Genome analysis reveals probiotic propensities of Paenibacillus polymyxa HK4.

The legislations on the usage of antibiotics as growth promoters and prophylactic agents have compelled to develop alternative tools to upsurge the animal protection and contain antibiotic usage. Probiotics have emerged as an effective antibiotic substitute in animal farming. The present study explores the probiotic perspective of Paenibacillus polymyxa HK4 interlinking the genotypic and phenotypic characteristics. The draft genome of HK4 revealed the presence of ORFs encoding the functions associated with tolerance to gastrointestinal stress and adhesion. The biosynthetic gene clusters encoding non-ribosomally synthesized peptides, polyketides and lanthipeptides such as fusaricidin, tridecaptin, polymyxin, paenilan and paenibacillin were annotated in HK4 genome. The strain harbored the chromosomal gene conferring the resistance to lincosamides. No functional gene encoding virulence or toxins could be identified in the genome of HK4. The genome analysis data was complemented by the in vitro experiments confirming its survival during gastrointestinal transit, antimicrobial potential and antibiotic sensitivity. NUCLEOTIDE SEQUENCE ACCESSION NUMBER: The draft-genome sequence of Paenibacillus polymyxa HK4 has been deposited as whole-genome shotgun project at GenBank under the accession number PRJNA603023.

Control of the polymyxin analog ratio by domain swapping in the nonribosomal peptide synthetase of Paenibacillus polymyxa.

Polymyxins are used as the last-line therapy against multidrug-resistant bacteria. However, their further clinical development needs to solve problems related to the presence of heterogeneous analogs, but there is still no platform or methods that can regulate the biosynthesis of polymyxin analogs. In this study, we present an approach to swap domains in the polymyxin gene cluster to regulate the production of different analogs. Following adenylation domain swapping, the proportion of polymyxin B1 increased from 41.36 to 52.90%, while that of B1-1 decreased from 18.25 to 3.09%. The ratio of polymyxin B1 and B3 following starter condensation domain swapping changed from 41.36 and 16.99 to 55.03 and 6.39%, respectively. The two domain-swapping strains produced 62.96% of polymyxin B1, 6.70% of B3 and 3.32% of B1-1. This study also revealed the presence of overflow fluxes between acetoin, 2,3-butanediol and polymyxin. To our best knowledge, this is the first report of engineering the polymyxin synthetase gene cluster in situ to regulate the relative proportions of polymyxin analogs. This research paves a way for regulating lipopeptide analogs and will facilitate the development of novel lipopeptide derivatives.

Plant growth promotion and suppression of bacterial leaf blight in rice by Paenibacillus polymyxa Sx3.

The effects and mechanisms of Paenibacillus polymyxa Sx3 on growth promotion and the suppression of bacterial leaf blight in rice were evaluated in this study. The results from a plate assay indicated that Sx3 inhibited the growth of 20 strains of Xanthomonas oryzae pv. oryzae (Xoo). Rice seedling experiments indicated that Sx3 promoted plant growth and suppressed bacterial leaf blight. In addition, bacteriological tests showed that Sx3 was able to fix nitrogen, solubilize phosphate and produce indole acetic acid, indicating that various mechanisms may be involved in the growth promotion by Sx3. The culture filtrate of P. polymyxa Sx3 reduced bacterial growth, biofilm formation and disrupted the cell morphology of Xoo strain GZ 0005, as indicated by the transmission and scanning electron microscopic observations. In addition, MALDI-TOF MS analysis revealed that Sx3 could biosynthesize two types of secondary metabolites fusaricidins and polymyxin P. In summary, this study clearly indicated that P. polymyxa Sx3 has strong in vitro and in vivo antagonistic activity against Xoo, which may be at least partially attributed to its production of secondary metabolites. SIGNIFICANCE AND IMPACT OF THE STUDY: Antagonistic bacteria can grow well in their originating environment. However, it is unclear whether antagonistic bacteria were able to survive in different ecological environments. This study revealed that Paenibacillus polymyxa Sx3 isolated from rhizosphere soil of cotton significantly promoted the plant growth and suppressed bacterial leaf blight in rice. Therefore, it could be inferred that P. polymyxa Sx3 has the potential to be used as biocontrol agents in plants grown in different ecological environments.

