Photoactive yellow protein from the halophilic bacterium Salinibacter ruber

A gene for photoactive yellow protein (PYP) was identified from the genome sequence of the extremely halophilic aerobic bacterium Salinibacter ruber (Sr). The sequence is distantly related to the prototypic PYP from Halorhodospira halophila (Hh) (37% identity) and contains most of the amino acid residues identified as necessary for function. However, the Sr pyp gene is not flanked by its two biosynthetic genes as in other species. To determine as to whether the Sr pyp gene encodes a functional protein, we cloned and expressed it in Escherichia coli, along with the genes for chromophore biosynthesis from Rhodobacter capsulatus. The Sr PYP has a 31-residue N-terminal extension as compared to other PYPs that appears to be important for dimerization; however, truncation of these extra residues did not change the spectral and photokinetic properties. Sr PYP has an absorption maximum at 431 nm, which is at shorter wavelengths than the prototypical Hh PYP (at 446 nm). It is also photoactive, being reversibly bleached by either blue or white light. The kinetics of dark recovery is slower than any of the PYPs reported to date (4.27 x 10(-4) s(-1) at pH 7.5). Sr PYP appears to have a normal photocycle with the I1 and I2 intermediates. The presence of the I2' intermediate is also inferred on the basis of the effects of temperature and alchohol on recovery. Sr PYP has an intermediate spectral form in equilibrium with the 431 nm form, similar to R. capsulatus PYP and the Y42F mutant of Hh PYP. Increasing ionic strength stabilizes the 431 nm form at the expense of the intermediate spectral form, and the kinetics of recovery is accelerated 6.4-fold between 0 and 3.5 M salt. This is observed with ions from both the chaotropic and the kosmotropic series. Ionic strength also stabilizes PYP against thermal denaturation, as the melting temperature is increased from 74 degrees C in buffer alone to 92 degrees C in 2 M KCl. Sr accumulates KCl in the cytoplasm, like Halobacterium, to balance osmotic pressure and has very acidic proteins. We thus believe that Sr PYP is an example of a halophilic protein that requires KCl to electrostatically screen the excess negative charge and stabilize the tertiary structure.     

Intraspecific comparative analysis of the species Salinibacter ruber

Salinibacter ruber is the first extremely halophilic member of the Bacteria domain of proven environmental relevance in hypersaline brines at or approaching NaCl saturation, that has been brought to pure culture. A collection of 17 strains isolated from five different geographical locations (Mallorca, Alicante, Ebro Delta, Canary Islands, and Peruvian Andes) were studied following the currently accepted taxonomic approach. Additionally, random amplification of genomic DNA led to the phenetic analysis of the intraspecific diversity. Altogether the taxonomic study indicated that S. ruber remained highly homogeneous beyond any geographical barrier. However, genomic fingerprints indicated that populations from different isolation sites could still be discriminated.     

Phylogenetic characterization of transport protein superfamilies: superiority of SuperfamilyTree programs over those based on multiple alignments

Transport proteins function in the translocation of ions, solutes and macromolecules across cellular and organellar membranes. These integral membrane proteins fall into >600 families as tabulated in the Transporter Classification Database (www.tcdb.org). Recent studies, some of which are reported here, define distant phylogenetic relationships between families with the creation of superfamilies. Several of these are analyzed using a novel set of programs designed to allow reliable prediction of phylogenetic trees when sequence divergence is too great to allow the use of multiple alignments. These new programs, called SuperfamilyTree1 and 2 (SFT1 and 2), allow display of protein and family relationships, respectively, based on thousands of comparative BLAST scores rather than multiple alignments. Superfamilies analyzed include: (1) Aerolysins, (2) RTX Toxins, (3) Defensins, (4) Ion Transporters, (5) Bile/Arsenite/Riboflavin Transporters, (6) Cation:Proton Antiporters, and (7) the Glucose/Fructose/Lactose superfamily within the prokaryotic phosphoenol pyruvate-dependent Phosphotransferase System. In addition to defining the phylogenetic relationships of the proteins and families within these seven superfamilies, evidence is provided showing that the SFT programs outperform programs that are based on multiple alignments whenever sequence divergence of superfamily members is extensive. The SFT programs should be applicable to virtually any superfamily of proteins or nucleic acids.     

