Primary structure of major outer-membrane protein I (ompF protein, porin) of Escherichia coli B/r.

In the outer membrane of Gram-negative bacteria hydrophilic pores exist, allowing the diffusion of various low-molecular-weight solutes. These pores are formed by proteins, the porins. In a preliminary communication [Chen, Krämer, Schmidmayr & Henning (1979) Proc. Natl. Acad. Sci. U.S.A. 76, 5014-5017] we presented the primary structure of one of these porins, the 340-amino-acid-residue protein I (ompF protein) from Escherichia coli B/r. In the present paper we give the experimental evidence for this sequence. Two tryptophan positions, one valine position, two aspartic acid positions and nine out of 82 amide determinations have been corrected. To aid further studies on this class of transmembrane proteins, the isolation of most of the constituent peptides is documented.     

Sequence evolution of the major satellite DNA of the genus Ctenomys (Octodontidae, Rodentia)

Sequence variability of RPCS (repetitive PuvII Ctenomys sequence), the major satellite DNA of octodontid Ctenomys rodents, was analysed in species belonging to three groups of species representing the two patterns of karyotypic evolution in the genus: stable and dynamic karyotypes among closely related species. The studied species represent the overall range of RPCS copy number (2000--6.6x10(6) copies per haploid genome) in the genus. RPCS sequence was characterised by PCR amplification of the genomic consensus sequence and cloned monomers. Our results suggest that RPCS genomic consensus sequence variability correlates with RPCS copy number stability and karyotypic stastis, but not with high or low RPCS copy number values. In contrast, the RPCS gcs shows a mutational profile that is similar across all analysed species. Our data suggest that an RPCS ancestral library of variants was maintained through the cladogenesis of the genus. There is also evidence pointing to the simultaneous contribution of processes of concerted evolution that resulted in a reduced representation of some ancestral variants and their partial replacement for new ones. In addition, analysis of distribution of the variability along the monomer suggests that subsequences of the RPCS are subject to some degree of constraint, probably driven by the recent replicative activity of RPCS in species with high copy number.     

Biogeographical contingency and the evolution of tropical anchovies (genus Cetengraulis) from temperate anchovies (genus Engraulis)

Aim Similar regimes of selection in different geographical settings can deterministically produce similar adaptive morphologies. We tested the hypothesis that the evolutionary trajectories of fish in upwelling zones can be altered by biogeographic contingencies in the biological and physical environment. Location Eastern Pacific and western Atlantic oceans. Methods We estimated phylogenetic relationships among eastern Pacific temperate anchovies (genus Engraulis) and tropical anchovies (genus Cetengraulis) with neighbour‐joining and Bayesian tree analysis of a 521‐bp segment of mitochondrial DNA cytochrome b. Available sequences for five additional engraulid taxa were included to establish polarity of the tree. Bayesian estimates (BEAST) of time to most recent common ancestor (TMRCA) for the nodes in the phylogeny were calibrated with divergence between Cetengraulis edentulus and Cetengraulis mysticetus precipitated by the rise of the Panama Isthmus 2.8–3.2 Ma. Results Neighbour‐joining and Bayesian trees indicate that South American Engraulis anchoita (Argentina) and Engraulis ringens (Chile) together are basal sister taxa to the California anchovy (Engraulis mordax) and Old World anchovies (Engraulis japonicas, Engraulis australis, Engraulis capensis and Engraulis encrasicolus). The two tropical species of Cetengraulis are sister‐taxa to Californian E. mordax, even though their phenotypes and ecologies differ markedly. A relaxed molecular clock indicates a TMRCA between Californian E. mordax and Cetengraulis at about 4.2 Ma (3.0–6.3 Ma 95% highest probability density). Main conclusions The TMRCA between the California anchovy, E. mordax, and tropical Cetengraulis coincides with the formation of the Gulf of California, which provided opportunities for allopatric isolation during climate oscillations. Mid‐Pliocene warming (3.1–2.9 Ma) may have trapped ancestors of Cetengraulis in the Gulf of California, where they evolved digestive tract morphologies to exploit inshore tropical habitats with low plankton productivities. While populations of several other temperate fishes have become isolated in the Gulf of California, few of these derived species show strong adaptive shifts from temperate sister taxa or range expansions into the tropical provinces of the western Atlantic and eastern Pacific.     

Lipopolysaccharides of bacterial pathogens from the genus Yersinia: a mini-review

This review summarizes the state of knowledge on the composition and structure of the lipopolysaccharides (LPS) from three species of Yersinia known to produce disease in humans: Y. pseudotuberculosis, Y. enterocolitica and Y. pestis. We also mention recent data on the genome sequence of Yersinia pestis and the role of LPS in relation to the virulence of this bacteria.     