Genome Mining of the Lipopeptide Biosynthesis of Paenibacillus polymyxa E681 in Combination with Mass Spectrometry: Discovery of the Lipoheptapeptide Paenilipoheptin.

Paenibacillus polymyxa strains are qualified for agro-biotechnological uses such as plant growth promotion and for biocontrol strategies against deleterious phytopathogenic competitors in the soil depending on their attractive arsenal of bioactive compounds. Moreover, they are potent producers of antibiotics for medical applications. To identify new products of such organisms, genome mining strategies in combination with mass spectrometry are the methods of choice. Herein, we performed such studies with the Paenibacillus strain E681. Bioinformatic evaluation of its genome sequence revealed four gene clusters A-D encoding nonribosomal peptide synthetases (NRPSs). Accordingly, four lipopeptide families were detected by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). Clusters A and D codify the well known fusaricidins and polymyxins. A yet-unknown lipoheptapeptide was discovered and structurally characterized by de novo sequencing by using MALDI-LIFT-TOF/TOF MS. It was designated as paenilipoheptin. From structure predictions we infer that the production of this agent is encoded by gene cluster C. Gene cluster B encodes the synthesis of tridecaptins, a family of open-chain lipotridecapeptides. Strain E681 produces new subspecies of such compounds (tridecaptins E) showing variations both in their fatty-acid part as well as in their peptide part.

Deciphering MCR-2 Colistin Resistance.

Antibiotic resistance is a prevalent problem in public health worldwide. In general, the carbapenem ß-lactam antibiotics are considered a final resort against lethal infections by multidrug-resistant bacteria. Colistin is a cationic polypeptide antibiotic and acts as the last line of defense for treatment of carbapenem-resistant bacteria. Very recently, a new plasmid-borne colistin resistance gene, mcr-2, was revealed soon after the discovery of the paradigm gene mcr-1, which has disseminated globally. However, the molecular mechanisms for MCR-2 colistin resistance are poorly understood. Here we show a unique transposon unit that facilitates the acquisition and transfer of mcr-2 Evolutionary analyses suggested that both MCR-2 and MCR-1 might be traced to their cousin phosphoethanolamine (PEA) lipid A transferase from a known polymyxin producer, Paenibacillus Transcriptional analyses showed that the level of mcr-2 transcripts is relatively higher than that of mcr-1 Genetic deletions revealed that the transmembrane regions (TM1 and TM2) of both MCR-1 and MCR-2 are critical for their location and function in bacterial periplasm, and domain swapping indicated that the TM2 is more efficient than TM1. Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) confirmed that all four MCR proteins (MCR-1, MCR-2, and two chimeric versions [TM1-MCR-2 and TM2-MCR-1]) can catalyze chemical modification of lipid A moiety anchored on lipopolysaccharide (LPS) with the addition of phosphoethanolamine to the phosphate group at the 4' position of the sugar. Structure-guided site-directed mutagenesis defined an essential 6-residue-requiring zinc-binding/catalytic motif for MCR-2 colistin resistance. The results further our mechanistic understanding of transferable colistin resistance, providing clues to improve clinical therapeutics targeting severe infections by MCR-2-containing pathogens.IMPORTANCE Carbapenem and colistin are the last line of refuge in fighting multidrug-resistant Gram-negative pathogens. MCR-2 is a newly emerging variant of the mobilized colistin resistance protein MCR-1, posing a potential challenge to public health. Here we report transfer of the mcr-2 gene by a unique transposal event and its possible origin. Distribution of MCR-2 in bacterial periplasm is proposed to be a prerequisite for its role in the context of biochemistry and the colistin resistance. We also define the genetic requirement of a zinc-binding/catalytic motif for MCR-2 colistin resistance. This represents a glimpse of transferable colistin resistance by MCR-2.

