Membrane transporters are a large group of proteins that span cell membranes and contribute to critical cell processes, including delivery of essential nutrients, ejection of waste products, and assisting the cell in sensing environmental conditions. Obtaining an accurate and specific annotation of the transporter proteins encoded by a micro-organism can provide details of its likely nutritional preferences and environmental niche(s), and identify novel transporters that could be utilized in small molecule production in industrial biotechnology. The Transporter Automated Annotation Pipeline (TransAAP) ( http://www.membranetransport.org/transportDB2/TransAAP_login.html ) is a fully automated web service for the prediction and annotation of membrane transport proteins in an organism from its genome sequence, by using comparisons with both curated databases such as the TCDB (Transporter Classification Database) and TDB, as well as selected Pfams and TIGRFAMs of transporter families and other methodologies. TransAAP was used to annotate transporter genes in the prokaryotic genomes in the National Center for Biotechnology Information (NCBI) RefSeq; these are presented in the transporter database TransportDB ( http://www.membranetransport.org ) website, which has a suite of data visualization and analysis tools. Creation and maintenance of a bioinformatic database specific for transporters in all genomic datasets is essential for microbiology research groups and the general research/biotechnology community to obtain a detailed picture of membrane transporter systems in various environments, as well as comprehensive information on specific membrane transport proteins.

The era of structural biology and genomics has opened new horizons in our understanding of complex biological questions. Three dimensional membrane transporter structures from X-ray crystallography, nuclear magnetic resonance, and increasingly, cryo-electron microscopy, have provided us invaluable information on the mechanisms of transporter function, while comparative genomic approaches for the analysis of membrane transport systems have provided insights into how microbes adapt to their environment. The observation that organisms with similar lifestyles and/or ecological niches display similar transporter profiles despite their phylogenetic and taxonomic differences strongly suggest the influence of the ambient environment on an organism's complement of membrane transport genes.

Participation of a polymeric media in light-emitting processes of composite nano-CdS/polyvinyl alcohol is studied by probing different absorption-emission routes via adjustment of excitation wavelengths. It is shown that the polymeric constituent of the composite contributes chiefly to the photoluminescence excitation processes via absorption and excitation transfer to the embedded CdS nanoparticles while the composite emission occurs mostly within the nanoparticles.

Abstract Introduction: Triple-negative invasive breast carcinoma (TNBC) does not over-express estrogen, progesterone receptors and HER2/neu. Approximately 15% breast cancers are “triple negative” and have poor overall survival with no targeted therapeutic options. HER2 positive patients also have a poor prognosis which is markedly improved with anti-HER2 therapy. ASCO/CAP guidelines have made substantial improvement in biomarker assessment by standardizing pre-analytic, analytic and post-analytic variables. Since 2009, we have performed HER2 fluorescence in situ hybridization (FISH) analysis in addition to immunohistochemistry (IHC) on all triple negative patients in an effort to accurately assess HER2 status. Materials and Methods: Pathology database at our institute was queried for triple negative breast cancer cases 2009-2012. 175 cases were identified with ER(SP1; Ventana)< = 1%, PR(1E2; Ventana)< = 1% and HER2 IHC (HercepTest® DAKO 2009-2011; 4B5-Ventana- 2012)< = 1+. FISH for Her2 was also performed on these cases. HER2/CEP17> = 2 was classified as HER2 positive. FISH for HER2 amplification was performed using the FDA-approved HER2 DNA Probe Kit (Vysis, Inc.). The hybridization results were recorded and analyzed by BioView Duet system (Allegro Plus Automated Scanner) with HER2 application software (BV-HER2-AF). Fisher's exact test was performed to correlate HER2 amplification with clinico-pathological parameters. Results: 175 breast cancers were classified as “triple-negative” by IHC between 2009-2012. The patients’ age ranged from 25 to 93 years old (mean = 58); 109 were Caucasian (62%), 33 African American (19%), 9 Asian (5%) and 4 Hispanic (2%). The majority of the cases (152, 86%) were invasive ductal carcinoma. The rest include 9 cases of medullary carcinoma, 3 cases of metaplastic carcinoma, 3 cases of apocrine carcinoma, 2 cases of papillary carcinoma and 2 cases of invasive lobular carcinoma. 34 had regional lymph node (axillary) metastasis (19%) and 9 had distant metastasis (5%). 23 of the 175 “triple-negative” cases (13%) showed HER2 amplification by HER2 FISH analysis (HER2/CEP17> = 2). 14.9% (20/134) TNBC cases were FISH positive 2009-11 (HercepTest®, DAKO) and 7.3% (3/41) cases were FISH positive in 2012 (4B5, Ventana). 14 of the 23 patients were Caucasian (61%), 4 African American (17%), 2 Asian and 1 Hispanic. 20 of the 23 cases (87%) had invasive ductal carcinoma, of which 18 (78%) were poorly differentiated. 8 of the 23 cases (35%) had lymph node metastasis and 1 case had distant metastasis. No statistically significant correlation was observed between the HER2 gene amplification and clinicopathological factors analyzed, including race (P = 1), age (P = 1), tumor size (P = 0.6), pathologic stage (P = 0.6), lymph node status (P = 0.08), and Ki-67 index (P = 0.8 with 20% cutoff; P = 0.1 with 70% cutoff). Conclusion: In this cohort, 13% TNBC were re-classified as HER2 positive by performing HER2 FISH test in addition to immunohistochemistry analysis. Rate of FISH positivity in TNBC declined over the four year study period. No clinicopathological variables are correlated with HER2 FISH amplification. These data suggest that dual testing of HER2 should be considered routinely in TNBC for accurate classification to guide therapy. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P3-05-01.

