Biomedical subjects

Nitin S Baliga

Publications and source records attributed to Nitin S Baliga.

15 recordsLinked to original sources

PubMed2006

An integrated systems approach for understanding cellular responses to gamma radiation.

Cellular response to stress entails complex mRNA and protein abundance changes, which translate into physiological adjustments to maintain homeostasis as well as to repair and minimize damage to cellular components. We have characterized the response of the halophilic archaeon Halobacterium salinarum NRC-1 to (60)Co ionizing gamma radiation in an effort to understand the correlation between genetic information processing and physiological change. The physiological response model we have constructed is based on integrated analysis of temporal changes in global mRNA and protein abundance along with protein-DNA interactions and evolutionarily conserved functional associations. This systems view reveals cooperation among several cellular processes including DNA repair, increased protein turnover, apparent shifts in metabolism to favor nucleotide biosynthesis and an overall effort to repair oxidative damage. Further, we demonstrate the importance of time dimension while correlating mRNA and protein levels and suggest that steady-state comparisons may be misleading while assessing dynamics of genetic information processing across transcription and translation.

Archaeal Proteins↗

PubMed2006

A systems view of haloarchaeal strategies to withstand stress from transition metals.

Given that transition metals are essential cofactors in central biological processes, misallocation of the wrong metal ion to a metalloprotein can have resounding and often detrimental effects on diverse aspects of cellular physiology. Therefore, in an attempt to characterize unique and shared responses to chemically similar metals, we have reconstructed physiological behaviors of Halobacterium NRC-1, an archaeal halophile, in sublethal levels of Mn(II), Fe(II), Co(II), Ni(II), Cu(II), and Zn(II). Over 20% of all genes responded transiently within minutes of exposure to Fe(II), perhaps reflecting immediate large-scale physiological adjustments to maintain homeostasis. At steady state, each transition metal induced growth arrest, attempts to minimize oxidative stress, toxic ion scavenging, increased protein turnover and DNA repair, and modulation of active ion transport. While several of these constitute generalized stress responses, up-regulation of active efflux of Co(II), Ni(II), Cu(II), and Zn(II), down-regulation of Mn(II) uptake and up-regulation of Fe(II) chelation, confer resistance to the respective metals. We have synthesized all of these discoveries into a unified systems-level model to provide an integrated perspective of responses to six transition metals with emphasis on experimentally verified regulatory mechanisms. Finally, through comparisons across global transcriptional responses to different metals, we provide insights into putative in vivo metal selectivity of metalloregulatory proteins and demonstrate that a systems approach can help rapidly unravel novel metabolic potential and regulatory programs of poorly studied organisms.

Adenosine Triphosphatases↗

PubMed2006

Integrated biclustering of heterogeneous genome-wide datasets for the inference of global regulatory networks.

BACKGROUND: The learning of global genetic regulatory networks from expression data is a severely under-constrained problem that is aided by reducing the dimensionality of the search space by means of clustering genes into putatively co-regulated groups, as opposed to those that are simply co-expressed. Be cause genes may be co-regulated only across a subset of all observed experimental conditions, biclustering (clustering of genes and conditions) is more appropriate than standard clustering. Co-regulated genes are also often functionally (physically, spatially, genetically, and/or evolutionarily) associated, and such a priori known or pre-computed associations can provide support for appropriately grouping genes. One important association is the presence of one or more common cis-regulatory motifs. In organisms where these motifs are not known, their de novo detection, integrated into the clustering algorithm, can help to guide the process towards more biologically parsimonious solutions. RESULTS: We have developed an algorithm, cMonkey, that detects putative co-regulated gene groupings by integrating the biclustering of gene expression data and various functional associations with the de novo detection of sequence motifs. CONCLUSION: We have applied this procedure to the archaeon Halobacterium NRC-1, as part of our efforts to decipher its regulatory network. In addition, we used cMonkey on public data for three organisms in the other two domains of life: Helicobacter pylori, Saccharomyces cerevisiae, and Escherichia coli. The biclusters detected by cMonkey both recapitulated known biology and enabled novel predictions (some for Halobacterium were subsequently confirmed in the laboratory). For example, it identified the bacteriorhodopsin regulon, assigned additional genes to this regulon with apparently unrelated function, and detected its known promoter motif. We have performed a thorough comparison of cMonkey results against other clustering methods, and find that cMonkey biclusters are more parsimonious with all available evidence for co-regulation.

Algorithms↗

PubMed2006

The Inferelator: an algorithm for learning parsimonious regulatory networks from systems-biology data sets de novo.

