Evolutionary process of amino acid biosynthesis in Corynebacterium at the whole genome level

Corynebacterium glutamicum, which is the closest relative of Corynebacterium efficiens, is widely used for the large scale production of many kinds of amino acids, particularly glutamic acid and lysine, by fermentation. Corynebacterium diphtheriae, which is well known as a human pathogen, is also closely related to these two species of Corynebacteria, but it lacks such productivity of amino acids. It is an important and interesting question to ask how those closely related bacterial species have undergone such significant functional differentiation in amino acid biosynthesis. The main purpose of the present study is to clarify the evolutionary process of functional differentiation among the three species of Corynebacteria by conducting a comparative analysis of genome sequences. When Mycobacterium and Streptomyces were used as out groups, our comparative study suggested that the common ancestor of Corynebacteria already possessed almost all of the gene sets necessary for amino acid production. However, C. diphtheriae was found to have lost the genes responsible for amino acid production. Moreover, we found that the common ancestor of C. efficiens and C. glutamicum have acquired some of genes responsible for amino acid production by horizontal gene transfer. Thus, we conclude that the evolutionary events of gene loss and horizontal gene transfer must have been responsible for functional differentiation in amino acid biosynthesis of the three species of Corynebacteria.     

The genome stability in Corynebacterium species due to lack of the recombinational repair system

Corynebacterium species are members of gram-positive bacteria closely related to Mycobacterium species, both of which are classified into the same taxonomic order Actinomycetales. Recently, three corynebacteria, Corynebacterium efficiens, Corynebacterium glutamicum, and Corynebacterium diphtheriae have been sequenced independently. We found that the order of orthologous genes in these species has been highly conserved though it has been disrupted in Mycobacterium species. This synteny suggests that corynebacteria have rarely undergone extensive genome rearrangements and have maintained ancestral genome structures even after the divergence of corynebacteria and mycobacteria. This is the first report that the genome structures have been conserved in free-living bacteria such as C. efficiens and C. glutamicum, although it has been reported that obligate parasites such as Mycoplasma and Chlamydia have the stable genomes. The comparison of recombinational repair systems among the three corynebacteria and Mycobacterium tuberculosis suggested that the absence of recBCD genes in corynebacteria be responsible for the suppression of genome shuffling in the species. The genome stability in Corynebacterium species will give us hints of the speciation mechanism with the non-shuffled genome, particularly the importance of horizontal gene transfer and nucleotide substitution in the genome.     

Genomic Islands in the Corynebacterium efficiens Genome

Corynebacterium efficiens is a gram-positive nonpathogenic bacterium which can grow and produce glutamate at 40°C or above. By using the cumulative GC profile method, we have identified four genomic islands which have many unifying genomic island-specific features in the C. efficiens genome. The presence of the gene encoding an aspartate kinase in a genomic island helps explain the unexpected low thermal stability of this enzyme; i.e., the adaptive mutations have not occurred extensively due to the recent horizontal gene transfer.     

钝齿棒杆菌N-乙酰谷氨酸激酶基因的克隆、序列分析及表达

以钝齿棒杆菌(Corynebacterium crenatum)野生株AS 1.542及产精氨酸突变株971.1的基因组为模板,用PCR方法扩增出N-乙酰谷氨酸激酶基因(argB)片段。核酸序列分析结果表明,该片段全长1505bp,包含一个ORF,推测此ORF区编码一条317个氨基酸的多肽,分子量为33.6kDa。C.crenatum野生株AS 1.542与突变株971.1的argB基因序列比较,发现只在结构区有一个核苷酸的差别但没有引起氨基酸变化。野生株AS 1.542argB基因的编码区核苷酸序列与C.glutamicumATCC 13032、Corynebacterium efficiensYS-314和Escherichia colik12的同源性分别是99.89%、76.62%和37.94%,而氨基酸同源性分别是100%、78.55%和25.25%。在C.crenatum argB基因上游存在启动子区域。经IPTG诱导该基因在棒杆菌中得到有效表达,野生株AS 1.542为宿主的重组子酶活明显提高。突变株971.1为宿主的重组菌酶活提高一倍,精氨酸积累提高约25%。     

Complete genome sequence and analysis of the multiresistant nosocomial pathogen Corynebacterium jeikeium K411, a lipid-requiring bacterium of the human skin flora

