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Tai, V, Poon AFY, Paulsen IT, Palenik B.  2011.  Selection in coastal Synechococcus (cyanobacteria) populations evaluated from environmental metagenomes. Plos One. 6:e24249. AbstractWebsite

Environmental metagenomics provides snippets of genomic sequences from all organisms in an environmental sample and are an unprecedented resource of information for investigating microbial population genetics. Current analytical methods, however, are poorly equipped to handle metagenomic data, particularly of short, unlinked sequences. A custom analytical pipeline was developed to calculate dN/dS ratios, a common metric to evaluate the role of selection in the evolution of a gene, from environmental metagenomes sequenced using 454 technology of flow-sorted populations of marine Synechococcus, the dominant cyanobacteria in coastal environments. The large majority of genes (98%) have evolved under purifying selection (dN/dS<1). The metagenome sequence coverage of the reference genomes was not uniform and genes that were highly represented in the environment (i.e. high read coverage) tended to be more evolutionarily conserved. Of the genes that may have evolved under positive selection (dN/dS>1), 77 out of 83 (93%) were hypothetical. Notable among annotated genes, ribosomal protein L35 appears to be under positive selection in one Synechococcus population. Other annotated genes, in particular a possible porin, a large-conductance mechanosensitive channel, an ATP binding component of an ABC transporter, and a homologue of a pilus retraction protein had regions of the gene with elevated dN/dS. With the increasing use of next-generation sequencing in metagenomic investigations of microbial diversity and ecology, analytical methods need to accommodate the peculiarities of these data streams. By developing a means to analyze population diversity data from these environmental metagenomes, we have provided the first insight into the role of selection in the evolution of Synechococcus, a globally significant primary producer.

Dyhrman, ST, Palenik B.  2001.  A single-cell immunoassay for phosphate stress in the dinoflagellate Prorocentrum minimum (Dinophyceae). Journal of Phycology. 37:400-410.   10.1046/j.1529-8817.2001.037003400.x   AbstractWebsite

Current techniques for studying phytoplankton physiology in the field, such as measurements of biochemical activities, nutrient addition bioassays, and determination of photosynthetic efficiency, are useful for assessing the physiology of the bulk community but suffer from a lack of specificity. This would be improved by the development of single-cell methods for monitoring in situ physiology, Here we develop and test an antibody-based assay for identifying phosphate stress in the model dinoflagellate Prorocentrum minimum (Pavillard) Schiller, Antiserum was raised against a cell-surface alkaline phosphatase purified from P, minimum. Western screening indicated that the antiserum reacted with phosphate-stressed cells but not nitrate-stressed or phosphate-replete cells in culture. Immunodepletion confirmed the identification of this protein as an alkaline phosphatase, Based on Western blots, the antiserum appeared to be specific for phosphate-regulated proteins in P, minimum because there is no discernible cross-reaction with closely related P, micans. A whole-cell immunofluorescence assay was used to identify phosphate stress in field populations of P, minimum from Narragansett Bay, Rhode Island. The percentage of labeled P, minimum cells in this environment during the summer of 1998 decreased through time as the inorganic phosphate concentration increased. The percentage of antibody-labeled cells significantly correlated with the percentage of ELF-97-labeled cells determined as another single-cell assay of phosphate stress. This is the first antibody-based method developed for monitoring cell-specific physiology in a dinoflagellate, and the method described here may serve as a model for developing similar tools in other species of phytoplankton.

Bradley, JM, Hill N, Le Brun NE, Stuart RK, Palenik B.  2014.  Spectroscopic investigation of iron mineralisation by a cyanobacterial ferritin. Journal of Biological Inorganic Chemistry. 19:S291-S291. AbstractWebsite
Thomas, EV, Phillippy KH, Brahamsha B, Haaland DM, Timlin JA, Elbourne LDH, Palenik B, Paulsen IT.  2009.  Statistical analysis of microarray data with replicated spots: a case study with Synechococcus WH8102. Comparative and Functional Genomics.   10.1155/2009/950171   AbstractWebsite

Until recently microarray experiments often involved relatively few arrays with only a single representation of each gene on each array. A complete genome microarray with multiple spots per gene (spread out spatially across the array) was developed in order to compare the gene expression of a marine cyanobacterium and a knockout mutant strain in a defined artificial seawater medium. Statistical methods were developed for analysis in the special situation of this case study where there is gene replication within an array and where relatively few arrays are used, which can be the case with current array technology. Due in part to the replication within an array, it was possible to detect very small changes in the levels of expression between the wild type and mutant strains. One interesting biological outcome of this experiment is the indication of the extent to which the phosphorus regulatory system of this cyanobacterium affects the expression of multiple genes beyond those strictly involved in phosphorus acquisition. Copyright (C) 2009 E. V. Thomas et al.

