Differentially expressed proteins in the interaction of Xanthomonas axonopodis pv. citri with leaf extract of the host plant

The present study reports the expression of proteins of Xanthomonas axonopodis pv. citri in response to different growth conditions. The bacterium was cultured in the basal medium MM1 and in the presence of leaf extracts from a susceptible host plant (sweet orange) as well as a resistant (ponkan) and a nonhost plant (passion fruit). The protein profiles were analyzed by two-dimensional gel electrophoresis (2-DE). Twelve differential spots (induced, up- and down-regulated and repressed) were observed in the protein profiles of the bacterium cultivated in citrus extract (susceptible host) when compared to that of MM1. The 2-DE profile of the bacterium cultured in the complex medium nutrient yeast glycerol was also obtained and the comparison with that of MM1 revealed 36 differential spots. Five proteins from the different treatments were successfully N-terminally sequenced and the putative functions were assigned by homology searches in databases. Two constitutively expressed proteins, B4 and B5, were identified as pseudouridine synthase and elongation factor P, respectively. The large subunit of ribulose 1,5-biphosphate carboxylase/oxygenase and a sulfate binding protein were found as specifically up-regulated in the presence of citrus extracts. Finally, the heat shock protein G was found exclusively in the complex medium and repressed in all other media.     

Group A streptococcus cell-associated pathogenic proteins as revealed by growth in hyaluronic acid-enriched media

Group A streptococcus (GAS), also know as Streptococcus pyogenes, is a human pathogen and can cause several fatal invasive diseases such as necrotising fasciitis, the so-called flesh-eating disease, and toxic shock syndrome. The destruction of connective tissue and the hyaluronic acid (HA) therein, is a key element of GAS pathogenesis. We therefore propagated GAS in HA-enriched growth media in an attempt to create a simple biological system that could reflect some elements of GAS pathogenesis. Our results show that several recognised virulence factors were up-regulated in HA-enriched media, including the M1 protein, a collagen-like surface protein and the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase, which has been shown to play important roles in streptococcal pathogenesis. Interestingly, two hypothetical proteins of unknown function were also up-regulated and detailed bioinformatics analysis showed that at least one of these hypothetical proteins is likely to be involved in pathogenesis. It was therefore concluded that this simple biological system provided a valuable tool for the identification of potential GAS virulence factors.     

Comparative analysis of 2,4,6-trinitrotoluene (TNT)-induced cellular responses and proteomes in <Emphasis Type="Italic">Pseudomonas</Emphasis> sp. HK-6 in two types of media

TNT-induced cellular responses and proteomes in Pseudomonas sp. HK-6 were comparatively analyzed in two different media: basal salts (BS) and Luria broth (LB). HK-6 cells could not degrade more than 0.5 mM TNT with BS medium, while in LB medium, they exhibited the enhanced capability to degrade as much as 3.0 mM TNT. Analysis of total cellular fatty acids in HK-6 cells suggested that the relative abundance of several saturated or unsaturated fatty acids is altered under TNT-mediated stress conditions. Scanning electron microscopy showed the presence of perforations, irregular rod formations, and wrinkled extracellular surfaces in cells under TNT stress. Proteomic analysis of soluble protein fractions from HK-6 cultures grown with TNT as a substrate revealed 11 protein spots induced by TNT. Among these, seven proteins (including Alg8, AlgB, NirB, and the AhpC/Tsa family) were detected only in LB medium containing TNT. The proteins AspS, Tsf, and assimilatory nitrate reductase were increasingly expressed only in BS medium containing TNT. The protein dGTPase was found to be induced and expressed when cells were grown in either type of TNT-containing media. These results provide a better understanding of the cytotoxicity and survival mechanism used by Pseudomonas sp. HK-6 when placed under TNT stress conditions.     

Analysis of the Pasteurella multocida outer membrane sub-proteome and its response to the in vivo environment of the natural host

This study describes the identification of outer membrane proteins (OMPs) of the bacterial pathogen Pasteurella multocida and an analysis of how the expression of these proteins changes during infection of the natural host. We analysed the sarcosine-insoluble membrane fractions, which are highly enriched for OMPs, from bacteria grown under a range of conditions. Initially, the OMP-containing fractions were resolved by 2-DE and the proteins identified by MALDI-TOF MS. In addition, the OMP-containing fractions were separated by 1-D SDS-PAGE and protein identifications were made using nano LC MS/MS. Using these two methods a total of 35 proteins was identified from samples obtained from organisms grown in rich culture medium. Six of the proteins were identified only by 2-DE MALDI-TOF MS, whilst 17 proteins were identified only by 1-D LC MS/MS. We then analysed the OMPs from P. multocida which had been isolated from the bloodstream of infected chickens (a natural host) or grown in iron-depleted medium. Three proteins were found to be significantly up-regulated during growth in vivo and one of these (Pm0803) was also up-regulated during growth in iron-depleted medium. After bioinformatic analysis of the protein matches, it was predicted that over one third of the combined OMPs predicted by the bioinformatics sub-cellular localisation tools PSORTB and Proteome Analyst, had been identified during this study. This is the first comprehensive proteomic analysis of the P. multocida outer membrane and the first proteomic analysis of how a bacterial pathogen modifies its outer membrane proteome during infection.     