Identification of Yersinia enterocolitica within the genus Yersinia

In this report we describe a PCR strategy for the unambigous identification of biochemically presumptive typed Yersinia (Y.) enterocolitica. A total of 269 isolates belonging to ten species of the genus Yersinia were investigated. In a first PCR only isolates classified as Y. enterocolitica (n = 113) gave rise to a specific amplification resulting in a sensitivity and a specificity of 100%. By sequencing the 269 amplicons of a second pan-Yersinia PCR spanning a distinct 16S rRNA gene region, 20 different sequence clusters could be identified within the genus. By this, Y. enterocolitica isolates of American and European origin could be distinguished safely and already described sequence clusters of the species Y. frederiksenii were confirmed. New 16S rRNA gene sequence clusters were detected for the species Y. frederiksenii, Y. intermedia, Y. mollaretii, Y. aldovae, Y. kristensenii, and Y. rohdei.     

Data reassessment in a phylogenetic context gives insight into chromosome evolution in the giant genus Solanum (Solanaceae)

Chromosome data are fundamental in evolution. However, there has been no attempt to synthesize and evaluate the significance of such information from a phylogenetic perspective in the giant genus Solanum, which was the aim of this work. New and published information of the main cytotaxonomic features (chromosome number, polyploidy, total length of the haploid complement, mean chromosome length, mean arm ratio, karyotype formula, nuclear DNA amount, number/position of rDNA sites) was compiled and mapped onto an embracing Solanaceae phylogeny, performing Ancestral States Reconstruction. There were 506 Solanum species with chromosome counts (49.7% from an estimated total of 1,018 spp.), with x?=?12 being the most frequent number (97%). Species with karyotypes represent 18.8%, while 8% have been studied with any molecular cytogenetic technique. Chromosome characters showed transitions associated with supported nodes, some of which have undergone fewer transitions than others. The common ancestor of all Solanum was a diploid with 2n?=?24, a karyotype with st and/or t chromosomes, 2C DNA content of 1–1.2 pg, one locus of 18–5.8–26S rDNA and one of 5S, both loci being asyntenic. The chromosomal variables behave as homoplastic, with reversions in all branches. The analysed characters were sorted from more to less conserved: asynteny of rDNA loci; number of sites of 18–5.8–26S; chromosome number; karyotype formula; number of 5S loci. This pattern of chromosomal evolution distinguishes Solanum from closely related genera and from genera from other families with a similar number of species.     

OmpC-like porin from Yersinia pseudotuberculosis: Molecular characteristics, physico-chemical and functional properties

Pore-forming protein from the outer membrane of Yersinia pseudotuberculosis cultured at 37°C has been isolated and characterized. Comparative analysis of the primary and three-dimensional structures of this protein and of OmpC porin from E. coli was carried out, functional properties of these proteins have been studied using bilayer lipid membranes (BLM) technique. The degree of homology, molecular mass and pore-forming properties of the isolated porin was found to be closer to those of OmpC porin from E. coli than OmpF porin from Y. pseudotuberculosis. The value of the most probable conductivity of OmpC porin from Y. pseudotuberculosis (0.18 pS) in BLM corresponded to the conductivity of the native trimer of this protein. Using CD spectroscopy, the porins investigated were shown to belong to the β-structured proteins. Data of the primary structure and intrinsic protein fluorescence revealed essential differences in localization and microenvironment of tryptophan residues in the porins investigated. Participation of external loops L2 and L6 in the formation of the antigenic structure of OmpC porin from Y. pseudotuberculosis was demonstrated. On the basis of crystal structure of osmoporin from Klebsiella pneumoniae, three-dimensional models of the monomer and trimer of the Y. pseudotuberculosis porin were obtained. Using Web server AGGRESCAN, the localization of protein structure sites with the increased aggregation capability (hot spots) has been deter-mined. It turned out that some of these zones localize in the region of intramonomeric contacts in the porin trimer; however, a large part of them is located on the external surface of the β-barrel. The process of thermal denaturation has been studied and the melting points of the porins were determined. It was found that significant changes in the microenvironment of the indole fluorophores (especially tryptophan residues of spectral class I) took place in the process of the thermodenaturation of the proteins. These changes preceded the irreversible conformational transition observed for the E. coli porin at 77°C and for the Y. pseudotuberculosis porin at 70°C.     

The protective properties of a porin from the outer membrane of Yersinia pseudotuberculosis

The protective properties of the species-specific pore-forming polypeptide of Y. pseudotuberculosis outer membrane (porin) were studied. The study showed that 80-90% of mice immunized with porin survived after challenge with 10-30 LD50 of Y. pseudotuberculosis, serovars 1 and 3. The LD50 of the preparation exceeded its ED50 more than 100-fold. Immunization made in two injections was more effective than immunization in one injection. The immunization of the animals with porin led to an increase in the activity of peritoneal exudate macrophages with respect to Y. pseudotuberculosis, serovars 1 and 3.     

