The incorporation of photoproteins into proteins of interest allows the study of either their localization or intermolecular interactions in the cell. Here we demonstrate the possibility of in vivo incorporating the photoprotein Aequorea victoria enhanced green fluorescent protein (EGFP) or Gaussia princeps luciferase (GLuc) into the tetradecameric quaternary structure of GroEL chaperonin and describe some physicochemical properties of the labeled chaperonin. Using size-exclusion and affinity chromatography, electrophoresis, fluorescent and electron transmission microscopy (ETM), small-angle X-ray scattering (SAXS), and bioluminescence resonance energy transfer (BRET), we show the following: (i) The GroEL14-EGFP is evenly distributed within normally divided E. coli cells, while gigantic undivided cells are characterized by the uneven distribution of the labeled GroEL14 which is mainly localized close to the cellular periplasm; (ii) EGFP and likely GLuc are located within the inner cavity of one of the two GroEL chaperonin rings and do not essentially influence the protein oligomeric structure; (iii) GroEL14 containing either EGFP or GLuc is capable of interacting with non-native proteins and the cochaperonin GroES.

Bioluminescence resonance energy transfer (BRET) seems to be a promising biophysical technique to study protein–protein interactions within living cells due to a very specific reaction of bioluminescence that essentially decreases the background of other cellular components and light-induced destruction of biomacromolecules. An important direction of the development of this technique is the study of known strong protein–protein complexes in vivo and the estimation of an average distance between chromophores of the donor and acceptor. Here, we demonstrate an in vivo interaction between barnase fused with luciferase (from Renilla reniformis, RLuc) and barstar fused with EGFP (enhanced green fluorescent protein of Aequorea victoria) monitored by BRET. The distance between the luciferase and EGFP chromophores within the complex has been evaluated as equal to (56 ± 2) Å.

A new series of heat-stable (st) mutants of bacteriophage T5, which contains deletions in the tRNA gene region, has been isolated. An accurate mapping of the deletion boundaries for more than 30 mutants of phage T5 has been carried out. As a result of the analysis of nucleotide sequences flanking the deleted regions in wild-type phage DNA, it has been shown that they all contain short, direct repeats of different lengths (2-35 nucleotide residues), and that only one repetition is retained in the mutant phage DNA. On the basis of the obtained results, it was suggested that deletion mutants of the phage T5 are formed as a result of illegal recombination occurring with the participation of short repeats in DNA (SHDIR). Based on the example of two mutants, it has been shown that the resistance to thermal inactivation depends on the size of the deleted region.

We report the complete genome sequencing of two Escherichia coli T5-related bacteriophages, DT57C and DT571/2, isolated from the same specimen of horse feces. These two isolates share 96% nucleotide sequence identity and can thus be considered representatives of the same novel species within the genus T5likevirus. The observed variation in the ltfA gene of these phages, resulting from a recent recombination event, may explain the observed host-range differences, suggesting that a modular mechanism makes a significant contribution to the short-term evolution (or adaptation) of T5-like phage genomes in the intestinal ecosystem. Comparison of our isolates to their closest relative, coliphage T5, revealed high overall synteny of the genomes and high conservation of the sequences of almost all structural proteins as well as of the other proteins with identified functions. At the same time, numerous alterations and non-orthologous replacements of non-structural protein genes (mostly of those with unknown functions) as well as substantial differences in tail fiber locus organization support the conclusion that DT57C and DT571/2 form a species-level group clearly distinct from bacteriophage T5.

