Flow cytometric analysys of Enterococcus faecalis pAD1(-) strains response to cAD1 pheromone

Conjugative plasmids transfer in Enterococcus faecalis is inducted by sex pheromones. The pheromone is excreted by recipient cells and induces expression of aggregation protein AS in donor cells. This protein is involved in formation of matting aggregates. Use of flow cytometry and anti-As monoclonal antibodies allowed collect of interesting data pheromone response. However, according to our knowledge, no study focused on unspecific influence on particular pheromone for plasmid-free recipient strains. Six pAD1 (-) and tree pAD1 (+) Enterococcus faecalis stains were cultivated for 18h in BHI, with and without cAD1 pheromone (Sigma, Germany), respectively. The bacteria were washed, stained with carboksyfluorescein (FCDA, and analyzed by flow cytometry in FACS BD scan cytometr. Relative fluorescence and size of aggregation was used to compare influence on particular strains. Surprisingly, the results shows divergence in fluorescence, size of aggregates and degree of correlation between fluorescence of aggregates and their sizes among pAD1(-) strains, allowing for distinguish of two groups. Three of studied strains have higher fluorescence than pAD (+) stains. Correlation between fluorescence and size of aggregates, significant higher than in pAD1(+) stains, decrease from r = 0.88 to r=0.74 in reaction to cAD1. The strains if other group fluorize with lower intensity than pAD1 (+). Furthermore, 30.4% pAD1 (-) of second group have no detectable fluorescence. In contrast to pAD1 (-) ) strains of the first group and pA1 (+) strains, low (r=0.55) correlation between fluorescence and size of aggregates of group II increase up to r=0.74 after incubation with cAD1 pheromone. Previous study of these pAD1 (-) strains, currently assigned to group II, shown their low frequency of collecting aph2" gene encoded on other conjugative plasmid, pMG. According to these results, such flow cytometric analysis may be used to predict ability of strain to collect unrelated conjugative plasmid.     

Resonance energy transfer study of membrane-bound aggregates of the sarcoplasmic reticulum calcium ATPase

The aggregation of the membrane-bound calcium ATPase from sarcoplasmic reticulum has been studied by resonance energy transfer. The temperature dependence of resonance energy transfer from a fluorescent membrane lipid donor to an acceptor covalently linked to the Ca2+ ATPase was observed for the native sarcoplasmic reticulum vesicles and for purified protein reconstituted into phospholipid vesicles. The efficiency of energy transfer in these systems increases as the size of protein aggregates decrease. This is due to the increased exposure of the protein in the lipid domain that results in the shortening of distances between donors and acceptors. The degree of aggregation was observed to decrease with increasing temperature. Aggregates rea h a limiting size at low temperature (5 degrees C) but not a high temperatures (45 degrees C). For the reconstituted system, the aggregate size showed a continuous, smooth decrease with increasing temperature. Sarcoplasmic reticulum vesicles showed a decrease in aggregation except for a region from 20 to 30 degrees C in which no change occurred. Arrhenius plots of the calcium transport activities for both systems do not reflect these differences, but instead show similar discontinuities and activation energies. A theoretical model is used to analyze the resonance energy transfer results for the reconstituted vesicles. The average radius of the ATPase aggregate is obtained from this analysis. The limiting, low temperature value of the aggregate radius is consistent with the formation of a tetramer. This structure breaks down to smaller, functional units at higher temperatures.     

Horizontal transfer of bait in the German cockroach: indoxacarb causes secondary and tertiary mortality

Horizontal transfer of indoxacarb in the German cockroach, Blattella germanica (L.), was examined under laboratory conditions. Results show that a single bait-fed adult cockroach (i.e., the donor) transferred indoxacarb to numerous primary recipients (secondary mortality),which then became secondary donors. These recipients subsequently became donors to other cockroaches and caused significant mortality in other members of the aggregation, resulting in tertiary kill. Indoxacarb was effectively transferred among adult cockroaches and resulted in significant secondary mortality. When adult males served as donors and vectored the insecticide to adult males, the donor:recipient ratio affected the mortality of the recipients and the rate of secondary mortality increased with increasing the ratio of donors to recipients. Furthermore, secondary mortality in the untreated cockroaches was significantly affected by the freshness of excretions from the donors, the presence of alternative food, and the duration of contact between the donors and the recipients. Ingested indoxacarb was most effectively translocated when the recipients interacted with freshly symptomatic donors in the absence of alternative food. The transfer of indoxacarb continued beyond secondary mortality and resulted in significant tertiary mortality. Excretions from a single bait-fed adult killed 38/50 (76%) nymphs within 72 h. The dead nymphs then vectored indoxacarb to 20 adult males and killed 16/20 (81%) recipients within 72 h. Behavioral mechanisms involved in the horizontal transfer of indoxacarb may include: contact with excretions, necrophagy, emetophagy, and ingestion of other excretions that originate from the donors.     

