DeepSig: Deep learning improves signal peptide detection in proteins

Motivation: The identification of signal peptides in protein sequences is an important step toward protein localization and function characterization. Results: Here, we present DeepSig, an improved approach for signal peptide detection and cleavage-site prediction based on deep learning methods. Comparative benchmarks performed on an updated independent dataset of proteins show that DeepSig is the current best performing method, scoring better than other available state-of-the-art approaches on both signal peptide detection and precise cleavage-site identification. Availability and implementation: DeepSig is available as both standalone program and web server at https://deepsig.biocomp.unibo.it. All datasets used in this study can be obtained from the same website

Phylogenetic and functional potential links pH and N2O emissions in pasture soils

This work was funded by the New Zealand Government through the New Zealand Fund for Global Partnerships in Livestock Emissions Research to support the objectives of the Livestock Research Group of the Global Research Alliance on Agricultural Greenhouse Gases (Agreement number: 16084) awarded to SEM and the University of Otago.peer-reviewedDenitrification is mediated by microbial, and physicochemical, processes leading to nitrogen loss via N2O and N2 emissions. Soil pH regulates the reduction of N2O to N2, however, it can also affect microbial community composition and functional potential. Here we simultaneously test the link between pH, community composition, and the N2O emission ratio (N2O/(NO + N2O + N2)) in 13 temperate pasture soils. Physicochemical analysis, gas kinetics, 16S rRNA amplicon sequencing, metagenomic and quantitative PCR (of denitrifier genes: nirS, nirK, nosZI and nosZII) analysis were carried out to characterize each soil. We found strong evidence linking pH to both N2O emission ratio and community changes. Soil pH was negatively associated with N2O emission ratio, while being positively associated with both community diversity and total denitrification gene (nir & nos) abundance. Abundance of nosZII was positively linked to pH, and negatively linked to N2O emissions. Our results confirm that pH imposes a general selective pressure on the entire community and that this results in changes in emission potential. Our data also support the general model that with increased microbial diversity efficiency increases, demonstrated in this study with lowered N2O emission ratio through more efficient conversion of N2O to N2.New Zealand Fund for Global Partnerships in Livestock Emissions Researc

Comprehensive proteomic profiling of outer membrane vesicles from Campylobacter jejuni

Gram-negative bacteria constitutively release outer membrane vesicles (OMVs) during cell growth that play significant roles in bacterial survival, virulence and pathogenesis. In this study, comprehensive proteomic analysis of OMVs from a human gastrointestinal pathogen Campylobacter jejuni NCTC11168 was performed using high-resolution mass spectrometry. The OMVs of C. jejuni NCTC11168 were isolated from culture supernatants then characterized using electron microscopy and dynamic light scattering revealing spherical OMVs of an average diameter of 50 nm. We then identified 134 vesicular proteins using high-resolution LTQ-Orbitrap mass spectrometry. Subsequent functional analysis of the genes revealed the relationships of the vesicular proteins. Furthermore, known N-glycoproteins were identified from the list of the vesicular proteome, implying the potential role of the OMVs as a delivery means for biologically relevant bacterial glycoproteins. These results enabled us to elucidate the overall proteome profile of pathogenic bacterium C. jejuni and to speculate on the function of OMVs in bacterial infections and communication

Isolation and genome sequencing of four Arctic marine Psychrobacter strains exhibiting multicopper oxidase activity

Moghadam MS, Albersmeier A, Winkler A, et al. Isolation and genome sequencing of four Arctic marine Psychrobacter strains exhibiting multicopper oxidase activity. BMC Genomics. 2016;17(1): 117

The Twin-Arginine Translocation Pathway in α-Proteobacteria Is Functionally Preserved Irrespective of Genomic and Regulatory Divergence

