Pathogenicity and identification of the lilac pathogen, Pseudomonas syringae pv. syringae van Hall 1902

Comparative in planta studies with Pseudomonas syringae pv. syringae have established optimum conditions for disease expression in lilac in terms of inoculum concentration, host age and post-inoculation conditions (temperature and day-length). Reproducible disease reactions required an inoculum concentration exceeding the ED⁵⁰, 5 × 10⁶ cfu/ml, and a temperature for post-inoculation incubation not exceeding 19°C. A revised host range of P. syringae pv. syringae, proposed on the basis of confirmation of pathogenicity of strains to lilac, comprises 44 species from monocotyledonous and dicotyledonous plants. Nine new hosts Abelmoschus esculentus, Bromus willdenowii, Camellia sinensis, Centrosema pubescens, Citrullus lanatus, Cotoneaster sp., Cucumis melo, Populus×euramericana and Triticum aestivum, are recorded. A comparative laboratory study was made of strains of P. syringae pv. syringae using more than 30 selected biochemical and nutritional tests. The pathovar could be characterised on the basis of 11 of these which may prove to be useful determinative tests.     

Conjugal transfer and mobilization capacity of the completely sequenced naphthalene plasmid pNAH20 from multiplasmid strain Pseudomonas fluorescens PC20

The complete 83 042-bp nucleotide sequence of the IncP-9 naphthalene degradation plasmid pNAH20 from Pseudomonas fluorescens PC20 exhibits striking similarity in size and sequence to another naphthalene (NAH) plasmid pDTG1. However, the positions of insertion sequence (IS) elements significantly alter both catabolic and backbone functions provided by the two plasmids. In pDTG1, insertion of a pCAR1 IS Pre1 -like element disrupts expression of the lower naphthalene operon and this strain utilizes the chromosomal pathway for complete naphthalene degradation. In pNAH20, this operon is intact and functional. The transfer frequency of pNAH20 is 100 times higher than that of pDTG1 probably due to insertion of the pCAR1 IS Pre2 -like element into the mpfR gene coding for a putative repressor of the mpf operon responsible for mating pilus formation. We also demonstrate in situ plasmid transfer – we isolated a rhizosphere transconjugant strain of pNAH20, P. fluorescens NS8. The plasmid pNS8, a derivative of pNAH20, lacks the ability to self-transfer as a result of an additional insertion event of IS Pre2 -like element that disrupts the gene coding for VirB2-like major pilus protein MpfA. The characteristics of the strain PC20 and the conjugal transfer/mobilization capacity of pNAH20 (or its backbone) make this strain/plasmid a potentially successful tool for bioremediation applications.     

Evidence for stable transformation of Pseudomonas aeruginosa with a pUC-based plasmid

Pseudomonas aeruginosa was transformed with pUC8:16, a pUC-based plasmid bearing the gene (vgb) encoding Vitreoscilla (bacterial) hemoglobin (VHb). Transformation was initially indicated by an increase in ampicillin resistance from 1500 to 2500 mg l^–1. Presence of the plasmid in P. aeruginosa was confirmed by amplification of a portion of vgb from and detection of VHb in the transformant but not the untransformed host. Southern blot analysis further indicated that pUC8:16 existed as an autonomous plasmid rather than integrated into the chromosome of the P. aeruginosa transformant.     

Host strain influences on supercoiled plasmid DNA production in Escherichia coli: Implications for efficient design of large-scale processes

We set out to investigate if E. coli genotype plays a significant role in host strain selection for optimal processing of plasmid DNA based on both quality and quantity of supercoiling. Firstly 17 E. coli commercial and non-commercial strains were selected and their available genetic backgrounds were researched in the open literature. Growth characteristics of all the strains were considered and made impartial by using a common medium and growth condition platform. By keeping the growth conditions constant for each strain/plasmid combination, we are only looking at one variable which is the host strain. The second step was to attempt to correlate the findings with common genotype characteristics (e.g. mutations such as endA or recA). We found that one can screen the number of strains which are likely to give good productivity early on, before any further optimisation and verification is performed, both for small and large plasmids. Also, it is worth noting that complex plasmid interactions with each strain prevent the use of genotype alone in making an intelligent choice for supercoiling optimisation. This leads to a third optimisation step selecting a few of the potentially high performing strains based on high DNA yield and supercoiling, with a view to identify the factors which need improvement in strain design and bioreactor optimisation. We found that high specific growth rates of some strains did not affect the level of DNA supercoiling but did influence the total plasmid yield, potentially an important aspect in the design of fermentation strategy. Interestingly, a few host/plasmid combinations result in what appears to be runaway plasmid replication.     