Distribution, abundance and diversity of the extremely halophilic bacterium Salinibacter ruber

Since its discovery in 1998, representatives of the extremely halophilic bacterium Salinibacter ruber have been found in many hypersaline environments across the world, including coastal and solar salterns and solar lakes. Here, we review the available information about the distribution, abundance and diversity of this member of the Bacteroidetes.     

Molecular adaptation: the malate dehydrogenase from the extreme halophilic bacterium Salinibacter ruber behaves like a non-halophilic protein

Malate dehydrogenase from the extreme halophilic bacterium, Salinibacter ruber (Sr MalDH) was purified and characterised as a tetramer by sedimentation velocity measurements, showing the enzyme belongs to the LDH-like group of MalDHs. In contrast to most other halophilic enzymes, which unfold when incubated at low salt concentration, Sr MalDH is completely stable in absence of salt. Its amino acid composition does not display the strong acidic character specific of halophilic proteins. The enzyme displays a strong KCl-concentration dependent variation in K(m) for oxaloacetate, but not for the NADH co-factor. Its activity is reduced by high salt concentration, but remains sufficient for the enzyme to sustain catalysis at approximately 30% of its maximal rates in 3 M KCl. The properties of the protein were compared with those from other LDH-like MalDHs of bacterial and archaeal origins, showing that Sr MalDH in fact behaves like a non-halophilic enzyme.     

Amino acid composition of bulk protein and salt relationships of selected enzymes of Salinibacter ruber,an extremely halophilic bacterium

The extremely halophilic bacterium Salinibacter ruber was previously shown to have a high intracellular potassium content, comparable to that of halophilic Archaea of the family Halobacteriaceae. The amino acid composition of its bulk protein showed a high content of acidic amino acids, a low abundance of basic amino acids, a low content of hydrophobic amino acids, and a high abundance of serine. We tested the level of four cytoplasmic enzymatic activities at different KCl and NaCl concentrations. Nicotinamide adenine dinucleotide (NAD)-dependent isocitrate dehydrogenase functioned optimally at 0.5-2 M KCl, with rates of 60% of the optimum value at 3.3 M. NaCl provided less activation: 70% of the optimum rates in KCl were found at 0.2-1.2 M NaCl, and above 3 M NaCl, activity was low. We also detected nicotinamide adenine dinucleotide phosphate (NADP)-dependent isocitrate activity, which remained approximately constant between 0-3.2 M NaCl and increased with increasing KCl concentration. NAD-dependent malate dehydrogenase functioned best in the absence of salt, but rates as high as 25% of the optimal values were measured in 3-3.5 M KCl or NaCl. NAD-dependent glutamate dehydrogenase, assayed by the reductive amination of 2-oxoglutarate, showed low activity in the absence of salt. NaCl was stimulatory with optimum activity at 3-3.5 M. However, no activity was found above 2.5 M KCl. Although the four activities examined all function at high salt concentrations, the behavior of individual enzymes toward salt varied considerably. The results presented show that Salinibacter enzymes are adapted to function in the presence of high salt concentrations.     

Characterization of polar membrane lipids of the extremely halophilic bacterium Salinibacter ruber and possible role of cardiolipin

The lipid composition of the extremely halophilic bacterium Salinibacter ruber (Bacteroidetes) was investigated by thin layer chromatography, gas chromatography, high performance liquid chromatography and electrospray ionization-mass spectrometry. Polar lipids represent about 80% of the total lipid extract. The main polar lipids are a sulfonic acid analogue of ceramide (or capnine analogue), phosphatidylcholine, phosphatidylserine, dimethylphosphatidylethanolamine, phosphatidylglycerol, cardiolipin or bisphosphatidylglycerol, and a glycolipid. The major acyl chains in the phospholipids are C16:1 Delta9cis and C18:1 Delta11cis, while the sulfonolipid contains an amide-bound iso C15:0 fatty acid. On changing the salinity of the culture medium, no significant differences were found in the lipid profile or the unsaturation of the lipid fatty acyl chains. The structure of the cardiolipin, which represents 20% of polar lipids, has been elucidated by gas chromatography and electrospray ionization mass spectrometry analysis.     

