Comparative Proteomic Analyses of <Emphasis Type="Italic">Streptococcus suis</Emphasis> Serotype 2 Cell Wall-Associated Proteins

Streptococcus suis serotype 2 (SS2) is a zoonotic pathogen that is distributed throughout the world. Virulence factors and/or markers of the virulent serotype 2 strains have not been fully identified. In this study a simple, rapid, and non-destructive method was used to extract cell wall-associated proteins from SS2 strains. Two virulent strains were compared with one avirulent strain by 2-dimensional electrophoresis (2DE). When the results of the 2DE analyses were combined with the results of mass spectrometry analyses, a total of 40 unique proteins were identified, including 26 antigens (2DE immunoblotting was used as a preliminary study). In addition to a known virulence factor, muramidase-released protein, two new proteins, catabolite control protein A and leucyl aminopeptidase, and nine potential virulence factors were also identified. The formers may be a potential virulence regulator or drug target, and the latter contains plasminogen-binding proteins and molecular chaperones. Our results complemented previous immunoproteomics studies of SS2 strains.     

Secretory delivery of heterologous proteins in attenuated <Emphasis Type="Italic">Vibrio anguillarum</Emphasis> for potential use in vaccine design

To synthesize and secrete heterologous proteins in an attenuated Vibrio anguillarum strain for potential multivalent live vaccine development, different antigen-delivery systems based on bacterial-originated secretion signal peptides (SPs) were designed and identified in this work. Four SPs were derived from hemolysin of Escherichia coli, RTX protein of V. cholerae, hemolysin of V. anguillarum, zinc-metalloprotease of V. anguillarum, respectively, and their abilities to support secretion of green fluorescent protein (GFP) in an attenuated V. anguillarum strain MVAV6203 were assayed. Immunodetection of GFP showed that the capability of the tested signal leaders to direct secretion of GFP varied greatly. Although all the four signal peptide-fused GFPs could be expressed correctly and trapped intracellularly in recombinant strains, only the EmpA signal peptide could confer efficient secretion to GFP. For the investigation of its potential application in live bacteria carrier vaccines, a heterologous protein EseB of Edwardsiella tarda was fused to the SP(empA) antigen-delivery system and introduced into the strain MVAV6203. Further analysis of EseB demonstrated that the constructed SP(empA) antigen-delivery system could be used to secrete foreign protein in attenuated V. anguillarum and be available for carrier vaccines development.     

Evolutionary Rate Covariation Identifies New Members of a Protein Network Required for Drosophila melanogaster Female Post-Mating Responses

Seminal fluid proteins transferred from males to females during copulation are required for full fertility and can exert dramatic effects on female physiology and behavior. In Drosophila melanogaster, the seminal protein sex peptide (SP) affects mated females by increasing egg production and decreasing receptivity to courtship. These behavioral changes persist for several days because SP binds to sperm that are stored in the female. SP is then gradually released, allowing it to interact with its female-expressed receptor. The binding of SP to sperm requires five additional seminal proteins, which act together in a network. Hundreds of uncharacterized male and female proteins have been identified in this species, but individually screening each protein for network function would present a logistical challenge. To prioritize the screening of these proteins for involvement in the SP network, we used a comparative genomic method to identify candidate proteins whose evolutionary rates across the Drosophila phylogeny co-vary with those of the SP network proteins. Subsequent functional testing of 18 co-varying candidates by RNA interference identified three male seminal proteins and three female reproductive tract proteins that are each required for the long-term persistence of SP responses in females. Molecular genetic analysis showed the three new male proteins are required for the transfer of other network proteins to females and for SP to become bound to sperm that are stored in mated females. The three female proteins, in contrast, act downstream of SP binding and sperm storage. These findings expand the number of seminal proteins required for SP''s actions in the female and show that multiple female proteins are necessary for the SP response. Furthermore, our functional analyses demonstrate that evolutionary rate covariation is a valuable predictive tool for identifying candidate members of interacting protein networks.     