Detection of IncP replicon-specific regions in DNA from Antarctic microbiota

Plasmids capable of horizontal transfer contribute to the adaptability of bacteria, as they may provide genes that enable their hosts to cope with different selective pressures. Only limited information is available on plasmids from Antarctic habitats, and up until now surveys have only used traditional methods of endogenous plasmid isolation. The method based on primer systems, designed on the basis of published sequences for plasmids from different incompatibility (Inc) groups, is appropriate to detect the replicon-specific regions of corresponding plasmids in cultured bacteria, or in total community DNA, which share sufficient DNA similarity with reference plasmids at the amplified regions. In this study, we applied broad-host-range plasmid-specific primers to DNA from microbial samples collected at six different locations in Northern Victoria Land (Antarctica). DNA preparations were used as targets for PCR (polymerase chain reaction) amplification with primers for the IncP (trfA2) and IncQ (oriV ) groups. PCR products were Southern blotted and hybridized with PCR-derived probes for trfA2 and oriV regions. This approach detected the occurrence of IncP-specific sequences in eight out of fifteen DNA samples, suggesting a gene-mobilizing capacity within the original habitats.     

Frequent conjugative transfer accelerates adaptation of a broad-host-range plasmid to an unfavorable Pseudomonas putida host

IncP-1 plasmids are known to be promiscuous, but it is not understood if they are equally well adapted to various species within their host range. Moreover, little is known about their fate in bacterial communities. We determined if the IncP-1beta plasmid pB10 was unstable in some Proteobacteria, and whether plasmid stability was enhanced after long-term carriage in a single host and when regularly switched between isogenic hosts. Plasmid pB10 was found to be very unstable in Pseudomonas putida H2, and conferred a high cost (c. 20% decrease in fitness relative to the plasmid-free host). H2(pB10) was then evolved under conditions that selected for plasmid maintenance, with or without regular plasmid transfer (host-switching). When tested in the ancestral host, the evolved plasmids were more stable and their cost was significantly reduced (9% and 16% for plasmids from host-switched and nonswitched lineages, respectively). Our findings suggest that IncP-1 plasmids can rapidly adapt to an unfavorable host by improving their overall stability, and that regular conjugative transfer accelerates this process.     

Complete sequence and genetic organization of pDTG1, the 83 kilobase naphthalene degradation plasmid from Pseudomonas putida strain NCIB 9816-4

The complete 83,042 bp sequence of the circular naphthalene degradation plasmid pDTG1 from Pseudomonas putida strain NCIB 9816-4 was determined in order to examine the process by which the nah and sal operons may have been compiled and distributed in nature. Eighty-nine open reading frames were predicted using computer analyses, comprising 80.0% of the pDTG1 DNA sequence. The most distinctive feature of the plasmid is the upper and lower naphthalene degradation operons, which occupy 9.5 kb and 13.4 kb regions, respectively, bordered by numerous defective mobile genetic element fragments. Identified on this plasmid were homologues of genes required for large plasmid replication, maintenance, and conjugation, as well as transposases, resolvases, and integrases, suggesting an evolution that involved the lateral transfer of DNA between bacterial species. Also found were genes that contain a high degree of sequence similarity to other known degradation genes, as well as genes involved in chemotaxis. Although the incompatibility group designation of pDTG1 remains unresolved, striking sequence organization and homology exists between the plasmid backbones of pDTG1 and the IncP-9 toluene-degradation plasmid pWW0, which suggests a divergent evolution from a progenitor plasmid prior to degradative gene incorporation.     

Structural rearrangements of a broad host range plasmid encoding mercury resistance from an epilithic isolate of Pseudomonas cepacia

Abstract A broad host range plasmid encoding mercury resistance (pQM3) underwent structural rearrangement when transferred into Pseudomonas aeruginosa . The rearrangement involved deletions and additions of DNA to the plasmid and resulted in the formation of smaller derivative plasmids. A 13 kb insert acquired in an epilithic Pseudomonas fluorescens isolate stabilised pQM3 and prevented rearrangements in P. aeruginosa . Integration of pQM3 into the chromosome of its natural host Pseudomonas cepacia PAR 161, and the P. aeruginosa chromosome reduced its transfer efficiency.     

