Heterologous expression of naturally evolved putative de novo proteins with chaperones

Over the past decade, evidence has accumulated that new protein‐coding genes can emerge de novo from previously non‐coding DNA. Most studies have focused on large scale computational predictions of de novo protein‐coding genes across a wide range of organisms. In contrast, experimental data concerning the folding and function of de novo proteins are scarce. This might be due to difficulties in handling de novo proteins in vitro, as most are short and predicted to be disordered. Here, we propose a guideline for the effective expression of eukaryotic de novo proteins in Escherichia coli. We used 11 sequences from Drosophila melanogaster and 10 from Homo sapiens, that are predicted de novo proteins from former studies, for heterologous expression. The candidate de novo proteins have varying secondary structure and disorder content. Using multiple combinations of purification tags, E. coli expression strains, and chaperone systems, we were able to increase the number of solubly expressed putative de novo proteins from 30% to 62%. Our findings indicate that the best combination for expressing putative de novo proteins in E. coli is a GST‐tag with T7 Express cells and co‐expressed chaperones. We found that, overall, proteins with higher predicted disorder were easier to express.StatementToday, we know that proteins do not only evolve by duplication and divergence of existing proteins but also arise from previously non‐coding DNA. These proteins are called de novo proteins. Their properties are still poorly understood and their experimental analysis faces major obstacles. Here, we aim to present a starting point for soluble expression of de novo proteins with the help of chaperones and thereby enable further characterization.     

Genes in S. cerevisiae encoding proteins with domains homologous to the mammalian ras proteins

The ras genes, which were first identified by their presence in RNA tumor viruses and which belong to a highly conserved gene family in vertebrates, have two close homologs in yeast, detectable by Southern blotting. We have cloned both genes (RAS1 and RAS2) from plasmid libraries and determined the complete nucleotide sequence of their coding regions. They encode proteins with nearly 90% homology to the first 80 positions of the mammalian ras proteins, and nearly 50% homology to the next 80 amino acids. Yeast RAS1 and RAS2 proteins are more homologous to each other, with about 90% homology for the first 180 positions. After this, at nearly the same position that the mammalian ras proteins begin to diverge from each other, the two yeast ras proteins diverge radically. The yeast ras proteins, like the proteins encoded by the mammalian genes, terminate with the sequence cysAAX, where A is an aliphatic amino acid. Thus the yeast ras proteins have the same overall structure and interrelationship as the family of mammalian ras proteins. The domains of divergence may correspond to functional domains of the ras proteins. Monoclonal antibody directed against mammalian ras proteins immunoprecipitates protein in yeast cells containing high copy numbers of the yeast RAS2 gene.     

Large-scale identification of putative exported proteins in Candida albicans by genetic selection

In all living organisms, secreted proteins play essential roles in different processes. Of special interest is the construction of the fungal cell wall, since this structure is absent from mammalian cells. The identification of the proteins involved in its biogenesis is therefore a primary goal in antifungal research. To perform a systematic identification of such proteins in Candida albicans, we carried out a genetic screening in which in-frame fusions with an intracellular allele of invertase gene SUC2 of Saccharomyces cerevisiae can be used to select and identify putatively exported proteins in the heterologous host S. cerevisiae. Eighty-three clones were selected, including 11 previously identified genes from C. albicans as well as 41 C. albicans genes that encode proteins homologous to already described proteins from related organisms. They include enzymes involved in cell wall synthesis and protein secretion. We also found membrane receptors and transporters presumably related to the interaction of C. albicans with the environment as well as extracellular enzymes and proteins involved in different morphological transitions. In addition, 11 C. albicans open reading frames (ORFs) identified in this screening encode proteins homologous to unknown or putative proteins, while 5 ORFs encode novel secreted proteins without known homologues in other organisms. This screening procedure therefore not only identifies a set of targets of interest in antifungal research but also provides new clues for understanding the topological locations of many proteins involved in processes relevant to the pathogenicity of this microorganism.     

