Characterization of heat-shock response of the marine bacterium Vibrio harveyi

We have investigated heat-shock response in a marine bacterium Vibrio harveyi. We have found that 39 C was the highest tempature at which V. harveyi was able to grow steadily. A shift from 30° C to 39° C caused increased synthesis of at least 10 proteins, as judged by SDS-PAGE, with molecular masses of 90, 70, 58, 41, 31, 27, 22, 15, 14.5 and 14kDa. The 70, 58, 41 and 14.5 kDa proteins were immunologically homologous to DnaK, GroEL, DnaJ and GroES heat-shock proteins of Escherichia coli, respectively. V. harveyi GroES protein had a lower molecular mass (14.5 kDa) than E. coli GroES, migrating in SDS-PAGE as 15 kDa protein. We showed that a protein of ～43 kDa, immunologically reactive with antiserum against E. coli sigma 32 subunit (σ³²) of RNA polymerase, was induced by heat-shock and co-purified with V. harveyi RNA polymerase. These results suggest that the 43 kDa protein is a heat-shock sigma protein of V. harveyi. Preparation containing the V. harveyi sigma 32 homologue, supplemented with core RNA polymerase of E. coli, was able to transcribe heat-shock promoters of E. coli in vitro.     

Expression and identification of a minor extracellular fibrinolytic enzyme (Vpr) from <Emphasis Type="Italic">Bacillus subtilis</Emphasis> KCTC 3014

Previously, three extracellular proteases, Vpr, PepT, and subtilisin were identified from Bacillus subtilis KCTC 3014. To confirm the activity of Vpr, two recombinant Vpr proteins, full Vpr with TTG (pGST-fTTG-Vpr) and full Vpr with ATG (pGST-fATG-Vpr) as an initiation codon were expressed using a pGEX-2T vector encoding glutathione S-transferase (GST) in Escherichia coli. Vpr was produced in two forms, occurring as four spots on a 2-DE gel, 68 and 75 kDa proteins with similar pI values (4.0 ∼ 4.5). Activity was detected in a fibrin zymography at the expected molecular size of 68 kDa (mature form) processed from full Vpr. However, the recombinant 75 kDa of GST-fVpr did not exhibit activity. Replacement of the TTG codon with ATG led to 1.9-fold increased enzyme activity in 68 kDa. Interestingly, the expression of GSTVpr resulted in the proteolytic degradation of the protein and no GST fusion Vpr protein was detected.     

Current two-dimensional electrophoresis technology for proteomics

Two-dimensional gel electrophoresis (2-DE) with immobilized pH gradients (IPGs) combined with protein identification by mass spectrometry (MS) is currently the workhorse for proteomics. In spite of promising alternative or complementary technologies (e.g. multidimensional protein identification technology, stable isotope labelling, protein or antibody arrays) that have emerged recently, 2-DE is currently the only technique that can be routinely applied for parallel quantitative expression profiling of large sets of complex protein mixtures such as whole cell lysates. 2-DE enables the separaration of complex mixtures of proteins according to isoelectric point (pI), molecular mass (M_r), solubility, and relative abundance. Furthermore, it delivers a map of intact proteins, which reflects changes in protein expression level, isoforms or post-translational modifications. This is in contrast to liquid chromatography-tandem mass spectrometry based methods, which perform analysis on peptides, where M_r and pI information is lost, and where stable isotope labelling is required for quantitative analysis. Today's 2-DE technology with IPGs (Görg et al., Electrophoresis 2000, 21, 1037–1053), has overcome the former limitations of carrier ampholyte based 2-DE (O'Farrell, J. Biol. Chem. 1975, 250, 4007–4021) with respect to reproducibility, handling, resolution, and separation of very acidic and/or basic proteins. The development of IPGs between pH 2.5–12 has enabled the analysis of very alkaline proteins and the construction of the corresponding databases. Narrow-overlapping IPGs provide increased resolution (δpI = 0.001) and, in combination with prefractionation methods, the detection of low abundance proteins. Depending on the gel size and pH gradient used, 2-DE can resolve more than 5000 proteins simultaneously (˜2000 proteins routinely), and detect and quantify < 1 ng of protein per spot. In this article we describe the current 2-DE/MS workflow including the following topics: sample preparation, protein solubilization, and prefractionation; protein separation by 2-DE with IPGs; protein detection and quantitation; computer assisted analysis of 2-DE patterns; protein identification and characterization by MS; two-dimensional protein databases.     