Phylogenetic estimation and morphological evolution of Alsineae (Caryophyllaceae) shed new insight into the taxonomic status of the genus Pseudocerastium

Pseudocerastium is a monotypic genus in Caryophyllaceae endemic to China. The genus has been widely accepted since it was described in 1998, however its phylogenetic position within Caryophyllaceae has never been studied. In the present study, the whole plastid genome and nuclear ribosomal internal transcribed spacer (ITS) sequences of Pseudocerastium stellarioides was obtained through genome skimming, and the phylogenetic position of the species was studied for the first time. Plastid phylogenomic analysis of Caryophyllaceae revealed that Pseudocerastium is clustered within the tribe Alsineae with strong support. Phylogenetic analyses based on an enlarged taxon sampling of Alsineae using five DNA regions (matK, rbcL, rps16 intron, trnL-F and ITS) revealed that P. stellarioides was nested deeply within Cerastium with strong support. Analyses of morphological character evolution suggest that the ancestral states in Alsineae include three styles and a six-lobed capsule at the apex, while both Cerastium and Pseudocerastium have five styles and ten lobes at the apex of the capsule, further supporting their close relationship. The species Pseudocerastium stellarioides is similar to Cerastium wilsonii in morphology, but differs in having villous indumentum on the lower part of the filaments and compressed globose seeds. Therefore, based on the present molecular and morphological evidence, the generic name Pseudocerastium is reduced here as a new synonym of Cerastium and the species P. stellarioides is transferred to Cerastium as C. jiuhuashanense.     

Phylogenetics and the evolution of major structural characters in the giant genus Euphorbia L. (Euphorbiaceae)

Euphorbia is among the largest genera of angiosperms, with about 2000 species that are renowned for their remarkably diverse growth forms. To clarify phylogenetic relationships in the genus, we used maximum likelihood, bayesian, and parsimony analyses of DNA sequence data from 10 markers representing all three plant genomes, averaging more than 16kbp for each accession. Taxon sampling included 176 representatives from Euphorbioideae (including 161 of Euphorbia). Analyses of these data robustly resolve a backbone topology of four major, subgeneric clades--Esula, Rhizanthium, Euphorbia, and Chamaesyce--that are successively sister lineages. Ancestral state reconstructions of six reproductive and growth form characters indicate that the earliest Euphorbia species were likely woody, non-succulent plants with helically arranged leaves and 5-glanded cyathia in terminal inflorescences. The highly modified growth forms and reproductive features in Euphorbia have independent origins within the subgeneric clades. Examples of extreme parallelism in trait evolution include at least 14 origins of xeromorphic growth forms and at least 13 origins of seed caruncles. The evolution of growth form and inflorescence position are significantly correlated, and a pathway of evolutionary transitions is supported that has implications for the evolution of Euphorbia xerophytes of large stature. Such xerophytes total more than 400 species and are dominants of vegetation types throughout much of arid Africa and Madagascar.     

OmpC-like porin from outer membrane of Yersinia enterocolitica: Molecular structure and functional activity

OmpC-like porin was isolated from the outer membrane (OM) of Yersinia enterocolitica cultured at 37°C (the “warm” variant) and its physicochemical and functional properties were studied. The amino acid sequence of OmpC porin was established, and the primary structure and transmembrane topology of this protein were analyzed in comparison with the OmpF porin isolated from Y. enterocolitica cultured at 6°C (the “cold” variant). Both porins of Y. enterocolitica had a high homology degree (65%) between themselves and with OmpC and OmpF porins from OM of Escherichia coli (58 and 76% homology, respectively). The secondary structure of OmpC and OmpF porins from OM of Y. enterocolitica consists of 16 β-strands connected by short “periplasmic” and longer “extracellular” loops with disordered structure, according to the topological model developed for porins of E. coli. The molecular structures of OmpC and OmpF porins of Y. enterocolitica have significant differences in the structure of the “extracellular” loops and in the position of one of three tryptophan residues. Using the bilayer lipid membrane (BLM) technique, pores formed by OmpC porin of Y. enterocolitica were shown to differ in electrophysiological characteristics from channels of OmpF protein of this microorganism. The isolated OmpC porin reconstructed into BLM displayed functional plasticity similarly to OmpF protein and nonspecific porins of other enterobacteria. The conductivity level of the channels formed by this protein in the BLM was regulated by value of the applied potential.     

