Kanamycin-resistant Transformants Research Articles

Preparation and characterization of a fluorogenic ddRFP-M biosensor as a specific SARS-CoV-2 main protease substrate

The conventional peptide substrates of SARS-CoV-2 main protease (Mpro) are frequently associated with high cost, unstable kinetics, and multistep synthesis. Hence, there is an urgent need to design affordable and stable Mpro substrates for pharmacological research. Herein, we designed a functional Mpro substrate based on a dimerization-dependent red fluorescent protein (ddRFP) for the evaluation of Mpro inhibitors in vitro. The codon-optimized DNA fragment encoding RFP-A1 domain, a polypeptide linker containing Mpro cleavage sequence (AVLQS), and the RFP-B1 domain was subcloned into the pET-28a vector. After transformation into Escherichia coli Rosetta(DE3) cells, the kanamycin resistant transformants were selected. Using a low temperature induction strategy, most of the target proteins (ddRFP-M) presented in the supernatant fractions were collected and purified by a HisTrapTM chelating column. Subsequently, the inhibition of Mpro by ensitrelvir and baicalein was assessed using ddRFP-M assay, and the biochemical properties of ddRFP-M substrate were analyzed. Our results showed that the fluorogenic substrate ddRFP-M was successfully prepared from E. coli cells, and this biosensor exhibited the expected specificity, sensitivity, and reliability. In conclusion, the production of the fluorogenic substrate ddRFP-M provides an expedient avenue for the assessment of Mpro inhibitors in vitro.

<i>Agrobacterium tumefaciens</i>-Mediated Transformation of Wild Tobacco Species <i>Nicotiana debneyi</i>, <i>Nicotiana clevelandii</i>, and <i>Nicotiana glutinosa</i>

Studies on Agrobacterium tumefaciens-mediated transformation of wild tobaccos Nicotiana debneyi, Nicotiana clevelandii, and Nicotiana glutinosa were conducted. Leaf disks were infected and co-cultivated with A. tumefaciens strain EHA105 carrying the binary vector pBISN1 with an intron interrupted β-glucuronidase (GUS) reporter gene (gusA) and the neomycin phosphotransferase gene (nptII). Selection and regeneration of kanamycin resistant shoots were conducted on regeneration medium containing 8.88 μM 6-benzylaminopurine (BAP), 0.57 μM indole-3-acetic acid (IAA), 50 mg·L-1 kanamycin and 250 mg·L-1 timentin. Kanamycin resistant shoots were rooted Murashige and Skoog (MS) medium containing 100 mg·L-1 kanamycin and 250 mg·L-1 timentin. Using this protocol, kanamycin-resistant plants were obtained from all three wild tobaccos at frequencies of 75.6% for N. debneyi, 25.0% for N. clevelandii, and 2.8% for N. glutinosa. Transcripts of nptII and gusA were detected in kanamycin-resistant T0 transformants (i.e., 2 for N. glutinosa and 5 for each of the N. debneyi and N. clevelandii) by the reverse transcript polymerase chain reaction (RT-PCR), and histochemical GUS assays confirmed expression of gusA in both T0 plants and T1 seedlings. The results indicate that the protocols are efficient for transformation of wild tobacco N. debneyi and N. clevelandii.

Identifying essential Streptococcus sanguinis genes using genome-wide deletion mutation.

Essential genes in pathogens are important for the development of antibacterial drugs. In this report, we described a protocol to identify essential genes in the Streptococcus sanguinis SK36 strain using genome-wide deletion mutation. A fusion PCR-based method is used to construct gene deletion fragments, which contain kanamycin resistance cassettes with two flanking arms of DNA upstream and downstream of the target gene. The linear fused PCR amplicons were transformed into S. sanguinis SK36 cells. No kanamycin-resistant transformants suggested the gene essentiality because the deletion of the essential gene renders a lethal phenotype of the transformants. The putative essential genes were further confirmed by independent transformations up to five attempts. The false nonessential genes were also identified by removing double-band mutants.

