Taxonomical composition and functional analysis of biofilms sampled from a nuclear storage pool

Abstract

Sampling small amounts of biofilm from harsh environments such as the biofilm present on the walls of a radioactive material storage pool offers few analytical options if taxonomic characterization and estimation of the different biomass contributions are the objectives. Although 16S/18S rRNA amplification on extracted DNA and sequencing is the most widely applied method, its reliability in terms of quantitation has been questioned as yields can be species-dependent. Here, we propose a tandem-mass spectrometry proteotyping approach consisting of acquiring peptide data and interpreting then against a generalist database without any a priori. The peptide sequence information is transformed into useful taxonomical information that allows to obtain the different biomass contributions at different taxonomical ranks. This new methodology is applied for the first time to analyze the composition of biofilms from minute quantities of material collected from a pool used to store radioactive sources in a nuclear facility. For these biofilms, we report the identification of three genera, namely Sphingomonas, Caulobacter, and Acidovorax, and their functional characterization by metaproteomics which shows that these organisms are metabolic active. Differential expression of Gene Ontology GOslim terms between the two main microorganisms highlights their metabolic specialization.

First Isolation and Characterization of Bacteria from the Core’s Cooling Pool of an Operating Nuclear Reactor

“…After isolation, the microorganisms were identified either by sequencing the DNA corresponding to 16S rRNA after amplification by PCR carried out on a colony, or by phylopeptidomics after extraction of proteins [29,31]. Amplification of 16S rDNA was performed with the universal primers 27F (5 -AGAGT TTGATCMTGGCTCAG-3 ) and 1492R (5 -TACGGYTACCTTGTTACGACTT-3 ) at an annealing temperature of 61 • C. Elongation was performed using DNA Phusion polymerase (Thermo Fisher, Waltham, MA, USA) at 72 • C for 45 s. The PCR products obtained after 35 cycles were purified on the gel (NucleoSpin ® Gel and PCR Clean-up, Macherey-Nagel).…”

Exploring Andean High-Altitude Lake Extremophiles through Advanced Proteotyping

“…Recently, tandem mass (MS/MS) spectrometry proteotyping has been documented as a competitive option to identify and characterize environmental isolates, as exemplified with marine isolates 29 and biofilms from extreme environments. 30 It utilizes the shotgun proteomics approach in which proteins are enzymatically cleaved into small peptides using trypsin. The resulting peptides are then separated by reverse-phase chromatography and sequenced by using MS/MS spectrometry.…”

A Simplified Label-Free Method for Proteotyping Sets of Six Isolates in a Single Liquid Chromatography-High-Resolution Tandem Mass Spectrometry Analysis

“…This information, thanks to its unparalleled discriminative power, makes it possible to rapidly identify atypical isolates and classify closely related microorganisms at the subspecies level. It is even compatible with mixtures of microorganisms which would represent a deadlock for whole-cell MALDI-TOF. , High-throughput application of this recent technology is feasible in 96-well plate format and is robust in terms of bacterial loads . As a result, tandem mass spectrometry proteotyping has been applied in clinical diagnostics to discriminate Pneumococcus species within the Mitis group, to identify Francisella directly from hare carcasses without the need to perform cultures, to detect SARS-CoV-2 coronaviruses, to screen marine isolates, to identify the microbial flora in samples from cystic fibrosis patients, to authenticate historical remains, and to diagnose infectious disease from 15-century-old bones .…”