In Situ Transfer of Antibiotic Resistance Genes from Transgenic (Transplastomic) Tobacco Plants to Bacteria

Interkingdom gene transfer is limited by a combination of physical, biological, and genetic barriers. The results of greenhouse experiments involving transplastomic plants (genetically engineered chloroplast genomes) cocolonized by pathogenic and opportunistic soil bacteria demonstrated that these barriers could be eliminated. The Acinetobacter sp. strain BD413, which is outfitted with homologous sequences to chloroplastic genes, coinfected a transplastomic tobacco plant with Ralstonia solanacearum and was transformed by the plant's transgene (aadA) containing resistance to spectinomycin and streptomycin. However, no transformants were observed when the homologous sequences were omitted from the Acinetobacter sp. strain. Detectable gene transfer from these transgenic plants to bacteria were dependent on gene copy number, bacterial competence, and the presence of homologous sequences. Our data suggest that by selecting plant transgene sequences that are nonhomologous to bacterial sequences, plant biotechnologists could restore the genetic barrier to transgene transfer to bacteria.     

Exploration of horizontal gene transfer between transplastomic tobacco and plant-associated bacteria

The likelihood of gene transfer from transgenic plants to bacteria is dependent on the transgene copy number and on the presence of homologous sequences for recombination. The large number of chloroplast genomes in a plant cell as well as the prokaryotic origin of the transgene may thus significantly increase the likelihood of gene transfer from transplastomic plants to bacteria. In order to assess the probability of such a transfer, bacterial isolates, screened for their ability to colonize decaying tobacco plant tissue and possessing DNA sequence similarity to the chloroplastic genes accD and rbcL flanking the transgene (aadA), were tested for their ability to take up extracellular DNA (broad host-range pBBR1MCS-3-derived plasmid, transplastomic plant DNA and PCR products containing the genes accD-aadA-rbcL) by natural or electrotransformation. The results showed that among the 16 bacterial isolates tested, six were able to accept foreign DNA and acquire the spectinomycin resistance conferred by the aadA gene on plasmid, but none of them managed to integrate transgenic DNA in their chromosome. Our results provide no indication that the theoretical gene transfer-enhancing properties of transplastomic plants cause horizontal gene transfer at rates above those found in other studies with nuclear transgenes.     

Transformation of Acinetobacter sp. strain BD413(pFG4DeltanptII) with transgenic plant DNA in soil microcosms and effects of kanamycin on selection of transformants

Here we show that horizontal transfer of DNA, extracted from transgenic sugar beets, to bacteria, based on homologous recombination, can occur in soil. Restoration of a 317-bp-deleted nptII gene in Acinetobacter sp. strain BD413(pFG4) cells incubated in sterile soil microcosms was detected after addition of nutrients and transgenic plant DNA encoding a functional nptII gene conferring bacterial kanamycin resistance. Selective effects of the addition of kanamycin on the population dynamics of Acinetobacter sp. cells in soil were found, and high concentrations of kanamycin reduced the CFU of Acinetobacter sp. cells from 10(9) CFU/g of soil to below detection. In contrast to a chromosomal nptII-encoded kanamycin resistance, the pFG4-generated resistance was found to be unstable over a 31-day incubation period in vitro.     

Intergeneric transfer of chromosomal and conjugative plasmid genes between Ralstonia solanacearum and Acinetobacter sp. BD413

Conjugative transfer of a broad-host range plasmid and transformation-mediated transfer of chromosomal genes were found to occur at significant frequencies between Ralstonia solanacearum and Acinetobacter sp. in planta. These intergeneric gene transfers are related to the conditions provided by the infected plant, including the extensive multiplication of these two bacteria in planta and the development of a competence state in Acinetobacter sp. Although interkingdom DNA transfer from nuclear transgenic plants to these bacteria was not detectable, plants infected by pathogens (e.g., Ralstonia solanacearum) and co-colonized by soil saprophyte bacteria (e.g., Acinetobacter sp.) can be considered as potential "hot spots" for gene transfer, even between phylogenetically remote organisms.     

