Corynebacterium efficiens

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A Microbial Biorealm page on the genus Corynebacterium efficiens

Classification

Higher order taxa

Cellular organisms; Bacteria; Actinobacteria; Actinobacteria (class); Actinobacteridae; Actinomycetales; Corynebacterineae; Corynebacteriaceae; Corynebacterium; Corynebacterium efficiens

Species

NCBI: Taxonomy

Corynebacterium efficiens

Description and significance

Monosodium Glutamate is mired in controversy today as both a unique flavor enhancer and potential cause of human neurologic disorders.[1] As sides spar over this issue, the reality is production of L-gluatamine aka l-gln (the pre-cursor amino acid to MSG) is in excess of 1 million tons.[2] For many companies, the choice method of l-gln production is via fermentation of sugars by microorganisms. Most notable are the microbes of the genus Corynebacterium. Species exploited for commercial production of l-gln have been Corynebacterium glutamicum and Corynebacterium callunae.[2,3] Ajinomoto, a Clinical Research Laboratory in Japan, has isolated three unique strains proven to be more efficient in l-gln production. (The details will be described in Biotech section) These three strains have been phylogenetically identified as a unique species of Corynebacterium, and are collectively named Corynebacterium efficiens.[2]

C. efficiens are gram-positive, non-motile cells. The isolates used to determine this new species were obtained from onion bulbs and soils of Kanagawa, Japan.[2] Grown on agar plates, the isolates are best grown between 30 and 40° C and appear as yellow, smooth, circular colonies.[2] As “coryneform” is literally translated to “club-shaped rods” in Greek, individual C. efficiens cells present as V-shaped rods caused by a “snapping” action during cell division.[2,4] C. efficiens is grown aerobically on simple media with glucose as the primary carbon source.

C. efficiens is of specific interest to companies involved in commercial production of amino acids because of its thermostability. The effectiveness of this species’ ability to grow efficiently at high temperatures has led to current genome sequencing to understand the genomic characteristics contributing to this organism’s thermostability. Scientists believe the key underlying C. efficiens thermostability will be invaluable for the development of thermostable protein synthesis.

Genome structure

C. efficiens has a single circular chromosome of approximately 3,147,090 bp and two plamids 23,743 bp and 48,672 bp in length.[2,5] The chromosome has a G+C content of 63% and the plasmids have a G+C content of 54% and 56%. This high G+C content is thought to play a factor in the thermostability of the organism.[2] The average Open Reading Frame for the chromosome is 981 bp. The average ORF for Plasmid 1 is 1,155 bp and plasmid 2 is 924 bp.

The genome structure plays an important role in identification of this specific species. Comparing 16S rDNA genome sequences of c. efficiens vs. the Corynebacterium genus reveal that the isolates of C. efficiens belong to the “glutamic-acid producing species".[2,6] Of these acid producing species, C. efficiens was 95.3 % similar to C. glutamicum. This is lower than the criteria used to define identical bacterial species (97%).[2] The importance of high G+C content will be discussed in “Cell structure and Metabolism”

C. efficiens genetic data is obtained from bacterial strain YS-314 [2]

Cell structure and metabolism

Hallmark to C. efficiens is its distinctive ability to produce l-gln at higher temperatures. Prior to C. efficiens identification, the microbe of choice for commercial production of l-gln is C. glutamicum. This species produce amino acid and grow effectively at a top temperature of ~30° C. C. efficiens has been found to grow and produce acid at 45° C.[2,7] The benefit of this advantage will be discussed in “application to biotechnology”. Comparative analysis of C. efficiens and other mesophilic corynebacterium (C. glutamicum & C. diphtheriae) reveal unique amino acid characteristics of C. efficiens proteins.[7] As mentioned above, C. efficiens has a higher G+C content which immediately indicate a higher “melting” temperature for DNA strands. Furthermore, studies have found that compared to C. glutamicum, C. efficiens has general amino acid substitutions throughout its proteins and cell structures. Notably arginine for lysine, and alanine/threonine for serine.[7] These amino acid substitutions correlate to protein thermostability as Arg maintains ion pairs more easily, and Ala/Thr strengthen hydrophobic interactions leading to stronger β-sheets for proteins.[7]

As mentioned many previously, C. efficiens is highly lauded for its ability to produce the amino acid l-glutamine. It produces this amino acid optimally when cultured aerobically at 45° C (in presence of 6% glucose).[2] It has been found that production also occurs with carbon sources such as fructose, mannose, ribose, maltose and dextrin. C. efficiens does not produce acid from xylose, mannitol, lactose, strarch or glycogen.[2]

Regarding nitrogen metabolism, studies have found certain gene clusters for nitrate uptake systems that indicate C. efficiens is better equipped for anaerobic nitrate and nitrite respiration compared to C. glutamicum.[10]

Ecology

C. efficiens is a soil living species. This habitat confers with studies that have found that C. efficiens along with fellow soil-living species C. glutamicum exhibit a broader spectrum of genes for nitrogen transport and metabolism compared to the pathogenic species C. diphtheriae and C. jeikeium.[10] The three specific isolates used to identify C. efficiens were isolated from onion bulbs and soils of Kanagawa, Japan. It is believed that C. efficiens along with other soil-dwelling Corynebacteria play primary roles in nitrogen levels of soil and atmosphere.[8]

As some Corynebacterium forms have been found to be pathogenic, it is apparent there is a diverse range of environments Corynebacterium can develop in. The thermostability of C. efficiens, for example, demonstrates the high adapatability of Corynebacterium.

