Expression of the Lantibiotic Mersacidin in Bacillus amyloliquefaciens FZB42

Lantibiotics are small peptide antibiotics that contain the characteristic thioether amino acids lanthionine and methyllanthionine. As ribosomally synthesized peptides, lantibiotics possess biosynthetic gene clusters which contain the structural gene (lanA) as well as the other genes which are involved in lantibiotic modification (lanM, lanB, lanC, lanP), regulation (lanR, lanK), export (lanT(P)) and immunity (lanEFG). The lantibiotic mersacidin is produced by Bacillus sp. HIL Y-85,54728, which is not naturally competent

Escherichia coli alpha-Hämolysin: Untersuchungen zur Struktur und Porenbildung

Escherichia coli α-Hämolysin (HlyA) ist ein Prototyp der RTX-Toxine, die zu den α-porenbildenden Toxinen gehören. HlyA bildet Poren in einer Vielzahl eukaryontischer Zielzellen. Das 107 kDa große Protein besteht aus 1024 Aminosäuren, die gemeinsam mit den Proteinen für posttranslationale Modifikation und Sekretion in einem Operon codiert werden. Die N-terminale Hälfte von HlyA besteht aus mehreren amphipathischen α –Helices, die mit der Porenbildung assoziiert werden, gefolgt von der Calcium-bindenden RTX-Domäne in der C-terminalen Hälfte des Moleküls. Über den porenbildenen Mechanismus ist wenig bekannt. Die vorliegende Arbeit fokussierte sich auf die Frage, ob dieser Prozess eine Oligomerisierung mehrerer HlyA-Moleküle beinhaltet, oder ob die membranschädigende Struktur von einem Monomer gebildet wird. Drei unabhängige biochemische Methoden wurden in dem Versuch eingesetzt, HlyA-Oligomere in permeabilisierten Membranen zu detektieren. In allen drei Ansätzen wurden negative Ergebnisse erreicht, was das Konzept bestätigt, dass die Pore von HlyA von einem Monomer gebildet wird. PCR-basierte Cysteinsubstitutionen wurden durchgeführt, um den N-terminus von HlyA zu charakterisieren. Einzelne Cysteinreste wurden an 21 Positionen innerhalb der Aminosäuresequenz 13-55 eingeführt, und mit dem umgebungssensitiven Fluorophor Badan markiert. Spektrofluorimetrische Messungen zeigten, dass alle untersuchten Aminosäuren innerhalb dieser Domäne unabhängig von der porenbildenden Aktivität in die Membran inserieren. Deletionen der Aminosäuren 1-50 hatten keinen Einfluß auf die lytische Aktivität, während die Deletion der Aminosäuren 1-100 in einer fast vollständig inaktiven Toxinmutante resultierte. r Die Einführung von Prolinen durch PCR-basierte Mutagenese wurde durchgeführt, um die Beteiligung vorhergesagter α-Helices innerhalb der N-terminalen Hälfte von HlyA an der hämolytischen Aktivität zu untersuchen. Die Ergebnisse deuten darauf hin, dass die Struktur von mindestens vier vorhergesagten Helices bedeutend für die hämolytische Aktivität ist.Escherichia coli α-hemolysin (HlyA) is a prototype of RTX-toxins belonging to α-pore forming toxins. HlyA formes pores in a wide range of eucaryotic target cells. The 107 kDa protein is composed of 1024 amino acids encoded in an operon together with proteins for posttranslational modification and secretion. The N-terminal domain of HlyA harbours a series of amphipathic α-helices that are associated with pore formation, which is followed by calcium-binding RTX-domain in the C-terminal half of the molecule. Little is known about the mechanism of pore formation. The present study was focussed on the question if this process includes oligomerization of several HlyA molecules, or if the membrane pertubating structure is formed by a monomer. Three independent biochemical approaches were employed in an effort to detect HlyA-oligomers in permeabilized membranes. Negative results were obtained in every case, in line with the concept that the pore generated by HlyA is monomeric. Cysteine scanning mutagenesis was performed to characterize the N-terminus of the molecule. A single cysteine residue was introduced at 21 positions within the sequence spanning residues 13-55 and labeled with the polarity-sensitive dye badan. Spectrofluorimetric analyses indicated that amino acids in this domain insert into the lipid bilayer, and that this insertion is independent from pore forming activity. Deletion of amino acids 1-50 did not affect lytic activity, while deletion of amino acids 1-100 resulted in a nearly inactive toxin-mutant. Introduction of two prolines using PCR based mutagenesis was employed to investigate the contributions to hemolysis of predicted α-helices within the N-terminal half of HlyA. Results suggest that structural integrity of at least four predicted helices is important for haemolytic activity

Strains and plasmids used in this study.