Characterization of the Polymyxin D Synthetase Biosynthetic Cluster and Product Profile of Paenibacillus polymyxa ATCC 10401.

The increasing prevalence of polymyxin-resistant bacteria has stimulated the search for improved polymyxin lipopeptides. Here we describe the sequence and product profile for polymyxin D nonribosomal peptide synthetase from Paenibacillus polymyxa ATCC 10401. The polymyxin D synthase gene cluster comprised five genes that encoded ABC transporters (pmxC and pmxD) and enzymes responsible for the biosynthesis of polymyxin D (pmxA, pmxB, and pmxE). Unlike polymyxins B and E, polymyxin D contains d-Ser at position 3 as opposed to l-α,γ-diaminobutyric acid and has an l-Thr at position 7 rather than l-Leu. Module 3 of pmxE harbored an auxiliary epimerization domain that catalyzes the conversion of l-Ser to the d-form. Structural modeling suggested that the adenylation domains of module 3 in PmxE and modules 6 and 7 in PmxA could bind amino acids with larger side chains than their preferred substrate. Feeding individual amino acids into the culture media not only affected production of polymyxins D1 and D2 but also led to the incorporation of different amino acids at positions 3, 6, and 7 of polymyxin D. Interestingly, the unnatural polymyxin analogues did not show antibiotic activity against a panel of Gram-negative clinical isolates, while the natural polymyxins D1 and D2 exhibited excellent in vitro antibacterial activity and were efficacious against Klebsiella pneumoniae and Acinetobacter baumannii in a mouse blood infection model. The results demonstrate the excellent antibacterial activity of these unusual d-Ser3 polymxyins and underscore the possibility of incorporating alternate amino acids at positions 3, 6, and 7 of polymyxin D via manipulation of the polymyxin nonribosomal biosynthetic machinery.

Biosynthesis of Polymyxins B, E, and P Using Genetically Engineered Polymyxin Synthetases in the Surrogate Host Bacillus subtilis.

The development of diverse polymyxin derivatives is needed to solve the toxicity and resistance problems of polymyxins. However, no platform has generated polymyxin derivatives by genetically engineering a polymyxin synthetase, which is a nonribosomal peptide synthetase. In this study, we present a two-step approach for the construction of engineered polymyxin synthetases by substituting the adenylation (A) domains of polymyxin A synthetase, which is encoded by the pmxABCDE gene cluster of Paenibacillus polymyxa E681. First, the seventh L-threonine-specific A-domain region in pmxA was substituted with the Lleucine- specific A-domain region obtained from P. polymyxa ATCC21830 to make polymyxin E synthetase, and then the sixth D-leucine-specific A-domain region (A6-D-Leu-domain) was substituted with the D-phenylalanine-specific A-domain region (A6-D-Phe-domain) obtained from P. polymyxa F4 to make polymyxin B synthetase. This step was performed in Escherichia coli on a pmxA-containing fosmid, using the lambda Red recombination system and the sacB gene as a counter-selectable marker. Next, the modified pmxA gene was fused to pmxBCDE on the chromosome of Bacillus subtilis BSK4dA, and the resulting recombinant strains BSK4-PB and BSK4-PE were confirmed to produce polymyxins B and E, respectively. We also succeeded in constructing the B. subtilis BSK4-PP strain, which produces polymyxin P, by singly substituting the A6-D-Leu-domain with the A6-D-Phe-domain. This is the first report in which polymyxin derivatives were generated by genetically engineering polymyxin synthetases. The two recombinant B. subtilis strains will be useful for improving the commercial production of polymyxins B and E, and they will facilitate the generation of novel polymyxin derivatives.

Antibacterial mechanisms of polymyxin and bacterial resistance.

Multidrug resistance in pathogens is an increasingly significant threat for human health. Indeed, some strains are resistant to almost all currently available antibiotics, leaving very limited choices for antimicrobial clinical therapy. In many such cases, polymyxins are the last option available, although their use increases the risk of developing resistant strains. This review mainly aims to discuss advances in unraveling the mechanisms of antibacterial activity of polymyxins and bacterial tolerance together with the description of polymyxin structure, synthesis, and structural modification. These are expected to help researchers not only develop a series of new polymyxin derivatives necessary for future medical care, but also optimize the clinical use of polymyxins with minimal resistance development.

