Antibiotic and genetic profiling of field-collected and laboratory wild-type strains reveals seven novel Bacillus spizizenii strains that produce the lanthipeptide subtilin

doi:10.21203/rs.3.rs-1327603/v1

Download PDF

Research Article

Antibiotic and genetic profiling of field-collected and laboratory wild-type strains reveals seven novel Bacillus spizizenii strains that produce the lanthipeptide subtilin

https://doi.org/10.21203/rs.3.rs-1327603/v1

This work is licensed under a CC BY 4.0 License

You are reading this latest preprint version

Two dozen field-collected Bacillus and a dozen laboratory Bacillus spizizenii wild-type strains were selected on the basis of their antagonistic properties against the Gram-positive strain Micrococcus luteus. Their genetic and antibiotic profiles were characterized (subtilin encoding spaS gene sequences, mass spectrometric and quantitative reversed phase rpHPLC analyses, as well as presence of the lanthionin cyclase protein SpaC by western blotting). We identified seven novel producer of the lanthipeptide subtilin. Phylogenetic analyses of the field collected (HS and N5) and laboratory (DSM 618, 1087, 6395, 6405, and 8439) subtilin producer strains on the basis of their 16S rDNA encoding rrn sequences showed that all seven strains can be classified to B. spizizenii. To the best of our knowledge, all B. spizizenii strains described so far are characterized by the fact that they can produce a lanthipeptide from the subtilin family. Furthermore, the potential of the analyzed strains with regard to the coproduction of the sactipeptide subtilosin and the lipopeptides surfactin and fengycin were determined.

Subtilin

lanthipeptide

subtilosin

surfactin

peptide antibiotic

Bacillus spizizenii

Gram-positive spore-forming bacteria of the genus Bacillus are among the most widespread bacteria worldwide; they can be found on the one hand in the soil, in fresh or sea water, as well as in the air (Ferrari et al. 1993; Moszer et al. 2002), on the other hand ind the gastrointestinal tract of ruminants and humans (Hong et al. 2005; Cutting 2011). Some B. subtilis strains share long history of safe use in fermented foods such as Nattō (B. subtilis natto, Japanese fermented soybeans) (Sun et al. 2016) and Doenjang (B. subtilis together with Aspergillus oryzae, Korean soybean paste Yue et al. 2021). For this reason, B. subtilis is generally classified as safe (GRAS). According to the currently accepted definition, probiotics are “live microorganisms which provide health benefits when consumed", for example by improving or restoring the gut flora (Hill et al. 2014). Very recently, it has been shown that lanthipeptide producing commensal strains of the human gastrointestinal tract reduce vancomycin-resistant Enterococcus faecium (VRE) colonization and represent potential probiotic agents (Kim et al. 2019). Lanthipeptides are gene-encoded small peptides (19-38 amino acids in length) that possess the unusual bridge-forming sulfur-containing amino acids meso-lanthionine and 3-methyl-lanthionine (Freund and Jung 1992; Stein 2005; Chatterjee et al. 2005; Arnison 2013; Letzel et al. 2014).

B. subtilis as well as very closely related B.spizizenii strains are able to produce more than two dozen antibiotics with an amazing variety of structures (Stein 2005). Peptide antibiotics represent the predominant class, among them lanthipeptides and non-ribosomal biosynthesized lipopeptide antibiotics from the surfactin, iturin, fengycin-class (Stein 2005; Zhao et al. 2018; Caulier et al. 2019). One of the one of the earliest described lanthipeptides is subtilin, a 32-amino-acid peptideproduced by a Bacillus strain (see Fig. 1a for a structure representation) that has been originally described 1944 (Jansen and Hirschmann 1944). The subtilin producing strain was originally isolated 1911-1912 by Karl Kellerman (Kellerman et al. 1912) and deposited as B. subtilis strain No. 6633 into the American Type Culture Collection (ATCC) by the “Bureau of Plant Industry, Soils, and Agricultural Engineering of the United States Department of Agriculture” (Garibaldi and Feeney 1949). Furthermore, several B. subtilis wild-type strains produce the macrocyclic sactipeptide subtilosin A (Fig. 1b; Zheng et al. 1999; Stein et al. 2004) with a series of unusual intramolecular thioether linkages (Marx et al. 2001; Kawulka et al. 2003; Stein 2020). The lipoheptapeptide lactone surfactin (Fig. 1c) is a powerful surfactant with potentantimicrobial activities of surfactin arebased on its detergent-like action on biological membranes (Heerklotz und Seelig 2001; Carrillo et al. 2003). Surfactin is distinguished by its exceptional emulsifying, foaming, antiviral, antitumor, antimycoplasma, and hypocholesterolemic activities (Peypoux et al. 1999; Kaspar et al. 2019).

The aim of this study was to analyze the potential of Bacillus strains to produce the lanthipeptide subtilin, the sactipeptide subtilosin, and the lipopeptide surfactin. Both, laboratory and field collected Bacillus strains were taxonomically classified by gene sequencing (SSU 16SrRNA encoding rrn and subtilin encoding spaS alleles). The antibiotic profiles were characterized under conditions optimal for the production of lanthi- and sactipeptides; MALDI mass spectrometry and reversed phase rpHPLC were used for qualitative and quantitative verification of the produced peptides. Furthermore, the presence of subtilin modifying enzymes (SpaC, the subtilin cyclase, for example) was verified by immunoblotting.

Bacterial strains and media

The Bacillus strains (for a complete list see Table 1) are from culture collections ATCC (American Type Culture Collection) and DSMZ (German Collection of Microorganisms and Cell Cultures; strain collections for microorganisms and links to them can be found in the Tab. S1, Supplement) or natural isolates (see below). For antibiotic production liquid Landy medium cultures were routinely grown aerobically at 37°C (Landy et al. 1948) supplemented with 0.5% yeast extract (Heinzmann et al. 2006; Stein 2020). Solid media contained TY agar (0.8% tryptone, 0.5% yeast extract, 0.5% NaCl, 1.5% agar) or Luria-Bertani (LB) agar (the commercial supplyer of the media components was Gibco, Neu-Isenburg, Germany), standard incubation conditions were overnight (15-20 h) at 37°C.

