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PubMed2003

Structure of the L1 protuberance in the ribosome.

The L1 protuberance of the 50S ribosomal subunit is implicated in the release/disposal of deacylated tRNA from the E site. The apparent mobility of this ribosomal region has thus far prevented an accurate determination of its three-dimensional structure within either the 50S subunit or the 70S ribosome. Here we report the crystal structure at 2.65 A resolution of ribosomal protein L1 from Sulfolobus acidocaldarius in complex with a specific 55-nucleotide fragment of 23S rRNA from Thermus thermophilus. This structure fills a major gap in current models of the 50S ribosomal subunit. The conformations of L1 and of the rRNA fragment differ dramatically from those within the crystallographic model of the T. thermophilus 70S ribosome. Incorporation of the L1-rRNA complex into the structural models of the T. thermophilus 70S ribosome and the Deinococcus radiodurans 50S subunit gives a reliable representation of most of the L1 protuberance within the ribosome.

Amino Acid Sequence↗
PubMed2003

Structural insight into the role of the ribosomal tunnel in cellular regulation.

Nascent proteins emerge out of ribosomes through an exit tunnel, which was assumed to be a firmly built passive path. Recent biochemical results, however, indicate that the tunnel plays an active role in sequence-specific gating of nascent chains and in responding to cellular signals. Consistently, modulation of the tunnel shape, caused by the binding of the semi-synthetic macrolide troleandomycin to the large ribosomal subunit from Deinococcus radiodurans, was revealed crystallographically. The results provide insights into the tunnel dynamics at high resolution. Here we show that, in addition to the typical steric blockage of the ribosomal tunnel by macrolides, troleandomycin induces a conformational rearrangement in a wall constituent, protein L22, flipping the tip of its highly conserved beta-hairpin across the tunnel. On the basis of mutations that alleviate elongation arrest, the tunnel motion could be correlated with sequence discrimination and gating, suggesting that specific arrest motifs within nascent chain sequences may induce a similar gating mechanism.

Amino Acid Sequence↗
PubMed1999

The identification of a new family of sugar efflux pumps in Escherichia coli.

Using a functional cloning strategy with an Escherichia coli genomic plasmid library, we have identified a new family of sugar efflux proteins with three highly homologous members in the E. coli genome. In addition, two open reading frames, one present in Yersinia pestis and the other in Deinococcus radiodurans, appear to encode closely related proteins. An in vitro transport assay using inside-out membrane vesicles prepared from overproducing strains was used to demonstrate that members of this new family can efflux [14C]-lactose. As sugar efflux phenomena have been reported previously in several bacterial species including E. coli, the identification of a new family of sugar efflux proteins may help to reveal the physiological role of sugar efflux in metabolism. It is proposed that the E. coli members of this family, whose functions were previously unknown, be given the gene family designation SET for sugar efflux transporter.

Antiporters↗
PubMed1999

Evolutionary relationships among photosynthetic prokaryotes (Heliobacterium chlorum, Chloroflexus aurantiacus, cyanobacteria, Chlorobium tepidum and proteobacteria): implications regarding the origin of photosynthesis.

The presence of shared conserved insertions or deletions in proteins (referred to as signature sequences) provides a powerful means to deduce the evolutionary relationships among prokaryotic organisms. This approach was used in the present work to deduce the branching orders of various eubacterial taxa consisting of photosynthetic organisms. For this purpose, portions of the Hsp60 and Hsp70 genes, covering known signature sequence regions, were PCR-amplified and sequenced from Heliobacterium chlorum, Chloroflexus aurantiacus and Chlorobium tepidum. This information was integrated with sequence data for several other proteins from numerous species to deduce the branching orders of different photosynthetic taxa. Based on signature sequences that are present in different proteins, it is possible to infer that the various eubacterial phyla evolved from a common ancestor in the following order: low G+C Gram-positive (H. chlorum) --> high G+C Gram-positive --> Deinococcus-Thermus --> green non-sulphur bacteria (Cf. aurantiacus ) --> cyanobacteria --> spirochaetes --> Chlamydia-Cytophaga-Aquifex-flavobacteria-green sulphur bacteria (Cb. tepidum) --> proteobacteria (alpha, delta and epsilon) and --> proteobacteria (beta and gamma). The members of the Heliobacteriaceae family that contain a Fe-S type of reaction centre (RC-1) and represent the sole photosynthetic phylum from the Gram-positive or monoderm group of prokaryotes are indicated to be the most ancestral of the photosynthetic lineages. Among the Gram-negative bacteria or diderm prokaryotes, green non-sulphur bacteria such as Cf. aurantiacus, which contains a pheophytin-quinone type of reaction centre (RC-2), are indicated to have evolved very early. Thus, the organisms containing either RC-1 or RC-2 existed before the evolution of cyanobacteria, which contain both these reaction centres to carry out oxygenic photosynthesis. The eubacterial divisions consisting of green sulphur bacteria and proteobacteria are indicated to have diverged after cyanobacteria. Some implications of these results concerning the origin of photosynthesis and the earliest prokaryotic fossils are discussed.

