Anaerobic degradation of p-ethylphenol by "Aromatoleum aromaticum" strain EbN1: pathway, regulation, and involved proteins

The denitrifying “Aromatoleum aromaticum” strain EbN1 was demonstrated to utilize p-ethylphenol under anoxic conditions and was suggested to employ a degradation pathway which is reminiscent of known anaerobic ethylbenzene degradation in the same bacterium: initial hydroxylation of p-ethylphenol to 1-(4-hydroxyphenyl)-ethanol followed by dehydrogenation to p-hydroxyacetophenone. Possibly, subsequent carboxylation and thiolytic cleavage yield p-hydroxybenzoyl-coenzyme A (CoA), which is channeled into the central benzoyl-CoA pathway. Substrate-specific formation of three of the four proposed intermediates was confirmed by gas chromatographic-mass spectrometric analysis and also by applying deuterated p-ethylphenol. Proteins suggested to be involved in this degradation pathway are encoded in a single large operon-like structure (~15 kb). Among them are a p-cresol methylhydroxylase-like protein (PchCF), two predicted alcohol dehydrogenases (ChnA and EbA309), a biotin-dependent carboxylase (XccABC), and a thiolase (TioL). Proteomic analysis (two-dimensional difference gel electrophoresis) revealed their specific and coordinated upregulation in cells adapted to anaerobic growth with p-ethylphenol and p-hydroxyacetophenone (e.g., PchF up to 29-fold). Coregulated proteins of currently unknown function (e.g., EbA329) are possibly involved in p-ethylphenol- and p-hydroxyacetophenone-specific solvent stress responses and related to other aromatic solvent-induced proteins of strain EbN1.     

Expression of "Dehydrin-Like" Proteins in Embryos and Seedlings of Zizania palustris and Oryza sativa during Dehydration

Proteins inducible by dehydration and abscisic acid (ABA), termed dehydrins or RAB (Responsive to ABA) proteins, have been identified in a number of species and have been suggested to play a role in desiccation tolerance, particularly during seed development. Seeds (caryopses) of North American wild rice (Zizania palustris var interior [Fassett] Dore) are tolerant of dehydration to <10% moisture content (fresh weight basis) only under restricted dehydration and rehydration conditions. In comparison, seeds of paddy rice (Oryza sativa L.) readily tolerate desiccation to <5% water content. Expression of “dehydrin-like” proteins in Zizania and Oryza seedlings and embryos was examined to investigate the relationship between the presence of such proteins and desiccation tolerance. [³⁵S]Methionine labeling of newly synthesized proteins showed that seedlings (first leaf stage) of both Zizania and Oryza synthesized a novel “heat-stable” protein of apparent molecular weight = 20,000 when dehydrated to <75% of their initial fresh weight. ABA (100 micromolar) induced synthesis of a protein with similar electrophoretic mobility in both species. Western blots using antiserum raised against maize (Zea mays L.) dehydrin detected a protein band from dehydrated Zizania shoots and mature embryonic axes that comigrated with the labeled 20-kilodalton polypeptide. Northern blots using a cDNA for an ABA-responsive protein from Oryza (rab 16a) showed that both seedlings and excised embryonic axes of Zizania accumulated RNA similar in sequence to rab 16a in response to water loss. Zizania seedlings and embryonic axes were also capable of ABA accumulation during dehydration. The intolerance of Zizania seeds to dehydration at low temperature is apparently not due to an absence of dehydrin-like proteins or an inability to accumulate ABA.     

Seed protein fraction electrophoresis in peanut (<Emphasis Type="Italic">Arachis hypogaea</Emphasis> L.) accessions and wild species

Total seed storage proteins were studied in 50 accessions of A. hypogaea (11 A. hypogaea ssp. hypogaea var hypogaea, 13 A. hypogaea ssp. hypogaea var hirsuta, 11 A. hypogaea ssp. fastigiata var fastigiata and 15 A. hypogaea ssp. fastigiata var. vulgaris accessions) in SDS PAGE. These accessions were also analysed for albumin and globulin seed protein fractions. Among the six seed protein markers presently used, it was found that globulin fraction showed maximum diversity (77.2%) in A. hypogaea accessions followed by albumin (52.3%), denatured total soluble protein fraction in embryo (33.3%) and cotyledon (28.5%). The cluster analysis based on combined data of cotyledons, embryos, albumins and globulins seed protein fractions demarcated the accessions of two subspecies hypogaea and fastigiata into two separate clusters supported by 51% bootstrap value, with few exceptions, suggesting the genotypes to be moderately diverse. Native and denatured total soluble seed storage proteins were also electrophoretically analysed in 27 wild Arachis species belonging to six sections of the genus. Cluster analysis using different methods were performed for different seed proteins data alone and also in combination. Section Caulorrhizae (C genome) and Triseminatae (T genome) formed one, distantly related group to A. hypogaea and other section Arachis species in the dendrogram based on denatured seed storage proteins data. The present analysis has maintained that the section Arachis species belong to primary and secondary genepools and, sections Procumbenetes and Erectoides belong to tertiary gene pools.     

