Demonstration of tra+ plasmid activity in bacteria indigenous to the phyllosphere of sugar beet; gene transfer to a recombinant pseudomonad

Abstract The presence of transfer proficient plasmids in bacteria isolated from the leaves of sugar beet ( Beta vulgaris L.) was studied. Of 435 bacteria sampled 79 (18%) contained plasmids. Pseudomonads (30%), Erwinia (12%) and Klebsiella (9%) were the largest populations sampled of which 22%, 33% and 29%, respectively, contained plasmids. The ability of these plasmids to self-transfer or mediate the mobilization of the tra⁻ mob⁺ broad host range IncQ plasmid R300B was determined. R300B was maintained in 61/79 natural plasmid containing isolates, the Gram positive isolates could not support R300B. Pseudomonas aureofaciens SBW25, isolated from sugar beet leaves, was chromosomally marked with a tetracycline resistance gene and used as a recipient (SBW25ETc). Five isolates of Erwinia herbicola and one of Erwinia salicis containing natural plasmids were able to mobilize R300B into the recombinant, SBW25ETc. These mobolizing ( tra⁺ ) plasmids were not maintained in transconjugant SBW25 cells. Analysis of the fragment patterns of Pst I digested plasmid DNA demonstrated that four (pSB139, pSB140, pSB142, pSB146; 110 kb) were identical, one (pSB153; 65 kb) was common to a subset of fragments in these four and another (pSB169; 100 kb) was unique. Other natural isolates were able to transfer copper resistance ( Erwinia rhapontici , 2 strains) or mercury resistance ( Pseudomonas fluorescens SBW340) to a rifampicin resistant recipient Pseudomonas putida UWC1 but not to SBW25ETc. These self-transferable plasmids were not able to mobilize R300B. These data demonstrate that the phyllosphere supports indigenous microbial populations which have the capacity to transfer genetic material between bacteria of different genera.     

Mobilization of CS fimbriae-associated plasmids of enterotoxigenic Escherichia coli of serotype O6: K15: H16 or H- into various wild-type hosts

Abstract CS fimbriae-associated plasmids of two enterotoxigenic Escherichia coli strains of serotype O6: K15: H16 or H- (biotypes A and F) with M _r values of 51 × 10⁶ and 72 × 10⁶, respectively, were mobilized into various alternative host bacteria. Expression of CS1 or CS2 fimbriae was obtained when either of the CS fimbriae-associated plasmids was introduced into CS Fim⁻, O6: K15: H16 or H- recipients with rhamnose-negative and rhamnose-positive fermentation phenotypes, respectively, whereas CS3 fimbriae were expressed irrespective of the biotype of the recipient. On transfer into a CS Fim⁻ variant of an enterotoxigenic O8: H9 strain and into two K-12 strains, a CS3-fimbriae-only phenotype was conferred by the presence of either of the plasmids. When a CS Fim⁻ variant of a Rha⁺ CS2-fimbriae-only strain of serotype O6: K15: H16 harboured either of the plasmids, both CS2 and CS3 fimbriae were expressed, indicating that the rare CS2-fimbriae-only wild-type phenotype is probably due to the presence of a defective plasmid in such strains. Mobilization of the 51 MDa CS fimbriae-associated plasmid into five non-enterotoxigenic Rha⁺ porcine isolates of E. coli with O6 serotypes other than O6: K15: H16 or H- yielded CS3-fimbriae-only transconjugants. Thus the correlation between a Rha⁺ fermentation phenotype and expression of CS2 fimbriae does not hold in general for O-group 6 strains.     

Plasmid and chromosomally encoded luminescence marker systems for detection of Pseudomonas fluorescens in soil

Luminescent strains of Pseudomonas fluorescens 10586 were constructed in which luciferase production was constitutive by introduction of Vibrio fischeri luxABE genes on the chromosome and on a multicopy plasmid. Light production in liquid batch culture was directly proportional to biomass concentration during exponential growth and enabled detection by luminometry of 1.7 × 10³ and 8.9 × 10⁴ cells/ml for the plasmid and chromosomally marked strains, respectively. Luminescent colonies of both strains were detectable by eye, enabling viable cell enumeration on solid media against a background of non-luminescent strains. Following inoculation into sterile and non-sterile soil lower levels of detection were increased but detection of 8.1–59 × 10³and 2.2–30 × 10³ cells per g of soil was possible for plasmid and chromosomally marked strains. Maximum specific growth rate in liquid culture was unaffected by introduction of lux marker genes on the chromosome, but was reduced in the plasmid marked strain. The chromosomally encoded marker was stable in both liquid culture and in soil, but the plasmid was unstable during continuous subculturing in liquid medium and during growth in soil. The chromosomally encoded luminescence-marker system therefore provides a convenient, non-extractive technique for quantification of genetically modified soil microbial inocula.     