Differentiation-related proteins of the broad bean rust fungus Uromyces viciae-fabae,as revealed by high resolution two-dimensional polyacrylamide gel electrophoresis

On artificial polyethylene membranes providing a thigmotropic signal, uredospores of the broad bean rust fungus Uromyces viciae-fabae differentiated a series of infection structures which in nature are necessary to invade the host tissue through the stomata. Within 24 h germ tubes, appressoria, substomatal vesicles, infection hyphae and haustorial mother cells were developed successively. Alterations in protein metabolism during infection structure differentiation of this obligate plant pathogen were analyzed in the absence of the host plant by high resolution two-dimensional polyacrylamide gel electrophoresis (2-DE) and silver staining. The norm pattern representing the 2-DE protein patterns of the whole developmental sequence of infection structures of U. viciae-fabae showed 733 spots. During infection structure differentiation 55 proteins were newly formed, altered in quantity, or disappeared. Major alterations in the protein pattern occurred during uredospore germination and when infection hyphae were formed. Uredospore germination was characterized by a decrease of acidic proteins and an increase mainly of proteins with isoelectric points ranging from weakly acidic to basic.Abbreviations 2-DE two-dimensional polyacrylamide gel electrophoresis - DAPI 4,6-diamino-phenylindol - kDa kilo Dalton - pl isoelectric point - PMSF phenylmethylsulfonyl fluoride - SDS-PAGE sodium dodecyl sulfate polyacrylamide gel electrophoresis     

Prefractionation enhances loading capacity and identification of basic proteins from human breast cancer tissues

Many basic proteins (pI > 7) and putative disease biomarkers are not identified using conventional proteomic methods. This study applied a new method to improve the identification of such proteins. Prefractionated basic proteins were compared with total tissue lysates from human ductal carcinoma in situ tissue loaded on basic immobilized pH gradient strips prior to two-dimensional gel electrophoresis (2-DE). Extraction of alkaline proteins was achieved in less than 20 min using a chromatofocusing resin and two buffers in a microcentrifuge tube. Prefractionation showed improved resolution and visualization of low-abundance proteins on 2-DE gels, allowing proteins to be excised, accumulated, trypsin-digested, and identified by liquid chromatography–tandem mass spectrometry. Proteins identified in the prefractionated samples had a higher number of peptides and three times the number of unique basic proteins when compared with total lysates. Low-molecular-weight (LMW, <26 kDa) unique alkaline proteins comprise 75% of those identified in prefractionated samples compared with 25% identified in total lysates, representing a 9-fold increase of LMW proteins due to prefractionation. Prefractionation ultimately increases loading capacity of samples onto the 2-DE gel and leads to better resolution, visualization, and identification of proteins with pI values greater than 7.     

Heat-Resistance and Heat-Shock Response in the Nosocomial Pathogen <Emphasis Type="Italic">Enterococcus faecium</Emphasis>

We have characterized the heat-shock response of the nosocomial pathogen Enterococcus faecium. The growth of E. faecium cells was analyzed at different temperatures; little growth was observed at 50°C, and no growth at 52°C or 55°C. In agreement, a marked decrease of general protein synthesis was observed at 52°C, and very light synthesis was detected at 55°C. The heat resistance of E. faecium cells was analyzed by measuring the survival at temperatures higher than 52°C and, after 2 h of incubation, viable cells were still observed at 70°C. By Western blot analysis, two heat-induced proteins were identified as GroEL (65 kDa) and DnaK (75 kDa). Only one isoform for either GroEL or DnaK was found. The gene expression of these heat-shock proteins was also analyzed by pulsed-labeled experiments. The heat-induced proteins showed an increased rate of synthesis during the first 5 min, reaching the highest level of induction after 10 min and returning to the steady-state level after 20 min of heat treatment. Received: 29 March 2002 / Accepted: 5 July 2002     