Potato transformation with the HvNHX3 gene and the improvement of transformant salt tolerance

Transformation by genes of various ion transporters is a tool to study plant salt tolerance mechanisms. In this work, the gene of a vacuolar NHX antiporter HvNHX3 was introduced in two potato Solanum tuberosum L. cultivars, Yubiley Zhukova and Skoroplodny-7. The binary vector pCambia-HvNHX3 was made and used for transformation. This vector carries HvNHX3 and NPTII for kanamycin selection each driven by 35S promoter from cauliflower mosaic virus. The presence of the target gene HvNHX3 and its mRNA were confirmed in 35 out of 48 kanamycin-resistant transformants. The growth parameters of 13 transgenic lines were measured in control and NaCl-containing media. The transformation of a relatively salt-tolerant cv. Yubiley Zhukova did not significantly change its growth characteristics and salt tolerance. In control conditions, most transgenic lines of cv. Skoroplodny-7 had the larger biomass and height. Unlike untransformed plants, they rooted and grew on 100 mM NaCl, indicating their greater salt tolerance.

PMdT1, a small ColE1-like plasmid mobilizing a new variant of the aac(6')-Ib-cr gene in Salmonella enterica serovar Typhimurium

To characterize a 5.9 kb aac(6')-Ib-cr-harbouring plasmid that was detected in a clinical Salmonella Typhimurium DT104B strain. Extraction and purification of plasmid DNA and electrotransformation assays were carried out in order to obtain kanamycin-resistant transformants. MICs of several fluoroquinolones and aminoglycosides were determined. DNA sequencing was performed by primer walking on purified plasmid preparations. The new plasmid nucleotide sequence was analysed and compared with available sequences using bioinformatic tools. pMdT1 is a 5.9 kb mobilizable ColE1-like plasmid that harbours aac(6')-Ib-cr4, a gene encoding a new variant of the AAC(6')-Ib-cr protein (225 amino acids). This active protein conferred resistance to tobramycin and kanamycin, and also decreased susceptibility to ciprofloxacin and norfloxacin in the transformant strain, as MICs demonstrated. The mobilization region, necessary for horizontal transfer and composed of the mobA, mobB, mobC and mobD genes, displayed a high degree of identity with those from representative ColE1-like plasmids. The basis of mobility (bom), oriT and origin of replication regions were also detected. Apart from the acetylase-encoding gene, three other open reading frames (ORFs) were determined. No similarities were found when the ORF1 sequence was compared with the sequences included in GenBank. The deduced ORF2 protein predicted a CopG-like structure characteristic of transcriptional regulators, and the deduced ORF3 protein was identical to macrophage stimulating factors. The pMdT1 is the smallest mobilizable ColE1-like plasmid containing an aac(6')-Ib-cr gene that has been described so far.

Novel Mechanism of RNA Repair by RtcB via Sequential 2′,3′-Cyclic Phosphodiesterase and 3′-Phosphate/5′-Hydroxyl Ligation Reactions

RtcB enzymes are a newly discovered family of RNA ligases, implicated in tRNA splicing and other RNA repair reactions, that seal broken RNAs with 2',3'-cyclic phosphate and 5'-OH ends. Parsimony and energetics would suggest a one-step mechanism for RtcB sealing via attack by the O5' nucleophile on the cyclic phosphate, with expulsion of the ribose O2' and generation of a 3',5'-phosphodiester at the splice junction. Yet we find that RtcB violates Occam's razor, insofar as (i) it is adept at ligating 3'-monophosphate and 5'-OH ends; (ii) it has an intrinsic 2',3'-cyclic phosphodiesterase activity. The 2',3'-cyclic phosphodiesterase and ligase reactions both require manganese and are abolished by mutation of the RtcB active site. Thus, RtcB executes a unique two-step pathway of strand joining whereby the 2',3'-cyclic phosphodiester end is hydrolyzed to a 3'-monophosphate, which is then linked to the 5'-OH end to form the splice junction. The energy for the 3'-phosphate ligase activity is provided by GTP, which reacts with RtcB in the presence of manganese to form a covalent RtcB-guanylate adduct. This adduct is sensitive to acid and hydroxylamine but resistant to alkali, consistent with a phosphoramidate bond.