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

Purification of mersacidin from culture supernatant of <i>B. amyloliquefaciens</i> mrs1 pPAR1.

<p>The culture supernatant of <i>B. amyloliquefaciens</i> mrs1 pPAR1 was applied to a Poros RP-HPLC column and eluted in a gradient of 30 % to 42 % acetonitrile (containing 0.1 % TFA). Active fractions were pooled and lyophilized. The resulting lyophilizate was resuspended in 5 % acetonitrile and applied to a Nucleosil RP-C18 column (<b>A</b>). The antimicrobial activity of the fractions was assayed in agar well diffusion tests against <i>M. luteus</i> (diameter 3.2 cm) (<b>C</b>) and analyzed by MALDI-TOF (<b>B</b>). Active fractions eluted after 20 min in a gradient of 50 – 65 % acetonitrile (0.1 % TFA) and were characterized by the presence of a peak with 1826.339 Da representing mersacidin.</p

Phylogenetic tree based on the partial nucleotide sequence of the <i>gyrA</i> gene.

<p>The tree was calculated based on the <i>gyrA</i> nucleotide sequences of the mersacidin producer (BHILY, marked by a red box) and different members of the genus <i>Bacillus</i> (NCBI accession numbers in brackets) [<i>B. amyloliquefaciens</i>  =  BAMY strains: FZB42 (CP000560), CAUB946 (FN652789), S23 (FN652780), ATCC15841 (FN662838), DSM7 (FN597644), NAUB3 (FN652783), NAUB55 (FN652801), UCMB5113 (AY212974); <i>B. licheniformis</i>  =  BLIC strains: MY75 (EU073420), DSM13 (BLi00007), CICC10085 (GQ355995); <i>B. subtilis</i>  =  BSUB strains: 168 (BSU00070), DV1-B1 (EF134416) and <i>B. cereus</i>  =  BCER strain: ATCC14579 (BC0006)]. The mersacidin wild type producer is placed among the members of the subspecies <i>B. amyloliquefaciens</i> subsp. <i>plantarum</i>; it does not belong to the subspecies <i>amyloliquefaciens</i> that consists of strains closely related to the type strain <i>B. amyloliquefaciens</i> DSM 7^T. It is also clearly not a member of the species <i>B. subtilis, B. cereus</i> or <i>B. licheniformis</i>.</p

Primers used in this study.

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

The (partial) mersacidin biosynthesis gene clusters of the mersacidin producer <i>B.</i> sp. HIL Y-85,54728, <i>B. amyloliquefaciens</i> FZB42 and its derivatives.

<p>The mersacidin gene cluster of the original producer strain <i>B.</i> sp. HIL Y-85,54728 (<b>A</b>) consists of the immunity genes <i>mrsFGE</i> (green colors), the structural gene <i>mrsA</i> (light blue), the modification enzymes <i>mrsD</i> and <i>mrsM</i> (dark blue colors), the exporter containing a protease domain <i>mrsT</i> (purple) and the regulatory genes <i>mrsR1, mrsR2</i> and <i>mrsK2</i> (yellow and orange colors). The genome of <i>B. amyloliquefaciens</i> FZB42 (<b>B</b>) harbors a partial mersacidin gene cluster consisting of the immunity genes <i>mrsFGE</i> and the regulatory genes <i>mrsK2</i> and <i>mrsR2</i>. The genes are found at the same site as in the original producer strain, i. e. between <i>ycdJ</i> and <i>fbaB</i>. In the mutant strain <i>B. amyloliquefaciens</i> mrs1 (<b>C</b>), a partial completion of the mersacidin gene cluster was reached by competence transformation using genomic DNA of a mersacidin deletion mutant (<i>B</i>. sp. HIL Y-85,54728 Rec1). An erythromycin resistance (<i>ermB</i>) cassette substituting <i>mrsA</i> served as selection marker. <i>mrsR1</i> is most probably not transcribed in this mutant because of a polar effect. The completion of the mersacidin gene cluster in <i>B. amyloliquefaciens</i> mrs1 pPAR1 (<b>D</b>) was achieved <i>in trans</i> by transformation with the plasmid pPAR1, carrying the structural gene <i>mrsA</i> and <i>mrsR1</i>, yielding <i>B. amyloliquefaciens</i> mrs1 pPAR1.</p

Results of discontiguous megablasts employing nucleotide sequences of <i>Bacillus</i> sp. HIL Y-85,54728.

<p>Results of discontiguous megablasts employing nucleotide sequences of <i>Bacillus</i> sp. HIL Y-85,54728.</p