Vladimir N. Ksenzenko

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

© 1997 Federation of European Biochemical Societies.

The pss4 gene from Rhizobium leguminosarum bv 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.

Elevated levels of synthesis of over 20 proteins results after mutation of the Rhizobium leguminosarum exopolysaccharide synthesis gene pssA.

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.

Cloning of new Rhodococcus extradiol dioxygenase genes and study of their distribution in different Rhodococcus strains

Four extradiol dioxygenase genes which encode enzymes active against catechol and substituted catechols were cloned from two different Rhodococcus strains, and their nucleotide sequences were determined. A catechol 2,3-dioxygenase gene (edoC) was shown to be identical to the previously described ipbC gene from the isopropylbenzene operon of Rhodococcus erythropolis. Amino acid sequences deduced from the three other genes (edoA, edoB and edoD) were shown to have various degrees of homology to different extradiol dioxygenases. The EdoA and EdoB dioxygenases were classified as belonging to the third family of type I oxygenases and represented two new subfamilies, whereas the EdoD dioxygenase was a type II enzyme. Analysis of six Rhodococcus strains revealed a wide distribution of the above dioxygenase genes. Rhodococcus sp. 11 was shown to harbour all four of the analysed dioxygenase genes. Nucleotide sequences homologous to the edoB gene were present in all of the strains, including R. erythropolis NCIMB 13065, which did not utilize any of the aromatic compounds analysed. The latter finding points to the existence of a silent pathway(s) for degradation of aromatic compounds in this Rhodococcus strain.

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.

Transfer RNAPhe isoacceptors possess non‐identical set of identity elements at high and low Mg2+ concentration

Primary structures of phage T5‐ and Escherichia coli‐encoded tRNAPhe 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 tRNAPhe gene transcript and mutants containing substitutions of the identity elements at positions 20, 34, 35 and 36 was compared with E. coli tRNAPhe 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 tRNAPhe 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 tRNAPhe 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.

Exopolysaccharide Biosynthesis in Rhizobium leguminosarum: From Genes to Functions

© 2012 Ivashina and Ksenzenko, licensee InTech. This is an open access chapter distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Exopolysaccharide Biosynthesis in Rhizobium leguminosarum: From Genes to Functions

Sequencing of Flagellin Genes from Natrialba magadii Provides New Insight into Evolutionary Aspects of Archaeal Flagellins

We have determined the nucleotide sequence of a flagellin gene locus from the haloalkaliphilic archaeon Natrialba magadii, identified the gene products among proteins forming flagella, and demonstrated cotranscription of the genes. Based on the sequence analysis we suggest that different regions of the genes might have distinct evolutionary histories including possible genetic exchange with bacterial flagellin genes.

[The dependence of stability of the green fluorescent protein-obelin hybrids on the nature of their constituent modules and the structure of the amino acid linker].

Recombinant plasmids containing genes for the green fluorescent protein (GFP) from Aequorea victoriaand the photoprotein obelin from Obelia longissimalinked in-frame by inserts differing in nucleotides sequences were constructed. The expression of the chimeric genes in Escherichia colicells 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.

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

Bacteriophage T5 BglII/HindIII 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 (tRNASer 1 and tRNASer 2) 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’.