Purification and characterization of the sopB gene product which is responsible for stable maintenance of mini-F plasmid

Summary The mini-F plasmid has the trans-acting sopA, sopB genes and the cis-acting sopC DNA which are essential for plasmid partitioning. In this paper, we report the purification of the sopB gene product from extracts of cells harboring a pBR322 derivative carrying the sopB gene. The purity of the final preparation was more than 95%, as determined by densitometry. The amino acid sequence of the amino-terminal region of the protein for the 17 residues identified was identical to that predicted from the DNA sequence of the sopB gene. Therefore, it was concluded that the protein was the sopB gene product. Using anti-SopB serum, the SopB protein was detected in the cell lysates of F⁺, F, and Hfr strains. The SopB protein bound to the plasmid DNA of a pBR322 derivative carrying the sopC DNA segment, but not to the vector plasmid pBR322.     

IS21 insertion in the trfA replication control gene of chromosomally integrated plasmid RP1: a property of stable Pseudomonas aeruginosa Hfr strains

Summary Broad host range IncP-1 plasmids are able to integrate into the chromosome of gram-negative bacteria. Strains carrying an integrated plasmid can be obtained when the markers of a temperature-sensitive (ts) plasmid derivative are selected at non-permissive temperature; in this way Hfr (high frequency) donor strains can be formed. The integrated plasmids, however, tend to be unstable in the absence of continuous selective pressure. In order to obtain stable Hfr donor strains of Pseudomonas aeruginosa PAO, we constructed a derivative of an RP1 (ts) plasmid, pME134, which was defective in the resolvase gene (tnpR) of transposon Tn801. Chromosomal integration of pME134 was selected in a recombination-deficient (rec-102) PAO strain at 43°C. Plasmid integration occurred at different sites resulting in a useful set of Hfr strains that transferred chromosomal markers unidirectionally. The tnpR and rec-102 mutations prevented plasmid excision from the chromosome. In several (but not all) Hfr strains that grew well and retained the integrated plasmid at temperatures below 43°C, the insertion element IS21 of RP1 was found to be inserted into the trfA locus (specifying an essential trans-acting replication funtion) of the integrated plasmid. One such Hfr strain was rendered rec ⁺; from its chromosome the pME134::IS21 plasmid (=pME14) was excised and transferred by conjugation to Escherichia coli where pME14 could replicate autonomously only when a helper plasmid provided the trfA ⁺ function in trans. Thus, it appears that trfA inactivation favours the stability of chromosomally integrated RP1 in P. aeruginosa.     

The Streptomyces ghanaensis low copy plasmid pSG2 and its use for vector construction

A plasmid, pSG2, was isolated from Streptomyces ghanaensis and characterized by electron microscopy, buoyant density measurement, and restriction enzyme analysis. The length of 13.8 kb, single restriction sites for HindIII, EcoRV and PvuII and the possibility of deleting non-essential regions of the plasmid made pSG2 a suitable basic replicon for vector development. pSG2 has a copy number of about four. Plasmid pSG2 was fused to a pACYC184 derivative modified to harbour a thiostrepton resistance gene. The resulting plasmid, designated pSW1, is a 16.6 kb shuttle plasmid which replicates in Escherichia coli and in several Streptomyces strains, including S. ghanaensis, S. lividans and S. viridochromogenes. Replacement of a Bg/II-fragment of plasmid pSG2 by a fragment encoding thiostrepton resistance resulted in a low copy 12.2 kb Streptomyces plasmid. This plasmid, designated pSW2, is a Streptomyces broad host range plasmid.     

Role of a small plasmid in the modification of carbon monoxide dehydrogenase in Pseudomonas carboxydovorans

Abstract Soluble fractions prepared from cells of Pseudomonas carboxydovorans bearing a small plasmid (1.76 × 10⁶) exhibited proteolytic activity on the β-subunit of CO dehydrogenase (CO-DH) in plasmid-cured cells of the same strain, implying that the plasmid carries gene(s) for processing the β subunit of the enzyme at the post-translational level. The protease was found to be a constitutive enzyme. It did not hydrolyze the β subunit of CO-DH in Pseudomonas carboxydohydrogena . Analysis of CO-DH after transformation of the cured cells with the small plasmid confirmed that the plasmid plays a role in the modification of the β subunit of CO-DH in P. carboxydovorans .     

