Preparation of bacteria--degraders of alkylnaphthalenesulfonate by a molecular breeding method

Transconjugant strain of bacteria degraders of alkylnaphthalenesulphonate(ANS)--P Pseudomonas alcaligenes TR (NPL-41) was obtained under conditions of ANS selective pressure and flow cultivation of bacteria destructors of alkylbenzene sulphonate (ABS) P. alcaligenes TR (pABS) and naphthalene P. putida BS438 (NPL-41) and their immobilization on an inert carrier. Transconjugant P. alcaligenes TR(NPL-41) was prepared by means of conjugative transfer of plasmid NPL-41 from P. putida BS438 to P. alcaligenes TR. The check out of transconjugant under conditions of flow cultivation showed their ability to degradation of ANS. Investigation of the stability of plasmid NPL-41 in the transconjugant P. alcaligenes TR(NPL-41), obtained by molecular breeding under conditions of flow cultivation and by conjugative transfer, showed the transconjugant prepared by the former method to be more stable.     

Direct detection and quantification of horizontal gene transfer by using flow cytometry and gfp as a reporter gene

A new cultivation-independent method for studying conjugal gene transfer between bacteria was evaluated. The method was based on direct detection and enumeration of donor and transconjugant bacterial cells by flow cytometry. Specific detection of transconjugants was obtained by using a conjugative plasmid tagged with a reporter gene (gfp) encoding green fluorescent protein. A chromosomal encoded repressor (lacI(ql)) repressed expression of GFP in the donor bacteria. Enumeration of the donor cells was performed after induction of GFP expression by the addition of inducer isopropyl-thio-beta-D-galactoside (IPTG). The method presented here provided simple and precise quantification of horizontal gene transfer between both Escherichia coli and Pseudomonas putida strains.     

Plasmid transfer between Bacillus thuringiensis subsp. israelensis strains in laboratory culture, river water, and dipteran larvae

Plasmid transfer between strains of Bacillus thuringiensis subsp. israelensis was studied under a range of environmentally relevant laboratory conditions in vitro, in river water, and in mosquito larvae. Mobilization of pBC16 was detected in vitro at a range of temperatures, pH values, and available water conditions, and the maximum transfer ratio was 10(-3) transconjugant per recipient under optimal conditions. Transfer of conjugative plasmid pXO16::Tn5401 was also detected under this range of conditions. However, a maximum transfer ratio of 1.0 transconjugant per recipient was attained, and every recipient became a transconjugant. In river water, transfer of pBC16 was not detected, probably as a result of the low transfer frequency for this plasmid and the formation of spores by the introduced donor and recipient strains. In contrast, transfer of plasmid pXO16::Tn5401 was detected in water, but at a lower transfer ratio (ca. 10(-2) transconjugant per donor). The number of transconjugants increased over the first 7 days, probably as a result of new transfer events between cells, since growth of both donor and recipient cells in water was not detected. Mobilization of pBC16 was not detected in killed mosquito larvae, but transfer of plasmid pXO16::Tn5401 was evident, with a maximum rate of 10(-3) transconjugant per donor. The reduced transfer rate in insects compared to broth cultures may be accounted for by competition from the background bacterial population present in the mosquito gut and diet or by the maintenance of a large population of B. thuringiensis spores in the insects.     

Sequence alignment with an appropriate substitution matrix.

A widely used algorithm for computing an optimal local alignment between two sequences requires a parameter set with a substitution matrix and gap penalties. It is recognized that a proper parameter set should be selected to suit the level of conservation between sequences. We describe an algorithm for selecting an appropriate substitution matrix at given gap penalties for computing an optimal local alignment between two sequences. In the algorithm, a substitution matrix that leads to the maximum alignment similarity score is selected among substitution matrices at various evolutionary distances. The evolutionary distance of the selected substitution matrix is defined as the distance of the computed alignment. To show the effects of gap penalties on alignments and their distances and help select appropriate gap penalties, alignments and their distances are computed at various gap penalties. The algorithm has been implemented as a computer program named SimDist. The SimDist program was compared with an existing local alignment program named SIM for finding reciprocally best-matching pairs (RBPs) of sequences in each of 100 protein families, where RBPs are commonly used as an operational definition of orthologous sequences. SimDist produced more accurate results than SIM on 50 of the 100 families, whereas both programs produced the same results on the other 50 families. SimDist was also used to compare three types of substitution matrices in scoring 444,461 pairs of homologous sequences from the 100 families.     

