[Bacteriophage T5 Mutants Carrying Deletions in tRNA Gene Region].

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.

Mutation in the pssM gene encoding ketal pyruvate transferase leads to disruption of Rhizobium leguminosarum bv. viciae-Pisum sativum symbiosis.

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 pssA gene encodes UDP-glucose: polyprenyl phosphate-glucosyl phosphotransferase initiating biosynthesis of Rhizobium leguminosarum exopolysaccharide].

Symbiotic nitrogen-fixing bacteria Rhizobium leguminosarum by. 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.

Cloning of genes for bacteriophage T5 stable RNAs.

One EcoRI-generated fragment (440 basepairs) and two EcoRI/HindIII fragments (220 and 960 basepairs) from the deletion region of T5 phage have been inserted into the phage lambda XIII and the plasmid pBR322 as vectors. Recombinant DNA molecules were studied by hybridization with in vivo 32P-labeled T5 4-5 S RNAs on nitrocellulose filters. Two-dimensional polyacrylamide gel electrophoretic fractionation and fingerprint analysis of the RNAs eluted from the filters were carried out to identify RNAs coded by cloned fragments. For the accurate localization of the genes for these RNAs, RNA-DNA hybrids were treated with T1 and pancreatic RNAases, and the eluted RNA fragments stable against RNAase action were electrophoresed. It was shown that the EcoRI 440 fragment contains the gene for tRNA 10 (tRNAAsp), the EcoRI/HindIII 220 fragment contains the gene for RNA III (107 bases) and parts of the genes for RNA I (107 bases) and tRNA 12 (tRNAHis), and the EcoRI/HindIII 960 fragment contains only a part of the gene for tRNA 9 (tRNAGln). The arrangement of these genes on the physical map of T5 phage was as follows: -tRNAGln-tRNAHis-RNA III-RNA I-...-tRNAAsp.

Processing of Escherichia coli alkaline phosphatase: role of the primary structure of the signal peptide cleavage region.

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.

Functional analysis of hybrid plasmids carrying genes for lambda site-specific recombination.

A number of hybrid plasmids, carrying lambda genes involved in site-specific integrative recombination, have been constructed in vitro. Analysis of protein synthesis in Escherichia coli minicells has shown that Int protein is synthesized only when int gene is expressed constitutively. The plasmids RSF2124::lambda-CD, RSF2124::lambda-Cint-c57, and pInt lambda were able to integrate into the chromosome of E.coli at the attB. The integration of hybrid plasmids into the genome of bacteria has also been shown for polA1 strains restricting the autonomous replication of ColE1 type plasmids. Genetic markers of hybrid plasmids are maintained in polA1 bacteria for at least 50 generations under nonselective conditions. The Southern blotting experiments using [32P]pBR322 DNA and EcoRI fragments of E. coli polA1 chromosome carrying integrated plasmid pInt lambda demonstrated that in this strain hybrid plasmids can be observed only when integrated into the attB of the chromosome according to Campbell's model of integration. In the cells, where autonomous replication of plasmids is possible, they can be observed both in extrachromosomal and integrated states. The integration of the ColE1 replication origin into the chromosome of bacteria is not lethal for the cells. Only attP and the int gene of lambda are necessary for the integration of hybrid plasmids under conditions of effective int gene expression. If the level of Int protein synthesis is high enough, the prophage excision can be observed in the absence of Xis product. The six-fold decrease of Int protein concentration in the cell (in case of pInt lambda 2 as compared to pInt lambda 1) is critical both for integration and excision.

The pss4 gene from Rhizobium leguminosarum by viciae VF39: cloning, sequence and the possible role in polysaccharide production and nodule formation.

The Tn5 insertion into the genome of Rhizobium leguminosarum bv viciae VF39, resulting in non-mucoid growth and formation of non-N2-fixing nodule-like structures on Vicia faba plants, was mapped within a 1.4-kb EcoRV-SacI fragment. Nucleotide sequence analysis revealed an ORF (pss4) of 263 amino acids (aa). Three transcription start points (tsp) were determined. Two of them were localized upstream from the first GTG codon; the third tsp was mapped in front of the second putative start codon (GTG) corresponding to Val64 of the Pss4 aa sequence. The expression of pss4 in a T7 RNA polymerase/promoter system produced a single approx. 29-kDa protein. Pss4 reveals similarity to several proteins involved in polysaccharide biosynthesis in various Rhizobium species. A nearly complete homology was found with PssA from Rl biovar phaseoli 8002 [Borthakur et al., Mol. Gen. Genet. 213 (1988) 155-162], except that Pss4 has an additional 63 aa on its N terminus.