A novel kanamycin/G418 resistance marker for direct selection of transformants in Escherichia coli and different yeast species

We have developed a set of cloning vectors possessing a modified Tn903 kanamycin resistance gene that enables the selection of both kanamycin-resistant transformants in Escherichia coli and G418-resistant transformants in the yeasts Saccharomyces cerevisiae, Hansenula polymorpha and Pichia pastoris. Expression of this gene in yeast is controlled by the H. polymorpha glyceraldehyde-3-phosphate dehydrogenase promoter, while expression in E. coli is governed by an upstream E. coli lacZ promoter. Applicability of the vectors for gene disruption in H. polymorpha and S. cerevisiae was demonstrated by inactivation of the HpMAL1 and URA3 genes, respectively. One of the vectors possesses a H. polymorpha ARS allowing plasmid maintenance in an episomal state. The small size of the vectors (2-2.5 kb) makes them convenient for routine DNA cloning. In addition, we report a novel approach for construction of gene disruption cassettes.

Abundance dynamics and sequence variation of neomycin phosphotransferase gene (nptII) homologs in river water

Monitoring the abundance and dynamics of antibiotic resistance genes is essential for assessing their potential effects on environmental and human health. The objectives of the present study were to investigate: (1) the abundance of neomycin phosphotransferase (nptII) gene homologs in water samples collected monthly from the South Saskatchewan River (Canada) for 24 mo, and (2) sequence variation of the cloned nptII gene homologs retrieved from the river. DNA from river micro- bial communities was used to transform a natural competent Pseudomonas stutzeri strain containing a truncated nptII gene on a plasmid (P. stutzeri pMR7). Of the 24 water samples, the recovery of kanamycin resistant (Km R ) transformants from DNA of 4 samples indicated the presence of nptII gene homologous sequences. However, the natural transformation process required ~3.2 × 10 4 copies of the nptII gene to recover a single Km R transformant. To overcome the limitation of detection, a real- time PCR assay using SYBR Green I was developed to quantify the abundance of nptII gene homolo- gous sequences in river microbial communities. The results showed that nptII gene homologous sequences were present in the river, and their abundance varied over the course of this study, rang- ing from undetectable levels to 4.36 × 10 6 copies l -1 water. Furthermore, nptII gene homologous sequences amplified from river microbial community DNA were cloned, and unique clones were sequenced. Comparison of the nucleotide and deduced amino acid sequences of the cloned frag- ments to those of the nptII gene on transposon Tn5 showed that they had over 96% homology.

Streptococcus pneumoniae Sheds Syndecan-1 Ectodomains through ZmpC, a Metalloproteinase Virulence Factor

Several microbial pathogens stimulate the ectodomain shedding of host cell surface proteins to promote their pathogenesis. We reported previously that Pseudomonas aeruginosa and Staphylococcus aureus activate the ectodomain shedding of syndecan-1 and that syndecan-1 shedding promotes P. aeruginosa pathogenesis in mouse models of lung and burned skin infections. However, it remains to be determined whether activation of syndecan-1 shedding is a virulence mechanism broadly used by pathogens. Here we show that Streptococcus pneumoniae stimulates syndecan-1 shedding in cell culture-based assays. S. pneumoniae-induced syndecan-1 shedding was repressed by peptide hydroxamate inhibitors of metalloproteinases but not by inhibitors of intracellular signaling pathways previously found to be essential for syndecan-1 shedding caused by P. aeruginosa, S. aureus, or other shedding agonists. A 170-kDa protein fraction with a peptide hydroxamate-sensitive shedding activity was purified by ammonium sulfate precipitation, DEAE chromatography, and size exclusion chromatography. Mass spectrometry analyses revealed that the 170-kDa fraction is composed of ZmpB and ZmpC, two metalloproteinase virulence factors of S. pneumoniae. Both the purified 170-kDa ZmpB/ZmpC fraction and unfractionated S. pneumoniae culture supernatant generated syndecan-1 ectodomains that are smaller than those released by endogenous shedding. Further, a mutant S. pneumoniae strain deficient in zmpC, but not zmpB, lost its capacity to stimulate syndecan-1 shedding. These data demonstrate that S. pneumoniae directly sheds syndecan-1 ectodomains through the action of ZmpC.