Phylogenetic position of the Hawaiian geraniums based on rbcL Sequences

The seven currently recognized species of Geranium endemic to the Hawaiian Islands are unusual in their shrubby or arborescent habit and unlobed, parallel-veined leaves rather than the palmately cleft or lobed leaves and herbaceous habit typical of the genus. Their placement within the genus and their biogeographic source have been obscured by this morphological distictiveness and the limited resolution of relationships on the basis of morphology in the very speciose subgenus Geranium. Phylogenetic analysis of rbcL gene sequences provides strong support for the monophyly of the Hawaiian group, and indicates that the Hawaiian clade is deeply nested within section Geranium rather than comprising a separate section. The continental relatives studied to date with the greatest similarity in sequence to the Hawaiian group are native to the Americas rather than Asia or the Pacific. The Hawaiian species are extremely similar to one another in rbcL sequence, while the tree topology obtained is consistent with a basal position for Geranium arboreum within the group.     

Phylogenetic position of Protoopalina intestinalis based on SSU rRNA gene sequence

A robust recognition of phylogenetic affinities of Opalinidae-the peculiar multinucleated intestine commensals of frogs-is hindered by the absence of reliable molecular data. Up to now all attempts to sequence opalinid genes failed, as the obtained sequences labeled as Protoopalina intestinalis, Cepedea virguloidea, and Opalina ranarum in GenBank apparently originate from a zygomycete contamination. In this paper, we present the first molecular data for the family Opalinidae-SSU rRNA gene of P. intestinalis. Our phylogenetic analyses undoubtedly show opalinids as a sister group to Proteromonas within the Stramenopila clade, confirming the monophyly of Patterson's order Slopalinida. The enigmatic genus Blastocystis is resolved with high statistical support as a sister group to Slopalinida. The information contained in the SSU rRNA gene proved insufficient to uncover broader affinities of this group to other groups of Stramenopila. Nevertheless, our analyses clearly demonstrate that Cavalier-Smith's phylum Bigyra, which comprises Oomycetes and their relatives together with Slopalinida and Blastocystis, is not monophyletic.     

Phylogenetic position of Dictyostelium inferred from multiple protein data sets

The phylogenetic position of Dictyostelium inferred from 18S rRNA data contradicts that from protein data. Protein trees always show the close affinity of Dictyostelium with animals, fungi, and plants, whereas in 18S rRNA trees the branching of Dictyostelium is placed at a position before the massive radiation of protist groups including the divergence of the three kingdoms. To settle this controversial issue and to determine the correct position of Dictyostelium, we inferred the phylogenetic relationship among Dictyostelium and the three kingdoms Animalia, Fungi, and Plantae by a maximum-likelihood method using 19 different protein data sets. It was shown at the significance level of 1 SE that the branching of Dictyostelium antedates the divergence of Animalia and Fungi, and Plantae is an outgroup of the Animalia-Fungi-Dictyostelium clade.Correspondence to: T. Miyata     

Phylogenetic assessment of alignments reveals neglected tree signal in gaps

Background  

The alignment of biological sequences is of chief importance to most evolutionary and comparative genomics studies, yet the two main approaches used to assess alignment accuracy have flaws: reference alignments are derived from the biased sample of proteins with known structure, and simulated data lack realism.     

Pfam: A comprehensive database of protein domain families based on seed alignments

Databases of multiple sequence alignments are a valuable aid to protein sequence classification and analysis. One of the main challenges when constructing such a database is to simultaneously satisfy the conflicting demands of completeness on the one hand and quality of alignment and domain definitions on the other. The latter properties are best dealt with by manual approaches, whereas completeness in practice is only amenable to automatic methods. Herein we present a database based on hidden Markov model profiles (HMMs), which combines high quality and completeness. Our database, Pfam, consists of parts A and B. Pfam-A is curated and contains well-characterized protein domain families with high quality alignments, which are maintained by using manually checked seed alignments and HMMs to find and align all members. Pfam-B contains sequence families that were generated automatically by applying the Domainer algorithm to cluster and align the remaining protein sequences after removal of Pfam-A domains. By using Pfam, a large number of previously unannotated proteins from the Caenorhabditis elegans genome project were classified. We have also identified many novel family memberships in known proteins, including new kazal, Fibronectin type III, and response regulator receiver domains. Pfam-A families have permanent accession numbers and form a library of HMMs available for searching and automatic annotation of new protein sequences. Proteins: 28:405–420, 1997. © 1997 Wiley-Liss, Inc.     

