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

The many faces of cGAS: how cGAS activation is controlled in the cytosol, the nucleus, and during mitosis

Psychosocial stress is one of the main environmental factors contributing to the development of psychiatric disorders. In humans and rodents, chronic stress is associated with elevated inflammatory responses, indicated by increased numbers of circulating myeloid cells and activation of microglia, the brain-resident immune cells. The endocannabinoid system (ECS) regulates neuronal and endocrine stress responses via the cannabinoid receptor 1 (CB1). CB1-deficient mice (Cnr

G-rich DNA-induced stress response blocks type-I-IFN but not CXCL10 secretion in monocytes

Excessive inflammation can cause damage to host cells and tissues. Thus, the secretion of inflammatory cytokines is tightly regulated at transcriptional, post-transcriptional and post-translational levels and influenced by cellular stress responses, such as endoplasmic reticulum (ER) stress or apoptosis. Here, we describe a novel type of post-transcriptional regulation of the type-I-IFN response that was induced in monocytes by cytosolic transfection of a short immunomodulatory DNA (imDNA), a G-tetrad forming CpG-free derivative of the TLR9 agonist ODN2216. When co-transfected with cytosolic nucleic acid stimuli (DNA or 3P-dsRNA), imDNA induced caspase-3 activation, translational shutdown and upregulation of stress-induced genes. This stress response inhibited the type-I-IFN induction at the translational level. By contrast, the induction of most type-I-IFN-associated chemokines, including Chemokine (C-X-C Motif) Ligand (CXCL) 10 was not affected, suggesting a differential translational regulation of chemokines and type-I-IFN. Pan-caspase inhibitors could restore IFN-beta secretion, yet, strikingly, caspase inhibition did not restore global translation but instead induced a compensatory increase in the transcription of IFN-beta but not CXCL10. Altogether, our data provide evidence for a differential regulation of cytokine release at both transcriptional and post-transcriptional levels which suppresses type-I-IFN induction yet allows for CXCL10 secretion during imDNA-induced cellular stress

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

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

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

Strains and plasmids used in this study.

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