Infection of primary fibroblasts with human cytomegalovirus (HCMV) causes a rapid stabilization of the cellular protein p53. p53 is a major effector of the cellular damage response, and activation of this transcription factor can lead either to cell cycle arrest or to apoptosis. Viruses employ many tactics to avoid p53-mediated effects. One method HCMV uses to counteract p53 is sequestration into its viral replication centers. In order to determine whether or not HCMV benefits from this sequestration, we infected a p53 ؊/؊ fibroblast line. We find that although these cells are permissive for viral infection, several parameters are substantially altered compared to wild-type (wt) fibroblasts.

The ability to express the genes of pathogenic human viruses, such as the acquired immune deficiency syndrome (AIDS) virus (also called human immunodeficiency virus) in bacterial cells affords the opportunity to study proteins that are ordinarily difficult or inconvenient to obtain in amounts sufficient for detailed analysis. A segment of the AIDS virus pol gene was expressed in Escherichia coli. Expression resulted in the appearance of reverse transcriptase activity in the bacterial cell extracts. The extracts contained two virus-related polypeptides that have the same apparent molecular weights as the two processed forms of virion-derived reverse transcriptase (p66 and p51). The formation of these two polypeptides depended on the coexpression of sequences located near the 5' end of the pol gene, a region that is thought to encode a viral protease. This bacterial system appears to generate mature forms of the AIDS virus reverse transcriptase by a proteolytic pathway equivalent to that which occurs during virus infection of human cells.

Mannheimia (Pasteurella) haemolytica is the only pathogen that consistently causes severe bronchopneumonia and rapid death of bighorn sheep (BHS; Ovis canadensis) under experimental conditions. Paradoxically, Bibersteinia (Pasteurella) trehalosi and Pasteurella multocida have been isolated from BHS pneumonic lungs much more frequently than M. haemolytica. These observations suggest that there may be an interaction between these bacteria, and we hypothesized that B. trehalosi overgrows or otherwise inhibits the growth of M. haemolytica. Growth curves (monoculture) demonstrated that B. trehalosi has a shorter doubling time (ϳ10 min versus ϳ27 min) and consistently achieves 3-log higher cell density (CFU/ml) compared to M. haemolytica. During coculture M. haemolytica growth was inhibited when B. trehalosi entered stationary phase (6 h) resulting in a final cell density for M. haemolytica that was 6 to 9 logs lower than expected with growth in the absence of B. trehalosi. Coculture supernatant failed to inhibit M. haemolytica growth on agar or in broth, indicating no obvious involvement of lytic phages, bacteriocins, or quorum-sensing systems. This observation was confirmed by limited growth inhibition of M. haemolytica when both pathogens were cultured in the same media but separated by a filter (0.4-m pore size) that limited contact between the two bacterial populations. There was significant growth inhibition of M. haemolytica when the populations were separated by membranes with a pore size of 8 m that allowed free contact. These observations demonstrate that B. trehalosi can both outgrow and inhibit M. haemolytica growth with the latter related to a proximity-or contact-dependent mechanism.The bighorn sheep (BHS; Ovis canadensis) population in North America has declined from an estimated two million at the beginning of the 19th century to fewer than 70,000 today (7, 30). The decline of BHS populations is presumably due to loss of habitat, competition for forage with domestic livestock, predation, and disease (9, 19). The most important disease that has limited the growth of BHS populations is pneumonia (13,14,19,31). Bacteria associated with BHS pneumonia are members of the genera Mannheimia and Pasteurella, particularly, the species Mannheimia (Pasteurella) haemolytica, Bibersteinia (Pasteurella) trehalosi, and Pasteurella multocida (6-9, 15, 20, 25, 31). Several independent studies have revealed that M. haemolytica is a major cause of BHS pneumonia. In fact, M. haemolytica is the only pathogen that has been shown to consistently cause severe bronchopneumonia and rapid death of BHS under experimental conditions (10, 14, 23). B. trehalosi has been isolated more often than M. haemolytica from the upper respiratory tract of healthy BHS (10, 12-14, 26, 31). Large numbers of B. trehalosi have also been isolated from the pneumonic lungs of BHS experimentally inoculated with M. haemolytica alone (10). Furthermore, our recent studies with M. haemolytica wild type and leukotoxin deletion mutants in BHS have revealed t...

