Genetic Analysis of Collective Motility of <i>Paenibacillus</i> sp. NAIST15-1

<div><p>Bacteria have developed various motility mechanisms to adapt to a variety of solid surfaces. A rhizosphere isolate, <i>Paenibacillus</i> sp. NAIST15-1, exhibited unusual motility behavior. When spotted onto 1.5% agar media, <i>Paenibacillus</i> sp. formed many colonies, each of which moved around actively at a speed of 3.6 μm/sec. As their density increased, each moving colony began to spiral, finally forming a static round colony. Despite its unusual motility behavior, draft genome sequencing revealed that both the composition and organization of flagellar genes in <i>Paenibacillus</i> sp. were very similar to those in <i>Bacillus subtilis</i>. Disruption of flagellar genes and flagellar stator operons resulted in loss of motility. <i>Paenibacillus</i> sp. showed increased transcription of flagellar genes and hyperflagellation on hard agar media. Thus, increased flagella and their rotation drive <i>Paenibacillus</i> sp. motility. We also identified a large extracellular protein, CmoA, which is conserved only in several <i>Paenibacillus</i> and related species. A <i>cmoA</i> mutant could neither form moving colonies nor move on hard agar media; however, motility was restored by exogenous CmoA. CmoA was located around cells and enveloped cell clusters. Comparison of cellular behavior between the wild type and <i>cmoA</i> mutant indicated that extracellular CmoA is involved in drawing water out of agar media and/or smoothing the cell surface interface. This function of CmoA probably enables <i>Paenibacillus</i> sp. to move on hard agar media.</p></div

<i>Paenibacillus</i> sp. strains used in this study.

<p><i>Paenibacillus</i> sp. strains used in this study.</p

CmoA has extracellular functions.

<p>(A) Schematic drawing of the structure of CmoA. The positions of the N-terminal signal sequence (required for secretion), the vWFA domain, and the IPT/TIG domains (IPT) are shown. (B) Gene organization of the <i>cmoA</i> locus. The positions of the transcriptional terminator sequences are indicated by downward arrows. (C) Extracellular complementation of the <i>cmoA</i> mutant. Secreted protein fractions were prepared from plate cultures of the indicated strains and spread over the surface of 1.5% agar plates. Wild-type and mutant strains were then inoculated onto the center of those plates and grown at 37°C for 18 h. Strains: WT, wild-type; Δ<i>cmoA</i>, P260; and Δ<i>hag</i>, P261 in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006387#pgen.1006387.t001" target="_blank">Table 1</a>. Plate diameter, 9 cm. (D) Localization of CmoA. Wild-type and <i>cmoA-mCherry</i> (P205) strains were spotted onto 1.5% agar media and grew until small colonies moved out from the inoculation site. Coverslips were placed directly onto the moving colonies on the plates and examined under a fluorescence microscope. Merged phase-contrast (false-colored red) and mCherry (false-colored green) images are shown. Scale bar, 5 μm. (E) The leading edge zones of expanding wild-type and <i>cmoA</i> mutant colonies on 0.6% agar media. The swarm colonies were spreading from the upper left to the lower right in these photographs. The leading edge of the <i>cmoA</i> mutant swarm colony is indicated by arrows. Scale bar, 20 μm. F. Northern blot analysis of <i>cmoA</i>. Total RNA samples were isolated from wild-type cells grown in liquid media, on 0.5% agar media, or on 1.5% agar media. The positions of 23S and 16S rRNAs are indicated by arrow heads. rRNAs stained with methylene blue are shown as a loading control.</p

Wandering colonies are sensitive to wetness.

<p>Dilution of wild-type culture was spread over the surface of a 1.5% agar plate and incubated at 37°C until wandering colonies appeared (A). Water was gently poured onto the plate (B), and then the plate was gently shaken three times (C). Scale bar, 2 mm.</p

Complementation test of <i>motCD</i> and <i>motABmotCD</i> mutants.

