Discrimination and Phylogenomic Classification of Bacillus anthracis-cereus-thuringiensis Strains Based on LC-MS/MS Analysis of Whole Cell Protein Digests

Abstract

Modern taxonomy, diagnostics, and forensics of bacteria benefit from technologies that provide data for genome-based classification and identification of strains; however, full genome sequencing is still costly, lengthy, and labor intensive. Therefore, other methods are needed to estimate genomic relatedness among strains in an economical and timely manner. Although DNA-DNA hybridization and techniques based on genome fingerprinting or sequencing selected genes like 16S rDNA, gyrB, or rpoB are frequently used as phylogenetic markers, analyses of complete genome sequences showed that global measures of genome relatedness, such as the average genome conservation of shared genes, can provide better strain resolution and give phylogenies congruent with relatedness revealed by traditional phylogenetic markers. Bacterial genomes are characterized by a high gene density; therefore, we investigated the integration of mass spectrometry-based proteomic techniques with statistical methods for phylogenomic classification of bacterial strains. For this purpose, we used a set of well characterized Bacillus cereus group strains isolated from poisoned food to describe a method that relies on liquid chromatography-electrospray ionization-tandem mass spectrometry of tryptic peptides derived from whole cell digests. Peptides were identified and matched to a prototype database (DB) of reference bacteria with fully sequenced genomes to obtain their phylogenetic profiles. These profiles were processed for predicting genomic similarities with DB bacteria estimated by fractions of shared peptides (FSPs). FSPs served as descriptors for each food isolate and were jointly analyzed using hierarchical cluster analysis methods for revealing relatedness among investigated strains. The results showed that phylogenomic classification of tested food isolates was in consonance with results from established genomic methods, thus validating our findings. In conclusion, the proposed approach could be used as an alternative method for predicting relatedness among microbial genomes of B. cereus group members and potentially may circumvent the need for whole genome sequencing for phylogenomic typing of strains.

Proteomic Analysis of Bacillus thuringiensis at Different Growth Phases by Using an Automated Online Two-Dimensional Liquid Chromatography-Tandem Mass Spectrometry Strategy

“…Hence, an in-depth study on the synthesis mechanisms of B. thuringiensis parasporal crystals and spores under different physiologically relevant conditions at the proteomic level is highly significant. To date, most B. thuringiensis proteomic studies have mainly focused on parasporal crystal purification and identification (7,52), spore composition and function (13,22), classification of Bacillus strains (16), and so on. There has been no report on the large-scale characterization of the B. thuringiensis proteome, which is becoming one of the international hot spots of research.…”

Proteomics in Food Science

“…The bottom-up proteomic approach , in which proteins are digested into peptides using proteases and the peptides are ionized, mass measured, and fragmented in a tandem mass spectrometer (Aebersold and Mann, 2003), has also been used for bacterial identification (Warscheid et al, 2003;Pribil et al, 2005). Recently, the discrimination and phylogenomic classification of Bacillus anthracis, Bacillus cereus, and Bacillus thuringiensis strains using LC-MS/MS of whole cell protein digests was reported (Dworzanski et al, 2010). In addition to the bottomup approach, we have the top-down proteomic approach that yields highly accurate masses and makes protein digestion unnecessary, providing a good approach for the characterization of proteins from microorganisms (Demirev et al, 2005;Fagerquist et al, 2009;Wynne et al, 2009).…”

Mass spectrometry and tandem mass spectrometry characterization of protein patterns, protein markers and whole proteomes for pathogenic bacteria

“…The same can be said for APEO n (alkylphenol polyethoxylate) -degrading bacteria e.g. strain BSN20 and Sphingopyxis terrae strain NBRC 15098, which share 99.9% sequence identity (Dworzanski et al, 2010, Hotta et al, 2012. It has been suggested that using less conserved genes (e.g.…”

“…It has been suggested that using less conserved genes (e.g. gyrase [gyrB], RNA polymerase [rpoB], aconitate hydratase and superoxide dismutase [sodA]) may yield a higher discriminatory power when trying to speciate certain bacterial strains (Dworzanski et al, 2010 in the absence of oxygen) with mass spectrometry by which lyophilized bacteria were directly inserted into the ion source of a mass spectrometer. Unfortunately, the complex and elegant monomeric, oligomeric and polymeric structures present in bacteria are converted into low mass volatiles, destroying most of the information present in the original cellular biochemistry.…”

“…In contrast, MALDI-TOF MS as mentioned above, takes a few minutes (Martens et al, 2008;Dworzanski et al, 2010;Karlsson et al, 2012 Many groups are creating in house databases in order to more accurately identify microbes. A technique, aptly named S10-GERMS (s10-spc-alpha operon gene-encoded ribosomal mass spectrum) was able to discriminate Sphingopyxis terrae strain NBRC 15098 from APEO n -degrading bacteria (alkylphenol polyethoxylate) strain BSN20 by using s14, a ribosomal subunit protein, as a biomarker.…”