Isolation and characterization of an antimicrobial lipopeptide produced by Paenibacillus ehimensis MA2012.

In this study, a novel lipopeptide antibiotic was isolated from the culture supernatant of Paenibacillus ehimensis strain MA2012. After analyses by mass spectrometry (MS), nuclear magnetic resonance (NMR), and high resolution mass spectrometry (HR-MS/MS) the compound was identified to be polypeptin C consisting of 3-hydroxy-4-methyl-hexanoic acid moiety and nine amino acids as peptide body. It has the same molecular mass (1115 Da) with that of polypeptin A and B but the amino acid positions differ. A relatively low concentration (125 ppm) of polypeptin C lowered the surface tension of water from 72.2 to 36.4 mN/m. It showed antimicrobial activity against several plant pathogenic bacteria and fungi. When the polypeptin C was applied to the ripe pepper fruits previously inoculated with conidia of Colletotrichum gloeosporioides, the hyphal growth on the fruit was significantly suppressed. Moreover, the hyphal morphology of C. gloeosporioides was greatly affected by the purified compound. All these data suggest the great potential of P. ehimensis MA2012 to control plant fungal and bacterial diseases.

[Construction of gene knock-out system for Paenibacillus polymyxa SC2].

OBJECTIVE: To construct an efficient gene knock-out system for Paenibacillus polymyxa SC2. METHODS: Temperature sensitive plasmid pRN5101 was transformed into P. polymyxa SC2 by electrotransformation. A mutant SC2-E was obtained, in which pmxE was disrupted by homologous recombination. To confirm whether pmxE was knocked out, we used antibacterial activity assay and high performance liquid chromatography to analyze the ability of mutants synthesizing polymyxin. RESULTS: We developed an efficient gene knock-out system for P. polymyxa SC2. Plasmid of pRN5101 could replicate at 28 degrees C and suicide at 39 degrees C in SC2. Mutants lost the ability of synthesizing polymyxin, indicating that pmxE gene was successfully knocked out. CONCLUSION: The constructed gene knock-out system for P. polymyxa provides a high-efficiency tool to detect genes function for P. polymyxa.

Efficient production of polymyxin in the surrogate host Bacillus subtilis by introducing a foreign ectB gene and disrupting the abrB gene.

In our previous study, Bacillus subtilis strain BSK3S, containing a polymyxin biosynthetic gene cluster from Paenibacillus polymyxa, could produce polymyxin only in the presence of exogenously added L-2,4-diaminobutyric acid (Dab). The dependence of polymyxin production on exogenous Dab was removed by introducing an ectB gene encoding the diaminobutyrate synthase of P. polymyxa into BSK3S (resulting in strain BSK4). We found, by observing the complete inhibition of polymyxin synthesis when the spo0A gene was knocked out (strain BSK4-0A), that Spo0A is indispensable for the production of polymyxin. Interestingly, the abrB-spo0A double-knockout mutant, BSK4-0A-rB, and the single abrB mutant, BSK4-rB, showed 1.7- and 2.3-fold increases, respectively, in polymyxin production over that of BSK4. These results coincided with the transcription levels of pmxA in the strains observed by quantitative real-time PCR (qRT-PCR). The AbrB protein was shown to bind directly to the upstream region of pmxA, indicating that AbrB directly inhibits the transcription of polymyxin biosynthetic genes. The BSK4-rB strain, producing high levels of polymyxin, will be useful for the development and production of novel polymyxin derivatives.

Paenibacillus polymyxa PKB1 produces variants of polymyxin B-type antibiotics.

