Characterization of the colistin (polymyxin E1 and E2) biosynthetic gene cluster.

Colistin is a mixture of polymyxin E1 and E2, bactericidal pentacationic lipopeptides used to treat infections caused by Gram-negative pathogens such as Pseudomonas aeruginosa and Klebsiella pneumoniae. Industrial production of colistin is obtained by a fermentation process of the natural producer Paenibacillus polymyxa var colistinus. NonRibosomal peptide synthetases (NRPS) coding the biosynthesis of polymyxins A, B and P have been recently described, rendering thereof the improvement of their production possible. However, the colistin biosynthesis pathway was not published so far. In this study, a Paenibacillus alvei has been identified by biochemical (Api 50 CH system) and molecular (16S rDNA sequencing) methods. Its culture supernatant displayed inhibitory activity against Gram-negative bacteria (P. aeruginosa, K. pneumoniae, Salmonella spp.). Two polymyxins, E1 and E2, were recovered from the supernatant and were characterized by high resolution LC-MS. A genomic library (960 clones) was constructed to identify the gene cluster responsible for biosynthesis of polymyxins. Selection of the clones harbouring the sequences of interest was obtained by a simple PCR-based screening. We used primers targeting NRPS sequences leading to the incorporation of amino acids present in polymyxins E. The sequences from three clones of interest were assembled on 50.4 kb. Thus, five open reading frames corresponding to a new NRPS gene cluster of 41 kb were identified. In silico, analyses revealed the presence of three NRPS implicated in the biosynthesis of polymyxins E. This work provides insightful information on colistin biosynthesis and might contribute to future drug developments in this group of antibiotics.

Lactococcus lactis Diversity Revealed by Targeted Amplicon Sequencing of purR Gene, Metabolic Comparisons and Antimicrobial Properties in an Undefined Mixed Starter Culture Used for Soft-Cheese Manufacture.

The undefined mixed starter culture (UMSC) is used in the manufacture of cheeses. Deciphering UMSC microbial diversity is important to optimize industrial processes. The UMSC was studied using culture-dependent and culture-independent based methods. MALDI-TOF MS enabled identification of species primarily from the Lactococcus genus. Comparisons of carbohydrate metabolism profiles allowed to discriminate five phenotypes of Lactococcus (n = 26/1616). The 16S sequences analysis (V1-V3, V3-V4 regions) clustered the UMSC microbial diversity into two Lactococcus operational taxonomic units (OTUs). These clustering results were improved with the DADA2 algorithm on the housekeeping purR sequences. Five L. lactis variants were detected among the UMSC. The whole-genome sequencing of six isolates allowed for the identification of the lactis subspecies using Illumina® (n = 5) and Pacbio® (n = 1) technologies. Kegg analysis confirmed the L. lactis species-specific niche adaptations and highlighted a progressive gene pseudogenization. Then, agar spot tests and agar well diffusion assays were used to assess UMSC antimicrobial activities. Of note, isolate supernatants (n = 34/1616) were shown to inhibit the growth of Salmonella ser. Typhimurium CIP 104115, Lactobacillus sakei CIP 104494, Staphylococcus aureus DSMZ 13661, Enterococcus faecalis CIP103015 and Listeria innocua CIP 80.11. Collectively, these results provide insightful information about UMSC L. lactis diversity and revealed a potential application as a bio-protective starter culture.

Complete Genome Sequence of the Polymyxin E (Colistin)-Producing Paenibacillus sp. Strain B-LR.

Paenibacillus bacteria are recovered from varied niches, including human lung, rhizosphere, marine sediments, and hemolymph. Paenibacilli can have plant growth-promoting activities and be antibiotic producers. They can produce exopolysaccharides and enzymes of industrial interest. Illumina and PacBio reads were used to produce a complete genome sequence of the colistin producer Paenibacillus sp. strain B-LR.

Novel nonribosomal peptide synthetase (NRPS) genes sequenced from intertidal mudflat bacteria.

Nonribosomal peptide synthetases (NRPS) are actively sought out, due to pharmacologically important activities of their metabolites. In marine environment, the most prevalent nonribosomal peptide antibiotic producers are sponges inhabiting microorganisms. Conversely, strains from marine sediments and more especially from intertidal mudflats have not been extensively screened for the presence of new NRPS. In this study, for the first time, a collection of one hundred intertidal mudflat bacterial isolates (Marennes-Oléron Bay, France) was assessed for (1) the presence of NRPS genes by degenerated PCR targeting conserved adenylation domains and (2) for their production of antimicrobial molecules. (1) Bacteria with adenylation domains (14 strains) were identified by 16S rRNA gene sequence analysis and grouped into Firmicutes (one strain) and Proteobacteria (13 strains). In silico analysis of the NRPS amino acid sequences (n = 7) showed 41-58% ID with sequences found in the NCBI database. Three new putative adenylation domain signatures were found. (2) The culture supernatant of one of these strains, identified as a Bacillus, was shown to strongly inhibit the growth of Staphylococcus aureus, S. epidermidis, and Enterococcus faecalis. This study portends that the intertidal mudflat niche could be of interest for the discovery of new NRPS genes and antimicrobial producing strains.

A Novel Depsipeptide Produced by Paenibacillus alvei 32 Isolated from a Cystic fibrosis Patient.

The important viscosity of the respiratory tract mucus of Cystic fibrosis (CF) patients impairs the mucociliary transport system and allows the growth of numerous micro-organisms. Among them, Pseudomonas aeruginosa and Staphylococcus aureus are known to be responsible for pulmonary infections. We imagined that CF microflora could also harbour micro-organisms naturally equipped to compete with these pathogens. A method was developed to recover these antibiotic-producing strains within 20 CF sputum. Using this approach, we have isolated an unusual Gram-positive bacterium identified as Paenibacillus alvei by Api galleries and 16S rRNA gene sequence analysis. This strain has inhibited the growth of P. aeruginosa, S. aureus and Klebsiella pneumoniae, in co-cultures. A liquid mineral medium named MODT50 was designed and optimised for the production and the recovery of the antimicrobial compounds. The supernatant has inhibited the growth of all Gram-positive strains tested, even Methicillin-resistant S. aureus. One antimicrobial compound with a peptide structure (mainly active against S. aureus, Micrococcus luteus, and Pseudomonas stutzeri) has been purified and characterised by liquid chromatography-mass spectrometry. The new active molecule (m/z 786.6) named depsipeptide L possesses a 15-guanidino-3-hydroxypentadecanoic acid side chain (m/z 298) linked on a cyclic part of four amino acids residues (Ser, two Leu/Ile, Arg). This work reports for the first time the production of such a molecule by a P. alvei strain in a mineral medium. The CF lung microflora might represent a valuable source for the discovery of new antimicrobial-producing strains.