Degradation of plasmid and plant DNA in water microcosms monitored by natural transformation and real-time polymerase chain reaction (PCR)

Extracellular DNA exists in the environment and can be taken up by competent bacterial cells, leading to horizontal gene transfer. The persistence of extracellular plasmid and plant DNA in water microcosms was monitored in this study. Water samples were two groundwater (GW1 and GW2) and one river water (RW) samples. Three treatments included: (1) intact, (2) 0.22 μm filter-sterilized, and (3) autoclaved water. DNA from a plasmid (pNS1) and a transgenic Bt ( Bacillus thuringiensis) corn line, both carrying a neomycin phosphotransferase gene ( nptII gene) conferring kanamycin and neomycin resistance, was inoculated into the microcosms at 0.4 and 0.8 μg/ml, respectively. By monitoring its ability to transform a competent Pseudomonas stutzeri strain harboring plasmid pMR7 ( P. stutzeri pMR7), plasmid DNA was degraded to undetectable levels in the intact and/or filter-sterilized water treatments within 48–96 h in GW1, GW2, and RW. Meanwhile, plasmid DNA persisted in the autoclaved treatment throughout the entire incubation period. For plant DNA, a highly sensitive real-time PCR method using SYBR Green I was developed to monitor the degradation dynamics of the nptII gene carried by the transgenic corn line in the microcosms. The results showed that the concentration of plant DNA was reduced by two orders of magnitude (from 0.8–0.008 μg/ml) within 96 h in the intact and filter-sterilized treatments of GW1, GW2, and RW, in contrast to its persistence in the autoclaved treatment. In addition, no kanamycin resistant (Km R) transformants were detected from in situ transformation of P. stutzeri pMR7 with plasmid pNS1 DNA.

Neisseria gonorrhoeae Type IV Pili Undergo Multisite, Hierarchical Modifications with Phosphoethanolamine and Phosphocholine Requiring an Enzyme Structurally Related to Lipopolysaccharide Phosphoethanolamine Transferases

The zwitterionic phospho-forms phosphoethanolamine and phosphocholine are recognized as influential and important substituents of pathogen cell surfaces. PilE, the major pilin subunit protein of the type IV pilus (Tfp) colonization factor of Neisseria gonorrhoeae undergoes unique, post-translational modifications with these moieties. These phospho-form modifications have been shown to be O-linked alternately to a specific, conserved serine residue of PilE. However, the enzymes and precursors involved in their addition are unknown, and the full spectrum of PilE post-translational modifications has yet to be defined. Here, an intact protein-based mass spectrometric approach was integrated with bioinformatics and reverse genetics to address these matters. Specifically we show that a protein limited in its distribution to pathogenic Neisseria species and structurally related to enzymes implicated in phosphoethanolamine modification of lipopolysaccharide is necessary for PilE covalent modification with phosphoethanolamine and phosphocholine. These findings strongly suggest that protein phospho-form modification is mechanistically similar to processes underlying analogous modifications of prokaryotic saccharolipid glycans. We also show that PilE undergoes multisite and hierarchical phospho-form modifications and that the stoichiometries of site occupancy can be influenced by PilE primary structure and the abundance of the pilin-like protein PilV. Together, these findings have important implications for the structure and antigenicity of PilE.

Transformation of Acinetobacter sp. BD413 with DNA from commercially available genetically modified potato and papaya

To estimate the likelihood of transfer of kanamycin-resistance gene (nptII) from commercially available genetically modified (GM) plants. Acinetobacter sp. BD413 carrying a plasmid containing an inactivated nptII gene was treated with DNA derived from GM potato and GM papaya. Kanamycin-resistant transformants were obtained at a frequency of 10-30 microg(-1) DNA. Calculation of the results suggested that 6-9 x 10(4) molecules of genomic DNA from GM plants were needed to obtain one transformant. However, such transformation events were not detectable in the absence of the plasmid in the host strain. Acinetobacter sp. BD413 was transformed with DNA derived from GM potato and GM papaya, in the presence of an inactivated nptII gene on a plasmid. However, the frequency of such events in the natural environment on wild-type strains, while evidently low, remains unknown. Our results may help to evaluate potential risks associated with the use of antibiotic-resistance determinants as genetic markers in GM plants. Complete risk assessment must consider factors other than transformation frequency alone, including the natural background of antibiotic resistance present in bacterial populations, and the spectrum and clinical use of the antimicrobial agents in question.

