Predictive model of conjugative plasmid transfer in the rhizosphere and phyllosphere.

A computer simulation model was used to predict the dynamics of survival and conjugation of Pseudomonas cepacia (carrying the transmissible recombinant plasmid R388:Tn1721) with a nonrecombinant recipient strain in simple rhizosphere and phyllosphere microcosms. Plasmid transfer rates were derived for a mass action model, and donor and recipient survival were modeled as exponential growth and decay processes or both. Rate parameters were derived from laboratory studies in which donor and recipient strains were incubated in test tubes with a peat-vermiculite solution or on excised radish or bean leaves in petri dishes. The model predicted donor, recipient, and transconjugant populations in hourly time steps. It was tested in a microcosm planted with radish seeds and inoculated with donor and recipient strains and on leaf surfaces of radish and bean plants also growing in microcosms. Bacteria were periodically enumerated on selective media over 7 to 14 days. When donor and recipient populations were 10(6) to 10(8) CFU/g (wet weight) of plant or soil, transconjugant populations of about 10(1) to 10(4) were observed after 1 day. An initial rapid increase and a subsequent decline in numbers of transconjugants in the rhizosphere and on leaf surfaces were correctly predicted.     

Predictive model of conjugative plasmid transfer in the rhizosphere and phyllosphere

A computer simulation model was used to predict the dynamics of survival and conjugation of Pseudomonas cepacia (carrying the transmissible recombinant plasmid R388:Tn1721) with a nonrecombinant recipient strain in simple rhizosphere and phyllosphere microcosms. Plasmid transfer rates were derived for a mass action model, and donor and recipient survival were modeled as exponential growth and decay processes or both. Rate parameters were derived from laboratory studies in which donor and recipient strains were incubated in test tubes with a peat-vermiculite solution or on excised radish or bean leaves in petri dishes. The model predicted donor, recipient, and transconjugant populations in hourly time steps. It was tested in a microcosm planted with radish seeds and inoculated with donor and recipient strains and on leaf surfaces of radish and bean plants also growing in microcosms. Bacteria were periodically enumerated on selective media over 7 to 14 days. When donor and recipient populations were 10(6) to 10(8) CFU/g (wet weight) of plant or soil, transconjugant populations of about 10(1) to 10(4) were observed after 1 day. An initial rapid increase and a subsequent decline in numbers of transconjugants in the rhizosphere and on leaf surfaces were correctly predicted.     

Conjugative transfer of plasmid DNA between bacteria in soil]

Conjugative transfer of plasmid RP4 between populations of azospirilla and between Escherichia coli and Azospirillum brasilense in nonsterile soil has been investigated. The process of genetic exchange was realized at the early stages of interpopulational interactions, further on the process intensity was obviously rather low. Population dynamics of azospirilla transconjugates in soil depends on the presence or the absence of additional food substrate.     

Role of sog polypeptides specified by plasmid ColIb-P9 and their transfer between conjugating bacteria.

The sog gene of the conjugative plasmid ColIb-P9 specifies two sequence-related polypeptides with the N-terminal third of the larger product having DNA primase activity. To resolve the function of the C-terminal portion of the polypeptides, we constructed a ColIb mutant containing a Tn5 insertion in the 3' region of sog. The mutation truncated sog gene products without inactivating DNA primase and rendered the plasmid defective in conjugation. Tests for the presence of conjugative pili, for complementation by a sog+ recombinant, and for mobilization of small origin of transfer (oriT) recombinant plasmids indicated that the mutant ColIb allows conjugative aggregation of cells but it is defective in DNA transfer at some stage subsequent to its initiation at oriT. Physical evidence is given that normal sog polypeptides are among a group of proteins transferred selectively from the donor to the recipient cell by a conjugation-specific process. No transfer of the mutant sog proteins was detected. It is proposed that the C-terminal region of sog polypeptides facilitates transfer of single-stranded ColIb DNA between conjugating cells following initiation of transfer at the oriT site, and that in this role the proteins are transmitted to the recipient cell.     

