High-throughput comparison of gene fitness among related bacteria

ABSTRACT: BACKGROUND: The contribution of a gene to the fitness of a bacterium can be assayed by whether and to what degree the bacterium tolerates transposon insertions in that gene. We use this fact to compare the fitness of syntenic homologous genes among related Salmonella strains to reveal differences not apparent at the gene sequence level. RESULTS: A transposon Tn5 derivative was used to construct mutants in Salmonella Typhimurium ATCC14028 (STM1) and Salmonella Typhi Ty2 (STY1), which were then grown in rich media. The locations of 234,152 and 53,556 integration sites, respectively, were mapped by sequencing. These data were compared to similar data available for a different Ty2 strain (STY2) and essential genes identified in E. coli K-12 (ECO). Of 277 genes considered essential in ECO, all had syntenic homologs in STM1, STY1, and STY2, and all but nine genes were either devoid of Tn insertions or had very few. For three of these nine genes, part of the annotated gene lacked Tn integrations (yejM, ftsN and murB). At least one of the other six genes, trpS, had a potentially functionally redundant gene encoded elsewhere in Salmonella but not in ECO. An additional 165 genes were almost entirely devoid of transposon integrations in all three Salmonella strains examined, including many genes associated with protein and DNA synthesis. Four of these genes (STM14_1498.L, STM14_2872, STM14_3360.RJ, and STM14_5442) are not found in E. coli. Notable differences in the extent of gene selection were also observed among the three different Salmonella isolates. Mutations in hns, for example, were selected against in STM1 but not in the two STY strains, which have a defect in rpoS rendering hns nonessential. CONCLUSIONS: Comparisons among transposon integration profiles from different members of a species and among related species, all grown in similar conditions, identify differences in gene fitness among syntenic homologous genes. Further differences in fitness profiles among shared genes can be expected in other selective environments, with potential relevance for comparative systems biology.     

Leptospira genomes are modified at 5'-GTAC.

Genomic DNAs of 14 strains from seven species of the spirochete Leptospira were resistant to cleavage by the restriction endonuclease RsaI (5'-GTAC). A modified base comigrating with m4C was detected by chromatography. Genomic DNAs from other spirochetes, Borrelia group VS461, and Serpulina strains were not resistant to RsaI digestion. Modification at 5'-GTAm4C may occur in most or all strains of all species of Leptospira but not in all genera of spirochetes. Genus-wide DNA modification has rarely been observed in bacteria.     

Tamoxifen-induced epigenetic silencing of oestrogen-regulated genes in anti-hormone resistant breast cancer

In the present study, we have taken the novel approach of using an in vitro model representative of tamoxifen-withdrawal subsequent to clinical relapse to achieve a greater understanding of the mechanisms that serve to maintain the resistant-cell phenotype, independent of any agonistic impact of tamoxifen, to identify potential novel therapeutic approaches for this disease state. Following tamoxifen withdrawal, tamoxifen-resistant MCF-7 cells conserved both drug resistance and an increased basal rate of proliferation in an oestrogen deprived environment, despite reduced epidermal growth-factor receptor expression and reduced sensitivity to gefitinib challenge. Although tamoxifen-withdrawn cells retained ER expression, a sub-set of ER-responsive genes, including pS2 and progesterone receptor (PgR), were down-regulated by promoter DNA methylation, as confirmed by clonal bisulphite sequencing experiments. Following promoter demethylation with 5-Azacytidine (5-Aza), the co-addition of oestradiol (E2) restored gene expression in these cells. In addition, 5-Aza/E2 co-treatment induced a significant anti-proliferative effect in the tamoxifen-withdrawn cells, in-contrast to either agent used alone. Microarray analysis was undertaken to identify genes specifically up regulated by this co-treatment. Several anti-proliferative gene candidates were identified and their promoters were confirmed as more heavily methylated in the tamoxifen resistant vs sensitive cells. One such gene candidate, growth differentiation factor 15 (GDF15), was carried forward for functional analysis. The addition of 5-Aza/E2 was sufficient to de-methylate and activate GDF15 expression in the tamoxifen resistant cell-lines, whilst in parallel, treatment with recombinant GDF15 protein decreased cell survival. These data provide evidence to support a novel concept that long-term tamoxifen exposure induces epigenetic silencing of a cohort of oestrogen-responsive genes whose function is associated with negative proliferation control. Furthermore, reactivation of such genes using epigenetic drugs could provide a potential therapeutic avenue for the management of tamoxifen-resistant breast cancer.     

