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J T Turner J S Lampel R S Stearman G W Sundin P Gunyuzlu J J Anderson 《Applied and environmental microbiology》1991,57(12):3522-3528
Deletion of chromosomally inserted gene sequences from Clavibacter xyli subsp. cynodontis, a xylem-inhabiting endophyte, was studied in vitro and in planta. We found that nonreplicating plasmid pCG610, which conferred resistance to kanamycin and tetracycline and contained segments of C. xyli subsp. cynodontis genomic DNA, integrated into a homologous sequence in the bacterial chromosome. In addition, pCG610 contains two copies of the gene encoding the CryIA(c) insecticidal protein of Bacillus thuringiensis subsp. kurstaki HD73. Using drug resistance phenotypes and specific DNA probes, we found that the loss of all three genes arose both in vitro under nonselective conditions and in planta. The resulting segregants are probably formed by recombination between the repeated DNA sequences flanking pCG610 that resulted from the integration event into the chromosome. Eventually, segregants predominated in the bacterial population. The loss of the integrated plasmid from C. xyli subsp. cynodontis revealed a possible approach for decreasing the environmental consequences of recombinant bacteria for agricultural use. 相似文献
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Stability of the delta-endotoxin gene from Bacillus thuringiensis subsp. kurstaki in a recombinant strain of Clavibacter xyli subsp. cynodontis. 总被引:2,自引:2,他引:0 下载免费PDF全文
J T Turner J S Lampel R S Stearman G W Sundin P Gunyuzlu J J Anderson 《Applied microbiology》1991,57(12):3522-3528
Deletion of chromosomally inserted gene sequences from Clavibacter xyli subsp. cynodontis, a xylem-inhabiting endophyte, was studied in vitro and in planta. We found that nonreplicating plasmid pCG610, which conferred resistance to kanamycin and tetracycline and contained segments of C. xyli subsp. cynodontis genomic DNA, integrated into a homologous sequence in the bacterial chromosome. In addition, pCG610 contains two copies of the gene encoding the CryIA(c) insecticidal protein of Bacillus thuringiensis subsp. kurstaki HD73. Using drug resistance phenotypes and specific DNA probes, we found that the loss of all three genes arose both in vitro under nonselective conditions and in planta. The resulting segregants are probably formed by recombination between the repeated DNA sequences flanking pCG610 that resulted from the integration event into the chromosome. Eventually, segregants predominated in the bacterial population. The loss of the integrated plasmid from C. xyli subsp. cynodontis revealed a possible approach for decreasing the environmental consequences of recombinant bacteria for agricultural use. 相似文献
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Transformation of the gram-positive bacterium Clavibacter xyli subsp. cynodontis by electroporation with plasmids from the IncP incompatibility group. 下载免费PDF全文
We report the transformation of a gram-positive bacterium, Clavibacter xyli subsp. cynodontis, with several plasmids in the IncP incompatibility group from gram-negative bacteria. Our results suggest that IncP plasmids may be transferable to other gram-positive organisms. After optimizing electroporation parameters, we obtained a maximum of 2 x 10(5) transformants per microgram of DNA. The availability of a transformation system for this bacteria will facilitate its use in indirectly expressing beneficial traits in plants. 相似文献
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The 4992-bp replicon of a large cryptic plasmid in the gram-positive bacterium Leifsonia xyli subsp. cynodontis was identified and sequenced. The replicon encoded two proteins essential for plasmid replication and stability. The putative replication protein (RepA) is homologous to that of the plasmids in mycobacterial pLR7 family, while the putative ParA protein immediately downstream of RepA is significantly homologous to the Walker-type ATPase required for partition of plasmid and chromosome of the gram-positive bacteria. These two proteins and other ORFs are clustered with the putative promoters and other regulatory sequences, illustrating an efficient organization of the replicon for this novel plasmid. 相似文献
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Integrative Cloning, Expression, and Stability of the cryIA(c) Gene from Bacillus thuringiensis subsp. kurstaki in a Recombinant Strain of Clavibacter xyli subsp. cynodontis 下载免费PDF全文