This chapter contains sections titled: Introduction Cloning a Putative Efflux Protein into a Plasmid Expression Vector Cultivation of Host Cells Containing the Plasmid Encoding the Target Protein Detection of Histidine-Tagged Efflux Proteins in E. coli Membrane Preparations Solubilization of the Efflux Proteins with Detergents Purification of Histidine-Tagged Efflux Proteins Confirmation of Integrity and Monodispersity of the Purified Protein Crystallization Trials on Purified Proteins Conclusions and Discussion Acknowledgements References

Pseudomonas aeruginosa PA7 is a non-respiratory human isolate from Argentina that is multiresistant to antibiotics. We first sequenced gyrA, gyrB, parC, parE, ampC, ampR, and several housekeeping genes and found that PA7 is a taxonomic outlier. We report here the complete sequence of the 6,588,339 bp genome, which has only about 95% overall identity to other strains. PA7 has multiple novel genomic islands and a total of 51 occupied regions of genomic plasticity. These islands include antibiotic resistance genes, parts of transposons, prophages, and a pKLC102-related island. Several PA7 genes not present in PAO1 or PA14 are putative orthologues of other Pseudomonas spp. and Ralstonia spp. genes. PA7 appears to be closely related to the known taxonomic outlier DSM1128 (ATCC9027). PA7 lacks several virulence factors, notably the entire TTSS region corresponding to PA1690-PA1725 of PAO1. It has neither exoS nor exoU and lacks toxA, exoT, and exoY. PA7 is serotype O12 and pyoverdin type II. Preliminary proteomic studies indicate numerous differences with PAO1, some of which are probably a consequence of a frameshift mutation in the mvfR quorum sensing regulatory gene.

BACKGROUND:Haloferax volcanii is an easily culturable moderate halophile that grows on simple defined media, is readily transformable, and has a relatively stable genome. This, in combination with its biochemical and genetic tractability, has made Hfx. volcanii a key model organism, not only for the study of halophilicity, but also for archaeal biology in general. METHODOLOGY/PRINCIPAL FINDINGS:We report here the sequencing and analysis of the genome of Hfx. volcanii DS2, the type strain of this species. The genome contains a main 2.848 Mb chromosome, three smaller chromosomes pHV1, 3, 4 (85, 438, 636 kb, respectively) and the pHV2 plasmid (6.4 kb). CONCLUSIONS/SIGNIFICANCE:The completed genome sequence, presented here, provides an invaluable tool for further in vivo and in vitro studies of Hfx. volcanii.