Table 1

*B. subtilis* trains used in this study and their profiles in subtilin (Sub), subtilosin (Sbo), and surfactin (Srf) production
Bacillus strain	Synonyms^a, properties	Sub^b,c,d,e	Sbo^b,c,e	Srf^e	Reference
spizizenii
ATCC 6633	DSM 347, NRS 231 (safe), IAM 1069, Sub⁺/Sbo⁺/Srf⁺ - reference strain	+	+	+	Garibaldi and Feeney (1946) Heinzmann et al. (2006)
DSM 618	Test strain for the detection of antibiotics in meat	+	+	+	this work
DSM 1087	W23	+	+	+	this work
DSM 6395	W23 2A2	+	+	+	this work
DSM 6405	mutant of W23 SR	+	+	+	this work
DSM 8439^T	W23, IAM 12021	+	+	+	this work
N5	soil isolate	+	+	+	this work
HS	soil isolate	+	+	+	this work
subtilis
168^f,g	DSM 402, ATCC 23857 Sub⁻ / Sbo⁺ / Srf⁻ - reference	-	+	-	Stein et al. (2004)
DSM 10^T	ATCC 6051^T, NCIB 3610	-	+	+	this work; Fuchs et al. (2011)
IP	soil isolate	-	+	+	this work
DSM 2109	ATCC 11774, NCTC 8236	-	+	+	this work
others
DSM 2109	ATCC 11774, NCTC 8236	-	+	+	this work
DSM 3256	IAM 1213	-	+	+	this work
DSM 3258	IAM 1260	-	-	+	this work
A1/3	ericin A/S	(+)	-	+	(Stein et al. 2002; Hofemeister et al. 2004)
HI-1	soil isolate	-	-	+	this work
DSM 15029	TU-B-10, entianin	(+)	+	+	Fuchs et al. (2011)
DSM 1088	B. natto	-	+	+	Stein et al. (2004)
DSM 2277	B. atrophaeus ATCC 51189, NCTC 10073	-	+	+	Fritze and Pukall (2001) Stein et al. (2004)
^a description an links to the strain collections are given in the Supplement (Tab. S1); ^b PCR, gene sequencing; ^c rp-HPLC
^d Western blotting (SpaC/SpaB protein); ^e MALDI-TOF MS; ^f genome sequence (NC_000964.3; Kunst et al. 1997)
^g B. subtilis 168 can be converted to a surfactin producer after introduction of the sfp gene (Lambalot et al. 1996)

Isolation of subtilin producing Bacilli

Soil samples with different nutrient content were isolated from nutrient-rich arable land (N, 50° 11'19”N; 08 ° 47'54''E) and forest (IP, 50° 08'22''N; 09° 16'33”E) in Hessen, Germany, and from alpine surroundings (HI, 47° 21'13”N; 10° 06’01”'E and HS, 47° 19’36'”N; 10° 11’02''E; both approx. 2000 m, Vorarlberg, Austria). Twenty five grams of soil were suspended in 100 mL of sterile water, pasteurized (10 min at 80 ° C) in order to accumulate spore-forming bacteria, diluted 1: 100, plated out on TY plates and incubated at 37°C. for 15 h. To monitor the antimicrobial activities, individual colonies were replica plated on TY agar plates in Petri dishes with cams in order to obtain optimal aerobic conditions for antibiotic production (Sarstedt, Nümbrecht, Germany) with Micrococcus luteus ATCC 9341 as the test organism (Stein 2004).

Molecular biology techniques

Established protocols were followed for molecular biology techniques; E. coli plasmids were isolated by the rapid alkaline extraction procedure. DNA amplification using Taq DNA polymerase was performed according to the instruction of the commercial supplier (Boehringer GmbH; Mannheim, Germany) in a DNA Thermal cycler (Eppendorf; Hamburg, Germany). The SSU rRNA encoding gene was amplified by PCR using conserved primers 16S_forward 5’-GAGAGTTTGATCCTGGCTCAG-3’ and 16S_reverse 5’-ACGACTTCACCCCAATCATC-3’) according to Heyrman et al. (2001). The nucleotide sequences of B. subtilis DSM 6405, 3258, N5, HS1, IP, and HI has been deposited under the NCBI GeneBank records DQ452508-13, respectively, as well as DSM618: DQ529249 (for NCBI GeneBank links see the supporting informations. The structural gene of subtilin spaS including 250 bp of the upstream non-coding region has been PCR-amplified with primers SpaS_Seq1 (5’-CTATGAATCAATGGAAGGG-3’) and SpaS_Seq2 (5’-CTTCATTTTCTTGTCCCG-3’); GeneBank records for B. subtilis DSM 618 (DQ452514: https://www.ncbi.nlm.nih.gov/nuccore/DQ452514), B. subtilis DSM 6405 (DQ452515: https://www.ncbi.nlm.nih.gov/nuccore/DQ452515), HS1 (DQ452516: https://www.ncbi.nlm.nih.gov/nuccore/DQ452516), and N5 (DQ452517:https://www.ncbi.nlm.nih.gov/nuccore/DQ452517). Subtilosin structural gene sbo sequencing has been performed with primers described previously (Stein 2004); GeneBank records for novel sbo sequences of the natural isolates B. subtilis HI-1, N5, and IP are deposited under accession numbers DQ452518-20, respectively. DNA cleavage and isolation was achieved with the QIAquick^TM purification kit (Qiagen GmbH; Hilden, Germany). Oligonucleotides were purchased from ARK (ARK Scientific GmbH Biosystems, Darmstadt, Germany). Sequencing was carried out by SRD (Scientific Research and Development, Oberursel, Germany); nucleotide sequences have determined at least two times for each DNA-strand.

DNA Sequence and phylogenetic analyses

Multiple sequence alignment (MSA) analyses were performed with Clustal Omega 1.2.4 that uses seeded guide trees and HMM profile-profile techniques to generate alignments (Waterhouse et al. 2009; Sievers et al. 2011) (https://www.ebi.ac.uk/Tools/msa/clustalo/) or MAFFT version 7 (Katoh et al. 2019; https://mafft.cbrc.jp/alignment/server/). Individual 16S rDNA sequences rrnO, rrnA, rrnJ, rrnW, rrnI, rrnH, rrnG, rrnE, rrnD, rrnB were extracted from the B. subtilis strains 168 (accession number NC_000964.3) and ATCC 6051 (NZ_CP020102.1). The SSU rDNA sequences of the B spizizenii strains ATCC 6633 (NZ_CP034943.1/CP034943.1), W23 (CP002183.1) and TU-B-10^T (CP002905.1) were extracted from Silva (https://www.arb-silva.de/) or manually. GC content calculation and GC profiling was performed with ENDMEMO (http://www.endmemo.com/bio/gc.php). Phylogenetic analyses were carried out by the neighborjoining method (Saitou and Nei 1987) using MAFFT (version 7; Kuraku et al. 2013) or Clustal Omega 1.2.4 software. The NCBI BLAST server (https://blast.ncbi.nlm.nih.gov/Blast.cgi) was used for homology searches.

