A sensitive, support-vector-machine method for the detection of horizontal gene transfers in viral, archaeal and bacterial genomes

In earlier work, we introduced and discussed a generalized computational framework for identifying horizontal transfers. This framework relied on a gene's nucleotide composition, obviated the need for knowledge of codon boundaries and database searches, and was shown to perform very well across a wide range of archaeal and bacterial genomes when compared with previously published approaches, such as Codon Adaptation Index and C + G content. Nonetheless, two considerations remained outstanding: we wanted to further increase the sensitivity of detecting horizontal transfers and also to be able to apply the method to increasingly smaller genomes. In the discussion that follows, we present such a method, Wn-SVM, and show that it exhibits a very significant improvement in sensitivity compared with earlier approaches. Wn-SVM uses a one-class support-vector machine and can learn using rather small training sets. This property makes Wn-SVM particularly suitable for studying small-size genomes, similar to those of viruses, as well as the typically larger archaeal and bacterial genomes. We show experimentally that the new method results in a superior performance across a wide range of organisms and that it improves even upon our own earlier method by an average of 10% across all examined genomes. As a small-genome case study, we analyze the genome of the human cytomegalovirus and demonstrate that Wn-SVM correctly identifies regions that are known to be conserved and prototypical of all beta-herpesvirinae, regions that are known to have been acquired horizontally from the human host and, finally, regions that had not up to now been suspected to be horizontally transferred. Atypical region predictions for many eukaryotic viruses, including the alpha-, beta- and gamma-herpesvirinae, and 123 archaeal and bacterial genomes, have been made available online at http://cbcsrv.watson.ibm.com/HGT_SVM/.     

A complementation method for functional analysis of mammalian genes

Our progress in understanding mammalian gene function has lagged behind that of gene identification. New methods for mammalian gene functional analysis are needed to accelerate the process. In yeast, the powerful genetic shuffle system allows deletion of any chromosomal gene by homologous recombination and episomal expression of a mutant allele in the same cell. Here, we report a method for mammalian cells, which employs a helper-dependent adenoviral (HD-Ad) vector to synthesize small hairpin (sh) RNAs to knock-down the expression of an endogenous gene by targeting untranslated regions (UTRs). The vector simultaneously expresses an exogenous version of the same gene (wild-type or mutant allele) lacking the UTRs for functional analysis. We demonstrated the utility of the method by using PRPF3, which encodes the human RNA splicing factor Hprp3p. Recently, missense mutations in PRPF3 were found to cause autosomal-dominant Retinitis Pigmentosa, a form of genetic eye diseases affecting the retina. We knocked-down endogenous PRPF3 in multiple cell lines and rescued the phenotype (cell death) with exogenous PRPF3 cDNA, thereby creating a genetic complementation method. Because Ad vectors can efficiently transduce a wide variety of cell types, and many tissues in vivo, this method could have a wide application for gene function studies.     

A simple method for predicting the functional differentiation of duplicate genes and its application to MIKC-type MADS-box genes

A simple statistical method for predicting the functional differentiation of duplicate genes was developed. This method is based on the premise that the extent of functional differentiation between duplicate genes is reflected in the difference in evolutionary rate because the functional change of genes is often caused by relaxation or intensification of functional constraints. With this idea in mind, we developed a window analysis of protein sequences to identify the protein regions in which the significant rate difference exists. We applied this method to MIKC-type MADS-box proteins that control flower development in plants. We examined 23 pairs of sequences of floral MADS-box proteins from petunia and found that the rate differences for 14 pairs are significant. The significant rate differences were observed mostly in the K domain, which is important for dimerization between MADS-box proteins. These results indicate that our statistical method may be useful for predicting protein regions that are likely to be functionally differentiated. These regions may be chosen for further experimental studies.     

ASRDb: A comprehensive resource for archaeal stress response genes

An organism''s survival strategy under the constantly changing environment depends on its ability to sense and respond to changes in its environment. Archaea, being capable to grow under various extreme environmental conditions, provide valuable model for exploring how single-celled organisms respond to environmental stresses. However, no such approach has ever been made to make an integrated classification of various archaeal stress responses.Archaeal Stress Response Database (ASRDb) is a web accessible (http://121.241.218.70/ASRDb) database that represents the first online available resource providing a comprehensive overview of stress response genes of 66 archaeal genomes. This database currently contains almost 6000 stress specific genes of 66 archaeal genomes. All the stress specific genes are grouped into 17 different stress categories. A user-friendly interface has been designed to examine data using query tools. This database provides an efficient search engine for random and advanced database search operations. We have incorporated BLAST search options to the resulting sequences retrieved from database search operations. A site map page representing the schematic diagram will enable user to understand the logic behind the construction of the database. We have also provided a very rich and informative help page to make user familiar with the database. We sincerely believe that ASRDb will be of particular interest to the life science community and facilitates the biologists to unravel the role of stress specific genes in the adaptation of microorganisms under various extreme environmental conditions.     