The Complex World of Polysaccharides 100 (reviewed by [9]).EPS refers to extracellular polysaccharides with slight or no cell association [9][10][11].Among the above mentioned polysaccharides, acidic EPSs attract attention due to their diverse functions both in free-living rhizobia and during the establishment of nitrogenfixing symbiosis with host legume plants.EPS forms a biofilm layer on the cell surface which is thought to contribute to the following processes: cellular protection against environmental stresses, attachment to surfaces, nutrient gathering, and the preferential absorption of flavonoids secreted by plants along the membrane surface [9].In addition, EPS biosynthesis is required for the effective nodulation of legumes such as Medicago, Pisum, Trifolium, Leucaena, and Vicia spp., which form indeterminate-type nodules (otherwise called meristematic or cylindrical) [12].EPS-deficient mutants of Rhizobium leguminosarum bv.viciae (hereafter Rlv), R. leguminosarum bv.trifolii (Rlt), and Sinorhizobium meliloti induce symbiotically defective phenotypes which include delayed root hair curling, nodules devoid of bacteria due to infection threads that abort within peripheral cells of the developing nodule, and small, partially infected, non-nitrogen-fixing nodules [12].The precise function(s) of the EPS molecules in these associations is still unclear; however, recent studies suggest that these extracellular polymers may function as symbiotic signaling molecules which regulate plant responses in the infection process [13][14][15][16][17].The current knowledge of EPS biosynthesis in rhizobia was based almost exclusively on the research with succinoglycan (EPS I) produced by S. meliloti [18].More than 26 genes needed for the synthesis, modification, polymerization, export, and processing of S. meliloti EPS I were identified, and a pathway for the polysaccharide biosynthesis was proposed [19][20][21].Recent discoveries on succinoglycan biosynthesis and its functioning in symbiosis are summarized in the exhaustive reviews [22,23].The EPSs produced by R. leguminosarum strains investigated to date structurally differ from those of S. meliloti.At present there is fragmentary information about the R. leguminosarum EPS assembly, modification, and processing, as well as the role of the individual gene products in these processes.These data were reviewed in [9][10][11]24].Here we report some recent and complementary data obtained in our group.On the basis of these data as well as the analysis of the sequenced R. leguminosarum and R. etli genomes we tried to outline main regularities in the EPS synthesis. Structural characteristics of exopolysaccharidesStructures of known R. leguminosarum and R. etli (Re) EPSs are presented in Table 1.It should be noted that EPSs secreted by the most of the Rlv and Rlt strains were shown to have repeating units of identical structure [25][26][27][28][29][30][31][32][33][34][35].These EPSs are branched heteropolysaccharides of the octasaccharide repeating unit consisting of a backbone of two glucose and two glucuronic acid residues, a side chain of three glucose and one galactose residues.The backbone contains 1-4 and 1-4 linkages only while the branch point is linked 1-6.The side chain contains 1-4 and 1-3 linkages.In general, the octasaccharide is modified by two pyruvyl, one or two non-stoichiometric O-acetyl, and one non-* EMBL/GenBank/DDBJ accession numbers: Rlt WSM2304 (CP001191), Re CFN42 (CP000133), Re CNPAF512 (AEYZ01000266) and Re CIAT 652 (CP001074)

AIMS:To study the question whether acidic exopolysaccharide (EPS) modification, e.g. pyruvylation, plays any role in the development of Rhizobium leguminosarum/Pisum sativum symbiosis.METHOD AND RESULTS:The amino acid sequence deduced from the pssM gene, localized within the pss (polysaccharide synthesis) gene locus, was shown to be homologous to several known and putative ketal pyruvate transferases, including ExoV from Sinorhizobium meliloti and GumL from Xanthomonas campestris. Rh. l. bv. viciae strain VF39 carrying a Km-cassette insertion into the pssM gene was obtained by the gene replacement technique. Knock-out of pssM led to the absence of the pyruvic acid ketal group at the subterminal glucose in the repeating unit of EPS as it was shown by (13)C and (1)H nuclear magnetic resonance (NMR) analysis. Complementation in trans restored the EPS modification in the pssM mutant. Disruption of the pssM gene resulted also in the formation of aberrant non-nitrogen-fixing nodules on peas. Ultrastructural studies of mutant nodules revealed normal nodule invasion and release of bacteria into the plant cell cytoplasm, but further differentiation of bacteroids was impaired, and the existing symbiosomes underwent lysis.CONCLUSION:PssM encodes ketal pyruvate transferase involved in the modification of the Rh. l. bv. viciae EPS. The absence of subterminal glucose pyruvylation in the EPS repeating units negatively influences (directly or indirectly) the formation of the nitrogen-fixing symbiosis with peas.SIGNIFICANCE AND IMPACT OF THE STUDY:Our finding that the absence of modification even at the single position of EPS is likely to be crucial for establishment of nitrogen-fixing symbiosis argues in favour of the idea concerning their specific signalling role in this process.

The “phiKMV-like viruses” comprise an important genus of T7 related phages infecting Pseudomonas aeruginosa. The genomes of these bacteriophages have localized single-strand interruptions (nicks), a distinguishing genomic trait previously thought to be unique for T5 related coliphages. Analysis of this feature in the newly sequenced phage φkF77 shows all four nicks to be localized on the non-coding DNA strand. They are present with high frequencies within the phage population and are introduced into the phage DNA at late stages of the lytic cycle. The general consensus sequence in the nicks (5′-CGACxxxxxCCTAoh pCTCCGG-3′) was shown to be common among all phiKMV-related phages.