Sequential Extraction Results in Improved Proteome Profiling of Medicinal Plant Pinellia ternata Tubers,Which Contain Large Amounts of High-Abundance Proteins

Pinellia ternata tuber is one of the well-known Chinese traditional medicines. In order to understand the pharmacological properties of tuber proteins, it is necessary to perform proteome analysis of P. ternata tubers. However, a few high-abundance proteins (HAPs), mainly mannose-binding lectin (agglutinin), exist in aggregates of various sizes in the tubers and seriously interfere with proteome profiling by two-dimensional electrophoresis (2-DE). Therefore, selective depletion of these HAPs is a prerequisite for enhanced proteome analysis of P. ternata tubers. Based on differential protein solubility, we developed a novel protocol involving two sequential extractions for depletion of some HAPs and prefractionation of tuber proteins prior to 2-DE. The first extraction using 10% acetic acid selectively extracted acid-soluble HAPs and the second extraction using the SDS-containing buffer extracted remaining acid-insoluble proteins. After application of the protocol, 2-DE profiles of P. ternata tuber proteins were greatly improved and more protein spots were detected, especially low-abundance proteins. Moreover, the subunit composition of P. ternata lectin was analyzed by electrophoresis. Native lectin consists of two hydrogen-bonded subunits (11 kDa and 25 kDa) and the 11 kDa subunit was a glycoprotein. Subsequently, major HAPs in the tubers were analyzed by mass spectrometry, with nine protein spots being identified as lectin isoforms. The methodology was easy to perform and required no specialized apparatus. It would be useful for proteome analysis of other tuber plants of Araceae.     

Temperature treatments of dark-grown pea seedlings cause an accelerated greening in the light at different levels of gene expression

We have previously shown that heat-shock in the dark evokes photomorphogenesis-like effects and circadian rhythmicity at the level of mRNAs when applied to emerging pea plantlets during several consecutive days [15]. Here we extend these findings by showing that a temperature shift to 10 °C above average and a single heat-shock are sufficient for induction of circadian rhythmicity and changes in morphogenesis. The maximum response to a single heat-shock occurs at days 2 to 3 after sowintreatments intensifies the morphogenetic effect. The heat-shocked plantlets have an elevated level of the xanthophyll lutein in the dark. Upon illumination of heat-shocked plantlets accumulation of chloroplast pigments as well as that of individual thylakoid membrane proteins and their corresponding mRNAs occur much faster than in the etiolated controls. This is reflected in an accelerated formation of grana stacks. Therefore, heat-shock seems to evoke a responsiveness of plantlets similar to that obtained earlier by other authors using pre-illumination. The working hypothesis is put forward that induction or synchronization of circadian rhythmicity by either light or heat-shock might be sufficient to explain the observed morphogenetic changes.Abbreviations CCI reaction center I core - CHS cyclic heat-shock - D1 protein 32 kDa psbA gene product - ELIP early light-inducible protein - LHCP light-harvesting chlorophyll a/b protein - PCOR protochlorophyllide oxidoreductase - SSU small subunit of ribulose-1,5-bisphosphate carboxylase - WSP proteins of the oxygen-evolving (water-splitting) complex     