Isolation of transposon TnA from plasmid RP4 carrying two copies of this element.

Employing heteroduplex and restriction analyses, two inverted copies of a 3.2.10(6) dalton transposable sequence, TnA, were found in RP4::TnA, a spontaneously arisen derivative of the plasmid RP4. Integration of the second copy of TnA causes loss of the conjugative properties of RP4. Both TnA sequences in RP4::TnA were localized and found to have opposite orientations. The DNA fragment corresponding to the individual transposon TnA was isolated after the endonuclease S1 digestion of RP4::TnA molecules annealed under conditions favoring intramolecular renaturation. The attempts to transform the cells of Escherichia coli QD5003, HB101[pCRI] and JC7623 with the isolated transposon were unsuccessful.

Aminoacylation of tRNA gene transcripts is strongly affected by 3'-extended and dimeric substrate RNAs.

Kinetic parameters of aminoacylation by E. coli phenylalanyl-tRNA synthetase vary for phage T5 tRNA(Phe) gene transcript from 0.950 to 2.545 microM for Km and from 550 to 400 min(-1) for kcat. To reveal the source of this variability for various RNA preparations, homogeneity of the transcripts has been examined. Presence of 3' extensions and dimer formation in transcript preparations reduced the catalytic efficiency kcat/Km several-fold. We have shown that the proportion of dimers and 3'-extended transcripts in tRNA preparations is sensitive to single-base substitutions in tRNA. While wild-type phage T5 tRNA(Phe) gene transcript contains about half of dimeric molecules, for some mutants this value increases up to 90% or drops to 0%. Phage T5 tRNA(Phe) gene with anticodon stem nucleotide substitutions used as a template in run-off transcription produces 5 times less 3'-extended molecules than the wild-type gene. In view of all these results kinetic parameters of aminoacylation reaction for many wild-type and mutant tRNA gene transcripts should be reevaluated.

'Red pigment' from ADE-2 mutants of S. cerevisiae prevents DNA cleavage by restriction endonucleases.

Protection of DNA from cleavage by restriction endonucleases EcoRI, HindIII, BamHI, and Bg/II with red pigment, produced by ADE-2 mutants of Saccharomyces cerevisiae is demonstrated. Purification of yeast DNA from pigment can be achieved by chromatography on hydroxyapatite columns.

The nucleotide sequence of the bacteriophage T5 ltf gene.

The nucleotide sequence of the bacteriophage T5 Bg/II-BamHI fragment (4,835 bp in length) known to carry a gene encoding the LTF protein which forms the phage L-shaped tail fibers was determined. It was shown to contain an open reading frame for 1,396 amino acid residues that corresponds to a protein of 147.8 kDa. The coding region of ltf gene is preceded by a typical Shine-Dalgarno sequence. Downstream from the ltf gene there is a strong transcription terminator. Data bank analysis of the LTF protein sequence reveals 55.1% identity to the hypothetical protein ORF 401 of bacteriophage lambda in a segment of 118 amino acids overlap.

Cloning and DNA sequence of the genes for two bacteriophage T5 tRNAsSer.

Bacteriophage T5 Bg/II/Hind III DNA fragment (803 basepairs), containing the genes for 2 tRNAs and 2 RNAs with unknown functions, was cloned in the plasmid pBR322. The analysis of DNA sequence indicates that tRNA genes code isoacceptor tRNAsSer (tRNA1Ser and tRNA2Ser) with anticodons UGA and GGA, respectively. The main unusual structural feature of these tRNAs is the presence of extra non-basepaired nucleotides in the joinings of stem 'b' with stems 'a' and 'c'.

Positively charged lysine at the N-terminus of the signal peptide of the Escherichia coli alkaline phosphatase provides the secretion efficiency and is involved in the interaction with anionic phospholipids.

Positively charged amino acid residues at the N-terminus of the signal peptide (SP) have been proposed to play a significant role in the initial step of protein secretion in bacteria. To test this hypothesis, Lys(-20) of the Escherichia coli alkaline phosphatase SP was replaced by other amino acid residues, and the effect of these substitutions on protein maturation was studied. The introduction of negatively charged and hydrophobic amino acids resulted in a decrease in secretion efficiency and impaired the SP-APL interaction, whereas His and Tyr had no significant effect. A structural analysis of the SP-APL interaction suggests that the positively charged Lys(-20) determines the stability of the complex.

Transfer RNA(Phe) isoacceptors possess non-identical set of identity elements at high and low Mg2+ concentration.