Amino Acid Sequence↗
PubMed2003

Acyl-homoserine lactone acylase from Ralstonia strain XJ12B represents a novel and potent class of quorum-quenching enzymes.

N-acylhomoserine lactones (AHLs) are used as signal molecules by many quorum-sensing Proteobacteria. Diverse plant and animal pathogens use AHLs to regulate infection and virulence functions. These signals are subject to biological inactivation by AHL-lactonases and AHL-acylases. Previously, little was known about the molecular details underlying the latter mechanism. An AHL signal-inactivating bacterium, identified as a Ralstonia sp., was isolated from a mixed-species biofilm. The signal inactivation encoding gene from this organism, which we call aiiD, was cloned and successfully expressed in Escherichia coli and inactivated three AHLs tested. The predicted 794-amino-acid polypeptide was most similar to the aculeacin A acylase (AAC) from Actinoplanes utahensis and also shared significant similarities with cephalosporin acylases and other N-terminal (Ntn) hydrolases. However, the most similar homologues of AiiD are deduced proteins of undemonstrated function from available Ralstonia, Deinococcus and Pseudomonas genomes. LC-MS analyses demonstrated that AiiD hydrolyses the AHL amide, releasing homoserine lactone and the corresponding fatty acid. Expression of AiiD in Pseudomonas aeruginosa PAO1 quenched quorum sensing by this bacterium, decreasing its ability to swarm, produce elastase and pyocyanin and to paralyze nematodes. Thus, AHL-acylases have fundamental implications and hold biotechnological promise in quenching quorum sensing.

4-Butyrolactone↗
PubMed2002

A poxvirus-like type IB topoisomerase family in bacteria.

We report that diverse species of bacteria encode a type IB DNA topoisomerase that resembles vaccinia virus topoisomerase. Deinococcus radiodurans topoisomerase IB (DraTopIB), an exemplary member of this family, relaxes supercoiled DNA in the absence of a divalent cation or ATP. DraTopIB has a compact size (346 aa) and is a monomer in solution. Mutational analysis shows that the active site of DraTopIB is composed of the same constellation of catalytic side chains as the vaccinia enzyme. Sequence comparisons and limited proteolysis suggest that their folds are conserved. These findings imply an intimate evolutionary relationship between the poxvirus and bacterial type IB enzymes, and they engender a scheme for the evolution of topoisomerase IB and tyrosine recombinases from a common ancestral strand transferase in the bacterial domain. Remarkably, bacteria that possess topoisomerase IB appear to lack DNA topoisomerase III.

Amino Acid Motifs↗
PubMed2005

Heptameric (L12)6/L10 rather than canonical pentameric complexes are found by tandem MS of intact ribosomes from thermophilic bacteria.