Polymyxins are cationic lipopeptide antibiotics active against many species of Gram-negative bacteria. We sequenced the gene cluster for polymyxin biosynthesis from Paenibacillus polymyxa PKB1. The 40.8 kb gene cluster comprises three nonribosomal peptide synthetase-encoding genes and two ABC transporter-like genes. Disruption of a peptide synthetase gene abolished all antibiotic production, whereas deletion of one or both transporter genes only reduced antibiotic production. Computational analysis of the peptide synthetase modules suggested that the enzyme system produces variant forms of polymyxin B (1 and 2), with D-2,4-diaminobutyrate instead of L-2,4-diaminobutyrate in amino acid position 3. Two antibacterial metabolites were resolved by HPLC and identified by high-resolution mass spectrometry and MS/MS sequencing as the expected variants 3 and 4 of polymyxin B(1) (1) and B(2) (2). Stereochemical analysis confirmed the presence of both D-2,4-diaminobutyrate and L-2,4-diaminobutyrate residues.

Isolation and identification of a Paenibacillus polymyxa strain that coproduces a novel lantibiotic and polymyxin.

A new bacterial strain, displaying potent antimicrobial properties against gram-negative and gram-positive pathogenic bacteria, was isolated from food. Based on its phenotypical and biochemical properties as well as its 16S rRNA gene sequence, the bacterium was identified as Paenibacillus polymyxa and it was designated as strain OSY-DF. The antimicrobials produced by this strain were isolated from the fermentation broth and subsequently analyzed by liquid chromatography-mass spectrometry. Two antimicrobials were found: a known antibiotic, polymyxin E1, which is active against gram-negative bacteria, and an unknown 2,983-Da compound showing activity against gram-positive bacteria. The latter was purified to homogeneity, and its antimicrobial potency and proteinaceous nature were confirmed. The antimicrobial peptide, designated paenibacillin, is active against a broad range of food-borne pathogenic and spoilage bacteria, including Bacillus spp., Clostridium sporogenes, Lactobacillus spp., Lactococcus lactis, Leuconostoc mesenteroides, Listeria spp., Pediococcus cerevisiae, Staphylococcus aureus, and Streptococcus agalactiae. Furthermore, it possesses the physico-chemical properties of an ideal antimicrobial agent in terms of water solubility, thermal resistance, and stability against acid/alkali (pH 2.0 to 9.0) treatment. Edman degradation, mass spectroscopy, and nuclear magnetic resonance were used to sequence native and chemically modified paenibacillin. While details of the tentative sequence need to be elucidated in future work, the peptide was unequivocally characterized as a novel lantibiotic, with a high degree of posttranslational modifications. The coproduction of polymyxin E1 and a lantibiotic is a finding that has not been reported earlier. The new strain and associated peptide are potentially useful in food and medical applications.

Isolation and partial characterization of antagonistic peptides produced by Paenibacillus sp. strain B2 isolated from the sorghum mycorrhizosphere.

Paenibacillus sp. strain B2, isolated from the mycorrhizosphere of sorghum colonized by Glomus mosseae, produces an antagonistic factor. This factor has a broad spectrum of activity against gram-positive and gram-negative bacteria and also against fungi. The antagonistic factor was isolated from the bacterial culture medium and purified by cation-exchange, reverse-phase, and size exclusion chromatography. The purified factor could be separated into three active compounds following characterization by amino acid analysis and by combined reverse-phase chromatography and mass spectrometry (liquid chromatography-mass spectrometry and mass spectrometry-mass spectrometry). The first compound had the same retention time as polymyxin B1, whereas the two other compounds were more hydrophobic. The molecular masses of the latter compounds are 1,184.7 and 1,202.7 Da, respectively, and their structure is similar to that of polymyxin B1, with a cyclic heptapeptide moiety attached to a tripeptide side chain and a fatty acyl residue. They both contain threonine, phenylalanine, leucine, and 2,4-diaminobutyric acid residues. The peptide with a molecular mass of 1,184.7 contains a 2,3-didehydrobutyrine residue with a molecular mass of 101 Da replacing a threonine at the A2 position of the polymyxin side chain. This modification could explain the broader range of antagonistic activity of this peptide compared to that of polymyxin B.

