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 acyl-AMP ligase FadD32 and AccD4-containing acyl-CoA carboxylase are required for the synthesis of mycolic acids and essential for mycobacterial growth: identification of the carboxylation product and determination of the acyl-CoA carboxylase components

Mycolic acids are major and specific long-chain fatty acids of the cell envelope of several important human pathogens such as Mycobacterium tuberculosis, M. leprae, and Corynebacterium diphtheriae. Their biosynthesis is essential for mycobacterial growth and represents an attractive target for developing new antituberculous drugs. We have previously shown that the pks13 gene encodes condensase, the enzyme that performs the final condensation step of mycolic acid biosynthesis and is flanked by two genes, fadD32 and accD4. To determine the functions of the gene products we generated two mutants of C. glutamicum with an insertion/deletion within either fadD32 or accD4. The two mutant strains were deficient in mycolic acid production and exhibited the colony morphology that typifies the mycolate-less mutants of corynebacteria. Application of multiple analytical approaches to the analysis of the mutants demonstrated the accumulation of a tetradecylmalonic acid in the DeltafadD32::km mutant and its absence from the DeltaaccD4::km strain. The parental corynebacterial phenotype was restored upon the transfer of the wild-type fadD32 and accD4 genes in the mutants. These data demonstrated that both FadD32 and AccD4-containing acyl-CoA carboxylase are required for the production of mycolic acids. They also prove that the proteins catalyze, respectively, the activation of one fatty acid substrate and the carboxylation of the other substrate, solving the long-debated question of the mechanism involved in the condensation reaction. We used comparative genomics and applied a combination of molecular biology and proteomic technologies to the analysis of proteins that co-immunoprecipitated with AccD4. This resulted in the identification of AccA3 and AccD5 as subunits of the acyl-CoA carboxylase. Finally, we used conditionally replicative plasmids to show that both the fadD32 and accD4 genes are essential for the survival of M. smegmatis. Thus, in addition to Pks13, FadD32 and AccD4 are promising targets for the development of new antimicrobial drugs against pathogenic species of mycobacteria and related microorganisms.     

Multiplex polymerase chain reaction to identify and determine the toxigenicity of Corynebacterium spp with zoonotic potential and an overview of human and animal infections

Corynebacterium diphtheriae, Corynebacterium ulcerans and Corynebacterium pseudotuberculosis constitute a group of potentially toxigenic microorganisms that are related to different infectious processes in animal and human hosts. Currently, there is a lack of information on the prevalence of disease caused by these pathogens, which is partially due to a reduction in the frequency of routine laboratory testing. In this study, a multiplex polymerase chain reaction (mPCR) assay that can simultaneously identify and determine the toxigenicity of these corynebacterial species with zoonotic potential was developed. This assay uses five primer pairs targeting the following genes: rpoB (Corynebacterium spp), 16S rRNA (C. ulcerans and C. pseudotuberculosis), pld (C. pseudotuberculosis), dtxR (C. diphtheriae) and tox [diphtheria toxin (DT) ]. In addition to describing this assay, we review the literature regarding the diseases caused by these pathogens. Of the 213 coryneform strains tested, the mPCR results for all toxigenic and non-toxigenic strains of C . diphtheriae, C. ulcerans and C. pseudotuberculosis were in 100% agreement with the results of standard biochemical tests and PCR-DT. As an alternative to conventional methods, due to its advantages of specificity and speed, the mPCR assay used in this study may successfully be applied for the diagnosis of human and/or animal diseases caused by potentially toxigenic corynebacterial species.     

Comparisons of potentials for L-lysine production among different Corynebacterium glutamicum strains

Corynebacterium glutamicum is an industrially important organism that is most widely used for the production of various amino acids. A defined L-lysine-producing mutant was generated by introduction of the lysC mutation (T311I) into each of six representative C. glutamicum strains. The resulting six isogenic mutants were compared for L-lysine production under traditional 30 degrees C conditions and industrially more advantageous 40 degrees C conditions. It was found that there were significant differences in yield and productivity, especially at 40 degrees C. These results indicate the diversity among C. glutamicum strains in fermentative characters, as well as the importance of selecting a strain with industrially best performance.     