The use of the replicating pDblet plasmid as a cloning vector with enhanced stability in Kluyveromyces marxianus

The replicating plasmid, pDblet, transformed the budding yeast Kluyveromyces marxianus to an efficiency of 10⁴ transformant g^–1 DNA. Transformed cells showed 1% of segregation rate without affecting their growth rate of 0.69 h^–1 and glucose consumption. These results were similar or better than the commonly used pE1 plasmid and suggests that pDblet can be used for cloning genes in K. marxianus.     

Heterogeneric conjugal transfer of the pheromone-responsive plasmid pIP964 (IncHlyI) of Enterococcus faecalis in the apparent absence of pheromone induction

Abstract Erythromycin-resistant derivatives of the pheromone-responsive plasmid pIP964 from Enterococcus faecalis were constructed to study its host range. This was done by inserting the integrative vector pAT112 and the related replicon pTCR1 harboring oriR of the broad host range plasmid pAMβ1 into the hemolysin-bacteriocin operon of pIP964, to give pTCR2 and pTCR3, respectively. Plasmid pTCR2 was transferred by filter matings from E. faecalis to Enterococcus faecium and Listeria monocytogenes at frequencies of 2×10⁻⁷ and 5×10⁻⁷ per donor, respectively, in the apparent absence of pheromone induction and cellular aggregation. In these hosts, pTCR2 remained intact as a self-replicating element and maintained its transfer capabilities. Plasmid pTCR3, but not pTCR2, was transferred at similar frequencies from E. faecalis to Lactococcus lactis and Streptococcus agalactiae . Thus, the transfer system of pIP964 possesses a broader host-range than its replication system.     

Use of a novel plasmid to monitor the fate of a genetically engineered Pseudomonas putida strain

Plasmid pSI30 was constructed to increase the sensitivity of detection of a genetically engineered micro-organism (GEM) and its recombinant DNA in environmental samples. This broad host-range, mobilizable plasmid contained chlorocatechol (clc) degradative genes, antibiotic resistance genes (ampicillin and kanamycin) and a fragment of eukaryotic DNA. The clc genes encode enzymes that convert 3-chlorocatechol to maleylacetic acid permitting the host, Pseudomonas putida RC-4, to grow on 3-chlorobenzoate. This catabolic phenotype was exploited using enrichment procedures to detect RC-4(pSI30) cells, free-living in the water column or when irreversibly bound to surfaces. The eukaryotic DNA sequence provided a unique target allowing positive identification by DNA:DNA hybridization. Using the eukaryotic DNA sequence as a probe, no transfer of the plasmid to indigenous bacteria was detected. Persistence of RC-4(pSI30) and its ability to multiply upon addition of 3-chlorobenzoate were demonstrated 78 days after its addition to natural freshwater. In flow-through microcosms RC-4(pSI30), undetectable as free-living cells, was found by enrichment as irreversibly bound sessile forms. These experiments revealed the stability of pSI30 and its utility in a 'combination' detection system for tracking the survival of a GEM and its DNA in environmental samples.     

Enhanced high copy number plasmid maintenance and heterologous protein production in an Escherichia coli biofilm

Escherichia coli has been widely used for heterologous protein production (HPP). To determine whether a biofilm environment could benefit E. coli HPP using high copy number plasmids, we compared plasmid maintenance and HPP by E. coli ATCC 33456 containing plasmid pEGFP (a pUC family vector) cultivated in biofilms and in suspended culture. Cells were grown with or without antibiotic selective pressure in flow cells or chemostats for up to 6 days. In biofilms, antibiotic selective pressure increased the plasmid copy number (PCN), but by 144 h, biofilms grown in antibiotic-free media had comparable plasmid concentrations. In the chemostat, the PCN declined steadily, although 100 ppm ampicillin in the medium slowed the rate of plasmid loss. Production of green fluorescent protein (GFP), a representative heterologous protein, was quantified by flow cytometry. In biofilms, at ampicillin concentrations >or=33 ppm, strongly fluorescent cells comprised more than half of the population by 48 h. In the chemostat, more than 50% of the population was non-fluorescent by 48 h in media containing 100 ppm ampicillin, and strongly fluorescent cells were <10% of the population. Biofilm structure was determined by confocal microscopy. Maximum biofilm thickness ranged from 30 to 45 microns, with no significant changes in biofilm structure after 48 h. Plasmid multimer percentages were similar to inocula for cells cultivated in either biofilms or the chemostat. The results indicate that the biofilm environment enhanced both plasmid maintenance and cellular GFP concentrations, and that low levels of antibiotic increased the beneficial effect.     