Horizontal transfer of a multi-drug resistance plasmid between coliform bacteria of human and bovine origin in a farm environment

Multi-drug-resistant coliform bacteria were isolated from feces of cattle exposed to antimicrobial agents and humans associated with the animals. Isolates from both cattle and humans harbored an R plasmid of 65 kb (pTMS1) that may have been transferred between them due to selective antibiotic pressure in the farm environment.     

Detection of plasmid RP4 transfer in soil and rhizosphere,and the occurrence of homology to RP4 in soil bacteria

Plasmid RP4 transfer between introduced pseudomonads was studied in non-rhizosphere and rhizosphere soil. The addition of nutrients to the non-rhizosphere soil stimulated plasmid transfers between introduced donor and recipient cells, and no transfer was detected in nonamended soil. Transfer was also detected in soil in a model rhizosphere, but not in corresponding non-rhizosphere soil. Colony hybridization with whole plasmid RP4 DNA as a probe was employed to detect transfers to indigenous organisms in soil. Although transfers to introduced recipient cells were easily detected in parallel controls, no indigenous organisms were identified that had received RP4. Background levels of soil organisms with the RP4 resistance pattern were considerable, and about 10% of these populations contained DNA sequences with homology to RP4. However, no plasmids could be detected in any of 20 isolates, nor was resistance transfer to aPseudomonas fluorescens recipient detected in filter matings.     

Conjugal plasmid transfer (pAM beta 1) in Lactobacillus plantarum.

The streptococcal plasmid pAM beta 1 (erythromycin resistance) was transferred via conjugation from Streptococcus faecalis to Lactobacillus plantarum and was transferred among L. plantarum strains. Streptococcus sanguis Challis was transformed with pAM beta 1 isolated from these transconjugants, and transformants harboring intact pAM beta 1 could conjugate the plasmid back to L. plantarum.     

The specific growth rate of Pseudomonas putida PAW1 influences the conjugal transfer rate of the TOL plasmid.

The kinetics of the conjugal transfer of a TOL plasmid were investigated by using Pseudomonas putida PAW1 as the donor strain and P. aeruginosa PAO 1162 as the recipient strain. Short-term batch mating experiments were performed in a nonselective medium, while the evolution of the different cell types was determined by selective plating techniques. The experimental data were analyzed by using a mass action model that describes plasmid transfer kinetics. This method allowed analysis of the mating experiments by a single intrinsic kinetic parameter for conjugal plasmid transfer. Further results indicated that the specific growth rate of the donor strain antecedent to the mating experiment had a strong impact on the measured intrinsic plasmid transfer rate coefficient, which ranged from 1 x 10(-14) to 5 x 10(-13) ml per cell per min. Preliminary analysis suggested that the transfer rates of the TOL plasmid are large enough to maintain the TOL plasmid in a dense microbial community without selective pressures.     

Frequency of horizontal gene transfer of a large catabolic plasmid (pJP4) in soil.

Limited work has been done to assess the bioremediation potential of transfer of plasmid-borne degradative genes from introduced to indigenous organisms in the environment. Here we demonstrate the transfer by conjugation of the catabolic plasmid pJP4, using a model system with donor and recipient organisms. The donor organism was Alcaligenes eutrophus JMP134 and the recipient organism was Variovorax paradoxus isolated from a toxic waste site. Plasmid pJP4 contains genes for mercury resistance and 2,4-dichlorophenoxyacetic (2,4-D) acid degradation. A transfer frequency of approximately 1/10(3) donor and recipient cells (parent cells) was observed on solid agar media, decreasing to 1/10(5) parent cells in sterile soil and finally 1/10(6) parent cells in 2,4-D-amended, nonsterile soil. Presumptive transconjugants were confirmed to be resistant to Hg, to be capable of degrading 2,4-D, and to contain a plasmid of size comparable to that of pJP4. In addition, we confirmed the transfer through PCR amplifications of the tfdB gene. Although transfer of pJP4 did occur at a high frequency in pure culture, the rate was significantly decreased by the introduction of abiotic (sterile soil) and biotic (nonsterile soil) stresses. An evaluation of the data from this model system implies that the reliance on plasmid transfer from a donor organism as a remediative strategy has limited potential.     

