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PubMed1986

Nucleotide sequences of two cellulase genes from alkalophilic Bacillus sp. strain N-4 and their strong homology.

Two genes for cellulases of alkalophilic Bacillus sp. strain N-4 (ATCC 21833) have been sequenced. From the DNA sequences the cellulases encoded in the plasmids pNK1 and pNK2 consist of 488 and 409 amino acids, respectively. The DNA and protein sequences of the pNK1-encoded cellulase are related to those of the pNK2-encoded cellulase. The pNK2-encoded cellulase lacks the direct repeat sequence of a stretch of 60 amino acids near the C-terminal end of the pNK1-encoded cellulase. The duplication of the cellulase genes and the formation of the direct repeat in the pNK1-encoded cellulase occurred at almost the same time.

Amino Acid Sequence↗
PubMed2026

Concurrent ecological and evolutionary processes contribute to mutualism breakdown between legumes and rhizobia.

Though they jointly shape community responses to environmental perturbations, ecology and evolution are often examined separately, even in microorganisms where both occur over short timescales. Here we examine ecological and evolutionary responses to 33 years of nitrogen fertilization using the legume-rhizobium mutualism. Pairing a manipulative inoculation study with full-length 16S rRNA gene amplicon sequencing and structural equation modeling allows us to synthesize across biological scales: whole bacterial community, genus Rhizobium, Rhizobium ASVs, and symbiosis plasmids. Clover's preferred partner decreases in N-addition soils, limiting host growth, while a diverse and largely uncharacterized Rhizobium community increases. This ecological change is compounded by a concurrent evolutionary degradation of symbiont partner quality via changing frequencies of symbiotic plasmids. Ecological (rarer symbionts) and evolutionary (inferior symbionts) processes each accounted for roughly half of this loss of host benefit, revealing that ecology and evolution jointly shape mutualism breakdown over the short timescales typical of microbial systems.

ecology↗
PubMed2005

RNA interference is ineffective as a routine method for gene silencing in chick embryos as monitored by fgf8 silencing.

The in vivo accessibility of the chick embryo makes it a favoured model system for experimental developmental biology. Although the range of available techniques now extends to miss-expression of genes through in ovo electroporation, it remains difficult to knock out individual gene expression. Recently, the possibility of silencing gene expression by RNAi in chick embryos has been reported. However, published studies show only discrete quantitative differences in the expression of the endogenous targeted genes and unclear morphological alterations. To elucidate whether the tools currently available are adequate to silence gene expression sufficiently to produce a clear and specific null-like mutant phenotype, we have performed several experiments with different molecules that trigger RNAi: dsRNA, siRNA, and shRNA produced from a plasmid coexpressing green fluorescent protein as an internal marker. Focussing on fgf8 expression in the developing isthmus, we show that no morphological defects are observed, and that fgf8 expression is neither silenced in embryos microinjected with dsRNA nor in embryos microinjected and electroporated with a pool of siRNAs. Moreover, fgf8 expression was not significantly silenced in most isthmic cells transformed with a plasmid producing engineered shRNAs to fgf8. We also show that siRNA molecules do not spread significantly from cell to cell as reported for invertebrates, suggesting the existence of molecular differences between different model systems that may explain the different responses to RNAi. Although our results are basically in agreement with previously reported studies, we suggest, in contrast to them, that with currently available tools and techniques the number of cells in which fgf8 gene expression is decreased, if any, is not sufficient to generate a detectable mutant phenotype, thus making RNAi useless as a routine method for functional gene analysis in chick embryos.

Animals↗
PubMed2006

The genome of Rhizobium leguminosarum has recognizable core and accessory components.