A new preferential medium for enumeration and isolation of desert actinomycetes

In order to facilitate the discovery of novel actinomycetes from the Egyptian deserts, which can be useful as new sources for bioactive metabolites, different media for enumeration and isolation of desert actinomycetes have been tested. For this purpose, 30 soil samples from different six sites representing the Western and Eastern deserts of Egypt were collected. The two deserts are considered hyper-arid and the soil characteristics were determined. The media used were glucose–yeast extract agar, soil extract agar and a new minimal medium (MM) containing glucose, yeast extract and mineral salts. The effects of the soil characteristics on the total viable actinomycete counts on the three media were evaluated. The results showed that the highest actinomycete count in samples from five out of six sites was obtained on MM. Also MM was more selective for actinomycetes and significantly decreased the number of fungal colonies and to a lower extent the number of bacterial colonies. Moreover, it supported the development of different and diverse groups of actinomycetes. From the results obtained in this study, MM is a new useful medium for enumeration and selective isolation of actinomycetes from the desert soils.     

Proteomics approach to identify wound-response related proteins from rice leaf sheath

In order to avoid the complex conditions of the intact plant for simple analysis of proteins in wound-response stress, we used the detached rice leaf sheath which is a very active part of the rice seedling. Proteins were extracted from rice leaf sheath at 0, 12, 24, 48 h after cutting and separated by two-dimensional (2-D) polyacrylamide gel electrophoresis. Changes in differentially displayed proteins were found in leaf sheaths after cutting in the 0-48 h time course. Ten proteins were up-regulated, while 19 proteins were down-regulated compared with those on the four 2-D gels. Among them, 14 proteins were analyzed by N-terminal, or internal amino acid sequence. The clear functions of nine proteins could be identified. Six proteins did not yield amino acid sequence information due to their blocked N-termini. Furthermore, 11 proteins were determined by matrix-assisted laser desorption/ionization-time of flight mass spectrometry, and identified protein database matching. It was shown that the down-regulated proteins were calreticulin (nos. 5, 6), histone H1 (no. 15) and hemoglobin (no. 17), putative peroxidase (no. 19); the up-regulated proteins were Bowman-Birk trypsin inhibitor (no. 23), putative receptor-like protein kinase (nos. 24, 25), calmodulin-related protein (no. 26), small subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase (no. 27), mannose-binding rice lectin (nos. 28, 29). Among all the above proteins, four (nos. 23, 24, 25, 26) have been confirmed to be wound-response proteins. The others cannot be excluded as also being related to wound-responses, such as the signal transduction-related proteins (nos. 5, 6), photosynthesis-related protein (no. 27), and stress-response proteins (nos. 19, 28, 29). This is the first time protein changes in response to wounding in rice leaf sheath have been shown.     

Cyclic Adenosine 3′,5′-Monophosphate and Morphogenesis in Mucor racemosus

Yeastlike cells of Mucor racemosus grown under 100% CO(2) underwent morphogenesis to hyphae after exposure to air. The addition of dibutyryl cyclic adenosine monophosphate (dbcAMP) to yeastlike cultures inhibited this morphogenesis in media containing 2% glucose. The maintenance of uniformly spherical, budding cells required 1 mM dbcAMP in a defined medium containing Casamino Acids, and 3 mM dbcAMP in a medium containing yeast extract and peptone. At these concentrations, dbcAMP also induced yeastlike development in young aerobic hyphae grown in media containing 2% glucose. Removal of dbcAMP resulted in hyphal development. The endogenous cyclic AMP (cAMP) content of yeastlike cultures was measured after a shift from CO(2) to air. A fourfold decrease in intracellular cAMP preceded the appearance of hyphal germ tubes. These results indicate that cAMP plays a role in the control of morphogenesis in Mucor racemosus.     