Symbiotic nitrogen-fixing bacteria Rhizobium leguminosarum bv. viciae VF39 secrete an acidic heteropolysaccharide, the biosynthesis of which involves the stage of polyprenyl diphosphate octasaccharide formation with its carbohydrate fragment corresponding to the repeating polymer unit. The amino acid analysis of the product of the pssA gene, we have earlier identified, showed its homology to bacterial polyisoprenyl phosphate hexose 1-phosphate transferases catalyzing the formation of phosphodiester bonds between polyprenyl phosphates and hexose 1-phosphates, whose donors are nucleotide sugars. The immunoblotting demonstrated that Rhizobium cells synthesize a protein with a molecular mass of 25 kDa, which implies the translation of the open reading frame occurring from the second initiating codon followed by the protein processing. It was shown that PssA is an integral membrane-bound protein involved in glucose 1-phosphate transfer from UDP-glucose to polyprenyl phosphate to form polyprenyl diphosphate glucose. These results suggest that the pssA gene encodes UDP-glucose:polyprenyl phosphate-glucosyl phosphotransferase.

The effective new variant of “sandwich” bioluminescent enzyme immunoassay (BEIA) for the sensitive detection of glycoprotein B (gB) of pseudorabies virus (PrV) was presently developed. The high affinity interaction of barnase–barstar protein pair and photoprotein obelin as bioluminescent marker were for the first time successfully applied to BEIA development. Preliminary the two monoclonal antibodies, 11/5 and 34/2, were raised against gB for ELISA PrV detection. Presently we used the same immuno-“sandwich” principle for BEIA. To do this the two different bioconjugates were elaborated. Recombinant barnase was chemically conjugated with monoclonal anti-PrV's gB IgG, and also barstar was fused in frame to obelin. The characteristics of BEIA method have been compared to ELISA PrV detection. We have shown the proposed here gB-BEIA was 40-fold more sensitive as opposed to gB-ELISA test. The construction might have a broad promise in multiple potential immunological applications.

Симбиотические азотфиксирующие бактерии Rhizobium leguminosarum bv. viciae VF39 секретируют кислый гетерополисахарид, биосинтез которого включает стадию образования полипренилдифосфатоктасахарида, углеводный фрагмент которого соответствует повторяющемуся звену полимера. Анализ аминокислотной последовательности продукта идентифицированного нами ранее гена pssA показал его гомологию с последовательностями бактериальных полиизопренилфосфат-гексозо-1-фосфат-трансфераз, катализирующих образование фосфодиэфирной связи между полипренилфосфатом и гексозо-1-фосфатами, донорами которых являются нуклеотидсахара. Методом иммуноблотинга показано, что в клетках ризобий синтезируется белок с молекулярной массой 25 кДа, что предполагает трансляцию открытой рамки считывания гена pssA со второго инициирующего кодона с последующей модификацией белка. Показано, что PssA является интегральным мембранным белком, участвующим в переносе глюкозо-1-фосфата с UDP-глюкозы на полипренилфосфат с образованием полипренилдифосфатглюкозы. На основании этих данных сделан вывод о том, что ген pssA кодирует UDP-глюкоза : полипренилфосфат–глюкозилфосфотрансферазу.

The bioluminescence emitted by Aequorea victoria jellyfish is greenish while its single bioluminescent photoprotein aequorin emits blue light. This phenomenon may be explained by a bioluminescence resonance energy transfer (BRET) from aequorin chromophore to green fluorescent protein (GFP) co-localized with it. However, a slight overlapping of the aequorin bioluminescence spectrum with the GFP absorption spectrum and the absence of marked interaction between these proteins in vitro pose a question on the mechanism providing the efficient BRET in A. victoria. Here we report the in vitro study of BRET between homologous Ca(2+)-activated photoproteins, aequorin or obelin (Obelia longissima), as bioluminescence energy donors, and GFP, as an acceptor. The fusions containing donor and acceptor proteins linked by a 19 aa peptide were purified after expressing their genes in Escherichia coli cells. It was shown that the GFP-aequorin fusion has a significantly greater BRET efficiency, compared to the GFP-obelin fusion. Two main factors responsible for the difference in BRET efficiency of these fusions were revealed. First, it is the presence of Ca(2+)-induced interaction between the donor and acceptor in the aequorin-containing fusion and the absence of the interaction in the obelin-containing fusion. Second, it is a red shift of GFP absorption toward better overlapping with aequorin bioluminescence induced by the interaction of aequorin with GFP. Since the connection of the two proteins in vitro mimics their proximity in vivo, Ca(2+)-induced interaction between aequorin and GFP may occur in A. victoria jellyfish providing efficient BRET in this organism.

