The sialidase gene from Clostridium septicum: cloning, sequencing, expression in Escherichia coli and identification of conserved sequences in sialidases and other proteins

Summary An oligonucleotide mixture corresponding to the codons for conserved and repeated amino acid sequences of bacterial sialidases (Roggentin et al. 1989) was used to clone a 4.3 kb PstI restriction fragment of Clostridium septicum DNA in Escherichia coli. The complete nucleotide sequence of the sialidase gene was determined from this fragment. The derived amino acid sequence corresponds to a protein of 110000 Da. The ribosomal binding site and promoter-like consensus sequences were identified upstream from the putative ATG initiation codon. The molecular and immunological properties of the sialidase expressed by E. coli are similar to those of the sialidase as isolated from C. septicum. The newly synthesized protein is assumed to include a leader peptide of 26 amino acids. On sequence alignment, the sialidases from C. septicum, C. sordellii and C. perfringens show significant homologies. As in other bacterial sialidases, conserved amino acid sequences occur at four positions in the protein. Aside from the consensus sequences, only poor homology to other bacterial and viral sialidases was found. The consensus sequence could be identified even in other, non-sialidase proteins, indicating a common function or the evolutionary relatedness of these proteins.     

Effects of site-specific mutations on the enzymatic properties of a sialidase fromClostridium perfringens

Three site-specific mutations were performed in two regions of a sialidase gene fromClostridium perfringens which are known to be conserved in bacterial sialidases. The mutant enzymes were expressed inEscherichia coli and, when measured with MU-Neu5Ac as substrate, exhibited variations in enzymatic properties compared with the wild-type enzyme. The conservative substitution of Arg 37 by Lys, located in a short conserved region upstream from the four repeated sequences common in bacterial sialidase genes, was of special interest, asK _M andV _max, as well asK _i measured with Neu5Ac2en, were dramatically changed. These data suggest that this residue may be involved in substrate binding. In addition to its low activity, this mutant enzyme has a lower temperature optimum and is active over a more limited pH range. This mutation also prevents the binding of an antibody able to inhibit the wild-type sialidase. The other mutations, located in one of the consensus sequences, were of lower influence on enzyme activity and recognition by antibodies.     

Selective cloning of genes encoding RNase H from Salmonella typhimurium, Saccharomyces cerevisiae and Escherichia coli rnh mutant

Summary We have cloned genes encoding RNase H from Escherichia coli rnh mutants, Salmonella typhimurium and Saccharomyces cerevisiae. Selection was accomplished by suppression of the temperature-sensitive growth phenotype of Escherichia coli strains containing the rnh-339::cat and either recB270 (Ts) or recC271 (Ts) mutations. RNases H from E. coli and S. typhimurium contained 155 amino acid residues and differed at only 11 positions. The S. cerevisiae and E. coli RNases H were about 30% homologous. A comparison of the amino acid sequences of several RNases H from cellular and retroviral sources revealed some strongly conserved regions as well as variable regions; the carboxyl-terminus was particularly variable. The overlapping, divergent promoter gene organization found in E. coli was observed to be present in S. typhimurium.     

The use of a non-coding region of chloroplast DNA in phylogenetic studies of the subtribeSonchinae (Asteraceae:Lactuceae)

The systematic utility of sequences from a non-coding region of chloroplast DNA (cpDNA) betweenpsbA andtrnH^(GUG) was examined by assessing phylogenetic relationships in subtribeSonchinae (Asteraceae:Lactuceae). Primers constructed against highly conserved regions of tRNA genes were used for PCR amplification and sequencing. ThepsbA-trnH intergenic spacer contains several insertions and deletions (indels) inSonchinae with the length varying from 385 to 450 bp. Sequence divergence ranges from 0.00% to 7.54% withinSonchinae, with an average of 2.4%. Average sequence divergence inSonchus subg.Sonchus is 2.0%, while the mean for subg.Dendrosonchus and its close relatives in Macaronesia (the woodySonchus alliance) is 1.0%. Our results suggest that this region does not evolve rapidly enough to resolve relationships among closely related genera or insular endemics in theAsteraceae. The phylogenetic utility ofpsbA-trnH sequences of the non-coding cpDNA was compared to sequences from the ITS region of nuclear ribosomal DNA. The results suggest that ITS sequences evolve nearly four times faster thanpsbA-trnH intergenic spacer sequences. Furthermore, the ITS sequences provide more variable and phylogenetically informative sites and generate more highly resolved trees with more strongly supported clades, and thus are more suitable for phylogenetic comparisons at lower taxonomic levels than thepsbA-trnH intergenic chloroplast sequences.     

