Ammonium assimilation and nitrogen control in Corynebacterium glutamicum and its relatives: an example for new regulatory mechanisms in actinomycetes

Nitrogen is an essential component of nearly all complex macromolecules in a bacterial cell, such as proteins, nucleic acids and cell wall components. Accordingly, most prokaryotes have developed elaborate control mechanisms to provide an optimal supply of nitrogen for cellular metabolism and to cope with situations of nitrogen limitation. In this review, recent advances in our knowledge of ammonium uptake, its assimilation, and related regulatory systems in Corynebacterium glutamicum, a Gram-positive soil bacterium used for the industrial production of amino acids, are summarized and discussed with respect to the situation in the bacterial model organisms, Escherichia coli and Bacillus subtilis, and in comparison to the situation in other actinomycetes, namely in mycobacteria and streptomycetes. The regulatory network of nitrogen control in C. glutamicum seems to be a patchwork of different elements. It includes proteins similar to the UTase/GlnK pathway of E. coli and expression regulation by a repressor protein as in B. subtilis, but it lacks an NtrB/NtrC two-component signal transduction system. Furthermore, the C. glutamicum regulation network has unique features, such as a new sensing mechanism. Based on its extremely well-investigated central metabolism, well-established molecular biology tools, a public genome sequence and a newly-established proteome project, C. glutamicum seems to be a suitable model organism for other corynebacteria, such as Corynebacterium diphtheriae and Corynebacterium efficiens.     

New insights into the biogenesis of the cell envelope of corynebacteria: identification and functional characterization of five new mycoloyltransferase genes in Corynebacterium glutamicum

Mycolic acids, the major lipid constituents of Corynebacterineae, play an essential role in maintaining the integrity of the bacterial cell envelope. We have previously characterized a corynebacterial mycoloyltransferase (PS1) homologous in its N-terminal part to the three known mycobacterial mycoloyltransferases, the so-called fibronectin-binding proteins A, B and C. The genomes of Corynebacterium glutamicum (ATCC13032 and CGL2005) and Corynebacterium diphtheriae were explored for the occurrence of other putative corynebacterial mycoloyltransferase-encoding genes (cmyt). In addition to csp1 (renamed cmytA), five new cmyt genes (cmytB-F) were identified in the two strains of C. glutamicum and three cmyt genes in C. diphtheriae. In silico analysis showed that each of the putative cMyts contains the esterase domain, including the three key amino acids necessary for the catalysis. In C. glutamicum CGL2005 cmytE is a pseudogene. The four new cmyt genes were disrupted in this strain and overexpressed in the inactivated strains. Quantitative analyses of the mycolate content of all these mutants demonstrated that each of the new cMyt-defective strains, except cMytC, accumulated trehalose monocorynomycolate and exhibited a lower content of covalently bound corynomycolate than did the parent strain. For each mutant, the mycolate content was fully restored by complementation with the corresponding wild-type gene. Finally, complementation of the cmytA-inactivated mutant by the individual new cmyt genes established the existence of two classes of mycoloyltransferases in corynebacteria.     

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.     

Nitrogen regulation in Corynebacterium glutamicum: isolation of genes involved and biochemical characterization of corresponding proteins

The regulation of nitrogen assimilation was investigated in the Gram-positive actinomycete Corynebacterium glutamicum. Biochemical studies and site-directed mutagenesis revealed that glutamine synthetase activity is regulated via adenylylation in this organism. The genes encoding the central signal transduction protein PH (glnB) and the primary nitrogen sensor uridylyltransferase (glnD) were isolated and sequenced. Additionally, genes putatively involved in the degradation of ornithine (ocd) and sarcosine (soxA), ammonium uptake (amtP) and protein secretion (ftsY, srp) were identified in C. glutamicum. Based on these observations, the mechanism of N regulation in C. glutamicum is similar to that of the Gram-negative Escherichia coli. As deduced from data base searches, the described regulation may also hold true for the important pathogen Mycobacterium glutamicum.     

