Structure-based functional inference in structural genomics

The dramatically increasing number of new protein sequences arising from genomics 4 proteomics requires the need for methods to rapidly and reliably infer the molecular and cellular functions of these proteins. One such approach, structural genomics, aims to delineate the total repertoire of protein folds in nature, thereby providing three-dimensional folding patterns for all proteins and to infer molecular functions of the proteins based on the combined information of structures and sequences. The goal of obtaining protein structures on a genomic scale has motivated the development of high throughput technologies and protocols for macromolecular structure determination that have begun to produce structures at a greater rate than previously possible. These new structures have revealed many unexpected functional inferences and evolutionary relationships that were hidden at the sequence level. Here, we present samples of structures determined at Berkeley Structural Genomics Center and collaborators laboratories to illustrate how structural information provides and complements sequence information to deduce the functional inferences of proteins with unknown molecular functions.Two of the major premises of structural genomics are to discover a complete repertoire of protein folds in nature and to find molecular functions of the proteins whose functions are not predicted from sequence comparison alone. To achieve these objectives on a genomic scale, new methods, protocols, and technologies need to be developed by multi-institutional collaborations worldwide. As part of this effort, the Protein Structure Initiative has been launched in the United States (PSI; www.nigms.nih.gov/funding/psi.html). Although infrastructure building and technology development are still the main focus of structural genomics programs [1–6], a considerable number of protein structures have already been produced, some of them coming directly out of semi-automated structure determination pipelines [6–10]. The Berkeley Structural Genomics Center (BSGC) has focused on the proteins of Mycoplasma or their homologues from other organisms as its structural genomics targets because of the minimal genome size of the Mycoplasmas as well as their relevance to human and animal pathogenicity (http://www.strgen.org). Here we present several protein examples encompassing a spectrum of functional inferences obtainable from their three-dimensional structures in five situations, where the inferences are new and testable, and are not predictable from protein sequence information alone.     

Antibacterial activities of peptides designed as hybrids of antimicrobial peptides

Hybrid peptides (HP-MA, HP-ME), each of 20 residues and incorporating 2–9 residues of Helicobacter pylori ribosomal protein L1 (HP) and 1–12 residues of magainin 2 and melittin, were designed. The antibiotic activities of these peptides were evaluated using bacterial, tumor and human erythrocyte cells. HP-MA had a stronger antibacterial activity against Gram-positive bacteria and Gram-negative bacteria than HP (2-20) and magainin 2, and HP-ME was similar to melittin. None of the hybrids had anti-tumor or hemolytic activity. These peptides were further investigated using an artificial liposomal vesicle and 1,6-diphenyl-1,3,5-hexatriene as a membrane probe, and confirmed to have similar antibacterial activities. The antibacterial effect of these hybrids is probably caused by their ability to damage the bacterial plasma membrane. Additional circular dichroism spectra suggested that the -helical structure of these peptides plays an important role in their antibiotic effect but that -helical property is less connected with the enhanced antibiotic activity.     

A reevaluation of the amino acid sequence of human follitropin beta-subunit

A collaborative study from two laboratories has been undertaken to re-evaluate the human follitropin -subunit sequence (hFSH), since areas of uncertainty remain in the wake of two earlier reports. The first report was by Shome and Parlow (1974). The second, by Saxena and Rathnam (1976), proposed revisions for sequence not definitively placed in the first study, as well as some differences in other placements. We have re-examined the sequence of the hFSH with more recent methodology. This has led to revision of certain areas of the sequence and resolution of differences between the two earlier proposals. Specifically, an-Ile-Ser- is established at 21–22, Asp at 41, Arg at 44, Lys at 46, and Glu at 111. These were areas of disagreement in the earlier proposals. A definitive placement of the residues around tryptophan-27 has now been obtained by three laboratories. C-terminal heterogeneity was observed with subunits ending at residue 107, 109, or 111. N-terminal heterogeneity has been observed in all preparations examined to date. A significant population of molecules with a proteolytic nick between residues 38–39 is noted. This is very likely an artifact of the collection and processing. The preparations examined in the present studies showed no evidence of residues 112–118 proposed by Saxena and Rathnam.     

Molecular strategies for overcoming antibiotic resistance in bacteria

Overuse of antibiotics in humans and livestock has led to the rapid evolution of bacteria that are resistant to multiple drugs such that even vancomycin, the drug of last resort, is no longer effective against some strains. Apart from the discovery and exploitation of the natural peptide antimicrobial agents that form part of the innate immune systems of plants and animals, there have been few new antibiotics developed in recent years. Here we review strategies designed to exploit recent advances in molecular biology, including recombinant DNA technology, molecular modelling and genomics to develop new antibacterial agents that overcome antibiotic resistance.     

