Field-based evaluation of a male-specific (F+) RNA coliphage concentration method.

Fecal contamination of water poses a significant risk to public health due to the potential presence of pathogens, including enteric viruses. Therefore, sensitive, reliable and easy to use methods for the concentration, detection and quantification of microorganisms associated with the safety and quality of water are needed. In this study, we performed a field evaluation of an anion exchange resin-based method to concentrate male-specific (F+) RNA coliphages (FRNA), fecal indicator organisms, from diverse environmental waters that were suspected to be contaminated with feces. In this system, FRNA coliphages are adsorbed to anion exchange resin and direct nucleic acid isolation is performed, yielding a sample amenable to real-time reverse transcriptase (RT)-PCR detection. Matrix-dependent inhibition of this method was evaluated using known quantities of spiked FRNA coliphages belonging to four genogroups (GI, GII, GII and GIV). RT-PCR-based detection was successful in 97%, 72%, 85% and 98% of the samples spiked (106 pfu/l) with GI, GII, GIII and GIV, respectively. Differential FRNA coliphage genogroup detection was linked to inhibitors that altered RT-PCR assay efficiency. No association between inhibition and the physicochemical properties of the water samples was apparent. Additionally, the anion exchange resin method facilitated detection of naturally present FRNA coliphages in 40 of 65 environmental water samples (61.5%), demonstrating the viability of this system to concentrate FRNA coliphages from water.

Tracing the interaction of bacteriophage with bacterial biofilms using fluorescent and chromogenic probes.

Phages T4 and E79 were fluorescently-labeled with rhodamine isothiocyanate (RITC), fluoroscein isothiocyanate (FITC), and by the addition of 4'6-diamidino-2-phenylindole (DAPI) to phage-infected host cells of Escherichia coli and Pseudomonas aeruginosa. Comparisons of electron micrographs with scanning confocal laser microscope (SCLM) images indicated that single RITC-labeled phage particles could be visualized. Biofilms of each bacterium were infected by labeled phage. SCLM and epifluorescence microscopy were used to observe adsorption of phage to single-layer surface-attached bacteria and thicker biofilms. The spread of the recombinant T4 phage, YZA1 (containing an rII-LacZ fusion), within a lac E. coli biofilm could be detected in the presence of chromogenic and fluorogenic homologs of galactose. Infected cells exhibited blue pigmentation and fluorescence from the cleavage products produced by the phage-encoded beta-galactosidase activity. Fluorescent antibodies were used to detect non-labeled progeny phage. Phage T4 infected both surface-attached and surface-associated E. coli while phage E79 adsorbed to P. aeruginosa cells on the surface of the biofilm, but access to cells deep in biofilms was somewhat restricted. Temperature and nutrient concentration did not affect susceptibility to phage infection, but lower temperature and low nutrients extended the time-to-lysis and slowed the spread of infection within the biofilm.

Lytic infection of Escherichia coli biofilms by bacteriophage T4.

Escherichia coli 3000 XIII formed biofilms on the surface of polyvinylchloride coupons in a modified Robbins device. Bacteriophage T4D+ infected cells in the biofilm and replicated. It is commonly held that bacteriophage cannot infect surface-attached bacteria (biofilms) because such bacteria are protected by an exopolymeric matrix that binds macromolecules and prevents their diffusion into the biofilm. To our knowledge this is the first observation that a bacteriophage can infect and multiply within cells growing as a biofilm.

Bacteriophage T2 and T4, dam+ and damh and Eco dam+ methylation: preference at different sites.

We present a method for determining preference for methylation at minor methylation sites. The target DNA sequence is first subjected to computer-assisted analysis to predict which restriction endonuclease(s) will generate fragments that will contain only one or two likely minor methylation site(s). The target DNA is then methylated in vitro with a radioactive methyl-group donor and subjected to digestion by the chosen restriction enzyme(s). The amount of radioactivity in the various fragments is determined, after separating them using polyacrylamide gel electrophoresis. We documented the effect of nearby bases on the methylation preference and the relative preference for methylation at some specific minor methylation sites.

Coincident chromosomal disomy in meiotic dyads from triploid yeast.

Among meiotic asci produced by triploid (3N) Saccharomyces cerevisiae are cases in which exactly two of the four ascospores proliferate into colonies. Given the unique asymmetry problems inherent in distributing three chromosome homologues in meiosis, these ascospore dyads are of special interest. We have tested 40 of these dyads (80 ascospores) for their chromosome content by ascertaining whether they have inherited one or two copies of each of the sixteen yeast chromosomes from the parental triploid. Overall, then, ascospores in these dyads can be either haploid (N) or disomic (N + 1) for each chromosome. The principal results of this analysis include: (1) Coincident disomy (inheritance of two copies of a given chromosome in both members of an ascospore dyad) was detected for 15 of the 16 yeast chromosomes, and at least once in every dyad. (2) Coincident disomy increased as a function of the mean number of disomic chromosomes per spore in each dyad, but this increase differed functionally from that expected if coincident disomy in the two ascospores were a simple, meiotically independent, concomitant of multiple disomy. We conclude from these results that: (1) The ascospore dyads, as the two proliferating spores of single meioses from the triploid, represent meiotic sisters. That is, they stem from the same half of the first meiotic division. (2) Multiply-disomic meiotic segregants of yeast triploids proliferate at the expense of their multiple disomy, as cells in spore colonies experience repeated and independent disomic chromosome losses (N + 1----N).(ABSTRACT TRUNCATED AT 250 WORDS)

Differential mitotic stability of yeast disomes derived from triploid meiosis.

The frequencies of recovered disomy among the meiotic segregants of yeast (Saccharomyces cerevisiae) triploids were assessed under conditions in which all 17 yeast chromosomes were monitored simultaneously. The studies employed inbred triploids, in which all homologous centromeres were identical by descent, and single haploid testers carrying genetic markers for all 17 linkage groups. The principal results include: (1) Ascospores from triploid meiosis germinate at frequencies comparable to those from normal diploids, but most fail to produce visible colonies due to the growth-retarding effects of high multiple disomy. (2) The probability of disome formation during triploid meiosis is the same for all chromosomes; disomy for any given chromosome does not exclude simultaneous disomy for any other chromosome. (3) The 17 yeast chromosomes fall into three frequency classes in terms of disome recovery. The results support the idea that multiply disomic meiotic segregants of the triploid experience repeated, nonrandom, post-germination mitotic chromosome losses (N + 1 leads to N) and that the observed variations in individual disome recovery are wholly attributable to inherent differences in disome mitotic stability.