System to study horizontal gene exchange among microorganisms without cultivation of recipients

Ribosomal RNA genes are characterized by highly conserved sequences and are present in multiple copies in most prokaryotic chromosomes. In principle, therefore, they might serve as sites for homologous recombination between unrelated microorganisms. Plasmids containing 23S ribosomal gene sequences, from different bacteria, which had been interrupted by insertion of a kanamycin-resistance gene, were used to transform Acinetobacter sp. DSM587 (former name: Acinetobacter calcoaceticus BD413-ivl10). In all cases, homologies between the 23S rRNA genes of phylogenetically distant bacteria and Acinetobac-ter sp. DSM587 were sufficient for replacement recombination events. The integration events, resulting in inactivation of any one of the seven rrn operons of Acinetobacter sp. DSM587, had no observable influence on cell growth. These results suggest the possibility of rRNA genes serving as natural vehicles for horizontal gene transfer. They also provide the basis of a novel strategy to analyse gene transfer without selection or cultivation of recipient cells. Because of the highly conserved structure of bacterial rrn operons, recombination events subsequent to gene transfer can be readily identified by polymerase chain reaction amplification of the recombinant sequence using a universal forward primer for the 16S rRNA gene and a reverse primer specific for the integrated marker gene.     

柑橘黄龙病赣南脐橙内生菌种群结构分析

【目的】分析赣南脐橙黄龙病植株和健康植株叶片内生菌,对比不同培养条件下培养出的内生菌,为筛选出对柑橘黄龙病原菌有影响的伴生菌奠定理论基础。【方法】通过PCR方法对脐橙中黄龙病菌进行验证,并基于16S r RNA基因高通量测序技术对患病与健康赣南脐橙叶片内生菌以及不同培养基富集培养后的内生菌进行多样性分析。【结果】所采集样品中有5株患病株,5株健康株。5株病株中共同含有的细菌属有13个,其中7个在5株健株中也共同存在。Defluviicoccus属和Granulicella属在病健株植物中都是优势菌属,且在健株中的平均含量高于病株。病株与健株的样品相似度存在明显界线。富集培养后不同样本和不同培养基中菌属分布不同。肠杆菌属(Enterobacter)、短小杆菌属(Curtobacterium)、假单胞菌属(Pseudomonas)和泛菌属(Pantoea)得到了大量富集,不动杆菌属和沙雷氏菌属等9个菌属富集量较少。另外,培养和未培养各样本间未分类菌(Unclassified)含量差异也较大。【结论】赣南脐橙患病植株和健康植株叶片内生菌有着明显差异,黄龙病菌的存在改变了脐橙叶片原有内生细菌的菌群结构。从活体植物组织内直接检测才能得到真正的植物内生菌群落分布情况。通过分析菌群的差异,有望找到与柑橘黄龙病菌生长相关的伴生菌。     

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.     

Gene silencing：Double－stranded RNA mediated mRNA degradation and gene inactivation

INTRODUCTIONThe genome structure of plants can be alteredby genetic transformation. During the process ofgene transfer, Agrobacterium tumefaCJens integratepart of their genome into the genome of susceptiblespecies. Recently, genetic transfOrmation techniqueshave been used to modify significantly the organi-zation of the genome. Introducing transgenes intop1ants can both modify the number of copies of agiven sequence and affect gene expression. Becausethe expression of a transgene cannot…     

Comparison of effectiveness of 5′-regulatory sequences in transplastomic tobacco chloroplasts

The development of tools which ensure the desired level of transgene expression in plastids is a prerequisite for the effective utilization of these plant organelles for the deployment of bioactive proteins. High-level accumulation of target proteins is considered as a positive feature of transplastomic plants, but excessive accumulation of foreign proteins may have deleterious effects on host plants. On the other hand, expression at low levels can result in ineffective phenotypes. We compared the effectiveness of different 5′-regulatory sequences in driving the expression of a reporter gene, β-glucuronidase (uidA), in tobacco chloroplasts. To achieve varying expression levels, we have chosen heterologous 5′-regulatory sequences which either differ significantly from their homologous counterparts or depend on specific nuclear encoded factors. The Medicago truncatula psbA promoter/5′-UTR supported the highest levels of protein accumulation, surpassing the other tested sequences by two to three orders of magnitude. The heterologous regulatory sequence of Phaseolus vulgaris rbcL gene was as efficient in tobacco chloroplasts as the corresponding homologous promoter/5′-UTR. The Arabidopsis thaliana ndhF promoter/5′-UTR supported as high reporter activity levels as the rbcL 5′-sequences, whereas the effectiveness of A. thaliana psbN promoter/5′-UTR was three fold lower. The characterized regulatory sequences can be utilized to establish transplastomic lines with desirable levels of target protein accumulation. The ability to control transgene expression should be useful for achieving appropriate levels of protein accumulation and thereby avoid their negative impacts on host plant physiology.     