Comparative Secretome Analysis of Magnaporthe oryzae Identified Proteins Involved in Virulence and Cell Wall Integrity

Plant fungal pathogens secrete numerous proteins into the apoplast at the plant–fungus contact sites to facilitate colonization. However, only a few secretory proteins were functionally characterized in Magnaporthe oryzae, the fungal pathogen causing rice blast disease worldwide. Asparagine-linked glycosylation 3 (Alg3) is an α-1,3-mannosyltransferase functioning in the N-glycan synthesis of N-glycosylated secretory proteins. Fungal pathogenicity and cell wall integrity are impaired in Δalg3 mutants, but the secreted proteins affected in Δalg3 mutants are largely unknown. In this study, we compared the secretomes of the wild-type strain and the Δalg3 mutant and identified 51 proteins that require Alg3 for proper secretion. These proteins were predicted to be involved in metabolic processes, interspecies interactions, cell wall organization, and response to chemicals. Nine proteins were selected for further validation. We found that these proteins were localized at the apoplastic region surrounding the fungal infection hyphae. Moreover, the N-glycosylation of these proteins was significantly changed in the Δalg3 mutant, leading to the decreased protein secretion and abnormal protein localization. Furthermore, we tested the biological functions of two genes, INV1 (encoding invertase 1, a secreted invertase) and AMCase (encoding acid mammalian chinitase, a secreted chitinase). The fungal virulence was significantly reduced, and the cell wall integrity was altered in the Δinv1 and Δamcase mutant strains. Moreover, the N-glycosylation was essential for the function and secretion of AMCase. Taken together, our study provides new insight into the role of N-glycosylated secretory proteins in fungal virulence and cell wall integrity.     

Changes in the N-glycome, glycoproteins with Asn-linked glycans, of Giardia lamblia with differentiation from trophozoites to cysts

Giardia lamblia is present in the intestinal lumen as a binucleate, flagellated trophozoite or a quadranucleate, immotile cyst. Here we used the plant lectin wheat germ agglutinin (WGA), which binds to the disaccharide di-N-acetyl-chitobiose (GlcNAc₂), which is the truncated Asn-linked glycan (N-glycan) of Giardia, to affinity purify the N-glycomes (glycoproteins with N-glycans) of trophozoites and cysts. Fluorescent WGA bound to the perinuclear membranes, peripheral acidified vesicles, and plasma membranes of trophozoites. In contrast, WGA bound strongly to membranes adjacent to the wall of Giardia cysts and less strongly to the endoplasmic reticulum and acidified vesicles. WGA lectin-affinity chromatography dramatically enriched secreted and membrane proteins of Giardia, including proteases and acid phosphatases that retain their activities. With mass spectroscopy, we identified 91 glycopeptides with N-glycans and 194 trophozoite-secreted and membrane proteins, including 42 unique proteins. The Giardia oligosaccharyltransferase, which contains a single catalytic subunit, preferred N glycosylation sites with Thr to those with Ser in vivo but had no preference for flanking amino acids. The most-abundant glycoproteins in the N-glycome of trophozoites were lysosomal enzymes, folding-associated proteins, and unique transmembrane proteins with Cys-, Leu-, or Gly-rich repeats. We identified 157 secreted and membrane proteins in the Giardia cysts, including 20 unique proteins. Compared to trophozoites, cysts were enriched in Gly-rich repeat transmembrane proteins, cyst wall proteins, and unique membrane proteins but had relatively fewer Leu-rich repeat proteins, folding-associated proteins, and unique secreted proteins. In summary, there are major changes in the Giardia N-glycome with the differentiation from trophozoites to cysts.     