The stress-shock response of the bacterium Methylophilus methylotrophus

A sudden increase in the growth temperature of Methylophilus methylotrophus results in the synthesis of a number of unique proteins. The major heat-shock proteins have apparent molecular masses of 83, 78, 63, 60, 16 and 14 kDa. Other stress conditions elicit a similar response, although there are significant differences in the sets of proteins produced under the various conditions. Addition of methanol induces proteins identical in size to the heat-shock 83, 79, 63 and 14 kDa proteins and also induces unique 94, 36 and 29 kDa species. Addition of ethanol induces proteins identical in size to the 78 and 20 kDa heat-shock proteins and the 94 and 36 kDa methanol-induced proteins and an apparently unique 13 kDa species. Simultaneous exposure to elevated temperature and either methanol or ethanol resulted in the synthesis of all of the proteins induced by the separate treatments. The stress-shock proteins are differentially located in cytoplasmic, periplasmic and membrane fractions.     

Heat-shock response in germinating pine pollen

Summary The in vitro culture of pine pollen at various temperatures reveals only a moderate degree of thermotolerance, with considerably reduced levels of growth at and above 35° C. Unlike the pollen of many previously studied species, pine pollen shows some ability to recover from short periods of growth at temperatures as high as 40° C, especially when such exposures occur during the early stages of pollen germination. The pollen of Pinus taeda, unlike that of most other species, shows both quantitative and qualitative changes in the proteins synthesized during germination in vitro following a switch to elevated temperatures (37° C). This response, which can be elicited both during the very early stages of germination as well as during the later stages of pollen tube growth, is reversible following a shift back to the lower temperatures. As previously shown with vegetative tissue of other plant species, the heat-shock response not only involves the induction of high-molecular-weight proteins (most notably 82 kDa and 70 kDa proteins), but also a number of low-molecular-weight (10–20 kDa) species. Two-dimensional gel electrophoretic analysis reveals a small number of qualitative differences in the types of low-molecular-weight heat-shock proteins synthesized in pollen versus vegetative tissue.     

Expression of CspA and GST by an Antarctic psychrophilic bacterium Colwellia sp. NJ341 at near-freezing temperature

Summary A psychrotrophic bacterium Colwellia sp. NJ341 from Antarctic sea ice could grow at −5 and 22 °C, and the extent of cellular protein content and growth were greater at low temperatures (0–10 °C) than at higher temperatures. SDS-PAGE analysis demonstrated the presence of a 7 kDa cold-shock protein. The further result of two-dimensional electrophoresis (2-DE) showed that two proteins a and c were newly synthesized at near-freezing temperatures. With matrix-assisted laser desorption/ionization mass spectrometry (MALDI-TOF MS) analysis, proteins a and c were identified as glutathione S-transferase (GST) and cold-shock protein A (CspA), respectively, which were involved in cold-adaptation at near-freezing temperature in an Antarctic psychrophilic bacterium Colwellia sp. NJ341.     

Application of two-dimensional electrophoresis for monitoring gastrointestinal digestion of milk

Summary. Two-dimensional electrophoresis (2-DE) was used for tracing in vivo gastrointestinal digestion of milk proteins in a rapid model system with rats. Contents of stomach and small intestine from digestion trials with rats given a single dose of milk powder were recovered after 1 hour. They were then subjected to 2-DE (IEF and SDS-PAGE). 2-DE showed undigested proteins in a MW range 13.0–66.0 kDa in stomach and 13.0–25.0 kDa in the small intestine, thus indicating that milk proteins are slowly digested. This approach may shed light on pattern of protein digestion and mechanism of amino acid and peptide assimilation.     