Construction and evaluation of a chromosomal expression platform (CEP) for ectopic, maltose-driven gene expression in Streptococcus pneumoniae

In this paper, the construction and evaluation of a chromosomal expression platform (CEP), which allows controlled gene expression following ectopic integration into the chromosome of Streptococcus pneumoniae, is described. CEP is based on the well-studied maltosaccharide-inducible system. To facilitate integration at CEP, a plasmid, pCEP, capable of replication in Escherichia coli, but not in S. pneumoniae, was assembled. This plasmid contains an expression/selection cassette flanked on each side by more than 2 kb of pneumococcal DNA. The cassette comprises a maltose-inducible promoter, P(M), separated from a kanamycin-resistance gene by NcoI and BamHI cloning sites. Clones harbouring the gene of interest integrated at CEP under the control of P(M) can be obtained through direct transformation of an S. pneumoniae recipient with ligation products between that gene and NcoI/BamHI-digested pCEP DNA, followed by selection for kanamycin-resistant transformants.

Spread of recombinant DNA by roots and pollen of transgenic potato plants, identified by highly specific biomonitoring using natural transformation of an Acinetobacter sp.

Transgenic potato plants with the nptII gene coding for neomycin phosphotransferase (kanamycin resistance) as a selection marker were examined for the spread of recombinant DNA into the environment. We used the recombinant fusion of nptII with the tg4 terminator for a novel biomonitoring technique. This depended on natural transformation of Acinetobacter sp. strain BD413 cells having in their genomes a terminally truncated nptII gene (nptII'; kanamycin sensitivity) followed by the tg4 terminator. Integration of the recombinant fusion DNA by homologous recombination in nptII' and tg4 restored nptII, leading to kanamycin-resistant transformants. DNA of the transgenic potato was detectable with high sensitivity, while no transformants were obtained with the DNA of other transgenic plants harboring nptII in different genetic contexts. The recombinant DNA was frequently found in rhizosphere extracts of transgenic potato plants from field plots. In a series of field plot and greenhouse experiments we identified two sources of this DNA: spread by roots during plant growth and by pollen during flowering. Both sources also contributed to the spread of the transgene into the rhizospheres of nontransgenic plants in the vicinity. The longest persistence of transforming DNA in field soil was observed with soil from a potato field in 1997 sampled in the following year in April and then stored moist at 4 degrees C in the dark for 4 years prior to extract preparation and transformation. In this study natural transformation is used as a reliable laboratory technique to detect recombinant DNA but is not used for monitoring horizontal gene transfer in the environment.

Bacterial disease resistance of transgenic hybrid poplar expressing the synthetic antimicrobial peptide D4E1.

Important losses in poplar productivity occur because of susceptibility to microbial pathogens. To enhance disease resistance in susceptible genotypes, the gene coding for D4E1, a synthetic antimicrobial peptide consisting of 17 amino acid residues, was introduced into poplar (Populus tremula L. x Populus alba L.) via Agrobacterium-mediated transformation. Four kanamycin-resistant transformants were selected based on significant accumulation of the D4E1 transcript and confirmed by reverse transcription-polymerase chain reaction and RNA dot-blot analysis. These transgenic poplar lines were tested for resistance to Agrobacterium tumefaciens, Xanthomonas populi pv. populi and Hypoxylon mammatum (Wahl.) Miller. One transgenic poplar line, Tr23, bearing the highest transcript accumulation for the D4E1 gene, showed a significant reduction in symptoms caused by A. tumefaciens and X. populi. However, none of the transgenic poplar lines showed a significant difference in disease response to the fungal pathogen H. mammatum.

