A microarray with sequences from the annotated open reading frames (ORFs) in Salmonella enterica subspecies 1, serovar Typhimurium was supplemented with annotated chromosomal ORFs from serovar Typhi that are divergent from Typhimurium (>10% DNA sequence divergence). This non- redundant array was used to (i) measure changes in gene copy number in DNA from actively growing versus stationary Typhi and (ii) to reveal the transcriptional response of Typhi to peroxide, a stress similar to that experienced when they are phagocytosed by macrophages. In S.enterica subspecies 1, pairs of genomes differ in the presence or absence of approximately 10% of their genes. An array twice the size of that needed to cover all ORFs for one genome could carry close homologs of all the ORFs for 10 genomes. Non-redundant DNA arrays could be constructed for any group of closely related organisms that differ by the presence and absence of a few genes.
Salmonella enterica serovar Typhimurium LT2 harbors four temperate prophages. The lytic cycle of these phages was induced with hydrogen peroxide or mitomycin C. Microarray analysis was used to monitor the increase in phage genome copy number and the changes in RNA expression. Phage gene transcription was classified temporally, and host genes that responded to hydrogen peroxide, mitomycin C, or phage induction were also identified. A region of the serovar Typhimurium LT2 host genome encompassing hundreds of genes, flanking the Fels-1 lambdoid prophage, was amplified manyfold during lytic induction, presumably due to Fels-1 runoff replication prior to excision, a phenomenon termed escape replication. An excisionase (xis) mutant of Fels-1 also induced escape replication but did not get packaged. Gifsy-1, a lambdoid prophage that does not normally produce escape replication, did so after deletion of either its integrase or excisionase genes. Escape replication is probably widespread; large regions of host genome amplification were also observed after phage induction in serovar Typhimurium strains SL1344 and 14028s at the suspected integration site of prophage genomes.Bacteriophages play an important role in the fitness of bacteria, in both negative and positive ways. Many phages of bacterial pathogens are known to carry virulence factors essential for successful pathogenesis of the bacterium (4). Bacteriophages are recognized as major vehicles for the lateral transfer of genes (6,7,10,17,18), and most sequenced bacterial genomes contain lysogenic phages, as well as remnants of phages. The life strategies and niches that can be occupied by bacterial strains are probably influenced by the collection of lysogenic phages associated with the strains and the flexibility and mobility of phenotypes that phages impart to the species.In previous studies workers mapped the boundaries of four known lysogenic phages in Salmonella enterica Typhimurium LT2 by hybridizing genomic DNA from strains cured of the phages to DNA microarrays containing all protein-encoding genes in the genome (16). Here we show that induction and replication of all four lysogenic phages in LT2 can be observed by microarray analysis of the DNA content after treatment with peroxide or mitomycin C. This analysis revealed large host genome amplifications adjacent to some known and suspected phage integration sites. These amplifications are presumably a consequence of escape replication, where phage replication occurs prior to excision. This phenomenon was observed genetically decades ago (9, 27), but until now its extent had not been measured by physical methods. Deletion of integrase and excisionase genes prevents independent phage genome replication and induces escape replication in lambdoid phage (2), and we monitored this on a microarray. Gene expression was also measured by microarray analysis during the lytic cycle to reveal changes in host gene expression caused by peroxide, by mitomycin C treatment, or by the replicating phage. Changes in transcriptio...
1. Understanding predator-prey interactions of the arthropod community in any given ecosystem is essential in pinpointing the biological control services provided by natural enemies. 2. Hence, four prey-specific polymerase chain reaction (PCR) assays were developed to analyse the gut contents of the cotton predator community. The four targeted prey included a herbivore/pest, omnivore/pest, omnivore/beneficial, and carnivore/beneficial. 3. First, prey retention tests were conducted to determine how long a prey item of each target species could be detected in a predator after ingestion. The assays yielded highly variable inter-assay and intra-assay prey detection efficiencies. 4. Then, a multifaceted field study was conducted to quantify the population dynamics of the cotton predator assemblage and to assess the frequencies of predation that each predator species exhibited on the targeted prey. In total, 1794 predators, representing 17 arthropod families, were collected over two seasons using both sweep net and whole plant sampling procedures. 5. The predator gut assays showed that there was substantial inter-guild predation occurring on the herbivore/pest, Bemisia tabaci (Gennadius); moderate intra-guild predation on the omnivore/pest, Lygus spp. (Lygus lineolaris (Palisot de Beauvois), L. hesperus Knight, and L. elisus Van Duzee) and the omnivore/beneficial, Geocoris spp. (Geocoris punctipes (Say), and G. pallens Stål); and very little intra-guild predation on the carnivore/beneficial, Collops vittatus (Say). 6. The gut assays also revealed that DNA of the targeted pests, B. tabaci and Lygus spp., were found more frequently in insect predators than spiders; whereas there were no significant differences in predation between the predatory insects and spiders for the beneficial insects, Geocoris spp. and C. vittatus. 7. Finally, there was a significantly higher frequency of predation events recorded for B. tabaci , Lygus spp., and Geocoris spp. in the sweep net samples. This indicates that the method of collection might influence the interpretation of the gut assay results.