2-DE proteomic analysis of HSP70 in mollusc Chamelea gallina

Bidimensional electrophoresis (2-DE) protocols were adapted on Chamelea gallina digestive glands studies by the analysis of Heat Shock Proteins (HSP) compared with monodimensional electrophoresis (1-DE) results. Because polycyclic aromatic hydrocarbons (PAH) act on HSPs, C. gallina specimens were exposed to 0.5 mg/L of benzo[a]pyrene (B[a]P) for 24 h, 7 and 12 days. Immunoblotting after 1-DE showed a single band of 70 kDa significantly induced after 7 days of B[a]P exposure. After 2-DE, eight major high-resolved spots between 17 and 98 kDa were revealed. Three spots fell within the range of 62–98 kDa and of 5–6 pI, parameters which could include HSP70. Two spots of 77 and 72 kDa, obtained after 2-DE immunoblotting, could correspond to constitutive HSC70 and to inducible HSP70 forms respectively. Changes observed in inducible and in constitutive forms might be related to an adaptation to stress and to a normal protein synthesis capability, respectively. Employment of 2-DE and relationship between HSP70 and HSC70 may be useful to clarify their role in molluscs subjected to stress events.     

Identification and characterization of adhesive factors of Clostridium difficile involved in adhesion to human colonic enterocyte-like Caco-2 and mucus-secreting HT29 cells in culture

Experiments reported in this communication showed that the highly toxinogenic Cd 79685, Cd 4784, and Wilkins Clostridium difficile strains and the moderately toxinogenic FD strain grown in the presence of blood adhere to polarized monolayers of two cultured human intestinal cell lines: the human colonic epithelial Caco-2 cells and the human mucus-secreting HT29-MTX cells. Scanning electron microscopy revealed that the bacteria interacted with well-defined apical microvilli of differentiated Caco-2 cells and that the bacteria strongly bind to the mucus layer that entirely covers the surface of the HT29-MTX cells. The binding of C. difficile to Caco-2 cells developed in parallel with the differentiation features of the Caco-2 cells, suggesting that the protein(s) which constitute C. difficile-binding sites are differentiation-related brush border protein(s). To better define this interaction, we tentatively characterized the mechanism(s) of adhesion of C. difficile with adherence assays. It was shown that heating of C. difficile grown in the presence of blood enhanced the bacterial interaction with the brush border of the enterocyte-like Caco-2 cells and the human mucus-secreting HT29-MTX cells. A labile surface-associated component was involved in C. difficile adhesion since washes of C. difficile grown in the presence of blood without heat shock decreased adhesion. After heating, washes of C. difficile grown in the presence of blood did not modify adhesion. Analysis of surface-associated proteins of C. difficile subjected to different culture conditions was con-ducted. After growth of C. difficile Cd 79685, Cd 4784, FD and Wilkins strains in the presence of blood and heating, two predominant SDS-extractable proteins with molecular masses of 12 and 27 kDa were observed and two other proteins with masses of 48 and 31 kDa disappeared. Direct involvement of the 12 and 27 kDa surface-associated proteins in the adhe-sion of C. difficile strains was demonstrated by using rat polycolonal antibodies pAb 12 and pAb 27 directed against the 12 and 27kDa proteins. Indeed, adhesion to Caco-2 cell monoiayers of C. difficiie strains grown in the presence of blood, without or with heat-shock, was blocked. Taken together, our results suggest that C. difficiie may utilize blood components as adhesins to adhere to human intestinal cultured cells.     

Soluble and membrane-associated heat shock proteins in soybean root

Summary Localization of heat shock proteins (Hsp) in endomembranes and determination of whether they are integral or peripheral membrane proteins will aid in understanding the physiological function of the heat shock response. Radiolabeled endomembranes (endoplasmic reticulum, Golgi, and plasma membrane), obtained by sucrose gradient centrifugation of heat-shocked soybean (Glycine max L.) root tissue were solubilized and the polypeptides separated by two-dimensional IEF-SDS-PAGE. Autoradiography revealed three groups of Hsp. A diverse group fo 25 low mol wt Hsp (18 to 24 kDa) with isoelectric point (pI) between 5 and 7; an intermediate mol wt group (30 to 47 kDa) with pI of 5.5 to 6.0; and a group of two high mol wt Hsp (75 to 80 kDa) with pI 4.8 to 5.2. The plasma membrane fraction lacked the Hsp pair of 47 kDa detected in the endoplasmic reticulum and Golgi fractions but possessed a unique Hsp of 30 kDa, pI 5.5.Comparison of soluble and microsome fractions revealed a difference in the pattern of the low mol wt Hsp class. The soluble fraction contained Hsp of 16–20 kDa with pI between 5 and 7.8 while the microsome fraction was characterized by Hsp of 18–24 kDa with pI between 5.8 and 6.5.The microsomal Hsp were not released by 1 M KCl. Treatment of the microsome fraction with Triton X-100 selectively released several Hsp, and Na₂CO₃ treatment removed additional Hsp from the membrane fraction.Abbreviations Hsp heat-shock protein(s) - GA Golgi apparatus - PM plasma membrane - 2 D two-dimensional     