Jay S. Lampel Gayle L. Canter Michael B. Dimock Jeffrey L. Kelly James J. Anderson Brenda B. Uratani James S. Foulke Jr. John T. Turner 《Applied microbiology》1994,60(2):501-508
A bacterial endophyte was engineered for insecticidal activity against the European corn borer. The cryIA(c) gene from Bacillus thuringiensis subsp. kurstaki was introduced into the chromosome of Clavibacter xyli subsp. cynodontis by using an integrative plasmid vector. The integration vectors pCG740 and pCG741 included the replicon pGEM5Zf(+), which is maintained in Escherichia coli but not in C. xyli subsp. cynodontis; tetM as a marker for selection in C. xyli subsp. cynodontis; and a chromosomal fragment of C. xyli subsp. cynodontis to allow for homologous recombination between the vector and the bacterial chromosome. Insertion of vector DNA into the chromosome was demonstrated by DNA hybridization. Recombinant strains MDR1.583 and MDR1.586 containing the cryIA(c) gene were shown to produce the 133,000-kDa protoxin and several smaller immunoreactive proteins. Both strains were equally toxic to insect larvae in bioassays. Significant insecticidal activity was demonstrated in planta. The cryIA(c) gene and the tetM gene introduced into strain MDR1.586 were shown to be deleted from some cells, thereby giving rise to a noninsecticidal segregant population. In DNA hybridization experiments and insect bioassays, these segregants were indistinguishable from the wild-type strain. Overall, these results demonstrate the plausibility of genetically engineered bacterial endophytes for insect control. 相似文献
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Here we report a toxin-antitoxin (TA) operon talAB identified from the Gram-positive bacterium Leifsonia xyli subsp. cynodontis. It is shown that talB encodes a broad-host cytotoxin functioning in different Gram-positive bacteria, while talA encodes its antidote. TalA and TalB form different hetero-oligomers in vitro; these hetero-oligomers, but not the antitoxin TalA, strongly bind to the talAB promoter region containing two inverted repeats. This represents a new mechanism of binding the promoter of a TA operon by the toxin and antitoxin complexes. 相似文献
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Suzuki KI Suzuki M Sasaki J Park YH Komagata KK 《The Journal of General and Applied Microbiology》1999,45(5):253-262
"Corynebacterium aquaticum" was first proposed by Leifson in 1962 but not included in the approved lists of bacterial names in 1980. This species has been left from reclassification of the genus Corynebacterium because of the unusual chemotaxonomic characteristics such as 2,4-diaminobutyric acid (DAB) in the peptidoglycan and menaquinones of MK-10 and MK-11. A close relationship of "C. aquaticum" to the genera Agromyces and Rathayibacter has been pointed out from the viewpoint of chemotaxonomic profiles and phylogeny based on the 16S rDNA sequences. An analysis of DAB isomers of the peptidoglycan distinguished "C. aquaticum" clearly from these genera by possessing both L-DAB and D-DAB. We also found that the type strain of Clavibacter xyli subsp. cynodontis and two strains of amine-decomposing bacteria showed the similar chemotaxonomic features and formed a cluster with "C. aquaticum" in the phylogenetic tree based on 16S rDNA sequences in the family Microbacteriaceae. Considering these results, we propose a new genus Leifsonia to accommodate the four strains. The four species, Leifsonia aquatica sp. nov., nom. rev., comb. nov. (type species, type strain=JCM 1368), Leifsonia shinshuensis sp. nov. (type strain=DB102=JCM 10591), Leifsonia naganoensis sp. nov. (type strain=DB103=JCM 10592), and Leifsonia cynodontis comb. nov. (type strain=JCM 9733=ICMP 8790), were proposed here for the strains. 相似文献
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Development and Application of Cloned DNA Probes for Clavibacter xyli subsp. xyli, the Causal Agent of Sugarcane Ratoon Stunting 总被引:1,自引:0,他引:1