A soil-based microbial biofilm exposed to 2,4-D: bacterial community development and establishment of conjugative plasmid pJP4

A soil suspension was used as a source to initiate the development of microbial communities in flow cells irrigated with 2,4-dichlorophenoxyacetic acid (2,4-D) (25 microg ml(-1)). Culturable bacterial members of the community were identified by 16S rRNA gene sequencing and found to be members of the genera Pseudomonas, Burkholderia, Collimonas and Rhodococcus. A 2,4-D degrading donor strain, Pseudomonas putida SM1443 (pJP4::gfp), was inoculated into flow cell chambers containing 2-day old biofilm communities. Transfer of pJP4::gfp from the donor to the bacterial community was detectable as GFP fluorescing cells and images were captured using confocal scanning laser microscopy (GFP fluorescence was repressed in the donor due to the presence of a chromosomally located lacI(q) repressor gene). Approximately 5-10 transconjugant microcolonies, 20-40 microm in diameter, could be seen to develop in each chamber. A 2,4-D degrading transconjugant strain was isolated from the flow cell system belonging to the genus Burkholderia.     

Computer-aided S-phase fraction determination in DNA static cytometry in breast cancer. A preliminary methodologic study on cytologic material.

This methodologic study was performed on a single-cell-cycle breast carcinoma to evaluate the feasibility of computer-aided S-phase fraction determination in DNA static cytometry. The investigation was performed on Feulgen-stained cytologic material in which the total optical density values of 1,000 consecutive, randomly selected nuclei were analyzed (MultiCycle software). A good correlation in the S-phase fraction value with flow cytometry was obtained when the G2/G1 ratio was fixed at 1.95, when the histogram data points were smoothed at least once and the coefficient of variation of the G2 peak was the same as that of G0-G1 or when a first-order S-phase polynomial model was used. The percentages of nuclei in G0-G1 and G2 were somewhat similar to those obtained with flow cytometry. The greatest discrepancy with flow cytometry was observed in the value of the coefficient of variation of the G0-G1 peak of the static cytometric data: it was at least twice as great. It always remained high despite the software options used. As for the influence of the sample size in the S-phase calculation, the software was also run on samples of 600 and 200 nuclei. When the G2/G1 ratio was fixed at 1.95, the data obtained from 600 nuclei did not differ from those obtained with 1,000 nuclei, whereas an analysis on 200 nuclei showed a substantial variation. The software also allowed calculation of the ratio of the G0-G1 peak of the neoplastic population against that of the diploid reference (DNA index), the value of which in flow cytometry was 1.0.(ABSTRACT TRUNCATED AT 250 WORDS)     

Role of the intraspecific competition in the regulation of Agrobacterium tumefaciens transconjugant population level in soil experiments

Abstract In microcosms of sterilized soil simultaneously inoculated with Pseudomonas aeruginosa carrying the plasmid R68-45 and the plasmid-free Agrobacterium tumefaciens , transconjugants were detectable after two days of incubation and their number remained constant thereafter. The growth of a transconjugant strain was monitored in sterile soil. When mixed together with the parental strains at high inoculum or when the soil was previously colonized by the donor, the transconjugant was able to grow. If the recipient was the first soil colonizer, the challenging population of transconjugant remained stable at its initial level. We demonstrated the possible role of intraspecific competition in the limitation of transconjugant numbers.     

Plant polyploidy and insect/plant interactions

We used flow cytometry and extensive geographic surveys of herbivore attack to test whether repeated evolution of autotetraploidy in the perennial herb Heuchera grossulariifolia Rydb. (Saxifragaceae) has created evolutionary barriers to attack by the specialist moth herbivore Greya politella (Prodoxidae). We found that the moth has colonized tetraploid as well as diploid populations, has colonized tetraploids of separate evolutionary origin, and, at least under some conditions, is more likely to attack tetraploids than diploids. Plant polyploidy therefore provides a potential route out of specialization as an evolutionary dead end in phytophagous insect taxa as well as a potentially important route to subsequent phylogenetic and geographic diversification of plant/insect interactions.     