Corynebacterium jeikeium is a "lipophilic" and multidrug-resistant bacterial species of the human skin flora that has been recognized with increasing frequency as a serious nosocomial pathogen. Here we report the genome sequence of the clinical isolate C. jeikeium K411, which was initially recovered from the axilla of a bone marrow transplant patient. The genome of C. jeikeium K411 consists of a circular chromosome of 2,462,499 bp and the 14,323-bp bacteriocin-producing plasmid pKW4. The chromosome of C. jeikeium K411 contains 2,104 predicted coding sequences, 52% of which were considered to be orthologous with genes in the Corynebacterium glutamicum, Corynebacterium efficiens, and Corynebacterium diphtheriae genomes. These genes apparently represent the chromosomal backbone that is conserved between the four corynebacteria. Among the genes that lack an ortholog in the known corynebacterial genomes, many are located close to transposable elements or revealed an atypical G+C content, indicating that horizontal gene transfer played an important role in the acquisition of genes involved in iron and manganese homeostasis, in multidrug resistance, in bacterium-host interaction, and in virulence. Metabolic analyses of the genome sequence indicated that the "lipophilic" phenotype of C. jeikeium most likely originates from the absence of fatty acid synthase and thus represents a fatty acid auxotrophy. Accordingly, both the complete gene repertoire and the deduced lifestyle of C. jeikeium K411 largely reflect the strict dependence of growth on the presence of exogenous fatty acids. The predicted virulence factors of C. jeikeium K411 are apparently involved in ensuring the availability of exogenous fatty acids by damaging the host tissue.     

The cytosolic, cell surface and extracellular proteomes of the biotechnologically important soil bacterium Corynebacterium efficiens YS-314 in comparison to those of Corynebacterium glutamicum ATCC 13032

Reference maps of the cytosolic, cell surface and extracellular proteome fractions of the amino acid-producing soil bacterium Corynebacterium efficiens YS-314 were established. The analysis window covers a pI range from 3 to 7 along with a molecular mass range from 10 to 130 kDa. After second-dimensional separation on SDS-PAGE and Coomassie staining, computational analysis detected 635 protein spots in the cytosolic proteome fraction, whereas 76 and 102 spots were detected in the cell surface and extracellular proteomes, respectively. By means of MALDI-TOF-MS and tryptic peptide mass fingerprinting, 164 cytosolic proteins, 49 proteins of the cell surface and 89 extracellular protein spots were identified, representing in total 177 different proteins. Additionally, reference maps of the three cellular proteome fractions of the close phylogenetic relative Corynebacterium glutamicum ATCC 13032 were generated and used for comparative proteomics. Classification according to the Clusters of Orthologous Groups of proteins scheme and abundance analysis of the identified proteins revealed species-specific differences. The high abundance of molecular chaperones and amino acid biosynthesis enzymes in C. efficiens points to environmental adaptations of this recently discovered amino acid-producing bacterium.     

Cloning, overexpression, purification, and characterization of S-adenosylhomocysteine hydrolase from Corynebacterium efficiens YS-314

The gene encoding S-adenosylhomocysteine hydrolase activity (SAHase: EC 3.3.1.1) from Corynebacterium efficiens (YS-314) was cloned and expressed as a fusion protein in Escherichia coli Rosetta (DE3). The analyzed nucleotide sequence of the cloned gene proved to be identical to those reported on the NCBI database. The recombinant enzyme is a tetramer, showing a molecular weight of approximately 210 kDa, as estimated by gel filtration. The K(M) values of the enzyme for S-adenosylhomocysteine (SAH), adenosine (Ado), and homocysteine (Hcy), were determined to be 1.4, 10, and 45 microM. The overexpression of the recombinant enzyme produced a high level of protein (>40 mg of protein per gram of wet cells) and revealed certain thermostability when characterized at temperatures above 40 degrees C. It also showed a high capacity for the synthesis of SAH, thermal stability, and high kinetic similarity to human SAHase, indicating a high biotechnological and pharmacological potential.     

Structure and expression of the human cystatin C gene.

The structural organization of the gene for the human cysteine-proteinase inhibitor cystatin C was studied. Restriction-endonuclease digests of human genomic DNA hybridized with human cystatin C cDNA and genomic probes produced patterns consistent with a single cystatin C gene and, also, the presence of six closely related sequences in the human genome. A 30 kb restriction map covering the genomic region of the cystatin C gene was constructed. The positions of three polymorphic restriction sites, found at examination of digests of genomic DNA from 79 subjects, were localized in the flanking regions of the gene. The gene was cloned and the nucleotide sequence of a 7.3 kb genomic segment was determined, containing the three exons of the cystatin C structural gene as well as 1.0 kb of 5'-flanking and 2.0 kb of 3'-flanking sequences. Northern-blot experiments revealed that the cystatin C gene is expressed in every human tissue examined, including kidney, liver, pancreas, intestine, stomach, antrum, lung and placenta. The highest cystatin C expression was seen in seminal vesicles. The apparently non-tissue-specific expression of this cysteine-proteinase inhibitor gene is discussed with respect to the structure of its 5'-flanking region, which shares several features with those of housekeeping genes.     