BACKGROUND: Rhizobium leguminosarum is an alpha-proteobacterial N2-fixing symbiont of legumes that has been the subject of more than a thousand publications. Genes for the symbiotic interaction with plants are well studied, but the adaptations that allow survival and growth in the soil environment are poorly understood. We have sequenced the genome of R. leguminosarum biovar viciae strain 3841. RESULTS: The 7.75 Mb genome comprises a circular chromosome and six circular plasmids, with 61% G+C overall. All three rRNA operons and 52 tRNA genes are on the chromosome; essential protein-encoding genes are largely chromosomal, but most functional classes occur on plasmids as well. Of the 7,263 protein-encoding genes, 2,056 had orthologs in each of three related genomes (Agrobacterium tumefaciens, Sinorhizobium meliloti, and Mesorhizobium loti), and these genes were over-represented in the chromosome and had above average G+C. Most supported the rRNA-based phylogeny, confirming A. tumefaciens to be the closest among these relatives, but 347 genes were incompatible with this phylogeny; these were scattered throughout the genome but were over-represented on the plasmids. An unexpectedly large number of genes were shared by all three rhizobia but were missing from A. tumefaciens. CONCLUSION: Overall, the genome can be considered to have two main components: a 'core', which is higher in G+C, is mostly chromosomal, is shared with related organisms, and has a consistent phylogeny; and an 'accessory' component, which is sporadic in distribution, lower in G+C, and located on the plasmids and chromosomal islands. The accessory genome has a different nucleotide composition from the core despite a long history of coexistence.

ATP-Binding Cassette Transporters↗
PubMed2000

The Bacillus subtilis AddAB helicase/nuclease is regulated by its cognate Chi sequence in vitro.

The AddAB enzyme is important to homologous DNA recombination in Bacillus subtilis, where it is thought to be the functional counterpart of the RecBCD enzyme of Escherichia coli. In vivo, AddAB responds to a specific five-nucleotide sequence (5'-AGCGG-3' or its complement) in a manner analogous to the response of the RecBCD enzyme to interaction with chi sequences. Here, we show that purified AddAB enzyme is able to load at a double-stranded DNA end and is both a DNA helicase and nuclease, whose combined action results in the degradation of both strands of the DNA duplex. During translocation, recognition of the properly oriented sequence 5'-AGCGG-3' causes attenuation of the AddAB enzyme nuclease activity that is responsible for degradation of the strand 3'-terminal at the entry site. Therefore, we conclude that 5'-AGCGG-3' is the B. subtilis Chi site and it is hereafter referred to as chi(Bs). After encountering chi(Bs), both the degradation of the 5'-terminal strand and the helicase activity persist. Thus, processing of a double-stranded DNA end by the AddAB enzyme produces a duplex DNA molecule with a protruding 3'-terminated single-stranded tail, a universal intermediate of the recombination process.

Attachment Sites, Microbiological↗
PubMed1991

Polymorphism within the nuclear and 2 micron genomes of Saccharomyces cerevisiae.

Seven strains of bakers' yeast were obtained as a representative sample of the Spanish baking industry. The nuclear genome was monitored for polymorphism by transverse alternating field electrophoresis (TAFE) and restriction maps of 2 micron DNA were produced. All seven strains were uniquely different when evaluated by their total chromosomal lengths whereas only two 2 micron variants were defined. There was no apparent correlation between chromosomal and plasmid polymorphism. The extensive chromosomal polymorphism within one 2 micron DNA type indicates the rapid and relatively recent evolution of the nuclear genome. The hybrid origin (S. cerevisiae-S. monacensis) of lager yeast was critically evaluated by TAFE analysis of S. cerevisiae and S. carlsbergensis chromosomes. The absence of corresponding S. cerevisiae chromosomes III and XIII in S. carlsbergensis argued against the hybrid origin of lager strains. We discuss limitations of the hybrid origin hypothesis of industrial yeasts and propose that the molecular coevolution observed in 2 micron DNA serves as a useful additional mechanism for rationalization of some of the structural polymorphism of the nuclear genome.

Chromosomes, Fungal↗
PubMed1999

The Arabidopsis transposon Tag1 is active in rice, undergoing germinal transposition and restricted, late somatic excision.

Tag1 is an autonomous transposable element of Arabidopsis thaliana that displays tight developmental control of its excision during shoot development. To determine how Tag1 behaves in a monocotyledonous species, Tag1 was inserted in a 35S-GUS marker gene and the construct was introduced into rice. Tag1 showed somatic excision activity in four out of eleven transgenic lines examined. In leaves, excision was primarily restricted to vascular bundles and produced sectors composed of only a few cells. Excision events in flowers occurred predominantly in or near the major veins of the palea and lemma to produce small sectors. In roots, small sectors were evident, but they were few in number. These data show that the timing of Tag1 excision during rice shoot development is late and mimics the late excision behavior of Tag1 in Arabidopsis. One of the transgenic rice lines, which had a high frequency of somatic excision, produced several germinal revertants, one of which was characterized by a new Tag1 insertion band. The pattern of Tag1 transcripts and the footprint sequences left behind after excision in rice were found to be very similar to those in Arabidopsis. These results show that key properties of Tag1 transposition and behavior are conserved between monocots and dicots and that Tag1 has the potential to serve as an insertional mutagen in rice.