Engineering of a xylose metabolic pathway in Corynebacterium glutamicum

The aerobic microorganism Corynebacterium glutamicum was metabolically engineered to broaden its substrate utilization range to include the pentose sugar xylose, which is commonly found in agricultural residues and other lignocellulosic biomass. We demonstrated the functionality of the corynebacterial xylB gene encoding xylulokinase and constructed two recombinant C. glutamicum strains capable of utilizing xylose by cloning the Escherichia coli gene xylA encoding xylose isomerase, either alone (strain CRX1) or in combination with the E. coli gene xylB (strain CRX2). These genes were provided on a high-copy-number plasmid and were under the control of the constitutive promoter trc derived from plasmid pTrc99A. Both recombinant strains were able to grow in mineral medium containing xylose as the sole carbon source, but strain CRX2 grew faster on xylose than strain CRX1. We previously reported the use of oxygen deprivation conditions to arrest cell replication in C. glutamicum and divert carbon source utilization towards product production rather than towards vegetative functions (M. Inui, S. Murakami, S. Okino, H. Kawaguchi, A. A. Vertès, and H. Yukawa, J. Mol. Microbiol. Biotechnol. 7:182-196, 2004). Under these conditions, strain CRX2 efficiently consumed xylose and produced predominantly lactic and succinic acids without growth. Moreover, in mineral medium containing a sugar mixture of 5% glucose and 2.5% xylose, oxygen-deprived strain CRX2 cells simultaneously consumed both sugars, demonstrating the absence of diauxic phenomena relative to the new xylA-xylB construct, albeit glucose-mediated regulation still exerted a measurable influence on xylose consumption kinetics.     

Integration of corynebacteriophages beta tox+, omega tox+, and gamma tox- into two attachment sites on the Corynebacterium diphtheriae chromosome.

The bacterial attachment sites of independently isolated Corynebacterium diphtheriae strains C7s and (belfanti)1030 lysogenic for corynebacteriophages beta tox+, omega tox+, and gamma tox- were determined by Southern blot analysis. Both corynebacterial strains contained two distinct bacterial attachment sites (attB1 and attB2). We found that infection by any of the three closely related corynebacteriophages may give rise to single, double, and triple lysogens. In the case of toxigenic C. diphtheriae strains C7s(beta tox+) and C7s(omega tox+), the final yields of diphtheria toxin produced under optimal conditions were equivalent and varied by one-, two-, or threefold depending upon the number of integrated prophage.     

Identification and characterization of the last two unknown genes,dapC and dapF,in the succinylase branch of the L-lysine biosynthesis of Corynebacterium glutamicum

The inspection of the complete genome sequence of Corynebacterium glutamicum ATCC 13032 led to the identification of dapC and dapF, the last two unknown genes of the succinylase branch of the L-lysine biosynthesis. The deduced DapF protein of C. glutamicum is characterized by a two-domain structure and a conserved diaminopimelate (DAP) epimerase signature. Overexpression of dapF resulted in an 8-fold increase of the specific epimerase activity. A defined deletion in the dapF gene led to a reduced growth of C. glutamicum in a medium with excess carbon but limited ammonium availability. The predicted DapC protein of C. glutamicum shared 29% identical amino acids with DapC from Bordetella pertussis, the only enzymatically characterized N-succinyl-aminoketopimelate aminotransferase. Overexpression of the dapC gene in C. glutamicum resulted in a 9-fold increase of the specific aminotransferase activity. A C. glutamicum mutant with deleted dapC showed normal growth characteristics with excess carbon and limited ammonium. Even a mutation of the two genes dapC and ddh, interrupting both branches of the split pathway, could be established in C. glutamicum. Overexpression of the dapF or the dapC gene in an industrial C. glutamicum strain resulted in an increased L-lysine production, indicating that both genes might be relevant targets for the development of improved production strains.     

Analysis of Corynebacterium glutamicum methionine biosynthetic pathway: isolation and analysis of metB encoding cystathionine gamma-synthase.

The metB gene encoding cystathionine y-synthase, the second enzyme of methionine biosynthetic pathway, was isolated from a pSL109-based Corynebacterium glutamicum gene library via complementation of an Escherichia coli metB mutant. A DNA-sequence analysis of the cloned DNA identified an open-reading frame of 1161 bp which encodes a protein with the molecular weight of 41,655 comprising of 386 amino acids. The putative protein product showed good amino acid-sequence homology to its counterpart in other organisms. Introduction of a plasmid carrying the cloned metB into the C. glutamicum resulted in a 10-fold increase in cystathionine gamma-synthase activities, demonstrating the identity of the cloned gene. The C. glutamicum metB mutant which was generated by the site-specific integration of the cloned DNA into its chromosome did not lose the ability to grow on glucose minimal medium lacking supplemental methionine. The growth rate of the mutant strain was also comparable to that of the parental strain. These data indicate that, in addition to the transsulfuration pathway, other methionine biosynthetic pathways may be present in C. glutamicum.     

