Overview of multi-species biofilms in different ecosystems: Wastewater treatment, soil and oral cavity.

In various natural ecosystems, bacteria most often live in a sessile state enchased in a self-produced extracellular matrix forming biofilms. Due to their either negative or positive impact on different aspects of our daily life, the number of studies devoted to biofilms is increasing. Most research is based on biofilms formed by a single bacterial species. These simple models allowed the understanding of the mechanisms involved in biofilms formation and regulation. This likewise helped the development of several means to control the biofilms formation. However, these models do not closely mimic the natural biofilms known as biochemically and microbiologically heterogeneous and dynamic structures. For this reason, current studies focus more on multispecies biofilms using complex models to best approximate the natural environment. In this review, we addressed on available examples of multispecies biofilms in different domains to illustrate the complexity and organization of life within a consortium. Finally, we review the most used analytical techniques to study multispecies biofilms highlighting the need of multi-scale strategies to better decipher this complex lifestyle.

Post-translational modifications in Pseudomonas aeruginosa revolutionized by proteomic analysis.

Pseudomonas aeruginosa is an opportunistic pathogen that causes severe infections in vulnerable individuals. It is known that post-translational modifications (PTMs) play a key role in bacterial physiology. Their characterization is still challenging and the recent advances in proteomics allow large-scale and high-throughput analyses of PTMs. Here, we provide an overview of proteomic data about the modified proteins in P. aeruginosa. We emphasize the significant contribution of proteomics in knowledge enhancement of PTMs (phosphorylation, N-acetylation and glycosylation) and we discuss their importance in P. aeruginosa physiology.

The marine bacteria Shewanella frigidimarina NCIMB400 upregulates the type VI secretion system during early biofilm formation.

Shewanella sp. are facultative anaerobic Gram-negative bacteria, extensively studied for their electron transfer ability. Shewanella frigidimarina has been detected and isolated from marine environments, and in particular, from biofilms. However, its ability to adhere to surfaces and form a biofilm is poorly understood. In this study, we show that the ability to adhere and to form a biofilm of S. frigidimarina NCIMB400 is significantly higher than that of Shewanella oneidensis in our conditions. We also show that this strain forms a biofilm in artificial seawater, whereas in Luria-Bertani, this capacity is reduced. To identify proteins involved in early biofilm formation, a proteomic analysis of sessile versus planktonic membrane-enriched fractions allowed the identification of several components of the same type VI secretion system gene cluster: putative Hcp1 and ImpB proteins as well as a forkhead-associated domain-containing protein. The upregulation of Hcp1 a marker of active translocation has been confirmed using quantitative reverse transcription polymerase chain reaction. Our data demonstrated the presence of a single and complete type VI secretion system in S. frigidimarina NCIMB400 genome, upregulated in sessile compared with planktonic conditions. The fact that three proteins including the secreted protein Hcp1 have been identified may suggest that this type VI secretion system is functional.

Protein lysine acetylation in bacteria: Current state of the art.

Post-translational modifications of proteins are key events in cellular metabolism and physiology regulation. Lysine acetylation is one of the best studied protein modifications in eukaryotes, but, until recently, ignored in bacteria. However, proteomic advances have highlighted the diversity of bacterial lysine-acetylated proteins. The current data support the implication of lysine acetylation in various metabolic pathways, adaptation and virulence. In this review, we present a broad overview of the current knowledge of lysine acetylation in bacteria. We emphasize particularly the significant contribution of proteomics in this field.

Proteomic profiling of lysine acetylation in Pseudomonas aeruginosa reveals the diversity of acetylated proteins.

Protein lysine acetylation is a reversible and highly regulated post-translational modification with the well demonstrated physiological relevance in eukaryotes. Recently, its important role in the regulation of metabolic processes in bacteria was highlighted. Here, we reported the lysine acetylproteome of Pseudomonas aeruginosa using a proteomic approach. We identified 430 unique peptides corresponding to 320 acetylated proteins. In addition to the proteins involved in various metabolic pathways, several enzymes contributing to the lipopolysaccharides biosynthesis were characterized as acetylated. This data set illustrated the abundance and the diversity of acetylated lysine proteins in P. aeruginosa and opens opportunities to explore the role of the acetylation in the bacterial physiology.