Comparison of Flow Cytometry,Fluorescence Microscopy and Spectrofluorometry for Analysis of Gene Electrotransfer Efficiency

In this study, we compared three different methods used for quantification of gene electrotransfer efficiency: fluorescence microscopy, flow cytometry and spectrofluorometry. We used CHO and B16 cells in a suspension and plasmid coding for GFP. The aim of this study was to compare and analyse the results obtained by fluorescence microscopy, flow cytometry and spectrofluorometry and in addition to analyse the applicability of spectrofluorometry for quantifying gene electrotransfer on cells in a suspension. Our results show that all the three methods detected similar critical electric field strength, around 0.55 kV/cm for both cell lines. Moreover, results obtained on CHO cells showed that the total fluorescence intensity and percentage of transfection exhibit similar increase in response to increase electric field strength for all the three methods. For B16 cells, there was a good correlation at low electric field strengths, but at high field strengths, flow cytometer results deviated from results obtained by fluorescence microscope and spectrofluorometer. Our study showed that all the three methods detected similar critical electric field strengths and high correlations of results were obtained except for B16 cells at high electric field strengths. The results also demonstrated that flow cytometry measures higher values of percentage transfection compared to microscopy. Furthermore, we have demonstrated that spectrofluorometry can be used as a simple and consistent method to determine gene electrotransfer efficiency on cells in a suspension.     

Streptococcus cremoris M12R transconjugants carrying the conjugal plasmid pTR2030 are insensitive to attack by lytic bacteriophages.

Conjugal transfer of lactose-fermenting ability (Lac+), nisin resistance (Nisr), and phage resistance (Hsp+) was demonstrated in matings between Streptococcus lactis ME2 (donor) and Streptococcus cremoris M43a (recipient), a derivative of M12R. Transconjugants were detected by transfer of Lac+ and were found to exhibit Nisr and harbor a 40-megadalton plasmid (pTR1040). Fifty-six percent of Lac+ transconjugants were resistant to the S. cremoris M12R lytic phage. Efficiency of plaquing for phage m12r . M12 on a phage-resistant transconjugant, T2r-M43a, was less than 4.3 X 10(-10). Five additional phages which were virulent for S. cremoris M12R and isolated from industrial sources failed to plaque on S. cremoris T2r-M43a. Mating experiments with T2r-M43a revealed that phage resistance was accompanied by high-frequency conjugation ability (Tra+) and the appearance of both pTR1040 and pTR2030 encoding Lac+ Nisr and Tra+ Hsp+, respectively, in transconjugants of S. lactis LM2302. Phage-sensitive Lac+ transconjugants of S. cremoris M43a (T2s-M43a) showed no conjugal ability. These observations confirmed that pTR2030 was present and responsible for the phage resistance and conjugal ability exhibited by the S. cremoris transconjugant T2r-M43a. Unlike the S. lactis LM2302 transconjugant carrying pTR2030, resistance of T2r-M43a to phage was not affected at high temperatures (35 to 40 degrees C) or destabilized in repeated transfers through a starter culture activity test. These results demonstrated that phage resistance conferred by pTR2030 in the S. cremoris transconjugant was effective against industrially significant phages under fermentation conditions normally encountered during cheese manufacture.     

四倍体不结球白菜的诱导及染色体倍性鉴定

用不同浓度秋水仙素处理子叶期不结球白菜生长点对其进行染色体倍性操作,根据形态解剖学、细胞学特征和流式细胞仪进行倍性鉴定.结果表明,浓度为0.2%的秋水仙素处理4次的效果最好,四倍体诱变率为8.42%.与二倍体相比,四倍体植株叶片、花器官、气孔等均表现巨大性;气孔密度和结实率降低;抽薹较晚.用流式细胞仪进行倍性鉴定,对照DNA相对含量为100,疑似株为200,表明是四倍体;疑似株有2个值与对照的比值约为1和2,表明是嵌合体(2x 4x).流式细胞仪鉴定结果与染色体计数法鉴定结果一致,表明流式细胞仪可以较准确地检测不结球白菜突变株倍性.     

Macrophage apoptosis associated with Salmonella enterica serovar Typhi plasmid

The present study was undertaken to investigate the relationship between plasmid isolated from S. enterica serovar Typhi (pR(ST98)) and macrophage apoptosis. pR(ST98) was transferred into an attenuated S. enterica serovar Typhimurium strain RIA to create a transconjugant pRsT98/RIA. Standard S. enterica serovar Typhimurium virulence strain SR-11 was used as a positive control, and RIA as a negative one. Murine macrophage-like cell line (J774A.1) was used as an infectious cell model in vitro. In order to determine the inhibition and bactericidal effect of amikacin (AMK) to extracellular bacteria and the best optimization co-culture ratio between Salmonella and J774A.1, the minimal inhibitory concentration (MIC) and minimal bactericidal concentration (MBC) of AMK to strains SR-11, pR(ST98)/RIA and RIA and multiplicity of infection (MOI) were detected first, and then J774A.1 was infected by the above three serovar Typhimurium strains. Apoptosis of J774A.1 was examined with electron microscopy and flow cytometry after annexin-V/propidium iodide labeling at 0, 1, 3, 6, 12 and 24 h. Mitochondrial membrane potential was detected by JC-1 staining method. It was demonstrated that MIC of AMK to the three strains was 10 microg/ml, MBC was 80 microg/ml, and optimal MOI was 100:1. pR(ST98)/RIA resulted in a higher apoptosis of J774A.1 than RIA, apoptotic features such as chromatin margination could be observed after 3 h, and death of J774A.1 cells was associated with the loss of mitochondrial membrane potential. These results indicated that pR(ST98) could enhance the virulence of its host bacteria, evidenced by increased macrophage apoptosis.     

