Salt-dependent regulation of archaellins in Haloarcula marismortui.

Microorganisms require a motility structure to move towards optimal growth conditions. The motility structure from archaea, the archaellum, is fundamentally different from its bacterial counterpart, the flagellum, and is assembled in a similar fashion as type IV pili. The archaellum filament consists of thousands of copies of N-terminally processed archaellin proteins. Several archaea, such as the euryarchaeon Haloarcula marismortui, encode multiple archaellins. Two archaellins of H. marismortui display differential stability under various ionic strengths. This suggests that these proteins behave as ecoparalogs and perform the same function under different environmental conditions. Here, we explored this intriguing system to study the differential regulation of these ecoparalogous archaellins by monitoring their transcription, translation, and assembly into filaments. The salt concentration of the growth medium induced differential expression of the two archaellins. In addition, this analysis indicated that archaellation in H. marismortui is majorly regulated on the level of secretion, by a still unknown mechanism. These findings indicate that in archaea, multiple encoded archaellins are not completely redundant, but in fact can display subtle functional differences, which enable cells to cope with varying environmental conditions.

Determinants of the species selectivity of oxazolidinone antibiotics targeting the large ribosomal subunit.

Oxazolidinone antibiotics bind to the highly conserved peptidyl transferase center in the ribosome. For developing selective antibiotics, a profound understanding of the selectivity determinants is required. We have performed for the first time technically challenging molecular dynamics simulations in combination with molecular mechanics Poisson-Boltzmann surface area (MM-PBSA) free energy calculations of the oxazolidinones linezolid and radezolid bound to the large ribosomal subunits of the eubacterium Deinococcus radiodurans and the archaeon Haloarcula marismortui. A remarkably good agreement of the computed relative binding free energy with selectivity data available from experiment for linezolid is found. On an atomic level, the analyses reveal an intricate interplay of structural, energetic, and dynamic determinants of the species selectivity of oxazolidinone antibiotics: A structural decomposition of free energy components identifies influences that originate from first and second shell nucleotides of the binding sites and lead to (opposing) contributions from interaction energies, solvation, and entropic factors. These findings add another layer of complexity to the current knowledge on structure-activity relationships of oxazolidinones binding to the ribosome and suggest that selectivity analyses solely based on structural information and qualitative arguments on interactions may not reach far enough. The computational analyses presented here should be of sufficient accuracy to fill this gap.

On the specificity of antibiotics targeting the large ribosomal subunit.

The peptidyltransferase center of the large ribosomal subunit is responsible for catalyzing peptide bonds. This active site is the target of a variety of diverse antibiotics, many of which are used clinically. The past decade has seen a plethora of structures of antibiotics in complex with the large ribosomal subunit, providing unprecedented insight into the mechanism of action of these inhibitors. Ten distinct antibiotics (chloramphenicol, clindamycin, linezolid, tiamulin, sparsomycin, and five macrolides) have been crystallized in complex with four distinct ribosomal species, three bacterial, and one archaeal. This review aims to compare these structures in order to provide insight into the conserved and species-specific modes of interaction for particular members of each class of antibiotics. Coupled with the wealth of biochemical data, a picture is emerging defining the specific functional states of the ribosome that antibiotics preferentially target. Such mechanistic insight into antibiotic inhibition will be important for the development of the next generation of antimicrobial agents.

Binding and action of CEM-101, a new fluoroketolide antibiotic that inhibits protein synthesis.

We characterized the mechanism of action and the drug-binding site of a novel ketolide, CEM-101, which belongs to the latest class of macrolide antibiotics. CEM-101 shows high affinity for the ribosomes of Gram-negative (Escherichia coli) and Gram-positive (Staphylococcus aureus) bacteria. The ketolide shows high selectivity in its inhibitory action and readily interferes with synthesis of a reporter protein in the bacterial but not eukaryotic cell-free translation system. Binding of CEM-101 to its ribosomal target site was characterized biochemically and by X-ray crystallography. The X-ray structure of CEM-101 in complex with the E. coli ribosome shows that the drug binds in the major macrolide site in the upper part of the ribosomal exit tunnel. The lactone ring of the drug forms hydrophobic interactions with the walls of the tunnel, the desosamine sugar projects toward the peptidyl transferase center and interacts with the A2058/A2509 cleft, and the extended alkyl-aryl arm of the drug is oriented down the tunnel and makes contact with a base pair formed by A752 and U2609 of the 23S rRNA. The position of the CEM-101 alkyl-aryl extended arm differs from that reported for the side chain of the ketolide telithromycin complexed with either bacterial (Deinococcus radiodurans) or archaeal (Haloarcula marismortui) large ribosomal subunits but closely matches the position of the side chain of telithromycin complexed to the E. coli ribosome. A difference in the chemical structure of the side chain of CEM-101 in comparison with the side chain of telithromycin and the presence of the fluorine atom at position 2 of the lactone ring likely account for the superior activity of CEM-101. The results of chemical probing suggest that the orientation of the CEM-101 extended side chain observed in the E. coli ribosome closely resembles its placement in Staphylococcus aureus ribosomes and thus likely accurately reflects interaction of CEM-101 with the ribosomes of the pathogenic bacterial targets of the drug. Chemical probing further demonstrated weak binding of CEM-101, but not of erythromycin, to the ribosome dimethylated at A2058 by the action of Erm methyltransferase.