A multicopy plasmid of the extremely thermophilic archaeon Sulfolobus effects its transfer to recipients by mating.

A plasmid of 45 kb, designated pNOB8, was found in high copy number in a new heterotrophic Sulfolobus isolate, NOB8H2, from Japan. Dissemination of the plasmid occurred in six cultures of nine different Sulfolobus strains when small amounts of the donor were added. These mixed cultures exhibited a high average copy number of the plasmid, between 20 and 40 per chromosome, and showed a marked growth retardation. Horizontal transfer of pNOB8 was proved by isolating transcipients from mating mixtures via single colonies. In these isolates, the copy number of the plasmid appeared to be subject to a control mechanism. Cell-free filtrates of donor cultures did not transmit the plasmid, and plating of the donor on lawns of recipients did not result in plaque formation, suggesting that the transfer was not mediated by a virus. Rapid formation of cell-to-cell contacts between differently stained donor and recipient partners was demonstrated after the two strains were mixed. Electron microscopic analysis of mating mixtures revealed many cell aggregates made up of 2 to 30 cells and intercellular cytoplasmic bridges connecting two or more cells. Cells that had been transformed with purified plasmid DNA as well as transcipients isolated from mating mixtures were shown to serve as donors for further transmission of pNOB8. The plasmid undergoes extensive genetic variations, since deletions and insertions were frequently observed in plasmid preparations from the donor strain and from mating mixtures.     

The Impact of Heterogeneity and Dark Acceptor States on FRET: Implications for Using Fluorescent Protein Donors and Acceptors

Förster resonance energy transfer (FRET) microscopy is widely used to study protein interactions in living cells. Typically, spectral variants of the Green Fluorescent Protein (FPs) are incorporated into proteins expressed in cells, and FRET between donor and acceptor FPs is assayed. As appreciable FRET occurs only when donors and acceptors are within 10 nm of each other, the presence of FRET can be indicative of aggregation that may denote association of interacting species. By monitoring the excited-state (fluorescence) decay of the donor in the presence and absence of acceptors, dual-component decay analysis has been used to reveal the fraction of donors that are FRET positive (i.e., in aggregates)._However, control experiments using constructs containing both a donor and an acceptor FP on the same protein repeatedly indicate that a large fraction of these donors are FRET negative, thus rendering the interpretation of dual-component analysis for aggregates between separately donor-containing and acceptor-containing proteins problematic. Using Monte-Carlo simulations and analytical expressions, two possible sources for such anomalous behavior are explored: 1) conformational heterogeneity of the proteins, such that variations in the distance separating donor and acceptor FPs and/or their relative orientations persist on time-scales long in comparison with the excited-state lifetime, and 2) FP dark states.     

Activation requirements of donor T cells and host T cell recruitment in adoptive transfer of murine experimental autoimmune orchitis (EAO)

The relative roles of donor and host T lymphocytes and the T cell activation requirements in adoptive transfer of experimental autoimmune orchitis (EAO) in (C57BL/6 x A/J)F1 mice were investigated in order to gain an understanding of the pathogenesis of this disease. Depletion of T cell subsets in recipients by adult thymectomy and treatment with monoclonal antibodies against CD4 or CD8 had no effect on the incidence of EAO following adoptive transfer of activated T cells from donors immunized with testis homogenate (TH) and adjuvants. In contrast, such depletion of CD4+ T cells inhibited development of EAO in actively immunized mice. Thus, CD4+ cells are required for induction of EAO, but donor CD4+ cells are sufficient by themselves without a comparable contribution from the recipient. Adoptive transfer of EAO required that donor splenic and lymph node T cells be activated in vitro before transfer. We found that exposure to antigen (TH) for as little as 4 hr allowed EAO to occur in 25% of recipients, and by 24 hr the cells were fully competent to induce disease. Proliferation of the cells could not be measured until 2 days later. In serial double-transfer experiments, it was found that the cells must be cultured with TH before each transfer in order for the secondary recipients to develop EAO. However, it was not necessary for the transferred T cells to "see" antigen in vivo in the primary recipients, since transfer to castrated primary recipients had no effect on EAO incidence in secondary recipients. Lymphocytes isolated from diseased testes of immunized donors were competent to transfer EAO without activation in vitro, suggesting that, unlike spleen and lymph node cells, these orchitic lymphocytes were already capable of trafficking to the testis.     