Small and lethal: searching for new antibacterial compounds with novel modes of action.

The discovery of drugs used to combat infectious diseases is in the process of constant change to address the ever-worsening problem of antibiotic resistance in pathogens and a lack of recent success in discovering new antibacterial drugs. In the past 2 decades, research in both academia and industry has made use of molecular biology, genetics, and comparative genomics, which has led to the development of key technologies for the discovery of novel antibacterial agents. Genome-scale efforts have led to the identification of numerous molecular targets. Chemical diversity from synthetic combinatorial libraries and natural products is being used to screen for new molecules. A wide variety of approaches are being used in the search for novel antibiotics, and these can be categorized as being either biochemically focused or cell based. The over-riding goal of all methods in use today is to discover new chemical matter with novel mechanisms of action against drug-resistant pathogens.     

Streptococcus pneumoniae as a genomics platform for broad-spectrum antibiotic discovery

Streptococcus pneumoniae is a useful tool for the discovery of broad-spectrum antibiotics because of its genetic malleability and importance as a pathogen. Recent publications of complete chromosomal DNA sequences for S. pneumoniae facilitate rapid and effective use of genomics-based technology to identify essential genes encoding new targets for antibacterial drugs. These methods include computational comparative genomics, gene disruption studies to determine essentiality or identify essential genes, and gene expression analysis using microarrays and gel-based proteomics. We review how genomics has transformed the use of the pneumococcus for the pursuit of new antibiotics, and made it the best species for the identification and validation of new antibiotic targets.     

Can ecology help genomics: the genome as ecosystem?

Ecologists study the rules that govern processes influencing the distribution and abundance of organisms, particularly with respect to the interactions of organisms with their biotic and abiotic environments. Over the past decades, using a combination of sophisticated mathematical models and rigorous experiments, ecologists have made considerable progress in understanding the complex web of interactions that constitute an ecosystem. The field of genomics runs on a path parallel to ecology. Like ecology, genomicists seek to understand how each gene in the genome interacts with every other gene and how each gene interacts with multiple, environmental factors. Gene networks connect genes as complex as the webs that connect the species in an ecosystem. In fact, genes exist in an ecosystem we call the genome. The genome as ecosystem is more than a metaphor – it serves as the conceptual foundation for an interdisciplinary approach to the study of complex systems characteristic of both genomics and ecology. Through the infusion of genomics into ecology and ecology into genomics both fields will gain fresh insight into the outstanding major questions of their disciplines.     

Lost in the world of functional genomics, systems biology, and translational research: is there life after the Milstein award?

We have always wanted to save the world from the scourges of virus infection by developing better drugs and vaccines. But fully understanding the intricacies of virus–host interactions, the first step in achieving this goal, requires the ability to view the process on a grand scale. The advent of high-throughput technologies, such as DNA microarrays and mass spectrometry, provided the first opportunities to obtain such a view. Here, we describe our efforts to use these tools to focus on the changes in cellular gene expression and protein abundance that occur in response to virus infection. By examining these changes in a comprehensive manner, we have been able to discover exciting new insights into innate immunity, interferon and cytokine signaling, and the strategies used by viruses to overcome these cellular defenses. Functional genomics may yet save the world from killer viruses.     

Antibiotic production by the immobilized cyanobacterium,Scytonema sp. TISTR 8208, in a seaweed-type photobioreactor

A photobioreactor was constructed using either anchored polyurethane foam strips (1 × 1 × 40 cm, PU-strips) fixed on a stainless-steel ring to prevent flotation, or free-floating polyurethane foam blocks (1 × 1 × 1 cm, PU-blocks) as biomass supporting materials (BSM). The cyanobacterium,Scytonema sp. TISTR 8208, which produces an antibiotic, was immobilized onto PU-strips or -blocks. The free-floating PU-blocks could immobilize only about 70% of the total cells, while the anchored PU-strips could immobilize as much as 97%. PU-strips were chosen as the BSM and we named this type of reactor, seaweed-type bioreactor (STB). Optimal physical conditions for antibiotic production were determined in the STB. Inoculum density was 0.4 g l^–1 and cells were sparged with air containing 5% CO₂ circulated at the gas flow rate of 250 ml min^–1 and illuminated at a light intensity of 200 mol photon m^–2 s^–1. The production of antibiotic could be increased 3-fold.Author for correspondence     