Large Pseudomonas phages isolated from barley rhizosphere

Abstract: Five bacteriophages infecting common fluorescent pseudomonads ( Pseudomonas fluorescens and Pseudomonas putida ) were isolated from barley rhizosphere soil. Morphological and molecular characteristics of the phages are described together with selected phage-host interactions. All phages belonged to the Myoviridae family with isometrical heads on contractile tails; 4 of them were unusually large and had complex protein and DNA profiles. The large phages had estimated genome sizes of 200 kb or more. Restriction enzyme analyses and DNA-DNA hybridizations showed that all isolates represented different phage species. None of the isolates were observed to establish lysogeny with the main host strain, P. putida MM1. The large phages multiplied slowly on their hosts, producing very small plaques; one-step growth experiments with one of the large phages (Psp 4) hence demonstrated a long latent period (2.5 h) and a very small burst size (10 particles). One of the large phages (Psp 3) was abundant in the rhizosphere (approx. 10⁴ pfu g⁻¹ soil) and had a particularly broad host range which extended to both fluorescent ( Pseudomonas aeruginosa, P. fluorescens, P. putida and Pseudomonas chlororaphis ) and non-fluorescent (Pseudomonas stutzeri) Pseudomonas spp. occurring in soil. The ecological importance of the large Pseudomonas phages must be further studied, but their slow multiplication rates suggested a possible mechanism of balanced phage-host co-existence in the rhizosphere.     

Stability and conjugal transfer kinetics of a TOL plasmid in Pseudomonas aeruginosa PAO 1162

Abstract Batch mating experiments were employed to study the kinetics of the conjugal transfer of a TOL plasmid, using the transconjugant strain Pseudomonas aeruginosa PAO 1162 (TOL) as the plasmid donor and Pseudomonas putida PB 2442 and Pseudomonas aeruginosa PAO 1162N as the plasmid recipients. Transfer rates from PAO 1162 (TOL) to PAO 1162N and PB 2442 measured for exponentially grown PAO 1162 (TOL) were 1.81 × 10⁻¹⁴ (standard error (S.E.) 1.25 × 10⁻¹⁵) ml·cell⁻¹min⁻¹ and 3.32 × 10⁻¹³ (S.E. 4.42 × 10⁻¹⁴) ml·cell⁻¹min⁻¹, respectively. The instability of the TOL plasmid in PAO 1162 (TOL) was evaluated under conditions that were non-selective for maintenance of the TOL catabolic functions. The measured rates of instability were 6.7 10⁻⁶ to 8.3 10⁻⁶ min⁻¹, and the loss of the catabolic functions was mainly caused by structural instability of the plasmid.     

Survival of genetically modified Pseudomonas fluorescens introduced into subtropical soil microcosms

Abstract A genetically modified strain of Pseudomonas fluorescens and its parent showed grossly similar decline rates following introduction into subtropical clay and sandy soils. In unplanted clay soit at pH 6.9 and 25°C, population densities declined progressively from about 10⁸ to 10³ colony forming units (cfu) g⁻¹ dry soil over 75 days, but in unplanted sandy soil the introduced populations could not be detected after 25 days. In clay soil at pH 8.7 or 4.7, or at environmental temperature, decay rates were enhanced as compared to those at pH 6.9 and 25°C. Counts of introduced strains in clay bulk soil and in rhizosphere and rhizoplane of maize suggested that the introduced bacteria competed well with the native bacteria, and colonized the roots at about 10⁶ cfu g⁻¹ dry root at 25°C, over 20 days. However, rhizoplane colonization was lower at environmental temperature. The decay rate of both strains was slower in planted than in unplanted sandy soil. The population densities in the rhizosphere and rhizoplane in the sandy soil were significantly lower than those in the clay soil. Both introduced strains colonized the maize roots in both soils, using seeds coated with bacteria in 1% carboxymethyl cellulose. Introduced cells were localized at different sites along the roots of plants developing in clay soil, with higher densities in the original (near the seeds) and root hair zones as compared to the intermediate zones. No significant difference was observed between the extent of root colonization of the genetically modified strain and its parent.     