Relationships between species ofLeymus,Psathyrostachys, andHordeum (Poaceae,Triticeae) inferred from Southern hybridization of genomic and cloned DNA probes

We have used total genomic DNA as a probe to size-fractionated restriction enzyme digests of genomic DNA from a range ofTriticeae species from the generaLeymus Hochst.,Psathyrostachys Nevski, andHordeum L., and hybrids betweenHordeum andLeymus to investigate their taxonomic relationships. Genomic Southern hybridization was found to be an effective and simple way to assess the distribution and diversity of essentially species-specific and common, repetitive DNA sequences, and is hence especially useful in evolutionary studies. The DNA sequences ofH. vulgare seem to diverge substantially from those ofH. brachyantherum, H. lechleri, H. procerum, andH. depressum. The genome ofThinopyron bessarabicum shows little homology to those of theLeymus species investigated, confirming thatT. bessarabicum is not an ancestral genome inLeymus. Although the genomes ofLeymus andPsathyrostachys share substantial proportions of DNA sequences, they include divergent repeated sequences as well. Hybridization with a ribosomal DNA probe (pTa 71) showed that the coding regions containing structural genes encoding the 18 S, 5.8 S, and 26 S ribosomal RNA were conserved among the species investigated, whereas the intergenic spacer region was more variable, presenting different sizes of restriction fragments and enabling a classification of the species. The rye heterochromatin probe pSc 119.2 hybridized to DNA fromH. lechleri andT. bessarabicum, but not to DNA from the other species investigated.     

Inhibition of sialidases from viral,bacterial and mammalian sources by analogues of 2-deoxy-2,3-didehydro-N-acetylneuraminic acid modified at the C-4 position

The inhibition of sialidase activity from influenza viruses A and B, parainfluenza 2 virus,Vibrio cholerae, Arthrobacter ureafaciens, Clostridium perfringens, and sheep liver by a range of 2-deoxy-2,3-didehydro-N-acetylneuraminic acid analogues modified at the C-4 position has been studied. All substitutions tested resulted in a decrease in the degree of inhibition of the bacterial and mammalian sialidases. For sialidases from influenza viruses A and B, on the other hand, most of the substitutions tested either had no significant effect on binding or, in the case of the basic amino and guanidino substituents, resulted in significantly stronger inhibition. The results for parainfluenza 2 virus sialidase were mostly intermediate, in that inhibition was neither significantly increased nor decreased by most of the modifications. We conclude that only the influenza A and B sialidase active sites possess acid groups correctly positioned to participate in charge-charge interactions in the region of C-4 of bound substrate, and that the C-4 binding pockets of the bacterial and mammalian sialidases examined are considerably smaller than is observed for either the influenza virus or parainfluenza virus sialidases.This paper is dedicated to the memory of Professor Dr E. Zbiral.     

Hordeum chilense repetitive sequences. Genome characterization using biotinylated probes

Summary A library of random DNA fragment clones of wild barley Hordeum chilense was screened for clones of repeated nucleotide sequences. Five clones were isolated that gave a stronger hybridization signal in colony and dot blot hybridization with total H. chilense DNA in comparison to Triticum aestivum DNA. Clones labelled with biotinylated nucleotides were used as probes to investigate the repeated sequences organization in the H. chilense genome. Tandemly arranged and interspersed sequences have been found, together with homology differences with related sequences present in T. Aestivum, which could allow the differentiation of H. chilense DNA when it is present in wheat. We show that biotin can replace the use of ³²P in preparing repeated sequence probes for Southern and DNA dot blot analyses.     