Camphor Plasmid-Mediated Chromosomal Transfer in Pseudomonas putida

Camphor-utilizing strains of Pseudomonas putida have been shown to carry the genetic information required for camphor degradation on a plasmid. The plasmid-carrying strains can serve as donors of both plasmid-borne and chromosomal genes. As recipients, plasmid-deleted strains are much superior to those carrying the camphor pathway genes. The transfer frequency of chromosomal, but not plasmid-borne, genes is markedly enhanced if the donor cells are irradiated with ultraviolet light followed by 3-h of growth on a rich medium in the dark. Recombinants selected for prototrophy are stable and most acquire the camphor (CAM) plasmid concomitantly; only a few of the Cam(+) recombinants inherit the donor's ability to transfer chromosomal genes at a high frequency. Transfer-defective mutations occur on the CAM plasmid, affecting both CAM and chromosomal gene transfer.     

High-frequency conjugal transfer of a gonococcal penicillinase plasmid.

Gonococci containing a 24 X 10(6)-dalton conjugal plasmid were able to mobilize for transfer a smaller, non-self-transmissible penicillinase (Pcr) plasmid with high frequency under appropriate conditions. In some strains, over 10% of donor colony-forming units transferred the Pcr plasmid in a mating of less than 2 h, which suggests that the conjugal system was naturally derepressed. Colony-opacity variants containing different quantities of an approximately 28,000-dalton outer membrane protein were altered in their ability to act as conjugal donors and recipients. Maximal transfer of the Pcr plasmid was observed between transparent donors and recipients, lacking appreciable amounts of the 28,000-dalton protein. Under conditions of high-frequency Pcr plasmid mobilization, no conjugal mobilization of chromosomal markers could be discerned.     

Collection and transfer of multiple embryos in the mare

A pituitary extract was used to induce multiple ovulations in mares to determine whether day-7 embryos from multiple ovulators were viable as indicated by their ability to develop when transferred to recipients. There were more ovulations/donor for induced multiple-ovulating mares than for control single-ovulating mares (4.6 +/- 0.5 vs 1.0 +/- 0.0; n=14). The embryo collection rate per ovulation was similar for multiple ovulators (0.6 +/- 0.1 embryos/ovulation) and single ovulators (0.7 +/- 0.1). The embryo collection rate per donor, therefore, was higher (P<0.01) for the multiple ovulators (2.9 +/- 0.7 vs 0.7 +/- 0.1). The transfer success rate per embryo at day 21 was different (P<0.05) among recipients which received an embryo from control single-ovulating donors (7 8 ), multiple ovulators from which a single embryo was recovered (2 2 ), and multiple ovulators from which multiple embryos were recovered (9 19 ). The recipient pregnancy rate/donor at day 21 was 88% (7 8 ) for single-ovulating controls and 138% (11 8 ) for induced multiple ovulators. Results indicate that the survivability of day-7 embryos from multiple-ovulating donors was reduced. However, despite the reduced survival rate/embryo, the number of pregnant recipients/donor was increased by induction of multiple ovulations because of the increased number of embryos available for transfer.     