The twin-arginine translocation (Tat) pathway exports fully folded proteins out of the cytoplasm of Gram-negative and Gram-positive bacteria. Although much progress has been made in unraveling the molecular mechanism and biochemical characterization of the Tat system, little is known concerning its functionality and biological role to confer adaptive skills, symbiosis or pathogenesis in the α-proteobacteria class. A comparative genomic analysis in the α-proteobacteria class confirmed the presence of tatA, tatB, and tatC genes in almost all genomes, but significant variations in gene synteny and rearrangements were found in the order Rickettsiales with respect to the typically described operon organization. Transcription of tat genes was confirmed for Anaplasma marginale str. St. Maries and Brucella abortus 2308, two α-proteobacteria with full and partial intracellular lifestyles, respectively. The tat genes of A. marginale are scattered throughout the genome, in contrast to the more generalized operon organization. Particularly, tatA showed an approximately 20-fold increase in mRNA levels relative to tatB and tatC. We showed Tat functionality in B. abortus 2308 for the first time, and confirmed conservation of functionality in A. marginale. We present the first experimental description of the Tat system in the Anaplasmataceae and Brucellaceae families. In particular, in A. marginale Tat functionality is conserved despite operon splitting as a consequence of genome rearrangements. Further studies will be required to understand how the proper stoichiometry of the Tat protein complex and its biological role are achieved. In addition, the predicted substrates might be the evidence of role of the Tat translocation system in the transition process from a free-living to a parasitic lifestyle in these α-proteobacteria

A New Suite of Plasmid Vectors for Fluorescence-Based Imaging of Root Colonizing Pseudomonads

In the terrestrial ecosystem, plant–microbe symbiotic associations are ecologically and economically important processes. To better understand these associations at structural and functional levels, different molecular and biochemical tools are applied. In this study, we have constructed a suite of vectors that incorporates several new elements into the rhizosphere stable, broad-host vector pME6031. The new vectors are useful for studies requiring multi-color tagging and visualization of plant-associated, Gram-negative bacterial strains such as Pseudomonas plant growth promotion and biocontrol strains. A number of genetic elements, including constitutive promoters and signal peptides that target secretion to the periplasm, have been evaluated. Several next generation fluorescent proteins, namely mTurquoise2, mNeonGreen, mRuby2, DsRed-Express2 and E2-Crimson have been incorporated into the vectors for whole cell labeling or protein tagging. Secretion of mTurquoise2 and mNeonGreen into the periplasm of Pseudomonas fluorescens SBW25 has also been demonstrated, providing a vehicle for tagging proteins in the periplasmic compartment. A higher copy number version of select plasmids has been produced by introduction of a previously described repA mutation, affording an increase in protein expression levels. The utility of these plasmids for fluorescence-based imaging is demonstrated by root colonization of Solanum lycopersicum seedlings by P. fluorescens SBW25 in a hydroponic growth system. The plasmids are stably maintained during root colonization in the absence of selective pressure for more than 2 weeks

Mechanisms and Evolution of Oxidative Sulfur Metabolism in Green Sulfur Bacteria

Green sulfur bacteria (GSB) constitute a closely related group of photoautotrophic and thiotrophic bacteria with limited phenotypic variation. They typically oxidize sulfide and thiosulfate to sulfate with sulfur globules as an intermediate. Based on genome sequence information from 15 strains, the distribution and phylogeny of enzymes involved in their oxidative sulfur metabolism was investigated. At least one homolog of sulfide:quinone oxidoreductase (SQR) is present in all strains. In all sulfur-oxidizing GSB strains except the earliest diverging Chloroherpeton thalassium, the sulfide oxidation product is further oxidized to sulfite by the dissimilatory sulfite reductase (DSR) system. This system consists of components horizontally acquired partly from sulfide-oxidizing and partly from sulfate-reducing bacteria. Depending on the strain, the sulfite is probably oxidized to sulfate by one of two different mechanisms that have different evolutionary origins: adenosine-5′-phosphosulfate reductase or polysulfide reductase-like complex 3. Thiosulfate utilization by the SOX system in GSB has apparently been acquired horizontally from Proteobacteria. SoxCD does not occur in GSB, and its function in sulfate formation in other bacteria has been replaced by the DSR system in GSB. Sequence analyses suggested that the conserved soxJXYZAKBW gene cluster was horizontally acquired by Chlorobium phaeovibrioides DSM 265 from the Chlorobaculum lineage and that this acquisition was mediated by a mobile genetic element. Thus, the last common ancestor of currently known GSB was probably photoautotrophic, hydrogenotrophic, and contained SQR but not DSR or SOX. In addition, the predominance of the Chlorobium–Chlorobaculum–Prosthecochloris lineage among cultured GSB could be due to the horizontally acquired DSR and SOX systems. Finally, based upon structural, biochemical, and phylogenetic analyses, a uniform nomenclature is suggested for sqr genes in prokaryotes

