Frameshift mutagenesis by ultraviolet and gamma radiations in Salmonella: SOS inducibility and effects of DNA polymerase I

Ultraviolet (UV) and gamma-induced mutagenesis have been studied using a doubly auxotrophic strain of Salmonella typhimurium carrying the amber leuA150 mutation (which reverts by base-pair substitution) and the frameshift hisC3076 marker (which reverts by compensating frameshifts). In the initially constructed LT2 background, both markers were poorly revertible by UV and essentially non-revertible by gamma-radiation. A derivative of this strain carrying the mutation-enhancing plasmid pKM101 was however readily reverted by both UV and gamma, with either Leu+ (base substitution) or His+ (frameshift) revertants being observed on appropriate selective media. Photoreactivation experiments suggested that the lesions leading to formation of the two types of mutagenic event were similar if not identical. Support for this suggestion was obtained when it was found that yields of both types of UV-induced revertant were significantly increased in an excision-deficient background, while no revertants of either type were found in a recA background. Yields of gamma-induced revertants were not greatly altered in a uvrB background, but were also reduced to zero (for both markers) in the recA background. These results are consistent with what has previously been well-documented for UV and gamma-induced base-pair substitution mutagenesis, and serve to emphasize the similarities between base-pair substitution mutagenesis and frameshift mutagenesis by these agents. There are differences, however, since although UV-induced reversion of the leuA150 marker was little affected and gamma-induced reversion of leuA150 was somewhat reduced in the presence of a polA mutation (polA3), the yields of His+ frameshift revertants were significantly increased in the polA3 background following treatment with either UV or gamma. Thus while inducible DNA repair (SOS repair) appears to be involved in generating both types of mutational event following either UV- or gamma-irradiation, at some stage in the processing of premutational lesions the level (or type) of DNA polymerase I activity in the cell seems to have an important role in determining whether or not frameshifts or base-pair substitutions will be produced at a particular frequency.     

Functional Amyloid and Other Protein Fibers in the Biofilm Matrix

Biofilms are ubiquitous in the natural and man-made environment. They are defined as microbes that are encapsulated in an extracellular, self-produced, biofilm matrix. Growing evidence from the genetic and biochemical analysis of single species biofilms has linked the presence of fibrous proteins to a functional biofilm matrix. Some of these fibers have been described as functional amyloid or amyloid-like fibers. Here we provide an overview of the biophysical and biological data for a wide range of protein fibers found in the biofilm matrix of Gram-positive and Gram-negative bacteria.     

Evaluation of methods for the isolation and detection of Escherichia coli O157 in minced beef

This study has evaluated enrichment and detection procedures for the isolation and detection of Escherichia coli O157 inoculated into minced beef. The use of a 24 h enrichment in modified EC broth containing novobiocin allowed low numbers of contaminating cells to multiply to levels detectable on culture media and by ELISA test kits. Total analysis time was reduced by the use of the Dynabead^TM immunomagnetic separation system. The use of the Petrifilm^TM Test Kit-HEC for E. coli O157: H7 and Organon Teknika EHEC-TEK system detected low numbers of contaminating cells following enrichment and reduced analysis time by 1 d. The incorporation of cefixime and tellurite into Sorbitol MacConkey Agar increased the rate and ease of isolation of E. coli O157 and its use is therefore recommended.     

Preparation of plasmid DNA by γ-irradiation of recA cells

If recA bacteria are exposed to appropriate doses of gamma-irradiation, nondividing cells which can sustain the multiplication of "small" plasmids are produced. The gamma-irradiation technique has a number of advantages over other methods for preparing pure plasmid DNA: (1) there is little, if any, contamination of DNA preparations by chromosomal DNA owing to extensive degradation of the irradiated DNA by endogenous nucleases, (2) there is no need to introduce a uvr mutation to the host bacteria (there is when UV is used to inactivate the chromosome), (3) the method is extremely simple to work with since operations are not limited by considerations of volume and cell density, and (4) there is no need to transfer material from container to container. Yields of plasmid DNA obtained by the gamma-irradiation technique compare favorably with those obtained by other methods.     

The effects of mutations in the polA and recA genes on mutagenesis by nitrosoguanidine in Salmonellatyphimurium

Results of semi-quantitative plate tests indicated that polA and recA mutants of Salmonella typhimurium strain LT2 trpB1 might be significantly less mutable by nitrosoguanidine (MNNG) than were their repair-proficient parents strains. Quantitative data obtained in treat-and-plate experiments showed that this was not the case, at least for low doses of MNNG, and also that the recA strain was significantly more mutable at low doses than its Rec⁺ parent. On the basis of these results it is suggested that cells of S. typhimurium may possess a recA⁺-dependent repair pathway capable of error-free removal of MNNG-induced pre-mutational lesions from their DNA.     

Molecular evolution of the sex peptide network in Drosophila

Successful reproduction depends on interactions between numerous proteins beyond those involved directly in gamete fusion. Although such reproductive proteins evolve in response to sexual selection pressures, how networks of interacting proteins arise and evolve as reproductive phenotypes change remains an open question. Here, we investigated the molecular evolution of the ‘sex peptide network’ of Drosophila melanogaster, a functionally well‐characterized reproductive protein network. In this species, the peptide hormone sex peptide (SP) and its interacting proteins cause major changes in female physiology and behaviour after mating. In contrast, females of more distantly related Drosophila species do not respond to SP. In spite of these phenotypic differences, we detected orthologs of all network proteins across 22 diverse Drosophila species and found evidence that most orthologs likely function in reproduction throughout the genus. Within SP‐responsive species, we detected the recurrent, adaptive evolution of several network proteins, consistent with sexual selection acting to continually refine network function. We also found some evidence for adaptive evolution of several proteins along two specific phylogenetic lineages that correspond with increased expression of the SP receptor in female reproductive tracts or increased sperm length, respectively. Finally, we used gene expression profiling to examine the likely degree of functional conservation of the paralogs of an SP network protein that arose via gene duplication. Our results suggest a dynamic history for the SP network in which network members arose before the onset of robust SP‐mediated responses and then were shaped by both purifying and positive selection.     

Environmental genomics of Late Pleistocene black bears and giant short-faced bears

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