IS elements in Aliivibrio salmonicida LFI1238: Occurrence,variability and impact on adaptability

Insertion sequence (IS) elements are short, self-replicating DNA sequences that are capable of efficiently spreading over the host genome. Possessing varied integration specificity IS elements are capable of the irreversible inactivation of genes, which diversifies the pool of intact genetic determinants in host populations. In the current study, we performed a complex analysis of IS elements (Vsa IS) in the previously sequenced genome of Aliivibrio salmonicida LFI1238 and proposed a model of the spread of the Vsa IS elements over the genome of this microorganism. Along with the prediction of the integration sites for Vsa IS elements, the current study provides an overview of the properties of A. salmonicida IS elements, as well as information regarding their occurrence in different bacterial classes. An analysis of individual alleles of the IS elements has allowed us to depict a history of the accumulation of mutations and to describe distinctive microevolution lines for actively transposing Vsa IS elements in the genome of A. salmonicida LFI1238. Our results demonstrate the high importance of the dead end microevolution of actively transposing Vsa IS elements for the inactivation of genes in A. salmonicida LFI1238.     

Accurate mass comparison coupled with two endopeptidases enables identification of protein termini

Protein termini play important roles in biological processes, but there have been few methods for comprehensive terminal proteomics. We have developed a new method that can identify both the amino and the carboxyl termini of proteins. The method independently uses two proteases, (lysyl endopeptidase) Lys-C and peptidyl-Lys metalloendopeptidase (Lys-N), to digest proteins, followed by LC-MS/MS analysis of the two digests. Terminal peptides can be identified by comparing the peptide masses in the two digests as follows: (i) the amino terminal peptide of a protein in Lys-C digest is one lysine residue mass heavier than that in Lys-N digest; (ii) the carboxyl terminal peptide in Lys-N digest is one lysine residue mass heavier than that in Lys-C digest; and (iii) all internal peptides give exactly the same molecular masses in both the Lys-C and the Lys-N digest, although amino acid sequences of Lys-C and Lys-N peptides are different (Lys-C peptides end with lysine, whereas Lys-N peptides begin with lysine). The identification of terminal peptides was further verified by examining their MS/MS spectra to avoid misidentifying pairs as termini. In this study, we investigated the usefulness of this method using several protein and peptide mixtures. Known protein termini were successfully identified. Acetylation on N-terminus and protein isoforms, which have different termini, was also determined. These results demonstrate that our new method can confidently identify terminal peptides in protein mixtures.     

Deletion of the prc (tsp) gene provides evidence for additional tail-specific proteolytic activity in Escherichia coli K-12

The Escherichia coli protease Prc (Tsp) exhibits specificity in vitro for proteins with nonpolar carboxyl termini. To determine whether Prc is responsible for the selective degradation in vivo of proteins with nonpolar carboxyl termini, we constructed a prc (tsp) deletion strain. Deletion of the prc gene did not prevent the rapid intracellular degradation of a variant of the amino-terminal domain of repressor with a nonpolar carboxyl terminus, even though this protein is a substrate for Prc in vitro. Our results indicate that at least one additional carboxy-terminal-specific proteolytic system must exist in E. coli.     

Some Photosystem II properties depending on the D1 protein variants in Thermosynechococcus elongatus

Cyanobacteria have multiple psbA genes encoding PsbA, the D1 reaction center protein of the Photosystem II complex which bears together with PsbD, the D2 protein, most of the cofactors involved in electron transfer reactions. The thermophilic cyanobacterium Thermosynechococcus elongatus has three psbA genes differently expressed depending on the environmental conditions. Among the 344 residues constituting each of the 3 possible PsbA variants there are 21 substitutions between PsbA1 and PsbA3, 31 between PsbA1 and PsbA2 and 27 between PsbA2 and PsbA3. In this review, we summarize the changes already identified in the properties of the redox cofactors depending on the D1 variant constituting Photosystem II in T. elongatus. This article is part of a Special Issue entitled: Photosynthesis Research for Sustainability: Keys to Produce Clean Energy.     

