to the aadA2a gene from the integron In6 of the broad-host-range plasmid pSa. The other one (C→G) is present in the extended −10 region of the integron promoter involved in expression of the antibiotic resistance gene. It was shown that this novel version of the integron promoter displays five times higher activity in both C. glutamicum and Escherichia coli than the original one.     

Studies on Insect Resistance of Bt Transplastomic Plants and the Phenotype of Their Progenies

Insecticidal protein gene CrylA (c) from Bacillus thuringiensis (Bt toxin gene) was placed under the control of psbA5'- and 3'- regulatory regions of rice (Oryza sativa L. ) chloroplast to construct Bt expression cassette, which was ligated with selectable marker aadA cassette and homology regions of tobacco ( Nicotiana tabacum L. ) chloroplast genome to generate transformation vector pTRS8. Leaves of tobacco plant cv. NC89 were transformed with particle bombardment method, plastid transformants were selected by their resistance to 500 mg/L of spectinomycin. Some transplastomic plants were toxic to the third-instar larvae of Helicoverpa zea, and the growth of the survived insects was remarkably inhibited. Genetic and molecular analyses of T1 and T2 progenies of plants with highly efficient insect resistance showed that Bt toxin gene had been inherited in progenies, and spectinomycin resistance was inherited maternally.     

Cloning of the BamHI methyl transferase gene from Bacillus amyloliquefaciens

We wish to report the initial characterization of a recombinant clone containing the BamHI methylase gene. Genomic chromosomal DNA purified from Bacillus amyloliquefaciens was partially cleaved with HindIII, fractionated by size, and cloned into pSP64. Plasmid DNA from this library was challenged with BamHI endonuclease and transformed into Escherichia coli HB101. A recombinant plasmid pBamM6.5 and a subclone pBamM2.5 were shown to contain the BamHI methylase gene based on three independent observations. Both plasmids were found to be resistant to BamHI endonuclease cleavage, and chromosomal DNA isolated from E. coli HB101 cells harboring either of the plasmids pBamM6.5 or pBamM2.5 was resistant to cleavage by BamHI endonuclease. In addition, DNA isolated from lambda phage passaged through E. coli HB101 containing either plasmid was also resistant to BamHI cleavage. Expression of the BamHI methylase gene is dependent on orientation in pSP64. In these clones preliminary evidence indicates that methylase gene expression may be under the direction of the plasmid encoded LacZ promoter.     

Nucleotide sequence of the transposon Tn7 gene encoding an aminoglycoside-modifying enzyme, 3"(9)-O-nucleotidyltransferase

The nucleotide sequence of a transposon Tn7 DNA fragment encoding a 3"(9)-O-nucleotidyltransferase, an aminoglycoside-modifying enzyme, which mediates bacterial resistance to spectinomycin and streptomycin, was determined. The aadA structural gene was 786 bases long and predicted a polypeptide of 262 amino acids with a calculated molecular weight of 29,207. Comparison of the DNA sequences of Tn7 and plasmid R538-1 indicated that their aadA genes were nearly identical. Comparison of the polypeptides predicted by the aadA genes of Tn7 and Tn554 indicated that the genes were related.     

Deletion analysis of the essentiality of penicillin-binding proteins 1A, 2B and 2X of Streptococcus pneumoniae

Abstract An internal fragment from each of the penicillinebinding protein (PBP) 1A, 2B and 2X genes of Streptococcus pneumoniae , which included the region encoding the active-site serine residue, was replaced by a fragment encoding spectinomycin resistance. The resulting constructs were tested for their ability to transform S. pneumoniae strain R6 to spectinomycin resistance. Spectinomycin-resistant transformants could not be obtained using either the inactivated PBP 2X or 2B genes, suggesting that deletion of either of these genes was a lethal event, but they were readily obtained using the inactivated PBP 1A gene. Analysis using the polymerase chain reaction confirmed that the latter transformants had replaced their chromosomal copy of the PBP 1A gene with the inactivated copy of the gene. Deletion of the PBP 1A gene was therefore tolerated under laboratory conditions and appeared to have little effect on growth or susceptibility to benzylpenicillin.     