The GacS/GacA signal transduction system is a central regulator in Pseudomonas spp., including the biological control strain P. fluorescens Pf-5, in which GacS/GacA controls the production of secondary metabolites and exoenzymes that suppress plant pathogens. A whole genome oligonucleotide microarray was developed for Pf-5 and used to assess the global transcriptomic consequences of a gacA mutation in P. fluorescens Pf-5. In cultures at the transition from exponential to stationary growth phase, GacA significantly influenced transcript levels of 635 genes, representing more than 10% of the 6147 annotated genes in the Pf-5 genome. Transcripts of genes involved in the production of hydrogen cyanide, the antibiotic pyoluteorin and the extracellular protease AprA were at a low level in the gacA mutant, whereas those functioning in siderophore production and other aspects of iron homeostasis were significantly higher in the gacA mutant than in wild-type Pf-5. Notable effects of gacA inactivation were also observed in the transcription of genes encoding components of a type VI secretion system and cytochrome c oxidase sub-units. Two novel gene clusters expressed under the control of gacA were identified from transcriptome analysis, and we propose global-regulator-based genome mining as an approach to decipher the secondary metabolome of Pseudomonas spp.

In order to enrich the phylogenetic diversity represented in the available sequenced bacterial genomes and as part of an "Assembling the Tree of Life" project, we determined the genome sequence of Thermomicrobium roseum DSM 5159. T. roseum DSM 5159 is a red-pigmented, rod-shaped, Gram-negative extreme thermophile isolated from a hot spring that possesses both an atypical cell wall composition and an unusual cell membrane that is composed entirely of long-chain 1,2-diols. Its genome is composed of two circular DNA elements, one of 2,006,217 bp (referred to as the chromosome) and one of 919,596 bp (referred to as the megaplasmid). Strikingly, though few standard housekeeping genes are found on the megaplasmid, it does encode a complete system for chemotaxis including both chemosensory components and an entire flagellar apparatus. This is the first known example of a complete flagellar system being encoded on a plasmid and suggests a straightforward means for lateral transfer of flagellum-based motility. Phylogenomic analyses support the recent rRNA-based analyses that led to T. roseum being removed from the phylum Thermomicrobia and assigned to the phylum Chloroflexi. Because T. roseum is a deep-branching member of this phylum, analysis of its genome provides insights into the evolution of the Chloroflexi. In addition, even though this species is not photosynthetic, analysis of the genome provides some insight into the origins of photosynthesis in the Chloroflexi. Metabolic pathway reconstructions and experimental studies revealed new aspects of the biology of this species. For example, we present evidence that T. roseum oxidizes CO aerobically, making it the first thermophile known to do so. In addition, we propose that glycosylation of its carotenoids plays a crucial role in the adaptation of the cell membrane to this bacterium's thermophilic lifestyle. Analyses of published metagenomic sequences from two hot springs similar to the one from which this strain was isolated, show that close relatives of T. roseum DSM 5159 are present but have some key differences from the strain sequenced.

ABSTRACT The complete genomes of three strains from the phylum Acidobacteria were compared. Phylogenetic analysis placed them as a unique phylum. They share genomic traits with members of the Proteobacteria, the Cyanobacteria, and the Fungi. The three strains appear to be versatile heterotrophs. Genomic and culture traits indicate the use of carbon sources that span simple sugars to more complex substrates such as hemicellulose, cellulose, and chitin. The genomes encode low-specificity major facilitator superfamily transporters and high-affinity ABC transporters for sugars, suggesting that they are best suited to low-nutrient conditions. They appear capable of nitrate and nitrite reduction but not N2 fixation or denitrification. The genomes contained numerous genes that encode siderophore receptors, but no evidence of siderophore production was found, suggesting that they may obtain iron via interaction with other microorganisms. The presence of cellulose synthesis genes and a large class of novel high-molecular-weight excreted proteins suggests potential traits for desiccation resistance, biofilm formation, and/or contribution to soil structure. Polyketide synthase and macrolide glycosylation genes suggest the production of novel antimicrobial compounds. Genes that encode a variety of novel proteins were also identified. The abundance of acidobacteria in soils worldwide and the breadth of potential carbon use by the sequenced strains suggest significant and previously unrecognized contributions to the terrestrial carbon cycle. Combining our genomic evidence with available culture traits, we postulate that cells of these isolates are long-lived, divide slowly, exhibit slow metabolic rates under low-nutrient conditions, and are well equipped to tolerate fluctuations in soil hydration.