The effect of electrical currents and tobramycin onPseudomonas aeruginosa biofilms

The combined use of antibiotics with low levels of electrical current has been reported to be more effective in controlling biofilms (the bioelectric effect) than antibiotics alone. An electrical colonisation cell was designed to study the effect of antibiotics on biofilms formed on a dialysis membrane away from the electrode surface. To avoid the electrochemical generation of toxic products,Pseudomonas aeruginosa biofilms were formed in minimal salts medium that excluded chloride-containing compounds. Under these conditions, electrical currents of up to 20 mA cm^–2 did not prevent biofilm formation or have any detrimental effect on an established biofilm. Tobramycin alone at concentrations of 10 g ml^–1 did not affect the biofilm, but were significantly enhanced by 9 mA cm^–2. The effect of tobramycin concentrations of 25 g ml^–1 were enhanced by a 15 mA cm^–2 electrical current. In both cases higher levels of electrical current, up to 20 mA cm^–2, did not further enhance the effect of the antibiotic. The possible mechanisms of action of the bioelectric effect have been reported to involve electrophoresis, iontophoresis and electroporesis, thus overcoming the biofilm biomass and cell wall barriers. Our results suggest that other factors may also be important, such as the metabolic activity and growth rate of the bacteria. Such factors may be critical in maximising antibiotic efficacy.     

Application of the “-Omic-” technologies in phytomedicine

The proof of efficacy of phytopreparations and the determination of their mode of action are permanent challenges for an evidence-based phytotherapy. The technology platform of genomics, proteomics and metabolomics ("-omic-" technologies) are high-throughput technologies. They increase substantially the number of proteins/genes that can be detected simultaneously and have the potential to relate complex mixtures to complex effects in the form of gene/protein expression profiles. Provided that phytopreparation-specific signatures in the form of gene/protein expression profiles can be developed, these technologies will be useful for the chemical and pharmacological standardization and the proof of the toxicological potential of a plant extract. Over a long-term perspective they may economize the proof of efficacy, the determination of the mode of action of phytomedicines and allow to investigate herbal extracts without prominent active principle(s). The application of this genomics revealed already that gene expression profiles induced by single drugs and the ones induced by the combination of the same drugs can be entirely different. These results make the information of the mode of action of isolated "active principles/lead substances" of phytopreparations questionable. The application of the "-omic-" technologies may lead to a change of paradigms towards the application of complex mixtures in medicine and open the new field of phytogenomics, -proteomics and -metabolomics.     

EST-based gene discovery in pig: virtual expression patterns and comparative mapping to human

A molecular understanding of porcine reproduction is of biological interest and economic importance. Our Midwest Consortium has produced cDNA libraries containing the majority of genes expressed in major female reproductive tissues, and we have deposited into public databases 21,499 expressed sequence tag (EST) gene sequences from the 3 end of clones from these libraries. These sequences represent 10,574 different genes, based on sequence comparison among these data, and comparison with existing porcine ESTs and genes indicate as many as 4652 of these EST clusters are novel. In silico analysis identified sequences that are expressed in specific pig tissues or organs and confirmed the broad expression in pig for many genes ubiquitously expressed in human tissues. Furthermore, we have developed computer software to identify sequence similarity of these pig genes with their human counterparts, and to extract the mapping information of these human homologues from genome databases. We demonstrate the utility of this software for comparative mapping by localizing 61 genes on the porcine physical map for Chromosomes (Chrs) 5, 10, and 14. The following Accession numbers were assigned to our deposited sequences: BF701840 – BF704551, BF708383, BF708386 – BF713604, BG322266 – BG322271, BI398567 – BI405235, BQ597354 – BQ605166.     

Understanding the industrial application potential of lactic acid bacteria through genomics

Lactic acid bacteria (LAB) are a heterogeneous group of bacteria contributing to various industrial applications, ranging from food and beverage fermentation, bulk and fine chemicals production to pharmaceuticals manufacturing. Genome sequencing is booming; hitherto, 25 genomes of LAB have been published and many more are in progress. Based on genomic content of LAB, this review highlights some findings related to applications revealed by genomics and functional genomics analyses. Finally, this review summarizes mathematical modeling strategies of LAB in the context of genomics, to further our understanding of industrial related features.     

Improved artemisinin accumulation in hairy root cultures of Artemisia annua by (22S, 23S)-homobrassinolide

When (22S, 23S)-homobrassinolide (SSHB) was added at 1 g l^–1 to hairy root cultures of Artemisia annua, the production of artemisinin reached to 14 mg l^–1, an increment of 57% over the control. SSHB treatment led concomitantly to an increased biomass production of 15 g l^–1. A stimulatory activity of SSHB on nucleic acids and soluble protein content in hairy roots was also observed at the growth stage.     