Cloning and characterization of a DNA region encoding a stress-sensitive restriction system from Corynebacterium glutamicum ATCC 13032 and analysis of its role in intergeneric conjugation with Escherichia coli.

RP4-mediated transfer of mobilizable plasmids in intergeneric conjugation of Escherichia coli donors with Corynebacterium glutamicum ATCC 13032 is severely affected by a restriction system in the recipient that can be inactivated by a variety of exogenous stress factors. In this study a rapid test procedure based on intergeneric conjugal plasmid transfer that permitted the distinction between restriction-negative and restriction-positive C. glutamicum clones was developed. By using this procedure, clones of the restriction-deficient mutant strain C. glutamicum RM3 harboring a plasmid library of the wild-type chromosome were checked for their restriction properties. A complemented clone with a restriction-positive phenotype was isolated and found to contain a plasmid with a 7-kb insertion originating from the wild-type chromosome. This plasmid, termed pRES806, is able to complement the restriction-deficient phenotype of different C. glutamicum mutants. Sequence analysis revealed the presence of two open reading frames (orf1 and orf2) on the complementing DNA fragment. The region comprising orf1 and orf2 displayed a strikingly low G+C content and was present exclusively in C. glutamicum strains. Gene disruption experiments with the wild type proved that orf1 is essential for complementation, but inactivation of orf2 also resulted in a small but significant increase in fertility. These results were confirmed by infection assays with the bacteriophage CL31 from Corynebacterium lilium ATCC 15990.     

Identification and functional analysis of six mycolyltransferase genes of Corynebacterium glutamicum ATCC 13032: the genes cop1, cmt1, and cmt2 can replace each other in the synthesis of trehalose dicorynomycolate,a component of the mycolic acid layer of the cell envelope

By data mining in the sequence of the Corynebacterium glutamicum ATCC 13032 genome, six putative mycolyltransferase genes were identified that code for proteins with similarity to the N-terminal domain of the mycolic acid transferase PS1 of the related C. glutamicum strain ATCC 17965. The genes identified were designated cop1, cmt1, cmt2, cmt3, cmt4, and cmt5 ( cmt from corynebacterium mycolyl transferases). cop1 encodes a protein of 657 amino acids, which is larger than the proteins encoded by the cmt genes with 365, 341, 483, 483, and 411 amino acids. Using bioinformatics tools, it was shown that all six gene products are equipped with signal peptides and esterase domains. Proteome analyses of the cell envelope of C. glutamicum ATCC 13032 resulted in identification of the proteins Cop1, Cmt1, Cmt2, and Cmt4. All six mycolyltransferase genes were used for mutational analysis. cmt4 could not be mutated and is considered to be essential. cop1 was found to play an additional role in cell shape formation. A triple mutant carrying mutations in cop1, cmt1, and cmt2 aggregated when cultivated in MM1 liquid medium. This mutant was also no longer able to synthesize trehalose di coryno mycolate (TDCM). Since single and double mutants of the genes cop1, cmt1, and cmt2 could form TDCM, it is concluded that the three genes, cop1, cmt1, and cmt2, are involved in TDCM biosynthesis. The presence of the putative esterase domain makes it highly possible that cop1, cmt1, and cmt2 encode enzymes synthesizing TDCM from trehalose monocorynomycolate.     

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 amrG1 gene is involved in the activation of acetate in Corynebacterium glutamicum

Corynebacterium glutamicum is an aerobic, Gram-positive microorganism, well known as a pro-ducer of several amino acids. Amino acid products are used on a large scale for food industry flavouring, feed additive, pharmaceutical and cosmetic purpose[1,2]. The organism is able to grow not only on glucose, fructose and lactose, but also on acetate, lactate as its sole carbon source. The growth on acetate requires its activation to acetyl-CoA. In C. glutamicum, acetate is activated in a two-step …