Generation of fertile transplastomic soybean

We describe here the development of a plastid transformation method for soybean, a leguminous plant of major agronomic interest. Chloroplasts from embryogenic tissue of Glycine max have been successfully transformed by bombardment. The transforming DNA carries a spectinomycin resistance gene (aadA) under the control of tobacco plastid regulatory expression elements, flanked by two adjacent soybean plastome sequences allowing its targeted insertion between the trnV gene and the rps12/7 operon. All generated spectinomycin resistant plants were transplastomic and no remaining wild type plastome copies were detected. No spontaneous mutants were obtained. The transformation efficiency is similar to that of tobacco plastids. All transplastomic T0 plants were fertile and T1 progeny was uniformly spectinomycin resistant, showing the stability of the plastid transgene. This is the first report on the generation of fertile transplastomic soybean.     

Degradation and transformability of DNA from transgenic leaves

The fate of transplastomic (chloroplast genome contains the transgene) tobacco plant DNA in planta was studied when the plant leaves were subjected to decay conditions simulating those encountered naturally, including grinding, incubation with cellulase or enzymes produced by Erwinia chrysanthemi, and attack by the plant pathogen Ralstonia solanacearum. Direct visualization of DNA on agarose gels, gene extraction yield (the number of amplifiable aadA sequences in extracted plant DNA), and the frequency that recipient bacteria can be transformed by plant DNA were used to evaluate the quality and quantity of plant DNA and the transgene. These measurements were used to monitor the physical and biological degradation of DNA inside decaying plant tissues. Our results indicate that while most of the DNA will be degraded inside plant cells, sufficient DNA persists to be released into the soil.     

不动杆菌属中aidE基因编码高丝氨酸内酯酶

群体感应(Quorum sensing,QS)是细菌在进化过程中形成的依赖于群体密度的细菌间交流方式。许多革兰氏阴性细菌以N-酰基高丝氨酸内酯(AHL)为信号分子,感应自身群体密度并调控致病基因表达。因此,淬灭AHLs信号分子可防治此类细菌引起的植物病害。本实验室前期已筛选得到了一株具有AHLs信号降解能力的不动杆菌菌株Acinetobacter sp.77,本研究通过基因组文库筛选,自菌株77中克隆得到具有AHLs降解活性的基因aidE。该基因编码268个氨基酸。序列一致性比较发现aidE的氨基酸序列与吉伦伯不动杆菌Acinetobacter gyllenbergii CIP110306中β-内酰胺酶一致性高达95%,但与已知的AHLs降解酶序列一致性较低,最高为缓黄分支杆菌Mycobacterium lentiflavum中AHL内酯酶Att M/Aii B家族蛋白(CQD23908.1),一致性仅为33%。通过高压液相色谱(HPLC)分析Aid E蛋白处理N-己酰基高丝氨酸内酯(C6-HSL)的反应产物,证明aidE为AHL内酯酶。序列比对研究发现,aidE基因在不动杆菌属中并不保守,其在菌株77基因组中的上下游的基因排列存在菌株水平的特异性,且aidE基因下游存在疑似IS插入序列,上述证据表明aidE基因有可能是通过水平转移进入Acinetobacter sp.77基因组中,或其在基因组中的位置发生过重排。表达aidE的软腐果胶杆菌Z3-3中完全检测不到AHLs信号产生,且致病力明显降低。综上所述,aidE为新发现的AHL内酯酶。在防治依赖QS系统表达致病性的细菌病害中具有应用潜力。     

Natural transformation of Acinetobacter sp. strain BD413 with cell lysates of Acinetobacter sp., Pseudomonas fluorescens, and Burkholderia cepacia in soil microcosms

