Corynebacterium efficiens is the closest relative of Corynebacterium glutamicum, a species widely used for the industrial production of amino acids. C. efficiens but not C. glutamicum can grow above 40°C. We sequenced the complete C. efficiens genome to investigate the basis of its thermostability by comparing its genome with that of C. glutamicum. The difference in GC content between the species was reflected in codon usage and nucleotide substitutions. Our comparative genomic study clearly showed that there was tremendous bias in amino acid substitutions in all orthologous ORFs. Analysis of the direction of the amino acid substitutions suggested that three substitutions are important for the stability of the C. efficiens proteins: from lysine to arginine, serine to alanine, and serine to threonine. Our results strongly suggest that the accumulation of these three types of amino acid substitutions correlates with the acquisition of thermostability and is responsible for the greater GC content of C. efficiens.

Background-Peripheral arterial disease (PAD) is common but commonly unrecognized. Improved recognition of PAD is needed. We used high-throughput proteomic profiling to find PAD-associated biomarkers. Methods and Results-Plasma was collected from PAD patients (ankle brachial index of Ͻ0.90; nϭ45) and subjects with risk factors but without PAD (nϭ43). Plasma was analyzed with surface-enhanced laser desorption/ionization time-of-flight mass spectrometry to quantify 1619 protein peaks. The peak intensity of a 12-kDa protein was higher in PAD patients. Western blot analyses and immunoaffinity studies confirmed that this protein was ␤2-microglobulin (B2M). In a validation study, B2M was measured by ELISA in plasma in age-and gender-matched PAD (nϭ20) and non-PAD (nϭ20) subjects. Finally, we studied a larger cohort of subjects (nϭ237) referred for coronary angiography but without known PAD. Plasma B2M levels were higher in PAD patients than in non-PAD patients with coronary artery disease. Plasma B2M correlated with ankle brachial index and functional capacity. Independent predictors of PAD were diabetes mellitus, age, and the combination of B2M and C-reactive protein level. Conclusions-In PAD patients, circulating B2M is elevated and correlates with the severity of disease independent of other risk factors. These findings might provide a needed biomarker for PAD and new insight into its pathophysiology. Further studies in other populations are needed to confirm the utility of measuring B2M in cardiovascular disease risk assessment.

Coryneform bacteria are rod-shaped, nonsporulating, grampositive bacteria with a high GC content. The nonpathogenic species, Corynebacterium glutamicum, Brevibacterium lactofermentum, and Brevibacterium flavum, are used for the fermentative production of nucleotides and amino acids, especially L-glutamate. Recently, B. lactofermentum and B. flavum have been reclassified as C. glutamicum (22), and the genome of C. glutamicum ATCC 13032 has been sequenced (12, 30). C. glutamicum was originally isolated as an L-glutamate-producing bacterium (20,36), although the wild type does not overproduce L-glutamate under ordinary culture conditions. Significant L-glutamate production is induced by incubating the biotin-auxotrophic wild type in a biotin-limited medium (32). Although L-glutamate overproduction is suppressed in the presence of an excess of biotin, the addition of certain fatty acid ester surfactants (7, 35) or penicillin (27) to the medium also induces L-glutamate overproduction. For example, as much as 220 mM L-glutamate is produced from 360 mM glucose by wild-type C. glutamicum cells under the L-glutamateproducing condition of adding 1 g of Tween 40 liter Ϫ1 , whereas only 22 mM L-glutamate is produced without Tween 40 addition (Table 1). This means that at least 10-fold more L-glutamate is metabolically synthesized under L-glutamate-producing conditions than under non-L-glutamate-producing conditions. From this perspective, metabolic flux change is expected to be crucial for L-glutamate overproduction in C. glutamicum.The molecular basis of the induction of L-glutamate overproduction remains unclear. One widely held view is that Lglutamate efflux is caused by an alteration in the chemical or physical characteristics of the cell membrane; this is because several changes in the bacterial membrane have been observed under L-glutamate-producing conditions, including a decrease in lipid content, a change in the amount of phospholipid (35), and altered fatty acid composition (31). Recently, alterations of the chemical and physical properties of the cytoplasmic membrane caused by biotin limitation and by genetic modification of lipid biosynthesis were shown to be necessary, but not sufficient, to achieve high L-glutamate efflux (11,25).Changes in metabolic flux have not been discussed as often as the basis of L-glutamate overproduction, although there have been suggestions that this process is important. The 2-oxoglutarate dehydrogenase complex (ODHC) catalyzes the oxidative decarboxylation of 2-oxoglutarate to succinyl coenzyme A (succinyl-CoA), and glutamate dehydrogenase (GDH) catalyzes ammonia assimilation of 2-oxoglutarate to form Lglutamate (Fig. 1). These two enzymes compete for 2-oxoglutarate at the branch point of the tricarboxylic acid (TCA) cycle and L-glutamate biosynthesis. It has been reported that ODHC activities are decreased under L-glutamate-producing conditions (14, 33). We and others previously compared ODHC and GDH activities and found a reduction in ODHC activity relative to GDH activity during...

