Application of Hidden Markov Model based methods for gaining insights into protein domain evolution and function

With the explosion in the amount of available sequence data, computational methods have become indispensable for studying proteins. Domains are the fundamental structural, functional and evolutionary units that make up proteins. Studying protein domains is an important part of understanding protein function and evolution. Hidden Markov Models (HMM) are one of the most successful methods that have been applied for protein sequence and structure analysis. In this study, HMM based methods were applied to study the evolution of sensory domains in microbial signal transduction systems as well as functional characterization and identification of cellulases in metagenomics datasets. Use of HMM domain models enabled identification of the ambiguity in sequence and structure based definitions of the Cache domain family. Cache domains are extracellular sensory domains that are present in microbial signal transduction proteins and eukaryotic voltage gated calcium channels. The ambiguity in domain definitions was resolved and more accurate HMM models were built that detected more than 50,000 new members. It was discovered that Cache domains constitute the largest family of extracellular sensory domains in prokaryotes. Cache domains were also found to be remotely homologous to PAS domains at the level of sequence, a relationship previously suggested purely based on structural comparisons. We used HMM-HMM comparisons to study the diversity of extracellular sensory domains in prokaryotic signal transductions systems. This approach allowed annotation of more than 46,000 sequences and reduced the percentage of unknown domains from 64% to 15%. New relationships were also discovered between domain families that were otherwise thought to be unrelated. Finally, HMM models were used to retrieve Family 48 glycoside hydrolases (GH48) from sequence databases. Analysis of these sequences, enabled the identification of distinguishing features of cellulases. These features were used to identify GH48 cellulases from metagenomics datasets. In summary, HMM based methods enabled domain identification, remote homology detection and functional characterization of protein domains

Application of comparative genomics in the identification and analysis of novel families of membrane-associated receptors in bacteria

BACKGROUND: A great diversity of multi-pass membrane receptors, typically with 7 transmembrane (TM) helices, is observed in the eukaryote crown group. So far, they are relatively rare in the prokaryotes, and are restricted to the well-characterized sensory rhodopsins of various phototropic prokaryotes. RESULTS: Utilizing the currently available wealth of prokaryotic genomic sequences, we set up a computational screen to identify putative 7 (TM) and other multi-pass membrane receptors in prokaryotes. As a result of this procedure we were able to recover two widespread families of 7 TM receptors in bacteria that are distantly related to the eukaryotic 7 TM receptors and prokaryotic rhodopsins. Using sequence profile analysis, we were able to establish that the first members of these receptor families contain one of two distinct N-terminal extracellular globular domains, which are predicted to bind ligands such as carbohydrates. In their intracellular portions they contain fusions to a variety of signaling domains, which suggest that they are likely to transduce signals via cyclic AMP, cyclic diguanylate, histidine phosphorylation, dephosphorylation, and through direct interactions with DNA. The second family of bacterial 7 TM receptors possesses an α-helical extracellular domain, and is predicted to transduce a signal via an intracellular HD hydrolase domain. Based on comparative analysis of gene neighborhoods, this receptor is predicted to function as a regulator of the diacylglycerol-kinase-dependent glycerolipid pathway. Additionally, our procedure also recovered other types of putative prokaryotic multi-pass membrane associated receptor domains. Of these, we characterized two widespread, evolutionarily mobile multi-TM domains that are fused to a variety of C-terminal intracellular signaling domains. One of these typified by the Gram-positive LytS protein is predicted to be a potential sensor of murein derivatives, whereas the other one typified by the Escherichia coli UhpB protein is predicted to function as sensor of conformational changes occurring in associated membrane proteins CONCLUSIONS: We present evidence for considerable variety in the types of uncharacterized surface receptors in bacteria, and reconstruct the evolutionary processes that model their diversity. The identification of novel receptor families in prokaryotes is likely to aid in the experimental analysis of signal transduction and environmental responses of several bacteria, including pathogens such as Leptospira, Treponema, Corynebacterium, Coxiella, Bacillus anthracis and Cytophaga

