Scanning electron micrographs of wild-type and mutant biofilms.

<p>Cells were grown for 24 h on LB plates and smeared on a silicon slice on the object stage. The samples were then imaged using a MIRA 3 scanning electron microscope at a magnification of 20000× times with a voltage of 15 KV. Scale bar = 1 μm.</p

Micrographs of wild type and <i>clp</i> mutants obtained by optical microscopy.

<p>The autoaggregation phenotype was visualized by oil microscopy (Olympus CX31) at 100×/1.25 after incubation for 24 h. Each image is representative of four replicate experiments. Scale bar = 10 μm.</p

Roles of CLP proteins in bacterial aggregation.

<p>(A) Cells were grown in liquid LB medium for 72 h in 96-well plates. The images were obtained by viewing from the top to the bottom. (B) Cells viewed from front to back after standing for 10 h following 24 h incubation in glass test tubes. (C) Cell sedimentation assy. WT, Δ<i>clpA</i>, Δ<i>clpB</i>, Δ<i>clpC</i>, and Δ<i>clpD</i> bacteria were grown until OD₆₀₀ = 0.7 and the bacterial precipitates were suspended by mixing, before the OD₆₀₀ values were measured at 1 h intervals.</p

Architecture and amino acid sequences of ClpA-ClpD in <i>B</i>.

<p>amyloliquefaciens FZB42. The first position of each Glycine-Xaa-Yaa repeat is shown in red. The repetitive sequences of Gly-Xaa-Thr are underlined. The results of the <i>in silico</i> analysis of the potential domains are shown in blue with rectangular boxes and the descriptions are shown in green ellipses.</p

Homology between the nucleic acid sequences of putative domains in the four <i>clp</i> genes.

<p>Schematic representation to scale of the <i>clpA</i>, <i>clpB</i>, <i>clpC</i>, and <i>clpD</i> sequences from <i>B</i>. <i>amyloliquefaciens</i> FZB42. Translated Glycine-Xaa-Threonine repeats within the collagen-like domain (CL) are show by the red band; N, amino-terminal domains are shown by the blue band; C, carboxyl-terminal domains are shown by the yellow band. The proportion of shared homology between the terminal domains of each protein are shown by dotted arrowed lines. The positions of the four genes are registered according to the numbered nucleic acid bases in the genome of <i>B</i>. <i>amyloliquefaciens</i> FZB42. The sequence lengths of <i>clpA</i>, <i>clpB</i>, <i>clpC</i>, and <i>clpD</i> range from 763771 bp to 764457 bp, from 764571 bp to 766568 bp, from 766695 bp to 76945 bp, and from 768125 bp to 769504 bp, respectively.</p

Bacterial strains and plasmids used.

<p>Bacterial strains and plasmids used.</p

Collagen-Like Proteins (ClpA, ClpB, ClpC, and ClpD) Are Required for Biofilm Formation and Adhesion to Plant Roots by <i>Bacillus amyloliquefaciens</i> FZB42

<div><p>The genes of collagen-like proteins (CLPs) have been identified in a broad range of bacteria, including some human pathogens. They are important for biofilm formation and bacterial adhesion to host cells in some human pathogenic bacteria, including several <i>Bacillus</i> spp. strains. Interestingly, some bacterial CLP-encoding genes (<i>clps</i>) have also been found in non-human pathogenic strains such as <i>B. cereus</i> and <i>B. amyloliquefaciens</i>, which are types of plant-growth promoting rhizobacteria (PGPR). In this study, we investigated a putative cluster of <i>clps</i> in <i>B. amyloliquefaciens</i> strain FZB42 and a collagen-related structural motif containing glycine-X-threonine repeats was found in the genes RBAM_007740, RBAM_007750, RBAM_007760, and RBAM_007770. Interestingly, biofilm formation was disrupted when these genes were inactivated separately. Scanning electron microscopy and hydrophobicity value detection were used to assess the bacterial cell shape morphology and cell surface architecture of <i>clps</i> mutant cells. The results showed that the CLPs appeared to have roles in bacterial autoaggregation, as well as adherence to the surface of abiotic materials and the roots of <i>Arabidopsis thaliana</i>. Thus, we suggest that the CLPs located in the outer layer of the bacterial cell (including the cell wall, outer membrane, flagella, or other associated structures) play important roles in biofilm formation and bacteria-plant interactions. This is the first study to analyze the function of a collagen-like motif-containing protein in a PGPR bacterium. Knocking out each <i>clp</i> gene produced distinctive morphological phenotypes, which demonstrated that each product may play specific roles in biofilm formation. Our <i>in silico</i> analysis suggested that these four tandemly ranked genes might not belong to an operon, but further studies are required at the molecular level to test this hypothesis. These results provide insights into the functions of <i>clps</i> during interactions between bacteria and plants.</p></div

Variations in biofilm formation by the wild type and <i>clp</i> mutants.

<p>Biofilm formation by wild type and <i>clp</i> mutants in LB medium (A) and on MSgg medium plates (B). The images of colonies were obtained after incubation for 48 h at 37°C. (C) The biofilm images are top-down views of 96-well plates, which were obtained after incubation for 24 h at 37°C in MSgg liquid medium. (D) Quantitative spectrophotometric biofilm assay following crystal violet staining in MSgg medium. Analysis of variance detected a significant main group effect between the wild type and <i>clp</i> mutants (b, <i>P</i> < 0.05).</p

Primers used in this study.

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

Adherence capacities of wild type and <i>clp</i> mutants on abiotic and root surfaces.

<p>(A) Adherence capacities of wild type and <i>clp</i> mutants on polystyrene surfaces. The OD₆₀₀ values indicate the biofilm adherence capacities of the wild type, <i>clpA</i> mutant, <i>clpB</i> mutant, <i>clpC</i> mutant, and <i>clpD</i> mutant to polystyrene surfaces. The cells were grown in MSgg medium, LB medium, and MS medium, respectively. (B) Adherence capacities of wild type and <i>clp</i> mutants to the roots of <i>A</i>. <i>thaliana</i>. The experiments were performed five times and similar results were obtained. The values represent the means ± standard deviations based on 12 measurements. Analysis of variance detected a significant main group effect between the wild type and <i>clp</i> mutants (b, <i>P</i> < 0.05). (C) Scanning electron microscopy (SEM) of wild type and <i>clp</i> mutants adhere to the roots surface of <i>A</i>. <i>thaliana</i>. SEM images were taken 20 min after bacterial soaking. The samples were imaged using a MIRA 3 scanning electron microscope at 10 KV. Scale bar = 5 μm.</p