Adaptations of archaeal and bacterial membranes to variations in temperature,pH and pressure

The cytoplasmic membrane of a prokaryotic cell consists of a lipid bilayer or a monolayer that shields the cellular content from the environment. In addition, the membrane contains proteins that are responsible for transport of proteins and metabolites as well as for signalling and energy transduction. Maintenance of the functionality of the membrane during changing environmental conditions relies on the cell’s potential to rapidly adjust the lipid composition of its membrane. Despite the fundamental chemical differences between bacterial ester lipids and archaeal ether lipids, both types are functional under a wide range of environmental conditions. We here provide an overview of archaeal and bacterial strategies of changing the lipid compositions of their membranes. Some molecular adjustments are unique for archaea or bacteria, whereas others are shared between the two domains. Strikingly, shared adjustments were predominantly observed near the growth boundaries of bacteria. Here, we demonstrate that the presence of membrane spanning ether-lipids and methyl branches shows a striking relationship with the growth boundaries of archaea and bacteria.

     

Pyrosequencing-derived bacterial, archaeal, and fungal diversity of spacecraft hardware destined for Mars

Spacecraft hardware and assembly cleanroom surfaces (233 m(2) in total) were sampled, total genomic DNA was extracted, hypervariable regions of the 16S rRNA gene (bacteria and archaea) and ribosomal internal transcribed spacer (ITS) region (fungi) were subjected to 454 tag-encoded pyrosequencing PCR amplification, and 203,852 resulting high-quality sequences were analyzed. Bioinformatic analyses revealed correlations between operational taxonomic unit (OTU) abundance and certain sample characteristics, such as source (cleanroom floor, ground support equipment [GSE], or spacecraft hardware), cleaning regimen applied, and location about the facility or spacecraft. National Aeronautics and Space Administration (NASA) cleanroom floor and GSE surfaces gave rise to a larger number of diverse bacterial communities (619 OTU; 20 m(2)) than colocated spacecraft hardware (187 OTU; 162 m(2)). In contrast to the results of bacterial pyrosequencing, where at least some sequences were generated from each of the 31 sample sets examined, only 13 and 18 of these sample sets gave rise to archaeal and fungal sequences, respectively. As was the case for bacteria, the abundance of fungal OTU in the GSE surface samples dramatically diminished (9× less) once cleaning protocols had been applied. The presence of OTU representative of actinobacteria, deinococci, acidobacteria, firmicutes, and proteobacteria on spacecraft surfaces suggests that certain bacterial lineages persist even following rigorous quality control and cleaning practices. The majority of bacterial OTU observed as being recurrent belonged to actinobacteria and alphaproteobacteria, supporting the hypothesis that the measures of cleanliness exerted in spacecraft assembly cleanrooms (SAC) inadvertently select for the organisms which are the most fit to survive long journeys in space.     

A genetically engineered protein domain binding to bacterial murein, archaeal pseudomurein, and fungal chitin cell wall material

The major murein and pseudomurein cell wall-binding domains, i.e., the Lysin Motif (LysM) (Pfam PF01476) and pseudomurein cell wall-binding (PMB) (Pfam PF09373) motif, respectively, were genetically fused. The fusion protein is capable of binding to both murein- and pseudomurein-containing cell walls. In addition, it also binds to chitin, the major polymer of fungal cell walls. Binding is influenced by pH and occurs at a pH close to the pI of the binding protein. Functional studies on truncated versions of the fusion protein revealed that murein and chitin binding is provided by the LysM domain, while binding to pseudomurein is achieved through the PMB domain.     

Differential recovery of bacterial and archaeal 16S rRNA genes from ruminal digesta in response to glycerol as cryoprotectant

Bacteria and archaea in frozen (− 20 °C) ruminal digesta were analysed by qPCR and cloning/sequencing of 16S rRNA genes. Samples frozen with and without glycerol as cryoprotectant indicated a major loss of Bacteroidetes in unprotected samples, resulting in higher proportions of Firmicutes. Archaeal numbers and diversity were unaffected.     