Signal Transduction in Membrane-Bound Adenylate Cyclases

Class III adenylate cyclases (ACs) are widespread signaling proteins, which translate diverse intracellular and extracellular stimuli into a uniform intracellular signal. They are typically composed of an N-terminal array of sensor domains and transducers, followed C-terminally by a catalytic domain, which, after dimerization, generates the second messenger cyclic adenosine monophosphate (cAMP). Many of the N-terminal domains are also found in other signaling proteins and can frequently be recombined between them. This work bioinformatically investigates the architectural and evolutionary principles that enable the productive interaction of a great diversity of upstream regulatory domains with the conserved AC catalytic domain. As part of this process, we have identified the novel cyclase transducer element (CTE), a pivotal hinge on the N-terminus of the AC catalytic domain. The element appears to convert unspecific signals moving along the coiled-coil backbone into specific conformational changes that determine AC activity. This suggests communication along a dimeric coiled coil as the structural rationale for the architectural similarities between many families of signaling proteins. Further, we bioinformatically classify the various six-helical transmembrane (6TM) domains observed in many bacterial and eukaryotic ACs. Recently, experimental results have indicated that these domains could function as regulatory receptors binding hydrophobic ligands inside the membrane. Our classification and the presence of a CTE in many 6TM ACs strongly support this hypothesis, concluding that some or all 6TM domains are regulatory receptors for as-yet-unknown ligands. Since mammalian ACs are important downstream messengers of G protein-coupled receptors, these findings suggest AC 6TM domains could be drug targets of possibly great pharmacological relevance

The Evolution of Two-Component Systems in Bacteria Reveals Different Strategies for Niche Adaptation

Two-component systems including histidine protein kinases represent the primary signal transduction paradigm in prokaryotic organisms. To understand how these systems adapt to allow organisms to detect niche-specific signals, we analyzed the phylogenetic distribution of nearly 5,000 histidine protein kinases from 207 sequenced prokaryotic genomes. We found that many genomes carry a large repertoire of recently evolved signaling genes, which may reflect selective pressure to adapt to new environmental conditions. Both lineage-specific gene family expansion and horizontal gene transfer play major roles in the introduction of new histidine kinases into genomes; however, there are differences in how these two evolutionary forces act. Genes imported via horizontal transfer are more likely to retain their original functionality as inferred from a similar complement of signaling domains, while gene family expansion accompanied by domain shuffling appears to be a major source of novel genetic diversity. Family expansion is the dominant source of new histidine kinase genes in the genomes most enriched in signaling proteins, and detailed analysis reveals that divergence in domain structure and changes in expression patterns are hallmarks of recent expansions. Finally, while these two modes of gene acquisition are widespread across bacterial taxa, there are clear species-specific preferences for which mode is used

Repurposing a chemosensory macromolecular machine

How complex, multi-component macromolecular machines evolved remains poorly understood. Here we reveal the evolutionary origins of the chemosensory machinery that controls flagellar motility in Escherichia coli. We first identify ancestral forms still present in Vibrio cholerae, Pseudomonas aeruginosa, Shewanella oneidensis and Methylomicrobium alcaliphilum, characterizing their structures by electron cryotomography and finding evidence that they function in a stress response pathway. Using bioinformatics, we trace the evolution of the system through γ-Proteobacteria, pinpointing key evolutionary events that led to the machine now seen in E. coli. Our results suggest that two ancient chemosensory systems with different inputs and outputs (F6 and F7) existed contemporaneously, with one (F7) ultimately taking over the inputs and outputs of the other (F6), which was subsequently lost