To elucidate the biological significance of dead bacterial cells in soil to the intra- and interspecies transfer of gene fragments by natural transformation, we have exposed the kanamycin-sensitive recipient Acinetobacter sp. strain BD413(pFG4) to lysates of the kanamycin-resistant donor bacteria Acinetobacter spp., Pseudomonas fluorescens, and Burkholderia cepacia. Detection of gene transfer was facilitated by the recombinational repair of a partially (317 bp) deleted kanamycin resistance gene in the recipient bacterium. The investigation revealed a significant potential of these DNA sources to transform Acinetobacter spp. residing both in sterile and in nonsterile silt loam soil. Heat-treated (80 degrees C, 15 min) cell lysates were capable of transforming strain BD413 after 4 days of incubation in sterile soil and for up to 8 h in nonsterile soil. Transformation efficiencies obtained in vitro and in situ with the various lysates were similar to or exceeded those obtained with conventionally purified DNA. The presence of cell debris did not inhibit transformation in soil, and the debris may protect DNA from rapid biological inactivation. Natural transformation thus provides Acinetobacter spp. with an efficient mechanism to access genetic information from different bacterial species in soil. The relatively short-term biological activity (e.g., transforming activity) of chromosomal DNA in soil contrasts the earlier reported long-term physical stability of DNA, where fractions have been found to persist for several weeks in soil. Thus, there seems to be a clear difference between the physical and the functional significance of chromosomal DNA in soil.     

Opportunistic colonization of Ralstonia solanacearum-infected plants by Acinetobacter sp. and its natural competence development

The behavior of the soil bacterium Acinetobacter sp. BD413 was monitored in Ralstonia solanacearum-infected and non-infected tomato plants after direct injection into the stem or natural infection by roots. In healthy plants, Acinetobacter sp. BD413 failed to colonize plant tissue. In plants infected simultaneously by the pathogen R. solanacearum,the Acinetobacter population increased linearly to about 3.1 x 10(7) cells per gram plant material and was maintained at a high level until the death of the plant. Moreover, Acinetobacter sp. BD413 was found to develop a competent state when multiplying in planta, indicating it could possibly be transformed by bacterial or plant DNA.     

Culture and identification of Desulfovibrio spp. from corals infected by black band disease on Dominican and Florida Keys reefs

Black band disease (BBD) of corals is characterized as a pathogenic microbial consortium composed of a wide variety of microorganisms. Together, many of these microorganisms contribute to an active sulfur cycle that produces anoxia and high levels of sulfide adjacent to the coral surface, conditions that are lethal to coral tissue. Sulfate-reducing bacteria, as sulfide producers, are an important component of the sulfur cycle and the black band community. Previous molecular survey studies have shown multiple Desulfovibrio species present in BBD but with limited consistency between bacterial species and infections. In this study we compared 16S rRNA gene sequences of sulfate-reducing bacteria selectively cultured from 6 BBD bands on 4 coral species, Diploria clivosa, D. strigosa, D. labyrinthiformes, and Siderastrea siderea, in the Florida Keys and Dominica. The 16S rRNA gene sequences were obtained through direct sequencing of PCR products or by cloning. A BLAST search revealed that 8 out of 10 cultures sequenced were highly homologous to Desulfovibrio sp. strain TBP-1, a strain originally isolated from marine sediment. Although the remaining 2 sequences were less homologous to Desulfovibrio sp. strain TBP-1, they did not match any other sulfate-reducing (or other) species in GenBank.     

Isolation and characterization of dibenzofuran-degrading bacteria

Two bacterial strains capable of utilizing dibenzofuran (DF) as a sole carbon source were isolated from soil samples of reclaimed land. The strains designated HL1 and HL7 were identified as Klebsiella sp. and Sphingomonas sp., respectively, on the basis of biochemical characteristics and the sequences of the 16S ribosomal DNA. Sphingomonas sp. strain HL7 degraded non-, mono- and also dichlorinated DF and dibenzo-p-dioxin (DD). Klebsiella sp. strain HL1 was able to degrade non- and monochlorinated DFs and DDs, but not dichlorinated ones. The metabolites formed from DF by strains HL1 and HL7 were similar to those by dioxin-degrading bacteria Sphingomonas sp. strain RW1 except for salicylic acid and catechol. Strain HL7 had a gene homologous to that encoding the dioxin dioxygenase alpha-subunit (dxnA1) gene of Sphingomonas sp. strain RW1. However, Southern hybridization analysis showed that the size of an EcoRV-digested genomic fragment involving the dioxin dioxygenase gene of strain HL7 was smaller than that of strain RW1, and that strain HL1 did not have the homologous gene. Strains HL1 and HL7 provided useful information regarding the dioxygenase genes.