Ability of extracellular proteinases of micromycetes <Emphasis Type="Italic">Aspergillus flavipes</Emphasis>, <Emphasis Type="Italic">Aspergillus fumigatus</Emphasis>, and <Emphasis Type="Italic">Aspergillus sydowii</Emphasis> to affect proteins of the human haemostatic system

The effect of extracellular proteinases of A. flavipes A17, A. fumigatus D1, and A. sydowii 1 on proteins of the human haemostasis system was studied. It was shown that A. fumigatus D1 proteinases are able to hydrolyze a wide range of chromogenic peptide substrates of specific human proteinases of the haemostatic system. Proteinases formed by A. flavipes A17 and A. sydowii 1 have a narrow specificity, mainly to thrombin and plasmin substrates. It was first shown that proteinase of A. flavipes A17 is capable to activate protein C and Factor X. Extracellular proteinase produced by A. sydowii 1 has greater fibrinolytic activity as compared with proteinases produced by A. flavipes A17 and A. fumigatus D1.     

<Emphasis Type="Italic">Deinococcus radiodurans</Emphasis> RecX and <Emphasis Type="Italic">Escherichia coli</Emphasis> RecX proteins are capable to replace each other in vivo and in vitro

A plasmid carrying the Deinococcus radiodurans recX gene under the control of a lactose promoter decreases the Escherichia coli cell resistance to UV irradiation and γ irradiation and also influences the conjugational recombination process. The D. radiodurans RecX protein functions in the Escherichia coli cells similarly to the E. coli RecX protein. Isolated and purified D. radiodurans RecX and E. coli RecX proteins are able to replace each other interacting with the E. coli RecA and D. radiodurans RecA proteins in vitro. Data obtained demonstrated that regulatory interaction of RecA and RecX proteins preserves a high degree of conservatism despite all the differences in the recombination reparation system between E. coli and D. radiodurans.     

Salinity induced changes in the chloroplast proteome of the aquatic pteridophyte <Emphasis Type="Italic">Azolla microphylla</Emphasis>

The growth of the nitrogen fixing aquatic pteridophyte Azolla microphylla is severely affected by salinity. Salinity exposure (0.5%) resulted in significant reduction in chlorophyll a and b content, altered chl a/b ratio and photosynthetic efficiency (Fv/Fm). Chloroplasts maintain photosynthesis but are highly sensitive to salinity stress. Chloroplast proteins extracted from A. microphylla was separated by two-dimensional electrophoresis (2DE) and approximately 200 proteins were observed on each gel. Forty two differentially expressed protein spots were detected and out of this 17 could be identified through MALDI-TOF-MS/MS analysis. Out of the 17 identified proteins, 15 were found to be down regulated and 2 proteins were up regulated. Most of the down regulated proteins were associated with Calvin cycle, ATP synthesis, oxygen evolution, photosystem I and ROS scavenging. The results show changes in proteome dynamics of the chloroplasts of A. microphylla and such changes may lead to reduction in growth and metabolism. The primary target of salinity in A. microphylla is photosynthesis and the changes in the proteome dynamics of the chloroplasts lead to reduced growth.     

Using S-adenosyl-l-homocysteine capture compounds to characterize S-adenosyl-l-methionine and S-adenosyl-l-homocysteine binding proteins

S-Adenosyl-l-methionine (SAM) is recognized as an important cofactor in a variety of biochemical reactions. As more proteins and pathways that require SAM are discovered, it is important to establish a method to quickly identify and characterize SAM binding proteins. The affinity of S-adenosyl-l-homocysteine (SAH) for SAM binding proteins was used to design two SAH-derived capture compounds (CCs). We demonstrate interactions of the proteins COMT and SAHH with SAH–CC with biotin used in conjunction with streptavidin–horseradish peroxidase. After demonstrating SAH-dependent photo-crosslinking of the CC to these proteins, we used a CC labeled with a fluorescein tag to measure binding affinity via fluorescence anisotropy. We then used this approach to show and characterize binding of SAM to the PR domain of PRDM2, a lysine methyltransferase with putative tumor suppressor activity. We calculated the K_d values for COMT, SAHH, and PRDM2 (24.1 ± 2.2 μM, 6.0 ± 2.9 μM, and 10.06 ± 2.87 μM, respectively) and found them to be close to previously established K_d values of other SAM binding proteins. Here, we present new methods to discover and characterize SAM and SAH binding proteins using fluorescent CCs.     