Changes in MCT 1, MCT 4, and LDH expression are tissue specific in rats after long-term hypobaric hypoxia.

Little is known about the effect of chronic hypobaric hypoxia on the enzymes and transporters involved in lactate metabolism. We looked at the protein expression of monocarboxylate transporters MCT 1, MCT 2, and MCT 4, along with total lactate dehydrogenase (LDH) and LDH isozymes in skeletal muscle, cardiac muscle, and liver. Expression of these components of the lactate shuttle affects the ability to transport and oxidize lactate. We hypothesized that the expression of MCTs and LDH would increase after acclimation to high altitude (HA). The response to acclimation to HA was, however, tissue specific. In addition, the response was different in whole muscle (Mu) and mitochondria-enriched (Mi) fractions. Heart, soleus, and plantaris muscles showed the greatest response to HA. Acclimation resulted in a 34% increase in MCT 4 in heart and a decrease in MCT 1 (-47%) and MCT 4 (-47%) in plantaris Mu. In Mi fractions, the heart had an increase (+40%) and soleus a decrease (-40%) in LDH. HA also had a significant effect on the LDH isozyme composition of both the Mu and Mi fractions. Mitochondrial density was decreased in both the soleus (-17%) and plantaris (-44%) as a result of chronic hypoxia. We conclude that chronic hypoxia had a tissue-specific effect on MCTs and LDH (that form the lactate shuttle) but did not produce a consistent increase in these components in all tissues.     

Phocanema decipiens: Molting in seals

Infective larvae of the anisakine nematode Phocanema decipiens from the muscle of Atlantic cod (Gadus morhua) were fed to harbor seals (Phoca vitulina) and gray seals (Halichoerus grypus). During maturation in the stomach of seal hosts, P. decipiens molted twice; these molts are the third and fourth of its life cycle. The third molt occurred between the second and fifth days of infection. The third stage, i.e., infective larva entering the third molt, had a cuticular tooth ventral to the mouth; the fourth stage larva emerging from the third molt had three bilobed lips with dentigerous ridges. The fourth molt occurred between the 5th and 15th days in seals. A female nematode emerging from the fourth molt possesses a vulva and a vagina; a male possesses caudal alae, pre- and postanal papillae. Significant morphometric changes in nematodes were associated with both molts. Females and males of P. decipiens reached maturity after 15 to 25 days in seals. Ova were detected in the feces of the seal hosts as early as the 16th day.     

Selection againstdam methylation sites in the genomes of DNA of enterobacteriophages

Summary Postreplicative methylation of adenine inEscherichia coli DNA to produce G^6m ATC (where^6mA is 6-methyladenine) has been associated with preferential daughter-strand repair and possibly regulation of replication. An analysis was undertaken to determine if these, or other, as yet unknown roles of GATC, have had an effect on the frequency of GATC inE. coli or bacteriophage DNA. It was first ascertained that the most accurate predictions of GATC frequency were based on the observed frequencies of GAT and ATC, which would be expected since these predictors take into account preferences in codon usage. The predicted frequencies were compared with observed GATC frequencies in all available bacterial and phage nucleotide sequences. The frequency of GATC was close to the predicted frequency in most genes ofE. coli and its RNA bacteriophages and in the genes of nonenteric bacteria and their bacteriophages. However, for DNA enterobacteriophages the observed frequency of DNA enterobacteriophages the observed frequency of GATC was generally significantly lower than predicted when assessed by the chi square test. No elevation in the rate of mutation of^6mA in GATC relative to other bases was found when pairs of DNA sequences from closely related phages or pairs of homologous genes from enterobacteria were compared, nor was any preferred pathway for mutation of^6mA evident in theE. coli DNA bacteriophages. This situation contrasts with that of 5-methylcytosine, which is hypermutable, with a preferred pathway to thymine. Thus, the low level of GATC in enterobacteriophages is probably due not to^6mA hypermutability, but to selection against GATC in order to bypass a GATC-mediated host function.     

Patterns of genetic variation in populations of infectious agents

Background  

The analysis of genetic variation in populations of infectious agents may help us understand their epidemiology and evolution. Here we study a model for assessing the levels and patterns of genetic diversity in populations of infectious agents. The population is structured into many small subpopulations, which correspond to their hosts, that are connected according to a specific type of contact network. We considered different types of networks, including fully connected networks and scale free networks, which have been considered as a model that captures some properties of real contact networks. Infectious agents transmit between hosts, through migration, where they grow and mutate until elimination by the host immune system.