Two Rieske Fe/S Proteins and TAT System in Mesorhizobium loti MAFF303099: Differential Regulation and Roles on Nodulation

Mesorhizobium loti MAFF303099 is a rhizobial strain that nodulates Lotus spp. A M. loti MAFF303099 mutant strain affected in the tatC gene was generated. This strain presented an altered protein secretion level to the culture supernatant and also a higher sensitivity to SDS. Its nodulation phenotype on Lotus showed the induction of small and colorless nodules, and in a larger number than those induced by the wild-type strain. In addition, these nodules presented defects in the degree of occupation by rhizobia. Two Rieske Fe/S proteins, encoded by the mll2707 and mlr0970 genes, were predicted as potential Tat substrates in M. loti MAFF303099. The transcriptional expression of mll2707 and mlr0970 genes was analyzed under different oxygen growth conditions. The mll2707 gene was expressed constitutively, while the expression of the mlr0970 gene was only detected under anaerobic and microaerophilic in vitro conditions. Both genes were down-regulated in the tatC mutant strain. mll2707 and mlr0970 mRNAs from the wild-type strain were detected in nodules. Using a translational reporter peptide fusion, we found that the Mll2707 protein was only detectable in the wild-type strain. On the other hand, although Mlr0970 protein was detected in wild-type and tatC mutant strains, its association with the membrane was favored in the wild-type strain. The tatC and the mll2707 mutant strains were affected in the cytochrome c oxidase activity. These results confirm that Mll2707 is required for cytochrome c-dependent respiration and that Tat functionality is required for the correct activity of Mll2707. The mll2707 mutant strain showed a nodulation phenotype similar to the tatC mutant strain, although it presented only a slight difference in comparison with wild-type strain in terms of nodule occupation. No defective phenotype was observed in the nodulation with the mlr0970 mutant strain. These results indicate that, of the two Rieske Fe/S proteins coded by M. loti MAFF303099, only Mll2707 expression is required for the induction of effective nodules, and that the functionality of the Tat system is necessary not only for the correct function of this protein, but also for some other protein required in an earlier stage of the nodulation process

In silico characterization of the global Geobacillus and Parageobacillus secretome