Proteomic analysis of sericin in <Emphasis Type="Italic">Bombyx mori</Emphasis> cocoons

Cocoon sericin plays an important role in the reeling of silk and serves as a valuable biomaterial in the field of biomedicine, skincare, and food industries; however, knowledge about cocoon sericin proteins has been limited. For a comprehensive study on sericin, cocoons of eight varieties of silkworm of different geographic origin and with varied cocoon color were analyzed utilizing proteomics and bioinformatics approaches. The electrophoresis pattern demonstrated some common protein bands for all silkworm varieties and distinctive protein bands for some of those examined in the present study. The Ser2 protein, a new Ser3 protein, and four other novel sericin proteins were identified in cocoons for the first time. Products of both Ser1 and Ser3 genes appear to be ubiquitous in the cocoon shell of Bombyx mori. In addition, cocoons with especially high-reelability produced by the mutant strain B84 had an unique protein product of the Ser2 gene, indicating that the protein may play an important role in cocoon reelability. A series of sequence conflicts and post-translational modifications (PTMs) were also revealed in sericin proteins. Lipid modifications of sericin proteins, which promote waterproofing of the cocoon shell, were observed. Further, hydroxylation was identified, which provided evidence for intermolecular bonds among neighboring molecules of sericin as found in collagens. The sericin proteome data obtained from this study illuminated the molecular complexity of cocoon sericin and contributed to our understanding of the properties of sericin in filature and biomaterials.     

The first protein map of Synechococcus sp. strain PCC 7942

The first protein map was developed of Synechococcus sp. strain PCC 7942, a model organism for studies of photosynthesis, prokaryotic circadian rhythms, cell division, carbon-concentrating mechanisms, and adaptive responses to a variety of stresses. The proteome was analyzed by two-dimensional gel electrophoresis with subsequent MALDI-TOF mass spectroscopy and database analysis. Of the 140 analyzed protein spots, 110 were successfully identified as 62 different proteins, many of which occurred as multiple spots on the gel. The identified proteins participate in the major metabolic and cellular processes in cyanobacterial cells during the exponential growth phase. In addition, 14 proteins which were previously either unknown or considered to be hypothetical were shown to be true gene products in Synechococcus sp. strain PCC 7942. These results may be helpful for the annotation of the recently sequenced genome of this cyanobacterium, as well as for biochemical and physiological studies of Synechococcus.     

Aerobic and anaerobic metabolism of squalene by a denitrifying bacterium isolated from marine sediment

The aerobic and anaerobic metabolism of the isoprenoid alkene squalene was investigated in a new type of marine denitrifying bacterium, strain 2sq31, isolated from marine sediment. Strain 2sq31 was identified as a species of Marinobacter. Under denitrifying conditions, the strain efficiently degraded squalene; of 0.7 mmol added per liter of medium, 77% was degraded within 120 days under anoxic conditions with nitrate as electron acceptor. Tertiary diols and methyl ketones were identified as metabolites, and an anaerobic pathway was suggested to explain the formation of such compounds. The first step in anaerobic degradation of squalene by strain 2sq31 involves hydration of double bonds to tertiary alcohols. Under oxic conditions, the degradation of squalene by strain 2sq31 was rapid and involved oxidative splitting of the C-10/C-11 or C-14/C-15 double bonds, in addition to the pathways observed under denitrifying conditions.     

Purification and characterization of thermostable serine proteases encoded by the genes <Emphasis Type="Italic">ttha0099</Emphasis> and <Emphasis Type="Italic">ttha01320</Emphasis> from <Emphasis Type="Italic">Thermus thermophilus</Emphasis> HB8

As an important class of proteases, serine proteases are required to show high activity under diverse conditions, especially at high temperatures. In the current study, two serine proteases SP348 and SP404 were analyzed by different bioinformatics tools. Both proteins are comprised of a trypsin domain and a PDZ domain, and belong to the trypsin family of proteases. The proteins were successfully expressed with Trx-tags as soluble proteins in the specialized Escherichia coli Rosetta-gami B(DE3)pLysS strain. A simple three-step purification protocol involving heat treatment, Ni–NTA purification and gel filtration was adopted to purify SP404. The molecular weight of recombinant SP404 was about 64 kDa. According to the circular dichroism spectroscopy analysis, SP404 is thermostable at 70 °C with alpha-helix, beta-sheet and random coil contents of about 8, 22 and 70 %, respectively. Our findings may broaden the range of microorganism-derived proteases and have a wide potential for industrial and fundamental studies.     