Survey of Surface Proteins from the Pathogenic Mycoplasma hyopneumoniae Strain 7448 Using a Biotin Cell Surface Labeling Approach

The characterization of the repertoire of proteins exposed on the cell surface by Mycoplasma hyopneumoniae (M. hyopneumoniae), the etiological agent of enzootic pneumonia in pigs, is critical to understand physiological processes associated with bacterial infection capacity, survival and pathogenesis. Previous in silico studies predicted that about a third of the genes in the M. hyopneumoniae genome code for surface proteins, but so far, just a few of them have experimental confirmation of their expression and surface localization. In this work, M. hyopneumoniae surface proteins were labeled in intact cells with biotin, and affinity-captured biotin-labeled proteins were identified by a gel-based liquid chromatography-tandem mass spectrometry approach. A total of 20 gel slices were separately analyzed by mass spectrometry, resulting in 165 protein identifications corresponding to 59 different protein species. The identified surface exposed proteins better defined the set of M. hyopneumoniae proteins exposed to the host and added confidence to in silico predictions. Several proteins potentially related to pathogenesis, were identified, including known adhesins and also hypothetical proteins with adhesin-like topologies, consisting of a transmembrane helix and a large tail exposed at the cell surface. The results provided a better picture of the M. hyopneumoniae cell surface that will help in the understanding of processes important for bacterial pathogenesis. Considering the experimental demonstration of surface exposure, adhesion-like topology predictions and absence of orthologs in the closely related, non-pathogenic species Mycoplasma flocculare, several proteins could be proposed as potential targets for the development of drugs, vaccines and/or immunodiagnostic tests for enzootic pneumonia.     

Identification of Novel Immunogenic Proteins of Neisseria gonorrhoeae by Phage Display

Neisseria gonorrhoeae is one of the most prevalent sexually transmitted diseases worldwide with more than 100 million new infections per year. A lack of intense research over the last decades and increasing resistances to the recommended antibiotics call for a better understanding of gonococcal infection, fast diagnostics and therapeutic measures against N. gonorrhoeae. Therefore, the aim of this work was to identify novel immunogenic proteins as a first step to advance those unresolved problems. For the identification of immunogenic proteins, pHORF oligopeptide phage display libraries of the entire N. gonorrhoeae genome were constructed. Several immunogenic oligopeptides were identified using polyclonal rabbit antibodies against N. gonorrhoeae. Corresponding full-length proteins of the identified oligopeptides were expressed and their immunogenic character was verified by ELISA. The immunogenic character of six proteins was identified for the first time. Additional 13 proteins were verified as immunogenic proteins in N. gonorrhoeae.     

Rainbow Vectors for Broad-Range Bacterial Fluorescence Labeling

Since their discovery, fluorescent proteins have been widely used to study protein function, localization or interaction, promoter activity and regulation, drug discovery or for non-invasive imaging. They have been extensively modified to improve brightness, stability, and oligomerization state. However, only a few studies have focused on understanding the dynamics of fluorescent proteins expression in bacteria. In this work, we developed a set plasmids encoding 12 fluorescent proteins for bacterial labeling to facilitate the study of pathogen-host interactions. These broad-spectrum plasmids can be used with a wide variety of Gram-negative microorganisms including Escherichia coli, Pseudomonas aeruginosa, Burkholderia cepacia, Bordetella bronchiseptica, Shigella flexneri or Klebsiella pneumoniae. For comparison, fluorescent protein expression and physical characteristics in Escherichia coli were analyzed using fluorescence microscopy, flow cytometry and in vivo imaging. Fluorescent proteins derived from the Aequorea Victoria family showed high photobleaching, while proteins form the Discosoma sp. and the Fungia coccina family were more photostable for microscopy applications. Only E2-Crimson, mCherry and mKeima were successfully detected for in vivo applications. Overall, E2-Crimson was the fastest maturing protein tested in E. coli with the best overall performance in the study parameters. This study provides a unified comparison and comprehensive characterization of fluorescent protein photostability, maturation and toxicity, and offers general recommendations on the optimal fluorescent proteins for in vitro and in vivo applications.     

