Synthase-selected sorting approach identifies a beta-lactone synthase in a nudibranch symbiotic bacterium.

BACKGROUND: Nudibranchs comprise a group of > 6000 marine soft-bodied mollusk species known to use secondary metabolites (natural products) for chemical defense. The full diversity of these metabolites and whether symbiotic microbes are responsible for their synthesis remains unexplored. Another issue in searching for undiscovered natural products is that computational analysis of genomes of uncultured microbes can result in detection of novel biosynthetic gene clusters; however, their in vivo functionality is not guaranteed which limits further exploration of their pharmaceutical or industrial potential. To overcome these challenges, we used a fluorescent pantetheine probe, which produces a fluorescent CoA-analog employed in biosynthesis of secondary metabolites, to label and capture bacterial symbionts actively producing these compounds in the mantle of the nudibranch Doriopsilla fulva. RESULTS: We recovered the genome of Candidatus Doriopsillibacter californiensis from the Ca. Tethybacterales order, an uncultured lineage of sponge symbionts not found in nudibranchs previously. It forms part of the core skin microbiome of D. fulva and is nearly absent in its internal organs. We showed that crude extracts of D. fulva contained secondary metabolites that were consistent with the presence of a beta-lactone encoded in Ca. D. californiensis genome. Beta-lactones represent an underexplored group of secondary metabolites with pharmaceutical potential that have not been reported in nudibranchs previously. CONCLUSIONS: Altogether, this study shows how probe-based, targeted sorting approaches can capture bacterial symbionts producing secondary metabolites in vivo. Video Abstract.

A centimeter-long bacterium with DNA contained in metabolically active, membrane-bound organelles.

Cells of most bacterial species are around 2 micrometers in length, with some of the largest specimens reaching 750 micrometers. Using fluorescence, x-ray, and electron microscopy in conjunction with genome sequencing, we characterized Candidatus (Ca.) Thiomargarita magnifica, a bacterium that has an average cell length greater than 9000 micrometers and is visible to the naked eye. These cells grow orders of magnitude over theoretical limits for bacterial cell size, display unprecedented polyploidy of more than half a million copies of a very large genome, and undergo a dimorphic life cycle with asymmetric segregation of chromosomes into daughter cells. These features, along with compartmentalization of genomic material and ribosomes in translationally active organelles bound by bioenergetic membranes, indicate gain of complexity in the Thiomargarita lineage and challenge traditional concepts of bacterial cells.

Examining the Relationship Between the Testate Amoeba Hyalosphenia papilio (Arcellinida, Amoebozoa) and its Associated Intracellular Microalgae Using Molecular and Microscopic Methods.

Symbiotic relationships between heterotrophic and phototrophic partners are common in microbial eukaryotes. Among Arcellinida (Amoebozoa) several species are associated with microalgae of the genus Chlorella (Archaeplastida). So far, these symbioses were assumed to be stable and mutualistic, yet details of the interactions are limited. Here, we analyzed 22 single-cell transcriptomes and 36 partially-sequenced genomes of the Arcellinida morphospecies Hyalosphenia papilio, which contains Chlorella algae, to shed light on the amoeba-algae association. By characterizing the genetic diversity of associated Chlorella, we detected two distinct clades that can be linked to host genetic diversity, yet at the same time show a biogeographic signal across sampling sites. Fluorescence and transmission electron microscopy showed the presence of intact algae cells within the amoeba cell. Yet analysis of transcriptome data suggested that the algal nuclei are inactive, implying that instead of a stable, mutualistic relationship, the algae may be temporarily exploited for photosynthetic activity before being digested. Differences in gene expression of H. papilio and Hyalosphenia elegans demonstrated increased expression of genes related to oxidative stress. Together, our analyses increase knowledge of this host-symbiont association and reveal 1) higher diversity of associated algae than previously characterized, 2) a transient association between H. papilio and Chlorella with unclear benefits for the algae, 3) algal-induced gene expression changes in the host.