Ribosomes are universal translators of the genetic code into protein and represent macromolecular structures that are asymmetric, often heterogeneous, and contain dynamic regions. These properties pose considerable challenges for modern-day structural biology. Despite these obstacles, high-resolution x-ray structures of the 30S and 50S subunits have revealed the RNA architecture and its interactions with proteins for ribosomes from Thermus thermophilus, Deinococcus radiodurans, and Haloarcula marismortui. Some regions, however, remain inaccessible to these high-resolution approaches because of their high conformational dynamics and potential heterogeneity, specifically the so-called L7/L12 stalk complex. This region plays a vital role in protein synthesis by interacting with GTPase factors in translation. Here, we apply tandem MS, an approach widely applied to peptide sequencing for proteomic applications but not previously applied to MDa complexes. Isolation and activation of ions assigned to intact 30S and 50S subunits releases proteins S6 and L12, respectively. Importantly, this process reveals, exclusively while attached to ribosomes, a phosphorylation of L12, the protein located in multiple copies at the tip of the stalk complex. Moreover, through tandem MS we discovered a stoichiometry for the stalk protuberance on Thermus thermophilus and other thermophiles and contrast this assembly with the analogous one on ribosomes from mesophiles. Together with evidence for a potential interaction with the degradosome, these results show that important findings on ribosome structure, interactions, and modifications can be discovered by tandem MS, even on well studied ribosomes from Thermus thermophilus.

Amino Acid Sequence↗
PubMed2006

Molecular analysis of the bacterial microbiota in the human stomach.

The microbiota of the human stomach and the influence of Helicobacter pylori colonization on its composition remain largely unknown. We characterized bacterial diversity within the human gastric mucosa by using a small subunit 16S rDNA clone library approach and analyzed 1,833 sequences generated by broad-range bacterial PCR from 23 gastric endoscopic biopsy samples. A diverse community of 128 phylotypes was identified, featuring diversity at this site greater than previously described. The majority of sequences were assigned to the Proteobacteria, Firmicutes, Actinobacteria, Bacteroidetes, and Fusobacteria phyla. Ten percent of the phylotypes were previously uncharacterized, including a Deinococcus-related organism, relatives of which have been found in extreme environments but not reported before in humans. The gastric clone libraries from 19 subjects contained H. pylori rDNA; however, only 12 of these subjects tested positive for H. pylori by conventional laboratory methods. Statistical analysis revealed a large degree of intersubject variability of the gastric ecosystem. The presence of H. pylori did not affect the composition of the gastric community. This gastric bacterial rDNA data set was significantly different from sequence collections of the human mouth and esophagus described in other studies, indicating that the human stomach may be home to a distinct microbial eco-system. The gastric microbiota may play important, as-yet-undiscovered roles in human health and disease.

Adult↗
PubMed2003

A lupus-like syndrome develops in mice lacking the Ro 60-kDa protein, a major lupus autoantigen.

Antibodies against a conserved RNA-binding protein, the Ro 60-kDa autoantigen, occur in 24-60% of all patients with systemic lupus erythematosus. Anti-Ro antibodies are correlated with photosensitivity and cutaneous lesions in these patients and with neonatal lupus, a syndrome in which mothers with anti-Ro antibodies give birth to children with complete congenital heart block and photosensitive skin lesions. In higher eukaryotes, the Ro protein binds small RNAs of unknown function known as Y RNAs. Because the Ro protein also binds misfolded 5S rRNA precursors, it is proposed to function in a quality-control pathway for ribosome biogenesis. Consistent with a role in the recognition or repair of intracellular damage, an orthologue of Ro in the radiation-resistant eubacterium Deinococcus radiodurans contributes to survival of this bacterium after UV irradiation. Here, we show that mice lacking the Ro protein develop an autoimmune syndrome characterized by anti-ribosome antibodies, anti-chromatin antibodies, and glomerulonephritis. Moreover, in one strain background, Ro-/- mice display increased sensitivity to irradiation with UV light. Thus, one function of this major human autoantigen may be to protect against autoantibody development, possibly by sequestering defective ribonucleoproteins from immune surveillance. Furthermore, the finding that mice lacking the Ro protein are photosensitive suggests that loss of Ro function could contribute to the photosensitivity associated with anti-Ro antibodies in humans.

Animals↗
PubMed2000

ROP-1, an RNA quality-control pathway component, affects Caenorhabditis elegans dauer formation.