We used a nonredundant microarray of the Salmonella enterica serovar Typhimurium LT2 and Typhi CT18 genomes to assess the genomic content of a diverse set of isolates of serovar Typhi. Comparative genomic hybridization revealed 13 regions of absent or divergent gene content in the eight Typhi strains examined compared to Typhi CT18. In particular, two Typhi CT18 prophage regions, STY1048 to STY1077 and STY2038 to STY2077, as well as a five-gene islet (STY3188 to STY3193) were absent or divergent in all other Typhi strains examined. Seven Typhi strains lacked most or all of the IS1 elements present in strain CT18, and three Typhi strains lacked a P4-like phage (STY4821 to STY4834). One strain was devoid of a 149-gene region (STY4521 to STY4680), which encodes numerous phage genes and the Vi antigen biosynthesis and export gene cluster, a type IV pilus, and numerous phage genes. In Typhi strain 26T25, an amplification of an entire inter-ribosomal region encompassing 31 genes has occurred. Furthermore, a 257-gene region (STY1360 to STY1639) showed an aberrant replication pattern in three Typhi isolates. Overall, these differences in gene content indicate that even within a highly clonal bacterial population the genomic reservoir is unstable.
Current standards, based on cell culture assay, indicate that poliovirus is inactivated by 0.5 mg of free chlorine per liter after 2 min; however, integrated cell culture-PCR detected viruses for up to 8 min of exposure to the same chlorine concentration, requiring 10 min for complete inactivation. Thus, the contact time for chlorine disinfection of poliovirus is up to five times greater than previously thought.The standard method of enterovirus assay and detection involves the use of animal cell culture. The presence of virus is indicated by the destruction of cells, referred to as the cytopathogenic effect (CPE), and requires from 3 to Ͼ14 days (8).Often secondary passage of the environmental samples onto a fresh monolayer of cells is necessary before CPE is produced, but secondary passage is not routinely conducted (3, 10). Integrated cell culture-PCR (ICC-PCR) combines the speed and sensitivity of PCR with a cultural viability assay to form a molecular detection method capable of isolating low levels of infectious virus (8). This study determined the efficacy of ICC-PCR to evaluate the chlorine contact time necessary to ensure that all target viruses were inactivated.Poliovirus type 1 (strain Lsc-2ab) was grown in buffalo green monkey kidney (BGM) cells (passages 135 to 195) at 37°C until CPE occurred. Poliovirus was isolated by freeze-thawing and centrifugation as previously described (8). The titers of virus stocks were determined at 2.3 ϫ 10 7 PFU/ml using conventional plaque assay methodology (4). All glassware was exposed to Ͼ5% sodium hypochlorite overnight, rinsed with high-performance liquid chromatography (HPLC) grade water, and exposed to UV light for at least 1 h to eliminate chlorine demand. Virus stock and sodium hypochlorite were added to 50 ml of chlorine demand-free HPLC grade water for a final concentration of 2.3 ϫ 10 2 PFU/ml and 0.5 mg/liter, respectively. Chlorine levels were measured at room temperature and pH 5.5 using a spectrophotometer (model 2000; Hach, Loveland, Colo.). Three replicate studies indicated that chlorine concentrations remained constant for longer than the duration of the experiments, Ն10 min.BGM continuous cell culture flasks (25 cm 2 ) were incubated with 1 ml of sample at 37°C for time intervals of 0, 1, 2, 3, 4, and 14 days before freezing at Ϫ80°C for PCR analysis. For conventional cell culture assays, flasks were incubated up to 14 days and observed daily for CPE. Secondary BGM assays were performed with lysate from the first passage, but the flasks were not frozen until CPE, or another 2-week incubation, occurred. Prior to PCR analysis, samples were centrifuged (model 5415C; Sorvall, Newtown, Conn.) for 15 min at 735 ϫ g and Ϫ5°C to remove cellular debris.Target sequences were reverse transcribed as previously described (8). Cell culture lysate template (5 l) was added to reverse transcription reagents and extracted by heat (5 min at 98°C), followed by a single cycle of 24°C for 10 min, 44°C for 60 min, 99°C for 5 min, and 5°C for 5 min. For cDNA amplification...
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