Co-incubation of the isopropanol cell wash extract and the culture supernatant.

<p>In agar well diffusion assays, the isopropanol extract (25 µl) showed an antimicrobial activity against <i>S. aureus</i> ATCC 33592 (blue bar) while the culture supernantant was inactive (see also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0006788#pone-0006788-g003" target="_blank">Fig. 3</a>). In order to test protease stability of the isopropanol extract, 25 µl of extract were mixed with 25 µl of culture supernatant (blue-green patterned bar), incubated for 2 h and then tested by agar diffusion. The co-incubation of both extracts had no effect on the activity of the isopropanol extract, indicating that the antimicrobial substance is stable against the proteases excreted by the producer strain.</p

Gene inactivation of the lantibiotic modifications enzyme LicM1 (A) and LicM2 (B) by homologous recombination.

<p>Gene inactivations of the modifications enzymes LicM1 and LicM2 were performed by plasmid integration using the recombinant plasmids pMADDelLicM1AC and pMADLicM2AC. The resulting insertion mutants <i>B. licheniformis</i> LicM1INT (A) and LicM2INT (B) harbour two copies of the <i>lanM</i> genes within the lantibiotic gene cluster: a copy that is transcribed but is truncated and a second copy that does not dispose of promoter, Shine Dalgarno sequence and start codon. Genes that derive from the plasmid pMADDelLicM1AC are marked in green colours and those deriving from pMADLicM2AC are marked in orange and red.</p

Amino acid alignments of two-peptide lantibiotics.

<p>(A) Amino acid sequence alignment of the LicA1 propeptide with the LanA1 propeptides of the two-peptide lantibiotics plantaricin (PlwA1, AAG02567), staphylococcin C55 (SacA1, BAB78438), lacticin 3147 (LtnA1, O87236), haloduracin (HalA1, BAB04173), BHT (BhtA1, AAZ76603) and Smb (SmbA1, BAD72777) and, for comparison, the propeptide of mersacidin (MrsA, Z47559). Amino acid identities of the LanA1 propeptides are highlighted in green (100%), pink (75%) and blue (35%). The thioether bridging pattern represents that of the Halα and Plwα peptide. (B) Amino acid sequence alignment of the LicA2 propeptide with the Lanβ/LanA2 propeptides of the two-peptide lantibiotics plantaricin (PlwA2, AAG02566), staphylococcin C55 (SacA2, BAB78439), lacticin 3147 (LtnA2, O87237), haloduracin (HalA2, BAB04172), BHT (BhtA2, AAZ76602) and Smb (SmbA2, BAD72776). Amino acid identities are highlighted in green (100%), pink (75%) and blue (35%). The thioether bridging pattern represents that of the Halβ and Plwβ peptides.</p

Stability assay: Treatment with proteases.

<p>No loss of both antimicrobial activities was detected after treatment with trypsin and chymotrypsin for 4 h for both extracts. However, incubation of the isopropanol cell wash extract (blue bar) with proteinase K and pronase E resulted in a total loss of activity against <i>M. luteus</i> while the antimicrobial activity of the culture supernant (green column) was unaffected by these proteases.</p

Oligonucleotides used in this study.

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

HPLC chromatogram of the <i>Bacillus licheniformis</i> isopropanol extract.

<p>The isopropanol extract was applied to a POROS RP-HPLC column and eluted in a gradient of 20% to 55% acetronitrile (containing 0.1% TFA). Maldi-TOF analysis of active fractions [+ medium activity, ++ strong activity and (+) poor activity] showed the presence of masses representing the Licα peptide or the Licβ peptide.</p

Agar well diffusion assays of the <i>B. licheniformis</i> DSM 13 culture supernatant and the isopropanol extract.

<p>The culture supernatant (green bars) as well as the isopropanol extract (blue bars) were active against Gram-positive bacteria but showed slightly different spectra of activity, indicating that supernatant and isopropanol cell wash extract contained different antibacterial compounds.</p

The lichenicidin gene cluster.