Molecular Studies on Entry Exclusion in Escherichia coli Minicells

Minicells produced by abnormal cell division in a strain of Escherichia coli (K-12) have been employed here to investigate the phenomenon of "entry exclusion." When purified minicells from strains containing F' or R factors, or both, are mated with radioactive thymidine-labeled Hfr or R(+) donors, the recipient minicells can be conveniently separated from normal-sized donors following mating, and the products of conjugation can be analyzed in the absence of donors and of further growth of the recipients. Transmissible plasmids or episomes are transferred less efficiently to purified minicells derived from strains carrying similar or related elements than to strains without them. Measurement of deoxyribonucleic acid (DNA) degradation and determination of weight-average molecular weights following transfer indicate that degradation of transferred DNA or transfer of smaller pieces cannot account for the comparative reduction in transfer to entry-excluding recipients. Therefore, we conclude that entry exclusion operates to prevent the physical entry of DNA into recipients expressing the exclusion phenotype. The R-produced repressor (product of the drd(+) gene), which represses fertility (i.e., ability to act as donor), reduces exclusion mediated by R or F factor, or both, in matings between strains carrying homologous elements. Furthermore, the data suggest that the presence of the F pilus or F-like R pilus on recipient cells ensures maximum expression of the exclusion phenotype but is not essential for its expression. In contrast to previous suggestions, we found no evidence for a reduction of entry exclusion attributable to the DNA temperature-sensitive chromosomal mutation dnaB(TS).     

Embryo transfer as a means of controlling the transmission of viral infections. X. The in vivo exposure of zona pellucida-intact porcine embryos to swine vesicular disease virus

Two experiments involving the transfer of embryos from donors infected with swine vesicular disease virus (SVDV) to "clean" recipients were carried out. In Experiment 1, 47 embryos were collected from 4 SVDV-infected donors and transferred to 2 recipients that subsequently produced 10 piglets. All of the recipients and piglets remained seronegative for SVDV. In addition to the transfers, 10 embryos and 58 unfertilized eggs from the infected donors were assayed in vitro and found to be negative for SVDV infectivity. A fifth donor was also inoculated with SVDV in this experiment, but it could not be demonstrated that infection had occurred. This SVDV-exposed donor provided two embryos for transfer and one embryo and two unfertilized eggs for in vitro assay. In Experiment 2, 158 embryos from 9 infected donors were transferred to 7 recipients, resulting in 12 piglets. A total of 7 embryos and 37 unfertilized eggs were assayed in vitro. The recipients, piglets, and embryos/eggs were all negative for SVDV infectivity. Although a final conclusion on the safety of using embryo transfer for the control of swine vesicular disease (SVD) is not possible, the results obtained justify additional studies.     

Antigen modulation of the secondary immune response. VI. Contribution of cells recruited from precursor pool to expression of memory.

The adoptive transfer system has been used extensively to study the ability of antigen triggered memory cells to become antibody forming cells and/or to proliferate and expand the memory cell population. Selective antigen triggering of the memory cells for low and high affinity antibody formation has also been studied in this way. One of the main counter-arguments to the interpretation of these data is that the presence of antigen in the adoptive host may lead to recruitment of new memory cells from either a host or donor precursor population. In this paper we examined the contribution of both host and donor precursor cells to the total antibody response in adoptive secondary recipients. The following donor-host combinations were used in which the recipients were given 1 mg fluid antigen intravenously: (A) normal (non-immune) donors to normal irradiated recipients; (B) normal donors to carrier primed irradiated recipients; (C) carrier primed donors to normal irradiated recipients; (D) normal donors to carrier primed recipients with challenge and subsequent transfer to additional carrier primed recipients; (E) carrier primed donor to normal recipients to carrier primed recipients; (F) repeat of B and C above with multiple antigen administration; (G) purified immune (DNP-BGG) donor T cells mixed with normal B cells transferred to normal irradiated recipients. In most cases recruitment was seen but this represented less than 4% of the responses seen with immune cells. Thus we conclude that this level of recruitment does not compromise the use of the adoptive transfer system for studying selective antigen triggering of memory cells.     

Influence of lipopolysaccharide and protein in the cell envelope on recipient capacity in conjugation of Salmonella typhimurium.