Arabidopsis↗
PubMed2026

A fitness advantage from the pLVPK plasmid fuels the global spread of a carbapenem-resistant hypervirulent Klebsiella pneumoniae high-risk clone: ST11-KL64.

BACKGROUND: The global emergence of carbapenem-resistant hypervirulent Klebsiella pneumoniae (CR-hvKP), particularly the ST11-KL64 subclone acquiring pLVPK-like virulence plasmids, represents a critical public health threat. This study investigates the epidemiological dominance and molecular mechanisms underlying ST11-KL64's fitness advantage over KL47 variants. METHODS: We performed comparative genomic analysis on 43,722 K. pneumoniae genomes (2011-2022) from 112 countries, focusing on ST11-CRKP strains. Capsular typing (KL64 vs. KL47), virulence gene profiling (aerobactin, RmpADC), and plasmid stability analysis were conducted using Kleborate, RAST, and PlasmidFinder. Plasmid-chromosome interactions were characterized through hybrid assembly approaches. RESULTS: ST11-KL64 demonstrated rapid expansion post-2016, surpassing KL47 as China's dominant CRKP subtype (40.5% vs. 28.9%), with regional predominance in Zhejiang (62.3%) and Sichuan (58.7%) provinces. Notably, 94.8% of KL64 strains maintained intact pLVPK plasmids with high aerobactin carriage (60.5%), while KL47 exhibited frequent plasmid fusion (58.8% with IncFIB[pNDM-Mar]) or chromosomal integration (41.4%), resulting in lower virulence potential (27.3% aerobactin+). Genomic analysis revealed KL64's superior plasmid stability (71.2% gene retention vs. KL47's 43.6%) and clinical correlation with severe outcomes (OR = 2.34, 95%CI 1.67-3.28). CONCLUSION: The ST11-KL64 subclone's epidemiological success stems from stable pLVPK plasmid maintenance, enabling simultaneous carbapenem resistance and hypervirulence. These findings highlight the urgent need for genomic surveillance targeting plasmid-mediated virulence in CRKP outbreaks, particularly in critical care settings where horizontal gene transfer may accelerate strain evolution.

Klebsiella pneumonia↗
PubMed2004

Exon junction sequences as cryptic splice sites: implications for intron origin.

Introns are flanked by a partially conserved coding sequence that forms the immediate exon junction sequence following intron removal from pre-mRNA. Phylogenetic evidence indicates that these sequences have been targeted by numerous intron insertions during evolution, but little is known about this process. Here, we test the prediction that exon junction sequences were functional splice sites that existed in the coding sequence of genes prior to the insertion of introns. To do this, we experimentally identified nine cryptic splice sites within the coding sequence of actin genes from humans, Arabidopsis, and Physarum by inactivating their normal intron splice sites. We found that seven of these cryptic splice sites correspond exactly to the positions of exon junctions in actin genes from other species. Because actin genes are highly conserved, we could conclude that at least seven actin introns are flanked by cryptic splice sites, and from the phylogenetic evidence, we could also conclude that actin introns were inserted into these cryptic splice sites during evolution. Furthermore, our results indicate that these insertion events were dependent upon the splicing machinery. Because most introns are flanked by similar sequences, our results are likely to be of general relevance.

Actins↗
PubMed2006

Cloning and characterization of zebrafish CTCF: Developmental expression patterns, regulation of the promoter region, and evolutionary aspects of gene organization.