Trans-sialidase genes expressed in mammalian forms of Trypanosoma cruzi evolved from ancestor genes expressed in insect forms of the parasite

The trans-sialidase of Trypanosoma cruzi mammalian forms transfers sialic acids from host's cell-surface glycoconjugates to acceptor molecules on parasite cell surface. To investigate the mechanism by which the mammalian stages of Trypanosoma cruzi have acquired their trans-sialidase, we compared the nucleotide and predicted amino acid sequences of trans-sialidase genes expressed in different developmental stages and strains of Trypanosoma cruzi with the sialidase gene of Trypanosoma rangeli and the sialidase genes of the prokaryotic genera Clostridium, Salmonella, and Actinomyces. The trans-sialidase gene products of Trypanosoma cruzi have a significant degree of structural and biochemical similarity to the sialidases found in bacteria and viruses, which would hint that horizontal gene transfer occurred in Trypanosome cruzi trans-sialidase evolutionary history. The comparison of inferred gene trees with species trees suggests that the genes encoding the T. cruzi trans-sialidase of mammalian forms might be derived from genes expressed in the insect forms of the genus Trypanosome. The branching order of trees inferred from T. cruzi trans-sialidase sequences, the sialidase from Trypanosoma rangeli, and bacterial sialidases parallels the expected branching order of the species and suggests that the divergence times of these sequences are remarkably long. Therefore, a vertical inheritance from a hypothetical eukaryotic trans-sialidase gene expressed in insect forms of trypanosomes is more likely to have occurred than the horizontal gene transfer from bacteria, and thus explains the presence of this enzyme in the mammalian infective forms of Trypanosoma cruzi.Correspondence to: M.R.S. Briones     

Comparative Genomics Reveals Long, Evolutionarily Conserved, Low-Complexity Islands in Yeast Proteins

Eukaryotic proteomes abound in low-complexity sequences, including tandem repeats and regions with significantly biased amino acid compositions. We assessed the functional importance of compositionally biased sequences in the yeast proteome using an evolutionary analysis of 2838 orthologous open reading frame (ORF) families from three Saccharomyces species (S. cerevisiae, S. bayanus, and S. paradoxus). Sequence conservation was measured by the amino acid sequence variability and by the ratio of nonsynonymous-to-synonymous nucleotide substitutions (K _a /K _s ) between pairs of orthologous ORFs. A total of 1033 ORF families contained one or more long (at least 45 residues), low-complexity islands as defined by a measure based on the Shannon information index. Low-complexity islands were generally less conserved than ORFs as a whole; on average they were 50% more variable in amino acid sequences and 50% higher in K _a /K _s ratios. Fast-evolving low-complexity sequences outnumbered conserved low-complexity sequences by a ratio of 10 to 1. Sequence differences between orthologous ORFs fit well to a selectively neutral Poisson model of sequence divergence. We therefore used the Poisson model to identify conserved low-complexity sequences. ORFs containing the 33 most conserved low-complexity sequences were overrepresented by those encoding nucleic acid binding proteins, cytoskeleton components, and intracellular transporters. While a few conserved low-complexity islands were known functional domains (e.g., DNA/RNA-binding domains), most were uncharacterized. We discuss how comparative genomics of closely related species can be employed further to distinguish functionally important, shorter, low-complexity sequences from the vast majority of such sequences likely maintained by neutral processes. [Reviewing Editor: Dr. Stuart Newfeld]     