Plasmid-determined resistance to chromate in Pseudomonas aeruginosa

Abstract Resistance to chromate in five independent Pseudomonas aeruginosa clinical isolates was transferred by conjugation to P. aeruginosa strain PU21. All chromate-resistant transconjugants contained large plasmids that also conferred resistance to inorganic mercury. One of these plasmids, pUM505, increased the resistance to CrO₄²⁻ and decreased the accumulation of intracellular ⁵¹CrO₄²⁻ by the host cells as compared to the plasmidless strain PU21.     

Effects of low concentrations of ampicillin in feed on the intestinal Escherichia coli of chicks

Ampicillin in low concentrations (1.7 and 5 g t^-1) was incorporated in the feed given to 1-d-old chicks for 2 weeks. This was sufficient to select, in the intestinal contents, resistant Escherichia coli strains for which the minimum inhibitory concentration of ampicillin was > 100 μg ml^-1. Different clones of E. coli were identified by their biotype, pattern of resistance to antibacterial agents and plasmid profile (designated P-P types). The experiment was repeated twice and the average proportion of ampicillin-resistant P-P types among 72 isolates of E. coli from chicks given feed containing 0, 1.7 and 5 g ampicillin t^-1 were 10%, 31% and 46% respectively.     

Effect of long-term vineyard monoculture on rhizosphere populations of pseudomonads carrying the antimicrobial biosynthetic genes phlD and/or hcnAB

The impact of repeated culture of perennial plants (i.e. in long-term monoculture) on the ecology of plant-beneficial bacteria is unknown. Here, the influence of extremely long-term monocultures of grapevine (up to 1603 years) on rhizosphere populations of fluorescent pseudomonads carrying the biosynthetic genes phlD for 2,4-diacetylphloroglucinol and/or hcnAB for hydrogen cyanide was determined. Soils from long-term and adjacent short-term monoculture vineyards (or brushland) in four regions of Switzerland were baited with grapevine or tobacco plantlets, and rhizosphere pseudomonads were studied by most probable number (MPN)-PCR. Higher numbers and percentages of phlD ⁺ and of hcnAB ⁺ rhizosphere pseudomonads were detected on using soil from long-term vineyards. On focusing on phlD , restriction fragment length polymorphism profiling of the last phlD -positive MPN wells revealed seven phlD alleles (three exclusively on tobacco, thereof two new ones). Higher numbers of phlD alleles coincided with a lower prevalence of the allele displayed by the well-studied biocontrol strain Pseudomonas fluorescens F113. The prevalence of this allele was 35% for tobacco in long-term monoculture soils vs. >60% in the other three cases. We conclude that soils from long-term grapevine monocultures represent an untapped resource for isolating novel biocontrol Pseudomonas strains when tobacco is used as bait.     

Distribution of plasmids in cyanobacteria of the LPP group

Abstract The distribution of plasmid DNA has been studied in 23 strains and variants of non-heterocystous filamentous cyanobacteria that are susceptible to infection by the LPP-1 cyanophage (archetype). 21 have identical plasmid profiles and contain 3 plasmids of M _rs 0.9, 10 and approx. 12 · 10⁶ respectively. In one strain, Plectonema FS180, the plasmids have been designated pMP1, pMP2, and pMP3, respectively. pMP1 shows sequence homology with pMP2 and pMP3 but not with DNA from an LPP-type cyanophage. Plectonema UTEX 598 lacks the small plasmid only, while Plectonema UTEX 1541 is distinct amongst all these strains with 3 plasmids of M _rs 3, 10, and > 30 · 10⁶, respectively. The findings support the view that the majority of these strains may be independent isolates of a single species.     

Identification and characterization of Bacillus anthracis by multiplex PCR analysis of sequences on plasmids pXO1 and pXO2 and chromosomal DNA

Abstract Bacillus anthracis can be identified on the basis of the detection of virulence factor genes located on two plasmids, pXO1 and pXO2. Thus isolates lacking both pXO1 and pXO2 are indistinguishable from closely related B. cereus group bacteria. We developed a multiplex PCR assay for characterization of B. anthracis isolates, and simultaneous confirmation of the species identity independent of plasmid content. The assay amplifies lef, cya, pag (pXO1) and cap (pXO2) genes, and a B. anthracis specific chromosomal marker, giving an easy-to-read profile. This system unambiguously identified virulent (pXO1⁺/2⁺) and avirulent (pXO1⁺/2⁻, pXO1⁻/2⁺ and pXO1⁻/2⁻) strains of B. anthracis and distinguished 'anthrax-like' strains from other B. cereus group bacteria.     

