High-throughput proteogenomics of Ruegeria pomeroyi: seeding a better genomic annotation for the whole marine Roseobacter clade

<p>Abstract</p> <p>Background</p> <p>The structural and functional annotation of genomes is now heavily based on data obtained using automated pipeline systems. The key for an accurate structural annotation consists of blending similarities between closely related genomes with biochemical evidence of the genome interpretation. In this work we applied high-throughput proteogenomics to <it>Ruegeria pomeroyi</it>, a member of the <it>Roseobacter </it>clade, an abundant group of marine bacteria, as a seed for the annotation of the whole clade.</p> <p>Results</p> <p>A large dataset of peptides from <it>R. pomeroyi </it>was obtained after searching over 1.1 million MS/MS spectra against a six-frame translated genome database. We identified 2006 polypeptides, of which thirty-four were encoded by open reading frames (ORFs) that had not previously been annotated. From the pool of 'one-hit-wonders', <it>i.e</it>. those ORFs specified by only one peptide detected by tandem mass spectrometry, we could confirm the probable existence of five additional new genes after proving that the corresponding RNAs were transcribed. We also identified the most-N-terminal peptide of 486 polypeptides, of which sixty-four had originally been wrongly annotated.</p> <p>Conclusions</p> <p>By extending these re-annotations to the other thirty-six <it>Roseobacter </it>isolates sequenced to date (twenty different genera), we propose the correction of the assigned start codons of 1082 homologous genes in the clade. In addition, we also report the presence of novel genes within operons encoding determinants of the important tricarboxylic acid cycle, a feature that seems to be characteristic of some <it>Roseobacter </it>genomes. The detection of their corresponding products in large amounts raises the question of their function. Their discoveries point to a possible theory for protein evolution that will rely on high expression of orphans in bacteria: their putative poor efficiency could be counterbalanced by a higher level of expression. Our proteogenomic analysis will increase the reliability of the future annotation of marine bacterial genomes.</p

Production of a full length Tat protein in E. coli and its purification

AbstractA full length tat gene was constructed by a combination of a polymerase chain reaction (PCR) for the first exon and chemical synthesis for the second exon. This gene was expressed in E. Coli under the control of the strongly regulated araB promoter, either directly or fused to a secretion signal encoding sequence. We the defined a rapid, three-step procedure for the purification of the Tat protei

Proteome data to explore the impact of pBClin15 on Bacillus cereus ATCC 14579

AbstractThis data article reports changes in the cellular and exoproteome of B. cereus cured from pBClin15.Time-course changes of proteins were assessed by high-throughput nanoLC-MS/MS. We report all the peptides and proteins identified and quantified in B. cereus with and without pBClin15. Proteins were classified into functional groups using the information available in the KEGG classification and we reported their abundance in term of normalized spectral abundance factor. The repertoire of experimentally confirmed proteins of B. cereus presented here is the largest ever reported, and provides new insights into the interplay between pBClin15 and its host B. cereus ATCC 14579. The data reported here is related to a published shotgun proteomics analysis regarding the role of pBClin15, “Deciphering the interactions between the Bacillus cereus linear plasmid, pBClin15, and its host by high-throughput comparative proteomics” Madeira et al. [1]. All the associated mass spectrometry data have been deposited in the ProteomeXchange Consortium (http://proteomecentral.proteomexchange.org) via the PRIDE partner repository (http://www.ebi.ac.uk/pride/), with the dataset identifier PRIDE: PXD001568, PRIDE: PXD002788 and PRIDE: PXD002789

Coriolopsis trogii MUT3379: A Novel Cell Factory for High-Yield Laccase Production

Coriolopsis trogii is a basidiomycete fungus which utilizes a large array of lignin-modifying enzymes to colonize and decompose dead wood. Its extracellular enzymatic arsenal includes laccases, i.e., polyphenol oxidases of relevant interest for different industrial applications thanks to their ability to oxidize a diverse range of natural and synthetic compounds. In this work, the production of laccases in C. trogii MUT3379 was explored and improved. From an initial production of ca. 10,000 U L−1, the fermentation process was gradually optimized, reaching a final yield of ca. 200,000 U L−1. An SDS-PAGE analysis of the secretome highlighted the presence of a main protein of ca. 60 kDa showing laccase activity, which was designated as Lac3379-1 once its primary sequence was established by tandem mass spectrometry. The characterization of Lac3379-1 revealed a remarkable enzymatic stability in the presence of surfactants and solvents and a diversified activity on a broad range of substrates, positioning it as an interesting tool for diverse biotechnological applications. The high-yield and robust production process indicates C. trogii MUT3379 as a promising cell factory for laccases, offering new perspectives for industrial applications of lignin-modifying enzymes

Nutrient recycling facilitates long-term stability of marine microbial phototroph–heterotroph interactions