Primary structures of phage T5- and Escherichia coli-encoded tRNA(Phe) are distinct at four out of 11 positions known as identity elements for E. coli phenylalanyl-tRNA synthetase (FRS). In order to reveal structural requirements for FRS recognition, aminoacylation of wild-type phage T5 tRNA(Phe) gene transcript and mutants containing substitutions of the identity elements at positions 20, 34, 35 and 36 was compared with E. coli tRNA(Phe) gene transcript. The wild-type phage T5 transcript can be aminoacylated with the same catalytic efficiency as the E. coli counterpart. However, the maximal aminoacylation rate for T5 and E. coli transcripts was reached at different Mg2+ concentrations: 4 and 15 mM, respectively. Aminoacylation assays with tRNA(Phe) mutants revealed that (i) phage transcripts with the substituted anticodon bases at positions 35 and 36 were efficient substrates for aminoacylation at 15 mM Mg2+ but not at optimal 4 mM Mg2+; (ii) any change of G34 in phage transcripts dramatically decreased the aminoacylation efficiency at both 4 and 15 mM Mg2+ whereas G34A mutation in the E. coli transcript exhibits virtually no influence on aminoacylation rate at 15 mM Mg2+; (iii) substitution of A20 with U in the phage transcript caused no significant change in the aminoacylation rate at both Mg2+ concentrations; (iv) phage transcripts with double substitutions A20U+A35C and A20U+A36C were very poor substrates for FRS. Collectively, the results indicate the non-identical mode of tRNA(Phe) recognition by E. coli FRS at low and high Mg2+ concentrations. Probably, along with identity elements, the local tRNA conformation is essential for recognition by FRS.

[Features of the structure of transfer RNA coded by bacteriophage T5]. / Osobennosti struktury transportnykh RNK, kodiruemykh bakteriofagom T5.

Nucleotide sequence of 24 genes for the bacteriophage T5 tRNAs specific for all amino acids involved in protein synthesis was determined. All of them, except tRNA(Pro), were shown to differ significantly from the generalized "clover-leaf" structure consisting in the displacement of invariant and semi-invariant residues, the absence of pairing in the stems and some other deviations from the canonical parameters of the model. Basing on the available information on the functional activity of the phage-specific tRNAs, one can put forward a suggestion that, at least for some of them, the above anomalies do not essentially influence their activity. A comparison of phage T5 tRNAs with the Escherichia coli and phage T4 counterparts was carried out. The majority of the known elements determining the specificity of E. coli tRNA aminoacylation was also found in all phage T5 tRNAs, except tRNAPhe).

[Study of the biogenesis and secretion of alkaline phosphatase and its mutant forms in Escherichia coli. I. Introduction of directed mutations into the alkaline phosphatase gene]. / Izuchenie biogeneza i sekretsii shchelochnoi fosfatazy i ee mutantnykhform u Excherichia coli. I. Vvedenie napravlennykh mutatsii v gen shchelochnoi fosfatazy.

Various mutations in E. coli alkaline phosphatase gene were obtained by oligonucleotide-directed mutagenesis. They result in amino acid substitutions in the signal peptide cleavage site [Val for Ala(-1)] and in the N terminus of mature polypeptide chain: Ala for Arg(+1) and Gln for Glu(+4); Gln for Glu(+4). Enzyme activity was observed in all E. coli strains transformed by plasmids with cloned mutant genes. In addition, an amber mutation was introduced into the Arg(+1) position, and the synthesis of mutant alkaline phosphatase was shown in E. coli strains containing suppressor tRNAs specific for Ser, Gln, Tyr, Leu, Ala, Glu, Phe, Gly, His, Pro, and Cys.

[Novel site-specific endonucleases F-TflI, F-TflII and F-TflIV encoded by the bacteriophage T5]. / Novye sait-spetsificheskie éndonukleazy F-TfI, F-TfII i F-TfIV, kodiruemye bakteriofagom T5.

Novel site-specific H-N-H endonucleases F-TflI, F-TflII and F-TflIV were identified. These endonucleases are encoded by open reading frames localized in the bacteriophage T5 tRNA gene region. The endonuclease F-TflIV was shown to introduce double-strand break into pseudo palindromic 17 bp DNA sequence yielding 1 bp extensions with 3'-overhangs. In contrast to F-TflIV, endonucleases F-TflI and F-TflII cleave only one strand of their asymmetric divergent DNA substrates. Each of these endonucleases introduces interruptions into only the particular strand (template or coding). Amino acid sequences of the endonucleases under study are highly homologous in the H-N-H motif regions and C-terminal sequences, forming putative catalytic domain. Endonuclease F-TflIV N-terminal region is homologous to the amino acid sequences representing H-T-H recognition domain found in LuxR family transcription regulators. Putative recognition NUMOD4 motif characteristic for a number of H-N-H endonucleases was shown to be also present in the endonuclease F-TflI and F-TflII N-terminal sequences. Two-domain structure was proposed for endonucleases F-TflI, F-TflII and F-TflIV with N-terminal recognition domain and C-terminal catalytic domain. A hypothesis of evolutionary origin of these endonucleases as a result of catalytic and recognition domains recombination was suggested.