Horizontal transfer of genetic determinants for degradation of phenol between the bacteria living in plant and its rhizosphere

Phenol and other monocyclic aromatic compounds (MACs) are highly water-soluble and volatile pollutants that plants are unable to completely degrade. Endophytic bacteria with MAC-degrading ability will facilitate phytoremediation, beneficial to plant survival in contaminated soil. Endophytic bacteria, strains FX1-FX3, and rhizosphere bacteria, strains FX0, FX4, and FX5, were isolated from the root tissue of a corn plant (Zea mays) and the corn rhizosphere near a chemical plant, respectively. The strains FX1-FX5 were able to grow on phenol and reduce phenol concentration, but the strain FX0 was unable to. The strains FX1, FX3, and FX4 were classified as Pseudomonas fluorescens and FX0, FX2, and FX5 as Burkholderia cepacia. The plasmids isolated from the strains FX1-FX5 were found to possess similar traits and to be loaded with a gene encoding the catechol 2, 3-dioxygenase (C23O), a key enzyme in the phenol degradation pathway. Alignment and phylogenetic analysis inferred that in situ horizontal transfer of the C23O gene might have occurred. The horizontal transfer of the C23O gene between endophytic and rhizosphere bacteria was proved by using conjugal matings experiment, in which the transconjugants were found to acquire the plasmid with the C23O gene, able to grow on phenol and degrade phenol.     

小麦-蚕豆间作条件下氮肥施用量对根际微生物区系的影响

通过田间小区试验,研究了小麦/蚕豆间作条件下4种施氮水平(0、90、180和270 kg·hm^-2)对根际微生物区系和多样性的影响.结果表明：在整个生育期,微生物数量有一定的波动,但均以开花期数量最高.与单作相比,间作显著增加了小麦和蚕豆根际的细菌、真菌、放线菌数量和微生物总量,而显著降低了开花期和成熟期蚕豆根际的微生物多样性.在不施氮（N₀）和低氮（N₉₀）水平下,间作与单作在微生物数量上的差异较大.间作对土壤微生物数量的促进效应在分蘖期和开花期最大,成熟期显著降低.小麦根际的微生物数量随施氮量的增加先增加后降低,以常规施氮处理（N₁₈₀）的微生物数量最多,氮肥用量对单作小麦的影响大于间作小麦. 施氮量对蚕豆根际细菌、真菌、放线菌数量和微生物多样性无显著影响,但降低了间作蚕豆根际微生物总量.适量施用氮肥能有效调节根际微生物区系,间作系统地上部植物多样性与地下部微生物区系间存在紧密联系.     

The modulation rate transfer function of a harbour porpoise (Phocoena phocoena)

During echolocation, toothed whales produce ultrasonic clicks at extremely rapid rates and listen for the returning echoes. The auditory brainstem response (ABR) duration was evaluated in terms of latency between single peaks: 5.5 ms (from peak I to VII), 3.4 ms (I–VI), and 1.4 ms (II–IV). In comparison to the killer whale and the bottlenose dolphin, the ABR of the harbour porpoise has shorter intervals between the peaks and consequently a shorter ABR duration. This indicates that the ABR duration and peak latencies are possibly related to the relative size of the auditory structures of the central nervous system and thus to the animal’s size. The ABR to a sinusoidal amplitude modulated stimulus at 125 kHz (sensitivity threshold 63 dB re 1 μPa rms) was evaluated to determine the modulation rate transfer function of a harbour porpoise. The ABR showed distinct envelope following responses up to a modulation rate of 1,900 Hz. The corresponding calculated equivalent rectangular duration of 263 μs indicates a good temporal resolution in the harbour porpoise auditory system similar to the one for the bottlenose dolphin. The results explain how the harbour porpoise can follow clicks and echoes during echolocation with very short inter click intervals.     