Eco RI restriction fragments of genomic DNA from Clavibacter xyli subsp. xyli (CXX) were ligated with plasmid pUC18 and cloned in Escherichia coli JM109. The cloned DNA inserts from recombinant plasmids were Eco RI-excised and labeled with non-radioactive digoxigenin and used as probes. Ten specific DNA probes, RSD3, 15, 30, 31, 32, 35, 37, 41, 71, and 73 were selected for disease detection and pathogen differentiation. In the specificity tests, all of the 10 CXX DNA, probes differentiated Clavibacter xyli from other bacteria specifically. Seven out of the 10 CXX probes crossreacted with C. x. subsp. cynodontis (CXC) very weakly under moderate stringency wash conditions of hybridization. In the sensitivity tests, all of the 10 DNA probes detected the homologous DNA of CXX from 0.19 to 0.75 ng. To detect various cell numbers of CXX, the DNA probes detected 104 to 105 cells effectively. In Southern hybridizations, distinctly different band patterns were shown when the probes hybridized with DNA from CXX and CXC. Among these probes, RSD3, 15, 30, 31, 35, 37, and 71, efficiently detected CXX present in the sap collected from symptomless sugarcane. 相似文献
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Contour-clamped homogeneous electric field gel analysis of genomic DNA of the plant pathogen Clavibacter michiganensis subsp. sepedonicus revealed the presence of a previously unreported extrachromosomal element. This new element was demonstrated to be a linear plasmid. Of 11 strains evaluated, all contained either a 90-kb (pCSL1) or a 140-kb (pCSL2) linear plasmid. 相似文献
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Brumbley SM Petrasovits LA Murphy RM Nagel RJ Candy JM Hermann SR 《Molecular plant-microbe interactions : MPMI》2004,17(2):175-183
Leifsonia xyli subsp. xyli, the causal agent of ratoon stunting disease in sugarcane, is a xylem-limited, nutritionally fastidious, slow growing, gram-positive coryneform bacterium. Because of the difficulties in growing this bacterium in pure culture, little is known about the molecular mechanisms of pathogenesis. Currently, the genome sequence of L. xyli subsp. xyli is being completed by the Agronomical and Environmental Genomes group from the Organization for Nucleotide Sequencing and Analysis in Brazil. To complement this work, we produced 712 Lxx::Tn4431 transposon mutants and sequenced flanking regions from 383 of these, using a rapid polymerase chain reaction-based approach. Tn4431 insertions appeared to be widespread throughout the L. xyli subsp. xyli genome; however, there were regions that had significantly higher concentrations of insertions. The Tn4431 mutant library was screened for individuals unable to colonize sugarcane, and one noncolonizing mutant was found. The mutant contained a transposon insertion disrupting two open reading frames (ORF), one of which had homology to an integral membrane protein from Mycobacterium leprae. Sequencing of the surrounding regions revealed two operons, pro and cyd, both of which are believed to play roles in disease. Complementation studies were carried out using the noncolonizing Lxx::Tn4431 mutant. The noncolonizing mutant was transformed with a cosmid containing 40 kbp of wild-type sequence, which included the two ORF disrupted in the mutant, and several transformants were subsequently able to colonize sugarcane. However, analysis of each of these transformants, before and after colonization, suggests that they have all undergone various recombinant events, obscuring the roles of these ORF in L. xyli subsp. xyli pathogenesis. 相似文献
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Plasmids have rarely been detected in organisms constituting the genus Mycoplasma. Recently, the isolation of a cryptic plasmid from Mycoplasma mycoides subsp. mycoides has been described, and we report here the isolation of a second cryptic plasmid from this species. Restriction map and Southern blot analyses show that the second plasmid is distinct from the previously described plasmid, although a limited region of homology was detected. The availability of mycoplasmal cryptic plasmids may lead to the development of cloning vectors that replicate in these organisms. 相似文献
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Isolation and characterization of Lactococcus lactis subsp. lactis promoters. 总被引:10,自引:5,他引:5 下载免费PDF全文
DNA fragments with promoter activity were isolated from the chromosome of Lactococcus lactis subsp. lactis. For the isolation, a promoter probe vector based on the cat gene was constructed, which allowed direct selection with chloramphenicol in Bacillus subtilis and L. lactis. Four of the putative promoters (P1, P2, P10, and P21) were analyzed further by sequencing, mapping of the 5' end of the mRNA, Northern (RNA blot) hybridization, and chloramphenicol acetyltransferase activity measurements. From these fragments, -10 and -35 regions resembling the consensus Escherichia coli sigma 70 and B. subtilis sigma 43 promoters were identified. Another set of promoters, together with a signal sequence, were also isolated from the same organism. These fragments promoted secretion of TEM beta-lactamase from L. lactis. When the two sets of promoters were compared, it was found that the ones isolated with the cat vector were more efficient (produced more mRNA). By changing the promoter part of the promoter-signal sequence fragment giving the best TEM beta-lactamase secretion into a more efficient one (P2), a 10-fold increase in enzyme production was obtained. 相似文献