Identification and characterization of C6orf37, a novel candidate human retinal disease gene on chromosome 6q14

We have identified a novel human gene, chromosome 6 open reading frame 37 (C6orf37), that is expressed in the retina and maps to human chromosome 6q14, a genomic region that harbors multiple retinal disease loci. The cDNA sequence contains an open reading frame of 1314 bp that encodes a 437-amino acid protein with a predicted molecular mass of 49.2 kDa. Northern blot analysis indicates that this gene is widely expressed, with preferential expression observed in the retina compared to other ocular tissues. The C6orf37 protein shares homology with putative proteins in R. norvegicus, M. musculus, D. melanogaster, and C. elegans, suggesting evolutionary conservation of function. Additional sequence analysis predicts that the C6orf37 gene product is a soluble, globular cytoplasmic protein containing several conserved phosphorylation sites. Furthermore, we have defined the genomic structure of this gene, which will enable its analysis as a candidate gene for chromosome 6q-associated inherited retinal disorders.     

Molecular characterization and chromosomal assignment of the canine protein C gene

Protein C is a precursor to a serine protease present in the plasma that plays an important physiological role in the regulation of blood coagulation. Mutations in the human protein C gene have been linked to some cases of Morbus Perthes disease, a thrombophilic condition that results in aseptic necrosis of the femur head and neck. We have cloned the canine protein C gene to investigate whether Morbus Perthes disease in dogs is also caused by mutations within this gene. A genomic λFIXII clone was isolated, and 11,420 bp of DNA sequence were determined containing the complete protein C gene (Acc No. AJ001979). As in humans, the gene consists of nine exons with the translation start codon located in the second exon. The 1.7-kb mRNA contains a 1368-bp open reading frame coding for 456 amino acids. With the genomic protein C clone as a probe in a FISH experiment, the canine protein C gene was assigned to Chromosome (Chr) 19q21-q22. To search for possible mutations, we amplified genomic DNA from one healthy and 15 clinically and pathohistologically confirmed Morbus Perthes patients. Sequence analysis did not reveal any amino acid differences between the affected dogs and the normal control. Several nucleotide polymorphisms were detected, which however, did not result in an amino acid exchange. From these data we conclude that in contrast to human, canine Morbus Perthes disease is most likely not caused by mutations within the protein C gene. Received: 24 June 1998 / Accepted: 18 September 1998     

Gene arrangements and branching orders of gram-positive bacteria

The availability of complete genomic sequence data allows one to develop new methods of reconstructing phylogenetic trees. A simple method of reconstructing branching orders based on gene transposition (or lateral transfer) is presented. It is argued that specific gene arrangements on four different genomes could determine a branching order. A computer search for such gene arrangements was carried out against gene order data of completely sequenced Gram-positive bacteria. Gene arrangements around ribosomal protein S4 gene, murC (UDP-N-acetylmuramate:alanine ligase) gene and dnaE (DNA polymerase III alpha chain) gene each suggest a branching order in which actinobacteria with a high genomic G+C content first branched off from other Gram-positives with a low G+C content and then a split occurred between Mycoplasma species and a group closely related to Bacillus subtilis. A recently sequenced thermophilic bacterium Thermoanaerobacter tengcongensis is suggested to have branched off from the lineage leading to the low G+C Gram-positives prior to the split between the Mycoplasma and Bacillus groups. By contrast to the indel analysis in which a single evolutionary event of insertion or deletion of a signature sequence is assumed, the present method does not necessarily require such a parsimonious assumption of gene transposition.     

Cloning and characterization of a novel gene (C8orf2), a human representative of a novel gene family with homology to C. elegans C42.C1.9.

Large-scale sequencing of selected genomic regions, coupled with in silico gene trapping, is a robust approach to identifying previously unknown genes. In this way we have found a gene (C8orf2) that is highly homologous to C. elegans C42C1.9. C8orf2 was situated on 8p11. 2 between STS markers NIB1979 (proximal) and AFMA295ZD5 (distal), oriented toward the centromere. C8orf2 consisted of 16 exons spanning more than 16.5 kb of genomic DNA, and was expressed ubiquitously in human tissues. The gene encoded 339-and 152-amino acid polypeptides by alternative splicing; the larger variant contained a region extremely rich in charged amino acids, in particular lysine and glutamic acid. C8orf2 also bore sequence homology to the human KE04p gene. Its conservation among highly divergent species suggests that C8orf2 belongs to a novel gene family.     

