Tracey Freitas

Genome sequence of the deep-sea γ-proteobacterium Idiomarina loihiensis reveals amino acid fermentation as a source of carbon and energy

We report the complete genome sequence of the deep-sea γ-proteobacterium, Idiomarina loihiensis, isolated recently from a hydrothermal vent at 1,300-m depth on the Lōihi submarine volcano, Hawaii. The I. loihiensis genome comprises a single chromosome of 2,839,318 base pairs, encoding 2,640 proteins, four rRNA operons, and 56 tRNA genes. A comparison of I. loihiensis to the genomes of other γ-proteobacteria reveals abundance of amino acid transport and degradation enzymes, but a loss of sugar transport systems and certain enzymes of sugar metabolism. This finding suggests that I. loihiensis relies primarily on amino acid catabolism, rather than on sugar fermentation, for carbon and energy. Enzymes for biosynthesis of purines, pyrimidines, the majority of amino acids, and coenzymes are encoded in the genome, but biosynthetic pathways for Leu, Ile, Val, Thr, and Met are incomplete. Auxotrophy for Val and Thr was confirmed by in vivo experiments. The I. loihiensis genome contains a cluster of 32 genes encoding enzymes for exopolysaccharide and capsular polysaccharide synthesis. It also encodes diverse peptidases, a variety of peptide and amino acid uptake systems, and versatile signal transduction machinery. We propose that the source of amino acids for I. loihiensis growth are the proteinaceous particles present in the deep sea hydrothermal vent waters. I. loihiensis would colonize these particles by using the secreted exopolysaccharide, digest these proteins, and metabolize the resulting peptides and amino acids. In summary, the I. loihiensis genome reveals an integrated mechanism of metabolic adaptation to the constantly changing deep-sea hydrothermal ecosystem.

Globin-coupled sensors: a class of heme-containing sensors in Archaea and Bacteria.

The recently discovered prokaryotic signal transducer HemAT, which has been described in both Archaea and Bacteria, mediates aerotactic responses. The N-terminal regions of HemAT from the archaeon Halobacterium salinarum (HemAT-Hs) and from the Gram-positive bacterium Bacillus subtilis (HemAT-Bs) contain a myoglobin-like motif, display characteristic heme–protein absorption spectra, and bind oxygen reversibly. Recombinant HemAT-Hs and HemAT-Bs shorter than 195 and 176 residues, respectively, do not bind heme effectively. Sequence homology comparisons and three-dimensional modeling predict that His-123 is the proximal heme-binding residue in HemAT from both species. The work described here used site-specific mutagenesis and spectroscopy to confirm this prediction, thereby providing direct evidence for a functional domain of prokaryotic signal transducers that bind heme in a globin fold. We postulate that this domain is part of a globin-coupled sensor (GCS) motif that exists as a two-domain transducer having no similarity to the PER-ARNT-SIM (PAS)-domain superfamily transducers. Using the GCS motif, we have identified several two-domain sensors in a variety of prokaryotes. We have cloned, expressed, and purified two potential globin-coupled sensors and performed spectral analysis on them. Both bind heme and show myoglobin-like spectra. This observation suggests that the general function of GCS-type transducers is to bind diatomic oxygen and perhaps other gaseous ligands, and to transmit a conformational signal through a linked signaling domain.

The diversity of globin-coupled sensors.

The recently discovered globin‐coupled sensors (GCSs) are heme‐containing two‐domain transducers distinct from the PAS domain superfamily. We have identified an additional 22 GCSs with varying multi‐domain C‐terminal transmitters through a search of the complete and incomplete microbial genome datasets. The GCS superfamily is composed of two major subfamilies: the aerotactic and gene regulators. We postulate the existence of protoglobin in Archaea as the predecessor to the chimeric GCS.