Heavy metal effects on Proteus mirabilis Superoxide dismutase production

Abstract Levels of genomic DNA relatedness were determined using a SI nuclease procedure for reference bacteria identified as biotypes of Corynebacterium diphtheriae , biovars of Corynebacterium pseudotuberculosis , and ' Corynebacterium ulcerans '. These results showed that the three species are separate taxa at the genomospecies level whereas biotypes and biovars are closely related genomically within each species. Phylogenetic analyses of small-subunit rDNA sequences revealed that ' Corynebacterium ulcerans ' forms a tight cluster with Corynebacterium pseudotuberculosis within the robust branch that groups all Corynebacterium sequenced to date. Therefore, we propose that the species incertae sedis ' C. ulcerans ' should be conclusively recognized as a distinct species within the genus Corynebacterium with strain CCUG 2708 = NCTC 7910 as type strain. This species is characterized by urease production and fermentation of glycogen.     

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.     

嘧啶核苷类物质乳清酸产生菌的选育

以谷氨酸棒杆菌JSIM-201菌株为出发菌株,通过紫外线和甲基磺酸乙酯诱变处理,得到了一株尿嘧啶营养缺陷型突变体U-12菌株,能以葡萄糖为碳源,硫酸铵为氮源,在发酵液中积累一种紫外吸收物质。对U-12菌株的发酵液分离提取结晶,经物理、化学分析鉴定,证明是乳清酸物质。发酵液中积累乳清酸8．6g/L。     

The complete Corynebacterium glutamicum ATCC 13032 genome sequence and its impact on the production of L-aspartate-derived amino acids and vitamins

The complete genomic sequence of Corynebacterium glutamicum ATCC 13032, well-known in industry for the production of amino acids, e.g. of L-glutamate and L-lysine was determined. The C. glutamicum genome was found to consist of a single circular chromosome comprising 3282708 base pairs. Several DNA regions of unusual composition were identified that were potentially acquired by horizontal gene transfer, e.g. a segment of DNA from C. diphtheriae and a prophage-containing region. After automated and manual annotation, 3002 protein-coding genes have been identified, and to 2489 of these, functions were assigned by homologies to known proteins. These analyses confirm the taxonomic position of C. glutamicum as related to Mycobacteria and show a broad metabolic diversity as expected for a bacterium living in the soil. As an example for biotechnological application the complete genome sequence was used to reconstruct the metabolic flow of carbon into a number of industrially important products derived from the amino acid L-aspartate.     

The Corynebacterium glutamicum gene pmt encoding a glycosyltransferase related to eukaryotic protein-O-mannosyltransferases is essential for glycosylation of the resuscitation promoting factor (Rpf2) and other secreted proteins

Two-dimensional gel electrophoresis and immunoassays revealed several proteins of the secretory subproteome of Corynebacterium glutamicum to be glycosylated. By genome-wide searches for genes involved in glycosylation, the C. glutamicum gene cg1014 was found to exhibit significant similarity to eukaryotic protein-O-mannosyltransferases (PMTs) and to a recently identified orthologue of Mycobacterium tuberculosis, Rv1002c, which is responsible for protein-O-mannosylation. The putative membrane protein Cg1014 showed the same predicted transmembrane topology as Saccharomyces cerevisiae PMT1 and M. tuberculosis Rv1002c along with conserved amino acid residues responsible for catalytic activity. Deletion of the C. glutamicum pmt gene (cg1014) caused a complete loss of glycosylation of secreted proteins including the resuscitation promoting factor 2 (Rpf2), which is involved in intercellular communication and growth stimulation of C. glutamicum. Because the gene pmt as well as rpf genes are present in the genomes of all actinobacteria sequenced so far, this work provides new insights into bacterial protein glycosylation and new opportunities to elucidate the molecular mechanisms of Rpf activity in pathogenic growth and infection.     

