High-throughput bacterial genome sequencing: an embarrassment of choice, a world of opportunity

Here, we take a snapshot of the high-throughput sequencing platforms, together with the relevant analytical tools, that are available to microbiologists in 2012, and evaluate the strengths and weaknesses of these platforms in obtaining bacterial genome sequences. We also scan the horizon of future possibilities, speculating on how the availability of sequencing that is 'too cheap to metre' might change the face of microbiology forever.     

Performance comparison of benchtop high-throughput sequencing platforms

Three benchtop high-throughput sequencing instruments are now available. The 454 GS Junior (Roche), MiSeq (Illumina) and Ion Torrent PGM (Life Technologies) are laser-printer sized and offer modest set-up and running costs. Each instrument can generate data required for a draft bacterial genome sequence in days, making them attractive for identifying and characterizing pathogens in the clinical setting. We compared the performance of these instruments by sequencing an isolate of Escherichia coli O104:H4, which caused an outbreak of food poisoning in Germany in 2011. The MiSeq had the highest throughput per run (1.6 Gb/run, 60 Mb/h) and lowest error rates. The 454 GS Junior generated the longest reads (up to 600 bases) and most contiguous assemblies but had the lowest throughput (70 Mb/run, 9 Mb/h). Run in 100-bp mode, the Ion Torrent PGM had the highest throughput (80–100 Mb/h). Unlike the MiSeq, the Ion Torrent PGM and 454 GS Junior both produced homopolymer-associated indel errors (1.5 and 0.38 errors per 100 bases, respectively).     

Phosphatidylinositol 3-kinase/AKT-mediated activation of estrogen receptor alpha: a new model for anti-estrogen resistance

Estrogen receptors (ERs) mediate most of the biological effects of estrogen in mammary and uterine epithelial cells by binding to estrogen response elements in the promoter region of target genes or through protein-protein interactions. Anti-estrogens such as tamoxifen inhibit the growth of ER-positive breast cancers by reducing the expression of estrogen-regulated genes. However, anti-estrogen-resistant growth of ER-positive tumors remains a significant clinical problem. Here we show that phosphatidylinositol (PI) 3-kinase and AKT activate ERalpha in the absence of estrogen. Although PI 3-kinase increased the activity of both estrogen-independent activation function 1 (AF-1) and estrogen-dependent activation function 2 (AF-2) of ERalpha, AKT increased the activity of only AF-1. PTEN and a catalytically inactive AKT decreased PI 3-kinase-induced AF-1 activity, suggesting that PI 3-kinase utilizes AKT-dependent and AKT-independent pathways in activating ERalpha. The consensus AKT phosphorylation site Ser-167 of ERalpha is required for phosphorylation and activation by AKT. In addition, LY294002, a specific inhibitor of the PI 3-kinase/AKT pathway, reduced phosphorylation of ERalpha in vivo. Moreover, AKT overexpression led to up-regulation of estrogen-regulated pS2 gene, Bcl-2, and macrophage inhibitory cytokine 1. We demonstrate that AKT protects breast cancer cells from tamoxifen-induced apoptosis. Taken together, these results define a molecular link between activation of the PI 3-kinase/AKT survival pathways, hormone-independent activation of ERalpha, and inhibition of tamoxifen-induced apoptotic regression.     

Metoclopramide-induced central nervous system depression in the chicken

Background

Metoclopramide is a dopamine D2-receptor antagonist used as an antiemetic and gastroprokinetic agent in man and animals. The drug causes sedation as a side effect in man. Such a sedative action of metoclopramide has not been documented in the chicken as the drug is not used clinically in this species. The present study examines the central nervous system depressant effects of metoclopramide in 7–14 days old broiler chicks.