Biological interactions underpin the functioning of marine ecosystems, be it via competition, predation, mutualism or symbiosis processes. Microbial phototroph–heterotroph interactions propel the engine that results in the biogeochemical cycling of individual elements, and they are critical for understanding and modelling global ocean processes. Unfortunately, studies thus far have focused on exponentially growing cultures in nutrient-rich media, meaning knowledge of such interactions under in situ conditions is rudimentary at best. Here, we have performed long-term phototroph–heterotroph co-culture experiments under nutrient-amended and natural seawater conditions, and show that it is not the concentration of nutrients but rather their circulation that maintains a stable interaction and a dynamic system. Using the Synechococcus–Roseobacter interaction as a model phototroph–heterotroph case study, we show that although Synechococcus is highly specialized for carrying out photosynthesis and carbon fixation, it relies on the heterotroph to remineralize the inevitably leaked organic matter, making nutrients circulate in a mutualistic system. In this sense we challenge the general belief that marine phototrophs and heterotrophs compete for the same scarce nutrients and niche space, and instead suggest that these organisms more probably benefit from each other because of their different levels of specialization and complementarity within long-term stable-state systems

Next-generation proteomics reveals a greater antioxidative response to drought in Coffea arabica than in Coffea canephora

Drought is a major threat to coffee, compromising the quality and quantity of its production. We have analyzed the core proteome of 18 Coffea canephora cv. Conilon Clone 153 and C. arabica cv. Icatu plants and assessed their responses to moderate (MWD) and severe (SWD) water deficits. Label-free quantitative shotgun proteomics identified 3000 proteins in both genotypes, but less than 0.8% contributed to ca. 20% of proteome biomass. Proteomic changes were dependent on the severity of drought, being stronger under SWD and with an enrolment of different proteins, functions, and pathways than under MWD. The two genotypes displayed stress-responsive proteins under SWD, but only C. arabica showed a higher abundance of proteins involved in antioxidant detoxification activities. Overall, the impact of MWD was minor in the two genotypes, contrary to previous studies. In contrast, an extensive proteomic response was found under SWD, with C. arabica having a greater potential for acclimation/resilience than C. canephora. This is likely supported by a wider antioxidative response and an ability to repair photosynthetic structures, being crucial to develop new elite genotypes that assure coffee supply under water scarcity levels.info:eu-repo/semantics/publishedVersio

Genome analysis and genome-wide proteomics of Thermococcus gammatolerans, the most radioresistant organism known amongst the Archaea

The genome sequence of Thermococcus gammatolerans, a radioresistant archaeon, is described; a proteomic analysis reveals that radioresistance may be due to unknown DNA repair enzymes

Next-Generation Proteomics Reveals a Greater Antioxidative Response to Drought in Coffea arabica Than in Coffea canephora

Drought is a major threat to coffee, compromising the quality and quantity of its production. We have analyzed the core proteome of 18 Coffea canephora cv. Conilon Clone 153 and C. arabica cv. Icatu plants and assessed their responses to moderate (MWD) and severe (SWD) water deficits. Label-free quantitative shotgun proteomics identified 3000 proteins in both genotypes, but less than 0.8% contributed to ca. 20% of proteome biomass. Proteomic changes were dependent on the severity of drought, being stronger under SWD and with an enrolment of different proteins, functions, and pathways than under MWD. The two genotypes displayed stress-responsive proteins under SWD, but only C. arabica showed a higher abundance of proteins involved in antioxidant detoxification activities. Overall, the impact of MWD was minor in the two genotypes, contrary to previous studies. In contrast, an extensive proteomic response was found under SWD, with C. arabica having a greater potential for acclimation/resilience than C. canephora. This is likely supported by a wider antioxidative response and an ability to repair photosynthetic structures, being crucial to develop new elite genotypes that assure coffee supply under water scarcity levelsinfo:eu-repo/semantics/publishedVersio

Exploring Andean High-Altitude Lake Extremophiles through Advanced Proteotyping

Quickly identifying and characterizing isolates from extreme environments is currently challenging while very important to explore the Earth′s biodiversity. As these isolates may, in principle, be distantly related to known species, techniques are needed to reliably identify the branch of life to which they belong. Proteotyping these environmental isolates by tandem mass spectrometry offers a rapid and cost-effective option for their identification using their peptide profiles. In this study, we document the first high-throughput proteotyping approach for environmental extremophilic and halophilic isolates. Microorganisms were isolated from samples originating from high-altitude Andean lakes (3700-4300 m a.s.l.) in the Chilean Altiplano, which represent environments on Earth that resemble conditions on other planets. A total of 66 microorganisms were cultivated and identified by proteotyping and 16S rRNA gene amplicon sequencing. Both the approaches revealed the same genus identification for all isolates except for three isolates possibly representing not yet taxonomically characterized organisms based on their peptidomes. Proteotyping was able to indicate the presence of two potentially new genera from the families of Paracoccaceae and Chromatiaceae/Alteromonadaceae, which have been overlooked by 16S rRNA amplicon sequencing approach only. The paper highlights that proteotyping has the potential to discover undescribed microorganisms from extreme environments

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

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