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We describe a novel proximity-dependent inhibition phenotype of Escherichia coli that is expressed when strains are cocultured in defined minimal media. When cocultures of "inhibitor" and "target" strains approached a transition between logarithmic and stationary growth, target strain populations rapidly declined >4 log CFU per ml over a 2-h period. Inhibited strains were not affected by exposure to conditioned media from inhibitor and target strain cocultures or when the inhibitor and target strains were incubated in shared media but physically separated by a 0.4-m-pore-size membrane. There was no evidence of lytic phage or extracellular bacteriocin involvement, unless the latter was only present at effective concentrations within immediate proximity of the inhibited cells. The inhibitory activity observed in this study was effective against a diversity of E. coli strains, including enterohemorrhagic E. coli serotype O157:H7, enterotoxigenic E. coli expressing F5 (K99) and F4 (K88) fimbriae, multidrug-resistant E. coli, and commensal E. coli. The decline in counts of target strains in coculture averaged 4.8 log CFU/ml (95% confidence interval, 4.0 to 5.5) compared to their monoculture counts. Coculture of two inhibitor strains showed mutual immunity to inhibition. These results suggest that proximity-dependent inhibition can be used by bacteria to gain a numerical advantage when populations are entering stationary phase, thus setting the stage for a competitive advantage when growth conditions improve.With the discovery of quorum sensing in the 1960s and 1970s, in comparison to the discovery of colicins in the 1920s, it became evident that populations of individual cells are capable of coordinating functions by using signaling molecules for communication. These communications can enhance fitness in a multispecies community, help exploit nutrients more efficiently, increase cooperation with neighboring cells, or harm competing bacteria. Some of the cell-to-cell communications have been well characterized. Streptococcus oralis 34 secretes an autoinducer that signals Actinomyces naeslundii T14V to congregate with S. oralis 34 and form a mutualistic biofilm in saliva (21). Vibrio cholerae uses autoinducers to shut down expression of virulence factors and biofilm formation at high cell densities (13). As an SOS response, certain bacteria can produce toxins, called bacteriocins, which can kill a narrow spectrum of competing cells that express suitable cell surface receptors (8)(9)(10)14). Bacteria can also produce inhibitory phage particles and iron-sequestering aerobactin to gain an advantage over competing bacteria (6,23).Many of these mechanisms enhance the fitness of bacterial strains in a given environment. Khachatryan et al. in 2004 observed a fitness trait allowing certain multidrug-resistant Escherichia coli in Holstein calves to dominate the enteric E. coli population (16). Neither antimicrobial drug use nor the presence of antimicrobial resistance genes was associated with the fitness trait observed in the ...

Recently, a rapidly amplifying family of mouse LINE-1 (L1) has been identified and named T(F). The evolutionary context surrounding the derivation of the T(F) family was examined through phylogenetic analysis of sequences in the 3' portion of the repeat. The Mus musculus domesticus T(F) family was found to be the terminal subfamily of the previously identified L1Md4 lineage. The L1Md4 lineage joins the other prototypical mouse LINE-1 lineage (the L1MdA2 lineage) approximately 1 MYA at about the time of the common ancestor of M. m. domesticus, Mus spicilegus, and Mus spretus. However, the T(F) family from M. m. domesticus was found to join to the previously reported M. spretus Ms475 and Ms7024 LINE-1 families at just 0.5 MYA, indicating horizontal transfer. The T(F) family from M. m. domesticus was then found to be even more recently related to LINE-1's from another species, M. spicilegus. A separate spretus A2 lineage was found through a directed search of a PCR library. This lineage, in contrast to the spretus T(F) lineage, does join domesticus at about 1 MYA, as would be expected in the absence of horizontal transfer. A third major family was also found that splits off from the L1Md4 lineage shortly after its departure from the L1MdA2 lineage. The new family, named the Z family, was found to contain the de novo LINE-1 inserts causing the beige and med mutations. Whether the split with the Z family was before or after the recombination that introduced the F-type promoters and defined the inception of T(F) as a lineage is unclear. In enumerating copies of the various LINE-1 families, we found that T(F) 3' ends were not much more numerous than the reported number of 5' ends, suggesting that T(F) may not be subjected to the 90% truncation pattern typical of LINE-1 as a whole.

We constructed and characterized a plasmid-based genetic system that reports the expression of a toluene-responsive promoter (PtbuA1) by effecting an irreversible, heritable change in the biosensor cell. Expression of the reporter gene gfp is strongly repressed in the absence of expression from the PtbuA1 promoter, and high level gfp expression in the original cell and its progeny is mediated by the site-specific recombination machinery of bacteriophage P22 to initiate removal of a repressor cassette. The reporter plasmid pTolLHB was functional in two soil saprophytes, Pseudomonas fluorescens A506 and Enterobacter cloacae JL1157, with the efficiency and sensitivity to low toluene concentrations being optimal in P. fluorescens A506. In culture, 80-100% of the A506 (pTolLHB) population expressed gfp following exposure to 0.2 micro m toluene for one to three hours. Compared to the response of A506 containing a plasmid-borne PtbuA1-gfp fusion, the recombination-based biosensor was more sensitive at detecting low toluene and trichloroethylene concentrations. An A506 (pTolLHB) inoculum, which had a background of 2.5% of the cells expressing gfp, was introduced onto barley roots in soil microcosms. If toluene was introduced into the microcosms, after 24 h, 72% of the A506 (pTolLHB) cells recovered from roots expressed gfp, indicating bioavailable toluene to rhizosphere bacteria. When toluene was not introduced, 16.5% of the A506 (pTolLHB) cells recovered from the roots expressed gfp, indicating that natural inducers of the PtbuA1 promoter were present in the barley rhizosphere. When introduced into rhizotrons containing barley plants and toluene vapours, the biosensor allowed localization of the availability of toluene along the seminal roots. In rhizotrons that were not exposed to toluene vapours, the biosensor exhibited high PtbuA1-promoter activity in distinct regions along the seminal roots, indicating spatial heterogeneity plant- or rhizosphere microbial community-derived inducers of the PtbuA1 promoter. This recombination-based toluene biosensor thus was useful in identifying bacterial exposure to transient or low levels of toluene, or related compounds, directly in the environment.

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