Caenorhabditis elegans dauer formation is an alternative larval developmental pathway that the worm can take when environmental conditions become detrimental. Animals can survive several months in this stress-resistant stage and can resume normal development when growth conditions improve. Although the worms integrate a variety of sensory information to commit to dauer formation, it is currently unknown whether they also monitor internal cellular damage. The Ro ribonucleoprotein complex, which was initially described as a human autoantigen, is composed of one major 60-kDa protein, Ro60, that binds to one of four small RNA molecules, designated Y RNAs. Ro60 has been shown to bind mutant 5S rRNA molecules in Xenopus oocytes, suggesting a role for Ro60 in 5S rRNA biogenesis. Analysis of ribosomes from a C. elegans rop-1(-) strain, which is null for the expression of Ro60, demonstrated that they contain a high percentage of mutant 5S rRNA molecules, thereby strengthening the notion of a link between the rop-1 gene product and 5S rRNA quality control. The Ro particle was recently shown to be involved in the resistance of Deinococcus radiodurans to UV irradiation, suggesting a role for the Ro complex in stress resistance. We have studied the role of rop-1 in dauer formation. We present genetic and biochemical evidence that rop-1 interacts with dauer-formation genes and is involved in the regulation of the worms' entry into the dauer stage. Furthermore, we find that the rop-1 gene product undergoes a proteolytic processing step that is regulated by the dauer formation pathway via an aspartic proteinase. These results suggest that the Ro particle may function in an RNA quality-control checkpoint for dauer formation.

Animals↗
PubMed1990

Thickness determination of biological samples with a zeta-calibrated scanning tunneling microscope.

A single-tube scanning tunneling microscope has been zeta-calibrated by using atomic steps of crystalline gold and was used for measuring the thickness of two biological samples, metal-coated as well as uncoated. The hexagonal surface layer of the bacterium Deinococcus radiodurans with an open network-type structure shows thickness values that are strongly influenced by the substrate and the preparation method. In contrast, the thickness of the purple membrane of Halobacterium halobium with its densely packed less-corrugated structure exhibits very little variation in thickness in coated preparations and the values obtained are in good agreement with x-ray data.

Bacteriorhodopsins↗
PubMed1994

Atomic force microscopy produces faithful high-resolution images of protein surfaces in an aqueous environment.

The atomic force microscope has the potential to monitor structural changes of a biological system in its native environment. To correlate them with the biological function at a molecular level, high lateral and vertical resolution are required. Here we demonstrate that the atomic force microscope is capable of imaging the surface of the hexagonally packed intermediate layer of Deinococcus radiodurans in buffer solution with a lateral resolution of 1 nm and a vertical resolution of 0.1 nm. On average, these topographs differ from those determined by electron microscopy by <0.5 nm.

Journal Article↗
PubMed1997

B12-dependent ribonucleotide reductases from deeply rooted eubacteria are structurally related to the aerobic enzyme from Escherichia coli.

The ribonucleotide reductases from three ancient eubacteria, the hyperthermophilic Thermotoga maritima (TM), the radioresistant Deinococcus radiodurans (DR), and the thermophilic photosynthetic Chloroflexus aurantiacus, were found to be coenzyme-B12 (class II) enzymes, similar to the earlier described reductases from the archaebacteria Thermoplasma acidophila and Pyrococcus furiosus. Reduction of CDP by the purified TM and DR enzymes requires adenosylcobalamin and DTT. dATP is a positive allosteric effector, but stimulation of the TM enzyme only occurs close to the temperature optimum of 80-90 degrees C. The TM and DR genes were cloned by PCR from peptide sequence information. The TM gene was sequenced completely and expressed in Escherichia coli. The deduced amino acid sequences of the two eubacterial enzymes are homologous to those of the archaebacteria. They can also be aligned to the sequence of the large protein of the aerobic E. coli ribonucleotide reductase that belongs to a different class (class I), which is not dependent on B12. Structure determinations of the E. coli reductase complexed with substrate and allosteric effectors earlier demonstrated a 10-stranded beta/alpha-barrel in the active site. From the conservation of substrate- and effector-binding residues we propose that the B12-dependent class II enzymes contain a similar barrel.

Amino Acid Sequence↗
PubMed1999

Controlled unzipping of a bacterial surface layer with atomic force microscopy.