<p>The genes of the prepeptides are black; the genes of the two modification enzymes are grey. The other genes are marked with the following patterns: processing transporter, vertical stripes; the peptidase gene, checkerboard pattern; the putative regulator gene, horizontal stripes; genes that might be involved in immunity and encode proteins that have similarity to transporters, dots. The numbers give the last two digits of the locus tags in the annotation of the <i>B. licheniformis</i> DSM 13 isolate. For the small white orfs, no functions could be assigned so far, however similar orfs are encoded in the vicinity of the haloduracin gene cluster.</p

MALDI-TOF mass spectra of the <i>B. licheniformis</i> MW3 wild type (A) and its insertion mutans LicM1INT (B) and LicM2INT (C).

<p>The isopropanol extracts of the insertion mutants <i>B. licheniformis</i> LicM1INT and LicM2INT were characterized by the loss of activity against <i>M. luteus</i>. MALDI-TOF spectra of these isopropanol extracts in comparison to the wildtype (A) showed the loss of a peak at 3251 Da in the case of the LicM1 insertion mutant (B) indicating that this peak represents the protonated form of the active Licα peptide. The insertion of a plasmid into the gene of the modification enzyme LicM2 inactivated this enzyme and most probably exerted a polar effect on the downstream genes, thus affecting production of both peptides. This mutant did not produce the Licα peptide and is further characterized by the absence of a 3021 Da peak, which might represent the protonated form of the mature Licβ peptide, harboring 12 dehydrated residues (C). In some cultivations a further peak of 3039 Da was observed, which probably denotes a Licβ peptide with only 11 of 15 possible dehydrations.</p

Cytoplasmic Sulfurtransferases in the Purple Sulfur Bacterium <em>Allochromatium vinosum:</em> Evidence for Sulfur Transfer from DsrEFH to DsrC

<div><p>While the importance of sulfur transfer reactions is well established for a number of biosynthetic pathways, evidence has only started to emerge that sulfurtransferases may also be major players in sulfur-based microbial energy metabolism. Among the first organisms studied in this regard is the phototrophic purple sulfur bacterium <em>Allochromatium vinosum</em>. During the oxidation of reduced sulfur species to sulfate this Gammaproteobacterium accumulates sulfur globules. Low molecular weight organic persulfides have been proposed as carrier molecules transferring sulfur from the periplasmic sulfur globules into the cytoplasm where it is further oxidized via the “Dsr” (<b><u>d</u></b>issimilatory <b><u>s</u></b>ulfite <b><u>r</u></b>eductase) proteins. We have suggested earlier that the heterohexameric protein DsrEFH is the direct or indirect acceptor for persulfidic sulfur imported into the cytoplasm. This proposal originated from the structural similarity of DsrEFH with the established sulfurtransferase TusBCD from <em>E. coli</em>. As part of a system for tRNA modification TusBCD transfers sulfur to TusE, a homolog of another crucial component of the <em>A. vinosum</em> Dsr system, namely DsrC. Here we show that neither DsrEFH nor DsrC have the ability to mobilize sulfane sulfur directly from low molecular weight thiols like thiosulfate or glutathione persulfide. However, we demonstrate that DsrEFH binds sulfur specifically to the conserved cysteine residue DsrE-Cys78 <em>in vitro</em>. Sulfur atoms bound to cysteines in DsrH and DsrF were not detected. DsrC was exclusively persulfurated at DsrC-Cys111 in the penultimate position of the protein. Most importantly, we show that persulfurated DsrEFH indeed serves as an effective sulfur donor for DsrC <em>in vitro.</em> The active site cysteines Cys78 of DsrE and Cys20 of DsrH furthermore proved to be essential for sulfur oxidation <em>in vivo</em> supporting the notion that DsrEFH and DsrC are part of a sulfur relay system that transfers sulfur from a persulfurated carrier molecule to the dissimilatory sulfite reductase DsrAB.</p> </div