Antibiotic Resistance Characteristics of Environmental Bacteria from an Oxytetracycline Production Wastewater Treatment Plant and the Receiving River

We characterized the bacterial populations in surface water receiving effluent from an oxytetracycline (OTC) production plant. Additional sampling sites included the receiving river water 5 km upstream and 20 km downstream from the discharge point. High levels of OTC were found in the wastewater (WW), and the antibiotic was still detectable in river water downstream (RWD), with undetectable levels in river water upstream (RWU). A total of 341 bacterial strains were isolated using nonselective media, with the majority being identified as Gammaproteobacteria. The MICs were determined for 10 antibiotics representing seven different classes of antibiotics, and the corresponding values were significantly higher for the WW and RWD isolates than for the RWU isolates. Almost all bacteria (97%) from the WW and RWD samples demonstrated multidrug-resistant (MDR) phenotypes, while in RWU samples, these were less frequent (28%). The WW and RWD isolates were analyzed for the presence of 23 tetracycline (tet) resistance genes. The majority of isolates (94.2% and 95.4% in WW and RWD, respectively) harbored the corresponding genes, with tet(A) being the most common (67.0%), followed by tet(W), tet(C), tet(J), tet(L), tet(D), tet(Y), and tet(K) (in the range between 21.0% and 40.6%). Class I integrons were detected in the majority of WW and RWD isolates (97.4% and 86.2%, respectively) but were not associated with the tet genes. We hypothesize that the strong selective pressure imposed by a high concentration of OTC contributes to the wide dissemination of tetracycline resistance genes and other antibiotic resistance genes, possibly through mobile genetic elements.The widespread emergence of antibiotic resistance, particularly multidrug resistance (MDR), among bacterial pathogens has become one of the most serious challenges in clinical therapy (22, 46). Some pathogens, such as MDR Klebsiella pneumoniae and Acinetobacter baumannii, are now virtually untreatable with current antibiotics (14, 30). Acquisition of resistance genes through horizontal transfer has been found to be ubiquitous in clinical pathogens (22). Environmental bacteria have been shown to be a reservoir of antibiotic resistance genes and a potential source of novel resistance genes in clinical pathogens (10, 12). Horizontal transfer of genes between bacterial strains could be facilitated by mobile genetic elements, such as plasmids, transposons, bacteriophages, integrons, insertion elements (IS), and genomic islands (13). Some elements, including class I integrons, conjugative plasmids, and transposons, are frequently linked to antibiotic resistance as they harbor rather diverse resistance genes and possibly promote the distribution of these genes in phylogenetically diverse bacteria (29). In light of the potential health risk, many studies have focused on antibiotic-resistant bacteria recovered from various ecosystems (1, 18, 45). Environments that contain antibiotic residues are particularly worrisome because antibiotics could exert selective pressure and might contribute to the appearance of resistant bacteria. Hospital sewage was once considered the major source of antibiotics in aquatic environments, followed by municipal, agricultural, and aquacultural wastewater (WW), which have also been shown to be important sources of these compounds and resistant bacteria (40). It has also been reported that treated antibiotic production wastewater contains much higher concentrations of antibiotic residues than other aquatic environments (20, 26, 27) and can serve as an important reservoir of resistant bacteria and genes (25).In the current study, we investigated resistance profiles of bacterial isolates from a unique wastewater treatment plant (WWTP) that is used solely for treating oxytetracycline (OTC), avermectin, and ivermectin production wastewater from the facility of the North China Pharmaceutical Group Corporation in Hebei Province, China. Avermectin and ivermectin are both broad-spectrum antiparasitic agents without any antibacterial or antifungal activities. The effluent of the WWTP is discharged directly into the receiving river without disinfection, and residual levels of OTC in wastewater and downstream sampling points nearly approach those in human blood after drug administration (15, 26). Thus, the characteristics of bacterial strains in heavily OTC-contaminated wastewater and surface water could be unique. In a 1-year (2002-2003) survey of antibiotic resistance in 140 clinical enterococcal isolates from a hospital in this region, the prevalence of tetracycline resistance ranged from 14.9% to 25.0% (47). This rate increased to 47.9 to 75.9% for 302 clinical enterococcal isolates from the same hospital over the following 3 years (2004-2006) (28), indicating that tetracycline resistance among human pathogens in this region is becoming more prevalent. Elucidation of the resistance characteristics of bacterial isolates from OTC wastewater and surface water might help explain the prevalence of increased resistance among human pathogens. To gain a comprehensive understanding of this relationship, we isolated bacterial strains from wastewater (WW) and river water downstream (RWD) and river water upstream (RWU) samples using nonselective culture media. Previously, it was shown that long-term administration of a single antibiotic can lead to MDR, a phenomenon that has been observed in bacteria obtained from the urinary tract, intestine, and other niches in both clinical therapy and livestock raising (11, 23, 34). Thus, the combination of long-term treatment and discharge of antibiotic production wastewater might contribute to MDR in environmental bacteria in this specific ecosystem. To test this hypothesis, we determined the resistance profiles of bacterial isolates for 10 antibiotics representing seven classes.OTC belongs to the tetracycline class of antibiotics, which includes tetracycline, chlortetracycline, doxycycline, minocycline, and glycylcyclines (6). This class of antibiotics represents broad-spectrum agents that act against a range of Gram-positive and Gram-negative bacteria by inhibiting protein synthesis. The tetracyclines have played an important role in human and veterinary medicine, and some have been used as growth promoters for livestock and aquaculture. However, resistance to tetracyclines in many commensal and pathogenic bacteria emerged soon after the widespread application of this class of antibiotic. More than 40 types of tetracycline resistance determinants have been described to date and grouped into three main classes: energy-dependent membrane-associated efflux proteins, which export tetracycline out of the cell; ribosomal protection proteins, which interact with the ribosome and disrupt the tetracycline binding site; and tetracycline inactivation enzymes (6, 37). Many studies of tetracycline resistance (tet) genes in various environmental niches have been reported. Resistance genes encoding efflux proteins and ribosomal protection proteins have been identified in phylogenetically diverse bacterial genera. Extensive reviews with detailed information about tet genes and their distribution are available (4, 6, 24, 37). Notably lacking, however, is information about tet genes in aquatic environments with high levels of antibiotic residue. Studies have shown that several tet genes are associated with mobile genetic elements (6, 37) and that the SOS response induced by antibiotics and other factors can promote horizontal dissemination of mobile genetic elements like genomic islands, as well as integron recombination, among bacterial populations (5, 17, 41). In the current study, the presence and distribution of 23 tet genes in bacterial isolates from wastewater and river water were determined by PCR. Class I integrons were also examined in the bacterial isolates to evaluate the possibility of horizontal transfer of these resistance genes and/or the promoted dissemination of mobile genetic elements in such heavily antibiotic-polluted environments. We also compared the characteristics of the recovered bacteria with those of isolates from samples from water upstream of the discharge site in the river. Our results reveal the potential environmental influence of wastewater discharge on acquired antibiotic resistance.     