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SUMMARY BackgroundChitinase 3-like-1 (CHI3L1) is up-regulated in the inflamed mucosa of inflammatory bowel disease (IBD).

The Corynebacterium glutamicum (' Brevibacterium lactohtmentum AJ12036)Laboratories', and Technology and Engineering La bo ra to r i es2, AJ I NO M OTO odhA gene, encoding 2-oxoglutarate dehydrogenase ( E l 0 subunit of the 2-oxoglutarate dehydrogenase complex), has been isolated and identified as an Co., Inc., 1-1 Suzuki-Cho, Kawasaki-ku, Kawasaki-shi 210, Japan homologous counterpart of the Escherichia coli sucA and Bacillus subtilis odhA genes. The nucleotide sequence of a 4394 bp chromosomal fragment containing the C. glutamicum odhA gene was determined. The odhA gene comprised 3771 bp (1257 codons, including the initiation codon) and a molecular mass of 138656 Da was predicted for the OdhA polypeptide. Northern blot analysis revealed a 3.9 kb transcript. The size of the transcript, together with the presence of a rho-independent terminator-like structure, suggests that C, glutamicum odhA is monocistronic. Cells harbouring plasmids carrying C. glutamicum odhA showed a threefold increase in specific 2-oxoglutarate dehydrogenase complex activity and expression of a protein with an apparent molecular mass of 136 kDa, in good agreement with the predicted size of the OdhA polypeptide. The C-terminal region of the C. glutamicum OdhA protein shows strong sequence similarity to Elos from other organisms. C. glutamicum OdhA has an N-terminal extension not found in previously reported Elos. The amino acid sequence of this extension shows similarity to that of the C-terminal region of dihydrolipoamide S-succinyltransferase (E2o) subunits of 2-oxoglutarate dehydrogenase complexes and dihydrolipoamide Sacetyltransferase (E2p) subunits of pyruvate dehydrogenase complexes. It suggests that the C. glutamicum odhA gene might encode a novel bifunctional protein with E l o and E2o activities.

Three glutamic-acid-producing coryneform strains were isolated from soil and vegetable samples. Chemotaxonomic investigations indicated that these strains belonged to the genus Corynebacterium. Phylogenetic studies, based on 16S rDNA analysis, demonstrated that the three strains formed a distinct cluster within the genus Corynebacterium and that their nearest relatives were Corynebacterium glutamicum and Corynebacterium callunae, also known as glutamic-acid-producing species. The data from 16S rDNA sequence and DNA-DNA relatedness studies clearly indicated that the three isolates represented a new species within the genus Corynebacterium. All of the isolates could grow at 45 degrees C and produced acid from dextrin; these were the most significant characteristics differentiating the three isolates from their neighbours. On the basis of the data presented here, it is proposed that the three glutamic-acid-producing isolates together be classified as Corynebacterium efficiens sp. nov., the type strain of which is YS-314T (= AJ 12310T = JCM 11189T = DSM 44549T).

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