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Additional file 1. Supplementary information

Cloning, Purification and Characterization of the Collagenase ColA Expressed by <i>Bacillus cereus</i> ATCC 14579

<div><p>Bacterial collagenases differ considerably in their structure and functions. The collagenases ColH and ColG from <i>Clostridium histolyticum</i> and ColA expressed by <i>Clostridium perfringens</i> are well-characterized collagenases that cleave triple-helical collagen, which were therefore termed as ´true´ collagenases. ColA from <i>Bacillus cereus</i> (<i>B</i>. <i>cereus</i>) has been added to the collection of true collagenases. However, the molecular characteristics of <i>B</i>. <i>cereus</i> ColA are less understood. In this study, we identified ColA as a secreted true collagenase from <i>B</i>. <i>cereus</i> ATCC 14579, which is transcriptionally controlled by the regulon phospholipase C regulator (PlcR). <i>B</i>. <i>cereus</i> ATCC 14579 ColA was cloned to express recombinant wildtype ColA (ColA^wt) and mutated to a proteolytically inactive (ColA^E501A) version. Recombinant ColA^wt was tested for gelatinolytic and collagenolytic activities and ColA^E501A was used for the production of a polyclonal anti-ColA antibody. Comparison of ColA^wt activity with homologous proteases in additional strains of <i>B</i>. <i>cereus sensu lato</i> (<i>B</i>. <i>cereus s</i>.<i>l</i>.) and related clostridial collagenases revealed that <i>B</i>. <i>cereus</i> ATCC 14579 ColA is a highly active peptidolytic and collagenolytic protease. These findings could lead to a deeper insight into the function and mechanism of bacterial collagenases which are used in medical and biotechnological applications.</p></div

Collection of annotated collagenases in <i>Bacillus</i> strains blasted against Q81BJ6 (ColA) as protein query.

<p>Collection of annotated collagenases in <i>Bacillus</i> strains blasted against Q81BJ6 (ColA) as protein query.</p

Collagenolytic activity of ColA.

<p>(<b>A</b>) ColA ΔPP from <i>B</i>. <i>cereus</i> ATCC 14579, its inactive version ColA ΔPP^E501A, ColG from <i>C</i>. <i>histolyticum</i> and the protease domain of ColT (ColT^PD) from <i>C</i>. <i>tetani</i> were tested for their peptidase activity using FALGPA as a substrate. * <i>p</i> = 0.0161 indicates statistical significance (Student´s t-test, paired, one-tailed). (<b>B</b>) In <i>in vitro</i> cleavage assays, the positive control ColG and ColA ΔPP^wt were incubated with tropocollagen type I for the indicated time periods and analyzed by coomassie stained SDS PAGE to analyze their collagenolytic activities. (<b>C</b>) As negative controls, ColT^PD and ColAΔPP^E501A were investigated. (<b>D</b>) As indicated, α–chymotrypsin was incubated with tropocollagen type I as an additional negative control and compared to untreated tropocollagen type I for the indicated time periods and analyzed by coomassie stained SDS PAGE.</p

Gelatinolytic activities expressed by different <i>Bacillus</i> species.

<p>(<b>A</b>) Equal amounts of proteins in lysates of <i>B</i>. <i>subtilis (Bs)</i>, <i>B</i>. <i>megaterium (Bm)</i>, <i>B</i>. <i>thuringiensis (Bt)</i>, <i>B</i>. <i>weihenstephanensis (Bw)</i> and <i>B</i>. <i>cereus</i> ATCC 14579 <i>(Bc)</i> were analyzed for proteolytic activity in gelatin zymography. Protein standard (m) indicated molecular weights of gelatinolytic activities. (<b>B</b>) Efficient disruption of bacteria and equal protein amounts were demonstrated by coomassie-stained SDS PAGEs. Protein standard (m) indicated molecular weights of proteins.</p

Cloning, overexpression and activity of <i>B</i>. <i>cereus</i> ATCC 14579 ColA.

<p>(<b>A</b>) ColA is expressed as a 110.1 kDa protein and consists of a 3.3 kDa signal peptide (aa 1–30), a 7.2 kDa propetide (aa 31–92) and a 99.6 kDa C-terminal part (aa 93–960) of ColA. Expression constructs for N-terminally GST-tagged ColA ΔSP (132.8 kDa) and ColA ΔPP (125.6 kDa) were cloned. Glutamic acid (E) 501 in the active center of ColA was exchanged by an alanine (E501A) to create proteolytically inactive ColA. (<b>B</b>) The expression, enrichment and activity of GST-ColA ΔPP^wt and GST-ColA ΔPP^E501A proteins in IPTG-induced <i>E</i>. <i>coli</i> lysates or purified via GST pull down (PD) experiments were analyzed by SDS-PAGE (left panel) and gelatin zymography (middle panel). To purify ColA ΔPP^wt, transformed <i>E</i>. <i>coli</i> (-) were induced by IPTG to stimulate GST-ColA ΔPP^wt expression. After lysing bacteria, GST-ColA ΔPP^wt was bound to GST sepharose and either eluted by glutathione (PD) as a GST fusion protein (GST-ColA ΔPP^wt) or cleaved and eluted with the PreScission protease (PreSc) to obtain the untagged protease ColA ΔPP^wt.</p

ColA is secreted by <i>B</i>. <i>cereus</i> ATCC 14579.

<p><i>B</i>. <i>cereus</i> ATCC 14579 wildtype (wt) and its isogenic <i>ΔplcR</i> deletion mutant were harvested and disrupted after growing in liquid cultures for indicated time periods. Equal protein amounts of bacterial lysates (upper panel) and equal volumes of supernatants were analyzed by gelatin zymography (upper and middle panel) and Western blotting using a polyclonal antibody directed against ColA ΔPP^E501A (lower panel). Recombinant ColA ΔPP (rColA) was used as control.</p