In crosses of Salmonella typhimurium FfinP301 lac+ to F- strains of S. typhimurium in broth, recipient strains which were rough mutants affected in the outer core region of the lipopolysaccharide gave an average of 1.4 Lac+ transconjugants per donor cell and over 50% of the donor and recipient cells in mating aggregates, whereas smooth recipient strains gave 0.08 Lac+ transconjugants and few cells in mating aggregates. Strains with mutations affecting the inner core of the lipopolysaccharide were usually poor recipients. When cells were mated on Millipore membrane filters, both smooth and rough strains gave ca. 1.0 Lac+ transconjugants per donor cell. Plasmids in Inc groups FI, FII, M, J, and I beta gave more transconjugants with rough than smooth strains, but there were no difference in crosses with plasmids in Inc groups T, L, P, N, and W. Strains with mutations in the ompA gene (deficient in Omp Ap = 33K = II* = conjugation protein) yielded only 0.02 Lac+ transconjugants per donor cell and few cells in mating aggregates. There was no indication of a deficiency of Omp Ap in smooth strains compared with rough strains. Reduced fertility of smooth recipients may occur because the O side chains of the lipopolysaccharide shield the recipient and reduce the frequency of stabilization of mating aggregates. However, gradient-of-transmission experiments indicated that once these mating aggregates are stabilized, they are equally stable in both smooth and rough recipients. Fertility was high in crosses of S. typhimurium Flac+ to Escherichia coli K-12 F- (0.75 Lac+ transconjugants per donor cell; over 50% of the cells in mating aggregates). In crosses of E. coli K-12 Flac+ to S. typhimurium smooth F-, ca. 10(-5) Lac+ transconjugants per donor cell were obtained; in crosses to rough recipient strains, fertility was increased 14-fold, and when the recipient was defective in the SA and LT host restriction systems, fertility was increased in additional 100-fold. Thus, both the lipopolysaccharide and the protein in the cell envelope of S. typhimurium were shown to be important in the recipient function in F-mediated conjugation.     

Recipient-induced transfer of the symbiotic plasmid pRL1JI in Rhizobium leguminosarum bv. viciae is regulated by a quorum-sensing relay

Analysis of the regulation of plasmid transfer genes on the symbiotic plasmid pRL1JI in Rhizobium leguminosarum bv. viciae has revealed a novel regulatory relay that is specifically poised to detect an N-acyl-homoserine lactone (AHL) made by different cells (potential recipients of pRL1JI). Adjacent to the traI-trbBCDEJKLFGHI plasmid transfer operon on pRL1JI are two regulatory genes, bisR and traR, which encode LuxR-type quorum-sensing regulators required for conjugation. Potential recipients of pRL1JI induce the traI-trb operon and plasmid transfer via a quorum-sensing relay involving BisR, TraR and the traI-trb operon in donor cells. BisR induces expression of traR in response to N-(3-hydroxy-7-cis-tetradecenoyl)-l-homoserine lactone (3-OH-C14:1-HSL), which is produced by CinI in potential recipient strains. In donor strains (carrying pRL1JI), BisR represses the expression of the chromosomal gene cinI; this repression results in a very low level of formation of 3-OH-C14:1-HSL and hence relatively low levels of expression of traR and the traI-trb operon in strains carrying pRL1JI. However, if 3-OH-C14:1-HSL from potential recipients is present, then traR and plasmid transfer are induced. The induction of traR occurs at very low concentrations of 3-OH-C14:1-HSL (around 1 nm). TraR then induces the traI-trb operon in a quorum-sensing dependent manner in re-sponse to the TraI-made AHLs, N-(3-oxo-octanoyl)-l-homoserine lactone and N-(octanoyl)-l-homoserine lactone. The resulting autoinduction results in high levels of expression of the traI-trb operon. Premature expression of the traI-trb operon is reduced by TraM, which probably titres out TraR preventing expression of traI when there are low levels of traR expression. Expression of traR in stationary phase cells is limited by feedback inhibition mediated by TraI-made AHLs.     

Mobilization of broad host range plasmid from Pseudomonas putida to established biofilm of Bacillus azotoformans. I. Experiments

A strain of Pseudomonas putida harboring plasmids RK2 and pDLB101 was exposed to a pure culture biofilm of Bacillus azotoformans grown in a rotating annular reactor under three different concentrations of the limiting nutrient, succinate. Experimental results demonstrated that the broad host range RSF1010 derivative pDLB101 was transferred to and expressed by B. azotoformans. At the lower concentrations, donor mediated plasmid transfer increased with increasing nutrient levels, but the highest nutrient concentration yielded the lowest rate of donor to recipient plasmid transfer. For transconjugant initiated transfer, the rate of transfer increased with increasing nutrient concentrations for all cases. At the lower nutrient concentrations, the frequency of plasmid transfer was higher between donors and recipients than between transconjugants and recipients. The reverse was true at the highest succinate concentration. The rates and frequencies of plasmid transfer by mobilization were compared to gene exchange by retrotransfer. The initial rate of retrotransfer was slower than mobilization, but then increased dramatically. Retrotransfer produced a plasmid transfer frequency more than an order of magnitude higher than simple mobilization.     

