Comparative proteomics using 2-D gel electrophoresis and mass spectrometry as tools to dissect stimulons and regulons in bacteria with sequenced or partially sequenced genomes

We propose two-dimensional gel electrophoresis (2-DE) and mass spectrometry to define the protein components of regulons and stimulons in bacteria, including those organisms where genome sequencing is still in progress. The basic 2-DE protocol allows high resolution and reproducibility and enables the direct comparison of hundreds or even thousands of proteins simultaneously. To identify proteins that comprise stimulons and regulons, peptide mass fingerprint (PMF) with matrix-assisted laser desorption ionization/time-of-flight mass spectrometry (MALDI-TOF-MS) analysis is the first option and, if results from this tool are insufficient, complementary data obtained with electrospray ionization tandem-MS (ESI-MS/MS) may permit successful protein identification. ESI-MS/MS and MALDI-TOF-MS provide complementary data sets, and so a more comprehensive coverage of a proteome can be obtained using both techniques with the same sample, especially when few sequenced proteins of a particular organism exist or genome sequencing is still in progress.     

Interactive effects of high stocking density and food deprivation on carbohydrate metabolism in several tissues of gilthead sea bream Sparus auratus

The influence of high stocking density (HSD) and food deprivation was assessed on carbohydrate metabolism of several tissues of gilthead sea bream Sparus auratus for 14 days. Fish were randomly assigned to one of four treatments: (1) fed fish under normal stocking density (NSD) (4 kg m(-3)); (2) fed fish under HSD (70 kg m(-3)); (3) food-deprived fish under NSD; and (4) food-deprived fish under HSD. After 14 days, samples were taken from the plasma, liver, gills, kidney and brain for the assessment of plasma cortisol, levels of metabolites and the activity of several enzymes involved in carbohydrate metabolism. HSD conditions alone elicited important changes in energy metabolism of several tissues that in some cases were confirmatory (5-fold increase in plama cortisol, 20% increase in plasma glucose, 60% decrease in liver glycogen and 20% increase in gluconeogenic potential in the liver) whereas in others provided new information regarding metabolic adjustments to cope with HSD in the liver (100% increase in glucose phosphorylating capacity), gills (30% decrease in capacity for phosphorylating glucose), kidney (80% increase in the capacity of phosphorylating glucose) and brain (2.5-fold increase in ATP levels). On the other hand, food deprivation alone resulted in increased plasma cortisol, and metabolic changes in the liver (enhanced gluconeogenic and glycogenolytic potential of 13% and 18%, respectively) and brain (10% increase in glycolytic capacity), confirmatory of previous studies, whereas new information regarding metabolic adjustments during food deprivation was obtained in the gills and kidney (decreased lactate levels in both tissues of 45% and 55%, respectively). Furthermore, the results obtained provided, for the first time in fish, information indicating that food deprivation increased the sensitivity of gilthead sea bream to the stress induced by HSD compared with the fed controls, as demonstrated by increased plasma cortisol levels (50% increase vs. fed fish) and a further increase in the capacity to export glucose mobilized from liver glycogen stores (70% decrease vs. fed fish). These results lend support for a cumulative effect of both stressors on plasma cortisol and parameters assessed on carbohydrate metabolism in the present experiments, and provide information regarding reallocation of metabolic energy to cope with simultaneous stressors in fish.     

Proteomic analysis of the Gallus gallus embryo at stage-29 of development

The chicken (Gallus gallus) is one of the primary models for embryological and developmental studies. In order to begin to understand the molecular mechanisms underlying the normal and abnormal development of the chicken, we used 2-DE to construct a whole-embryo proteome map. Proteins were separated by IEF on IPG strips, and by 11% SDS-PAGE) gels. Protein identification was performed by means of PMF with MALDI-TOF-MS. In all, 105 protein spots were identified, 35 of them implicated in embryo development, 10 related with some diseases, and 16, finally, being proteins that have never been identified, purified or characterized in the chicken before. This map will be updated continuously and will serve as a reference database for investigators, studying changes at the protein level under different physiological conditions.     