MALDI-TOF mass spectrometry (MALDI-TOF MS)

Cell free aliquots of 500 µL culture supernatants of overnight grown

B. subtilis strains in Landy media were extracted with 500 µL 1-butanol, 400 µL of the organic phase was dried in a speed-vac evaporator, and extracted peptides were dissolved in 5 µL 50% acetonitrile and 1% trifluoroacetic acid (v/v in H₂O). 0.3-0.5 µL aliquots of the solutions were mixed directly on the target with 1.5 µL matrix solution, and the mixture was dried with the help of hot air. The matrix was 20 mg/mL DHBs (9:1 mixture of 2,5-dihydroxybenzoic acid and 2-hydroxy-5-methoxybenzoic acid) complemented with solubilized in aqueous solution of 50% acetonitrile and 1% trifluoroacetic acid (v/v in H₂O). Generally, if sample spots are readily crystallized mass spectra with sufficient signal-to-noise values were obtained from the edge of the crystals (Stein 2008). Delayed extraction^TM (DE) MALDI time-of-flight (TOF) mass spectra were recorded on a Voyager-DE STR instrument (Applied Biosystems Instruments) using a nitrogen laser (λ = 336 nm, repetition rate = 20 Hz) for desorption and ionization with an acquisition mass range from 600 to 15 000 m/z and the low mass gate set to 550 m/z. The total acceleration voltage was 20 kV with 68.5% grid voltage on the first grid, 0.02% guide wire voltage, 200 ns delay, and a mirror voltage ratio of 1.12. All experiments were carried out with the reflector positive ion mode. Between 500 and 1 000 laser shots were accumulated for each mass spectrum.

Subtilin quantitation

Similar growth profiles were obtained for all B. subtilis strains tested after growth in 2 mL Landy medium (37°C, 175 rpm). In standard determinations B. subtilis was grown for 16 h, the cultures were centrifuged (4°C, 15 min, 15,000 g), and the subtilin amount was determined in 500 µL aliquots by RP-HPLC using a Beckman Gold HPLC System and an analytical ODS Hypersil column (particle size: 5 µm, width & length: 2 x 250 mm, Maisch, Ammerbuch, Germany) as described previously (Heinzmann et al. 2006).

SDS-PAGE and Western blotting

SDS-PAGE (10% Tris-Glycine gels) and Western blot analyses were performed with an immuno-purified SpaC-directed immunoserum as described previously (Helfrich et al 2007). Molecular standards were purchased from Biorad (München, Germany).

Identification of antibiotic producing B. subtilis and field collected bacterial strains

Initially we tested twelveB. subtilis laboratory strains (B. subtilis subsp. subtilis and subsp. spizizenii, respectively), including well established strains from the German Collection of Microorganisms (DSMZ) on their ability to inhibit the growth of M. luteus. Interestingly, a number of B. subtilis DSM strains: DSM 618, 1087, 6395, 6405, and 8439 showed promising antimicrobial activities with growth inhibition activities comparable to the well-characterized subtilin-producing strain B. subtilis subsp. spizizenii ATCC 6633 (representative examples are given in Fig. 2a). The laboratory strains B. subtilis DSM 3256 and DSM 3258 exhibited semi-large inhibition zones. To get more insight into B. subtilis strains without laboratory history we took random soil samples from various environmental habitats, nutrient-rich farmland and forest (200 m altitude, Hesse, Germany) and alpine environments (2000 m altitude, Vorarlberg, Austria). In a first pasteurization step, we were able to select the spore-forming microorganism out of the natural probes, which were single colony streaked on agar plates (40 single colonies, TY medium). Antibiotic producing strains were identified using M. luteus as a highly sensitive Gram-positive target strain using optimized conditions for the detection of lanthipeptides (Heinzmann et al. 2006) and sactipeptides (Stein et al. 2002). Notably, 20 to 25 percent of the isolated aerobically grown spore-formers inhibited the growth of M. luteus (not shown). Twelve probable antibiotic producing strains were selected: Six strains from the farmland (N), one from the forest (IP), and five strains from alpine environments (HS and HI). The strains with large and clear inhibition zones HS and N5, (Fig. 2a), were later identified as producer of subtilin (Sub⁺, for a summary see Table 1). In contrast, the strains HI1 and IP exhibited relatively small inhibition zones, both strains were found to be Sub⁻ in further investigations (for a summary see Tab.1).

Phylogenetic analyses of laboratory and field-collected Bacillus strains; genomic organization of SSU rRNA encoding rrn alleles of B. spizizenii and B. subtilis

The 16S rDNA of all spore-forming microorganisms examined (laboratory and field-collected strains) were PCR amplified, sequenced, and their nucleotide sequences were taxonomically classified. B. subtilis strains 168 and ATCC 6051 have a total of ten rrn alleles (Tab. S2, Supplement), each 1552-1554 nucleotides in length; MSA analyzes indicated a few (two to three) nucleotide variations within these individual genomes (Tab. S3, Supplement). It is noteworthy, that B. spizizenii ATCC 6633 has ten identical rrn alleles in both length and nucleotide sequence, whereas for its close relative, the W23 strain, only eight rrn alleles (1552-1554 nucleotides) were found. The B. spizizeni typing strain TU-B-10 contains ten rrn genes, but the genome seems to be organized differently: The organization of the first five rrn alleles is similar to the organization of B. subtilis 168 or B. spizizenii ATCC 663, but the positions of alleles 6-10 indicate extensive genetic re-organization (Tab. S2 Supplement).

Notably, the rrn sequences of the field-collected Bacillus strains HS1 and HS2 (hereinafter referred to as strain HS) and N1, N5, and N6 (referred to as strain N5), as well as the laboratory strains DSM 618 and DSM 6405 were identical to rrn sequences of B. spizizenii ATCC 6633 and W23 (data not shown), suggesting their classification as B.spizizenii (Nakamura et al. 1999; Zeigler et al. 2008; Zeigler 2011). On the other hand, the field-collected strains IP and HI1 (referred to as strain HI) as well as the laboratory strain DSM 3258 are classified as further members of B. subtilis (see Fig. 3a for phylogenetic analyses). As indicated in Fig. 3b, MSA analyses revealed that position 181 of the individual rrn alleles can be used for clear species differentiation between B. subtilis and B. spizizenii. Interestingly, position 600 of the B. spizizenii typing strain TU-B-10 is identical to the B. subtilis sequence, whereas a transition (T/C exchange) was found at this position for the B. spizizenii ATCC 6633 and W23 rrn alleles.

BLAST searches revealed, that the field collected Bacillus strains HI2 and HI3 belong to B. macroides and B. licheniformis, respectively. Both species are known for members with a high potential to produce antibiotics. The strain N2 was classified as B. thuringiensis, a class of Bacillus strains some members of which produce the lipopeptide kurstakin (Hathout et al. 2000). No sequence was obtained for strain N3, strain N4 was discarded because no further significant antibiotic activities could be identified under the test conditions used.