A single-cell genome perspective on studying intracellular associations in unicellular eukaryotes.

Single-cell genomics (SCG) methods provide a unique opportunity to analyse whole genome information at the resolution of an individual cell. While SCG has been extensively used to investigate bacterial and archaeal genomes, the technique has been rarely used to access the genetic makeup of uncultivated microbial eukaryotes. In this regard, the use of SCG can provide a wealth of information; not only do the methods allow exploration of the genome, they can also help elucidate the relationship between the cell and intracellular entities extant in nearly all eukaryotes. SCG enables the study of total eukaryotic cellular DNA, which in turn allows us to better understand the evolutionary history and diversity of life, and the physiological interactions that define complex organisms. This article is part of a discussion meeting issue 'Single cell ecology'.

A Putative Bacterial ABC Transporter Circumvents the Essentiality of Signal Peptidase.

UNLABELLED: The type I signal peptidase of Staphylococcus aureus, SpsB, is an attractive antibacterial target because it is essential for viability and extracellularly accessible. We synthesized compound 103, a novel arylomycin-derived inhibitor of SpsB with significant potency against various clinical S. aureus strains (MIC of ~1 µg/ml). The predominant clinical strain USA300 developed spontaneous resistance to compound 103 with high frequency, resulting from single point mutations inside or immediately upstream of cro/cI, a homolog of the lambda phage transcriptional repressor cro These cro/cI mutations led to marked (>50-fold) overexpression of three genes encoding a putative ABC transporter. Overexpression of this ABC transporter was both necessary and sufficient for resistance and, notably, circumvented the essentiality of SpsB during in vitro culture. Mutation of its predicted ATPase gene abolished resistance, suggesting a possible role for active transport; in these bacteria, resistance to compound 103 occurred with low frequency and through mutations in spsB Bacteria overexpressing the ABC transporter and lacking SpsB were capable of secreting a subset of proteins that are normally cleaved by SpsB and instead were cleaved at a site distinct from the canonical signal peptide. These bacteria secreted reduced levels of virulence-associated proteins and were unable to establish infection in mice. This study reveals the mechanism of resistance to a novel arylomycin derivative and demonstrates that the nominal essentiality of the S. aureus signal peptidase can be circumvented by the upregulation of a putative ABC transporter in vitro but not in vivo IMPORTANCE: The type I signal peptidase of Staphylococcus aureus (SpsB) enables the secretion of numerous proteins by cleavage of the signal peptide. We synthesized an SpsB inhibitor with potent activity against various clinical S. aureus strains. The predominant S. aureus strain USA300 develops resistance to this inhibitor by mutations in a novel transcriptional repressor (cro/cI), causing overexpression of a putative ABC transporter. This mechanism promotes the cleavage and secretion of various proteins independently of SpsB and compensates for the requirement of SpsB for viability in vitro However, bacteria overexpressing the ABC transporter and lacking SpsB secrete reduced levels of virulence-associated proteins and are unable to infect mice. This study describes a bacterial resistance mechanism that provides novel insights into the biology of bacterial secretion.

De Novo Guanine Biosynthesis but Not the Riboswitch-Regulated Purine Salvage Pathway Is Required for Staphylococcus aureus Infection In Vivo.