Direct transformation of the liverwort <Emphasis Type="Italic">Marchantia polymorpha</Emphasis> L. by particle bombardment using immature thalli developing from spores

The liverwort, Marchantia polymorpha L., belongs to a group of basal land plants and is an emerging model for plant biology. We established a procedure to prepare sporangia of M. polymorpha under laboratory conditions by promoting its transition to reproductive development by far-red light irradiation. Here we report an improved direct transformation system of M. polymorpha using immature thalli developing from spores. Hygromycin-resistant transformants were obtained on selective media by transformation with a plasmid carrying the hygromycin-phosphotransferase gene (hpt) conferring hygromycin resistance in 4 weeks. The aminoglycoside-3'-adenyltransferase gene (aadA) conferring spectinomycin resistance was also successfully used as an additional selectable marker for nuclear transformation of M. polymorpha. The availability of the aadA gene in addition to the hpt gene should make M. polymorpha a versatile host for genetic manipulation. DNA gel-blot analyses indicated that transformed thalli carried a variable number of copies of the transgene integrated into the genome. Although the previous system using thalli grown from gemmae required a two-step selection in liquid and solid media for 8 weeks, the system reported here using thalli developing from spores allows generation of transformants in half the time by direct selection on solid media, facilitating genetic analyses in this model plant.     

Generation and Analysis of Soybean Plastid Transformants Expressing <Emphasis Type="Italic">Bacillus thuringiensis</Emphasis> Cry1Ab Protoxin

We describe the generation of fertile and homoplasmic soybean plastid transformants, expressing the Bacillus thuringiensis insecticidal protoxin Cry1Ab. Transgenes were targeted in the intergenic region of Glycine max plastome, between the rps12/7 and trnV genes and selection was carried out using the aadA gene encoding spectinomycin resistance. Molecular analysis confirmed the integration of the cry1Ab and aadA expression cassettes at the expected location in the soybean plastome, and the transmission of the transgenes to the next generation. Western blot analyses showed that the Cry1Ab protoxin is highly expressed in leaves, stems and seeds, but not in roots. Its expression confers strong insecticidal activity to the generated transgenic soybean, as exemplified with velvetbean caterpillar (Anticarsia gemmatalis).     

Characterisation of two new gene cassettes, aadA5 and dfrA17

Escherichia coli INS33 was isolated from the urinary tract of an infected patient. It was resistant to ampicillin, chloramphenicol, spectinomycin, streptomycin, sulfafurazole, tetracycline and trimethoprim. PCR screening revealed the presence of a class 1 integron that harboured two new gene cassettes, designated dfrA17 and aadA5. The new dfrA17 cassette was 91% identical to the known dfrA7 cassette. The aadA5 cassette was 95% identical over the first 830 bp to aadA4, but lacked the IS26 element found at the 3' end of this truncated cassette. Cloning and expression of the cassette region demonstrated that dfrA17 conferred high level resistance to trimethoprim but aadA5 conferred resistance to spectinomycin but not to streptomycin.     

The region of the IncN plasmid R46 coding for resistance to beta-lactam antibiotics, streptomycin/spectinomycin and sulphonamides is closely related to antibiotic resistance segments found in IncW plasmids and in Tn21-like transposons.