Light-sensing in bacteria

Thesis (Ph.D.) - Indiana University, Biochemistry and Molecular Biology, 2015Light reception plays an important role in regulating lifestyle changes in bacteria. My research focused on a number of different light-sensing systems in different bacteria: PpaA in Rhodobacter sphaeroides, PixD-PixE in Synechocystis sp. PCC, and the known light-receptors in Rhodospirillum centenum. PpaA from R. sphaeroides had previously been shown to be a heme-binding protein, despite its sequence similarity to cobalamin-binding proteins. My research showed that PpaA is in fact a bona fide cobalamin-binding protein. PpaA binds specifically hydroxy-cobalamin, but not other forms of cobalamin. PpaA does have some ability to bind heme, but a mutant form of PpaA that showed better heme-binding was inactive in vivo. This suggests that PpaA functional cofactor is cobalamin, rather than heme. We also tested cobalamin-binding in a number of homologs of PpaA and found that almost all are indeed cobalamin-binding proteins. The genome of Rhodospirillum centenum contains four reading frames that encode light sensing proteins. We identified possible role for each of these light-receptors by making deletion mutants, and testing the impact of these deletion on the transcription levels. Our results suggest that the PYP-phytochrome hybrid Ppr plays a role as a global regulator of transcription. A BLUF and a bacteriophytochrome on the other hand showed changes in expression levels of a number of genes involved in motility. Lastly, deletion of a LOV domain containing protein did not result in any significant changes in gene expression levels. This suggests that the LOV protein does not regulate life-style changes, but rather controls immediate responses. PixD is a short BLUF protein that has been shown to play a role in regulating phototaxis in Synechocystis sp. PCC6803. We were able to show that Slr1692, encoded by an ORF upstream of pixE-pixD might play a role in regulating phototaxis

D-Glucose-6-phosphate stimulates SagS-dependent biofilm formation in Pseudomonas aeruginosa

The SagS protein is a two-component regulatory system in Pseudomonas aeruginosa that works to independently regulate biofilm formation and antibiotic tolerance. Previous work found that these two pathways are controlled by two distinct sets of amino acids within the sensory domain of SagS that are thought to be potential ligand binding sites. Despite the extensive research done on the structure and function of SagS, the signals that activate this protein have yet to be identified. In this study we aimed to identify ligands that stimulate SagS-dependent biofilm formation. To do this we utilized ΔsagS mutants, one harboring wild-type sagS under the control of its native promoter and the other harboring the empty vector, attachment assays and COMSTAT analysis. Initially, we found that Mn2+ stimulates SagS-dependent attachment, but not SagS-dependent biofilm formation. While we ultimately identified D-glucose-6-phosphate as a ligand that enhances SagS-dependent biofilm formation. These findings provide a better understanding of how SagS works to control biofilm formation in P. aeruginosa and points to potential new method for treating P. aeruginosa infections

CACHD1 is an α2δ-like protein that modulates CaV3 voltage-gated calcium channel activity

The putative cache (Ca2+ channel and chemotaxis receptor) domain containing 1 (CACHD1) protein has predicted structural similarities to members of the alpha2delta voltage-gated Ca2+ channel (VGCC) auxiliary subunit family. CACHD1 mRNA and protein were highly expressed in the male mammalian CNS, in particular in the thalamus, hippocampus and cerebellum, with a broadly similar tissue distribution to CaV3 subunits, in particular, CaV3.1. In expression studies, CACHD1 increased cell-surface localization of CaV3.1 and these proteins were in close proximity at the cell surface consistent with the formation of CACHD1-CaV3.1 complexes. In functional electrophysiological studies, co-expression of human CACHD1 with CaV3.1, CaV3.2 and CaV3.3 caused a significant increase in peak current density and corresponding increases in maximal conductance. By contrast, alpha2delta-1 had no effect on peak current density or maximal conductance in either CaV3.1, CaV3.2 or CaV3.3. Comparison of CACHD1-mediated increases in CaV3.1 current density and gating currents revealed an increase in channel open probability. In hippocampal neurons from male and female E19 rats, CACHD1 overexpression increased CaV3-mediated action potential (AP) firing frequency and neuronal excitability. These data suggest that CACHD1 is structurally an alpha2delta-like protein that functionally modulates CaV3 voltage-gated calcium channel activity

Chemoperception of Specific Amino Acids Controls Phytopathogenicity in Pseudomonas syringae pv. tomato