Functional analysis of two divalent metal-dependent regulatory genes dmdR1 and dmdR2 in Streptomyces coelicolor and proteome changes in deletion mutants

In Gram-positive bacteria, the expression of iron-regulated genes is mediated by a class of divalent metal-dependent regulatory (DmdR) proteins. We cloned and characterized two dmdR genes of Streptomyces coelicolor that were located in two different nonoverlapping cosmids. Functional analysis of dmdR1 and dmdR2 was performed by deletion of each copy. Deletion of dmdR1 resulted in the derepression of at least eight proteins and in the repression of three others, as shown by 2D proteome analysis. These 11 proteins were characterized by MALDI-TOF peptide mass fingerprinting. The proteins that show an increased level in the mutant correspond to a DNA-binding hemoprotein, iron-metabolism proteins and several divalent metal-regulated enzymes. The levels of two other proteins--a superoxide dismutase and a specific glutamatic dehydrogenase--were found to decrease in this mutant. Complementation of the dmdR1-deletion mutant with the wild-type dmdR1 allele restored the normal proteome profile. By contrast, deletion of dmdR2 did not affect significantly the protein profile of S. coelicolor. One of the proteins (P1, a phosphatidylethanolamine-binding protein), overexpressed in the dmdR1-deleted mutant, is encoded by ORF3 located immediately upstream of dmdR2; expression of both ORF3 and dmdR2 is negatively controlled by DmdR1. Western blot analysis confirmed that dmdR2 is only expressed when dmdR1 is disrupted. Species of Streptomyces have evolved an elaborated regulatory mechanism mediated by the DmdR proteins to control the expression of divalent metal-regulated genes.     

Production and characterization of a noncytotoxic deletion variant of the Aspergillus fumigatus allergen Aspf1 displaying reduced IgE binding

Aspergillus fumigatus is responsible for many allergic respiratory diseases, the most notable of which - due to its severity - is allergic bronchopulmonary aspergillosis. Aspf1 is a major allergen of this fungus: this 149-amino acid protein belongs to the ribotoxin family, whose best characterized member is alpha-sarcin (EC 3.1.27.10). The proteins of this group are cytotoxic ribonucleases that degrade a unique bond in ribosomal RNA impairing protein biosynthesis. Aspf1 and its deletion mutant Aspf1Delta(7-22) have been produced as recombinant proteins; the deleted region corresponds to an exposed beta-hairpin. The conformation of these two proteins has been studied by CD and fluorescence spectroscopy. Their enzymatic activity and cytotoxicity against human rhabdomyosarcoma cells was also measured and their allergenic properties have been studied by using 58 individual sera of patients sensitized to Aspergillus. Aspf1Delta(7-22) lacks cytotoxicity and shows a remarkably reduced IgE reactivity. From these studies it can be concluded that the deleted beta-hairpin is involved in ribosome recognition and is a significant allergenic region.     

Gene conversion tracts associated with crossovers in Rhizobium etli

Gene conversion has been defined as the nonreciprocal transfer of information between homologous sequences. Despite its broad interest for genome evolution, the occurrence of this mechanism in bacteria has been difficult to ascertain due to the possible occurrence of multiple crossover events that would mimic gene conversion. In this work, we employ a novel system, based on cointegrate formation, to isolate gene conversion events associated with crossovers in the nitrogen-fixing bacterium Rhizobium etli. In this system, selection is applied only for cointegrate formation, with gene conversions being detected as unselected events. This minimizes the likelihood of multiple crossovers. To track the extent and architecture of gene conversions, evenly spaced nucleotide changes were made in one of the nitrogenase structural genes (nifH), introducing unique sites for different restriction endonucleases. Our results show that (i) crossover events were almost invariably accompanied by a gene conversion event occurring nearby; (ii) gene conversion events ranged in size from 150 bp to 800 bp; (iii) gene conversion events displayed a strong bias, favoring the preservation of incoming sequences; (iv) even small amounts of sequence divergence had a strong effect on recombination frequency; and (v) the MutS mismatch repair system plays an important role in determining the length of gene conversion segments. A detailed analysis of the architecture of the conversion events suggests that multiple crossovers are an unlikely alternative for their generation. Our results are better explained as the product of true gene conversions occurring under the double-strand break repair model for recombination.     

