GanR represses the <i>gan</i> operon and the <i>ganR</i> gene.

<p><b>(A)</b> Assays of β-galactosidase activities by the reporter strains bearing either P_<i>ganS</i>-<i>lacZ</i>, or P_<i>ganR</i>-<i>lacZ</i>, or P_<i>ganB</i>-<i>lacZ</i> in the wild type strain (blue bars; YC1073, YC1085, and YC1088) and the <i>ganR</i> mutant (red bars; YC1074, YC1086, and YC1089). A deletion mutation in <i>ganA</i> was also introduced into the above strains. Cells were grown in LB shaking broth to OD₆₀₀ = 1 before harvest and analyses. Assays were done in triplicates and error bars represent standard deviations. <b>(B-C)</b> Assays of ß-galactosidase activities by the wild type reporter strains bearing either P_<i>ganS</i>-<i>lacZ</i>(YC1073, panel B) or P_<i>ganR</i>-<i>lacZ</i>(YC1085, panel C). Cells were grown in LB shaking culture over a period of 5.5 hours after inoculation. Both culture densities (red squares, right-hand y-axis) and ß-galactosidase activities of cells (blue diamonds, left-hand y-axis) were measured. Assays were repeated multiple times and representative data was shown here. <b>(D)</b> Assays of ß-galactosidase activities by the P_<i>ganS</i>-<i>lacZ</i> reporter strains in the wild type background (YC1071), the Δ<i>sinR</i> (YC1091), Δ<i>spo0A</i> (YC1092), Δ<i>degU</i> (YC1248), and Δ<i>ccpA</i> (YC1249) mutants. The <i>ganA</i> deletion mutation was not introduced into the above strains. In some mutants, an <i>epsH</i> deletion mutation was also introduced to prevent cell aggregation during shaking growth [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0179761#pone.0179761.ref045" target="_blank">45</a>]. Cells were grown in LB shaking culture to OD₆₀₀ = 1 before harvest and analyses. Error bars represent standard deviations. <b>(E)</b> Display of the promoter regions of <i>ganS</i> and <i>ganR</i> from <i>B</i>. <i>subtilis</i> NCIB3610 and <i>B</i>. <i>licheniformis</i> ATCC8480. The inverted repeats are highlighted in red, the -35 and -10 motifs of the sigma A-dependent promoter are underlined and shown in italic. ATG or GTG start codons of <i>ganS</i> or <i>ganR</i> are highlighted in blue. The <i>cre</i> box for putative CcpA binding sequences in the <i>ganS</i> promoter regions is highlighted in green. The transcriptional start of the <i>ganS</i> gene in <i>B</i>. <i>subtilis</i> was determined in a very recent study [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0179761#pone.0179761.ref029" target="_blank">29</a>] and labeled as +1. <b>(F)</b> The consensus DNA motif logo was generated from a multiple sequence alignment of the putative motifs from the selected promoters using WebLogo [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0179761#pone.0179761.ref031" target="_blank">31</a>]. The height of each stack, displayed in bits, is representative of the frequency of the nucleotide in the motif.</p

A negative feedback regulation on GanA by its catalytic product galactose.

<p><b>(A)</b> The wild type strain (3610) and the <i>ganR</i> mutant (YC222S) were streaked out on LB plates supplemented with 40 μg ml^-1 X-gal, and without (upper panel) or with (lower panel) galactose (0.5%, w/v). Plates were incubated at 37°C overnight before images were taken. <b>(B-C)</b> Assays of ß-galactosidase activities of protein lysates from cells expressing <i>ganA</i> (panel B, YC222S) or <i>lacZ</i> (panel C, YC1074). Assays were done in the presence of 2.5 mM ONPG and a gradient of galactose (from 2.5 to 20 mM). Error bars represent standard deviations from multiple trials. <b>(D)</b> An overview of complex regulations on galactan utilization involving both (1) a positive feedback mechanism on the transcription of the <i>gan</i> operon by ß-1,4-galactobiose and (2) a negative feedback mechanism at the protein level on GanA by its catalytic product galactose.</p

Strains used in this study.

<p>Strains used in this study.</p

The role of the <i>gan</i> operon in galactan utilization and biofilm formation in <i>B</i>. <i>subtilis</i>.

<p><b>(A)</b> A working model for the complete galactan utilization pathway in <i>B</i>. <i>subtilis</i>. UDP-Glu and UDP-Gal are the expected end products of galactan utilization carried out by products of the <i>gan</i> and <i>gal</i> genes. UDP-Glu and UDP-Gal are also two essential sugar nucleotide precursors for biosynthesis of exopolysaccharides (EPS) [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0179761#pone.0179761.ref020" target="_blank">20</a>]. The Leloir pathway consists of <i>galK</i>, <i>galT</i>, and <i>galE</i>, whose products convert galactose to UDP-Glu [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0179761#pone.0179761.ref021" target="_blank">21</a>]. EPS biosynthesis is carried out by enzymes encoded in the <i>espA-O</i> operon, which is indirectly activated by the master regulator Spo0A. Activation of Spo0A by protein phosphorylation in turn depends on multiple sensory histidine kinases including KinB [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0179761#pone.0179761.ref008" target="_blank">8</a>]. It is hypothesized in this study that hydrolyzed products of galactan (e.g. galactobiose) can induce <i>kinB</i> expression via the action of GanR (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0179761#sec023" target="_blank">discussion</a>). <b>(B)</b> Genetic organization of the galactan utilization genes in <i>B</i>. <i>subtilis</i> NCIB3610 and <i>B</i>. <i>licheniformis</i> ATCC8480. Putative promoters and transcription terminators are indicated. Different from <i>B</i>. <i>licheniformis</i>, in the genome of <i>B</i>. <i>subtilis</i>, the <i>galTK</i> genes and the <i>galE</i> gene are separated from the <i>gan</i> operon. In <i>B</i>. <i>subtilis</i>, the <i>gan</i> operon is also only four genes away from the <i>epsA-O</i> operon. Known or proposed functions of the <i>gan</i> and <i>gal</i> genes are as follows: <i>ganSPQ</i> encodes a permease for uptake of galactoligosaccharides; <i>ganA</i> encodes a ß-galactosidase; <i>ganB</i> encodes an endo ß-1,4-galactanse; <i>ganR</i> encodes a transcription repressor; <i>galK</i> encodes a galactokinase; <i>galT</i> for galactose-1-phosphate uridyltransferase; <i>galE</i> for the UDP-galactose-4-epimerase. <b>(C)</b> Development of pellicle biofilms by <i>B</i>. <i>licheniformis</i> ATCC8480, <i>B</i>. <i>subtilis</i> NCIB3610, and <i>B</i>. <i>cereus</i> AR156 in LB supplemented with 0.5% galactan (w/v). LB itself is a biofilm-inert medium for the above strains and used as a control. Images were taken after incubation at 30°C for 3 days. The scale bar represents 0.5 cm.</p