SpaS sequence of different B. spizizenii strains, presence of the SpaC protein

For rapid PCR amplification of the subtilin structural gene spaS oligonucleotide primers (SpaS-Seq1 and SpaS_Seq2) complementary to the –35 region of the spaS promoter and the spaS-spaI intergenic region were used. The presence of the spaS gene was verified in the case of B. subtilis strains DSM 618, 1087, 6395, 6405 and 8439, as well as the natural isolates HS and N5 (Fig. 2b; for a summary see Tab.1). Sequences of both the spaS genes and their flanking regions (ribosomal binding site, -10-region) were identical to their counterpart in the ATCC 6633 strain (the GeneBank records for B. subtilis DSM 618, 6405, HS1, and N5 are DQ452514-17, respectively; ATCC 6633: NZ_CP034943.1, and TU-B-10: NC_016047.1). Since the spaS gene was detected within the DNA of all B. spizizenii, we expected also the subtilin maturing proteins in the cultures of these strains. This hypothesis was confirmed by detection of the subtilin cylase SpaC (Fig. 2c) and subtilin dehydratase (SpaB, not shown) within PAGE-separated cell extracts of the W23 strains using SpaC or SpaB specific immunosera, respectively. Most likely, all observed W23-type SpaC proteins are closely related, as EriC of B. subtilis A1/3 with only 85% sequence identity to the SpaC counterpart from the ATCC 6633 strain (Stein et al. 2002a) gave only weak immunoblotting signals. Consistent with these observations is that for the strains which lack spaS (Fig. 2b, strains 168, 3258, HI, and IP), no SpaC protein could be detected in the associated cell extracts either (Fig. 2b); the same applies for SpaB (not shown).

MALDI-TOFMS profiling of B. subtilis culture supernatants

MALDI-TOFMS analyses of butanolic extracts of B. subtilis culture supernatants resulted in well resolved peak clusters (Fig. 4). Prominent cluster between m/z 3280 and 3520 representH⁺-, Na⁺- and K⁺-adducts of the lanthipeptide subtilin and its succinylated species (Chan et al. 1993; Heinzmann et al. 2006) and the sactipeptide subtilosin, respectively (see Tab. 1 for summary). Furthermore, the nonribosomal generated lipoheptapeptide surfactin was identified by MALDI-MS experiments due to characteristic m/z values of its different isoforms. Remarkably, all investigated B. subtilis strains produced surfactin (see Tab. 1 for a summary), notably, with the exception of the laboratory-adapted B. subtilis strain 168, which is known as a surfactin non-producer due to a mutation within the 4’-phosphopantetheine transferase sfp gene which posttranslationally modifies the required surfactin synthetase enzymes (Lambalot et al. 1996). Further MS/MS-fragmentation experiments according to Leenders et al. (1999) unambiguously assign the observed m/z values to the lipoheptapeptide sequence Glu-Leu-Leu-Asp-Val-Leu-Leu which can be clearly assigned to the peptide moiety of surfactin (data not shown). However, the detection of surfactin within the culture supernatants of all investigated B. subtilis strains, and the widespread frequent appearance of the potential to produce surfactin among strains of the genus Bacillus (Peypoux et al. 1999; Kalinovskaya et al. 2002; Torres et al. 2016) restricts the usage of the phenotype “surfactin production” as biomarker for subspecies classification/differentiation (e.g. between B. subtilis and B. spizizenii).

Quantitative determination of subtilin

Subtilin was quantitatively determined in culture supernatants of stationary grown Bacillus cells in Landy medium (Fig. 5a/b). Whereas the production yields of B. subtilis ATCC 6633 (4.9 mg/mL) and the field-collected strainN5 (4.2 mg/mL) were comparable, the strain DSM 618 produces three-fold higher amounts (14.9 mg/mL). The largest subtilin yield was obtained from the laboratory strain B. subtilis DSM 6405 (33 mg/mL) and the field-collected B. subtilis HS (30 mg/mL). Representative chromatograms for these strains are shown in Fig. 5a. A further observation was that the proportion of N-terminally succinylated subtilin also increased with increasing yields of subtilin, especially for the high producer strains 618 and 6405. The production yields of the DSM strains 1087, 6395 and 8439 were similar to the ATCC 6633 strain (data not shown).

GC-Content of the subtilin gene cluster spa

Our results show that for all B. spizizenii strains characterized so far, the Sub⁺ phenotype is a characteristic feature. The analysis of the base compositions of a given genome is a common strategy to investigate gene history (Garcia-Vallvé et al. 2000; Popa et al. 2001). Remarkably, in all analyzed

B. spizizenii genomes the average GC-content of the subtilin gene cluster with 36 % is significantly lower (about 8 %) than the GG content of about 44 % of respective GC-content average of the respective B. spizizenii host genome (Fig. 6 and Tab S3, supporting information). This observation is a strong hint that B. spizizenii acquired the subtilin gene cluster most likely from another organism by a recent horizontal gene transfer event as is hypothesized for a number of lanthipeptide producers (Zhang et al. 2012).

Nakamura et al. (1999) proposed the classification of Bacillus subtilis strains into two classes: The 168-type strains into (1) B subtilis subsp. subtilis, and W23-type strains into (2) B. subtilis subsp. spizizenii. Classical chemotaxonomy differentiates between both classes by the composition of their cell wall teichoic acids: Whereas 168-type strains are endowed with the essential major teichoic acids poly(glycerol phosphate) and the non-essential minor teichoic acids poly(glucopyranosyl N-acetylgalactosamine 1-phosphate), the W-23-type mainly consists of poly(ribitol phosphate) (Lazarevic et al. 2002). Very recently, B. subtilis subsp. spizizenii was promoted to species status on the basis of comparative genomics and secondary metabolite (mycosubtilin and bacillaene) production (Dunlap et al. 2020). The experiments presented in this study have revealed that all B. spizizenii strains from both laboratory (ATCC 6633, DSM 618, 1087, 6395, 6405, and 8439) as well as natural origin (HS and N5) produced identical mixtures of antibiotics including the lanthipeptide subtilin, the sactipeptide subtilosin, as well as nonribosomally biosynthesized lipopeptides from the surfactin class (Tab. 1). However, the lanthipeptide subtilin cannot be used as a characteristic for closely related species differentiation since the production of subtilin and subtilin-related structures has also been found in B. subtilis A1/3 (Stein et al. 2002a), and other Bacillus strains (Bacillus sp., Velho et al. 2013, (B. mojavensis, Reva et al. 2020; B. vallismortis, Kim et al. 2018; B. intestinalis, Xu et al. 2015).