Comparison of flow cytometry and epifluorescence microscopy for counting bacteria in aquatic ecosystems.

Flow cytometry was used to count bacterial cells from diverse origins: one strain of E. coli, one sample of lake water, and 18 samples of estuary water. To verify the accuracy and the precision of this technique, total bacteria counts made by flow cytometry were compared with counts by direct observation using epifluorescence microscopy. The results of this study showed that flow cytometry was a reliable technique for counting a mixture of bacteria in samples from aquatic ecosystems.     

An automated technique for most-probable-number (MPN) analysis of densities of phagotrophic protists with lux AB labelled bacteria as growth medium.

An automated modification of the most-probable-number (MPN) technique has been developed for enumeration of phagotrophic protozoa. The method is based on detection of prey depletion in micro titre plates rather than on presence of protozoa. A transconjugant Pseudomonas fluorescens DR54 labelled with a luxAB gene cassette was constructed, and used as growth medium for the protozoa in the micro titre plates. The transconjugant produced high amounts of luciferase which was stable and allowed detection for at least 8 weeks. Dilution series of protozoan cultures and soil suspensions were inoculated into micro titre plates amended with a suspension of the transconjugant. After 45 days measurement of light emission allowed detection of individual wells in the titre plates, where protozoan grazing had removed the inoculated bacteria.     

Studies on the Characterization of Root Morphology of White Clover Infected by Transconjugant of Rhizobium leguminosarum

White clover plants were inoculated with transconjugant strain' 290 which was obtained from introduction of host specific nodulation genes of wild-type Rhizobium trifolii strain ANU 843 to Rhizobium leguminosarum strain 300. The characterization of root morphology of white clover induced by the transconjugant was observed and compared to the plants induced by the parent strains. White clover started tO form a typical root hair curling inoculated with transconjugant strain 290 24h after inoculation, at 48h a part of cell wall of root hair was degradated, infection thread was observed in the infected root hair cell, cortical cell divisions occurred extensively. All these characterizations were similar to that infected by strain ANU 843. Plant inoculation test indicated that no nodule was formed when inoculated by R. leguminosarum strain 300, while plants nodulated when inoculated with transconjugant strain 290 as well as R. trifolii ANU 843. This suggests that introduction of host specific nodulation genes of R. trifolii results in conferring the nodulation ability of R. leguminosarum on white clover.     

Streptococcus cremoris M12R transconjugants carrying the conjugal plasmid pTR2030 are insensitive to attack by lytic bacteriophages

Conjugal transfer of lactose-fermenting ability (Lac+), nisin resistance (Nisr), and phage resistance (Hsp+) was demonstrated in matings between Streptococcus lactis ME2 (donor) and Streptococcus cremoris M43a (recipient), a derivative of M12R. Transconjugants were detected by transfer of Lac+ and were found to exhibit Nisr and harbor a 40-megadalton plasmid (pTR1040). Fifty-six percent of Lac+ transconjugants were resistant to the S. cremoris M12R lytic phage. Efficiency of plaquing for phage m12r . M12 on a phage-resistant transconjugant, T2r-M43a, was less than 4.3 X 10(-10). Five additional phages which were virulent for S. cremoris M12R and isolated from industrial sources failed to plaque on S. cremoris T2r-M43a. Mating experiments with T2r-M43a revealed that phage resistance was accompanied by high-frequency conjugation ability (Tra+) and the appearance of both pTR1040 and pTR2030 encoding Lac+ Nisr and Tra+ Hsp+, respectively, in transconjugants of S. lactis LM2302. Phage-sensitive Lac+ transconjugants of S. cremoris M43a (T2s-M43a) showed no conjugal ability. These observations confirmed that pTR2030 was present and responsible for the phage resistance and conjugal ability exhibited by the S. cremoris transconjugant T2r-M43a. Unlike the S. lactis LM2302 transconjugant carrying pTR2030, resistance of T2r-M43a to phage was not affected at high temperatures (35 to 40 degrees C) or destabilized in repeated transfers through a starter culture activity test. These results demonstrated that phage resistance conferred by pTR2030 in the S. cremoris transconjugant was effective against industrially significant phages under fermentation conditions normally encountered during cheese manufacture.     