Davis, AK, Hildebrand M, Palenik B.  2005.  A stress-induced protein associated with the girdle band region of the diatom Thalassiosira pseudonana (Bacillariophyta). Journal of Phycology. 41:577-589.   10.1111/j.1529-8817.2005.00076.x   AbstractWebsite

We report the characterization of a cell-surface protein isolated from the centric diatom Thalassiosira pseudonana Hasle and Heimdal. This protein has an apparent molecular weight of 150 kDa, is highly acidic, and is intimately associated with the cell wall. Although originally identified in cells experiencing copper toxicity, it is also induced by silicon and iron limitation but not by phosphate or nitrate limitation. Using immunofluorescence techniques, the 150-kDa protein was localized to the girdle band region and covered the elongated girdle band region of morphologically aberrant cells suffering from copper toxicity. Although having biochemical similarities to girdle band associated proteins identified in pennate diatoms known as pleuralins, the 150-kDa protein is not a sequence homolog and is predicted to have a number of unique features, such as chitin binding domains and a possible RGD cell attachment motif. Results presented here suggest that this protein is normally cell cycle regulated and may be involved in stabilizing cells during the division process.

Snyder, DS, Brahamsha B, Azadi P, Palenik B.  2009.  Structure of compositionally simple lipopolysaccharide from marine Synechococcus. Journal of Bacteriology. 191:5499-5509.   10.1128/jb.00121-09   AbstractWebsite

Lipopolysaccharide (LPS) is the first defense against changing environmental factors for many bacteria. Here, we report the first structure of the LPS from cyanobacteria based on two strains of marine Synechococcus, WH8102 and CC9311. While enteric LPS contains some of the most complex carbohydrate residues in nature, the full-length versions of these cyanobacterial LPSs have neither heptose nor 3-deoxy-D-manno-octulosonic acid (Kdo) but instead 4-linked glucose as their main saccharide component, with low levels of glucosamine and galacturonic acid also present. Matrix-assisted laser desorption ionization mass spectrometry of the intact minimal core LPS reveals triacylated and tetraacylated structures having a heterogeneous mix of both hydroxylated and nonhydroxylated fatty acids connected to the diglucosamine backbone and a predominantly glucose outer core-like region for both strains. WH8102 incorporated rhamnose in this region as well, contributing to differences in sugar composition and possibly nutritional differences between the strains. In contrast to enteric lipid A, which can be liberated from LPS by mild acid hydrolysis, lipid A from these organisms could be produced by only two novel procedures: triethylamine-assisted periodate oxidation and acetolysis. The lipid A contains odd-chain hydroxylated fatty acids, lacks phosphate, and contains a single galacturonic acid. The LPS lacks any limulus amoebocyte lysate gelation activity. The highly simplified nature of LPSs from these organisms leads us to believe that they may represent either a primordial structure or an adaptation to the relatively higher salt and potentially growth-limiting phosphate levels in marine environments.

Toledo, G, Palenik B, Brahamsha B.  1999.  Swimming marine Synechococcus strains with widely different photosynthetic pigment ratios form a monophyletic group. Applied and Environmental Microbiology. 65:5247-5251. AbstractWebsite

Unicellular marine cyanobacteria are ubiquitous in both coastal and oligotrophic regimes. The contribution of these organisms to primary production and nutrient cycling is substantial on a global scale. Natural populations of marine Synechococcus strains include multiple genetic lineages, but the link, if any, between unique phenotypic traits and specific genetic groups is still not understood. We studied the genetic diversity (as determined by the DNA-dependent RNA polymerase rpoC1 gene sequence) of a set of marine Synechococcus isolates that are able to swim, Our results show that these isolates form a monophyletic group. This finding represents the first example of correspondence between a physiological trait and a phylogenetic group in marine Synechococcus. In contrast, the phycourobilin (PUB)/phycoerythrobilin (PEB) pigment ratios of members of the motile clade varied considerably. An isolate obtained from the California Current (strain CC9703) displayed a pigment signature identical to that of nonmotile strain WH7803, which is considered a model for low-PUB/PEB-ratio strains, whereas several motile strains had higher PUB/PEB ratios than strain WH8103, which is considered a model for high-PUB/PEB-ratio strains. These findings indicate that the PUB/FEB pigment ratio is not a useful characteristic for defining phylogenetic groups of marine Synechococcus strains.

Pantoja, S, Lee C, Marecek JF, Palenik BP.  1993.  Synthesis and use of fluorescent molecular probes for measuring cell-surface enzymatic oxidation of amino-acids and amines in seawater. Analytical Biochemistry. 211:210-218.   10.1006/abio.1993.1259   AbstractWebsite

A method for investigating cell-surface enzymatic oxidative deamination of amino acids and amines in sea water was developed. This technique used synthetic fluorescent Lucifer Yellow derivatives of the amino acid lysine and the amine cadaverine as molecular probes to investigate oxidation pathways and rates. The probes were chemically stable under the conditions used and did not adsorb to container surfaces. The oxidative deamination of the fluorescent probes added to phyto-plankton cultures and the subsequent production of their fluorescent oxidation products could be selectively detected by HPLC at 250 pM levels. This approach allows selective investigation of cell-surface enzymatic oxidation since neither transport of the probes across the cell membrane nor chemical transformation of the probes occurs. Bacteria were also capable of oxidizing the fluorescent amino acid probe.