Vitellogenin motifs conserved in nematodes and vertebrates

Summary Caenorhabditis elegans vitellogenins are encoded by a family of six genes, one of which,vit-5, has been previously sequenced and shown to be surprisingly closely related to the vertebrate vitellogenin genes. Here we report an alignment of the amino acid sequences of vitellogenins from frog and chicken with those from threeC. elegans genes:vit-5 and two newly sequenced genes,vit-2 andvit-6. The four introns ofvit-6 are all in different places from the four introns ofvit-5, but three of these eight positions are identical or close to intron locations in the vertebrate vitellogenin genes. The encoded polypeptides have diverged from one another sufficiently to allow us to draw some conclusions about conserved positions. Many cysteine residues have been conserved, suggesting that vitellogenin structure has been maintained over a long evolutionary distance and is dependent upon disulfide bonds. In addition, a 20-residue segment shows conservation between the vertebrate and the nematode vitellogenins. This sequence may play a highly conserved role in vitellogenesis, such as specific recognition by oocytes. On the whole, however, selection may be acting more strongly on amino acid composition and codon usage than on amino acid sequence, as might be expected for abundant storage proteins: The amino acid compositions ofvit-2, vit-5, andvit-6 products are remarkably similar, despite the fact that the sequence of thevit-2 protein is only 22% and 50% identical to the sequences ofvit-6 andvit-5 proteins, respectively.     

Ribosomal DNA repeat unit polymorphism in 25 Hordeum species

Summary Tandemly repeated DNA sequences containing structural genes encoding ribosomal RNA (rDNA) were investigated in 25 species of Hordeum using the wheat rDNA probe pTA71. The rDNA repeat unit lengths were shown to vary between 8.5 and 10.7 kb. The number of length classes (1–3) per accession generally corresponded to the number of nucleolar organizing regions (NORs). Intraspecific variation was found in H. parodii, H. spontaneum and H. leporinum, but not in H. bulbosum. Restriction analysis showed that the positions of EcoRI, SacI and certain BamHI cleavage sites in the rRNA structural genes were highly conserved, and that repeat unit length variation was generally attributable to the intergenic spacer region. Five rDNA BamHI restriction site maps corresponded to the following groups of species: Map A — H. murinum, H. glaucum, H. leporinum, H. bulbosum, H. marinum, H. geniculatum; Map B — H. leporinum; Map C — H. vulgare, H. spontaneum, H. agriocrithon; Map D — H. chilense, H. bogdanii; and Map E — remaining 14 Hordeum species. The repeat unit of H. bulbosum differed from all other species by the presence of a HindIII site. The closer relationship of H. bulbosum to H. leporinum, H. murinum and H. glaucum than to H. vulgare was indicated by their BamHI restriction maps.Contribution No. 1169, Plant Research Centre     

Rhizosphere-Bacterial Community in <Emphasis Type="Italic">Eperua falcata</Emphasis> (Caesalpiniaceae) a Putative Nitrogen-Fixing Tree from French Guiana Rainforest

The rainforest of French Guiana is still largely unaffected by human activity. Various pristine sites like the Paracou Research Station are devoted to study this tropical ecosystem. We used culture-independent techniques, like polymerase chain reaction-temperature gradient gel electrophoresis, and construction of clone libraries of partial 16S rRNA and nifH genes, to analyze the composition of the bacterial community in the rhizosphere of mature trees of Eperua falcata and Dicorynia guianensis, both species within the Caesalpiniaceae family. E. falcata is one of the more abundant pioneer tree species in this ecosystem and so far, no root nodules have ever been found. However, its nitrogen-fixing status is regarded as “uncertain”, whereas D. guianensis is clearly considered a non-nitrogen-fixing plant. The rhizospheres of these mature trees contain specific bacterial communities, including several currently found uncultured microorganisms. In these communities, there are putative nitrogen-fixing bacteria specifically associated to each tree: D. guianensis harbors several Rhizobium spp. and E. falcata members of the genera Burkholderia and Bradyrhizobium. In addition, nifH sequences in the rhizosphere of the latter tree were very diverse. Retrieved sequences were related to bacteria belonging to the α-, β-, and γ-Proteobacteria in the E. falcata rhizoplane, whereas only two sequences related to γ-Proteobacteria were found in D. guianensis. Differences in the bacterial communities and the abundance and diversity of nifH sequences in E. falcata rhizosphere suggest that this tree could obtain nitrogen through a nonnodulating bacterial interaction.     