We present a method (the Inferelator) for deriving genome-wide transcriptional regulatory interactions, and apply the method to predict a large portion of the regulatory network of the archaeon Halobacterium NRC-1. The Inferelator uses regression and variable selection to identify transcriptional influences on genes based on the integration of genome annotation and expression data. The learned network successfully predicted Halobacterium's global expression under novel perturbations with predictive power similar to that seen over training data. Several specific regulatory predictions were experimentally tested and verified.

Adenosine Triphosphatases↗

PubMed2006

The Gaggle: an open-source software system for integrating bioinformatics software and data sources.

BACKGROUND: Systems biologists work with many kinds of data, from many different sources, using a variety of software tools. Each of these tools typically excels at one type of analysis, such as of microarrays, of metabolic networks and of predicted protein structure. A crucial challenge is to combine the capabilities of these (and other forthcoming) data resources and tools to create a data exploration and analysis environment that does justice to the variety and complexity of systems biology data sets. A solution to this problem should recognize that data types, formats and software in this high throughput age of biology are constantly changing. RESULTS: In this paper we describe the Gaggle -a simple, open-source Java software environment that helps to solve the problem of software and database integration. Guided by the classic software engineering strategy of separation of concerns and a policy of semantic flexibility, it integrates existing popular programs and web resources into a user-friendly, easily-extended environment. We demonstrate that four simple data types (names, matrices, networks, and associative arrays) are sufficient to bring together diverse databases and software. We highlight some capabilities of the Gaggle with an exploration of Helicobacter pylori pathogenesis genes, in which we identify a putative ricin-like protein -a discovery made possible by simultaneous data exploration using a wide range of publicly available data and a variety of popular bioinformatics software tools. CONCLUSION: We have integrated diverse databases (for example, KEGG, BioCyc, String) and software (Cytoscape, DataMatrixViewer, R statistical environment, and TIGR Microarray Expression Viewer). Through this loose coupling of diverse software and databases the Gaggle enables simultaneous exploration of experimental data (mRNA and protein abundance, protein-protein and protein-DNA interactions), functional associations (operon, chromosomal proximity, phylogenetic pattern), metabolic pathways (KEGG) and Pubmed abstracts (STRING web resource), creating an exploratory environment useful to 'web browser and spreadsheet biologists', to statistically savvy computational biologists, and those in between. The Gaggle uses Java RMI and Java Web Start technologies and can be found at http://gaggle.systemsbiology.net.

Computational Biology↗

PubMed2005

Catabolite control protein A (CcpA) contributes to virulence and regulation of sugar metabolism in Streptococcus pneumoniae.

We characterized the role of catabolite control protein A (ccpA) in the physiology and virulence of Streptococcus pneumoniae. S. pneumoniae has a large percentage of its genome devoted to sugar uptake and metabolism, and therefore, regulation of these processes is likely to be crucial for fitness in the nasopharynx and may play a role during invasive disease. In many bacteria, carbon catabolite repression (CCR) is central to such regulation, influencing hierarchical sugar utilization and growth rates. CcpA is the major transcriptional regulator in CCR in several gram-positive bacteria. We show that CcpA functions in CCR of lactose-inducible beta-galactosidase activity in S. pneumoniae. CCR of maltose-inducible alpha-glucosidase, raffinose-inducible alpha-galactosidase, and cellobiose-inducible beta-glucosidase is unaffected in the ccpA strain, suggesting that other regulators, possibly redundant with CcpA, control these systems. The ccpA strain is severely attenuated for nasopharyngeal colonization and lung infection in the mouse, establishing its role in fitness on these mucosal surfaces. Comparison of the cell wall fraction of the ccpA and wild-type strains shows that CcpA regulates many proteins in this compartment that are involved in central and intermediary metabolism, a subset of which are required for survival and multiplication in vivo. Both in vitro and in vivo defects were complemented by providing ccpA in trans. Our results demonstrate that CcpA, though not a global regulator of CCR in S. pneumoniae, is required for colonization of the nasopharynx and survival and multiplication in the lung.

Animals↗

PubMed2004

Comprehensive de novo structure prediction in a systems-biology context for the archaea Halobacterium sp. NRC-1.

BACKGROUND: Large fractions of all fully sequenced genomes code for proteins of unknown function. Annotating these proteins of unknown function remains a critical bottleneck for systems biology and is crucial to understanding the biological relevance of genome-wide changes in mRNA and protein expression, protein-protein and protein-DNA interactions. The work reported here demonstrates that de novo structure prediction is now a viable option for providing general function information for many proteins of unknown function. RESULTS: We have used Rosetta de novo structure prediction to predict three-dimensional structures for 1,185 proteins and protein domains (<150 residues in length) found in Halobacterium NRC-1, a widely studied halophilic archaeon. Predicted structures were searched against the Protein Data Bank to identify fold similarities and extrapolate putative functions. They were analyzed in the context of a predicted association network composed of several sources of functional associations such as: predicted protein interactions, predicted operons, phylogenetic profile similarity and domain fusion. To illustrate this approach, we highlight three cases where our combined procedure has provided novel insights into our understanding of chemotaxis, possible prophage remnants in Halobacterium NRC-1 and archaeal transcriptional regulators. CONCLUSIONS: Simultaneous analysis of the association network, coordinated mRNA level changes in microarray experiments and genome-wide structure prediction has allowed us to glean significant biological insights into the roles of several Halobacterium NRC-1 proteins of previously unknown function, and significantly reduce the number of proteins encoded in the genome of this haloarchaeon for which no annotation is available.