DNA sequence homology between attB-related sites of Corynebacterium diphtheriae, Corynebacterium ulcerans, Corynebacterium glutamicum, and the attP side of γ-Cornephage

Chromosomal restriction fragments of Corynebacterium ulcerans and C. diphtheriae, containing an integration site for corynephages of the beta family, show homology on Southern blots. Homologous DNA in also found in the soil isolate C. glutamicum, although this strain is not susceptible to beta-corynephages. Three of these DNA fragments, one for each bacterial strain, and a fragment of gamma-corynephage DNA previously shown to contain the phage integration site, were cloned and sequenced. Alignment of the 3 bacterial sequences shows a very high degree of homology in a stretch of ca 120 nucleotides, whereas the rest of the sequences is generally non-homologous. Within this common bacterial portion, a segment of ca. 96 nucleotides (core sequence) is also highly homologous to the phage sequence. The first half (ca. 50 bp) of the core sequence is identical in all aligned sequences whereas the second half, which is largely occupied by a stem-and-loop structure, contains point mutations peculiar to each clone. The described sequences are likely to be involved in phage integration/excision processes.     

外源精氨酸对谷氨酸棒杆菌在高葡萄糖胁迫下生长和发酵特性的影响

【目的】探究外源添加不同氨基酸和相容性溶质对谷氨酸棒杆菌(Corynebacterium glutamicum)在高糖胁迫环境下生长的影响及可能的作用机理。【方法】通过在培养基中外源添加各种氨基酸和相容性溶质,研究其对谷氨酸棒杆菌在高葡萄糖和高蔗糖胁迫下生长的影响,并分析添加精氨酸对高葡萄糖胁迫下菌株糖转运和代谢途径中关键酶转录水平的影响,以及对菌株发酵产氨基酸的影响。进一步探究了碱性氨基酸在其它棒状杆菌属中抵御高葡萄糖胁迫的潜在作用。【结果】在高葡萄糖胁迫条件下,外源添加赖氨酸、精氨酸和组氨酸后谷氨酸棒杆菌的生物量分别提高54.7%、50.0%和37.6%;而在高蔗糖胁迫条件下,添加脯氨酸和四氢嘧啶后菌株生物量增加20%以上。进一步研究表明,在高葡萄糖胁迫下,外源添加精氨酸后谷氨酸棒杆菌的葡萄糖利用速率提高约2.5倍,谷氨酸的发酵产量也增加了127.5%。此外,碱性氨基酸对其它4种棒状杆菌也具有一定的渗透保护效应。【结论】精氨酸对谷氨酸棒杆菌在高葡萄糖胁迫下具有良好的渗透保护作用,可能归因于其能促进葡萄糖的转运和代谢能力,同时发现碱性氨基酸的渗透保护效应对棒状杆菌属具有一定的普遍性。     

赖氨酸-尸胺反向转运蛋白cadB基因谷氨酸棒杆菌表达载体的构建及转化

旨在提高谷氨酸棒杆菌合成尸胺的能力,将CadB克隆至谷氨酸棒杆菌中,与LDC共表达,在谷氨酸棒杆菌合成尸胺的同时,帮助尸胺转运至细胞外,解除尸胺的反馈抑制作用。谷氨酸棒杆菌能够高产赖氨酸脱羧酶的底物L-赖氨酸,但不含ldc和cadB基因,因而不能够直接合成尸胺。从E.coliK12中克隆出赖氨酸-尸胺反向转运蛋白基因,与绿色荧光蛋白基因gfp融合构建成融合表达载体pXBG,并转化至谷氨酸棒杆菌进行诱导表达,结果表明表达的CadB蛋白可以正确的定位于谷氨酸棒杆菌的细胞膜上。将基因cadB连接到含有赖氨酸脱羧酶基因的pXMJ19-ldc上,构建成能够共表达赖氨酸脱羧酶和赖氨酸-尸胺反向转运蛋白的重组质粒pXLB,并转化到谷氨酸棒杆菌中。