Murine transfer factor. IV. Studies with genetically regulated immune responses

Transfer factor-containing dialysates from mice that were either high or low responders to GAT10, GLA5, or ovalbumin were assayed for their ability to transfer delayed hypersensitivity to murine recipients of either high or low responder phenotype. Dialysates from high responder strains contained transfer factor that would transfer delayed hypersensitivity to both high and low responder recipients. These transfers were not restricted by disparities at the MHC or Igh loci. Identically prepared materials from low responder donors contained little or no transfer factor activity and would not transfer delayed hypersensitivity to either high or low responder recipients. Thus, administration of transfer factor transfers the high responder phenotype to low responder recipients. The data also suggest that production of transfer factor is regulated by Ir genes but that the immunologic activities of transfer factor are not.     

Aggregation of smooth muscle membranes and its use in the preparation of plasma membrane enriched fraction from gastric fundus smooth muscle

Microsomal membranes isolated from rat gastric fundus smooth muscle by differential centrifugation aggregate substantially in the presence of the divalent metal ion Mg2+ or Ca2+. The magnitude of cation-induced membrane aggregation is higher for Ca2+ than for Mg2+, but the ion concentration required for half-maximum membrane aggregation (K0.5 value) is similar for Mg2+ and Ca2+. Cation-induced membrane aggregation is suppressed by high ionic strength and low pH of the medium. Cation-induced membrane aggregation of mitochondrial membrane and plasma membrane enriched fractions differ in the rate of aggregate formation, metal ion concentration dependence, and pH dependence. Such different properties of membrane aggregation were used to prepare a plasma membrane enriched fraction by conventional differential centrifugation. Subfractionation of the heterogeneous microsomal membranes by free-flow electrophoresis indicated that smooth muscle plasma membranes showed a higher electrophoretic mobility than the intracellular membranes. These results suggest that ionic interactions on the cell membrane surfaces differ from those on the intracellular membrane surfaces and that induction of membrane aggregation by Ca2+ or Mg2+ is a useful procedure for an effective and rapid preparation of plasma membrane enriched fraction from smooth muscle.     

Genetic analysis of the pAD1 pheromone response in Streptococcus faecalis, using transposon Tn917 as an insertional mutagen

The conjugative plasmid pAD1 (56.7 kilobases) in Streptococcus faecalis has been shown to confer a mating response to the sex pheromone cAD1 excreted by recipient strains. The response is characterized by the synthesis of a proteinaceous adhesin which coats the surface of the pAD1 -containing donor cell and facilitates the formation of mating aggregates. Donors exposed to cAD1 -containing filtrates of recipients undergo self-aggregation (clumping), an event believed to be associated with an interaction between the adhesin and a binding substance always present on the surface of both recipients and donors. To analyze the molecular processes involved in the mating response, mutants were generated by the erythromycin resistance transposon Tn917 . Transpositions to pAD1 in S. faecalis DS16 gave rise to a number of derivatives that exhibited "constitutive clumping" and the ability to transfer at high frequencies in short (10-min) matings. These mutants fell into two subclasses, which exhibited colony morphologies that were "dry" or "normal". The Tn917 insertions were mapped by restriction enzyme analysis to two separate clusters, designated traA and traB. The dry colony subclass corresponded to traA and represented a span of 1.5 kilobases, whereas the normal subclass corresponded to traB and spanned 1.3 kilobases. The two clusters were separated by 1.7 kilobases in which insertions of Tn917 did not affect the ability to respond normally to cAD1 . Neither type of constitutive clumper produced cAD1 . Another series of insertions exhibited reduced donor potential. In two cases, the reduction in transfer was three to four orders of magnitude; these mapped in traA . In two other cases, the reduction was one to two orders of magnitude. These mapped outside of traA and traB, and one was associated with an increase in plasmid copy number.