Conservation of upstream regulators of scute on the notum of cyclorraphous Diptera

Bristles on the notum of many cyclorraphous flies are arranged into species-specific stereotyped patterns. Differences in the spatial expression of the proneural gene scute correlate with the positions of bristles in those species looked at so far. However, the examination of a number of genes encoding trans-regulatory factors, such as pannier, stripe, u-shaped, caupolican and wingless, indicates that they are expressed in conserved domains on the prospective notum. This suggests that the function of a trans-regulatory network of genes is relatively unchanged in derived Diptera, and that many differences are likely to be due to changes in cis-regulatory sequences of scute. In contrast, in Anopheles gambiae, a basal species with no stereotyped bristle pattern, the expression patterns of pannier and wingless are not conserved, and expression of AgASH, the Anopheles proneural gene, does not correlate in a similar manner with the bristle pattern. We discuss the possibility that independently acting cis-regulatory sequences at the scute locus may have arisen in the lineage giving rise to cyclorraphous flies.     

Molecular cloning and expression of perhydrolase genes from Pseudomonas aeruginosa and Burkholderia cepacia in Escherichia coli

Two bacterial perhydrolase genes, perPA and perBC, were cloned from Pseudomonas aeruginosa and Burkholderia cepacia, respectively, using PCR amplification with primers designed to be specific for conserved amino acid sequences of the already-known perhydrolases. The amino acid sequence of PerPA was identical to a putative perhydrolase of P. aeruginosa PAO1 genome sequences, whereas PerBC of B. cepacia was a novel bacterial perhydrolase showing similarity of less than 80% with all other existing perhydrolases. Most importantly, the perPA gene was expressed as a soluble intracellular form to an extent of more than 50% of the total protein content in Escherichia coli. Two perhydrolase enzymes were confirmed to exhibit the halogenation activity towards Phenol Red and monochlorodimedone. These results suggested that we successfully obtained the newly identified members of the bacterial perhydrolase family, expanding the pool of available perhydrolases.     

Sequence Comparison of Plant Ornithine Decarboxylases Reveals High Homology and Lack of Introns

We have designed and constructed four oligonucleotides corresponding to the most conserved regions of ornithine decarboxylases (ODC; EC 4.1.1.17) of plant origin. These oligonucleotides were used for the amplification of homologous fragments from several plants (Zea mays, Capsicum annuum, Sorghum bicolor, Phaseolus vulgaris, Carica papaya and Daucus carota). The amplified fragments were cloned and sequenced, revealing high homology to other ODCs. Peptide sequences coded by these fragments were compared by Clustal analyses. These analyses identified the location of the conserved sequences corresponding to the binding sites of substrate and cofactor. Data demonstrated that the plant ODCs fragments lacked intron sequences and were extremely homologous (over 80 %), constituting a compact group separated from other eukaryotic ODCs. This revised version was published online in July 2006 with corrections to the Cover Date.     

念珠藻属植物nifH基因的适应性进化分析

念珠藻(Nostoc)固氮过程关键在于固氮酶的催化,而固氮酶复合物中的铁蛋白(NifH)是由高度保守的nifH基因编码的,该基因是进化史上现存最古老的功能基因之一。该研究选取念珠藻属及近缘类群的nifH基因序列共40条,采用最大似然法构建系统发育树;运行PAML4.9软件,对nifH基因编码蛋白进行生物信息学分析,并使用分支模型、位点模型和分支-位点模型检测该基因的选择位点,探讨nifH基因的适应性进化特征。结果表明:(1)最大似然树显示内类群中该研究物种共分为6个分支(A、B、C、D、E和F),其中D和E是2个大的分支,每个大分支中又各包含2个特殊的小分支A、F和B、C,其中F分支包含新疆古尔班通古特沙漠采集到的9株念珠藻,A分支包含F分支及该研究测定序列的4株葛仙米,B分支包含本研究测定序列的4株地皮菜和3株未定种的念珠藻,C分支包含NCBI数据库中下载的5株念珠藻、鱼腥藻序列和本研究测定序列的1株念珠藻。(2)在所分析的3种进化模型中,仅通过分支-位点模型检测出14个统计学上显著的正选择位点,即1F、2S、3S、4T、5A、6F、7F、8I、9S、10C、17I、27Y、29D和31R位点,表明念珠藻属植物的nifH基因发生了适应性变化,分支-位点模型是研究藻类基因适应性进化较好的模型。     