UNLABELLED: De novo guanine biosynthesis is an evolutionarily conserved pathway that creates sufficient nucleotides to support DNA replication, transcription, and translation. Bacteria can also salvage nutrients from the environment to supplement the de novo pathway, but the relative importance of either pathway during Staphylococcus aureus infection is not known. In S. aureus, genes important for both de novo and salvage pathways are regulated by a guanine riboswitch. Bacterial riboswitches have attracted attention as a novel class of antibacterial drug targets because they have high affinity for small molecules, are absent in humans, and regulate the expression of multiple genes, including those essential for cell viability. Genetic and biophysical methods confirm the existence of a bona fide guanine riboswitch upstream of an operon encoding xanthine phosphoribosyltransferase (xpt), xanthine permease (pbuX), inosine-5'-monophosphate dehydrogenase (guaB), and GMP synthetase (guaA) that represses the expression of these genes in response to guanine. We found that S. aureus guaB and guaA are also transcribed independently of riboswitch control by alternative promoter elements. Deletion of xpt-pbuX-guaB-guaA genes resulted in guanine auxotrophy, failure to grow in human serum, profound abnormalities in cell morphology, and avirulence in mouse infection models, whereas deletion of the purine salvage genes xpt-pbuX had none of these effects. Disruption of guaB or guaA recapitulates the xpt-pbuX-guaB-guaA deletion in vivo In total, the data demonstrate that targeting the guanine riboswitch alone is insufficient to treat S. aureus infections but that inhibition of guaA or guaB could have therapeutic utility. IMPORTANCE: De novo guanine biosynthesis and purine salvage genes were reported to be regulated by a guanine riboswitch in Staphylococcus aureus We demonstrate here that this is not true, because alternative promoter elements that uncouple the de novo pathway from riboswitch regulation were identified. We found that in animal models of infection, the purine salvage pathway is insufficient for S. aureus survival in the absence of de novo guanine biosynthesis. These data suggest targeting the de novo guanine biosynthesis pathway may have therapeutic utility in the treatment of S. aureus infections.

Global gene expression of methicillin-resistant Staphylococcus aureus USA300 during human and mouse infection.

Little is known about the expression of methicillin-resistant Staphylococcus aureus (MRSA) genes during infection conditions. Here, we described the transcriptome of the clinical MRSA strain USA300 derived from human cutaneous abscesses, and compared it with USA300 bacteria derived from infected kidneys in a mouse model. Remarkable similarity between the transcriptomes allowed us to identify genes encoding multiple proteases and toxins, and iron- and peptide-transporter molecules, which are upregulated in both infections and are likely important for establishment of infection. We also showed that disruption of the global transcriptional regulators agr and sae prevents in vivo upregulation of many toxins and proteases, protecting mice from lethal infection dose, and hinting at the role of these transcriptional regulators in the pathology of MRSA infection.

Analysis of the nucleotide sequence of the guinea pig cytomegalovirus (GPCMV) genome.

In this report we describe the genomic sequence of guinea pig cytomegalovirus (GPCMV) assembled from a tissue culture-derived bacterial artificial chromosome clone, plasmid clones of viral restriction fragments, and direct PCR sequencing of viral DNA. The GPCMV genome is 232,678 bp, excluding the terminal repeats, and has a GC content of 55%. A total of 105 open reading frames (ORFs) of > 100 amino acids with sequence and/or positional homology to other CMV ORFs were annotated. Positional and sequence homologs of human cytomegalovirus open reading frames UL23 through UL122 were identified. Homology with other cytomegaloviruses was most prominent in the central approximately 60% of the genome, with divergence of sequence and lack of conserved homologs at the respective genomic termini. Of interest, the GPCMV genome was found in many cases to bear stronger phylogenetic similarity to primate CMVs than to rodent CMVs. The sequence of GPCMV should facilitate vaccine and pathogenesis studies in this model of congenital CMV infection.

The Rosetta stone method.

Analysis of amino acid sequences from different organisms often reveals cases in which two or more proteins encoded for separately in a genome also appear as fusions, either in the same genome or that of some other organism. Such fusion proteins, termed Rosetta stone sequences, help link disparate proteins together, and suggest the likelihood of functional interactions between the linked entities, describing local and global relationships within the proteome. These relationships help us understand the role of proteins within the context of their associations, and facilitate assignment of putative functions to uncharacterized proteins based on their linkages with proteins of known function.

Phylogenetic profiling.