BACKGROUND : Geobacillus and Parageobacillus are two ecologically diverse thermophilic genera within the phylum Firmicutes. These taxa have long been of biotechnological interest due to their ability to secrete thermostable enzymes and other biomolecules that have direct applications in various industrial and clinical fields. Despite the commercial and industrial interest in these microorganisms, the full scope of the secreted protein, i.e. the secretome, of Geobacillus and Parageobacillus species remains largely unexplored, with most studies focusing on single enzymes. A genome-wide exploration of the global secretome can provide a platform for understanding the extracellular functional “protein cloud” and the roles that secreted proteins play in the survival and adaptation of these biotechnologically relevant organisms. RESULTS : In the present study, the global secretion profile of 64 Geobacillus and Parageobacillus strains, comprising 772 distinct proteins, was predicted using comparative genomic approaches. Thirty-one of these proteins are shared across all strains used in this study and function in cell-wall/membrane biogenesis as well as transport and metabolism of carbohydrates, amino acids and inorganic ions. An analysis of the clustering patterns of the secretomes of the 64 strains according to shared functional orthology revealed a correlation between the secreted profiles of different strains and their phylogeny, with Geobacillus and Parageobacillus species forming two distinct functional clades. CONCLUSIONS : The in silico characterization of the global secretome revealed a metabolically diverse set of secreted proteins, which include proteases, glycoside hydrolases, nutrient binding proteins and toxins.Additional file 1: Table S1. Similarity matrix. Table showing the percentage of orthology between the secretomes of the 64 genomes used in this study. This similarity matrix was used to generate the orthology dendogram in Fig. 6.Additional file 2: Table S2. Presence/absence matrix of the global secretome of Geobacillus and Parageobacillus. Table showing the presence or absence of the 772 protein sequences constituting the global secretome (annotated in the first row) across the 64 genomes used in this study (annotated in the first column). Presence/absence is indicated using a binary code of 1 and 0 to represent presence and absence, respectively.Additional file 3: Figure S1. Distribution of GH families across the 51 glycoside hydrolases present in the global secretome. Pie-chart showing the distribution of glycoside hydrolase families in the global secretome of Geobacillus and Parageobacillus. The four most abundant families represented in the dataset include beta-galactosidases (GH2), alpha-amylases (GH13), chitinases (GH18), and lytic transglycosylases (GH23). The following families were also found to be present in the global secretome: GH1– beta-glucosidases and beta-galactosidases; GH 3–beta-d-glucosidases, alpha-l-arabinofuranosidases; GH5–cellulases; GH10–endo-beta-1,3-xylanases; GH19–chitinases; GH25–chalaropsis-type lysozymes; GH27–alphagalactosidases and alpha-N-acetylgalactosaminidases; GH32–invertases; GH43–endo-alpha-l-arabinanases and beta-d-xylosidases; GH52–betaxylosidases; GH53–beta-1,4-galactanases; GH70–transglucosylases; GH73–beta-N-acetylglucosaminidases.Additional file 4: Figure S2. Xylanase activity assay of Geobacillus and Parageobacillus type strains on Oat Spelt Xylan. Bar-plot showing the xylan degrading activity of the supernatant of selected Geobacillus and Parageobacillus strains, as measured using the DNS protocol [91]. The concentration of reduced sugars was determined by measuring the average absorbance of each sample against a xylose standard. Strains were labelled as follow: T1–P. thermoglucosidasius DSM 2542T; T2–G. subterraneus DSM 15332T; T3–P. caldoxylosilyticus DSM 12041T; T4–G. thermodenitrificans DSM 465T; T5–G. stearothermophilus ATCC 12980T; T6–G. kaustophilus DSM 7263T; T7–P. thermoantarcticus M1T; T8 - P. toebii DSM 14590T.Additional file 5: Figure S3. Qualitative amylase activity plate assays. Description of data: 1% Starch agar plates showing the starch-degrading activity of the supernatant of the Geobacilus and Parageobacillus strains tested. The plates were stained with iodine tincture (2.5% w/v Iodine, 2.5% Potassium Iodide), and the areas of clearance represent starch degradation and corresponding amylase activity. The strains were labelled as described for Figure S2, and the positive control used in this assay (+) is α-amylase from Aspergillus oryzae, provided by Sigma-Aldrich® (Product Code: 9001-19-8).Additional file 6: Figure S4. PNPB Lipase activity assay of Geobacillus and Parageobacillus strains. Description of data: Bar-plot showing the degradation rates of PNPB by the supernatant of the eight Geobacillus and Parageobacillus strains tested. The labelling for the different strains is the same as described for Additional file 4: Figure S2.Additional file 7: Table S3. Blast results for proteins with homology to biotechnologically relevant enzymes. Description of data: Table showing the blast results for the most significant hits between protein sequences from the global secretome and enzymes from the Uniprot database that have been previously highlighted as being of biotechnological relevance. The scores and e-values, as well as the accession numbers were obtained using the Blast function against the UniprotDB.This study was funded through a University of Pretoria (UP) Postdoctoral Researcher fellowship.A University of Pretoria (UP) Postdoctoral Researcher fellowship.http://www.microbialcellfactories.comam2019BiochemistryGeneticsMicrobiology and Plant Patholog