Flagellin (FliC) protein sequence diversity among <Emphasis Type="Italic">Bacillus thuringiensis</Emphasis> does not correlate with H serotype diversity

In Escherichia coli, the fliC gene encodes flagellin, the protein responsible for eliciting the immunological reaction in H serotyping. Here, the presence of the flagellin fliC gene was studied in 86 Bacillus thuringiensis strains encompassing 67 H serotypes. Nineteen strains from four additional species in the B. cereus sensu lato group, B. cereus, B. anthracis, B. mycoides, and B. weihenstephanensis, were added for comparison purposes. The fliC genes were amplified, cloned and their nucleotide sequences determined and translated into amino acid sequences. A bootstrapped neighbor-joining tree was generated from the alignment of the translated amino acid sequences of the amplicons. Although most B. thuringiensis H serotypes had different flagellin amino acid sequences, some different B. thuringiensis serovars shared identical flagellin amino acid sequences. In addition, although serovars from the same H serotype were sometimes found clustered together, several serovars from the same H serotype carried flagellins with sufficiently different amino acid sequences as to be located on distant clusters. No correlations could be established between flagellin (FliC) protein sequence diversity among B. thuringiensis H serotypes and H serotype diversity. These suggest that the B. thuringiensis fliC gene does not code for the flagellin copy responsible for eliciting the immunological reaction in H serotyping. In a previous study, the authors have shown that the B. thuringiensis hag gene codes for the flagellin copy responsible for eliciting the immunological reaction in H serotyping. It is proposed that the B. thuringiensis fliC gene studied here be renamed and that the so-called hag gene studied before be renamed fliC, both in accordance with the E. coli nomenclature. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Organisation of nodulation and nitrogen fixation genes on a Rhizobium trifolii symbiotic plasmid

A Rhizobium trifolii symbiotic plasmid specific gene library was constructed and the physical organisation of regions homologous to nifHDK, nifA and nod genes was determined. These symbiotic gene regions were localised to u 25 kb region on the sym-plasmid, pPN1. In addition four copies of a reiterated sequence were identified on this plasmid, with one copy adjacent to nifH. No rearrangement of these reiterated sequences was observed between R. trifolii bacterial and bacteroid DNA. Analysis of a deletion derivative of pPN1 showed that these sequences were spread over a 110 kb region to the left of nifA.     

Phylogenetic and Exon–Intron Structure Analysis of Fungal Subtilisins: Support for a Mixed Model of Intron Evolution

Phylogenetic and exon–intron structure analyses of intra- and interspecific fungal subtilisins in this study provided support for a mixed model of intron evolution: a synthetic theory of introns-early and introns-late speculations. Intraspecifically, there were three phase zero introns in Pr1A and its introns 1 and 2 located at the highly conserved positions were phylogentically congruent with coding region, which is in favor of the view of introns-early speculation, while intron 3 had two different sizes and was evolutionarily incongruent with coding region, the evidence for introns-late speculation. Noticeably, the subtilisin Pr1J gene from different strains of M. ansiopliae contained different number of introns, the strong evidence in support of introns-late theory. Interspecifically, phylogenetic analysis of 60 retrievable fungal subtilisins provided a clear relationship between amino acid sequence and gene exon–intron structure that the homogeneous sequences usually have a similar exon–infron structure. There were 10 intron positions inserted by highly biased phase zero introns across examined fungal subtilisin genes, half of these positions were highly conserved, while the others were species-specific, appearing to be of recent origins due to intron insertion, in favor of the introns-late theory. High conservations of positions 1 and 2 inserted by the high percentage of phase zero introns as well as the evidence of phylogenetic congruence between the evolutionary histories of intron sequences and coding region suggested that the introns at these two positions were primordial.Reviewing Editor:Dr. Manyuan Long