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DNA fragments with promoter activity were isolated from the chromosome of Lactococcus lactis subsp. lactis. For the isolation, a promoter probe vector based on the cat gene was constructed, which allowed direct selection with chloramphenicol in Bacillus subtilis and L. lactis. Four of the putative promoters (P1, P2, P10, and P21) were analyzed further by sequencing, mapping of the 5' end of the mRNA, Northern (RNA blot) hybridization, and chloramphenicol acetyltransferase activity measurements. From these fragments, -10 and -35 regions resembling the consensus Escherichia coli sigma 70 and B. subtilis sigma 43 promoters were identified. Another set of promoters, together with a signal sequence, were also isolated from the same organism. These fragments promoted secretion of TEM beta-lactamase from L. lactis. When the two sets of promoters were compared, it was found that the ones isolated with the cat vector were more efficient (produced more mRNA). By changing the promoter part of the promoter-signal sequence fragment giving the best TEM beta-lactamase secretion into a more efficient one (P2), a 10-fold increase in enzyme production was obtained. 相似文献
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Monteiro-Vitorello CB Camargo LE Van Sluys MA Kitajima JP Truffi D do Amaral AM Harakava R de Oliveira JC Wood D de Oliveira MC Miyaki C Takita MA da Silva AC Furlan LR Carraro DM Camarotte G Almeida NF Carrer H Coutinho LL El-Dorry HA Ferro MI Gagliardi PR Giglioti E Goldman MH Goldman GH Kimura ET Ferro ES Kuramae EE Lemos EG Lemos MV Mauro SM Machado MA Marino CL Menck CF Nunes LR Oliveira RC Pereira GG Siqueira W de Souza AA Tsai SM Zanca AS Simpson AJ Brumbley SM Setúbal JC 《Molecular plant-microbe interactions : MPMI》2004,17(8):827-836
The genome sequence of Leifsonia xyli subsp. xyli, which causes ratoon stunting disease and affects sugarcane worldwide, was determined. The single circular chromosome of Leifsonia xyli subsp. xyli CTCB07 was 2.6 Mb in length with a GC content of 68% and 2,044 predicted open reading frames. The analysis also revealed 307 predicted pseudogenes, which is more than any bacterial plant pathogen sequenced to date. Many of these pseudogenes, if functional, would likely be involved in the degradation of plant heteropolysaccharides, uptake of free sugars, and synthesis of amino acids. Although L. xyli subsp. xyli has only been identified colonizing the xylem vessels of sugarcane, the numbers of predicted regulatory genes and sugar transporters are similar to those in free-living organisms. Some of the predicted pathogenicity genes appear to have been acquired by lateral transfer and include genes for cellulase, pectinase, wilt-inducing protein, lysozyme, and desaturase. The presence of the latter may contribute to stunting, since it is likely involved in the synthesis of abscisic acid, a hormone that arrests growth. Our findings are consistent with the nutritionally fastidious behavior exhibited by L. xyli subsp. xyli and suggest an ongoing adaptation to the restricted ecological niche it inhabits. 相似文献
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Analysis of the promoters for the two immunity genes present in the ColE3-CA38 plasmid using two new promoter probe vectors. 总被引:5,自引:0,他引:5 下载免费PDF全文
We have constructed two new promoter probe vectors which carry a polylinker derived from plasmid pUC19 proximal to the 5' end of a promoter-less galactokinase gene. Using these two vectors we have demonstrated that the ColE3imm gene and the ColE8imm gene present on the ColE3-CA38 plasmid have their own promoters, independent of the SOS promoter of the colicin E3 structural gene. The activity of two terminators, one located proximal to the 5' end of the ColE8imm gene, the other located proximal to the 5' end of the lys gene, were shown by a comparison of the galactokinase activity conferred by several of the recombinant plasmids. 相似文献
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