Molecular cloning and expression in yeast of 2,3–oxidosqualene– triterpenoid cyclases from Arabidopsis thaliana

A vast array of triterpenes are found in living organisms in addition to lanosterol and cycloartenol, which are involved in sterol biosynthesis in non–photosynthetic and photosynthetic eukaryotes respectively. The chemical structure of these triterpenes is determined by a single step catalysed by 2,3–oxidosqualene–triterpene cyclases. The present study describes cloning and functional expression in yeast of several OS–triterpene cyclases. Three Arabidopsis thaliana cDNAs encoding proteins (ATLUP1, ATLUP2, ATPEN1) 57%, 58% and 49% identical to cycloartenol synthase from the same plant were isolated. Expression of these cDNAs in yeast showed that the recombinant proteins catalyse the synthesis of various pentacyclic triterpenes. Whereas ATLUP1 is essentially involved in the synthesis of lupeol, ATLUP2 catalyses the production of lupeol, – and –amyrin (in a 15:55:30 ratio). ATLUP2 is therefore a typical multifunctional enzyme. Under the same conditions, ATPEN1 did not lead to any product. Systematic sequencing of the Arabidopsis genome has led to genomic sequences encoding proteins identical to the above triterpene synthases. ATLUP1 and ATLUP2 are representative of a small subfamily (A) of at least five genes, whereas ATPEN1 is representative of a subfamily (B) of at least seven genes. The number of introns is characteristic of each subfamily. Whereas genes of family A possess 17 exons and 16 introns, genes of the subfamily B contain 14 exons and 13 introns. The size of each exon is remarkably conserved within each subfamily whereas that of each intron appears to be highly variable. Organization of the genes, sequences and functions of the deduced proteins are discussed in evolutionary terms.