Investigation of subpopulation heterogeneity and plasmid stability in recombinant escherichia coli via a simple segregated model

Many microbial and cell cultures exhibit phenomena that can best be described using a segregated modeling approach. Heterogeneties are more marked in recombinant cell cultures because subpopulations, which often exhibit different growth and productivity characteristics, are more easily identified by selective markers. A simple segregated mathematical model that simulates the growth of recombinant Escherichia coli cells is developed. Subpopulations of different growth rate, plasmid replication rate, and plasmid segregation probability are explicitly considered. Results indicate that a third mechanism of plasmid instability, referred to here as a "downward selective pressure," is significant when describing plasmid loss in batch and chemostat cultures. Also, the model agrees well with experimental data from cultures under antibiotic selective pressure. Finally, model simulations of chemostat cultures reveal the importance of initial conditions on culture stability and the possible presence of nonrandom partitioning functions. (c) 1993 John Wiley & Sons, Inc.     

Relationship between carbon monoxide dehydrogenase and a small plasmid in Pseudomonas carboxydovorans

Abstract Isolation of plasmid DNA followed by plasmid curing was carried out to examine the relationship of plasmid to carbon monoxide dehydrogenase (CO-DH) production in carboxydobacteria. A small plasmid of almost identical size (1.52−1.76 × 10⁶) was present in Pseudomonas carboxydovorans, Azotobacter sp.1, and Azomonas sp.2. Azomonas sp.1 contained two kinds of plasmids (1.5 × 10⁶ and 2.47 × 10⁶). No plasmids were found in Pseudomonas carboxydohydrogena , JC1, and HY1. A plasmid-cured clone of P. carboxydovorans was obtained by growing the cells at 37°C. The cured cell was able to grow CO autotrophically on solid, but not in liquid, medium. CO-DH of the cured cell was active and consisted of three subunits similar to those found in the wild-type enzyme, with the exception that the β subunit of the enzyme was larger than that of the wild-type enzyme. These results suggest that the small plasmids do not carry genes encoding CO-DH but may have gene(s) for processing the β subunit of the enzyme.     

Transfer and expression of the herbicide-degrading plasmid pJP4 in aerobic autotrophic bacteria

Plasmid pJP4 encoding the ability to degrade the herbicide 2,4-dichlorophenoxyacetic acid (Tfd⁺) was transferred by conjugation from Escherichia coli JMP397 to various lithoautotrophic strains of Alcaligenes eutrophus and to the autotrophic bacterium Pseudomonas oxalaticus. The herbicide-degrading function of the plasmid was phenotypically expressed in all of the recipients. The majority of Tfd⁺ transconjugants also exhibited additional plasmid-encoded properties such as 3-chlorobenzoate degradation, resistance to mercuric ions, and sensitivity to the male-specific bacteriophage PR11. Furthermore, Tfd⁺ transconjugants were able to act as donors of plasmid pJP4. Physical evidence is presented by agarose gel electrophoresis showing that plasmid pJP4 coexisted with the resident plasmids widely distributed in this group of bacteria. However, in some of the hosts plasmid pJP4 was not stably maintained, had a reduced size and tended to form multimers.     

DH10B菌株高效电转化条件探究

以pUC19、pECBAC1、pCLD04541DNA以及3个不同大小的BACDNA为材料,研究了E.coli DH10B菌株在5个不同脉冲电场下的转化效率。研究发现,随着DNA片段大小的增加,最高转化效率和最适场强迅速减小。利用DH10B细胞转化pUC19 DNA的最适场强是21kV/cm,而190kb BAC DNA仅为13kV/cm;在最适场强下,40kb BAC DNA的转化效率约是190kb BAC DNA的50倍。通过大量数据绘制了不同因素影响下转化效率的变化曲线,优化了E.coli DH10B菌株电转化条件,为质粒的重组转化以及大片段基因组文库的构建奠定了基础。