Our finding that different strains produce different amounts of subtilin - the yield of the HS strain was 7-fold superior to the original subtilin producer B. subtilis ATCC 6633 (Heinzmann et al. 2006), imply differential efficiencies in subtilin production. The examined B. spizizenii strains may have developed different genetic elements for the regulation of the extremely complex system of subtilin biosynthesis, such as repressor (AbrB) or activator (Sigma factor H) elements or variations in the promoter regions (-35 region) (Stein et al. 2002b and 2003; Kleerebezem 2004; Kleerebezem et al. 2004; Spieß et al. 2015). Furthermore, also the subsequent steps in subtilin biosynthesis for example post-translational dehydration of serine (threonine), addition of neighboring cysteines (Kiesau et al. 1997; Helfrich et al. 2007), and final processing (Stein and Entian 2002; Corvey et al. 2003) might exhibit differential efficiency in the investigated strains.

Recently, it was shown that succinylation of subtilin is a consequence of high glucose concentrations in the culture medium, for example the Landy medium (Bochmann et al. 2015). We found especially for the strains with very high production rates of subtilin - HS and DSM 6405, that the proportion of succinylated subtilin species were significantly increased compared to the unsuccinylated species (data not shown). Since N-terminal succinylated subtilin shows less antimicrobial activity, this could also be a necessary self-protection mechanism of B. subtilis cells against the antibacterial effect of their own product (Heinzmann et al. 2006; Geiger et al. 2019). The B. spizizenii strain TU-B-10 turned out to be an extraordinary exception, here the production of a subtilin isoform EtnS (entianin) was identified, which differs from subtilin in three conservative amino acid exchanges: L6V, A15L, and L24I (Fuchs et al. 2011).

In an age in which bacterial resistance to antibiotics is becoming increasingly important, systematic screening for new antibiotic agents with new mechanisms of action is still an important strategy. Microorganisms from the genus Bacillus are able to produce a large number of different antimicrobial substances (Stein 2005; Zhao et al. 2018; Tran et al 2022). It is to be expected that a large number of new active antimicrobial agents and isoforms of known antibiotics with minor chemical modifications will be found in the future through systematic screening, a strategy which is strongly supported by subsequent genome sequencing and novel genome mining bioinformatic tools (Walker et al. 2020). The findings from this work show that many B. spizizenii strains always produce a cocktail of antibiotic agents (Stein 2004 and 2020; Mülner et al. 2020), in particular always several lipophilic membrane-active agents like subtilin, subtilosin, surfactin, and fengycin. As a result, future studies should increasingly consider the synergistic effects of these interesting drug classes which have in common an amphiphilic structure with effects either in the cytoplasmic membrane itself (surfactin, fengycin, or the sactibiotic subtilosin) or in membrane-associated processes like cell wall biosynthesis (lanthipeptide subtilin). In particular, production strains of such antibiotic cocktails or strains that are suitable for use as probiotics should be examined under more realistic growth conditions, such as co-cultures of different microorganisms in the same habitat (microbiome), in more natural behavior and production conditions, e.g. in biofilms (Kolter 2010). These aspects should be considered when further developing B. subtilis or other relates microorganisms as a biosynthetic platform for lanthipeptides (Schmitt et al. 2019), a very promising strategy to genetically engineer lanthipeptide structures to obtain drugs with novel properties.

ACKNOWLWDGEMENTS

We greatly acknowledge Michael Karas, University Frankfurt, for the opportunity to use his MALDI-TOF mass spectrometric equipment.

Contributions

M.H. performed research, interpreted the data and wrote the paper, K.-D. E. conceived of or designed study, and reviewed the manuscript, and T. S. conceived of or designed study, performed research interpreted the data and wrote the paper. All authors read, reviewed, and approved the final version of the manuscript.

Corresponding author

Correspondence to Torsten Stein

Ethics approval

Not applicable.

Consent for publication

The authors declare that they agree to publishing.

Competing Interest

The authors have no relevant financial or non-financial interests to disclose.

Data availability statement

All data generated or analysed during this study are included in this published article and the
supplementary information files.