Comparative muscle proteomics/phosphoproteomics analysis provides new insight for the biosafety evaluation of <Emphasis Type="Italic">fat</Emphasis>-<Emphasis Type="Italic">1</Emphasis> transgenic cattle

The biosafety of fat-1 transgenic cattle has been a focus of our studies since the first fat-1 transgenic cow was born. In this study, we used tandem mass tag labeling, TiO₂ enrichment, and nanoscale liquid chromatography coupled with tandem mass spectrometry (nanol LC–MS/MS) to compare proteomic and phosphoproteomic profiling analyses of muscle between fat-1 transgenic cows and wild-type cows. A total of 1555 proteins and 900 phosphorylation sites in 159 phosphoproteins were identified in the profiling assessments, but only four differentially expressed proteins and nine differentially expressed phosphopeptides were detected in fat-1 transgenic cows relative to wild-type cows. Bioinformatics analyses showed that all of the identified proteins and phosphoproteins were mainly related to the metabolic processes of three major nutrients: carbohydrates, lipids, and proteins. All of these differentially expressed proteins might take part in DNA recombination, repair, and regulation of the immune system. In conclusion, most of the identified proteins and phosphoproteins exhibited few changes. Our results provide new insights into the biosafety of fat-1 transgenic cattle.     

Isolation of the sodium-dependent d-glucose transport protein from brush-border membranes

Rabbit kidney brush-border membrane vesicles were exposed to bacterial protease which cleaves off a large number of externally oriented proteins. Na⁺-dependent d-glucose transport is left intact in the protease-treated vesicles. The protease-treated membrane was solubilized with deoxycholate and the deoxycholate-extracted proteins were further resolved by passage through Con A-Sepharose columns. Sodium-dependent d-glucose activity was found to reside in a fraction containing a single protein band of M_r ? 165000 which is apparently a dimer of M_r ? 85 000. When reconstituted and tested for transport, this protein showed Na⁺-dependent, stereo-specific and phlorizin-inhibitable glucose transport. Transport activity is completely recovered and is 20-fold increased in specific activity. A similar isolate was obtained from rabbit small intestinal brush-border membranes and kidneys from several other species of animals.     

Synergism and Antagonism between Bacillus thuringiensis Vip3A and Cry1 Proteins in Heliothis virescens,Diatraea saccharalis and Spodoptera frugiperda

Second generation Bt crops (insect resistant crops carrying Bacillus thuringiensis genes) combine more than one gene that codes for insecticidal proteins in the same plant to provide better control of agricultural pests. Some of the new combinations involve co-expression of cry and vip genes. Because Cry and Vip proteins have different midgut targets and possibly different mechanisms of toxicity, it is important to evaluate possible synergistic or antagonistic interactions between these two classes of toxins. Three members of the Cry1 class of proteins and three from the Vip3A class were tested against Heliothis virescens for possible interactions. At the level of LC₅₀, Cry1Ac was the most active protein, whereas the rest of proteins tested were similarly active. However, at the level of LC₉₀, Cry1Aa and Cry1Ca were the least active proteins, and Cry1Ac and Vip3A proteins were not significantly different. Under the experimental conditions used in this study, we found an antagonistic effect of Cry1Ca with the three Vip3A proteins. The interaction between Cry1Ca and Vip3Aa was also tested on two other species of Lepidoptera. Whereas antagonism was observed in Spodoptera frugiperda, synergism was found in Diatraea saccharalis. In all cases, the interaction between Vip3A and Cry1 proteins was more evident at the LC₉₀ level than at the LC₅₀ level. The fact that the same combination of proteins may result in a synergistic or an antagonistic interaction may be an indication that there are different types of interactions within the host, depending on the insect species tested.     