We have combined high-resolution atomic force microscopy (AFM) imaging and force spectroscopy to gain insight into the interaction forces between the individual protomers of the hexagonally packed intermediate (HPI) layer of Deinococcus radiodurans. After imaging the HPI layer, the AFM stylus was attached to individual protomers by enforced stylus-sample contact to allow force spectroscopy experiments. Imaging of the HPI layer after recording force-extension curves allowed adhesion forces to be correlated with structural alterations. By using this approach, individual protomers of the HPI layer were found to be removed at pulling forces of approximately 300 pN. Furthermore, it was possible to sequentially unzip entire bacterial pores formed by six HPI protomers. The combination of high-resolution AFM imaging of individual proteins with the determination of their intramolecular forces is a method of studying the mechanical stability of supramolecular structures at the level of single molecules.

Gram-Positive Cocci↗
PubMed2002

Cysteine activation is an inherent in vitro property of prolyl-tRNA synthetases.

Aminoacyl-tRNA synthetases are well known for their remarkable precision in substrate selection during aminoacyl-tRNA formation. Some synthetases enhance the accuracy of this process by editing mechanisms that lead to hydrolysis of incorrectly activated and/or charged amino acids. Prolyl-tRNA synthetases (ProRSs) can be divided into two structurally divergent groups, archaeal-type and bacterial-type enzymes. A striking difference between these groups is the presence of an insertion domain (approximately 180 amino acids) in the bacterial-type ProRS. Because the archaeal-type ProRS enzymes have been shown to recognize cysteine, we tested selected ProRSs from all three domains of life to determine whether cysteine activation is a general property of ProRS. Here we show that cysteine is activated by recombinant ProRS enzymes from the archaea Methanocaldococcus jannaschii and Methanothermobacter thermautotrophicus, from the eukaryote Saccharomyces cerevisiae, and from the bacteria Aquifex aeolicus, Borrelia burgdorferi, Clostridium sticklandii, Cytophaga hutchinsonii, Deinococcus radiodurans, Escherichia coli, Magnetospirillum magnetotacticum, Novosphingobium aromaticivorans, Rhodopseudomonas palustris, and Thermus thermophilus. This non-cognate amino acid was efficiently acylated in vitro onto tRNA(Pro), and the misacylated Cys-tRNA(Pro) was not edited by ProRS. Therefore, ProRS exhibits a natural level of mischarging that is to date unequalled among the aminoacyl-tRNA synthetases.

Amino Acid Sequence↗
PubMed2006

Bacterial flavodoxins support nitric oxide production by Bacillus subtilis nitric-oxide synthase.

Unlike animal nitric-oxide synthases (NOSs), the bacterial NOS enzymes have no attached flavoprotein domain to reduce their heme and so must rely on unknown bacterial proteins for electrons. We tested the ability of two Bacillus subtilis flavodoxins (YkuN and YkuP) to support catalysis by purified B. subtilis NOS (bsNOS). When an NADPH-utilizing bacterial flavodoxin reductase (FLDR) was added to reduce YkuP or YkuN, both supported NO synthesis from either L-arginine or N-hydroxyarginine and supported a linear nitrite accumulation over a 30-min reaction period. Rates of nitrite production were directly dependent on the ratio of YkuN or YkuP to bsNOS. However, the V/Km value for YkuN (5.2 x 10(5)) was about 20 times greater than that of YkuP (2.6 x 10(4)), indicating YkuN is more efficient in supporting bsNOS catalysis. YkuN that was either photo-reduced or prereduced by FLDR transferred an electron to the bsNOS ferric heme at rates similar to those measured for heme reduction in the animal NOSs. YkuN supported a similar NO synthesis activity by a different bacterial NOS (Deinococcus radiodurans) but not by any of the three mammalian NOS oxygenase domains nor by an insect NOS oxygenase domain. Our results establish YkuN as a kinetically competent redox partner for bsNOS and suggest that FLDR/flavodoxin proteins could function physiologically to support catalysis by bacterial NOSs.

Animals↗
PubMed2003

A novel complexity measure for comparative analysis of protein sequences from complete genomes.