Cell size does not always correspond to genome size: phylogenetic analysis in geckos questions optimal DNA theories of genome size evolution

At higher taxonomic levels, a significant correlation between genome size (GS) and erythrocyte size (ES) has been reported for many taxa. Under optimal DNA theories, several mechanisms presuming a causative link between GS and ES have been proposed to explain this seemingly general pattern. The correlation between GS and ES has been rarely tested among closely related organisms within an explicit phylogenetic framework. Eyelid geckos (family Eublepharidae) serve as a proper group to conduct such an analysis. We used flow cytometry to measure GS in 15 forms of eublepharids and conducted a phylogenetic reconstruction of GS and ES to test the successiveness of evolutionary shifts in these traits. Most parsimoniously, there were two independent increases and two decreases in GS during the evolution of eublepharids. Nevertheless, changes in GS and ES were not phylogenetically associated in a manner predicted by optimal DNA theories. Our results question the generality of causative bonds between DNA content and cell size and demonstrate that cell size cannot always serve as a proxy of GS. We suggest there is no need to expect a direct causative link between GS and ES to explain the correlation between GS and cell size at higher taxonomic levels. Such a correlation can be explained by simple mechanistic constraints and a combination of the population-genetic model of genome complexity with cell-size-metabolic rate relationship.     

Primate chromosome evolution: with reference to marker order and neocentromeres

Establishing chromosomal homology in comparative cytogenetics remained speculative until the advent of molecular cytogenetics. Chromosome sorting by flow cytometry and degenerate oligonucleotide primed-PCR (DOP-PCR) brought a significant simplification and impetus to chromosome painting. Comparative chromosome painting has permitted reasonable hypotheses for ancestral karyotypes at many points on the phylogenetic tree of mammals. Derived associations often provided landmarks that showed the route evolution took. More recently hybridization with cloned DNA has provided information on intrachromosomal rearrangements. BAC-FISH allows marker order, in addition to syntenies and associations, to be added to the ancestral karyotypes. Comparisons of marker order across species revealed that centromere shifts (evolutionary new centromeres) are frequent and important phenomena of chromosome evolution. Further comparison between evolutionary new centromeres and clinical neocentromeres shows that an evolutionary perspective can provide compelling, underlying, explicative grounds for contemporary genomic phenomena.     

Comparison of five clustering algorithms to classify phytoplankton from flow cytometry data.

BACKGROUND: Artificial neural networks (ANNs) have been shown to be valuable in the analysis of analytical flow cytometric (AFC) data in aquatic ecology. Automated extraction of clusters is an important first stage in deriving ANN training data from field samples, but AFC data pose a number of challenges for many types of clustering algorithm. The fuzzy k-means algorithm recently has been extended to address nonspherical clusters with the use of scatter matrices. Four variants were proposed, each optimizing a different measure of clustering "goodness." METHODS: With AFC data obtained from marine phytoplankton species in culture, the four fuzzy k-means algorithm variants were compared with each other and with another multivariate clustering algorithm based on critical distances currently used in flow cytometry. RESULTS: One of the algorithm variants (adaptive distances, also known as the Gustafson--Kessel algorithm) was found to be robust and reliable, whereas the others showed various problems. CONCLUSIONS: The adaptive distances algorithm was superior in use to the clustering algorithms against which it was tested, but the problem of automatic determination of the number of clusters remains to be addressed.     

三叶草素基因(tfx)在快生型大豆根瘤菌(Sinorhizobium fredii)H12-2中的表达与稳定性测定

将含有三叶草条基因的重组质粒pT2TFXK和pT2TX3K以接合转移的方式导入快生型大豆根瘤菌H12－2。转移接合子H12－2（pXK）能产三叶草素并具有抑菌活性;H12－2（P3K）表现出对三叶草素的抗性。抑菌谱试验结果表明：82％的供试快生型大豆根瘤菌菌株对三叶草素敏感;所有供试慢生型大豆根瘤菌则表现抗性。稳定性检测结果表明：在共生与人工培养条件下,导入的pXK和p3K质粒均可在宿主菌中稳定存在和表达。