Immunoproteomic assay of secreted proteins of <Emphasis Type="Italic">Streptococcus suis</Emphasis> serotype 9 with convalescent sera from pigs

Streptococcus suis is an important pathogen of pigs. In China, in addition to S. suis serotype 2, S. suis serotype 9 (SS9) is also a prevalent serotype. There is no vaccine available for SS9. An immunoproteome-based approach was developed to identify SS9 immunogenic proteins for vaccine development. Secreted proteins extracted from SS9 strain GZ0565 were screened by two-dimensional Western blotting using convalescent sera from pigs. Protein spots were excised from preparative gels and were identified by matrix-assisted laser desorption ionization time-of-flight mass spectrometry, which led to the identification of ten immunogenic proteins (sortases, ABC transporter substrate-binding protein–maltose/maltodextrin, ABC transporter periplasmic protein, CHAP domain containing protein, peptidoglycan-binding LysM, elongation factor Tu, elongation factor G, thymidine kinase, molecular chaperone DnaK, hypothetical protein SSU98_2184). These novel immunogenic proteins, which are encoded by genes that are reasonably conserved among SS9 strains, may be developed as antigens for further study of SS9 vaccine.     

Dual Localization of Squalene Epoxidase, Erg1p, in Yeast Reflects a Relationship between the Endoplasmic Reticulum and Lipid Particles

Squalene epoxidase, encoded by the ERG1 gene in yeast, is a key enzyme of sterol biosynthesis. Analysis of subcellular fractions revealed that squalene epoxidase was present in the microsomal fraction (30,000 × g) and also cofractionated with lipid particles. A dual localization of Erg1p was confirmed by immunofluorescence microscopy. On the basis of the distribution of marker proteins, 62% of cellular Erg1p could be assigned to the endoplasmic reticulum and 38% to lipid particles in late logarithmic-phase cells. In contrast, sterol Δ²⁴-methyltransferase (Erg6p), an enzyme catalyzing a late step in sterol biosynthesis, was found mainly in lipid particles cofractionating with triacylglycerols and steryl esters. The relative distribution of Erg1p between the endoplasmic reticulum and lipid particles changes during growth. Squalene epoxidase (Erg1p) was absent in an erg1 disruptant strain and was induced fivefold in lipid particles and in the endoplasmic reticulum when the ERG1 gene was overexpressed from a multicopy plasmid. The amount of squalene epoxidase in both compartments was also induced approximately fivefold by treatment of yeast cells with terbinafine, an inhibitor of the fungal squalene epoxidase. In contrast to the distribution of the protein, enzymatic activity of squalene epoxidase was only detectable in the endoplasmic reticulum but was absent from isolated lipid particles. When lipid particles of the wild-type strain and microsomes of an erg1 disruptant were mixed, squalene epoxidase activity was partially restored. These findings suggest that factor(s) present in the endoplasmic reticulum are required for squalene epoxidase activity. Close contact between lipid particles and endoplasmic reticulum may be necessary for a concerted action of these two compartments in sterol biosynthesis.     

Proteomic analysis of irregular,bullet‐shaped magnetosomes in the sulphate‐reducing magnetotactic bacterium Desulfovibrio magneticus RS‐1

Recent molecular studies on magnetotactic bacteria have identified a number of proteins associated with bacterial magnetites (magnetosomes) and elucidated their importance in magnetite biomineralisation. However, these analyses were limited to magnetotactic bacterial strains belonging to the α‐subclass of Proteobacteria. We performed a proteomic analysis of magnetosome membrane proteins in Desulfovibrio magneticus strain RS‐1, which is phylogenetically classified as a member of the δ‐Proteobacteria. In the analysis, the identified proteins were classified based on their putative functions and compared with the proteins from the other magnetotactic bacteria, Magnetospirillum magneticum AMB‐1 and M. gryphiswaldense MSR‐1. Three magnetosome‐specific proteins, MamA (Mms24), MamK, and MamM, were identified in strains RS‐1, AMB‐1, and MSR‐1. Furthermore, genes encoding ten magnetosome membrane proteins, including novel proteins, were assigned to a putative magnetosome island that contains subsets of genes essential for magnetosome formation. The collagen‐like protein and putative iron‐binding proteins, which are considered to play key roles in magnetite crystal formation, were identified as specific proteins in strain RS‐1. Furthermore, genes encoding two homologous proteins of Magnetococcus MC‐1 were assigned to a cryptic plasmid of strain RS‐1. The newly identified magnetosome membrane proteins might contribute to the formation of the unique irregular, bullet‐shaped crystals in this microorganism.     