The genome of Phytophthora infestans, the pathogen that triggered the Irish potato famine in the nineteenth century, has been sequenced. It remains a devastating pathogen, with late blight destroying crops worth billions of dollars each year. Blight is difficult to control, in part because it adapts so quickly to genetically resistant potato strains. Comparison with two other Phytophthora genomes shows rapid turnover and extensive expansion of specific families of secreted disease effector proteins, including many genes induced during infection that have activities thought to alter host physiology. These fast evolving effector genes are found in highly dynamic and expanded regions of the genome, a factor that may contribute to its rapid adaptability to host plants. The P. infestans genome is the biggest so far sequenced, at about 240 megabases, with an extremely high repeat content of close to 75%. It is a model organism for the oomycetes, a distinct lineage of fungus-like eukaryotes related to organisms such as brown algae and diatoms. Phytophthora infestans is a fungus-like eukaryote and the most destructive pathogen of potato, with current annual worldwide potato crop losses due to late blight estimated at $6.7 billion. Here, the sequence of the P. infestans genome is reported. Comparison with two other Phytophthora genomes showed rapid turnover and extensive expansion of certain secreted disease effector proteins, probably explaining the rapid adaptability of the pathogen to host plants. Phytophthora infestans is the most destructive pathogen of potato and a model organism for the oomycetes, a distinct lineage of fungus-like eukaryotes that are related to organisms such as brown algae and diatoms. As the agent of the Irish potato famine in the mid-nineteenth century, P. infestans has had a tremendous effect on human history, resulting in famine and population displacement1. To this day, it affects world agriculture by causing the most destructive disease of potato, the fourth largest food crop and a critical alternative to the major cereal crops for feeding the world’s population1. Current annual worldwide potato crop losses due to late blight are conservatively estimated at $6.7 billion2. Management of this devastating pathogen is challenged by its remarkable speed of adaptation to control strategies such as genetically resistant cultivars3,4. Here we report the sequence of the P. infestans genome, which at ∼240 megabases (Mb) is by far the largest and most complex genome sequenced so far in the chromalveolates. Its expansion results from a proliferation of repetitive DNA accounting for ∼74% of the genome. Comparison with two other Phytophthora genomes showed rapid turnover and extensive expansion of specific families of secreted disease effector proteins, including many genes that are induced during infection or are predicted to have activities that alter host physiology. These fast-evolving effector genes are localized to highly dynamic and expanded regions of the P. infestans genome. This probably plays a crucial part in the rapid adaptability of the pathogen to host plants and underpins its evolutionary potential.

The identification and annotation of protein-coding genes is one of the primary goals of whole-genome sequencing projects, and the accuracy of predicting the primary protein products of gene expression is vital to the interpretation of the available data and the design of downstream functional applications. Nevertheless, the comprehensive annotation of eukaryotic genomes remains a considerable challenge. Many genomes submitted to public databases, including those of major model organisms, contain significant numbers of wrong and incomplete gene predictions. We present a community-based reannotation of the Aspergillus nidulans genome with the primary goal of increasing the number and quality of protein functional assignments through the careful review of experts in the field of fungal biology.