Phylogenetic profiles describe the presence or absence of a protein in a set of reference genomes. Similarity between profiles is an indicator of functional coupling between gene products: the greater the similarity, the greater the likelihood of proteins sharing membership in the same pathway or cellular system. By virtue of this property, uncharacterized proteins can be assigned putative functions, based on the similarity of their profiles with those of known proteins. Profile comparisons, when extended to the entire genome, have the power to reveal functional linkages on a genome-wide scale (the functional "interactome"), elucidating both known and novel pathways and cellular systems.

Estimating protein function using protein-protein relationships.

Many newly identified gene products from completely sequenced genomes are difficult to characterize in the absence of sequence homology to known proteins. In such a scenario, the context of the proteins' functional associations can be used for annotation; overrepresented functional linkages with a certain class of proteins or members of a pathway allow putative function assignments based on the "guilt-by-association" principle. Two computational functional genomics methods, phylogenetic profiling and identification of Rosetta stone linkages, are described in this chapter, which allow assessment of functional linkages between proteins, consequently facilitating annotation. Phylogenetic profiling involves measuring similarity between profiles that describe the presence or absence of a protein in a set of reference genomes, whereas Rosetta stone fusion sequences help link two or more independently transcribed and translated proteins. Both methods can be applied to investigate functional associations between individual proteins, and can also be extended to reconstruct the genome-wide network of functional linkages by querying the entire protein complement of an organism.

Computational modeling of the Plasmodium falciparum interactome reveals protein function on a genome-wide scale.

Many thousands of proteins encoded by the genome of Plasmodium falciparum, the causal organism of the deadliest form of human malaria, are of unknown function. It is of utmost importance that these proteins be characterized if we are to develop combative strategies against malaria based on the biology of the parasite. In an attempt to infer protein function on a genome-wide scale, we computationally modeled the P. falciparum interactome, elucidating local and global functional relationships between gene products. The resulting interaction network, reconstructed by integrating in silico and experimental functional genomics data within a Bayesian framework, covers approximately 68% of the parasite genome and provides functional inferences for more than 2000 uncharacterized proteins, based on their associations. Network reconstruction involved the use of a novel strategy, where we incorporated continuously updated, uniform reference priors in our Bayesian model. This method for generating interaction maps is thus also well suited for application to other genomes, where pre-existing interactome knowledge is sparse. Additionally, we superimposed this map on genomes of three apicomplexan pathogens--Plasmodium yoelii, Toxoplasma gondii, and Cryptosporidium parvum--describing relationships between these organisms based on retained functional linkages. This comparison provided a glimpse of the highly evolved nature of P. falciparum; for instance, a deficit of nearly 26% in terms of predicted interactions is observed against P. yoelii, because of missing ortholog partners in pairs of functionally linked proteins.

Protein function prediction using the Protein Link EXplorer (PLEX).

UNLABELLED: We introduce the Protein Link EXplorer (PLEX), a web-based environment that allows the construction of a phylogenetic profile for any given amino acid sequence, and its comparison with profiles of approximately 350,000 predicted genes from 89 genomes, as a means of interactively identifying functionally linked genes and predicting protein function. PLEX can be searched iteratively and also enables searches for chromosomal gene neighbors and Rosetta Stone linkages. PLEX search results are accompanied by quantitative estimates of linkage confidence, enabling users to take advantage of coinheritance, operon and gene fusion-based methods for inferring gene function and reconstructing cellular systems and pathways. AVAILABILITY: http://bioinformatics.icmb.utexas.edu/plex

Genome sequence of Haloarcula marismortui: a halophilic archaeon from the Dead Sea.