Protein mapping of Chaetomium globosum,a potential biological control agent through proteomics approach

Chaetomium globosum is a ubiquitous filamentous fungus having biological control properties. The potential isolates mycoparasitize the pathogen and produce antifungal metabolites which suppress the growth of pathogenic fungi. A proteomics approach was undertaken to separate and identify proteins from a mycoparasitic strain Cg1 of C. globosum under normal and heat shock conditions in order to identify differentially expressed proteins. We developed and standardized the procedure for extraction of total proteins and 2D gel electrophoresis, which resulted in profiling of more than 100 protein spots. 48 proteins were identified by a combination of matrix-assisted laser desorption/ ionization time-of-flight mass spectrometry (MALDI-TOF) and liquid chromatography mass spectrometry (LCMS/MS). Out of total proteins identified, 79 % were hypothetical proteins and 21 % proteins were functionally characterized. Out of total 79 % hypothetical proteins 24 % proteins matched with C. globosum while 18 % proteins matched with Aspergillus spp., 13 % with Coprinopsis cinerea, 10 % with Giberrella zaea, 8 % with Magnaporthe grisea and 5 % with Neurospora crassa and Lodderomyces elonisporus. Some of the functionally characterized proteins included MAP kinase, maltose permease, GTP binding protein, dyenin heavy chain, HET- C2, vacuolar Dig A protein, polyketide synthase, peptide prolyl cis trans isomerase and translation elongation factor. This study has generated a protein reference map for Chaetomium globosum, and being the first report on proteomics studies would greatly help to unravel biocontrol mechanism and its survival under heat stress conditions.     

Comparative proteomics of adult Paragonimus kellicotti excretion/secretion products released in vitro or present in the lung cyst nodule

Paragonimus kellicotti is a zoonotic lung fluke infection, the agent of North American paragonimiasis, and an excellent model for other Paragonimus infections. The excretory/secretory proteins (ESP) released by parasites and presented at the parasite-host interface are frequently proposed to be useful targets for drugs and/or vaccines In vitro culture conditions may alter ESP compared to those produced in vivo. In order to investigate ESPs produced in vivo we took advantage of the fact that adult P. kellicotti reproduce in the lungs of experimentally infected gerbils in tissue cysts. We performed a mass-spectrometric analysis of adult P. kellicotti soluble somatic protein (SSPs) extracts, excreted/secreted proteins (ESPs) produced by adult worms during in vitro culture, and lung cyst fluid proteins (CFPs) from experimentally infected gerbils. We identified 2,137 P. kellicotti proteins that were present in at least two of three biological replicates and supported by at least two peptides. Among those were 1,914 proteins found in SSP, 947 in ESP and 37 in CFP. In silico analysis predicted that only 141 of the total 2,137 proteins were secreted via classical or non-classical pathways. The most abundant functional categories in SSP were storage and oxidative metabolism. The most abundant categories in ESP were proteins related to metabolism and signal transduction. The 37 parasite-related proteins in CFP belonged to 11 functional categories. The largest groups were proteins with unknown function, cytoskeletal proteins and proteasome machinery. 29 of these 37 proteins were shared among all three sample types. To our knowledge, this is the first study that compares in vitro and in vivo ESP for any Paragonimus species. This study has provided new insights into ESPs of food-borne trematodes that are produced and released in vivo. Proteins released at the host-parasite interface may help the parasite evade host immunity and may represent new targets for novel treatments or diagnostic tests for paragonimiasis.     

Differential Subcellular Localization of Leishmania Alba-Domain Proteins throughout the Parasite Development

Alba-domain proteins are RNA-binding proteins found in archaea and eukaryotes and recently studied in protozoan parasites where they play a role in the regulation of virulence factors and stage-specific proteins. This work describes in silico structural characterization, cellular localization and biochemical analyses of Alba-domain proteins in Leishmania infantum. We show that in contrast to other protozoa, Leishmania have two Alba-domain proteins, LiAlba1 and LiAlba3, representative of the Rpp20- and the Rpp25-like eukaryotic subfamilies, respectively, which share several sequence and structural similarities but also important differences with orthologs in other protozoa, especially in sequences targeted for post-translational modifications. LiAlba1 and LiAlba3 proteins form a complex interacting with other RNA-binding proteins, ribosomal subunits, and translation factors as supported by co-immunoprecipitation and sucrose gradient sedimentation analysis. A higher co-sedimentation of Alba proteins with ribosomal subunits was seen upon conditions of decreased translation, suggesting a role of these proteins in translational repression. The Leishmania Alba-domain proteins display differential cellular localization throughout the parasite development. In the insect promastigote stage, Alba proteins co-localize predominantly to the cytoplasm but they translocate to the nucleolus and the flagellum upon amastigote differentiation in the mammalian host and are found back to the cytoplasm once amastigote differentiation is completed. Heat-shock, a major signal of amastigote differentiation, triggers Alba translocation to the nucleolus and the flagellum. Purification of the Leishmania flagellum confirmed LiAlba3 enrichment in this organelle during amastigote differentiation. Moreover, partial characterization of the Leishmania flagellum proteome of promastigotes and differentiating amastigotes revealed the presence of other RNA-binding proteins, as well as differences in the flagellum composition between these two parasite lifestages. Shuttling of Alba-domain proteins between the cytoplasm and the nucleolus or the flagellum throughout the parasite life cycle suggests that these RNA-binding proteins participate in several distinct regulatory pathways controlling developmental gene expression in Leishmania.     