Mutations in inverted DNA repeats in P_<i>ganS</i> decrease GanR binding.

<p><b>(A-C)</b> Gel mobility shift assays to determine binding of His₆-GanR proteins to the wild type DNA sequence <b>(A)</b>, the Mut1 mutagenic sequence <b>(B)</b>, and Mut2 mutagenic sequence <b>(C)</b> of the <i>ganS</i> promoter. In all lanes, 16 ng (approximately 5 nM) fluorescent DNA probe was added. His₆-GanR proteins were added in a range of concentrations (from 4, 1.3, 0.8, 0.4, to 0.08 μM). In each panel, the right-most lane is the fluorescent probe alone. The left-most lane contains 0.8 μM His₆-GanR proteins, 16 ng of fluorescent probe, and 160 ng of unlabeled cold probe for competitive binding. In the upper section in each gel, shifted DNA bands were indicated by arrows. <b>(D)</b> The ratio of shifted versus total DNA was quantified from panels A-C, and graphed to show percent probe shifted versus protein concentration using WT, Mut1, and Mut2 probes.</p

Characterization of the regulation of a plant polysaccharide utilization operon and its role in biofilm formation in <i>Bacillus subtilis</i>

<div><p>The soil bacterium <i>Bacillus subtilis</i> is often found in association with plants in the rhizosphere. Previously, plant polysaccharides have been shown to stimulate formation of root-associated multicellular communities, or biofilms, in this bacterium, yet the underlying mechanism is not fully understood. A five-gene <i>gan</i> operon (<i>ganSPQAB</i>) in <i>B</i>. <i>subtilis</i> has recently been shown to be involved in utilization of the plant-derived polysaccharide galactan. Despite these findings, molecular details about the regulation of the operon and the role of the operon in biofilm formation remain elusive. In this study, we performed comprehensive genetic analyses on the regulation of the <i>gan</i> operon. We show that this operon is regulated both by a LacI-like transcription repressor (GanR), which directly binds to pairs of inverted DNA repeats in the promoter region of the operon, and by the catabolite control protein A (CcpA). Derepression can be triggered by the presence of the inducer β-1,4-galactobiose, a hydrolysis product of galactan, or <i>in situ</i> when <i>B</i>. <i>subtilis</i> cells are associated with plant roots. In addition to the transcriptional regulation, the encoded ß-galactosidase GanA (by <i>ganA</i>), which hydrolyzes ß-1,4-galactobiose into galactose, is inhibited at the enzymatic level by the catalytic product galactose. Thus, the galactan utilization pathway is under complex regulation involving both positive and negative feedback mechanisms in <i>B</i>. <i>subtilis</i>. We discuss about the biological significance of such complex regulation as well as a hypothesis of biofilm induction by galactan via multiple mechanisms.</p></div

GanR directly binds to the promoters of <i>ganS</i> and <i>ganR</i>.

<p><b>(A)</b> Gel mobility shift assays to probe binding of purified His₆-GanR to the DNAs containing the promoter sequence of <i>ganS</i>, <i>ganR</i>, or <i>yvaB</i>. Approximately 1 μg (approximately 0.3 μM) of DNA was added to each lane, His₆-GanR was added at increasing concentrations from 1, 3, 10, to 30 μM, no protein was added in the control lanes (left-most). Mobility retarded DNA bands were indicated by arrows. <b>(B)</b> The DNA sequence of the <i>ganS</i> promoter in <i>B</i>. <i>subtilis</i>. The -35 and -10 motifs of the sigma A-dependent promoter are highlighted in italic. The putative GanR binding motifs are labeled from Box1 to Box4. <b>(C)</b> A schematic display of site-directed mutagenesis on the putative GanR binding sites in the <i>ganS</i> promoter (mut1, mut2, and mut3). Letters in red are the introduced nucleotide changes in each of the boxes. Mutagenesis in Box4 was avoided due to overlap with the -35 motif of the promoter. <b>(D)</b> Assays of ß-galactosidase activities by the P_<i>ganS</i>-<i>lacZ</i> reporter strains with either the wild type promoter sequence of <i>ganS</i>, or with various sited-directed mutations shown in (C). Cells were grown in LB shaking culture to OD₆₀₀ = 1 before harvest and analyses. Error bars represent standard deviations from four independent analyses.</p