Survival of κ-carrageenan-encapsulated and unencapsulated Pseudomonas aeruginosa UG2Lr cells in forest soil monitored by polymerase chain reaction and spread plating

Abstract A method was developed for direct extraction, purification and amplification of DNA from forest soil. Eighty-two % of the DNA in Pseudomonas aeruginosa UG2Lr introduced into soil was recovered. The detection limit for the strain was approximately 800 cfu g⁻¹ of dry soil based on the polymerase chain reaction (PCR). Survival of κ-carrageenan-encapsulated and unencapsulated UG2Lr was monitored by antibiotic selective and bioluminescence-based nonselective plating and PCR-amplification of a tnsA fragment. After freeze-thaw treatment of soil samples, the unencapsulated UG2Lr declined from an initial population density of 1 × 10⁹ cfu g⁻¹ of dry soil to below the detection threshold of both selective (14 cfu g⁻¹ of dry soil) and nonselective (1 × 10³ cfu g⁻¹ of dry soil) plating. However, presence of nonculturable UG2Lr cells in the soil was revealed by PCR and resuscitation of the bacteria. Population density of the encapsulated UG2Lr increased from 2.7 × 10⁶ to 2.9 × 10⁸ cfu g⁻¹ of dry soil after a 3-week incubation at 22°C and declined to 6.3 × 10⁶ cfu g⁻¹ of dry soil after the freeze-thaw treatment.     

Plasmid-borne mercury resistance in aquatic bacteria

Abstract Bacteria isolated from the River Mersey were analysed for their tolerance to mercury (HgCl₂). About 40% of the population was tolerant to mercury and in 13 of 52 mercury-tolerant isolates tested the mercury resistance (Hg^®) was transferred to Escherichia coli in conjugal matings. These 13 isolates represented a range of gram-negative genera and in each case mercury resistance was coded by a conjugative plasmid. These plasmids (75 kb to > 250 kb in size) all expressed mercury resistance of the narrow spectrum variety, volatilised HgCl₂ to elemental Hg° vapour and showed some degree of temperature sensitivity of transfer. None expressed resistance to nine different antibiotics. These 13 Hg^R plasmids were classified by restriction mapping into three distinct groups typified by pMER11, pMER327 and pMER610. The eight pMER610 group plasmids are identical and belong to the IncHI-2 group. Two of the four pMER327 group plasmids are closely related while the other two contain some common restriction fragments. pMER11 is quite distinct from the other groups. These results imply that within this aquatic environment plasmids play an important role in the response of bacteria to contaminating mercury and that there is widespread plasmid transfer and considerable genetic rearrangement.     

Antibiotic resistance and plasmid profile of Aeromonas hydrophila isolates from cultured fish, Telapia (Telapia mossambica)

Strains of Aeromonas hydrophila isolates from skin lesions of the common freshwater fish, Telapia mossambica , were screened for the presence of plasmid DNA by agarose gel electrophoresis and tested for susceptibility to 10 antimicrobial agents. Of the 21 fish isolates examined, all were resistant to ampicillin and sensitive to gentamycin. Most isolates were resistant to streptomycin (57%), tetracycline (48%) and erythromycin (43%). While seven of 21 isolates harboured plasmids, with sizes ranging from 3 to 63·4 kilobase pair (kb), it was only possible to associate the presence of a plasmid with antibiotic resistance (ampicillin and tetracycline) in strain AH11. Both the plasmid and the associated antimicrobial resistance could be transferred to an Escherichia coli recipient by single-step conjugation at a frequency of 4·3×10⁻³ transconjugants per donor cell.     

Monitoring the fate of genetically engineered bacteria sprayed on the phylloplane of bush beans and grass

Abstract The fate of a Bacillus amyloliquefaciens with the recombinant plasmid pSB20 sprayed on the phyllosphere of grass, and of a Tn 5 marked Pseudomonas syringae sprayed on the phyllosphere of bush beans was studied in planted soil microcosms. B. amyloliquefaciens showed a decline from 1.5×10⁸ to 3.1×10² cfu g⁻¹ on the phylloplane of grass in the course of the experiment. B. amyloliquefaciens was easy to follow by selective cultivation due to the complete absence of bacterial background growth. Southern blot hybridization of Hin dIII digested genomic DNA showed plasmid restriction patterns identical with pSB20 indicating high plasmid stability. In total DNA extracts from phyllosphere bacteria the recombinant plasmid was detectable by Southern blot hybridization up to 6×10⁴ cfu g⁻¹ (wet weight). Counts of hybridizing colonies showed that P. syringae established on the phyllosphere of bush beans at between 5×10³ and 4×10⁶ cfu g⁻¹ fresh weight. During senescence of the bean plants the strain was no longer detectable by selective cultivation and subsequent colony hybridization. In contrast, Tn5 marked DNA was detected after PCR amplification over the whole period of the experiment.     