Archaeal Proteins↗

PubMed2004

Systems level insights into the stress response to UV radiation in the halophilic archaeon Halobacterium NRC-1.

We report a remarkably high UV-radiation resistance in the extremely halophilic archaeon Halobacterium NRC-1 withstanding up to 110 J/m2 with no loss of viability. Gene knockout analysis in two putative photolyase-like genes (phr1 and phr2) implicated only phr2 in photoreactivation. The UV-response was further characterized by analyzing simultaneously, along with gene function and protein interactions inferred through comparative genomics approaches, mRNA changes for all 2400 genes during light and dark repair. In addition to photoreactivation, three other putative repair mechanisms were identified including d(CTAG) methylation-directed mismatch repair, four oxidative damage repair enzymes, and two proteases for eliminating damaged proteins. Moreover, a UV-induced down-regulation of many important metabolic functions was observed during light repair and seems to be a phenomenon shared by all three domains of life. The systems analysis has facilitated the assignment of putative functions to 26 of 33 key proteins in the UV response through sequence-based methods and/or similarities of their predicted three-dimensional structures to known structures in the PDB. Finally, the systems analysis has raised, through the integration of experimentally determined and computationally inferred data, many experimentally testable hypotheses that describe the metabolic and regulatory networks of Halobacterium NRC-1.

Animals↗

PubMed2004

Specificity of anion binding in the substrate pocket of bacteriorhodopsin.

The structure of the D85S mutant of bacteriorhodopsin with a nitrate anion bound in the Schiff base binding site and the structure of the anion-free protein have been obtained in the same crystal form. Together with the previously solved structures of this anion pump, in both the anion-free state and bromide-bound state, these new structures provide insight into how this mutant of bacteriorhodopsin is able to bind a variety of different anions in the same binding pocket. The structural analysis reveals that the main structural change that accommodates different anions is the repositioning of the polar side chain of S85. On the basis of these X-ray crystal structures, the prediction is then made that the D85S/D212N double mutant might bind similar anions and do so over a broader pH range than does the single mutant. Experimental comparison of the dissociation constants, K(d), for a variety of anions confirms this prediction and demonstrates, in addition, that the binding affinity is dramatically improved by the D212N substitution.

Amino Acid Substitution↗

PubMed2004

Low-pass sequencing for microbial comparative genomics.

BACKGROUND: We studied four extremely halophilic archaea by low-pass shotgun sequencing: (1) the metabolically versatile Haloarcula marismortui; (2) the non-pigmented Natrialba asiatica; (3) the psychrophile Halorubrum lacusprofundi and (4) the Dead Sea isolate Halobaculum gomorrense. Approximately one thousand single pass genomic sequences per genome were obtained. The data were analyzed by comparative genomic analyses using the completed Halobacterium sp. NRC-1 genome as a reference. Low-pass shotgun sequencing is a simple, inexpensive, and rapid approach that can readily be performed on any cultured microbe. RESULTS: As expected, the four archaeal halophiles analyzed exhibit both bacterial and eukaryotic characteristics as well as uniquely archaeal traits. All five halophiles exhibit greater than sixty percent GC content and low isoelectric points (pI) for their predicted proteins. Multiple insertion sequence (IS) elements, often involved in genome rearrangements, were identified in H. lacusprofundi and H. marismortui. The core biological functions that govern cellular and genetic mechanisms of H. sp. NRC-1 appear to be conserved in these four other halophiles. Multiple TATA box binding protein (TBP) and transcription factor IIB (TFB) homologs were identified from most of the four shotgunned halophiles. The reconstructed molecular tree of all five halophiles shows a large divergence between these species, but with the closest relationship being between H. sp. NRC-1 and H. lacusprofundi. CONCLUSION: Despite the diverse habitats of these species, all five halophiles share (1) high GC content and (2) low protein isoelectric points, which are characteristics associated with environmental exposure to UV radiation and hypersalinity, respectively. Identification of multiple IS elements in the genome of H. lacusprofundi and H. marismortui suggest that genome structure and dynamic genome reorganization might be similar to that previously observed in the IS-element rich genome of H. sp. NRC-1. Identification of multiple TBP and TFB homologs in these four halophiles are consistent with the hypothesis that different types of complex transcriptional regulation may occur through multiple TBP-TFB combinations in response to rapidly changing environmental conditions. Low-pass shotgun sequence analyses of genomes permit extensive and diverse analyses, and should be generally useful for comparative microbial genomics.