Linkage between the intramembrane H-bond network around aspartic acid 83 and the cytosolic environment of helix 8 in photoactivated rhodopsin

Understanding the coupling between conformational changes in the intramembrane domain and at the membrane-exposed surface of the bovine photoreceptor rhodopsin, a prototypical G protein-coupled receptor (GPCR), is crucial for the elucidation of molecular mechanisms in GPCR activation. Here, we have combined Fourier transform infrared (FTIR) and fluorescence spectroscopy to address the coupling between conformational changes in the intramembrane region around the retinal and the environment of helix 8, a putative cytosolic surface switch region in class-I GPCRs. Using FTIR/fluorescence cross-correlation we show specifically that surface alterations monitored by emission changes of fluorescein bound to Cys316 in helix 8 of rhodopsin are highly correlated with (i) H-bonding to Asp83 proximal of the retinal Schiff base but not to Glu122 close to the beta-ionone and (ii) with a metarhodopsin II (MII)-specific 1643 cm(-1) IR absorption change, indicative of a partial loss of secondary structure in helix 8 upon MII formation. These correlations are disrupted by limited C-terminal proteolysis but are maintained upon binding of a transducin alpha-subunit (G(talpha))-derived peptide, which stabilizes the MII state. Our results suggest that additional C-terminal cytosolic loop contacts monitored by an amide II absorption at 1557 cm(-1) play a functionally crucial role in keeping helix 8 in the position in which its environment is strongly coupled to the retinal-binding site near the Schiff base. In the intramembrane region, this coupling is mediated by the H-bonding network that connects Asp83 to the NPxxY(x)F motif preceding helix 8.     

The carboxy-terminal extension of the D1-precursor protein is dispensable for a functional photosystem II complex in Chlamydomonas reinhardtii

The D1-precursor protein of the photosystem II reaction centre contains a carboxy-terminal extension whose proteolytic removal is necessary for oxygen-evolving activity. To address the question of the role of the carboxy-terminal extension in the green alga Chlamydomonas reinhardtii, we truncated D1 by converting codon Ser345 of the psbA gene into a stop codon. Particle gun transformation of an in vitro modified psbA gene fragment also carrying mutations conferring herbicide resistance yielded a homoplasmic transformant containing the stop codon. Since oxygen evolution capacity is not affected in this mutant as compared with herbicide-resistant control cells, the carboxy-terminal extension is dispensable for a functional photosystem II complex under normal growth conditions.     

DNA sequence analysis of the recA genes from Proteus vulgaris, Erwinia carotovora, Shigella flexneri and Escherichia coli B/r

Summary The complete nucleotide sequences of therecA genes fromEscherichia coli B/r,Shigella flexneri, Erwinia carotovora andProteus vulgaris were determined. The DNA sequence of the coding region of theE. coli B/r gene contained a single nucleotide change compared with theE. coli K12 gene sequence whereas theS. flexneri gene differed at 7 residues. In both cases, the predicted proteins were identical in primary structure to theE. coli K12 RecA protein. The DNA sequences of the recA genes fromE. carotovora andP. vulgaris were 80% and 74% homologous, respectively, to theE. coli K12 gene. The predicted amino acid sequences of theE. carotovora andP. vulgaris RecA proteins were 91% and 85% identical respectively, to that ofE. coli K12. The RecA proteins from bothP. vulgaris andE. carotovora diverged significantly in sequence in the last 50 residues whereas they showed striking conservation throughout the first 300 amino acids which include an ATP-binding region and a subunit interaction domain. A putative LexA repressor binding site was localized upstream of each of the heterologous genes.     

Morpholinone mediated oxazolone‐free C‐terminus amide coupling permitting a convergent strategy for peptide synthesis

3‐Substituted‐5‐phenylmorpholinones have been demonstrated to act as N‐protected C‐terminus activated α‐amino acids capable of undergoing solution phase N‐terminus peptide extension following standard coupling procedures. The N‐acylated morpholinones do not undergo epimerisation of the stereocentre of the C‐terminus amino acid residue as oxazolone formation is sterically prevented, although C‐terminus peptide coupling is still possible. This convergent approach to peptide synthesis is exemplified by the preparation of L‐ala‐L‐ala‐L‐ala and L‐ala‐D‐ala‐L‐ala. Copyright © 2008 European Peptide Society and John Wiley & Sons, Ltd.     

Flexible interwoven termini determine the thermal stability of thermosomes

Group II chaperonins, which assemble as double-ring complexes, assist in the refolding of nascent peptides or denatured proteins in an ATP-dependent manner. The molecular mechanism of group II chaperonin assembly and thermal stability is yet to be elucidated. Here, we selected the group II chaperonins (cpn-α and cpn-β), also called thermosomes, from Acidianus tengchongensis and investigated their assembly and thermal stability. We found that the binding of ATP or its analogs contributed to the successful assembly of thermosomes and enhanced their thermal stabilities. Cpn-β is more thermally stable than cpn-α, while the thermal stability of the hetero thermosome cpn-αβ is intermediate. Cryo-electron microscopy reconstructions of cpn-α and cpn-β revealed the interwoven densities of their non-conserved fl exible N/C-termini around the equatorial planes. The deletion or swapping of their termini and pH-dependent thermal stability assays revealed the key role of the termini electrostatic interactions in the assembly and thermal stability of the thermosomes.