Structure of the N-Linked Glycan Present on Multiple Glycoproteins in the Gram-negative Bacterium, Campylobacter jejuni

Mass spectrometry investigations of partially purified Campylobacter jejuni protein PEB3 showed it to be partially modified with an Asn-linked glycan with a mass of 1406 Da and composed of one hexose, five N-acetylhexosamines and a species of mass 228 Da, consistent with a trideoxydiacetamidohexose. By means of soybean lectin affinity chromatography, a mixture of glycoproteins was obtained from a glycine extract, and two-dimensional gel proteomics analysis led to the identification of at least 22 glycoproteins, predominantly annotated as periplasmic proteins. Glycopeptides were prepared from the glycoprotein mixture by Pronase digestion and gel filtration. The structure of the glycan was determined by using nano-NMR techniques to be GalNAc-alpha1,4-GalNAc-alpha1,4-[Glcbeta1,3-]GalNAc-alpha1,4-GalNAc-alpha1,4-GalNAc-alpha1,3-Bac-beta1,N-Asn-Xaa, where Bac is bacillosamine, 2,4-diacetamido-2,4,6-trideoxyglucopyranose. Protein glycosylation was abolished when the pglB gene was mutated, providing further evidence that the enzyme encoded by this gene is responsible for formation of the glycopeptide N-linkage. Comparison of the pgl locus with that of Neisseria meningitidis suggested that most of the homologous genes are probably involved in the biosynthesis of bacillosamine.

Stable transformation of the Xylella fastidiosa citrus variegated chlorosis strain with oriC plasmids.

Xylella fastidiosa is a gram-negative, xylem-limited bacterium affecting economically important crops (e.g., grapevine, citrus, and coffee). The citrus variegated chlorosis (CVC) strain of X. fastidiosa is the causal agent of this severe disease of citrus in Brazil and represents the first plant-pathogenic bacterium for which the genome sequence was determined. Plasmids for the CVC strain of X. fastidiosa were constructed by combining the chromosomal replication origin (oriC) of X. fastidiosa with a gene which confers resistance to kanamycin (Kan(r)). In plasmid p16KdAori, the oriC fragment comprised the dnaA gene as well as the two flanking intergenic regions, whereas in plasmid p16Kori the oriC fragment was restricted to the dnaA-dnaN intergenic region, which contains dnaA-box like sequences and AT-rich clusters. In plasmid p16K, no oriC sequence was present. In the three constructs, the promoter region of one of the two X. fastidiosa rRNA operons was used to drive the transcription of the Kan(r) gene to optimize the expression of kanamycin resistance in X. fastidiosa. Five CVC X. fastidiosa strains, including strain 9a5c, the genome sequence of which was determined, and two strains isolated from coffee, were electroporated with plasmid p16KdAori or p16Kori. Two CVC isolates, strains J1a12 and B111, yielded kanamycin-resistant transformants when electroporated with plasmid p16KdAori or p16Kori but not when electroporated with p16K. Southern blot analyses of total DNA extracted from the transformants revealed that, in all clones tested, the plasmid had integrated into the host chromosome at the promoter region of the rRNA operon by homologous recombination. To our knowledge, this is the first report of stable transformation in X. fastidiosa. Integration of oriC plasmids into the X. fastidiosa chromosome by homologous recombination holds considerable promise for functional genomics by specific gene inactivation.

The random mutational analysis of the structure and function of photosystem II reaction center targeting to the psbAII gene of Synechocystis sp. PCC 6803

In order to analyze the structure?function relationships in the photosystem II (PSII), mutants of Synechocystis sp. PCC 6803 having random amino acid substitution(s) in the D1 subunit were created in this study. The targeted random mutagenesis covered 210 amino acid segment of the D1 protein (S148-A357). The kanamycin-resistant transformants carrying the mutated fragment of psbAII gene, created by in vitro random PCR, were screened based on the loss of photoautotrophy, either by direct inspection of growth on agar plates or systematic nitrofurantoin selection under illumination diminishing photosynthetically active cells (Yamasato and Satoh, 2001). By this method, we have succeeded in isolating approximately 150 photosynthesis-deficient mutants, moreover, 73 of them were heterotrophic mutants and did not contain any stop codon or codon shift. In the 73 mutants, the amino acid substitutions were distributed almost evenly in the targeted region. However, the frequency of mutation was high at specific sites, which have established to be important in the PSII, such as Y161, H198 and H272, proving the effectiveness of this method in identifying amino acid residues of functional importance in the PSII. On the other hand, some novel sites, such as Q165, Q187, N298 and W278, were identified to be important to the manifestation of photoautotrophy of this organism. The analysis of these random mutants provides entirely new aspects in understanding the structure?function relationships in the PSII, which could not be elucidated by the crystallographic analysis. The preliminary characterization of these mutants will be presented in the accompanying poster.