Identifying molecularly defined antigens for a Histoplasma capsulatum-specific interferon gamma release assay

BackgroundIn a previous work we showed the feasibility of an interferon gamma release assay (IGRA) for detecting latent infection by Histoplasma capsulatum. While in that proof-of-concept study we used crude fungal extracts as antigens, the newest IGRAs developed for other infections are based on molecularly defined antigens, mostly on mixtures of immunogenic peptides.AimsTo identify proteins in H. capsulatum that might serve as molecularly defined antigens for an IGRA test.MethodsWe surveyed the literature looking for known H. capsulatum-immunogenic proteins and assayed two of them as antigens in an IGRA test, in a study that involved 80 volunteers. Furthermore, we used several bioinformatics tools to identify specific H. capsulatum proteins and to analyze possible strategies for the design of H. capsulatum-specific immunogenic peptides.ResultsSeven H. capsulatum-immunogenic proteins were retrieved from the literature. IGRA tests using either the heat shock protein 60 or the M antigen showed high sensitivities but low specificities, most likely due to the high sequence similarity with the corresponding orthologs in other pathogenic microorganisms. We identified around 2000 H. capsulatum-specific proteins, most of which remain unannotated. Class II T-cell epitope predictions for a small number of these proteins showed a great variability among different alleles, prompting for a “brute force” approach for peptide design.ConclusionsThe H. capsulatum genome encodes a large number of distinctive proteins, which represent a valuable source of potential specific antigens for an IGRA test. Among them, the Cfp4 protein stands out as a very attractive candidate.     

Conservation and variation of ribosomal proteins in several species of the cellular slime molds Dictyostelium and Polysphondylium

Genus- and species-specific composition of ribosomal proteins was investigated in four species of the genus Dictyostelium (D. discoideum, D. purpureum, D. murcoroides and D. giganteum) and two species of the genus Polysphondylium (P. pallidum and P. violaceum). Ribosomal proteins were resolved by a high-resolution, two-dimensional gel method. In general, the numbers and distributions for the majority of ribosomal proteins were similar within the species of each genus, although some differences were detected. More differences were observed between Dictyostelium and Polysphondylium than among the individual species within each genus. Stage-specific ribosomal proteins previously demonstrated in D. discoideum were found to be developmentally regulated in other Dictyostelium species, and in both Polysphondylium species. The study shows that ribosomal proteins may be a potentially useful new biochemical parameter for the molecular taxonomy of the cellular slime molds.     

Proteins Related to the Type I Secretion System Are Associated with Secondary SecA_DEAD Domain Proteins in Some Species of Planctomycetes,Verrucomicrobia, Proteobacteria,Nitrospirae and Chlorobi