Site-specific endonucleases F-TflI, F-TflII, and F-TflIV have been revealed, which belong to the H-N-H family and are encoded by ORFs located in the tRNA gene region of bacteriophage T5. It has been shown that endonuclease F-TflIV introduces a double-strand break in a 17-bp pseudopalindromic DNA sequence to yield 1-nt 3′-protruding ends. Unlike F-TflIV, F-TflI, and F-TflII introduce single-strand breaks in asymmetrical, highly degenerate sequences, each cleaving only one (template or coding) strand. Amino acid sequence analysis has revealed a high homology of the enzymes in the region of the H-N-H motif and in the putative C-terminal catalytic domain. The N-terminal region of F-TflIV proved to be homologous to the HTH domain of LuxR-related transcriptional regulators, which is responsible for DNA recognition and binding. The N-terminal regions of F-TflI and F-TflII contain a composite motif NUMOD4, which is characteristic of a putative recognition domain of some H-N-H endonucleases. A two-domain structure, with the N-terminal recognition and C-terminal catalytic domains, and evolutionary origin via recombination of the catalytic and recognition domain-coding regions are proposed for F-TflI, F-TflII, and F-TflIV.

ABSTRACT The Pseudomonas fluorescens 23F phosphonoacetate hydrolase gene ( phnA ) encodes a novel carbon-phosphorus bond cleavage enzyme whose expression is independent of the phosphate status of the cell. Analysis of the regions adjacent to the phosphonoacetate hydrolase structural gene ( phnA ) indicated the presence of five open reading frames (ORFs). These include one ( phnR ) whose putative product shows high levels of homology to the LysR family of positive transcriptional regulators. Its presence was shown to be necessary for induction of the hydrolase activity. 2-Phosphonopropionate was found to be an inducer (and poor substrate) for phosphonoacetate hydrolase. Unlike phosphonoacetate, which is also an inducer of phosphonoacetate hydrolase, entry of 2-phosphonopropionate into cells appeared to be dependent on the presence of a gene ( phnB ) that lies immediately downstream of phnA and whose putative product shows homology to the glycerol-3-phosphate transporter. RNA analysis revealed transcripts for the phnAB and phnR operons, which are transcribed divergently; the resulting mRNAs overlapped by 29 nucleotide bases at their 5′ ends. Transcripts of phnAB were detected only in cells grown in the presence of phosphonoacetate, whereas transcripts of phnR were observed in cells grown under both induced and uninduced conditions. The expression of three additional genes found in the phnA region did not appear necessary for the degradation of phosphonoacetate and 2-phosphonopropionate by either Pseudomonas putida or Escherichia coli cells.

Recombinant plasmids containing genes for the green fluorescent protein (GFP) from Aequorea victoria and the photoprotein obelin from Obelia longissima linked in-frame by inserts differing in nucleotides sequences were constructed. The expression of the chimeric genes in Escherichia coli cells resulted in synthesis of the GFP–obelin hybrid proteins. These proteins were purified to homogeneity and subjected to limited trypsinolysis. It was shown that the resistance of GFP–obelin hybrid proteins to trypsin depends on the nature of their constituent modules and the amino acid sequences of linkers between the modules. The kinetics of accumulation of full-length hybrid proteins during the growth of bacterial cells does not depend on the structure of the peptide linkers. Most of the full-length product accumulates in cells in the form of inclusion bodies resistant to endogenous proteases. The soluble fraction of the protein undergoes considerable proteolysis regardless of the linker structure.