Brucella ovis is a veterinary pathogen associated with epididymitis in sheep. Despite its genetic similarity to the zoonotic pathogens B. abortus, B. melitensis and B. suis, B. ovis does not cause zoonotic disease. Genomic analysis of the type strain ATCC25840 revealed a high percentage of pseudogenes and increased numbers of transposable elements compared to the zoonotic Brucella species, suggesting that genome degradation has occurred concomitant with narrowing of the host range of B. ovis. The absence of genomic island 2, encoding functions required for lipopolysaccharide biosynthesis, as well as inactivation of genes encoding urease, nutrient uptake and utilization, and outer membrane proteins may be factors contributing to the avirulence of B. ovis for humans. A 26.5 kb region of B. ovis ATCC25840 Chromosome II was absent from all the sequenced human pathogenic Brucella genomes, but was present in all of 17 B. ovis isolates tested and in three B. ceti isolates, suggesting that this DNA region may be of use for differentiating B. ovis from other Brucella spp. This is the first genomic analysis of a non-zoonotic Brucella species. The results suggest that inactivation of genes involved in nutrient acquisition and utilization, cell envelope structure and urease may have played a role in narrowing of the tissue tropism and host range of B. ovis.

The deluge of data generated by genome sequencing has led to an increasing reliance on bioinformatic predictions, since the traditional experimental approach of characterizing gene function one at a time cannot possibly keep pace with the sequence-based discovery of novel genes. We have utilized Biolog phenotype MicroArrays to identify phenotypes of gene knockout mutants in the opportunistic pathogen and versatile soil bacterium Pseudomonas aeruginosa in a relatively high-throughput fashion. Seventy-eight P. aeruginosa mutants defective in predicted sugar and amino acid membrane transporter genes were screened and clear phenotypes were identified for 27 of these. In all cases, these phenotypes were confirmed by independent growth assays on minimal media. Using qRT-PCR, we demonstrate that the expression levels of 11 of these transporter genes were induced from 4- to 90-fold by their substrates identified via phenotype analysis. Overall, the experimental data showed the bioinformatic predictions to be largely correct in 22 out of 27 cases, and led to the identification of novel transporter genes and a potentially new histamine catabolic pathway. Thus, rapid phenotype identification assays are an invaluable tool for confirming and extending bioinformatic predictions.

The human malaria parasite Plasmodium vivax is responsible for 25–40% of the ∼515 million annual cases of malaria worldwide. Although seldom fatal, the parasite elicits severe and incapacitating clinical symptoms and often causes relapses months after a primary infection has cleared. Despite its importance as a major human pathogen, P. vivax is little studied because it cannot be propagated continuously in the laboratory except in non-human primates. We sequenced the genome of P. vivax to shed light on its distinctive biological features, and as a means to drive development of new drugs and vaccines. Here we describe the synteny and isochore structure of P. vivax chromosomes, and show that the parasite resembles other malaria parasites in gene content and metabolic potential, but possesses novel gene families and potential alternative invasion pathways not recognized previously. Completion of the P. vivax genome provides the scientific community with a valuable resource that can be used to advance investigation into this neglected species. Four distinct Plasmodium species are known to regularly infect humans: Plasmodium falciparum, P. vivax, P. malariae and P. ovale. The genome sequence of P. falciparum, the cause of the most severe type of human malaria, was completed in 2002 at the same time as the mosquito vector, Anopheles gambiae. In this week's Nature, which focuses on the malaria parasite, two further malaria genome sequences are described. First that of P. vivax, which contributes significant numbers to malaria incidence in humans, though in contrast to P. falciparum, the resulting disease is usually not fatal. The genome of this rather neglected species is presented together with a comparative analysis with the genomes of other Plasmodium species. Second, we publish the genome sequence of Plasmodium knowlesi. For long regarded as a monkey malaria parasite, it is increasingly becoming recognized as the fifth human-infecting Plasmodium species. In particular, it is prevalent in South East Asia where it is often misdiagnosed as another human malaria parasite P. malariae. As a model organism P. knowlesi stands out: not only is it a primate system, useful for work on vaccines, but it can be cultured in vitro and subjected to efficient transfection and gene knockouts. In a Review Article, Elizabeth Winzeler considers the progress made towards using the genome sequence to understand basic malaria parasite biology, and in particular the work on developing rational therapeutic approaches to combat P. falciparum infections. See also the Editorial. For a comprehensive collection of resources visit Nature's past malaria specials: Malaria killer blow ; Outlook on malaria ; Malaria web focus ; Malaria Insight ; Nature Medicine focus on malaria ; Focus on malaria