Isolation and characterization of recombinant OmpF-like porin from the Yersinia pseudotuberculosis outer membrane

The encoding sequence of the pore-forming OmpF-like protein from the Yersinia pseudotuberculosis outer membrane was cloned and expressed in Escherichia coli cells. Conditions for isolation and refolding of recombinant monomer and porin trimer were selected. Their spatial structures were characterized by the intrinsic protein fluorescence and CD spectroscopy. It was shown that recombinant porins are similar in the composition of secondary structure elements to isolated porins, but have a considerably less compact tertiary structure. The pore-forming activities of the recombinant proteins are similar to those of Y. pseudotuberculosis native porins.     

Isolation and characterization of recombinant OmpF-like porin from the Yersinia pseudotuberculosis outer membrane

The encoding sequence of the pore-forming OmpF-like protein from the Yersinia pseudotuberculosis outer membrane was cloned and expressed in Escherichia coli cells. Conditions were selected for isolation and refolding of recombinant monomer and porin trimer. Their spatial structures were characterized by the intrinsic protein fluorescence and CD spectroscopy. It was shown that the recombinant porins are similar in the composition of secondary structure elements to the isolated porins, but have a considerably less compact tertiary structure. The pore-forming activities of the recombinant proteins are similar to those of Y. pseudotuberculosis native porins. The English version of the paper: Russian Journal of Bioorganic Chemistry, 2008, vol. 34, no. 2; see also http://www.maik.ru.     

Cloning and expression of the major porin protein gene opcP of Burkholderia (formerly Pseudomonas) cepacia in Escherichia coli

The outer membrane protein OpcP1 of Burkholderia (formerly Pseudomonas) cepacia is one of the subunits forming the porin oligomer OpcPO by non-covalent association. OpcP1 was cleaved with lysyl endopeptidase and the N-terminal amino acid (aa) sequences of the polypeptide fragments were determined. Based on the sequence information, we cloned the opcP gene on a 10-kb EcoRI DNA fragment of the B. cepacia ATCC25416 chromosome. Nucleotide (nt) sequencing revealed a 1086-bp open reading frame (ORF), encoding a 361-aa polypeptide with a signal sequence of 20 residues. The predicted opcP gene encoded a mature protein of M_r 35 696, which agrees well with the value observed previously on SDS-PAGE. The opcP was sub-cloned into pTrc99A and introduced into Escherichia coli. Immunoblot analysis using murine antiserum specific to OpcP1 visualized the protein expressed in the E. coli cells after induction by isopropyl β-d-thiogalactopyranoside (IPTG).     

Genome evolution in the genus Sorghum (Poaceae)

BACKGROUND AND AIMS: The roles of variation in DNA content in plant evolution and adaptation remain a major biological enigma. Chromosome number and 2C DNA content were determined for 21 of the 25 species of the genus Sorghum and analysed from a phylogenetic perspective. METHODS: DNA content was determined by flow cytometry. A Sorghum phylogeny was constructed based on combined nuclear ITS and chloroplast ndhF DNA sequences. KEY RESULTS: Chromosome counts (2n = 10, 20, 30, 40) were, with few exceptions, concordant with published numbers. New chromosome numbers were obtained for S. amplum (2n = 30) and S. leiocladum (2n = 10). 2C DNA content varies 8.1-fold (1.27-10.30 pg) among the 21 Sorghum species. 2C DNA content varies 3.6-fold from 1.27 pg to 4.60 pg among the 2n = 10 species and 5.8-fold (1.52-8.79 pg) among the 2n = 20 species. The x = 5 genome size varies over an 8.8-fold range from 0.26 pg to 2.30 pg. The mean 2C DNA content of perennial species (6.20 pg) is significantly greater than the mean (2.92 pg) of the annuals. Among the 21 species studied, the mean x = 5 genome size of annuals (1.15 pg) and of perennials (1.29 pg) is not significantly different. Statistical analysis of Australian species showed: (a) mean 2C DNA content of annual (2.89 pg) and perennial (7.73 pg) species is significantly different; (b) mean x = 5 genome size of perennials (1.66 pg) is significantly greater than that of the annuals (1.09 pg); (c) the mean maximum latitude at which perennial species grow (-25.4 degrees) is significantly greater than the mean maximum latitude (-17.6) at which annual species grow. CONCLUSIONS: The DNA sequence phylogeny splits Sorghum into two lineages, one comprising the 2n = 10 species with large genomes and their polyploid relatives, and the other with the 2n = 20, 40 species with relatively small genomes. An apparent phylogenetic reduction in genome size has occurred in the 2n = 10 lineage. Genome size evolution in the genus Sorghum apparently did not involve a 'one way ticket to genomic obesity' as has been proposed for the grasses.