Inhibiting mild steel corrosion from sulfate-reducing bacteria using antimicrobial-producing biofilms in Three-Mile-Island process water.

Biofilms were used to produce gramicidin S (a cyclic decapeptide) to inhibit corrosion-causing, sulfate-reducing bacteria (SRB). In laboratory studies these biofilms protected mild steel 1010 continuously from corrosion in the aggressive, cooling service water of the AmerGen Three-Mile-Island (TMI) nuclear plant, which was augmented with reference SRB. The growth of both reference SRB (Gram-positive Desulfosporosinus orientis and Gram-negative Desulfovibrio vulgaris) was shown to be inhibited by supernatants of the gramicidin-S-producing bacteria as well as by purified gramicidin S. Electrochemical impedance spectroscopy and mass loss measurements showed that the protective biofilms decreased the corrosion rate of mild steel by 2- to 10-fold when challenged with the natural SRB of the TMI process water supplemented with D. orientis or D. vulgaris. The relative corrosion inhibition efficiency was 50-90% in continuous reactors, compared to a biofilm control which did not produce the antimicrobial gramicidin S. Scanning electron microscope and reactor images also revealed that SRB attack was thwarted by protective biofilms that secrete gramicidin S. A consortium of beneficial bacteria (GGPST consortium, producing gramicidin S and other antimicrobials) also protected the mild steel.

Novel pyrazine metabolites found in polymyxin biosynthesis by Paenibacillus polymyxa.

A complex mixture of methyl-branched alkyl-substituted pyrazines was found in the growth medium of the polymyxin-producing bacterium Paenibacillus polymyxa, and of these, seven are new natural compounds. A total of 19 pyrazine metabolites were identified. The dominant metabolite was 2,5-diisopropylpyrazine as identified using a combination of high-resolution mass spectrometry, (1)H- and (13)C-nuclear magnetic resonance, gas chromatography-mass spectrometry as well as co-elution with an authentic standard. Its biosynthesis was correlated with growth and production was strongly stimulated by valine supplementation. The other pyrazine metabolites, all related pyrazines with either one, two or three alkyl substituents, were identified by means of their mass spectral data and/or co-elution with authentic standards.

[Isolation and identification of an antibiotic from culture 8-86]. / Vydelenie i identifikatsiia antibiotika iz kul'tury 8-86.

An antibiotic complex was isolated from culture 8-86 referred to Bacillus. The complex consisted of components 8-86A and 8-86B active against gram-negative organisms. By its physico-chemical properties such as IR and UV spectra, amino acid composition, specific rotation and fatty acid composition component 8-86B was shown to be close to polymyxin F.

[Enzyme activity of Bacillus polymyxa 153, the producer of polymyxin B, during sporulation and biosynthesis]. / Aktivnost' nekotorykh fermentov Bacillus polymyxa 153--produtsenta polimiksina B v protsesse sporoobrazovaniia i biosinteza.

Metabolic properties of Bacillus polymyxa 153 were studied during vegetative growth, polymyxin B biosynthesis and active sporulation. In the cell extracts there was detected activity of exoproteases, endoproteases, tricarboxylic acid cycle dehydrogenases and pyruvate dehydrogenase. The enzymes activity in the cells growing into spores was higher than that in the cells of the vegetative developmental type. The activity of the enzymes depended on the culture age.

[Polymyxin biosynthesis by Bacillus polymyxa during growth limitation by the nutritional sources]. / Biosintez polimiksina Bacillus polymyxa pri limitatsii rosta istochnikami pitaniia.

The synthesis of the antibiotic polymyxin M was studied under the conditions of batch and continuous cultivation of Bacillus polymyxa var. Ross whose growth was limited with glucose, phosphate and ammonium nitrogen. Polymyxin M was synthesized when the culture growth decelerated as a result of its limitation with the above compounds. Different amounts of the antibiotic were synthesized depending on the type of a limiting factor. The highest productiveness was found in the case of glucose limitation. The optimal conditions for polymyxin M synthesis were established under the conditions of one-step continuous cultivation.