Analysis of Protein Expression Profiles of <Emphasis Type="Italic">Halobacillus dabanensis</Emphasis> D-8<Superscript>T</Superscript> Under Optimal and High Salinity Conditions

Two-dimensional (2-D) gel electrophoresis was employed to display the expression profiles of proteins of Halobacillus dabanensis D-8^T under 1%, 10%, and 20% salinities. Approximately 700 protein spots could be detected in the 2-D gels by Imagemaster™ 2D Platinum software. The molecular masses of the majority of intracellular proteins were distributed in the range of 17.5 kDa–66 kDa and isoelectric points of 4.0–5.9. In total 133 protein spots were observed with a changed expression level under different salinity conditions. Sixty-two protein spots showed upregulation and 26 new protein spots were found under high salinity conditions, while 25 protein spots were downregulated and 20 spots disappeared. Twenty-seven proteins with a markedly changed expression in hypersaline environments were identified by matrix-assisted laser desorption/ionization time-of-flight mass spectroscopy (MALDI-TOF/MS) and MASCOT. A changed expression pattern was observed for proteins related to energy-producing pathways, stress regulators, and proteins involved in the survival of strain D-8^T under high salt challenges. Many proteins play necessary roles in the adaptation to high salt or as a general stress protein, and some proteins are salt-stressed specific proteins that improve the capability of salt-tolerance of strain D-8^T growth under extremely hypersaline condition.     

Resuscitation-promoting factors: distribution among actinobacteria, synthesis during life-cycle and biological activity

We have identified new members of the resuscitation-promoting factor (Rpf) protein family in numerous as yet unsequenced actinobacteria suggesting that Rpfs are common in this bacterial group. Formation and final delivery of the Rpf proteins varied from strain to strain and were dependent on growth conditions. Rpfs were found to act complementary to resuscitate other actinobacteria and might therefore be useful tools for the isolation of new actinobacterial species, especially from soil samples in which lots of microorganisms may have adopted dormant states.     

Sterol carrier protein hypothesis: requirement for three substrate-specific soluble proteins in liver cholesterol biosynthesis.

The 105,000 x g supernatant (S₁₀₅) of liver is required for the conversion of squalene to cholesterol by microsomal membranes. Substantial controversy has existed concerning the properties of what was originally considered to be a single sterol carrier protein present in S₁₀₅ and required for this conversion. We have now resolved this controversy by the discovery that S₁₀₅ contains several sterol carrier proteins. Based upon experiments with three substrates, three substrate-specific soluble proteins (with different properties) have been identified which operate at distinct points in microsomal cholesterol synthesis. These proteins are provisionally designated sterol carrier protein₁ (SCP₁), sterol carrier protein₂ (SCP₂), and sterol carrier protein₃ (SCP₃). SCP₁ is required for the microsomal conversion of squalene to lanosterol, SCP₂ for the microsomal conversion of 4,4-dimethyl-Δ⁸-cholesterol to C₂₇-sterols, and SCP₃ for the microsomal conversion of 7-dehydrocholesterol to cholesterol. Available evidence is consistent with the proposal that a given sterol carrier protein is a soluble constituent of a single microsomal enzyme or enzyme complex, and that it participates both as a carrier for the water-insoluble substrate and as an essential enzyme constituent facilitating catalysis. It may well be that enzymatic transformations of water-insoluble substrates require both microsomal membranes and substrate-specific soluble proteins. This requirement could be a common biological mechanism for water-insoluble substrates.     

The complete nucleotide sequence of PEBV RNA2 reveals the presence of a novel open reading frame and provides insights into the structure of tobraviral subgenomic promoters.

The 3374 nucleotide sequence of RNA2 from the British PEBV strain SP5 has been determined. The RNA includes three open reading frames flanked by 5' and 3' noncoding regions of 509 and 480 nucleotides. The open reading frames specify coat protein, a 29.6K product homologous to the 29.1K product of TRV(TCM) RNA2 and a 23K product not homologous to any previously described protein. The homology demonstrated between the coat proteins of PRV, TRV and PEBV indicates a common evolutionary origin for these proteins. Upstream of each ORF are located sequences homologous to those with which subgenomic RNAs of other tobraviruses start. Subgenomic RNAs for the expression of the three ORFs may start at these points. On all five tobraviral RNA2 molecules sequenced to date, these sequences were found upstream of the coat protein ORF in association with a strongly-conserved potential secondary structural element. Similar potential structures were identified upstream of other tobraviral ORFs. These structures may contribute to the activity of the tobraviral subgenomic promoter.     