Functional genomic approaches using the nematode Caenorhabditis elegans as a model system

Since the completion of the genome project of the nematode C. elegans in 1998, functional genomic approaches have been applied to elucidate the gene and protein networks in this model organism. The recent completion of the whole genome of C. briggsae, a close sister species of C. elegans, now makes it possible to employ the comparative genomic approaches for identifying regulatory mechanisms that are conserved in these species and to make more precise annotation of the predicted genes. RNA interference (RNAi) screenings in C. elegans have been performed to screen the whole genome for the genes whose mutations give rise to specific phenotypes of interest. RNAi screens can also be used to identify genes that act genetically together with a gene of interest. Microarray experiments have been very useful in identifying genes that exhibit co-regulated expression profiles in given genetic or environmental conditions. Proteomic approaches also can be applied to the nematode, just as in other species whose genomes are known. With all these functional genomic tools, genetics will still remain an important tool for gene function studies in the post genome era. New breakthroughs in C. elegans biology, such as establishing a feasible gene knockout method, immortalized cell lines, or identifying viruses that can be used as vectors for introducing exogenous gene constructs into the worms, will augment the usage of this small organism for genome-wide biology.     

The Exon/Intron Organization and Chromosomal Mapping of the Mouse ADAMTS-1 Gene Encoding an ADAM Family Protein with TSP Motifs

ADAM is a recently discovered gene family that encodes proteins with a disintegrin and metalloproteinase. ADAMTS-1 is a gene encoding a new member protein of the ADAM family with the thrombospondin (TSP) type I motif, the expression of which is associated with inflammatory processes. In the present study, we have characterized the exon/intron organization of the mouse ADAMTS-1 gene. The ADAMTS-1 gene is composed of nine exons, all of which are present within the 9.2-kb genomic region. Among the nine exons, exons 1, 5, and 6 encode a proprotein domain, a disintegrin-like domain, and a TSP type I motif, respectively, of the ADAMTS-1 protein, suggesting that there is a correlation between exon/intron organization and functional domains. In addition, the exon/ intron organization of the ADAMTS-1 gene is very different from that of the metalloproteinase-like/disintegrin-like/cysteine-rich protein gene (MDC) (ADAM11), suggesting that the genomic structure of ADAM family genes is not necessarily conserved. Furthermore, fluorescencein situhybridization revealed that the ADAMTS-1 gene is located in region C3–C5 of chromosome 16, to which none of the previously identified ADAM genes have been mapped.     

Cloning and genomic organization of the mouse gene slc23a1 encoding a vitamin C transporter.

Vitamin C is known to exist in particularly high concentrations in brain tissue, and its free radical scavenging function is thought to represent a major antioxidative defense system. We have cloned, sequenced and analyzed the genomic structure of a mouse sodium-dependent vitamin C transporter gene, Slc23a1 (also known as Svct2). The mouse Slc23a1 cDNA is 6.4 kb long and was cloned directly from a mouse brain RNA preparation. Hybridization screening of a mouse genomic BAC library identified BAC 53L21 which contains at least the entire coding sequence of the mouse Slc23a1 gene. Determination of the exon-intron structure of the gene revealed 17 exons ranging from 58 bp to 4407 bp extending over 50 kb of the mouse genome, with the translation start codon located in exon 3. Its 1944 nucleotide open reading frame encodes a polypeptide of 647 aa, which is highly similar to rat and human orthologs. The mouse gene was assigned to chromosome 2qG2 by fluorescence in situ hybridization analysis. Expression of this gene was demonstrated in a wide range of tissues, with especially high levels in brain. Neurodegenerative diseases with an established role for oxidative stress in the cytoplasm may therefore be conditions of SLC23A1 dysfunction. Key words: gene structure; Vitamin C; transporter; oxidative stress     

A Candida albicans homolog of a human cyclophilin gene encodes a peptidyl-prolyl cis-trans isomerase

A Candida albicans cDNA and its genomic counterpart were isolated from lambda phage libraries using a human T-cell cyclophilin (Cyp) cDNA as a hybridization probe. The clones contain a 486-bp open reading frame predicting a 162-amino acid, approx. 18 kDa protein which is similar in size to, and which shares 68 and 81% homology with, human T-cell Cyp and cytosolic Saccharomyces cerevisiae Cyp, respectively. Northern blots show the presence of a single mRNA species of about 800 bp. However, genomic Southern blots suggest the presence of at least one other Cyp-related gene in C. albicans. The cDNA was engineered for expression in Escherichia coli, and the resulting recombinant protein, like mammalian Cyps, exhibited a peptidyl-prolyl cis-trans isomerase (PPIase) activity which was sensitive to inhibition by cyclosporin A in vitro. These results indicate that the gene which we have cloned encodes a C. albicans Cyp. We designate this gene CYP1 (cyclophilin). Interestingly, the predicted C. albicans protein contains only two cysteine residues which do not align with any of the four cysteines conserved among mammalian Cyps. This suggests that the PPIase catalytic mechanism may not involve an enzyme-bound hemithioorthoamide, as previously reported for porcine Cyp.