First evidence for the salt-dependent folding and activity of an esterase from the halophilic archaea Haloarcula marismortui.

A gene encoding an esterase from Haloarcula marismortui, a halophilic archaea from the Dead Sea, was cloned, expressed in Escherichia coli, and the recombinant protein (Hm EST) was biochemically characterized. The enzymatic activity of Hm EST was shown to exhibit salt dependence through salt-dependent folding. Hm EST exhibits a preference for short chain fatty acids and monoesters. It is inhibited by phenylmethylsulfonyl fluoride, diethyl-p-nitrophenyl phosphate, and 5-methoxy-3-(4-phenoxyphenyl)-3H-[1,3,4]oxadiazol-2-one, confirming the conclusion from sequence alignments that Hm EST is a serine carboxylesterase belonging to the hormone-sensitive lipase family. The activity of Hm EST is optimum in the presence of 3 M KCl and no activity was detected in the absence of salts. Far-UV circular dichroism showed that Hm EST is totally unfolded in salt-free medium and secondary structure appears in the presence of 0.25-0.5 M KCl. After salt depletion, the protein was able to recover 60% of its initial activity when 2 M KCl was added. A 3D model of Hm EST was built and its surface properties were analyzed, pointing to an enrichment in acidic residues paralleled by a depletion in basic residues. This peculiar charge repartition at the protein surface supports a better stability of the protein in a high salt environment.

Negamycin binds to the wall of the nascent chain exit tunnel of the 50S ribosomal subunit.

Negamycin, a small-molecule inhibitor of protein synthesis, binds the Haloarcula marismortui 50S ribosomal subunit at a single site formed by highly conserved RNA nucleotides near the cytosolic end of the nascent chain exit tunnel. The mechanism of antibiotic action and the function of this unexplored tunnel region remain intriguingly elusive.

Antibiotics targeting ribosomes: resistance, selectivity, synergism and cellular regulation.

Antibiotics target ribosomes at distinct locations within functionally relevant sites. They exert their inhibitory action by diverse modes, including competing with substrate binding, interfering with ribosomal dynamics, minimizing ribosomal mobility, facilitating miscoding, hampering the progression of the mRNA chain, and blocking the nascent protein exit tunnel. Although the ribosomes are highly conserved organelles, they possess subtle sequence and/or conformational variations. These enable drug selectivity, thus facilitating clinical usage. The structural implications of these differences were deciphered by comparisons of high-resolution structures of complexes of antibiotics with ribosomal particles from eubacteria resembling pathogens and from an archaeon that shares properties with eukaryotes. The various antibiotic-binding modes detected in these structures demonstrate that members of antibiotic families possessing common chemical elements with minute differences might bind to ribosomal pockets in significantly different modes, governed by their chemical properties. Similarly, the nature of seemingly identical mechanisms of drug resistance is dominated, directly or via cellular effects, by the antibiotics' chemical properties. The observed variability in antibiotic binding and inhibitory modes justifies expectations for structurally based improved properties of existing compounds as well as for the discovery of novel drug classes.

AMP-forming acetyl-CoA synthetase from the extremely halophilic archaeon Haloarcula marismortui: purification, identification and expression of the encoding gene, and phylogenetic affiliation.

Halophilic archaea activate acetate via an (acetate)-inducible AMP-forming acetyl-CoA synthetase (ACS), (Acetate+ATP+CoA --> Acetyl-CoA+AMP+PP(i)). The enzyme from Haloarcula marismortui was purified to homogeneity. It constitutes a 72-kDa monomer and exhibited a temperature optimum of 41 degrees C and a pH optimum of 7.5. For optimal activity, concentrations between 1 M and 1.5 M KCl were required, whereas NaCl had no effect. The enzyme was specific for acetate (100%) additionally accepting only propionate (30%) as substrate. The kinetic constants were determined in both directions of the reaction at 37 degrees C. Using the N-terminal amino acid sequence an open reading frame - coding for a 74 kDa protein - was identified in the partially sequenced genome of H. marismortui. The function of the ORF as acs gene was proven by functional overexpression in Escherichia coli. The recombinant enzyme was reactivated from inclusion bodies, following solubilization in urea and refolding in the presence of salts, reduced and oxidized glutathione and substrates. Refolding was dependent on salt concentrations of at least 2 M KCl. The recombinant enzyme showed almost identical molecular and catalytic properties as the native enzyme. Sequence comparison of the Haloarcula ACS indicate high similarity to characterized ACSs from bacteria and eukarya and the archaeon Methanosaeta. Phylogenetic analysis of ACS sequences from all three domains revealed a distinct archaeal cluster suggesting monophyletic origin of archaeal ACS.