A number of bacteria belonging to the PVC (Planctomycetes-Verrucomicrobia-Chlamydiae) super-phylum contain unusual ribosome-bearing intracellular membranes. The evolutionary origins and functions of these membranes are unknown. Some proteins putatively associated with the presence of intracellular membranes in PVC bacteria contain signal peptides. Signal peptides mark proteins for translocation across the cytoplasmic membrane in prokaryotes, and the membrane of the endoplasmic reticulum in eukaryotes, by highly conserved Sec machinery. This suggests that proteins might be targeted to intracellular membranes in PVC bacteria via the Sec pathway. Here, we show that canonical signal peptides are significantly over-represented in proteins preferentially present in PVC bacteria possessing intracellular membranes, indicating involvement of Sec translocase in their cellular targeting. We also characterized Sec proteins using comparative genomics approaches, focusing on the PVC super-phylum. While we were unable to detect unique changes in Sec proteins conserved among membrane-bearing PVC species, we identified (1) SecA ATPase domain re-arrangements in some Planctomycetes, and (2) secondary SecA_DEAD domain proteins in the genomes of some Planctomycetes, Verrucomicrobia, Proteobacteria, Nitrospirae and Chlorobi. This is the first report of potentially duplicated SecA in Gram-negative bacteria. The phylogenetic distribution of secondary SecA_DEAD domain proteins suggests that the presence of these proteins is not related to the occurrence of PVC endomembranes. Further genomic analysis showed that secondary SecA_DEAD domain proteins are located within genomic neighborhoods that also encode three proteins possessing domains specific for the Type I secretion system.     

Leishmania infantum chagasi: A genome-based approach to identification of excreted/secreted proteins

The parasitic protozoan, Leishmania, survives in harsh environments within its mammalian and sand fly hosts. Secreted proteins likely play critical roles in the parasite’s interactions with its environment. As a preliminary identification of the spectrum of potential excreted/secreted (ES) proteins of Leishmania infantum chagasi (Lic), a causative agent of visceral leishmaniasis, we used standard algorithms to screen the annotated L. infantum genome for genes whose predicted protein products have an N-terminal signal peptide and lack transmembrane domains and membrane anchors. A suite of 181 candidate ES proteins were identified. These included several that were documented in the literature to be released by other Leishmania spp. Six candidate ES proteins were selected for further validation of their expression and release by different parasite stages. We found both amastigote-specific and promastigote-specific released proteins. The ES proteins of Lic are candidates for future studies of parasite virulence determinants and host protective immunity.     

Investigations on the action of Phytophthora quercina,P. citricola and P. gonapodyides toxins on tobacco plants

Phytophthora quercina, P. citricola and P. gonapodyides isolated from declining oak roots and from soil rhizosphere in the field, released proteins into their culture medium. The proteins of P. quercina and P. gonapodyides caused severe chlorosis and necrosis on tobacco leaves. Only few symptoms were seen for the P. citricola protein. Surprisingly leaf chlorosis and necrosis were only visible in the light, whereas wilt symptoms were light-independent. The proteins were characterized on SDS gels as small peptides with basic and acidic isoelectric points. All proteins were heat stable. Even boiling for 15 min did not affect their activity. However, pronase treatment totally destroyed their activity. Transmission electron microscopy studies clearly showed that membrane structures especially of chloroplasts were damaged. The proteins of P. quercina and P. gonapodyides strongly crossreacted with the antibody raised against the P. cryptogea protein cryptogein. Therefore, these proteins might belong to the family of Phytophthora leaf necrotic proteins called elicitins.     

Homology between the HrpO protein of Pseudomonas solanacearum and bacterial proteins implicated in a signal peptide-independent secretion mechanism