<p>Multicopy plasmids carrying <i>motAB</i> (pHY<i>motAB</i>) or <i>motCD</i> (pHY<i>motCD</i>) were introduced into the <i>motCD</i> deletion mutant or the <i>motABmotCD</i> deletion mutant, and the motility ability of resultant strains was examined at 37°C for 18 h on 2×YT agar media. pHY300PLK is a parental plasmid. Plate diameter, 9 cm.</p

Binding of Promoter DNA to SoxR Protein Decreases the Reduction Potential of the [2Fe–2S] Cluster

The [2Fe–2S] transcriptional factor SoxR, a member of the MerR family, functions as a sensor of oxidative stress in <i>Escherichia coli</i>. The transcriptional activity of SoxR is regulated by the reversible oxidation and reduction of [2Fe–2S] clusters. Electrochemistry measurements on DNA-modified electrodes have shown a dramatic shift in the reduction potential of SoxR from −290 to +200 mV with the promoter DNA-bound [Gorodetsky, A. A., Dietrich, L. E. P., Lee, P. E., Demple, B., Newman, D. K., and Barton, J. K. (2008) DNA binding shifts the reduction potential of the transcription factor SoxR, Proc. Natl. Acad. Sci. U.S.A. 105, 3684−3689]. To determine the change of the SoxR reduction potential using the new condition, the one-electron oxidation–reduction properties of [2Fe–2S] cluster in SoxR were investigated in the absence and presence of the DNA. The [2Fe–2S] cluster of SoxR was completely reduced by nicotinamide adenine dinucleotide phosphate (NADPH)–cytochrome P450 reductase (CRP) in the presence of a NADPH generating system (glucose 6-dehydrogenase and glucose-6 phosphate), indicating that CRP can serve as an NADPH-dependent electron carrier for SoxR. The reduction potential of SoxR was measured from equilibrium data coupled with NADPH and CRP in the presence of electron mediators. The reduction potentials of DNA-bound and DNA-free states of SoxR were −320 and −293 mV versus NHE (normal hydrogen electrode), respectively. These results indicate that DNA binding causes a moderate shift in the reduction potential of SoxR

CmoA levels in the wild-type and mutant strains.

<p>Strains (WT, wild-type; <i>cmoA-mCherry</i>, P205, Δ<i>hag</i>, P261; Δ<i>cmoA</i>, P198 in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006387#pgen.1006387.t001" target="_blank">Table 1</a>) were grown at 37°C for 5 h on 1.5% agar media. The secreted protein (lanes 1, 3, 5, and 7) and cell-surface associated protein (lanes 2, 4, 6, and 8) fractions were prepared and separated by SDS-PAGE. The positions of CmoA-mCherry, CmoA, and flagellin are indicated by arrows.</p

Organization of flagellar genes in <i>Paenibacillus</i> sp. and <i>Bacillus subtilis</i>.

<p>Five flagellar gene loci are shown for each bacterium. Flagellar structural and regulatory genes are shown in yellow. Genes specific for <i>Paenibacillus</i> sp. and <i>Bacillus subtilis</i> are shown in green and blue, respectively. Genes not directly linked to motility are in white. Genes described in text are denoted by large letters. The gene map is not to scale.</p

Motility-defective mutants.

<p>Wild-type and mutant strains were inoculated onto the center of 2×YT solidified with the indicated agar concentrations. Plates were incubated at 37°C for 18 h. Plate diameter, 9 cm.</p

Motility of <i>Paenibacillus</i> sp.

<p>(A) Colony spreading pattern of <i>Paenibacillus</i> sp. The wild-type strain was inoculated onto the center of 2×YT plates containing a variety of agar concentrations and grown at 37°C for 18 h. Plate diameter, 9 cm. (B) Morphology of motile cells. The wild-type strain was inoculated onto the center of plates containing 0.3%, 0.5%, or 1.5% agar and incubated at 37°C for 6 h. Coverslips were placed directly on the surface of the leading edge zones of the colonies and cell morphology observed under a light microscope. Stacks of cells can be seen in the lower-right panel, and are indicated by an arrow. Scale bar, 5 μm.</p