Analysis of sequence complexities of proteins is an important step in the characterization and classification of new genomes. A new measure has been proposed to compute sequence complexity in protein sequences based on linguistic complexity. The algorithm requires a single parameter, is computationally simple and provides a framework for comparative genomic analysis. Protein sequences were classified into groups of high or low complexity based on a quantitative measure termed F(c), which is proportional to the fraction of low complexity sequence present in the protein. The algorithm was tested on sequences of 196 non-homologous proteins whose crystal structures are available at </=2.0 A resolution. Protein sequences of high complexity had 'globular' structures (95% agreement), whereas those of low complexity had non-globular structures (80% agreement). Application of this measure to proteins of unknown structure/function from different genomes revealed that the sequences of high complexity constitute the majority in all genomes (about 90% in Archaea, about 93% in Eubacteria, 89% in Saccharomyces cerevisiae and 90% in Caenorhabditis elegans). Aeropyrum pernix among Archaeae and Deinococcus radiodurans among Eubacteria have the lowest fraction of high complexity proteins (75% and 80% respectively). Further, it was observed that a few bacterial pathogens (Mycobacterium tuberculosis, Pseudomonas aeruginosa) have high fraction of low complexity proteins. The program ScanCom is available from the authors as a PERL script (UNIX system).

Algorithms↗
PubMed2000

The natural evolutionary relationships among prokaryotes.

Two contrasting and very different proposals have been put forward to account for the evolutionary relationships among prokaryotes. The currently widely accepted three domain proposal by Woese et al. (Proc. Natl. Acad. Sci. USA (1990) 87: 4576-4579) calls for the division of prokaryotes into two primary groups or domains, termed archaebacteria (Archaea) and eubacteria (Bacteria), both of which are suggested to have originated independently from a universal ancestor. However, this proposal, which is based primarily on genes involved in the information transfer processes, is inconsistent with the ultrastructural characteristics of prokaryotes as well as with many gene phylogenies and provides no explanation as to how the structural and molecular differences seen between these groups arose and how other prokaryotic taxa are related or evolved from the common ancestor. It also postulates that the last common ancestor of all organisms was a hypothetical entity lacking a cell membrane, which is contrary to the basic requirement of a cell membrane to define and separate all forms of life from the surrounding environment. A second alternate proposal for the evolutionary relationships among prokaryotes has emerged from extensive analyses of numerous conserved inserts and deletions found in various proteins (Gupta, R. S., Microbiol. Mol. Biol. Rev. (1998)62: 1435-1491; FEMS Microbiol. Rev. (2000) 24: in press. This proposal points to a specific relationship between archaebacteria and gram-positive bacteria, both of which are prokaryotes bounded by a single cell membrane (monoderm prokaryotes). Gram-negative bacteria, which are bounded by two different membranes (diderm prokaryotes), are indicated to comprise a structurally and phylogenetically distinct taxa originating from gram-positive bacteria. This proposal postulates that the earliest prokaryote was a gram-positive bacteria from which both archaebacteria and diderm prokaryotes evolved by normal evolutionary mechanisms in response to the strong selection pressure exerted by antibiotics produced by certain groups of gram-positive bacteria. This proposal accounts for both the molecular as well structural differences seen among the main groups of prokaryotes by known evolutionary mechanisms without invoking any hypothetical process or entity and thus is a closer representation of the natural relationships among prokaryotes than the proposal for two distinct domains. Based on this new proposal, it is now possible to logically deduce the branching order of different prokaryotic taxa from the common ancestor, which is as follows: Gram-positive bacteria (Low G + C) (<=> Archaebacteria) => Gram-positive bacteria (High G + C) (<=> Archaebacteria)=> Deinococcus-Thermus => Green nonsulfur bacteria => Cyanobacteria => Spirochetes => Chlamydia- Cytophaga-Green sulfur bacteria => Proteobacteria-1 (epsilon, delta)=> Proteobacteria-2 (alpha) => Proteobacteria-3 (beta) => Proteobacteria-4 (gamma). A surprising but very important aspect of the relationship deduced here is that the main eubacterial phyla are related to each other linearly rather than in a tree-like manner, suggesting that the major evolutionary changes within prokaryotes (bacteria) have occurred in a directional manner.

Archaea↗