A region of approximately 22 kb of DNA defines the large hrp gene cluster of strain GMI1000 of Pseudomonas solanacearum. The majority of mutants that map to this region have lost the ability to induce disease symptoms on tomato plants and are no longer able to elicit a hypersensitive reaction (HR) on tobacco, a nonhost plant. In this study we present the complementation analysis and nucleotide sequence of a 4772 by region of this hrp gene cluster. Three complete open reading frames (ORFs) are predicted within this region. The corresponding putative proteins, HrpN, HrpO and HpaP, have predicted sizes of 357, 690 and 197 amino acids, respectively, and predicted molecular weights of 38607, 73 990 and 21959 dalton, respectively. HrpN and HrpO are both predicted to be hydrophobic proteins with potential membrane-spanning domains and HpaP is rich in proline residues. A mutation in hpaP (for hrp associated) does not affect the HR on tobacco or the disease on tomato plants. None of the proteins is predicted to have an N-terminal signal sequence, which would have indicated that the proteins are exported. Considerable sequence similarities were found between HrpO and eight known or predicted prokaryotic proteins: LcrD of Yersinia pestis and Y. enterocolitica, FlbF of Caulobacter crescentus, F1hA of Bacillus subtilis, MxiA and VirH of Shigella flexneri, InvA of Salmonella typhimurium and HrpC2 of Xanthomonas campestris pv. vesicatoria. These homologies suggest that certain hrp genes of phytopathogenic bacteria code for components of a secretory system, which is related to the systems for secretion of flagellar proteins, Ipa proteins of Shigella flexneri and the Yersinia Yop proteins. Furthermore, these homologous proteins have the common feature of being implicated in a distinct secretory mechanism, which does not require the cleavage of a signal peptide. The sequence similarity between HrpO and HrpC2 is particularly high (66% identity and 81 % similarity) and the amino acid sequence comparison between these two proteins presented here reveals the first such sequence similarity to be shown between Hrp proteins of P. solanacearum and X. campestris. An efflux of plant electrolytes was found to be associated with the interactions between P. solanacearum and both tomato and tobacco leaves. This phenomenon may be part of the mechanism by which hrp gene products control and determine plant-bacterial interactions, since hrpO mutants induced levels of leakage which were significantly lower than those induced by the wild type on each plant.     

Recent advances in recombinant protein expression by Corynebacterium, Brevibacterium, and Streptomyces: from transcription and translation regulation to secretion pathway selection

Gram-positive bacteria are widely used to produce recombinant proteins, amino acids, organic acids, higher alcohols, and polymers. Many proteins have been expressed in Gram-positive hosts such as Corynebacterium, Brevibacterium, and Streptomyces. The favorable and advantageous characteristics (e.g., high secretion capacity and efficient production of metabolic products) of these species have increased the biotechnological applications of bacteria. However, owing to multiplicity from genes encoding the proteins and expression hosts, the expression of recombinant proteins is limited in Gram-positive bacteria. Because there is a very recent review about protein expression in Bacillus subtilis, here we summarize recent strategies for efficient expression of recombinant proteins in the other three typical Gram-positive bacteria (Corynebacterium, Brevibacterium, and Streptomyces) and discuss future prospects. We hope that this review will contribute to the development of recombinant protein expression in Corynebacterium, Brevibacterium, and Streptomyces.     

Differential Expression of In Vivo and In Vitro Protein Profile of Outer Membrane of Acidovorax avenae Subsp. avenae

Outer membrane (OM) proteins play a significant role in bacterial pathogenesis. In this work, we examined and compared the expression of the OM proteins of the rice pathogen Acidovorax avenae subsp. avenae strain RS-1, a Gram-negative bacterium, both in an in vitro culture medium and in vivo rice plants. Global proteomic profiling of A. avenae subsp. avenae strain RS-1 comparing in vivo and in vitro conditions revealed the differential expression of proteins affecting the survival and pathogenicity of the rice pathogen in host plants. The shotgun proteomics analysis of OM proteins resulted in the identification of 97 proteins in vitro and 62 proteins in vivo by mass spectrometry. Among these OM proteins, there is a high number of porins, TonB-dependent receptors, lipoproteins of the NodT family, ABC transporters, flagellins, and proteins of unknown function expressed under both conditions. However, the major proteins such as phospholipase and OmpA domain containing proteins were expressed in vitro, while the proteins such as the surface anchored protein F, ATP-dependent Clp protease, OmpA and MotB domain containing proteins were expressed in vivo. This may indicate that these in vivo OM proteins have roles in the pathogenicity of A. avenae subsp. avenae strain RS-1. In addition, the LC-MS/MS identification of OmpA and MotB validated the in silico prediction of the existance of Type VI secretion system core components. To the best of our knowledge, this is the first study to reveal the in vitro and in vivo protein profiles, in combination with LC-MS/MS mass spectra, in silico OM proteome and in silico genome wide analysis, of pathogenicity or plant host required proteins of a plant pathogenic bacterium.