IMPORTANCE There is substantive evidence that chemotaxis is a key requisite for efficient pathogenesis in plant pathogens. However, information regarding particular bacterial chemoreceptors and the specific plant signal that they sense is scarce. Our work shows that the phytopathogenic bacterium Pseudomonas syringae pv. tomato mediates not only chemotaxis but also the control of pathogenicity through the perception of the plant abundant amino acids Asp and Glu. We describe the specificity of the perception of L- and D-Asp and L-Glu by the PsPto-PscA chemoreceptor and the involvement of this perception in the regulation of pathogenicity-related traits. Moreover, a saturating concentration of D-Asp reduces bacterial virulence, and we therefore propose that ligand-mediated interference of key chemoreceptors may be an alternative strategy to control virulence.Supplemental material for this article may be found at https://doi.org/10.1128/mBio .01868-19.We acknowledge M. Trini Gallegos for kindly provide plasmid pCdrA::gfpS and S. Nebreda for technical assistance.Chemotaxis has been associated with the pathogenicity of bacteria in plants and was found to facilitate bacterial entry through stomata and wounds. However, knowledge regarding the plant signals involved in this process is scarce. We have addressed this issue using Pseudomonas syringae pv. tomato, which is a foliar pathogen that causes bacterial speck in tomato. We show that the chemoreceptor P. syringae pv. tomato PscA (PsPto-PscA) recognizes specifically and with high affinity L-Asp, L-Glu, and D-Asp. The mutation of the chemoreceptor gene largely reduced chemotaxis to these ligands but also altered cyclic di-GMP (c-di-GMP) levels, biofilm formation, and motility, pointing to cross talk between different chemosensory pathways. Furthermore, the PsPto-PscA mutant strain showed reduced virulence in tomato. Asp and Glu are the most abundant amino acids in plants and in particular in tomato apoplasts, and we hypothesize that this receptor may have evolved to specifically recognize these compounds to facilitate bacterial entry into the plant. Infection assays with the wild-type strain showed that the presence of saturating concentrations of D-Asp also reduced bacterial virulence.This work was supported by grants AGL2015-63851-R and RTI2018-095222-B100 (to E.L.-S.) and BIO2016-76779-P (to T.K.) from the Ministerio de Economía y Competitividad, Spain. J.P.C.-V. was supported by the FPI program (BES-2016-076452, MINECOSpain)

Application of Bioinformatics to Protein Domain, Protein Network, and Whole Genome Studies.

Bioinformatics primarily focuses on the study of sequence data. Analyzing both nucleotide and protein sequence data provides valuable insight into their function, evolution, and importance in organism adaptation. For this dissertation, I have applied bioinformatics to the study sequence data on three levels of complexity: protein domain, protein network, and whole genome. In the protein domain study, I used sequence similarity searches to identify a novel FIST (F-box and intracellular signal transduction proteins) domain. The domain was found to exist in all three kingdoms of life, pointing to its functional importance. Due to its presence exclusively with transducer and output domains, it was deduced that FIST functions as an input/sensory domain involved in signal transduction. Further functional characterization revealed FIST\u27s proximity to amino acid metabolism and transport genes. This suggested that FIST functions as a small ligand sensor. In the protein network study, I examined the evolution of the chemotaxis system within the clade of Escherichia. Our study confirmed previous results demonstrating that many urinary pathogenic Escherichia coli have lost two of their five chemotaxis receptors. However, sequence analysis demonstrates that this loss occurred as an ancestral event and was not a result of adaptive evolution. The retention of the core of the system in the vast majority of Escherichia confirms that chemotaxis is important for survival in all of Escherichia\u27s habitats. However analysis of the loss and gain of chemotaxis receptors suggests that the array of compounds that Escherichia needs to sense often does not require all 5 canonical receptors. In the genome study, I used comparative genomic analysis to examine the evolutionary history of Azospirillum, agriculturally important plant growth-promoting bacteria. Taxonomic and genomic studies have revealed that Azospirillum are very distinct from their closest relatives in both habitat and genome structure. Comparative genomic analysis revealed that Azospirillum had undergone massive horizontal gene transfer. Among acquired genes were many of those implicated in survival in the rhizosphere and in plant growth-promotion. It is proposed that this bacteria\u27s unique genome plasticity and ability to uptake large amounts of foreign DNA allowed azospirilla to transition from an aquatic to terrestrial environment