Marine bacterial,archaeal and protistan association networks reveal ecological linkages

Microbes have central roles in ocean food webs and global biogeochemical processes, yet specific ecological relationships among these taxa are largely unknown. This is in part due to the dilute, microscopic nature of the planktonic microbial community, which prevents direct observation of their interactions. Here, we use a holistic (that is, microbial system-wide) approach to investigate time-dependent variations among taxa from all three domains of life in a marine microbial community. We investigated the community composition of bacteria, archaea and protists through cultivation-independent methods, along with total bacterial and viral abundance, and physico-chemical observations. Samples and observations were collected monthly over 3 years at a well-described ocean time-series site of southern California. To find associations among these organisms, we calculated time-dependent rank correlations (that is, local similarity correlations) among relative abundances of bacteria, archaea, protists, total abundance of bacteria and viruses and physico-chemical parameters. We used a network generated from these statistical correlations to visualize and identify time-dependent associations among ecologically important taxa, for example, the SAR11 cluster, stramenopiles, alveolates, cyanobacteria and ammonia-oxidizing archaea. Negative correlations, perhaps suggesting competition or predation, were also common. The analysis revealed a progression of microbial communities through time, and also a group of unknown eukaryotes that were highly correlated with dinoflagellates, indicating possible symbioses or parasitism. Possible ‘keystone'' species were evident. The network has statistical features similar to previously described ecological networks, and in network parlance has non-random, small world properties (that is, highly interconnected nodes). This approach provides new insights into the natural history of microbes.     

A simple, rapid method for demonstrating bacterial flagella

We developed a simple, rapid method for demonstrating flagellation of bacteria using the fluorescent protein stain NanoOrange (Molecular Probes, Eugene, Oreg.). The NanoOrange reagent binds to hydrophobic regions of proteins, which results in substantial enhancement of fluorescence. Unbound reagent is essentially nonfluorescent. NanoOrange fluorescently stained bacterial cell bodies, as well as flagella and other appendages, which could be directly observed by epifluorescence microscopy. Detection of flagella was further improved by using a charge-coupled device camera for image capture and processing. The reliability of the method was tested by using 37 pure cultures of marine bacteria. Detection of flagella on the isolates by NanoOrange staining was compared to detection by transmission electron microscopy (TEM). For 36 of 37 cultures, the two methods yielded the same results. In one case, flagella were detected by TEM but not by NanoOrange, although the difference may be attributable to differences between the culture preparations. NanoOrange staining is rapid (10 to 15 min) and does not require fixation or dehydration, so live samples can be stained. Since NanoOrange is a general protein stain and works directly in seawater, it may also prove to be useful for staining other proteinaceous material that is of interest to aquatic microbial ecologists.     

A sensitive method for computing GO-based functional similarities among genes with 'shallow annotation'

Methods for computing similarities among genes have attracted increasing attention for their applications in gene clustering, gene expression data analysis, protein interaction prediction and evaluation. To address the need for automatically computing functional similarities of genes, an important class of methods that computes functional similarities by comparing Gene Ontology (GO) annotations of genes has been developed. However, all of the currently available methods have some drawbacks; for example, they either ignore the specificity of the GO terms or do not consider the information contained within the GO structure. As a result, the existing methods perform weakly when the genes are annotated with 'shallow annotations'. Here, we propose a new method to compute functional similarities among genes based on their GO annotations and compare it with the widely-used G-SESAME method. The results show that the new method reliably distinguishes functional similarities among genes and demonstrate that the method is especially sensitive to genes with 'shallow annotations'. Moreover, our method has high correlations with sequence and EC similarities.     

A generic method for assignment of reliability scores applied to solvent accessibility predictions

Background  

Estimation of the reliability of specific real value predictions is nontrivial and the efficacy of this is often questionable. It is important to know if you can trust a given prediction and therefore the best methods associate a prediction with a reliability score or index. For discrete qualitative predictions, the reliability is conventionally estimated as the difference between output scores of selected classes. Such an approach is not feasible for methods that predict a biological feature as a single real value rather than a classification. As a solution to this challenge, we have implemented a method that predicts the relative surface accessibility of an amino acid and simultaneously predicts the reliability for each prediction, in the form of a Z-score.     

Expression of bacterial hemoglobin genes to improve astaxanthin production in a methanotrophic bacterium Methylomonas sp

Astaxanthin has been widely used as a feed supplement in poultry and aquaculture industries. One challenge for astaxanthin production in bacteria is the low percentage of astaxanthin in the total carotenoids. An obligate methanotrophic bacterium Methylomonas sp. 16a was engineered to produce astaxanthin. Astaxanthin production appeared to be dramatically affected by oxygen availability. We examined whether astaxanthin production in Methylomonas could be improved by metabolic engineering through expression of bacterial hemoglobins. Three hemoglobin genes were identified in the genome of Methylomonas sp. 16a. Two of them, thbN1 and thbN2, belong to the family of group I truncated hemoglobins. The third one, thbO, belongs to the group II truncated hemoglobins. Heterologous expression of the truncated hemoglobins in Escherichia coli improved cell growth under microaerobic conditions by increasing final cell densities. Co-expression of the hemoglobin genes along with the crtWZ genes encoding astaxanthin synthesis enzymes in Methylomonas showed higher astaxanthin production than expression of the crtWZ genes alone on multicopy plasmids. The hemoglobins likely improved the activity of the oxygen-requiring CrtWZ enzymes for astaxanthin conversion. A plasmid-free production strain was constructed by integrating the thbN1–crtWZ cassette into the chromosome of an astaxanthin-producing Methylomonas strain. It showed higher astaxanthin production than the parent strain.     