Microbial Transformation of Squalene: Terminal Methyl Group Oxidation by Corynebacterium sp

Corynebacterium sp. strain SY-79 was isolated from soil, using squalene as a carbon source. Microbiological properties of this bacterium are described. The metabolic product of this bacterium from squalene was identified as 2,6,10,15,19, 23-hexamethyl-2,6,10,14,18,22-tetracosahexaenedioic acid (squalenedioic acid).     

Search for novel stress-responsive protein components using a yeast mutant lacking two cytosolic Hsp70 genes, SSA1 and SSA2

Heat shock proteins (Hsp) 70 are a ubiquitous family of molecular chaperones involved in many cellular processes. A yeast strain, ssa1/2, with two functionally redundant cytosolic Hsp70s (SSA1 and SSA2) deleted shows thermotolerance comparable to mildly heat-shocked wild type yeast, as well as increased protein synthesis and ubiquitin-proteasome protein degradation. Since mRNA abundance does not always correlate well with protein expression levels it is essential to study proteins directly. We used a gel-based approach to identify stress-responsive proteins in the ssa1/2 mutant and identified 43 differentially expressed spots. These were trypsin-digested and analyzed by nano electrospray ionization liquid chromatography tandem mass spectrometry (nESI-LC-MS/MS). A total of 22 non-redundant proteins were identified, 11 of which were confirmed by N-terminal sequencing. Nine proteins, most of which were up-regulated (2-fold or more) in the ssa1/2 mutant, proved to be stress-inducible proteins such as molecular chaperones and anti-oxidant proteins, or proteins related to carbohydrate metabolism. Interestingly, a translational factor Hyp2p up-regulated in the mutant was also found to be highly phosphorylated. These results indicate that the cytosolic Hsp70s, Ssa1p and Ssa2p, regulate an abundance of proteins mainly involved in stress responses and protein synthesis.     

Short panicle1 encodes a putative PTR family transporter and determines rice panicle size

The architecture of the rice inflorescence, which is determined mainly by the number and length of primary and secondary inflorescence branches, is of importance in both agronomy and developmental biology. The position and number of primary branches are established during the phase transition from vegetative to reproductive growth, and several of the genes identified as participating in this process do so by regulating the meristemic activities of inflorescence. However, little is known about the molecular mechanism that controls inflorescence branch elongation. Here, we report on a novel rice mutant, short panicle1 ( sp1 ), which is defective in rice panicle elongation, and thus leads to the short-panicle phenotype. Gene cloning and characterization indicate that SP1 encodes a putative transporter that belongs to the peptide transporter (PTR) family. This conclusion is based on the findings that SP1 contains a conserved PTR2 domain consisting of 12 transmembrane domains, and that the SP1-GFP fusion protein is localized in the plasma membrane. The SP1 gene is highly expressed in the phloem of the branches of young panicles, which is consistent with the predicted function of SP1 and the sp1 phenotype. Phylogenetic analysis implies that SP1 might be a nitrate transporter. However, neither nitrate transporter activity nor any other compounds transported by known PTR proteins could be detected in either a Xenopus oocyte or yeast system, in our study, suggesting that SP1 may need other component(s) to be able to function as a transporter, or that it transports unknown substrates in the monocotyledonous rice plant.     

In silico and in vivo analysis of signal peptides effect on recombinant glucose oxidase production in nonconventional yeast <Emphasis Type="Italic">Yarrowia lipolytica</Emphasis>

Signal peptide (SP) is an important factor and biobrick in the production and secretion of recombinant proteins. The aim of this study was in silico and in vivo analysis of SPs effect on the production of recombinant glucose oxidase (GOX) in Yarrowia lipolytica. Several in silico softwares, namely SignalP4, Signal-CF, Phobius, WolfPsort 0.2, SOLpro and ProtParam, were used to analyse the potential of 15 endogenous and exogenous SPs for the secretion of recombinant GOX in Y. lipolytica. According to in silico results, the SP of GOX was predicted as suitable in terms of high secretory potential and of protein solubility and stability which is chosen for in vivo analysis. The recombinant Y. lipolytica strain produced 280 U/L of extracellular GOX after 7 days in YPD medium. The results show that the SP of GOX can be applied to efficient production of extracellular heterologous proteins and metabolic engineering in Y. lipolytica.