Mineralization of small saline and soda lakes can vary significantly depending on the season and weather conditions. The ability to rapidly adjust intracellular concentrations of key osmolytes (also known as compatible solutes) to changes in external salinity is an important property of microorganisms inhabiting these biotopes (8,18,34). Ectoine (1,4,5,6-tetrahydro-2-methyl-4-pyrimidine carboxylic acid) was found to be a major compatible solute in many halophilic or halotolerant bacteria isolated from alkaline, moderately hypersaline environments (14). This organic solute can be synthesized de novo or taken up from the environment when available (15, 18). The biochemistry and genetics of ectoine synthesis have been described for several bacteria (15,28,29,32). However, little is known about the transcriptional regulation of the ectoine biosynthetic pathway. Comprehensive analysis of the ectoine gene cluster ectABC in Chromohalobacter salexigens showed four putative transcription initiation sites upstream of the ectA start codon. Two 70 -dependent, one S -dependent, and one 32 -dependent promoter were identified and shown to be involved in ectABC transcription in this bacterium (6). Transcription of the ectA, ectB, and ectC genes from Marinococcus halophilus was initiated from three individual 70 / A -dependent promoter sequences located upstream of each gene (3). In Bacillus pasteurii, the ectABC genes are organized in a single operon preceded by a typical A -dependent promoter region (21). The halotolerant obligate methanotroph Methylomicrobium alcaliphilum 20Z is capable of growth at a salinity as high as 2 M NaCl (19). It was demonstrated that in response to the elevated salinity of the growth medium, M. alcaliphilum cells accumulate ectoine as a major osmoprotective compound (20). The ectoine biosynthesis pathway in M. alcaliphilum 20Z is similar to the pathway employed by halophilic/halotolerant heterotrophs and involves three specific enzymes: diaminobutyric acid (DABA) aminotransferase (EctB), DABA acetyltransferase (EctA), and ectoine synthase (EctC) (7,21,24,30,32,49). In M. alcaliphilum 20Z, the ectoine biosynthetic genes were shown to be organized in the ectABC-ask operon containing the additional ask gene, encoding aspartokinase (32). Here we describe the transcriptional organization of the ectoine biosynthetic genes in M. alcaliphilum 20Z. We identify a new MarR-like transcriptional regulator (EctR1) and show that EctR1 represses the expression of the ectABC-ask operon from the ectAp 1 promoter by binding at the putative Ϫ10 sequence. These results demonstrate the presence of a new, previously uncharacterized regulatory system for ectoine biosynthesis in the salt-tolerant methanotroph. MATERIALS AND METHODSBacterial strains and culture conditions. The M. alcaliphilum and Escherichia coli strains, plasmids, and primers used in this study are listed in Tables S1 and S2 in the supplemental material. M. alcaliphilum strains were grown at 30°C under a methane-air atmosphere (1:1) or in the presence of 0....
Bacteriophage capsids constitute icosahedral shells of exceptional stability that protect the viral genome. Many capsids display on their surface decoration proteins whose structure and function remain largely unknown. The decoration protein pb10 of phage T5 binds at the centre of the 120 hexamers formed by the major capsid protein. Here we determined the 3D structure of pb10 and investigated its capsid-binding properties using NMR, SAXS, cryoEM and SPR. Pb10 consists of an α-helical capsid-binding domain and an Ig-like domain exposed to the solvent. It binds to the T5 capsid with a remarkably high affinity and its binding kinetics is characterized by a very slow dissociation rate. We propose that the conformational exchange events observed in the capsid-binding domain enable rearrangements upon binding that contribute to the quasi-irreversibility of the pb10-capsid interaction. Moreover we show that pb10 binding is a highly cooperative process, which favours immediate rebinding of newly dissociated pb10 to the 120 hexamers of the capsid protein. In extreme conditions, pb10 protects the phage from releasing its genome. We conclude that pb10 may function to reinforce the capsid thus favouring phage survival in harsh environments.
The most attractive and methodologically convenient way to enhance protein stability is via the introduction of disulphide bond(s). However, the effect of the artificially introduced SS-bond on protein stability is often quite unpredictable. This raises the question of how to choose the protein sites in an intelligent manner, so that the 'fastening' of these sites by the SS-bond(s) would provide maximal protein stability. We hypothesize that the successful design of a stabilizing SS-bond requires finding highly mobile protein regions. Using GFP as an illustrative example, we demonstrate that the knowledge of the peculiarities of the intramolecular hydrophobic interactions, combined with the understanding of the local intrinsic disorder propensities (that can be evaluated by various disorder predictors, e.g., PONDRFIT), is sufficient to find the candidate sites for the introduction of stabilizing SS-bridge(s). In fact, our analysis revealed that the insertion of the engineered SS-bridge between two highly flexible regions of GFP noticeably increased the conformational stability of this protein toward the thermal and chemical unfolding. Therefore, our study represents a novel approach for the rational design of stabilizing disulphide bridges in proteins.
The most complex problem in studying multi-state protein folding is the determination of the sequence of formation of protein intermediate states. A far more complex issue is to determine at what stages of protein folding its various parts (secondary structure elements) develop. The structure and properties of different intermediate states depend in particular on these parts. An experimental approach, named μ-analysis, which allows understanding the order of formation of structural elements upon folding of a multi-state protein was used in this study. In this approach the same elements of the protein secondary structure are “tested” by substitutions of single hydrophobic amino acids and by incorporation of cysteine bridges. Single substitutions of hydrophobic amino acids contribute to yielding information on the late stages of protein folding while incorporation of ss-bridges allows obtaining data on the initial stages of folding. As a result of such an μ-analysis, we have determined the order of formation of beta-hairpins upon folding of the green fluorescent protein.
Bacterial S1 protein is a functionally important ribosomal protein. It is a part of the 30S ribosomal subunit and is also able to interact with mRNA and tmRNA. An important feature of the S1 protein family is a strong tendency towards aggregation. To study the amyloidogenic properties of S1, we isolated and purified the recombinant ribosomal S1 protein of Pseudomonas aeruginosa. Using the FoldAmyloid, Waltz, Pasta 2.0, and AGGRESCAN programs, amyloidogenic regions of the protein were predicted, which play a key role in its aggregation. The method of limited proteolysis in combination with high performance liquid chromatography and mass spectrometric analysis of the products, made it possible to identify regions of the S1 protein from P. aeruginosa that are protected from the action of proteinase K, trypsin, and chymotrypsin. Sequences of theoretically predicted and experimentally identified amyloidogenic regions were used to synthesize four peptides, three of which demonstrated the ability to form amyloid-like fibrils, as shown by electron microscopy and fluorescence spectroscopy. The identified amyloidogenic sites can further serve as a basis for the development of new antibacterial peptides against the pathogenic microorganism P. aeruginosa.
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