Arnison PG, Bibb MJ, Bierbaum G, Bowers AA et al (2013) Ribosomally synthesized and post-translationally modified peptide natural products: Overview and recommendations for a universal nomenclature. Nat Prod Rep 30:108–160. https://doi.org/10.1039/C2NP20085F
Bochmann SM, Spieß T, Kötter P, Entian KD (2015) Synthesis and succinylation of subtilin-like lantibiotics are strongly influenced by glucose and transition state regulator AbrB. Appl Environ Microbiol 81:614–622. https://aem.asm.org/content/81/2/614
Carrillo C, Teruel JA, Aranda FJ, Ortiz A (2003) Molecular mechanism of membrane permeabilization by the peptide antibiotic surfactin. Biochim Biophys Acta 1611:91–97. https://doi.org/10.1016/S0005-2736(03)00029-4
Caulier S, Nannan C, Gillis A, Licciardi F, Bragard C, Mahillon J (2019) Overview of the antimicrobial compounds produced by members of the Bacillus subtilis group. Front Microbiol 10:302. https://doi.org/10.3389/fmicb.2019.00302
Chan WC, Bycroft BW, Leyland ML, Lian LY, Roberts GC (1993) A novel post-translational modification of the peptide antibiotic subtilin: isolation and characterization of a natural variant from Bacillus subtilis A.T.C.C. 6633. Biochem J 291:23–27. https://doi.org/10.1042/bj2910023
Chatterjee C, Paul M, Xie L, van der Donk WA (2005) Biosynthesis and mode of action of lantibiotics. Chem Rev 105:633–684. https://doi.org/10.1021/cr030105v
Corvey C, Stein T, Düsterhus S, Karas M, Entian K-D (2003) Activation of subtilin precursors by Bacillus subtilis extracellular serine proteases subtilisin (AprE), WprA, and Vpr. Biochem Biophys Res Commun 304:48–54. https://doi.org/10.1016/S0006-291X(03)00529-1
Cutting SM (2011) Bacillus probiotics. Food Microbiol 28:214–220. https://doi.org/10.1016/j.fm.2010.03.007
Dunlap CA, Bowman MJ, Zeigler DR (2020) Promotion of Bacillus subtilis subsp. inaquosorum, Bacillus subtilis subsp. spizizenii and Bacillus subtilis subsp. stercoris to species status. Antonie Van Leeuwenhoek 113:1-12. https://doi.org/10.1007/s10482-019-01354-9
Ferrari E, Jarnagin A, Schmidt BF (1993) Commercial production of extracellular enzymes. In: Sonenshein AL, Hoch JA, Losick R (eds) Bacillus subtilis and Other Gram-positive bacteria. American Society for Microbiology, Washington, DC, pp 917–937
Freund S, Jung G (1992) Lantibiotics: An Overview and Conformational Studies on Gallidermin and Pep5. In: James R, Lazdunski C, Pattus F (eds) Bacteriocins, Microcins and Lantibiotics. NATO ASI Series (Series H: Cell Biology, vol 65. Springer, Berlin, Heidelberg. https://doi.org/10.1007/978-3-642-76974-0_9
Fritze D, Pukall R (2001) Reclassification of bioindicator strains Bacillus subtilis DSM 675 and Bacillus subtilis DSM 2277 as Bacillus atrophaeus. Int J Syst Evol Microbiol 51:35–37. https://doi.org/10.1099/00207713-51-1-35
Fuchs SW, Jaskolla TW, Bochmann S, Kötter P, Wichelhaus T, Karas M, Stein T, Entian K-D (2011) Entianin, a novel subtilin-like lantibiotic from Bacillus subtilis subsp. spizizenii DSM 15029T with high antimicrobial activity. Appl Environ Microbiol 77:1698–1707. https://doi.org/10.1128/aem.01962-10
Garcia-Vallvé S, Romeu A, Palau J (2000) Horizontal gene transfer in bacterial and archaeal complete genomes. Genome Res 10(11):1719–1725.
Garibaldi JA, Feeney RF (1949) Subtilin production. Ind Eng Chem 41:432–435. https://doi.org/10.1021/ie50470a040
Geiger C, Korn SM, Häsler M, Peetz O, Martin J, Kötter P, Morgner N, Entian KD (2019) LanI-Mediated Lantibiotic Immunity in Bacillus subtilis: Functional Analysis. Appl Environ Microbiol 85:e00534–e00519. https://doi.org/10.1128/AEM.00534-19
Hathout Y, Ho YP, Ryzhov V, Demirev P, Fenselau C (2000) Kurstakins: a new class of lipopeptides isolated from Bacillus thuringiensis. J Nat Prod 63:1492–1496. https://pubs.acs.org/doi/10.1021/np000169q
Heerklotz H, Seelig J (2001) Detergent-like action of the antibiotic peptide surfactin on lipid membranes. Biophys J 81:1547–1554. https://doi.org/10.1016/S0006-3495(01)75808-0
Heinzmann S, Entian K-D, Stein T (2006) Engineering Bacillus subtilis ATCC 6633 for improved production of the lantibiotic subtilin. Appl Microbiol Biotechnol 69:532–536. https://doi.org/10.1007/s00253-005-0023-9
Helfrich M, Entian K-D, Stein T (2007) Structure-function relationships of the lanthionine cyclase SpaC involved in biosynthesis of the Bacillus subtilis peptide antibiotic subtilin. Biochemistry 46:3224–3233. https://pubs.acs.org/doi/10.1021/bi062124f
Heyrman J, Swings J (2001) 16S rDNA sequence analysis of bacterial isolates from biodeteriorated mural paintings in the Servilia tomb (Necropolis of carmona, Seville, Spain). Syst Appl Microbiol 24:417–422. https://doi.org/10.1078/0723-2020-00048
Hill C, Guarner F, Reid G, Gibson GR, Merenstein DJ, Pot B, Morelli L, Canani RB, Flint HJ, Salminen S, Calder PC, Sanders ME (2014) The international scientific association for probiotics and prebiotics consensus statement on the scope and appropriate use of the term probiotic. Nat Rev Gastroenterol Hepatol 11:506–514. https://doi.org/10.1038/nrgastro.2014.66
Hofemeister J, Conrad B, Adler B, Hofemeister B, Feesche J, Kucheryava N, Steinborn G, Franke P, Grammel N, Zwintscher A, Leenders F, Hitzeroth G, Vater J (2004) Genetic analysis of the biosynthesis of non-ribosomal peptide- and polyketide-like antibiotics, iron uptake and biofilm formation by Bacillus subtilis A1/3. Mol Genet Genomics 272:363–378. https://doi.org/10.1007/s00438-004-1056-y
Hong HA, Le HD, Cutting SM (2005) The use of bacterial spore formers as probiotics. FEMS Microbiol Rev 29:813–835. https://doi.org/10.1016/j.femsre.2004.12.001
Jansen EF, Hirschmann DJ (1944) Subtilin - an antibacterial product of Bacillus subtilis: Culturing conditions and properties. Arch Biochem 4:297–309
Kalinovskaya NI, Kuznetsova TA, Ivanova EP, Romanenko LA, Voinov VG, Huth F, Laatsch H (2002) Characterization of surfactin-like cyclic depsipeptides synthesized by Bacillus pumilus from ascidian Halocynthiarantium. Mar Biotechnol (NY) 4:179–188. https://doi.org/10.1007/s10126-001-0084-4
Kaspar F, Neubauer P, Gimpel M (2019) Bioactive Secondary Metabolites from Bacillus subtilis: A Comprehensive Review. J Nat Prod 82. https://doi.org/10.1021/acs.jnatprod.9b00110. :2038-2053
Katoh K, Rozewicki J, Yamada KD (2019) MAFFT online service: multiple sequence alignment, interactive sequence choice and visualization. Brief Bioinform 20:1160–1166. https://doi.org/10.1093/bib/bbx108