Surface-associated, PrfA-regulated proteins of Listeria monocytogenes synthesized under stress conditions

The expression of the five clustered genes of Listeria monocytogenes: plcA, hly, mpl, actA and plcB is under the control of the positive regulation factor PrfA. Listeriolysin, encoded by the hly gene, is the only prominent PrfA-controlled gene product observed when L. monocytogenes strain NCTC 7973 is cultured in a rich medium at 37°C to the logarithmic growth phase. Stress conditions such as heat-shock or stationary culture conditions lead to the induction of additional PrfA-dependent proteins (PdPs): ActA (92 kDa), a 38kDa protein of unknown function and a 34kDa protein which probably represents PlcA. Under nutrient-stress conditions PdPs are preferentially synthesized and in addition to the already known PdPs at least five new, not yet functionally identified PdPs are detected. All PdPs are either secreted or are localized at the cell surface. Differences in the amount as well as the sizes of the PdPs are observed in different L. monocytogenes strains.     

Protein Profile of <Emphasis Type="Italic">Acidithiobacillus ferrooxidans</Emphasis> Strains Exhibiting Different Levels of Tolerance to Metal Sulfates

Strains of Acidithiobacillus ferrooxidans exhibited differences in the inhibition of Fe²⁺ oxidation in the presence of 250 mM of cadmium, zinc, and manganese sulfates in respirometric assays. Strains LR and I35 were practically not inhibited, whereas strains SSP and V3 showed significant inhibition (30–70%). Analysis by SDS-PAGE of total proteins from cells grown in the absence of metal sulfates showed different profiles between the more tolerant strains (LR and I35) and the more susceptible ones (SSP and V3). Total proteins of strains LR and V3 were also resolved by two-dimensional polyacrylamide gel electrophoresis (2-DE). A set of major proteins (40, 32, 22, and 20 kDa) could be identified only in the more tolerant strain LR. Our results show that protein profiles analysis could differentiate A. ferrooxidans strains that considerably differ in the tolerance to metal sulfates and present low genomic similarity as revealed by Random Amplified Polymorphic DNA (RAPD) data obtained previously in our laboratory.     

Resolution of Escherichia coli O157:H7 That Contaminated Radish Sprouts in Two Outbreaks by Two-Dimensional Gel Electrophoresis

Two-dimensional gel electrophoresis (2-DE) was performed to examine exoproteins and periplasmic proteins of Shiga toxin-producing Escherichia coli (STEC) O157:H7 strains isolated from cases associated with radish sprouts in two outbreaks. We found that STEC O157:H7 released a large number of proteins into the medium during the stationary phase of growth, as observed with 2-DE. Although pulsed-field gel electrophoresis (PFGE) patterns of STECs NGY9 (RIMD0509894), a Sakai isolate; NGY33, a Gamagoori isolate; and NGY120, a Kanagawa isolate, were all the same, comparison of 2-DE patterns of exoproteins and periplasmic proteins clarified that NGY9 was distinct from NGY33, whereas NGY33 and NGY120 were of close lineage. We therefore suggest that 2-DE analysis of exoproteins and periplasmic proteins is a powerful epidemiological method with high resolution. Received: 21 August 2000 / Accepted: 9 March 2001