Relationship Between Messenger Ribonucleic Acid and Enzyme Levels Specified by the Leucine Operon of Escherichia coli K-12

The levels of leucine-forming enzymes in Escherichia coli K-12 varied over a several thousand-fold range, depending upon conditions of growth. The highest levels were achieved by growing auxotrophs in a chemostat under conditions of leucine limitation. Under such conditions, enzyme levels were increased 45- to 90-fold relative to cells grown in minimal medium containing leucine (the latter values arbitrarily called 1). Leucine operon-specific messenger ribonucleic acid levels were elevated to about the same extent as enzyme levels in cells grown in a chemostat. Growth in media of greater complexity resulted in progressively lower levels of leucine-forming enzymes, reaching a value of less than 0.02 for growth in a medium containing tryptone broth and yeast extract. The levels of leucine operon-specified enzymes and messenger ribonucleic acid were also measured in strains containing about 25 copies of plasmid pCV1(ColE1-leu) per chromosome. For such strains grown in minimal medium, enzyme levels were proportional to the number of plasmids per cell. Furthermore, they followed the same trends as those described above upon derepression in a chemostat or upon repression following growth in rich media. Leucine messenger ribonucleic acid, measured both by pulse-labeling and hybridization-competition experiments, was roughly proportional to enzyme levels over this entire range. For a plasmid-containing strain grown in a chemostat under conditions of leucine limitation (about 100 plasmids per chromosome), about 27% of pulse-labeled ribonucleic acid was coded for by genes in or adjacent to the leucine operon, and 10% of the total protein was β-isopropylmalate dehydrogenase.     

Influence of sugars and light on rhizosecretion of α-glucosidase and acid phosphatase during micropropagation of potato

The effects of carbohydrate supply and light on rhizosecretion during micropropagation of potato plantlets (Solanum tuberosum L., cv. ‘Iwa’) in liquid medium were investigated. Soluble protein content was higher in the spent medium for plantlets grown under light conditions than in the dark. For those plantlets grown under light conditions and on different sugar-supplemented media, they rhizosecreted the highest amount of soluble protein when grown in the presence of maltose, while they rhizosecreted the lowest amount of soluble protein when grown on medium containing glucose. Moreover, plantlets grown under light and on a medium containing sucrose were the most vigorous, and exhibited the highest levels of rhizosecreted acid phosphatase activity. However, there was no direct relationship between plantlet growth and rhizosecretion. When plantlets were grown in the dark and on medium containing maltose, a higher α-glucosidase activity was detected than those grown on medium containing sucrose. These results suggested that rhizosecretion of certain proteins from plantlets grown in vitro might not require exposure to light conditions.     

Production of chitinolytic enzymes by a strain (BM17) of Paenibacillus pabuli isolated from crab shells samples collected in the east sector of central Tyrrhenian Sea

Nineteen bacterial isolates were grown in shaken cultures in media containing chitin as carbon source and different additional nitrogen sources such as yeast nitrogen base (YNB), yeast extract (YE), corn steep liquor (CSL) and ammonium sulfate. Strain BM17 showed the highest activity (200 U/l) in medium containing Chitin (1%) and YNB (0.5%). Molecular analysis of the 16S rRNA gene showed that strain BM17 belongs to the species Paenibacillus pabuli (99.72% homology). The enzyme activity started after 12-24 h; exponential enzyme production was recorded from the 24th h and lasted till the 96th h of incubation when activity peaked to decrease thereafter. Medium optimisation was carried out by Response Surface Methodology (RSM) considering the effects of chitin, corn steep liquor and yeast extract. BM17 chitinolytic activity was induced by chitin but the increase of its concentration did not have significant effects on the enzyme activity. By contrast, the nitrogen source, particularly YE, strongly affected the enzyme production.     

Micropropagation of Clerodendrum aculeatum through adventitious shoot induction and production of consistent amount of virus resistance inducing protein

Rapid micropropagation through adventitious shoot induction from in vitro raised leaf explants of Clerodendrum aculeatum (Verbenaceae), was successfully achieved for the first time. Basal portion of the leaves showed highest regeneration potential when grown on MS medium supplemented with BA (5.0 mg/l) and NAA and IBA (0.5 mg/l of each). Shoots after elongation in growth regulator-free medium, were rooted in MS medium containing 0.5 mg/l of NAA and IBA. Aqueous leaf extract of in vitro raised plants, induced high degree of resistance against viruses in susceptible healthy hosts when applied prior to virus inoculation. Upon purification from leaves of cultured plants, the resistance inducing protein, showed molecular mass of 34 kDa. Amount of resistance inducing protein obtained from leaves of cultured plants, was consistent throughout the year, as compared to the protein isolated from leaves of field grown plants, which showed marked seasonal fluctuation. The purified 34 kDa protein from in vitro raised plants, was serologically related to field grown plants and possessed similar characteristics. The micropropagated plants were successfully established in earthen pots under greenhouse conditions.     