CTCF is a nuclear phosphoprotein capable of using different subsets of its 11 Zn fingers (ZF) for sequence-specific binding to many dissimilar DNA CTCF-target sites. Such sites were identified in the genomic DNA of various multicellular organisms, in which the CTCF gene was cloned, including insects, birds, rodents, and primates. CTCF/DNA-complexes formed in vivo with different 50-bp-long sequences mediate diverse functions such as positive and negative regulation of promoters, and organization of all known enhancer-blocking elements ("chromatin insulators") including constitutive and epigenetically regulated elements. Abnormal functions of certain CTCF sites are implicated in cancer and in epigenetic syndromes such as BWS and skewed X-inactivation. We describe here the cloning and characterization of the CTCF cDNA and promoter region from zebrafish, a valuable vertebrate model organism. The full-length zebrafish CTCF cDNA clone is 4244 bp in length with an open reading frame (ORF) of 2391 bp that encodes 797 amino acids. The zebrafish CTCF amino acid sequence shows high identity (up to 98% in the zinc finger region) with human CTCF, and perfect conservation of exon-intron organization. Southern blot analyses indicated that the zebrafish genome contains a single copy of the CTCF gene. In situ hybridization revealed the presence of zebrafish CTCF transcripts in all early stages of embryogenesis. Transfection assays with luciferase reporter-constructs identified a core promoter region within 146 bp immediately upstream of the transcriptional start site of zebrafish CTCF that is located at a highly conserved YY1/Initiator element.

Amino Acid Sequence↗
PubMed2003

Organisation and sequence determination of glutamine-dependent carbamoyl phosphate synthetase II in Toxoplasma gondii.

Carbamoyl phosphate synthetase II encodes the first enzymic step of de novo pyrimidine biosynthesis. Carbamoyl phosphate synthetase II is essential for Toxoplasma gondii replication and virulence. In this study, we characterised the primary structure of a 28kb gene encoding Toxoplasma gondii carbamoyl phosphate synthetase II. The carbamoyl phosphate synthetase II gene was interrupted by 36 introns. The predicted protein encoded by the 37 carbamoyl phosphate synthetase II exons was a 1,687 amino acid polypeptide with an N-terminal glutamine amidotransferase domain fused with C-terminal carbamoyl phosphate synthetase domains. This bifunctional organisation of carbamoyl phosphate synthetase II is unique, so far, to protozoan parasites from the phylum Apicomplexa (Plasmodium, Babesia, Toxoplasma) or zoomastigina (Trypanosoma, Leishmania). Apicomplexan parasites possessed the largest carbamoyl phosphate synthetase II enzymes due to insertions in the glutamine amidotransferase and carbamoyl phosphate synthetase domains that were not present in the corresponding gene segments from bacteria, plants, fungi and mammals. The C-terminal allosteric regulatory domain, the carbamoyl phosphate synthetase linker domain and the oligomerisation domain were also distinct from the corresponding domains in other species. The novel C-terminal regulatory domain may explain the lack of activation of Toxoplasma gondii carbamoyl phosphate synthetase II by the allosteric effector 5-phosphoribosyl 1-pyrophosphate. Toxoplasma gondii growth in vitro was markedly inhibited by the glutamine antagonist acivicin, an inhibitor of glutamine amidotransferase activity typically associated with carbamoyl phosphate synthetase II, guanosine monophosphate synthetase, or CTP synthetase.

Animals↗
PubMed1999

Functional assessment of surface loops: deletion of eukaryote-specific peptide inserts in thymidylate synthase of Saccharomyces cerevisiae.

The primary structure is known for at least 29 thymidylate synthases and the crystal structure is known for several from both prokaryotes and eukaryotes. All these are markedly similar making thymidylate synthase one of the most highly conserved enzymes known. There are, however, two surface loops, one near the active site and the other near the dimer interface, which exist in distinctly prokaryotic and eukaryotic versions. Specifically, in eukaryotes these two surface loops have small peptide inserts conserved in size and partly conserved in sequence, that are not present in the prokaryotic thymidylate synthases. To address the possibility that these inserts provide eukaryote-specific functions the Saccharomyces cerevisiae loops were individually modified to mimic their prokaryotic counterparts. Altering the surface loop near the active site increased Km for the nucleotide substrate and decreased apparent Vmax. Mutant variants with alterations in the other surface loop were unable to dimerize. Therefore these surface loops have acquired, perhaps by way of the eukaryotic inserts, characteristics that are important for catalytic activity and quaternary structure respectively.

Alleles↗
PubMed2000

Directed evolution of a (beta alpha)8-barrel enzyme to catalyze related reactions in two different metabolic pathways.