The LE1 bacteriophage replicates as a plasmid within Leptospira biflexa: construction of an L. biflexa-Escherichia coli shuttle vector.

We have discovered that LE1, one of the plaque-forming phages previously described as lytic for the Leptospira biflexa saprophytic spirochete (I. Saint Girons, D. Margarita, P. Amouriaux, and G. Baranton, Res. Microbiol. 141:1131-1138, 1990), was indeed temperate. LE1 was found to be unusual, as Southern blot analysis indicated that it is one of the few phages to replicate in the prophage state as a circular plasmid. The unavailability of such small endogenous replicons has hindered genetic experimentation in Leptospira. We have developed a shuttle vector with DNA derived from LE1. Random LE1 DNA fragments were cloned into a pGEM 7Zf(+) derivative devoid of most of the bla gene but carrying a kanamycin resistance marker from the gram-positive bacterium Enterococcus (Streptococcus) faecalis. These constructs were transformed into L. biflexa strain Patoc 1 by electroporation, giving rise to kanamycin-resistant transformants. A 2.2-kb fragment from LE1 was responsible for replication of the vector in L. biflexa. However, a larger region including an intact parA gene homologue was necessary for the stability of the shuttle vector. Direct repeats and AT-rich regions characterized the LE1 origin of replication. Our data indicate that the replicon derived from the LE1 leptophage, together with the kanamycin resistance gene, is a promising tool with which to develop the genetics of Leptospira species.

A second T-region of the soybean-supervirulent chrysopine-type Ti plasmid pTiChry5, and construction of a fully disarmed vir helper plasmid.

Agrobacterium tumefaciens Chry5, which is particularly virulent on soybeans, induces tumors that produce a family of Amadori-type opines that includes deoxyfructosyl glutamine (Dfg) and its lactone, chrysopine (Chy). Cosmid clones mapping to the right of the known oncogenic T-region of pTiChry5 conferred Amadori opine production on tumors induced by the nopaline strain C58. Sequence analysis of DNA held in common among these cosmids identified two 25-bp, direct repeats flanking an 8.5-kb segment of pTiChry5. These probable border sequences are closely related to those of other known T-regions and define a second T-region of pTiChry5, called T-right (TR), that confers production of the Amadoriopines. The oncogenic T-left region (TL) was located precisely by identifying and sequencing the likely border repeats defining this segment. The two T-regions are separated by approximately 15 kb of plasmid DNA. Based on these results, we predicted that pKYRT1, a vir helper plasmid derived from pTiChry5, still contains all of TR and the leftmost 9 kb of TL. Consistent with this hypothesis, transgenic Arabidopsis thaliana plants selected for with a marker encoded by a binary plasmid following transformation with KYRT1 co-inherited production of the Amadori opines at high frequency. All opine-positive transgenic plants also contained TR-DNA, while those plants that lacked TR-DNA failed to produce the opines. Moreover, A. thaliana infected with KYRT1 in which an nptII gene driven by the 35S promoter of Cauliflower mosaic virus was inserted directly into the vir helper plasmid yielded kanamycin-resistant transformants at a low but detectable frequency. These results demonstrate that pKYRT1 is not disarmed, and can transfer Ti plasmid DNA to plants. A new vir helper plasmid was constructed from pTiChry5 by two rounds of sacB-mediated selection for deletion events. This plasmid, called pKPSF2, lacks both of the known T-regions and their borders. pKPSF2 failed to transfer Ti plasmid DNA to plants, but mobilized the T-region of a binary plasmid at an efficiency indistinguishable from those of pKYRT1 and the nopaline-type vir helper plasmid pMP90.