Characterization of N-terminal protein modifications in Pseudomonas aeruginosa PA14.

Even though protein initiator methionine excision (NME) and N-terminal acetylation (NTA) have been relatively well investigated in eukaryotic proteomes, few studies were dedicated to these modifications in bacteria up to now. In this work, we investigated, for the first time, the N-terminal proteome of the bacterium Pseudomonas aeruginosa PA14 by studying the NME and NTA processes using proteomic approaches. For NME, most of proteins had their initiator Met cleaved (63%) and the nature of the penultimate residue seems to be essential for this cleavage. Concerning NTA, two methods were applied (protein fractionation and peptide enrichment). This allowed us to identify 117 Nα-acetylated proteins, among them 113 have not yet been described as modified in bacteria. Most often, the non-acetylated form was over-represented compared to the acetylated form, arguing that this latter was a minor part of the total abundance of a given protein. Furthermore, some proteins with acetylated initiator methionine were observed. The present work significantly enlarges the number of N-terminally modified proteins in bacteria and confirms that these modifications are a general and fundamental process, not only restricted to eukaryotes. BIOLOGICAL SIGNIFICANCE: Protein modifications in prokaryotes have been detected more recently than in eukaryotes. Methionine cleavage and N-terminal acetylation are two common protein N-terminal modifications. Despite their importance in bacterial processes, they are less investigated. The characterization of N-terminal acetylation in bacteria is a challenge because no antibody exists and it is a less frequent modification than in eukaryotes. We used proteomic approaches (enrichment, fractionation, nanoLC-MS/MS, and bioinformatic analyses) to investigate the N-terminal methionine excision and to profile the N-terminal acetylome of P. aeruginosa strain PA14. From our results, around 60% of the proteins had their iMet cleaved. In total, 117 proteins were identified constituting the largest dataset in prokaryotes. Among them, proteins kept their initiator methionine and were acetylated. These results may facilitate the design of experiments to better understand the role of acetylation at the protein N-terminus of P. aeruginosa PA14.

Potential of liquid-isoelectric-focusing protein fractionation to improve phosphoprotein characterization of Pseudomonas aeruginosa PA14.

Protein phosphorylation on serine, threonine, and tyrosine is known to be involved in a wide variety of cellular processes and signal transduction in bacteria. Bacterial-proteome analysis is required to determine which proteins have been conditionally expressed and whether any post-translational modifications are present. One of the greatest challenges of proteome analysis is the fractionation of these complex protein mixtures to detect low-abundance phosphoproteins. Liquid-phase isoelectric focusing (IEF) is a promising analytical tool in proteomics, but as far as we are aware no work has studied the reproducibility of this approach. In this study, we investigated the phosphoproteome of Pseudomonas aeruginosa strain PA14. We first tested in-solution IEF protein fractionation, and then used this technique to fractionate the proteins in the complex mixture. Next, phosphopeptides were enriched with titanium dioxide and analyzed by high-resolution, high-accuracy liquid chromatography-mass spectrometry. With this approach, we succeeded in characterizing 73 unique phosphorylated peptides belonging to 63 proteins. Interestingly, we observed a higher percentage of modified tyrosine, revealing the importance of this phosphorylated residue in bacteria.

Extracellular Ser/Thr/Tyr phosphorylated proteins of Pseudomonas aeruginosa PA14 strain.

Protein phosphorylation on serine, threonine, and tyrosine is known to be involved in a wide variety of cellular processes, signal transduction, and bacterial virulence. We characterized, for the first time, the extracellular phosphoproteins of the Pseudomonas aeruginosa PA14 strain. We identified 28 phosphoproteins (59 phosphosites) including enzymes, with various phosphorylation sites, known as potent secreted virulence factors in P. aeruginosa. The high phosphorylation level of these virulence factors might reflect a relationship between Ser/Thr/Tyr phosphorylation and virulence.