Archaea↗

PubMed2004

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.

Archaeal Proteins↗

PubMed2003

Proteomic analysis of an extreme halophilic archaeon, Halobacterium sp. NRC-1.

Halobacterium sp. NRC-1 insoluble membrane and soluble cytoplasmic proteins were isolated by ultracentrifugation of whole cell lysate. Using an ion trap mass spectrometer equipped with a C18 trap electrospray ionization emitter/micro-liquid chromatography column, a number of trypsin-generated peptide tags from 426 unique proteins were identified. This represents approximately one-fifth of the theoretical proteome of Halobacterium. Of these, 232 proteins were found only in the soluble fraction, 165 were only in the insoluble membrane fraction, and 29 were in both fractions. There were 72 and 61% previously annotated proteins identified in the soluble and membrane protein fractions, respectively. Interestingly, 57 of previously unannotated proteins found only in Halobacterium NRC-1 were identified. Such proteins could be interesting targets for understanding unique physiology of Halobacterium NRC-1. A group of proteins involved in various metabolic pathways were identified among the expressed proteins, suggesting these pathways were active at the time the cells were collected. This data containing a list of expressed proteins, their cellular locations, and biological functions could be used in future studies to investigate the interaction of the genes and proteins in relation to genetic or environmental perturbations.

Archaeal Proteins↗

PubMed2003

Cytoscape: a software environment for integrated models of biomolecular interaction networks.

Cytoscape is an open source software project for integrating biomolecular interaction networks with high-throughput expression data and other molecular states into a unified conceptual framework. Although applicable to any system of molecular components and interactions, Cytoscape is most powerful when used in conjunction with large databases of protein-protein, protein-DNA, and genetic interactions that are increasingly available for humans and model organisms. Cytoscape's software Core provides basic functionality to layout and query the network; to visually integrate the network with expression profiles, phenotypes, and other molecular states; and to link the network to databases of functional annotations. The Core is extensible through a straightforward plug-in architecture, allowing rapid development of additional computational analyses and features. Several case studies of Cytoscape plug-ins are surveyed, including a search for interaction pathways correlating with changes in gene expression, a study of protein complexes involved in cellular recovery to DNA damage, inference of a combined physical/functional interaction network for Halobacterium, and an interface to detailed stochastic/kinetic gene regulatory models.

Algorithms↗

PubMed2002

Coordinate regulation of energy transduction modules in Halobacterium sp. analyzed by a global systems approach.

The extremely halophilic archaeon Halobacterium NRC-1 can switch from aerobic energy production (energy from organic compounds) to anaerobic phototrophy (energy from light) by induction of purple membrane biogenesis. The purple membrane is made up of multiple copies of a 1:1 complex of bacterioopsin (Bop) and retinal called bacteriorhodopsin that functions as a light-driven proton pump. A light- and redox-sensing transcription regulator, Bat, regulates critical genes encoding the biogenesis of the purple membrane. To better understand the regulatory network underlying this physiological state, we report a systems approach using global mRNA and protein analyses of four strains of Halobacterium sp.: the wild-type, NRC-1; and three genetically perturbed strains: S9 (bat+), a purple membrane overproducer, and two purple membrane deficient strains, SD23 (a bop knockout) and SD20 (a bat knockout). The integrated DNA microarray and proteomic data reveal the coordinated coregulation of several interconnected biochemical pathways for phototrophy: isoprenoid synthesis, carotenoid synthesis, and bacteriorhodopsin assembly. In phototrophy, the second major biomodule for ATP production, arginine fermentation, is repressed. The primary systems level insight provided by this study is that two major energy production pathways in Halobacterium sp., phototrophy and arginine fermentation, are inversely regulated, presumably to achieve a balance in ATP production under anaerobic conditions.

Adenosine Triphosphate↗

PubMed2001

Promoter analysis by saturation mutagenesis.

Gene expression and regulation are mediated by DNA sequences, in most instances, directly upstream to the coding sequences by recruiting transcription factors, regulators, and a RNA polymerase in a spatially defined fashion. Few nucleotides within a promoter make contact with the bound proteins. The minimal set of nucleotides that can recruit a protein factor is called a cis-acting element. This article addresses a powerful mutagenesis strategy that can be employed to define cis-acting elements at a molecular level. Technical details including primer design, saturation mutagenesis, construction of promoter libraries, phenotypic analysis, data analysis, and interpretation are discussed.

Journal Article↗