Global Gene Expression Analysis of Long-Term Stationary Phase Effects in E. coli K12 MG1655

Global gene expression was monitored in long-term stationary phase (LSP) cells of E. coli K12 MG1655 and compared with stationary phase (SP) cells that were sub-cultured without prolonged delay to get an insight into the survival strategies of LSP cells. The experiments were carried out using both LB medium and LB supplemented with 10% of glycerol. In both the media the LSP cells showed decreased growth rate compared to SP cells. DNA microarray analysis of LSP cells in both the media resulted in the up- and down-regulation of several genes in LSP cells compared to their respective SP cells in the corresponding media. In LSP cells grown in LB 204 genes whereas cells grown in LB plus glycerol 321 genes were differentially regulated compared to the SP cells. Comparison of these differentially regulated genes indicated that irrespective of the medium used for growth in LSP cells expression of 95 genes (22 genes up-regulated and 73 down-regulated) were differentially regulated. These 95 genes could be associated with LSP status of the cells and are likely to influence survival and growth characteristics of LSP cells. This is indeed so since the up- and down-regulated genes include genes that protect E. coli LSP cells from stationary phase stress and genes that would help to recover from stress when transferred into fresh medium. The growth phenotype in LSP cells could be attributed to up-regulation of genes coding for insertion sequences that confer beneficial effects during starvation, genes coding for putative transposases and simultaneous down-regulation of genes coding for ribosomal protein synthesis, transport-related genes, non-coding RNA genes and metabolic genes. As yet we still do not know the role of several unknown genes and genes coding for hypothetical proteins which are either up- or down-regulated in LSP cells compared to SP cells.     

Direct regeneration from in vitro leaf and petiole tissues of <Emphasis Type="Italic">Populus tremula</Emphasis> ‘Erecta’

Dormant buds from a mature tree of Populus tremula ‘Erecta’ were incubated on a Murashige and Skoog (MS) medium supplemented with 1.0 μM thidiazuron (TDZ). Induced shoots were then proliferated on medium of MS or Woody Plant Medium (WPM), or Driver and Kuniyuki Walnut (DKW) supplemented with varying levels of benzyladenine (BA). Overall, shoots grown on MS medium supplemented with 1.25–2.5 μM BA exhibited the highest frequency of shoot proliferation (>95%) and more than 60% of responding explants produced more than five shoots per explant. Shoot organogenesis was induced from both leaf and petiole explants incubated on WPM medium containing BA, or TDZ, or zeatin. Among the different cytokinins tested, zeatin induced the highest frequency (average 72.1%) of shoot organogenesis. None of explants survived on media containing no cytokinins within 6–8 weeks following culture. Overall, a higher frequency of shoot regeneration was obtained from petioles than from leaf explants. The highest frequency of regeneration was achieved when petioles were incubated on WPM containing 10–20 μM zeatin. Addition of naphthaleneacetic acid (NAA) did not have a significant effect on shoot regeneration in all treatments. Shoot organogenesis was directly induced from petiole explants without intervening callus. Regenerated shoots were easily rooted on all tested media supplemented with 0.5 μM NAA. Rooted plants were transferred to potting mix and grown in the greenhouse.     

Growth of Metarhizium anisopliae on non-preferred carbon sources yields conidia with increased UV-B tolerance

Conidia of the insect-pathogenic fungus Metarhizium anisopliae var. anisopliae produced on different growth substrates (culture media or insect cadavers) demonstrate reproducibly altered tolerance to UV-B radiation [Rangel, D.E.N., Braga, G.U.L., Flint, S.D., Anderson, A.J., Roberts, D.W., 2004. Variations in UV-B tolerance and germination speed of M. anisopliae conidia produced on artificial and natural substrates. J. Invertebr. Pathol. 87, 77-83]. In the current study, the fungus was grown on potato dextrose agar with yeast extract (PDAY), on minimal medium [(MM)=Czapek medium without saccharose], or on MM with one of 16 different carbon sources. The conidia produced on these media were exposed to UV-B radiation. Great amplitude in phenotypic plasticity for UV-B tolerance was demonstrated, viz., conidia produced under nutritive stress [MM or MM supplemented with non-preferred carbon sources (e.g., fructose, galactose, lactose etc.)] had at least two times higher tolerance than conidia produced on the rich medium (PDAY). Endogenous trehalose and mannitol accumulated at least two times more in conidia produced on MM (or MM with lactose, a non-preferred carbon source), as compared to conidia from MM plus glucose. High accumulations of these two carbohydrates in fungal spores are known to protect them against a wide range of stresses. Sporulation, however, was most profuse on PDAY, second best on MM plus d-mannose and least on MM or MM containing non-preferred carbon sources. Taken together, the results illustrate that nutritive stress generated by MM or MM plus a non-preferred carbon source greatly improved UV-B tolerance, but reduced conidial yield; while, on the other hand, preferred carbon sources improved conidial yield, but reduced UV-B tolerance.     