The protein expression profiles of Rhizobium leguminosarum strains in response to specific genetic perturbations in exopolysaccharide (EPS) biosynthesis genes were examined using two-dimensional gel electrophoresis. Lesions in either pssA, pssD, or pssE of R. leguminosarum bv. viciae VF39 or in pssA of R. leguminosarum bv. trifolii ANU794 not only abolished the capacity of these strains to synthesize EPS but also had a pleiotropic effect on protein synthesis levels. A minimum of 22 protein differences were observed for the two pssA mutant strains. The differences identified in the pssD and pssE mutants of strain VF39 were a distinct subset of the same protein synthesis changes that occurred in the pssA mutant. The pssD and pssE mutant strains shared identical alterations in the proteins synthesized, suggesting that they share a common function in the biosynthesis of EPS. In contrast, a pssC mutant that produces 38% of the EPS level of the parental strain showed no differences in its protein synthesis patterns, suggesting that the absence of EPS itself was contributing to the changes in protein synthesis and that there may be a complex interconnection of the EPS biosynthetic pathway with other metabolic pathways. Genetic complementation of pssA can restore wild-type protein synthesis levels, indicating that many of the observed differences in protein synthesis are also a specific response to a dysfunctional PssA. The relevance of these proteins, which are grouped as members of the pssA mutant stimulon, remains unclear, as the majority lacked a homologue in the current sequence databases and therefore possibly represent a novel functional network(s). These findings have illustrated the potential of proteomics to reveal unexpected higher-order processes of protein function and regulation that arise from mutation. In addition, it is evident that enzymatic pathways and regulatory networks are more interconnected and more sensitive to structural changes in the cell than is often appreciated. In these cases, linking the observed phenotype directly to the mutated gene can be misleading, as the phenotype could be attributable to downstream effects of the mutation.

The 7181-nt XhoI-MluI fragment of Rhizobium leguminosarum by. viciae VF39 chromosomal DNA was sequenced. Mutations in this region disturb the synthesis of an extracellular polysaccharide and nodulation in Vicia faba roots. Computer analysis revealed eight open reading frames designated pssE,D,C,F,G,H,I and orf8. All genes showed expression in a T7 promoter/RNA polymerase system. Comparison of the deduced amino acid sequences of their protein products with known protein sequences and analysis of exopolysaccharides in mutants showed that the pssE,D,C,EG,H,I genes code for glycosyltransferases consecutively involved in the synthesis of the octasaccharide unit of the polysaccharide.

15 strains belonging to genera Pseudomonas. Xanthomonas. Erwinia, Rhizobium and Cory nebacterium were analysed for their ability to synthesize indolyl-3-acetic aced (IAA) involving tryptophane-2-monooxygenase and indolyl-3-ocetamide hydrolase. As a result of DNA-DNA hybridization and polymerase chain reaction. iaaM gene encoding tryptophane-2-monooxygenase was revealed in 11 tested strains, with 9 attributed to phytopathogens. Activity of iaaH gene controlling further transformation of indolyl-3-acetamide into IAA was demonstrated by thin-layer chromatography method. The IAA biosynthesis pathway via indolyl-3-acetamide was supposed to be typical primarily for phytopathogenic bacteria.

A wide range (69) of mutant Escherichia coli alkaline phosphatases with single amino acid substitutions at positions from -5 to +1 of the signal peptide were obtained for studying protein processing as a function of the primary structure of the cleavage region. Amber suppressor mutagenesis, used to create mutant proteins, included: (i) introduction of amber mutations into respective positions of the phoA gene; and (ii) expression of each mutant phoA allele in E. coli strains producing amber suppressor tRNAs specific to Ala, Cys, Gln, Glu, Gly, His, Leu, Lys, Phe, Pro, Ser and Tyr. Most amino acid substitutions at positions -3 and -1 resulted in a complete block of protein processing. These data give new experimental support for the "-3, -1 rule". Only Ala, Gly and Ser at position -1 allowed protein processing, and Ala provided the highest rate of processing. The results revealed the more conservative nature of the amino acids at the -1 position of signal peptides of Gram-negative bacteria as compared with those of eukaryotic organisms. Position -3 was less regular, since not only Ala, Ser and Gly, but also Leu and Cys at this position, allowed the processing. Mutations at position -4 had an insignificant effect on the processing. Surprisingly, efficient processing was provided mainly by large amino acid residues at position -2 and by middle-sized residues at position -5, indicating that the processing rate is affected by the size of amino acid residues not only at positions -1 and -3. Conformation analysis of the cleavage site taken together with the mutation and statistical data suggests an extended beta-conformation of the -5 to -1 region in the signal peptidase binding pocket.