In-depth proteomic analyses of <Emphasis Type="Italic">Haliotis laevigata</Emphasis> (greenlip abalone) nacre and prismatic organic shell matrix

Background

The shells of various Haliotis species have served as models of invertebrate biomineralization and physical shell properties for more than 20 years. A focus of this research has been the nacreous inner layer of the shell with its conspicuous arrangement of aragonite platelets, resembling in cross-section a brick-and-mortar wall. In comparison, the outer, less stable, calcitic prismatic layer has received much less attention. One of the first molluscan shell proteins to be characterized at the molecular level was Lustrin A, a component of the nacreous organic matrix of Haliotis rufescens. This was soon followed by the C-type lectin perlucin and the growth factor-binding perlustrin, both isolated from H. laevigata nacre, and the crystal growth-modulating AP7 and AP24, isolated from H. rufescens nacre. Mass spectrometry-based proteomics was subsequently applied to to Haliotis biomineralization research with the analysis of the H. asinina shell matrix and yielded 14 different shell-associated proteins. That study was the most comprehensive for a Haliotis species to date.

Methods

The shell proteomes of nacre and prismatic layer of the marine gastropod Haliotis laevigata were analyzed combining mass spectrometry-based proteomics and next generation sequencing.

Results

We identified 297 proteins from the nacreous shell layer and 350 proteins from the prismatic shell layer from the green lip abalone H. laevigata. Considering the overlap between the two sets we identified a total of 448 proteins. Fifty-one nacre proteins and 43 prismatic layer proteins were defined as major proteins based on their abundance at more than 0.2% of the total. The remaining proteins occurred at low abundance and may not play any significant role in shell fabrication. The overlap of major proteins between the two shell layers was 17, amounting to a total of 77 major proteins.

Conclusions

The H. laevigata shell proteome shares moderate sequence similarity at the protein level with other gastropod, bivalve and more distantly related invertebrate biomineralising proteomes. Features conserved in H. laevigata and other molluscan shell proteomes include short repetitive sequences of low complexity predicted to lack intrinsic three-dimensional structure, and domains such as tyrosinase, chitin-binding, and carbonic anhydrase. This catalogue of H. laevigata shell proteins represents the most comprehensive for a haliotid and should support future efforts to elucidate the molecular mechanisms of shell assembly.

     

Occurrence in vivo of "hidden breaks" at specific sites of 26 S ribosomal RNA of Musca carnaria.

A fragmentation process occurs in 26 S ribosomal RNA of mature cytoplasmic ribosomes of Musca carnaria. It consists of the sequential appearance of three “hidden breaks” that fragment 26 S rRNA (M_r = 1.42 × 10⁶) into four pieces with approximate molecular weights of 0.68 × 10⁶, 0.35 × 10⁶, 0.29 × 10⁶ and 0.096 × 10⁶, respectively. This fragmentation was not observed in 17 S rRNA (M_r = 0.74 × 10⁶).Extremely mild treatment of newly assembled ribosomes with pancreatic RNAase reproduces the 26 S rRNA fragmentation phenomenon in vitro in the same way as it occurs in vivo.This evidence is discussed in relation to the secondary structure of 26 S rRNA and its binding with specific ribosomal proteins.     

Characterization of a <Emphasis Type="Italic">Pinus sylvestris</Emphasis> thaumatin-like protein gene and determination of antimicrobial activity of the in vitro expressed protein

Thaumatin-like proteins (TLPs) are pathogenesis-related proteins, which are involved in plant defense responses to pathogen infection. Expression of the Pinus sylvestris L. TLP gene is up-regulated by methyl jasmonate treatment and inoculation with Heterobasidion annosum. A full-length Pinus taeda TLP gene sequence was used to design PCR primers for amplification of the full-length TLP gene from P. sylvestris. A putative 705-bp open reading frame of TLP gene was cloned into Escherichia coli cells, and then subcloned into the overexpression vector pET100 using BL21 Star expression bacteria. Optimization of the expression of recombinant TLP was achieved by decreasing both expression temperature and IPTG concentration. The purified 24.6-kDa TLP shows antimicrobial activity against 12 fungal species.     

Characterization of novel 2-oxoglutarate dependent dioxygenases converting l-proline to cis-4-hydroxy-l-proline

Hydroxyprolines are valuable chiral building blocks for organic synthesis of pharmaceuticals. Several microorganisms producing l-proline trans-4- and cis-3-hydroxylase were discovered and these enzymes were applied to the industrial production of trans-4- and cis-3-hydroxy-l-proline, respectively. Meanwhile, other hydroxyproline isomers, cis-4- and trans-3-hydroxy-l-proline, were not easily available because the corresponding hydroxylase have not been discovered. Herein we report novel l-proline cis-4-hydroxylases converting free l-proline to cis-4-hydroxy-l-proline. Two genes encoding uncharacterized proteins from Mesorhizobium loti and Sinorhizobium meliloti were cloned and overexpressed in Escherichia coli, respectively. The functions of purified proteins were investigated in detail, and consequently we detected l-proline cis-4-hydroxylase activity in both proteins. Likewise l-proline trans-4-, cis-3-hydroxylase and prolyl hydroxylase, these enzymes belonged to a 2-oxoglutarate dependent dioxygenase family and required a non-heme ferrous ion. Although their reaction mechanisms were similar to other hydroxylases, the amino acid sequence homology was not observed (less than 40%).     