Kawulka K, Sprules T, McKay RT, Mercier P, Diaper CM, Zuber P, Vederas JC (2003) Structure of subtilosin A, an antimicrobial peptide from Bacillus subtilis with unusual posttranslational modifications linking cysteine sulfurs to alpha-carbons of phenylalanine and threonine. J Am Chem Soc 125:4726–4727. https://doi.org/10.1021/ja029654t
Kellerman KF, McBeth IG ( (1912) The fermentation of cellulose. Zentralblatt für Bakteriologie. Parasitenkunde Infektionskrankheiten und Hygiene Abt II 34:485–494)
Kiesau P, Eikmanns U, Gutowski-Eckel Z, Weber S, HammelmannM, Entian K-D (1997) Evidence for a multimeric subtilin synthetase complex. J Bacteriol 179:1475–1481. https://doi.org/10.1128/jb.179.5.1475-1481.1997
Kim J-Y, Park M-K, Yi LeeY-J, Kim HBahnY-S, Lee JF D-W (2018) The whole genome sequencing and assembly of Bacillus vallismortis DSM 11031T strain EMBL/GenBank/DDBJ databases. CP026362.1. Accessed 29 January 2022.
Kim SG, Becattini S, Moody TU et al (2019) Microbiota-derived lantibiotic restores resistance against vancomycin-resistant Enterococcus. Nature 572:665–669. https://doi.org/10.1038/s41586-019-1501-z
Kleerebezem M (2004) Quorum sensing control of lantibiotic production; nisin and subtilin autoregulate their own biosynthesis. Peptides 25:1405–1414. https://doi.org/10.1016/j.peptides.2003.10.021
Kleerebezem M, Bongers R, Rutten G, de Vos WM, Kuipers OP (2004) Autoregulation of subtilin biosynthesis in Bacillus subtilis: the role of the spa-box in subtilin-responsive promoters. Peptides 25:1415–1424. https://doi.org/10.1016/j.peptides.2003.11.025
Kolter R (2010) Biofilms in lab and nature: a molecular geneticist's voyage to microbial ecology. Int Microbiol 13:1–7. https://doi.org/10.2436/20.1501.01.105
Kowall M, Vater J, Kluge B, Stein T, Franke P, Ziessow D (1998) Separation and Characterization of Surfactin Isoforms Produced by Bacillus subtilis OKB 105. J Colloid Interface Sci 204:1–8. https://doi.org/10.1006/jcis.1998.5558
Kunst F, Ogasawara N, Moszer I et al (1997) The complete genome sequence of the Gram-positive bacterium Bacillus subtilis. Nature 390:249–256. https://doi.org/10.1038/36786
Kuraku S, Zmasek CM, Nishimura O, Katoh K (2013) aLeaves facilitates on-demand exploration of metazoan gene family trees on MAFFT sequence alignment server with enhanced interactivity. Nucleic Acid Res. https://doi.org/10.1093/nar/gkt389. W22-W28
Lambalot RH, Gehring AM, Flugel RS, Zuber P, LaCelle M, Marahiel MA, Reid R, Khosla C, Walsh CT (1996) A new enzyme superfamily - the phosphopantetheinyl transferases. Chem Biol 3:923–936. https://doi.org/10.1042/bj2910023
Landy M, Warren GH, Rosenman SB, Colio LG (1948) Bacillomycin: An antibiotic from Bacillus subtilis active against pathogenic fungi. Proc Soc Exp Biol Med 67:539–541. https://doi.org/10.3181%2F00379727-67-16367
Lazarevic V, Abellan FX, Möller SB, Karamata D, Mauël C (2002) Comparison of ribitol and glycerol teichoic acid genes in Bacillus subtilis W23 and 168: identical function, similar divergent organization, but different regulation. Microbiology 148:815–824. https://doi.org/10.1099/00221287-148-3-815
Leenders F, Stein T, Kablitz B, Franke P, Vater J (1999) Rapid Typing of Bacillus subtilis Strains by their Secondary Metabolites Using Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry of Intact Cells. Rapid Commun Mass Spectrom 13:943–949
Letzel AC, Pidot SJ, Hertweck C (2014) Genome mining for ribosomally synthesized and post-translationally modified peptides (RiPPs) in anaerobic bacteria. BMC Genomics 15:983. https://doi.org/10.1186/1471-2164-15-983
Marx R, Stein T, Entian KD, Glaser SJ (2001) Structure of the Bacillus subtilis peptide antibiotic subtilosin A determined by 1H-NMR and matrix assisted laser desorption/ionization time-of-flight mass spectrometry. J Protein Chem 20:501–506. https://doi.org/10.1023/A:1012562631268
Moszer I, Jones LM, Moreira S, Fabry C, Danchin A (2002) SubtiList: the reference database for the Bacillus subtilis genome. Nucleic Acids Res 30:62–65. https://doi.org/10.1093/nar/30.1.62
Mülner P, Schwarz E, Dietel K, Junge H, Herfort S, Weydmann M, Lasch P, Cernava T, Berg G, Vater J (2020) Profiling for Bioactive Peptides and Volatiles of Plant Growth Promoting Strains of the Bacillus subtilis Complex of Industrial Relevance. Front Microbiol 11:1432. https://doi.org/10.3389/fmicb.2020.01432
Nakamura LK, Roberts MS, Cohan FM (1999) Relationship of Bacillus subtilis clades associated with strains 168 and W23: a proposal for Bacillus subtilis subsp. subtilis subsp. nov. and Bacillus subtilis subsp. spizizenii subsp. nov. Int J Syst Bacteriol 49:1211–1215. https://doi.org/10.1099/00207713-49-3-1211
Peypoux F, Bonmatin JM, Wallach J (1999) Recent trends in the biochemistry of surfactin. Appl Microbiol Biotechnol 51:553–563. https://doi.org/10.1007/s002530051432
Popa O, Hazkani-Covo E, Landan G, Martin W, Dagan T (2011) Directed networks reveal genomic barriers and DNA repair bypasses to lateral gene transfer among prokaryotes. Genome Res 21(4):599–609. https://genome.cshlp.org/content/21/4/599
Reva ON, Lutz S, Ahrens CH (2020) Plant growth promoting and environmental Bacillus: genomic and epigenetic comparison. EMBL/GenBank/DDBJ databases. GCA_012648005.1 Accessed 29 January 2022
Saitou N, Nei M (1987) The neighbor-joining method: a new method for reconstructing phylogenetic trees. Mol Biol Evol 4:406–425. https://doi.org/10.1093/oxfordjournals.molbev.a040454
Schmitt S, Montalbán-López M, Peterhoff D et al (2019) Analysis of modular bioengineered antimicrobial lanthipeptides at nanoliter scale. Nat Chem Biol 15:437–443. https://doi.org/10.1038/s41589-019-0250-5
Sievers F, Wilm A, Dineen D, Gibson TJ, Karplus K, Li W, Lopez R, McWilliam H, Remmert M, Söding J, Thompson JD, Higgins DG (2011) Fast, scalable generation of high-quality protein multiple sequence alignments using Clustal Omega. Mol Syst Biol 7:539. https://doi.org/10.1038/msb.2011.75
Spieß T, Korn SM, Kötter P, Entian K-D (2015) Autoinduction Specificities of the Lantibiotics Subtilin and Nisin. Appl Environ Microbiol 81:7914–7923. https://doi.org/10.1128/AEM.02392-15