Comparative proteomic analysis of alcoholic fermentation employing a new environmental strain of Saccharomyces cerevisiae

The aim of this study was to investigate the biochemical pathways induced during must fermentation employing the Saccharomyces cerevisiae, strain Z622, isolated from the traditional wine area of Zitsa (Epirus, Greece). Proteomic analysis (two-dimensional gel electrophoresis followed by nano-LC ESI-MS/MS) was used to assess the protein profile response of strain Z622 cultured in Debina grape must, yeast extract–malt extract (YM) or minimal medium (MM) under fermentation conditions, at the beginning and at the end of fermentation. The proteomic results and their analysis with Gene Ontology (GO) terms revealed that the intracellular proteins induced in yeast cells grown exclusively in Debina grape must during the early stage of fermentation were mainly involved in amino acid metabolism, glycolysis and sterol formation. These three pathways reflect the adaptation of the cell to the fermentative metabolism. During the late stage, several enzymes were induced due to starvation conditions or to participation in defence mechanisms against oxidative stress and protein degradation. Several of the identified enzymes have not previously been reported to be induced during wine fermentation and thus the results presented here can contribute to a better understanding of how S. cerevisiae cells adapt to wine fermentation.     

Proteomic analysis of bacterial-blight defense-responsive proteins in rice leaf blades

Plants exhibit resistance against incompatible pathogens, via localized and systemic responses as part of an integrated defense mechanism. To study the compatible and incompatible interactions between rice and bacteria, a proteomic approach was applied. Rice cv. Java 14 seedlings were inoculated with compatible (Xo7435) and incompatible (T7174) races of Xanthomonas oryzae pv. oryzae (Xoo). Cytosolic and membrane proteins were fractionated from the leaf blades and separated by 2-D PAGE. From 366 proteins analyzed, 20 were differentially expressed in response to bacterial inoculation. These proteins were categorized into classes related to energy (30%), metabolism (20%), and defense (20%). Among the 20 proteins, ribulose-1,5-bisphosphate carboxylase/oxygenase large subunit (RuBisCO LSU) was fragmented into two smaller proteins by T7174 and Xo7435 inoculation. Treatment with jasmonic acid (JA), a signaling molecule in plant defense responses, changed the level of protein accumulation for 5 of the 20 proteins. Thaumatin-like protein and probenazole-inducible protein (PBZ) were commonly up-regulated by T7174 and Xo7435 inoculation and JA treatment. These results suggest that synthesis of the defense-related thaumatin-like protein and PBZ are stimulated by JA in the defense response pathway of rice against bacterial blight.     

Initiation, Growth and Nuclear Characteristics of Tissue Cultures of Paeonia suffruticosa

Tissue cultures of the garden paeony, Paeonia suffruticosa have been established using explants of etiolated stems. Callus formation was induced on agar-solidified media containing ammonium ions or amino acids together with the hormones 2,4-dichlorophenoxyacetic acid and kinetin, but not on media lacking the reduced nitrogen component. Attempts to induce callus from explants from green plants were completely unsuccessful and were characterized by the production of intense brown colorations, both of the explant and the medium. Subcultured tissue without the added hormones produced roots, both on solid and liquid media. Growth was tested on a range of liquid media, SH/2, SH, SH × 2 and SH—M, containing 1250, 2500, 5000 and 2500 mg/l potassium nitrate. The SH—M medium also contained 1650 mg/l ammonium nitrate. Growth measured as increased fresh weight was best in the SH/2, SH and SH—M media and was curtailed in the SH × 2 medium. Soluble protein content was highest at the lowest nitrogen concentration. A histochemical comparison of tissue grown on the SH/2, SH—M and SH—M lacking hormones showed that the cells in all the cultures remained diploid while differences were established in total nuclear protein, measured using the ninhydrin-Schiff procedure. Nuclei from SH—M grown cells have a higher protein content than those from the SH/2 medium while cells from the SH—M medium lacking hormones show a further increase in nuclear protein. This raises the question whether this change in nuclear protein is related to the morphogenesis of roots which occurs in this medium.