[Localization of canonical single-strand breaks in DNA of the Pseudomonas aeruginosa bacteriophage phi kF77]. / Lokalizatsiia kanonicheskikh odnonitevykh razryvov v DNK faga phi kF77 Pseudomonas aueruginoza.

Bacteriophages phi k of P. aeruginosa were characterized by the presence of T4 DNA-ligase-repaired, single-chain breaks in their genome. A restriction map was constructed for one of these phages (phi kF77) with restriction endonucleases SalI, HindIII, EcoRI, MluI, XbaI and ClaI. phi kF77 DNA was resistant to the cleavage by BamHI, BglII, HpaI, PstI, PvuII and XhoI endonucleases. Single-chain breaks were mapped by means of electron microscopy of partially denatured DNA molecules, electrophoretic studies of denatured DNA and S1-analysis. Four major nicks were thus located which were revealed in 33 to 83% of DNA molecules. On the basis of mutual hybridization of single-strand DNA fragments it was shown that all nicks are located in one of the phi kF77 DNA chains. S1-treated hybrids of 32P-labeled single-strand fragments with intact DNA chain were used for DNA orientation. The physical map of phi kF77 DNA was constructed.

[Biogenesis and secretion of alkaline phosphatase and its mutant forms in Escherichia coli. II. Effect of replacing amino acids at the processing site and N-terminal domain of the mature polypeptide chain of alkaline phosphatase on its biogenesis]. / Izuchenie biogeneza i sekretsii shchelochnoi fosfatazy i ee mutantnykh form u Escherichia coli. II. Vliianie zamen aminokislot v saite protsessinga i N-kontsevom domene zreloi polipeptidnoi tsepi shchelochnoi fosfatazy na ee biogenez.

The effect of amino acid substitutions in E. coli alkaline phosphatase on its biogenesis has been studied. The substitution of Val for Ala(-1) in the signal peptide cleavage site completely inhibits all stages of posttranslational modification: processing and formation of isozymes. The absence of processing does not prevent translocation of the precursor across the cytoplasmic membrane and formation of an active enzyme macromolecule. The precursor of the above mutant protein was found in the periplasm and in the cytoplasmic membrane. The substitution of Gln for Glu(+4), as well as the double substitution of Ala for Arg(+1) and Gln for Glu(+4), in the N-terminus of mature polypeptide chain result in the change in the isozyme spectrum. Differences in the rates of processing in vivo of both mutant proteins were not revealed. However, the double amino acid substitution significantly increases the efficiency of in vitro processing. All amino acid substitutions studied have no effect on the peculiarities of biogenesis which are conditioned by oversynthesis of the enzyme encoded by the phoA gene in the plasmid: secretion into the culture medium and accumulation of precursor as insoluble aggregates in the cytoplasm. However, extracellular activities of mutant proteins differ from that of the wild-type protein, which may result from the change either in the efficiency of their secretion or in their catalytic properties.

[Cloning of bacteriophage T5 DNA fragments in the plasmid pBR322. Analysis of recombinant plasmids by the method of bonding with RNA-polymerase from Escherichia coli on nitrocellulose filters]. / Klonirovanie fragmentov DNK bakteriofaga T5 v plazmide pBR322. Analiz rekombinantnykh plazmid metodom sviazyvaniia s RNK-polimerazoi Escherichia coli na nitrotselliuloznykh fil'trakh.

DNA of bacteriophage T5 was hydrolyzed with restriction endonucleases HindIII and BamHI, and subjected to the combined hydrolysis with BamHI+EcoRI and BamHI+ +HindIII. Fragments obtained were cloned in the plasmid pBR322. About 17% of T5 genome were recovered in recombinant plasmids. Cloned fragments were localized on the physical map of the phage by restriction analysis and Southern hybridization. With the aim of direct cloning of T5 promoters, PstI/HindIII fragments were inserted into pBR322 followed by selection of recombinants on ApsTCr phenotype. Binding of BsuRI and AluI fragments of hybrid plasmids with E. coli RNA polymerase was studied by nitrocellulose filter assay. The fragments, which were capable to form heparin resistant complexes were identified.