Schematic representations of <i>Mp</i>AFP and other large adhesion proteins.

<p>(A) Proteins are drawn to scale. Each Ig-like unit of the large repetitive region is shown as a green rectangle. The RTX-repeats in the C-terminal region are illustrated as brown blocks. The schematic diagrams are based on the following sequences from NCBI: <i>Pseudomonas putida</i> KT2440 LapA (NP_742337) and LapF (NP_742967), <i>M. sp. MWYL1</i> hemolysin-type calcium-binding protein (YP_001340713), <i>M. posidonica</i> outer membrane adhesin-like protein (YP_004481992) and the <i>M. mediterranea</i> homologues (YP_004313542+YP_004313541) that are separated by one nucleotide preceding an irregular GTG start codon. (B) Weblogo representations of 9-aa RTX repeats. Consensus plots are shown for: i) 12 RTX repeats in <i>Mp</i>AFP_RIV; ii) 29 RTX repeats from homologs of <i>Mp</i>AFP from three other <i>Marinomonas</i> species shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0048805#pone-0048805-g005" target="_blank">Fig. 5A; iii</a>) 10 RTX repeats in adhesion proteins LapA and LapF. As shown in the weblogo plots, the RTX repeats from the three homologs of <i>Mp</i>AFP and the LAPs follow the consensus of the conventional nine-residue RTX repeats of GGxGxDxUx (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0048805#pone-0048805-g005" target="_blank">Fig. 5Bii and 5Biii</a>). The RTX-like repeats in <i>Mp</i>AFP_RIV deviate from the conventional RTX repeats by introducing conserved ice-binding residues at positions 3 and 5 (Thr and Asx, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0048805#pone-0048805-g005" target="_blank">Fig. 5Bi</a>). Residues are colored black except for Gly (orange), Thr (red), and Ala (green).</p

Re-Evaluation of a Bacterial Antifreeze Protein as an Adhesin with Ice-Binding Activity

<div><p>A novel role for antifreeze proteins (AFPs) may reside in an exceptionally large 1.5-MDa adhesin isolated from an Antarctic Gram-negative bacterium, <em>Marinomonas primoryensis</em>. <em>Mp</em>AFP was purified from bacterial lysates by ice adsorption and gel electrophoresis. We have previously reported that two highly repetitive sequences, region II (RII) and region IV (RIV), divide <em>Mp</em>AFP into five distinct regions, all of which require mM Ca²⁺ levels for correct folding. Also, the antifreeze activity is confined to the 322-residue RIV, which forms a Ca²⁺-bound beta-helix containing thirteen Repeats-In-Toxin (RTX)-like repeats. RII accounts for approximately 90% of the mass of <em>Mp</em>AFP and is made up of ∼120 tandem 104-residue repeats. Because these repeats are identical in DNA sequence, their number was estimated here by pulsed-field gel electrophoresis. Structural homology analysis by the Protein Homology/analogY Recognition Engine (Phyre2) server indicates that the 104-residue RII repeat adopts an immunoglobulin beta-sandwich fold that is typical of many secreted adhesion proteins. Additional RTX-like repeats in RV may serve as a non-cleavable signal sequence for the type I secretion pathway. Immunodetection shows both repeated regions are uniformly distributed over the cell surface. We suggest that the development of an AFP-like domain within this adhesin attached to the bacterial outer surface serves to transiently bind the host bacteria to ice. This association would keep the bacteria within the upper reaches of the water column where oxygen and nutrients are potentially more abundant. This novel envirotactic role would give AFPs a third function, after freeze avoidance and freeze tolerance: that of transiently binding an organism to ice.</p> </div

Statistics of homology modeling studies on <i>Mp</i>AFP domains.

<p>Statistics of homology modeling studies on <i>Mp</i>AFP domains.</p

Lambda library clones encoding <i>Mp</i>AFP.

<p>(A) Schematic layout of the lambda clone containing DNA coding for the C-terminal portion of <i>Mp</i>AFP and nine downstream genes, 12–20. The flanking genes (blue arrows) are identified by their numbers in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0048805#pone.0048805.s005" target="_blank">Table S2</a>. The arrows point in the direction of transcription. The left and right arms of lambda phage (not to scale) are indicted by the tan-colored rectangles. Green boxes represent the tandem, identical 312-bp repeats of <i>Mp</i>AFP. The question mark represents the unknown number of these repeats present within the clone. The location of the initial hybridization probe is indicated by the horizontal red line. The scale is indicated by the bracket representing DNA coding for 1600 amino acids (aa's). (B) Schematic layout of a second lambda clone that contains DNA coding for the N-terminal portion of <i>Mp</i>AFP and eleven upstream genes (1–11). Symbols are as described in (A), with the bracket representing DNA coding for 600 amino acids and the red bar indicates the location of the second hybridization probe.</p

Amino acid sequence of <i>Mp</i>AFP.