Results

Injection of metoclopramide at 50, 100 and 200 mg/kg, subcutaneously (s.c.) induced sedation in the chicks in a dose dependent manner. The chicks manifested, within 3.6–19 minutes of metoclopramide injection, signs of sedation characterized by drooping of the head and wings, closed eyelids, reduced motility and decreased distress calls. The duration of sedation ranged between 37.2 to 163.4 minutes. Metoclopramide at 100 and 200 mg/kg induced, within 12.2 and 6.2 minutes, sleep (loss of righting reflex) for 43.8 and 158.6 minutes, respectively. The median effective doses of metoclopramide for induction of sedation and sleep in the chicks were 11 and 53 mg/kg, s.c., respectively. Lower doses of metoclopramide (5 and 10 mg/kg, s.c.) significantly decreased the open-field activity of the chicks and increased the durations of their tonic immobility. All treated-chicks recovered from the central nervous system depressant effect of metoclopramide without any observable adverse effects.

Conclusion

The data suggest that metoclopramide induces central nervous system depression in chicks, and the drug could have potential clinical applications as a sedative-hypnotic agent in avian species not intended for human consumptions.     

Cytogenetic study for possible mutagenic activity induced by ice-nucleation bacteria or their metabolic products in human lymphocytes in vitro

A means for eliminating ice-nucleation-active (INA) bacteria, the microorganisms responsible for frost damage to plants at mild freezing temperatures, is the use as competitors of other naturally occurring, non-nucleating strains. Inactive mutants (INA-) of INA bacteria have been produced by genetic or chemical methods and proposed for biological control of INA populations. Since, however, the application of these INA- mutants in the field may create health hazards to animals, we have studied the possible mutagenic activity of the INA- mutants by examining chromosome aberrations, sister-chromatid exchange (SCE) frequencies, and proliferation kinetics of human lymphocyte cultures. These cultures were treated with: (a) a naturally occurring INA- bacterium (p 767), (b) 2 parental strains (cit 7 and cit 13) of INA bacteria isolated from Citrus orchards, and (c) 2 INA- mutant strains (cit 7 del 1b and cit 13-12), produced, respectively, by chemical modification and by deletion of the corresponding parental strains. Neither whole bacteria nor infiltrates of bacterial growth media, in which toxic metabolic bacterial products might have been released, induced elevation of either chromosome aberrations and SCEs or a cell-division delay. Negative results were also obtained when sonicated bacteria were tested for possible intracellular mutagenic components.     

Exploring Biodiversity in the Bacterial Community of the Mediterranean Phyllosphere and its Relationship with Airborne Bacteria

We studied the structure and diversity of the phyllosphere bacterial community of a Mediterranean ecosystem, in summer, the most stressful season in this environment. To this aim, we selected nine dominant perennial species, namely Arbutus unedo, Cistus incanus, Lavandula stoechas, Myrtus communis, Phillyrea latifolia, Pistacia lentiscus, Quercus coccifera (woody), Calamintha nepeta, and Melissa officinalis (herbaceous). We also examined the extent to which airborne bacteria resemble the epiphytic ones. Genotype composition of the leaf and airborne bacteria was analysed by using denaturing gradient gel electrophoresis profiling of a 16S rDNA gene fragment; 75 bands were cloned and sequenced corresponding to 28 taxa. Of these, two were found both in the air and the phyllosphere, eight only in the air, and the remaining 18 only in the phyllosphere. Only four taxa were found on leaves of all nine plant species. Cluster analysis showed highest similarity for the five evergreen sclerophyllous species. Aromatic plants were not grouped all together: the representatives of Lamiaceae, bearing both glandular and non-glandular trichomes, formed a separate group, whereas the aromatic and evergreen sclerophyllous M. communis was grouped with the other species of the same habit. The epiphytic communities that were the richest in bacterial taxa were those of C. nepeta and M. officinalis (Lamiaceae). Our results highlight the remarkable presence of lactic acid bacteria in the phyllosphere under the harsh conditions of the Mediterranean summer, the profound dissimilarity in the structure of bacterial communities in phyllosphere and air, and the remarkable differences of leaf microbial communities on neighbouring plants subjected to similar microbial inocula; they also point to the importance of the leaf glandular trichome in determining colonization patterns.