The nucleotide sequence of a 2.5 kb segment of the pKM101 (R46) genome has been determined. The 1.3 kb from a BamHI site at 153 to base 1440 differs by only 2 bases from a part of the published sequence of the aadB (gentamicin resistance) gene region including the coding region for the N-terminal 70 amino acids of the predicted aadB product. The same sequence has been found 5'-to the dhfrII gene of R388 and to the aadA gene of Tn21 (R538-1). Three open reading frames are located in this region, two on the same strand as the resistance genes and one on the complementary strand. The latter predicts a polypeptide of 337 amino acids, whose N-terminal segment is 40% homologous to the predicted product of an open reading frame of 179 amino acids located next to the dhfrI gene of Tn7. The oxa2 (oxacillin resistance) gene predicts a long polypeptide commencing with (the N-terminal) 70 amino acids of the aadB product. A similar arrangement is found in the aadA gene of R538-1. The N-terminal segment of an aadA gene is located 3'- to oxa2, separated by 36 bases. Sequences surrounding the BamHI site are identical to sequences 5'- to the tnpM gene of Tn21 and homology ceases where homology between Tn21 and Tn501 commences. The possibility that this antibiotic resistance segment is a discrete mobile DNA element is discussed.     

aadA confers streptomycin resistance in Borrelia burgdorferi

To enhance genetic manipulation of the Lyme disease spirochete Borrelia burgdorferi, we assayed the aadA gene for the ability to confer resistance to the antibiotics spectinomycin and streptomycin. Using the previously described pBSV2 as a backbone, a shuttle vector, termed pKFSS1, which carries the aadA open reading frame fused to the B. burgdorferi flgB promoter was constructed. The hybrid flgB promoter-aadA cassette confers resistance to spectinomycin and streptomycin in both B. burgdorferi and Escherichia coli. pKFSS1 has a replication origin derived from the 9-kb circular plasmid and can be comaintained in B. burgdorferi with extant shuttle vector pCE320, which has a replication origin derived from a 32-kb circular plasmid, or pBSV2, despite the fact that pKFSS1 and pBSV2 have the same replication origin. Our results demonstrate the availability of a new selectable marker and shuttle vector for genetically dissecting B. burgdorferi at the molecular level.     

Cloning the BamHI restriction modification system.

BamHI, a Type II restriction modification system from Bacillus amyloliquefaciensH recognizes the sequence GGATCC. The methylase and endonuclease genes have been cloned into E. coli in separate steps; the clone is able to restrict unmodified phage. Although within the clone the methylase and endonuclease genes are present on the same pACYC184 vector, the system can be maintained in E. coli only with an additional copy of the methylase gene present on a separate vector. The initial selection for BamHI methylase activity also yielded a second BamHI methylase gene which is not homologous in DNA sequence and hybridizes to different genomic restriction fragments than does the endonuclease-linked methylase gene. Finally, the interaction of the BamHI system with the E. coli Dam and the Mcr A and B functions, have been studied and are reported here.     

Nucleotide sequence analysis of a gene encoding a streptomycin/spectinomycin adenylyltransferase

The nucleotide sequence of 1400 bp from R-plasmid R538-1 containing the streptomycin/spectinomycin adenyltransferase gene (aadA) was determined, and the location of the aadA gene was identified by a combination of insertion and deletion mutants. Its gene product, aminoglycoside 3"-adenylyltransferase (AAD(3")(9), has a Mr of 31,600.     

Evolutionarily conserved and functionally important residues in the I-CeuI homing endonuclease.

Two approaches were used to discern critical amino acid residues for the function of the I- Ceu I homing endonuclease: sequence comparison of subfamilies of homologous proteins and genetic selection. The first approach revealed residues potentially involved in catalysis and DNA recognition. Because I- Ceu I is lethal in Escherichia coli , enzyme variants not perturbing cell viability were readily selected from an expression library. A collection of 49 variants with single amino acid substitutions at 37 positions was assembled. Most of these positions are clustered within or around the LAGLI-DADG dodecapeptide and the TQH sequence, two motifs found in all protein subfamilies examined. The Km and kcat values of the wild-type and nine variant enzymes synthesized in vitro were determined. Three variants, including one showing a substitution of the glutamine residue in the TQH motif, revealed no detectable endonuclease activity; five others showed reduced activity compared to the wild-type enzyme; whereas the remaining variant cleaved the top strand about three times more efficiently than the wild-type. Our results not only confirm recent reports indicating that amino acids in the LAGLI-DADG dodecapeptide are functionally critical, but they also suggest that some residues outside this motif directly participate in catalysis.