<p>The complete amino acid sequence of <i>Mp</i>AFP is divided into five distinct regions (RI–RV). Because it was not possible to sequence through the DNA encoding the highly repetitive RII (∼120 identical 104-aa repeats), the protein was deposited in NCBI with two accession codes: ABL74377 and ABL74378. The two segments of <i>Mp</i>AFP are separated by a line of “Xs”. The first segment of <i>Mp</i>AFP contains RI and two 104-aa repeats of RII. The residues are identified with asterisks (1*–602*). The second segment begins with two 104-aa repeats of RII and continues through RIII, RIV and RV, with the residues identified by regular numbers (1–1567). The color scheme for the highlighted residues corresponds to that of <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0048805#pone-0048805-g006" target="_blank">Fig. 6</a>. The second 104-aa repeat of RII is indicated in bold underlined letters in both segments of <i>Mp</i>AFP. The nine-residue RTX-like repeats in RIV and RV are represented by bold double-underlined letters. The boxed sequences EADATFEAANISVGR and IDAGTGNDEIYIK are the two sequenced tryptic peptides identified by tandem MS/MS (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0048805#pone.0048805.s004" target="_blank">Table S1</a>) that were used to design degenerate PCR primers that amplified the first nucleic acid probe used for the isolation of the <i>Mp</i>AFP gene.</p

Localization of <i>Mp</i>AFP by immunofluorescence.

<p>Immobilized <i>M. primoryensis</i> cells were fixed in 1% paraformaldehyde before being incubated with anti-RII (A, B and C) or anti-RIV (D, E and F) polyclonal antibodies, followed by Alexa Fluor 350 - conjugated goat anti-rabbit secondary antibody (blue in A and D) and SYTO 9 (green in B and E). Images (C) and (F) are composite images of (A) and (B), and (D) and (E), respectively. A 10-micron scale marker is shown by the white horizontal line in panels C and F.</p

Estimation of 312-bp repeat copy number by Southern blotting.

<p>(A) Schematic diagram of the <i>Mp</i>AFP coding region illustrating the relative positions of restriction enzyme cut sites. Cross-hatched lines indicate the break between the two segments of <i>Mp</i>AFP coding region described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0048805#pone-0048805-g003" target="_blank">Fig. 3</a>. Four restriction enzymes, including <i>Mse</i>I, <i>Ase</i>I, <i>Pst</i>I and <i>Alu</i>I cut only outside the 312-bp repeats. Since the cut site of <i>Mse</i>I (T/TAA) is present within that of <i>Ase</i>I (AT/TAAT), <i>Mse</i>I is indicated in parentheses beside <i>Ase</i>I. <i>Msp</i>I is the only restriction enzyme in this set that cuts within the 312-bp repeats in RII, and it also cuts once in RI. (B) Southern blot of the digests using a 2×312-bp repeat from RII as the probe. Undigested DNA (lane 6) remained near the well, whereas the four restriction enzymes <i>Alu</i>I, <i>Pst</i>I, <i>Ase</i>I and <i>Mse</i>I (lane 1, 2, 4 and 5) that cut outside the repeats produced a fragment of approximately 37,500 bp in length. The <i>Msp</i>I (lane 3) partial digest produced a ladder of bands at 312-bp intervals and those containing between 2 (624 bp) and 13 (4056 bp) repeats are marked on this blot. DNA length markers (kb) are indicated on the left of the blot.</p

Homology models produced for domains of <i>Mp</i>AFP using Phyre2.

<p>The linear map of the regions of <i>Mp</i>AFP is colored as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0048805#pone-0048805-g003" target="_blank">Fig. 3</a>. Hatched lines indicate the break between the two contiguously sequenced segments of <i>Mp</i>AFP. Uncolored regions could only be modeled <i>ab initio</i> by the Phyre2 server and did not produce reliable results. Colored regions were modeled at greater than 90% confidence and their structures in ribbon representation are shown above and below the map of <i>Mp</i>AFP. The X-ray crystal structure of RIV is shown <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0048805#pone.0048805-Garnham2" target="_blank">[9]</a>. Residues for each model/structure are numbered according to the sequence in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0048805#pone-0048805-g003" target="_blank">Fig. 3</a>. The protein folds are shown in the colors of the rainbow from the N terminus (blue) to the C terminus (red).</p

Polyacrylamide gel electrophoresis of <i>Mp</i>AFP-enriched fractions.

<p>(A) The ice-bound fractions from one (lane 1) and two (lane 2) cycles of ice affinity purification were subjected to SDS-PAGE and stained with Coomassie blue. <i>Mp</i>AFP barely entered the resolving section of the gel. The locations of molecular mass markers (kDa) are indicated on the left. A right-pointing arrow indicates the junction between the stacking and resolving. (B) The ice-bound fraction from the second cycle of ice affinity purification was electrophoresed on a non-denaturing gel and stained with Stains-all. <i>Mp</i>AFP was still present within the stacking section of the gel when the bromophenol blue marker had reached the bottom of the separating gel. A left-pointing arrow indicates the junction between the stacking and resolving gels.</p

Additional file 1 of Genome-wide identification of B-box zinc finger (BBX) gene family in Medicago sativa and their roles in abiotic stress responses

Supplementary Material 1: Table S1. Paralogous gene pairs in segmental duplication events of alfalfa MsBBX genes. Table S2. Collinear genes of MsBBXs between alfalfa and Arabidopsis, alfalfa and O. sativa, alfalfa and M. truncatula. Table S3. The functions of Cis-regulatory elements in MsBBX gene promoters. Table S4. The primers used in this stud