Molecular dynamics and in vitro analysis of Connexin43: A new 14-3-3 mode-1 interacting protein

The interaction of cellular proteins with the gap junction protein Connexin43 (Cx43) is thought to form a dynamic scaffolding complex that functions as a platform for the assembly of signaling, structural, and cytoskeletal proteins. A high stringency Scansite search of rat Cx43 identified the motif containing Ser373 (S373) as a 14‐3‐3 binding site. The S373 motif and the second best mode‐1 motif, containing Ser244 (S244), are conserved in rat, mouse, human, chicken, and bovine, but not in Xenopus or zebrafish Cx43. Docking studies of a mouse/rat 14‐3‐3θ homology model with the modeled phosphorylated S373 or S244 peptide ligands or their serine‐to‐alanine mutants, S373A or S244A, revealed that the pS373 motif facilitated a greater number of intermolecular contacts than the pS244 motif, thus supporting a stronger 14‐3‐3 binding interaction with the pS373 motif. The alanine substitution also reduced more than half the number of intermolecular contacts between 14‐3‐3θ and the S373 motif, emphasizing the phosphorylation dependence of this interaction. Furthermore, the ability of the wild‐type or the S244A GST‐Cx43 C‐terminal fusion protein, but not the S373A fusion protein, to interact with either 14‐3‐3θ or 14‐3‐3ζ in GST pull‐down experiments clearly demonstrated that the S373 motif mediates the direct interaction between Cx43 and 14‐3‐3 proteins. Blocking growth factor–induced Akt activation and presumably any Akt‐mediated phosphorylation of the S373 motif in ROSE 199 cells did not prevent the down‐regulation of Cx43‐mediated cell–cell communication, suggesting that an Akt‐mediated interaction with 14‐3‐3 was not involved in the disruption of Cx43 function.

Protoglobin and Globin-coupled Sensors

The strategy for detecting oxygen, carbon monoxide, nitric oxide, and sulfides is predominantly through heme-based sensors utilizing either a globin domain or a PAS domain. Whereas PAS domains bind various cofactors, globins bind only heme. Globin-coupled sensors (GCSs) couple an N-terminal sensor globin domain to varied C-terminal signaling domains to effect aerotaxis and gene regulation. Having descended from an ancient protoglobin, GCSs are now ubiquitous and are encoded in the genome of several extremophiles (temperature, salt, and pH). We postulate that their role in regulating gene expression governs microbial processes critical to elemental recycling, bioremediation, and cellulose degradation. Functional and evolutionary analyses of the GCSs, their protoglobin ancestor, and their relationship to the Last Universal Common Ancestor (LUCA) are also discussed in the context of globin-based signal transduction.

Tessellation Analysis of Glomerular Spatial Arrangement in Mice with Heritable Renal Hypoplasia

Renal hypoplasia results from an insufficient kidney volume caused, in part, by a deficient number of glomeruli. The purpose of this study was to apply tessellation analysis to determine whether glomerular point patterns differed between adult normal (WT) and mutant (Br) mice with heritable renal hypoplasia and to delineate a spatial distribution accounting for the observed patterns. Kidneys from adult WT and Br mice were collected, processed with routine light histology and representative transverse sections were photographed. Cortical area and perimeter were calculated from traced tissue contours and glomeruli were identified and digitized. Voronoi tessellations were constructed and average parameters for Voronoi polygon number, area, perimeter and edge counts as well as spatial metrics comprising nearest neighbor and centroidal distances were calculated and compared. Point distributions were simulated by randomizing glomerular coordinates from each section and plotting the new points utilizing uniform random, Gaussian random, or isotropic functions. Average nearest neighbor distances were generated for each specimen and ranked with respect to corresponding values generated from 1,000 iterations for each simulated set. Results showed that WT and Br were significantly different for each parameter suggesting that WT kidneys possessed more glomeruli, but these were less clustered compared to Br. Simulations suggested that WT and Br demonstrated similar, but not identical, underlying glomerular spatial distributions. Defective gene expression in Br is important for determining glomerular number and the defective pattern likely results from a heterochronic disturbance consisting of a truncated growth trajectory during embryonic kidney development. Anat Rec, 2010.

Cloning, expression, and purification of the N-terminal heme-binding domain of globin-coupled sensors.

Globin-coupled sensors (GCSs) are multidomain proteins, consisting of an N-terminal globin domain fused to a variety of C-terminal transmitter domains. Functional classification of GCSs is based on the transmitter domain(s) they possess, broadly falling under either aerotaxis or gene regulation. This chapter describes methods and strategies for cloning, expression, and purification of GCSs for spectroscopic analysis and determination of the minimum heme-binding domain, together with bioinformatic approaches for database searching and examination of domain architectures.