We report the complete sequence of the 4,274,642-bp genome of Haloarcula marismortui, a halophilic archaeal isolate from the Dead Sea. The genome is organized into nine circular replicons of varying G+C compositions ranging from 54% to 62%. Comparison of the genome architectures of Halobacterium sp. NRC-1 and H. marismortui suggests a common ancestor for the two organisms and a genome of significantly reduced size in the former. Both of these halophilic archaea use the same strategy of high surface negative charge of folded proteins as means to circumvent the salting-out phenomenon in a hypersaline cytoplasm. A multitiered annotation approach, including primary sequence similarities, protein family signatures, structure prediction, and a protein function association network, has assigned putative functions for at least 58% of the 4242 predicted proteins, a far larger number than is usually achieved in most newly sequenced microorganisms. Among these assigned functions were genes encoding six opsins, 19 MCP and/or HAMP domain signal transducers, and an unusually large number of environmental response regulators-nearly five times as many as those encoded in Halobacterium sp. NRC-1--suggesting H. marismortui is significantly more physiologically capable of exploiting diverse environments. In comparing the physiologies of the two halophilic archaea, in addition to the expected extensive similarity, we discovered several differences in their metabolic strategies and physiological responses such as distinct pathways for arginine breakdown in each halophile. Finally, as expected from the larger genome, H. marismortui encodes many more functions and seems to have fewer nutritional requirements for survival than does Halobacterium sp. NRC-1.

A probabilistic functional network of yeast genes.

A conceptual framework for integrating diverse functional genomics data was developed by reinterpreting experiments to provide numerical likelihoods that genes are functionally linked. This allows direct comparison and integration of different classes of data. The resulting probabilistic gene network estimates the functional coupling between genes. Within this framework, we reconstructed an extensive, high-quality functional gene network for Saccharomyces cerevisiae, consisting of 4681 (approximately 81%) of the known yeast genes linked by approximately 34,000 probabilistic linkages comparable in accuracy to small-scale interaction assays. The integrated linkages distinguish true from false-positive interactions in earlier data sets; new interactions emerge from genes' network contexts, as shown for genes in chromatin modification and ribosome biogenesis.

LGL: creating a map of protein function with an algorithm for visualizing very large biological networks.

Networks are proving to be central to the study of gene function, protein-protein interaction, and biochemical pathway data. Visualization of networks is important for their study, but visualization tools are often inadequate for working with very large biological networks. Here, we present an algorithm, called large graph layout (LGL), which can be used to dynamically visualize large networks on the order of hundreds of thousands of vertices and millions of edges. LGL applies a force-directed iterative layout guided by a minimal spanning tree of the network in order to generate coordinates for the vertices in two or three dimensions, which are subsequently visualized and interactively navigated with companion programs. We demonstrate the use of LGL in visualizing an extensive protein map summarizing the results of approximately 21 billion sequence comparisons between 145579 proteins from 50 genomes. Proteins are positioned in the map according to sequence homology and gene fusions, with the map ultimately serving as a theoretical framework that integrates inferences about gene function derived from sequence homology, remote homology, gene fusions, and higher-order fusions. We confirm that protein neighbors in the resulting map are functionally related, and that distinct map regions correspond to distinct cellular systems, enabling a computational strategy for discovering proteins' functions on the basis of the proteins' map positions. Using the map produced by LGL, we infer general functions for 23 uncharacterized protein families.

Discovery of uncharacterized cellular systems by genome-wide analysis of functional linkages.

We introduce a general computational method, applicable on a genome-wide scale, for the systematic discovery of uncharacterized cellular systems. Quantitative analysis of the coinheritance of pairs of genes among different organisms, calculated using phylogenetic profiles, allows the prediction of thousands of functional linkages between the corresponding proteins. A comparison of these functional linkages to known pathways reveals that calculated linkages are comparable in accuracy to genome-wide yeast two-hybrid screens or mass spectrometry interaction assays. In aggregate, these linkages describe the structure of large-scale networks, with the resulting yeast network composed of 3,875 linkages among 804 proteins, and the resulting pathogenic Escherichia coli network composed of 2,043 linkages among 828 proteins. The search of such networks for groups of uncharacterized, linked proteins led to the identification of 27 novel cellular systems from one nonpathogenic and three pathogenic bacterial genomes.