Studies with a reconstituted muscle glycolytic system. The rate and extent of glycolysis in simulated post-mortem conditions

The stimulation of succinate-cytochrome c reductase in Jerusalem artichoke mitochondria by lowering osmolarity was found to be associated with conformational changes in the inner membrane rather than with rupture of the outer membrane. This conclusion is based on the following evidence. (1) When the activation of succinate dehydrogenase was measured by using either K(3)Fe(CN)(6) or exogenous cytochrome c as an electron acceptor, electron flow to cytochrome c was always 7% of that to K(3)Fe(CN)(6) throughout the activation process. (2) The rate of exogenous cytochrome c reduction by succinate and NADH was directly related to the maximum rate of electron flow as determined by oxygen utilization. These two observations are not consistent with the low rate of succinate-cytochrome c reductase being limited by a permeability barrier at the outer membrane. (3) In addition to stimulating the succinate-cytochrome c reductase, lowering the osmolarity caused simultaneous changes in the permeability of the inner membrane to ferricyanide and NADH. The data show that lowering the osmolarity results in progressive changes in the permeability of the inner membrane. The first change detected was an increased permeability to K(3)Fe(CN)(6), then a simultaneous increase in accessibility of the respiratory chain to exogenous cytochrome c and an increased permeability to NADH, followed finally by rupture as measured by the release of malate dehydrogenase.     

Measuring the rate of conjugal plasmid transfer in a bacterial population using quantitative PCR

Horizontal transfer of genes between species is an important mechanism for bacterial genome evolution. In Escherichia coli, conjugation is the transfer from a donor (F⁺) to a recipient (F⁻) cell through cell-to-cell contact. We demonstrate what we believe to be a novel qPCR method for quantifying the transfer kinetics of the F plasmid in a population by enumerating the relative abundance of genetic loci unique to the plasmid and the chromosome. This approach allows us to query the plasmid transfer rate without the need for selective culturing with unprecedented single locus resolution. We fit the results to a mass action model where the rate of plasmid growth includes the lag time of newly formed F⁺ transconjugants and the recovery time between successive conjugation events of the F⁺ donors. By assaying defined mixtures of genotypically identical donor and recipient cells at constant inoculation densities, we extract an F plasmid transfer rate of 5 × 10⁻¹⁰ (cells/mL · min)⁻¹. We confirm a plasmid/chromosome ratio of 1:1 in homogenous F⁺ populations throughout batch growth. Surprisingly, in some mixture experiments we observe an excess of F plasmid in the early saturation phase that equilibrates to a final ratio of one plasmid per chromosome.     

The enhancement of conjugal plasmid pBHR1 transfer between bacteria in the presence of extracellular metabolic products produced by Microcystis aeruginosa

Conjugal plasmid transfer from Escherichia coli S17-1 (pBHR1) to Pseudomonas stutzeri was investigated in the presence of a cyanophyta Microcystis aeruginosa. The plasmid transfer frequency increased with higher densities of M. aeruginosa. The extracellular metabolic products (EMPs) from M. aeruginosa were found to enhance the plasmid transfer between bacteria. Furthermore, the plasmid transfer frequency in medium containing EMPs was significantly higher than that in culture medium with or without glucose. These results suggest that M. aeruginosa enhances conjugal plasmid transfer between bacteria through its EMPs, and that identity of the carbon source is an important factor affecting conjugal plasmid transfer in aquatic environments.     

Electron transfer between globular proteins. Dependence of the rate of transfer on distance

Based on the assumption that electron transfer between globular proteins occurs by a collective excitation of polaron type, the dependence of the rate of this process on the distance between the donor and acceptor centers with regard to their detailed electron structure was calculated. The electron structure of the heme was calculated by the quantum-chemical MNDO-PM3 method. The results were compared with experimental data on interprotein and intraglobular electron transfer. It is shown that, in the framework of this model, the electron transfer is not exponential and does not require a particular transfer pathway since the whole protein macromolecule is involved in the formation of the electron excited state.     