A hidden reservoir of integrative elements is the major source of recently acquired foreign genes and ORFans in archaeal and bacterial genomes

Background  

Archaeal and bacterial genomes contain a number of genes of foreign origin that arose from recent horizontal gene transfer, but the role of integrative elements (IEs), such as viruses, plasmids, and transposable elements, in this process has not been extensively quantified. Moreover, it is not known whether IEs play an important role in the origin of ORFans (open reading frames without matches in current sequence databases), whose proportion remains stable despite the growing number of complete sequenced genomes.     

A method for numbering binary rooted and unrooted phylogenies and their hypothetical taxonomic units

A set of 2n−2 relations (edges) and a set ofn−1 hypothetical taxonomic units (HTUs) derive from the estimation of a binary phylogeny of a set ofn operational taxonomic units (OTUs). We propose an easy way for numbering thesen−1 hypothetical taxonomic units, as well as for then−2 interior points of an unrooted binary phylogeny. We also present an alternative method to the one proposed by Rohlf (Bull. math. Biol. 45, 33–40, 1983) for numbering the π _i=1 ⁿ (2i−3) possible rooted binary phylogenies and the π _i=1 ⁿ⁻¹ (2i−3) possible unrooted binary phylogenies conerning a set ofn operational taxonomic units. An illustrative example of the method is presented. It is hoped that some studies in phylogenetics will become more accessible, from the viewpoint of computational economy, by the use of this method.     

A rapid bioluminescence method for quantifying bacterial adhesion to polystyrene

Bioluminescence ATP analysis has been used to assess bacterial adhesion with hydrophobic polystyrene tubes as the attachment surface. The assay was performed at 37 degrees C and pH 6.8 with a 10 min incubation period. A variation of more than 200-fold was observed in the adherence capacity of 34 urinary isolates of Escherichia coli, and organisms could be classified as strongly or weakly adherent. All strains capable of strong adhesion possessed both type 1 fimbriae and flagella, and maximum adhesion was expressed during the exponential growth phase. Attachment was in all cases virtually eliminated by addition of 2.5% (w/v) D-mannose to the incubation buffer. Conversely, strains which were deficient in type 1 fimbriae or flagella, or both, were weakly adherent during all phases of growth. There was no correlation between adherence of E. coli to polystyrene and adherence to buccal or uroepithelial cells, but there was a significant association with adherence to uromucoid (P less than 0.002).     

A rapid, simple method for staining bacterial flagella.

A simple modification of Gray's flagellar staining procedure is described. It can be used on air-dried smears or directly on wet mounts of motile bacteria. The stained bacterial flagella can be observed with phase-contrast or bright-field optics. No rigorous cleaning of slides, counterstains, or any washing procedures are required with the staining method, making it very suitable for routine examinations.     

A mutation spectra database for bacterial and mammalian genes.

Each mutation spectrum in this database is a dataset of changes in DNA base sequence in mutations induced in a gene by a particular mutagen (including spontaneous processes) under defined conditions. There are 240 datasets with 24 500 mutants in nine bacterial genes, two phage genes, five mammalian genes and one yeast gene. The database is available on the Web at http://info.med.yale.edu/mutbase/ . The data tables can be viewed on the Web and downloaded in text form for local use. The data are also available in dBASE III, a format which can be utilized by essentially any desktop computer database program or spreadsheet, and makes feasible analyses of a large number of mutants. Researchers are invited to submit additional data. A data entry program, MUTSIN, diagrams each mutation on the computer screen as the data are entered and alerts the user to any discrepancies between the entry and the gene sequence.     

Application of quantitative real-time PCR for enumeration of total bacterial,archaeal, and yeast populations in kimchi

Kimchi is a Korean traditional fermented food made of brined vegetables, with a variety of spices. Various microorganisms are associated with the kimchi fermentation process. This study was undertaken in order to apply quantitative real-time PCR targeting the 16S and 26S rRNA genes for the investigation of dynamics of bacterial, archaeal, and yeast communities during fermentation of various types of kimchi. Although the total bacterial and archaeal rRNA gene copy numbers increased during kimchi fermentation, the number of yeasts was not significantly altered. In 1 ng of bulk DNA, the mean number of rRNA gene copies for all strains of bacteria was 5.45×10⁶ which was 360 and 50 times greater than those for archaea and yeast, respectively. The total gene copy number for each group of microorganisms differed among the different types of kimchi, although the relative ratios among them were similar. The common dominance of bacteria in the whole microbial communities of various types of kimchi suggests that bacteria play a principal role in the kimchi fermentation process.