Post-transcriptional control of protein abundance is a highly important, underexplored regulatory process by which organisms respond to their environments. Here we describe an important and previously unidentified regulatory pathway involving the ribosomal modification protein RimK, its regulator proteins RimA and RimB, and the widespread bacterial second messenger cyclic-di-GMP (cdG). Disruption of rimK affects motility and surface attachment in pathogenic and commensal Pseudomonas species, with rimK deletion significantly compromising rhizosphere colonisation by the commensal soil bacterium P. fluorescens, and plant infection by the pathogens P. syringae and P. aeruginosa. RimK functions as an ATP-dependent glutamyl ligase, adding glutamate residues to the C-terminus of ribosomal protein RpsF and inducing specific effects on both ribosome protein complement and function. Deletion of rimK in P. fluorescens leads to markedly reduced levels of multiple ribosomal proteins, and also of the key translational regulator Hfq. In turn, reduced Hfq levels induce specific downstream proteomic changes, with significant increases in multiple ABC transporters, stress response proteins and non-ribosomal peptide synthetases seen for both ΔrimK and Δhfq mutants. The activity of RimK is itself controlled by interactions with RimA, RimB and cdG. We propose that control of RimK activity represents a novel regulatory mechanism that dynamically influences interactions between bacteria and their hosts; translating environmental pressures into dynamic ribosomal changes, and consequently to an adaptive remodeling of the bacterial proteome.

Rapid but yet sensitive, specific, and high-throughput detection of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in clinical samples is key to diagnose infected people and to better control the spread of the virus. Alternative methodologies to PCR and immunodiagnostics that would not require specific reagents are worthy to investigate not only for fighting the COVID-19 pandemic but also to detect other emergent pathogenic threats. Here, we propose the use of tandem mass spectrometry to detect SARS-CoV-2 marker peptides in nasopharyngeal swabs. We documented that the signal from the microbiota present in such samples is low and can be overlooked when interpreting shotgun proteomic data acquired on a restricted window of the peptidome landscape. In this proof-of-concept study, simili nasopharyngeal swabs spiked with different quantities of purified SARS-CoV-2 viral material were used to develop a nanoLC–MS/MS acquisition method, which was then successfully applied on COVID-19 clinical samples. We argue that peptides ADETQALPQR and GFYAQGSR from the nucleocapsid protein are of utmost interest as their signal is intense and their elution can be obtained within a 3 min window in the tested conditions. These results pave the way for the development of time-efficient viral diagnostic tests based on mass spectrometry.

Detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a crucial tool for fighting the COVID-19 pandemic. This dataset brief presents the exploration of a shotgun proteomics dataset acquired on SARS-CoV-2 infected Vero cells. Proteins from inactivated virus samples were extracted, digested with trypsin, and the resulting peptides were identified by data-dependent acquisition tandem mass spectrometry. The 101 peptides reporting for six viral proteins were specifically analyzed in terms of their analytical characteristics, species specificity and conservation, and their proneness to structural modifications. Based on these results, a shortlist of 14 peptides from the N, S, and M main structural proteins that could be used for targeted mass-spectrometry method development and diagnostic of the new SARS-CoV-2 is proposed and the best candidates are commented.The world is facing a tremendous pandemic caused by the SARS-CoV-2 virus. [1] The mutations naturally-occurring in the viral genome upon its spread may challenge molecular biology diagnostic tests. [2,3] Mass-spectrometry (MS) based detection of organisms could be an alternative for fast deployment of novel detection means in case of emergence of new pathogens. More specifically, proteotyping based on peptide/protein mass measurement can be performed with either a targeted strategy or without any a priori. [4,5] For such purpose sample preparation, acquisition modes and parameters should be optimized depending on the nature of the samples and the target. Targeted mass spectrometry approaches can be applied for the routine detection of pathogens with high sensitivity. [6] Previous knowledge of

Shotgun proteomics analysis of SARS-CoV-2-infected cells and how it can optimize whole viral particle antigen production for vaccines,

Tandem mass spectrometry-based proteotyping allows characterizing microorganisms in terms of taxonomy and is becoming an important tool for investigating microbial diversity from several ecosystems. Fast and automatable sample preparation for obtaining peptide pools amenable to tandem mass spectrometry is necessary for enabling proteotyping as a high-throughput method. First, the protocol to increase the yield of lysis of several representative bacterial and eukaryotic microorganisms was optimized by using a long and drastic bead-beating setting with 0.1 mm silica beads, 0.1 and 0.5 mm glass beads, in presence of detergents. Then, three different methods to obtain greater digestion yield from these extracts were tested and optimized for improve efficiency and reduce application time: denaturing electrophoresis of proteins and in-gel proteolysis, suspension-trapping filter-based approach (S-Trap) and, solid-phase-enhanced sample preparation named SP3. The latter method outperforms the other two in terms of speed and delivers also more peptides and proteins than with the in-gel proteolysis (2.2 fold for both) and S-trap approaches (1.3 and 1.2 fold, respectively). Thus, SP3 directly improves tandem mass spectrometry proteotyping.

Effective regulation of primary carbon metabolism is critically important for bacteria to successfully adapt to different environments. We have identified an uncharacterised transcriptional regulator; RccR, that controls this process in response to carbon source availability. Disruption of rccR in the plant-associated microbe Pseudomonas fluorescens inhibits growth in defined media, and compromises its ability to colonise the wheat rhizosphere. Structurally, RccR is almost identical to the Entner-Doudoroff (ED) pathway regulator HexR, and both proteins are controlled by the same ED-intermediate; 2-keto-3-deoxy-6-phosphogluconate (KDPG). Despite these similarities, HexR and RccR control entirely different aspects of primary metabolism, with RccR regulating pyruvate metabolism (aceEF), the glyoxylate shunt (aceA, glcB, pntAA) and gluconeogenesis (pckA, gap). RccR displays complex and unusual regulatory behaviour; switching repression between the pyruvate metabolism and glyoxylate shunt/gluconeogenesis loci depending on the available carbon source. This regulatory complexity is enabled by two distinct pseudo-palindromic binding sites, differing only in the length of their linker regions, with KDPG binding increasing affinity for the 28 bp aceA binding site but decreasing affinity for the 15 bp aceE site. Thus, RccR is able to simultaneously suppress and activate gene expression in response to carbon source availability. Together, the RccR and HexR regulators enable the rapid coordination of multiple aspects of primary carbon metabolism, in response to levels of a single key intermediate.

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Pseudomonas species have evolved dynamic and intricate regulatory networks to fine-tune gene expression, with complex regulation occurring at every stage in the processing of genetic information. This approach enables Pseudomonas to generate precise individual responses to the environment in order to improve their fitness and resource economy. The weak correlations we observe between RNA and protein abundance highlight the significant regulatory contribution of a series of intersecting post-transcriptional pathways, influencing mRNA stability, translational activity and ribosome function, to Pseudomonas environmental responses. This review examines our current understanding of three major post-transcriptional regulatory systems in Pseudomonas spp.; Gac/Rsm, Hfq and RimK, and presents an overview of new research frontiers, emerging genome-wide methodologies, and their potential for the study of global regulatory responses in Pseudomonas.