Stein T, Borchert S, Conrad B, Feesche J, Hofemeister B, Hofemeister J, Entian K-D (2002a) Two different lantibiotic-like peptides originate from the ericin gene cluster of Bacillus subtilis A1/3. J Bacteriol 184:1703–1711. https://jb.asm.org/content/184/6/1703
Stein T, Borchert S, Kiesau P, Heinzmann S, Klöss S, Klein C, Helfrich M, Entian K-D (2002b) Dual control of subtilin biosynthesis and immunity in Bacillus subtilis. Mol Microbiol 44:403–416. https://doi.org/10.1046/j.1365-2958.2002.02869.x
Stein T, Entian K-D (2002) Maturation of the lantibiotic subtilin: matrix-assisted laser desorption/ionization time-of-flight mass spectrometry to monitor precursors and their proteolytic processing in crude bacterial cultures. Rapid Commun Mass Spectrom 16:103–110. https://doi.org/10.1002/rcm.552
Stein T, Heinzmann S, Kiesau P, Himmel B, Entian K-D (2003) The spa-box for transcriptional activation of subtilin biosynthesis and immunity in Bacillus subtilis. Mol Microbiol 47:1627–1636. https://onlinelibrary.wiley.com/doi/full/ 10.1046/j.1365-2958.2003.03374.x
Stein T, Düsterhus S, Stroh A, Entian K-D (2004) Subtilosin Production by Two Bacillus subtilis Subspecies and Variance of the sbo-alb Cluster. Appl Environ Microbiol 70:2349–2353. https://doi.org/10.1128/AEM.70.4.2349-2353.2004
Stein T (2005) Bacillus subtilis antibiotics: structures, syntheses, and specific functions. Mol Microbiol 56:845–857. https://doi.org/10.1111/j.1365-2958.2005.04587.x
Stein T, Heinzmann S, Düsterhus S, Borchert S, Entian K-D (2005) Expression and functional analysis of the subtilin immunity genes spaIFEG in the subtilin-sensitive host Bacillus subtilis MO1099. J Bacteriol 187:822–828. https://doi.org/10.1128/JB.187.3.822-828.2005
Stein T (2008) Whole-cell matrix-assisted laser desorption/ionization mass spectrometry for rapid identification of bacteriocin/lantibiotic-producing bacteria. Rapid Commun Mass Spectrom 22:1146–1152. https://onlinelibrary.wiley.com/doi/full/ 10.1002/rcm.3481
Stein T (2020) Oxygen-limiting growth conditions and deletion of the transition state regulator protein Abrb in Bacillus subtilis 6633 result in an increase in subtilosin production and a decrease in subtilin production. Probiotics Antimicrob Proteins 12:725–731. https://doi.org/10.1007/s12602-019-09547-4
Sun P, Li J, Bu D, Nan X, Du H (2016) Effects of Bacillus subtilis natto and Different Components in Culture on Rumen Fermentation and Rumen Functional Bacteria In Vitro. Curr Microbiol 72:589–595. https://doi.org/10.1007/s00284-016-0986-z
Torres MJ, Brandan CP, Petroselli G, Erra-Balsells R, Audisio MC (2016) Antagonistic effects of Bacillus subtilis subsp. subtilis and B. amyloliquefaciens against Macrophomina phaseolina: SEM study of fungal changes and UV-MALDI-TOF MS analysis of their bioactive compounds. Microbiol Res 182:31-39. https://doi.org/10.1016/j.micres.2015.09.005
Tran C, Cock IE, Chen X, Feng Y (2022) Antimicrobial Bacillus: Metabolites and Their Mode of Action. Antibiotics 11:88. https://doi.org/10.3390/antibiotics11010088
Velho RV, Basso AP, Segalin J, Costa-Medina LF, Brandelli A (2013) The presence of sboA and spaS genes and antimicrobial peptides subtilosin A and subtilin among Bacillus strains of the Amazon basin. Genet Mol Biol 36:101–104. https://doi.org/10.1590/S1415-47572013000100014
Walker MC, Eslami SM, Hetrick KJ, Ackenhusen SE, Mitchell DA, van der Donk WA (2020) Precursor peptide-targeted mining of more than one hundred thousand genomes expands the lanthipeptide natural product family. BMC Genomics 21:387. https://doi.org/10.1186/s12864-020-06785-7
Waterhouse AM, Procter JB, Martin DMA, Clamp M, Barton GJ (2009) Jalview Version 2-a multiple sequence alignment editor and analysis workbench. Bioinformatics 25:1189–1191. https//DOI.org/10.1093/bioinformatics/btp033
Xu SY, Boitano M, Clark TA, Vincze T, Fomenkov A, Kumar S, Too PH, Gonchar D, Degtyarev SK, Roberts RJ (2015) Complete Genome Sequence Analysis of Bacillus subtilis T30. Genome announcements vol. 3 e00395-15. https://doi.org/10.1128/genomeA.00395-15
Yue X, Li, Liu Y, Zhang X, Zheng Y (2021) Microbial diversity and function of soybean paste in EastAsia: what we know and what we don’t. Curr Opin Food Sci 37:145–152. https://doi.org/10.1016/j.cofs.2020.10.012
Zeigler DR, Prágai Z, Rodriguez S, Chevreux B, Muffler A, Albert T, Bai R, Wyss M, Perkins JB
The origins of 168, W23, and other Bacillus subtilis legacy strains.J Bacteriol190:6983–9695. https://jb.asm.org/content/190/21/6983
Zeigler DR (2011) The genome sequence of Bacillus subtilis subsp. spizizenii W23: insights into speciation within the B. subtilis complex and into the history of B. subtilis genetics. Microbiology 157:2033–2041. https://doi.org/10.1099/mic.0.048520-0
Zhao P, Xue Y, Gao W, Li J, Zu X, Fu D et al (2018) Bacillaceae-derived peptide antibiotics since 2000. Peptides 101:10–16. https://doi.org/10.1016/j.peptides.2017.12.018
Zheng G, Yan LZ, Vederas JC, Zuber P (1999) Genes of the sbo-alb locus of Bacillus subtilis are required for production of the antilisterial bacteriocin subtilosin. J Bacteriol 181:7346–7355. https://doi.org/10.1128/JB.181.23.7346-7355.1999
Zhang Q, Yu Y, Vélasquez JE, van der Donk WA (2012) Evolution of lanthipeptide synthetases. Proc Natl Acad Sci U S A 109:18361–18366. https://www.pnas.org/content/109/45/18361
Zhang Q, Yu Y, Vélasquez JE, van der Donk WA (2012) Evolution of lanthipeptide synthetases. Proc Natl Acad Sci U S A. 109:18361-18366. https://www.pnas.org/content/109/45/18361

Helfrichetal.Supportinginformation.pdf

Download PDF

Reviewers invited by journal
12 Feb, 2022
Editor invited by journal
10 Feb, 2022
Editor assigned by journal
06 Feb, 2022
First submitted to journal
04 Feb, 2022

You are reading this latest preprint version

Antibiotic and genetic profiling of field-collected and laboratory wild-type strains reveals seven novel Bacillus spizizenii strains that produce the lanthipeptide subtilin

Status:

Version 1

Abstract

Figures

Introduction

Materials And Methods

Isolation of subtilin producing Bacilli

Molecular biology techniques

DNA Sequence and phylogenetic analyses

MALDI-TOF mass spectrometry (MALDI-TOF MS)

Subtilin quantitation

SDS-PAGE and Western blotting

Results And Discussion

Quantitative determination of subtilin

Conclusion

Declarations

Contributions

Corresponding author

Ethics approval

Consent for publication

References

Supplementary Files

Status:

Version 1