Plasmid and pathogenicity in Xanthomonas oryzae pathovar oryzae, the bacterial blight pathogen of Oryza sativa

Xanthomonas oryzae pv. oryzae , the causative agent for bacterial leaf blight of rice, comprises diverse groups of strains differing in biochemical and pathological characteristics. A collection of X.o . pv. oryzae strains differing in geographical origin was screened for the presence of plasmids. Out of 17 isolates of X.o. pv. oryzae , 14 harboured plasmids of which two isolates (XoP5, XoC26) had two plasmids each and one isolate (XoR20) had three. The remaining isolates contained a single plasmid of identical mobility. Finger print analysis of plasmids was carried out using Eco RI for 10 isolates. The restriction fragment pattern was distinct for each isolate. They were classified under three groups based on cluster analysis using the unweighted pair group method with averages (UPGMA). Of the 18 plasmids, the plasmid pMA36 ( X.o. pv. oryzae XoC36) was further characterized. This plasmid was cured by acridine orange at the frequency rate of 10%. The cured strain was transformed with pMA36 at a frequency of 2.3 times 10² transformants μg^-1 of plasmid DNA. The plasmid-cured strain was virulent on rice but symptom development was delayed when compared to wild and transformed strains. The wild type strain ( X.o. pv. oryzae XoC36) was resistant to ampicillin, carbenicillin and rifampicin whereas the cured strain was resistant to carbenicillin and rifampicin but sensitive to ampicillin. The transformant was resistant to the three antibiotics indicating that the plasmid pMA36 codes for ampicillin resistance. The plasmid influenced the pathogenicity of X.o. pv. oryzae.     

Antimicrobial resistance and in vitro gene transfer in bacteria isolated from the ulcers of EUS-affected fish in India

Aims:  The occurrence of drug resistance and plasmid-mediated transferability was investigated in 15 Aeromonas isolates collected from the ulcers of epizootic ulcerative syndrome (EUS)-affected fishes Katla ( Catla catla ), Mrigel ( Cirrhinus mrigala ) and Punti ( Puntius sp.).
Methods and Results:  Disc diffusion assay showed that all the strains were resistant to ampicillin and sensitive to streptomycin. Of the 15 isolates examined, 93·3% isolates were resistant to erythromycin, sulfadiazine and novobiocin, while 66% were resistant to rifampin and 20% to chloramphenicol. All isolates harboured plasmids with sizes ranging from 64 to 23 kbp with a 23-kbp plasmid in common. Plasmids from 11 Aeromonas strains were transferred to Escherichia coli DH5α recipient strain along with the transfer of ampicillin, erythromycin and chloramphenicol resistance determinants with frequencies ranging from 7·0 × 10⁻⁶ to 1·8 × 10⁻⁵ transconjugants per recipient cell.
Conclusions:  The resistance to ampicillin, erythromycin, sulfadiazine, novobiocin and chloramphenicol is prevalent among the bacteria isolated from EUS-affected fish, and resistant determinants of some of these antibiotics have been transferred to the bacteria of other origin.
Significance and Impact of the Study:  The emergence of antibiotic resistance bacteria and gene transfer in vitr o suggests that antibiotics should be used more cautiously to treat Aeromonas infections in aquaculture.     

Isolation and characterization of Azotobacter chroococcum from the roots of Zea mays

Abstract Bacteria showing rapid growth on a nitrogenfree medium and acetylene-reducing activity were isolated from maize roots collected from agricultural soils in Spain. The isolates were Gram-negative motile rods and were identified as Azotobacter chroococcum . Acetylene-reducing activity and microbial counts were determined on root segments from 7- and 30-day-old plants. Rates obtained were in the range of 0.0053–0.848 nmol C₂H₂· g⁻¹· h⁻¹. Root populations were 1.4–6.0 × 10⁴ micro-organisms · g⁻¹. These results showed that there was an association between A. chroococcum strains and roots of maize planted in some Spanish soils.     

Interactions between the soil-borne bacteriophage Fo-l and Pseudomonas spp. on sugarbeet roots

The large plaquing (24 mm²) soilborne bacteriophage, Fo-l, did not affect the colonization ability on sugarbeet roots of its host, fluorescent Pseudomonas sp. strain B26. Phage Fo-l did not increase in numbers on sugarbeet roots when seeds were coated with less than 10⁶ CFU (colony forming units) of B26 and when less than 300 PFU (plaque forming units) of phage Fo-l was added per g of soil (dry weight). Above these threshold values, phage replication occurred and up to 2 × 10⁶ PFU per root system could be recovered.