GroEL-Proteotyping of Bacterial Communities Using Tandem Mass Spectrometry.

Profiling bacterial populations in mixed communities is a common task in microbiology. Sequencing of 16S small subunit ribosomal-RNA (16S rRNA) gene amplicons is a widely accepted and functional approach but relies on amplification primers and cannot quantify isotope incorporation. Tandem mass spectrometry proteotyping is an effective alternative for taxonomically profiling microorganisms. We suggest that targeted proteotyping approaches can complement traditional population analyses. Therefore, we describe an approach to assess bacterial community compositions at the family level using the taxonomic marker protein GroEL, which is ubiquitously found in bacteria, except a few obligate intracellular species. We refer to our method as GroEL-proteotyping. GroEL-proteotyping is based on high-resolution tandem mass spectrometry of GroEL peptides and identification of GroEL-derived taxa via a Galaxy workflow and a subsequent Python-based analysis script. Its advantage is that it can be performed with a curated and extendable sample-independent database and that GroEL can be pre-separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) to reduce sample complexity, improving GroEL identification while simultaneously decreasing the instrument time. GroEL-proteotyping was validated by employing it on a comprehensive raw dataset obtained through a metaproteome approach from synthetic microbial communities as well as real human gut samples. Our data show that GroEL-proteotyping enables fast and straightforward profiling of highly abundant taxa in bacterial communities at reasonable taxonomic resolution.

Characterization of phage vB_EcoS-EE09 infecting E. coli DSM613 Isolated from Wastewater Treatment Plant Effluent and Comparative Proteomics of the Infected and Non-Infected Host.

Phages influence microbial communities, can be applied in phage therapy, or may serve as bioindicators, e.g., in (waste)water management. We here characterized the Escherichia phage vB_EcoS-EE09 isolated from an urban wastewater treatment plant effluent. Phage vB_EcoS-EE09 belongs to the genus Dhillonvirus, class Caudoviricetes. It has an icosahedral capsid with a long non-contractile tail and a dsDNA genome with an approximate size of 44 kb and a 54.6% GC content. Phage vB_EcoS-EE09 infected 12 out of the 17 E. coli strains tested. We identified 16 structural phage proteins, including the major capsid protein, in cell-free lysates by protein mass spectrometry. Comparative proteomics of protein extracts of infected E. coli cells revealed that proteins involved in amino acid and protein metabolism were more abundant in infected compared to non-infected cells. Among the proteins involved in the stress response, 74% were less abundant in the infected cultures compared to the non-infected controls, with six proteins showing significant less abundance. Repressing the expression of these proteins may be a phage strategy to evade host defense mechanisms. Our results contribute to diversifying phage collections, identifying structural proteins to enable better reliability in annotating taxonomically related phage genomes, and understanding phage-host interactions at the protein level.