Comparative proteomic analysis of leaf tissue from tomato plants colonized with <Emphasis Type="Italic">Rhizophagus irregularis</Emphasis>

A comparative proteomic approach was performed to analyze the differential accumulation of leaf proteins in response to the symbiosis between Solanum lycopersicum and the arbuscular mycorrhizal fungus (AMF) Rhizophagus irregularis. Protein profiling was examined in leaves from tomato plants colonized with AMF (M), as well as non-colonized plants fertilized with low phosphate (20 μM P; NM-LP) and non-colonized plants fertilized with regular phosphate Hoagland’s solution (200 μM P; NM-RP). Comparisons were made between these groups, and 2D-SDS-PAGE revealed that 27 spots were differentially accumulated in M vs. NM-LP. Twenty-three out of the 27 spots were successfully identified by mass spectrometry. Two of these proteins, 2-methylene-furan-3-one reductase and auxin-binding protein ABP19a, were up-accumulated in M plants. The down-accumulated proteins in M plants were associated mainly with photosynthesis, redox, and other molecular functions. Superoxide dismutase, harpin binding protein, and thioredoxin peroxidase were down-accumulated in leaves of M tomato plants when compared to NM-LP and NM-RP, indicating that these proteins are responsive to AMF colonization independently of the phosphate regime under which they were grown. 14-3-3 protein was up-accumulated in NM-RP vs. NM-LP plants, whereas it was down-accumulated in M vs. NM-LP and M vs. NM-RP, regardless of their phosphate nutrition. This suggests a possible regulation by P nutrition and AMF colonization. Our results demonstrate AMF-induced systemic changes in the expression of tomato leaf proteins, including the down-accumulation of proteins related to photosynthesis and redox function.     

The "cytochrome b5 fold": structure of a novel protein superfamily

Selective proteolysis allows the isolation of a heme-binding fragment spectrally similar to microsomal cytochrome b₅ from both baker's yeast flavocytochrome b₂ (a flavohemoprotein) and liver sulfite oxidase (a molybdoprotein). The amino acid sequences of these two fragments have been published separately (Guiard &; Lederer, 1976,1979). We present in this paper an alignment of those sequences with that of microsomal cytochrome b₅. The structural consequences of the similarity between the three primary structures are discussed in the light of the cytochrome b₅ three-dimensional model (Mathews et al., 1971,1972,1975; Mathews &; Czerwinski, 1976).It is concluded that the three heme-binding proteins are in all probability the products of a divergent evolution from a common ancestor and that they must present a basically similar backbone with some surface alterations. We propose to name this backbone the “cytochrome b₅ fold”. The comparison of the three proteins suggests hypotheses concerning the molecular surface areas involved in the recognition of cytochrome c (the common acceptor) and of the respective reductase (flavo- or molybdoprotein).In addition, our results suggest that at some point in evolution, several copies of an initial hemoprotein gene were formed in the cellular genome. Subsequently, one copy was fused with the gene for another function: a flavoreductase in yeast cells or a molybdoreductase in hepatic cells.     

Comparative analysis of diploid species of <Emphasis Type="Italic">Avena</Emphasis> L. using cytogenetic and biochemical markers: <Emphasis Type="Italic">Avena canariensis</Emphasis> Baum et Fedak and <Emphasis Type="Italic">A. longiglumis</Emphasis> Dur.

The diploid oat species containing the A genome of two types (Al and Ac) were studied by electrophoresis of grain storage proteins (avenins), chromosome C-banding, and in situ hybridization with probes pTa71 and pTa794. The karyotypes of the studied species displayed similar C-banding patterns but differed in size and morphology of several chromosomes, presumably, resulting from structural rearrangements that took place during the divergence of A genomes from a common ancestor. In situ hybridization demonstrated an identical location of the 45S and 5S rRNA gene loci in Avena canariensis and A. longiglumis similar to that in the A. strigosa genome. However, the 5S rDNA locus in A. longiglumis (5S rDNA1) was considerably decreased in the chromosome 3Al long arm. The analysis demonstrated that these oat species were similar in the avenin component composition, although individual accessions differed in the electrophoretic mobilities of certain components. A considerable similarity of A. canariensis and A. longiglumis to the Avena diploid species carrying the As genome variant was demonstrated.