Over the past decade, modern methods of mass spectrometry (MS) have emerged that allow reliable, fast and cost-effective identification of pathogenic microorganisms. While MALDI-TOF MS has already revolutionized the way microorganisms are identified, recent years have witnessed also substantial progress in the development of liquid chromatography (LC)-MS based proteomics for microbiological applications. For example, LC-tandem mass spectrometry (LC-MS²) has been proposed for microbial characterization by means of multiple discriminative peptides that enable identification at the species, or sometimes at the strain level. However, such investigations can be laborious and time-consuming, especially if the experimental LC-MS² data are tested against sequence databases covering a broad panel of different microbiological taxa. In this proof of concept study, we present an alternative bottom-up proteomics method for microbial identification. The proposed approach involves efficient extraction of proteins from cultivated microbial cells, digestion by trypsin and LC-MS measurements. Peptide masses are then extracted from MS¹ data and systematically tested against an in silico library of all possible peptide mass data compiled in-house. The library has been computed from the UniProt Knowledgebase covering Swiss-Prot and TrEMBL databases and comprises more than 12,000 strain-specific in silico profiles, each containing tens of thousands of peptide mass entries. Identification analysis involves computation of score values derived from correlation coefficients between experimental and strain-specific in silico peptide mass profiles and compilation of score ranking lists. The taxonomic positions of the microbial samples are then determined by using the best-matching database entries. The suggested method is computationally efficient – less than two minutes per sample - and has been successfully tested by a test set of 39 LC-MS¹ peak lists obtained from 19 different microbial pathogens. The proposed method is rapid, simple and automatable and we foresee wide application potential for future microbiological applications.

We have previously reported the draft genome sequences of 59 endospore-forming Gram-positive bacterial strains isolated from Vietnamese crop plants due to their ability to suppress plant pathogens. Based on their draft genome sequence, eleven of them were assigned to the Brevibacillus and one to the Lysinibacillus genus. Further analysis including full genome sequencing revealed that several of these strains represent novel genomospecies. In vitro and in vivo assays demonstrated their ability to promote plant growth, as well as the strong biocontrol potential of Brevibacilli directed against phytopathogenic bacteria, fungi, and nematodes. Genome mining identified 157 natural product biosynthesis gene clusters (BGCs), including 36 novel BGCs not present in the MIBiG data bank. Our findings indicate that plant-associated Brevibacilli are a rich source of putative antimicrobial compounds and might serve as a valuable starting point for the development of novel biocontrol agents.

Seventeen bacterial strains able to suppress plant pathogens have been isolated from healthy Vietnamese crop plants and taxonomically assigned as members of the Bacillus cereus group. In order to prove their potential as biocontrol agents, we perform a comprehensive analysis that included the whole-genome sequencing of selected strains and the mining for genes and gene clusters involved in the synthesis of endo- and exotoxins and secondary metabolites, such as antimicrobial peptides (AMPs). Kurstakin, thumolycin, and other AMPs were detected and characterized by different mass spectrometric methods, such as MALDI-TOF-MS and LIFT-MALDI-TOF/TOF fragment analysis. Based on their whole-genome sequences, the plant-associated isolates were assigned to the following species and subspecies: B. cereus subsp. cereus (6), B. cereus subsp. bombysepticus (5), Bacillus tropicus (2), and Bacillus pacificus. These three isolates represent novel genomospecies. Genes encoding entomopathogenic crystal and vegetative proteins were detected in B. cereus subsp. bombysepticus TK1. The in vitro assays revealed that many plant-associated isolates enhanced plant growth and suppressed plant pathogens. Our findings indicate that the plant-associated representatives of the B. cereus group are a rich source of putative antimicrobial compounds with potential in sustainable agriculture. However, the presence of virulence genes might restrict their application as biologicals in agriculture.