Enzymes participating in different metabolic pathways often have similar catalytic mechanisms and structures, suggesting their evolution from a common ancestral precursor enzyme. We sought to create a precursor-like enzyme for N'-[(5'-phosphoribosyl)formimino]-5-aminoimidazole-4-carboxamide ribonucleotide (ProFAR) isomerase (HisA; EC ) and phosphoribosylanthranilate (PRA) isomerase (TrpF; EC ), which catalyze similar reactions in the biosynthesis of the amino acids histidine and tryptophan and have a similar (betaalpha)(8)-barrel structure. Using random mutagenesis and selection, we generated several HisA variants that catalyze the TrpF reaction both in vivo and in vitro, and one of these variants retained significant HisA activity. A more detailed analysis revealed that a single amino acid exchange could establish TrpF activity on the HisA scaffold. These findings suggest that HisA and TrpF may have evolved from an ancestral enzyme of broader substrate specificity and underscore that (betaalpha)(8)-barrel enzymes are very suitable for the design of new catalytic activities.

Aldose-Ketose Isomerases↗
PubMed1983

Partial mRNA sequences for human A alpha, B beta, and gamma fibrinogen chains: evolutionary and functional implications.

Using rat cDNA and genomic probes to screen a human liver cDNA library, we have isolated clone of 2,274, 855, and 736 base pairs (bp) coding for the A alpha, B beta and gamma chains of human fibrinogen. Sequence analysis reveals a hitherto unrecognized extension of 15 amino acids at the carboxyl terminus of the A alpha chain, the terminal residue of which is proline. This brings the known length of the human A alpha chain to 625 amino acids. The 13-amino-acid repeated region in the midportion of the A alpha chain clearly has arisen through an 8-fold duplication of a 39-bp genetic element, which itself appears to have been constructed from smaller 6-bp repeating units. Greater than 50% sequence homology between B beta- and gamma-chain coding regions confirms postulates that these genes have arisen by duplication and subsequent divergence of an ancestral gene. A comparison of human and rat gamma-chain cDNAs shows more than 88% sequence homology over the carboxyl-terminal 162 amino acids, implying strong selective pressures on these portions of the gamma-chain gene.

Adult↗
PubMed1998

Directed evolution of a thermostable esterase.

We have used in vitro evolution to probe the relationship between stability and activity in a mesophilic esterase. Previous studies of these properties in homologous enzymes evolved for function at different temperatures have suggested that stability at high temperatures is incompatible with high catalytic activity at low temperatures through mutually exclusive demands on enzyme flexibility. Six generations of random mutagenesis, recombination, and screening stabilized Bacillus subtilis p-nitrobenzyl esterase significantly (>14 degreesC increase in Tm) without compromising its catalytic activity at lower temperatures. Furthermore, analysis of the stabilities and activities of large numbers of random mutants indicates that these properties are not inversely correlated. Although enhanced thermostability does not necessarily come at the cost of activity, the process by which the molecule adapts is important. Mutations that increase thermostability while maintaining low-temperature activity are very rare. Unless both properties are constrained (by natural selection or screening) the evolution of one by the accumulation of single amino acid substitutions typically comes at the cost of the other, regardless of whether the two properties are inversely correlated or not correlated at all.

Amino Acid Substitution↗
PubMed1998

Genetic code in evolution: switching species-specific aminoacylation with a peptide transplant.

The genetic code is established in aminoacylation reactions whereby amino acids are joined to tRNAs bearing the anticodons of the genetic code. Paradoxically, while the code is universal there are many examples of species-specific aminoacylations, where a tRNA from one taxonomic domain cannot be acylated by a synthetase from another. Here we consider an example where a human, but not a bacterial, tRNA synthetase charges its cognate eukaryotic tRNA and where the bacterial, but not the human, enzyme charges the cognate bacterial tRNA. While the bacterial enzyme has less than 10% sequence identity with the human enzyme, transplantation of a 39 amino acid peptide from the human into the bacterial enzyme enabled the latter to charge its eukaryotic tRNA counterpart in vitro and in vivo. Conversely, substitution of the corresponding peptide of the bacterial enzyme for that of the human enabled the human enzyme to charge bacterial tRNA. This peptide element discriminates a base pair difference in the respective tRNA acceptor stems. Thus, functionally important co-adaptations of a synthetase to its tRNA act as small modular units that can be moved across taxonomic domains and thereby preserve the universality of the code.

Amino Acyl-tRNA Synthetases↗