Effect of simulated altitude erythrocythemia in women on hemoglobin flow rate during exercise

The effect of simulated altitude erythrocythemia on hemoglobin flow rate and maximal O2 uptake (VO2max) was determined for nine women sea-level residents. Test conditions included normoxia and normobaric hypoxia (16% O2-84% N2). Cycle tests were performed under normoxia (T1-N) and hypoxia (T1-H) at prereinfusion control and under hypoxia 48 h after a placebo infusion (T2-H) and 48 h after autologous infusion of 334 ml of erythrocytes (T3-H). Hematocrit (38.1-44.9%) and hemoglobin concentration (12.7-14.7 g.dl-1) increased from control to postreinfusion. At peak exercise, VO2max decreased from T1-N (2.40 l.min-1) to T1-H (2.15 l.min-1) then increased at T3-H (2.37 l.min-1). Maximal arterial-mixed venous O2 difference decreased from T1-N to T1-H and increased at T3-H. Cardiac output (Q), stroke volume, heart rate, and total peripheral resistance during maximal exercise were unchanged from T1-N through T3-H. Hemoglobin flow rate (Hb flow) at maximum did not change from T1-N to T1-H but increased at T3-H. When compared with submaximal values for T1-N, VO2 was unchanged at T1-H and T3-H; Q increased at T1-H and decreased at T3-H; arterial-mixed venous O2 difference decreased at T1-H and increased at T3-H; Hb flow did not change at T1-N but increased at T3-H. For young women, simulated altitude erythrocythemia increased peak Hb flow and decreased physiological altitude (227.8 m) but did not affect maximum cardiac output (Qmax).     

Survival of,and plasmid transfer between modified Enterobacter cloacae strains under long-term starvation conditions in a continuous-flow system

Continuous-flow, packed-bed column reactors, which provide an experimental model of a soil profile, were used to investigate survival of, and plasmid transfer between, strains of Enterobacter cloacae. When columns, inoculated with nutrient-sufficient donor and recipient strains, were provided with a minimal salts medium with no added carbon source, transconjugant cells appeared in their effluents. During the first few days of such experiments, the concentration of cells in the effluent declined but then the donor population stabilized, while the recipient and transconjugant populations continued to decrease. The results indicate that the amount of nutrient required to maintain and transfer plasmids is very low. No transconjugants were observed in the effluent from columns inoculated with pre-starved donor and recipient strains.     

The conjugal transfer system of Agrobacterium tumefaciens octopine-type Ti plasmids is closely related to the transfer system of an IncP plasmid and distantly related to Ti plasmid vir genes.

We have determined the DNA sequences of two unlinked regions of octopine-type Ti plasmids that contain genes required for conjugal transfer. Both regions previously were shown to contain sequences that hybridize with tra genes of the nopaline-type Ti plasmid pTiC58. One gene cluster (designated tra) contains a functional oriT site and is probably required for conjugal DNA processing, while the other gene cluster (designated trb) probably directs the synthesis of a conjugal pilus and mating pore. Most predicted Tra and Trb proteins show relatively strong sequence similarity (30 to 50% identity) to the Tra and Trb proteins of the broad-host-range IncP plasmid RP4 and show significantly weaker sequence similarity to Vir proteins found elsewhere on the Ti plasmid. An exception is found in the Ti plasmid TraA protein, which is predicted to be a bifunctional nickase-helicase that has no counterpart in IncP plasmids or among Vir proteins but has homologs in at least six other self-transmissible and mobilizable plasmids. We conclude that this Ti plasmid tra system evolved by acquiring genes from two or three different sources. A similar analysis of the Ti plasmid vir region indicates that it also evolved by appropriating genes from at least two conjugal transfer systems. The widely studied plasmid pTiA6NC previously was found to be nonconjugal and to have a 12.65-kb deletion of DNA relative to other octopine-type Ti plasmids. We show that this deletion removes the promoter-distal gene of the trb region and probably accounts for the inability of this plasmid to conjugate.