Polymorphism and double hexamer structure in the archaeal minichromosome maintenance (MCM) helicase from Methanobacterium thermoautotrophicum

Methanobacterium thermoautotrophicum minichromosome maintenance complex (mtMCM), a cellular replicative helicase, is a useful model for the more complex eukaryotic MCMs. Biochemical and crystallographic evidence indicates that mtMCM assembles as a double hexamer (dHex), but previous electron microscopy studies reported only the presence of single heptamers or single hexamers (Pape, T., Meka, H., Chen, S., Vicentini, G., Van Heel, M., and Onesti, S. (2003) EMBO Rep. 4, 1079-1083; Yu, X., VanLoock, M. S., Poplawski, A., Kelman, Z., Xiang, T., Tye, B. K., and Egelman, E. H. (2002) EMBO Rep. 3, 792-797). Here we present the first three-dimensional electron microscopy reconstruction of the full-length mtMCM dHex in which two hexamers contact each other via the structurally well defined N-terminal domains. The dHex has obvious side openings that resemble the side channels of LTag (large T antigen). 6-fold and 7-fold rings were observed in the same mtMCM preparation, but we determined that assembly as a double ring favors 6-fold structures. Additionally, open rings were also detected, which suggests a direct mtMCM loading mechanism onto DNA.     

Expression of apolipoprotein AI mRNA in peripheral white blood cells of patients with alcoholic liver disease

Because (i) changes in plasma and liver mRNA of apolipoprotein (apo) AI have been observed in patients with alcoholic liver disease, (ii) apo AI mRNA can be induced in non-hepatic tissues, and (iii) apolipoproteins expression is influenced by plasma colloid osmotic pressure (P(CO)) and viscosity (eta), we analyzed the Apo AI mRNA expression in the peripheral white blood cells (PWBC), P(CO), and eta in control volunteers (C), patients with liver cirrhosis (LC), and cirrhotic patients with superimposed alcoholic hepatitis (LC+AH). We found that apo AI mRNA is expressed in the PWBC in 20% of C and it is induced 1.5 fold in 66.6% of LC and 1.95 fold in 85% of LC+AH. A significant decrease of P(CO) in LC and LC + AH (14.8 +/- 2.4 and 16.2 +/- 2.4 mm Hg, respectively) compared to C (27.9 +/- 2 mm Hg) was observed. By contrast, eta was mildly increased from 1.7389 +/- 0.07 in C to 1.8022 +/- 0.154 in LC and 1.9030 +/- 0.177 in LC+AH. No significant correlation was found between P(CO) and eta with apo AI mRNA but with lipid profile. In conclusion, apo AI mRNA expression in PWBC is associated to liver disease severity and could be an indirect indicator of alcoholic liver damage.     

A novel protease from Entamoeba histolytica homologous to members of the family S28 of serine proteases

Serine proteases are one of the biologically most important and widely distributed enzyme families. A protease capable of degrading the substrate Suc-AAF-AMC was isolated from axenically grown trophozoites of Entamoeba histolytica. The enzyme was purified by ion-exchange chromatography and electroelution, and appeared on 2D-PAGE as a spot of 60 kDa and pI of 4.65. Data obtained from zymogram suggest the active protease is present either as homodimer (130 kDa) or homotetramer (250 kDa). The optimal temperature of the enzyme was 37 degrees C, and it exhibited activity over a broad pH range. The protease was strongly inhibited by TPCK and chelating agents. The enzymatic activity was restored upon addition of calcium. BLAST analysis with the sequence of internal peptides of the protein revealed two open reading frames within the genome of E. histolytica, homologous to members of the family S28, clan SC of serine proteases.