Phylogenetic position of the Tardigrada based on the 18S ribosomal RNA gene sequences

The phylogenetic position of the Tardigrada remains uncertain. This is due to the limited information available, and the uncertainty of whether some characters are homologous or analogous with other taxa. Based on some morphological characters, current discussion centres on whether the taxon branches from the annelid-arthropod lineage, or lies within the arthropod complex. The molecular data presented here from an analysis of the 18S rRNA gene sequences are used to test the validity of these two hypotheses. Phylogenetic inference by the maximum parsimony and distance (neighbour-joining) methods suggests that the Tardigrada is a sister group of the major protostome eucoelomate assemblage that emerged before the arthropods, annelids, molluscs, and sipunculids evolved. The tardigrade clade also appears as an independent lineage separate from the nematode clade, thus supporting the current idea that tardigrades do not have a close aschelminth relationship. The molecular data also imply that several morphological features, considered significant in determining the phylogenetic relationships of tardigrades, are not synapomorphic characters.     

Phylogenetic position of Dunaliella acidophila (Chlorophyceae) based on ITS and rbcL sequences

Dunaliella acidophila is one of the most extreme acidophiles on earth and is able to survive in highly acidic habitats. This characteristic has made this organism the universal model for the study of abiotic stress. Although D. acidophila is currently circumscribed to the subgenus Pascheria within Dunaliella Teodoresco (Chlorophyceae), its taxonomic position has stirred controversy. The comparison of D. acidophila CCAP19/35 internal transcribed spacers (including ITS2 secondary structure analysis) and RuBisCo large subunit (rbcL) sequences with other Dunaliella species confirms that D. acidophila should maintain its phylogenetic position within the genus Dunaliella, suggesting its inclusion within the subgenus Dunaliella. Furthermore, the ITS1 and ITS2 data revealed that D. acidophila was highly divergent from the other freshwater species assessed, D. lateralis, with which it barely shares a 56.8% similarity.     

Phylogenetic relations in section Arachis based on seed protein profile

Summary Seed protein profiles of nine diploid species (2n = 20), ten tetraploid accessions, two synthetic amphidiploids and two autotetraploids (2n = 40) were studied using SDS-polyacrylamide gel electrophoresis. While the general profiles suggested considerable homology among these taxa in spite of speciation and ploidy differences, appreciable genetic differences were present to support the existing genomic divisions and sub-divisions in the section Arachis. A high degree of relationship was indicated between the two diploid species (A. duranensis containing the A genome and A. batizocoi (ICG 8210) containing the B genome) and tetraploids A. monticola/ A. hypogaea (2n = 40) containing AABB genome. Similar relationships were recorded between the AABB synthetic amphidiploid and the profile obtained from the mixture of protein of A. duranensis and A. batizocoi, suggesting that these two diploid species were the donors of the A and B genome, respectively, to tetraploid A. monticola/A. hypogaea.Submitted as Journal Article No. 1114 by International Crops Research Institute for the Semi-Arid Tropics (ICRISAT)     

Twilight zone of protein sequence alignments

Sequence alignments unambiguously distinguish between protein pairs of similar and non-similar structure when the pairwise sequence identity is high (>40% for long alignments). The signal gets blurred in the twilight zone of 20-35% sequence identity. Here, more than a million sequence alignments were analysed between protein pairs of known structures to re-define a line distinguishing between true and false positives for low levels of similarity. Four results stood out. (i) The transition from the safe zone of sequence alignment into the twilight zone is described by an explosion of false negatives. More than 95% of all pairs detected in the twilight zone had different structures. More precisely, above a cut-off roughly corresponding to 30% sequence identity, 90% of the pairs were homologous; below 25% less than 10% were. (ii) Whether or not sequence homology implied structural identity depended crucially on the alignment length. For example, if 10 residues were similar in an alignment of length 16 (>60%), structural similarity could not be inferred. (iii) The 'more similar than identical' rule (discarding all pairs for which percentage similarity was lower than percentage identity) reduced false positives significantly. (iv) Using intermediate sequences for finding links between more distant families was almost as successful: pairs were predicted to be homologous when the respective sequence families had proteins in common. All findings are applicable to automatic database searches.