Comparing C. efficiens genomically to C. glutamicum and C. diptheriae demonstrates thermoadaptability. C. diptheriae is not considered a glutamic acid producing strain and was found to be more closely related to C. glutamicum rather than C. efficiens. This suggests that C. glutamicum is closer to the ancestral genome strucure of Corynebacterium and C. efficiens must have acquired thermostability through divergence from sister species.[7] This, in turn, allows C. efficiens to grow in habitats of higher temperature.[8]

Pathology

Currently, C. efficiens is not known to be a human pathogen. Monosodium glutamate, the highly controversial compound produced from the L-gln of C. efficiens, is argued to be cause of numbness, and tingling when consumed. More specifically, monosodium glutamate is proposed to affect the hypothalamus and may even be a major cause of obesity.[1] The microorganism C. efficiens however has not been proven to be a direct human or plant pathogen.

Application to Biotechnology

C. efficiens holds the attention of biotechnology companies for its ability to remain stable at high temperatures. Motivation for these companies is the mantra “to produce high quality l-gln at a low cost, it is of prime importance to obtain a strain of microorganism with food production efficiency.”[9,2] Prior to fermentation as the primary method of extraction, the methods used were extraction from hydolysates of plant or animal protein, and chemical synthesis.[9] Fermentation however has emerged as the choice form for large scale production. The advantage that C. efficiens holds is its ability to grow substantially at 45°C, a temperature where C. glutamicum or C. callunae cannot grow.[2] To sustain bacterial activity in production of l-gln, a cooling system must be used, a costly endeavor for companies. If fermentation at high temperatures could proceed without significant cooling, cooling costs (loss of bacterial activity) would be reduced and endow economic advantage for the company.[2] For this reason, ‘’C. efficiens’’ thrives as a strain of choice for the production of l-gln. Partnered with the increasing demand for monosodium glutamate, and l-gln as a pharmaceutical grade supplement, C. efficiens faces a future of promising growth in the biotechnology field.[2,9]?

Current Research

1.) Paired with the constant advancement of genomic analysis technology is the emergence of bioinformatics. Bioinformatics is defined as “the use of computer science, mathematics, and information theory to model and analyze biological systems, especially systems involving genetic material.”[12] One component of bioinformatics is software programs that help to further analyze genomic sequences. Currently, one project is CoryneRegNet: An Integrative Bioinformatics Platform for the Analysis of Transcription Factors and Regulatory Networks. [13] This program is based on analysis from Corynebacterium to provide further information on transcriptional regulatory networks. C. efficiens is one species used as a bacterial model to “allow systematic analysis of network [transcriptional] behavior in response to environmental conditions.” [13]

2.) As biotech companies recognized the economic advantage of growing glutamate strains at higher temperatures, researchers wanted to further study C. efficiens to discover properties that allow for its thermostability. As these studies progressed, characteristics for thermo-stable proteins were discovered as well as specific metabolism pathways catered to increased glutamate production. Current research is shedding light on the effect of 2-oxoglutamate dehydrogenase complex on glutamate production.[14] An initial comparative study done comparing C. glutamicum and C. efficiens indicate that glutamate production is improved with a decrease in 2-oxoglutamate dehydrogenase complex activity.[14] This conclusion paired with other current studies are springboards for further development of more efficient strains for amino acid production.

3.) Most current research on C. efficiens is focused on the thermostability of its components. As the G+C content of the genome was shown, the amino acid substitutions discovered by Nishio and his group prove to be the most promising in a growing field of protein synthesis.[7] Most information and subsequent studies of C. efficiens are associated with the pilot company linked to its discovery, Ajinomoto of Japan. C. efficiens is leading the way for development of efficient and economical bacterial strains for fermentative processes.[2,7]

References

1. TRUTH IN LABELING CAMPAIGN. “Effects of Processed Glutamic Acid (MSG).” 10 Aug 2007. <http://www.truthinlabeling.org/>

2. R. Fudou, Y. Jojima, A. Seto, K. Yamada, E. Kimura, T. Nakamatsu, A. Hiraishi and S. Yamanaka. “Corynebacterium efficiens sp. nov., a glutamic-acid-producing species from soil and vegetables”. International Journal of Systematic and Evolutionary Microbiology, Vol 52, 1127-1131, Copyright © 2002 by Society for General Microbiology. <http://ijs.sgmjournals.org/cgi/content/abstract/52/4/1127>

3. Liebl, W. (1992). “The genus Corynebacterium – nonmedical”. The Prokaryotes, 2nd ed., vol. 2. pp. 1157-1171.

4. Akers, A. “Corynebacterium – Soil Microbiology”. 24 Aug 2007 <http://filebox.vt.edu/users/chagedor/biol_4684/Microbes/coryne.html>

5. Kanehisa Laboratories - Kyoto Encyclopedia of Genes and Genomes. “Corynebacterium efficiens”. Kanehisa Laboratory, Bioinformatics Center, Institute for Chemical Research, Kyoto University. Kanehisa Laboratory, Human Genome Center, Institute of Medical Science, University of Tokyo. 10 July 2007. <http://www.genome.jp/kegg-bin/show_organism?org=cef>

6. Pascual, L., Lawson, P.A., Farrow, J., Navarro, M., Collins, M. (1995). “Phylogenetic analysis of the genus ‘’Corynebacterium’’ based on 16s rRNA gene sequences”. International Journal of Systematic Bacteriology. 45, 724-728. Oct 1995

7. Nishio,Y., Nakamura, Y., Kawarabayasi, Y., Usuda, Y., Kimura, E., Sugimoto, S., Matsui, K., Yamagishi, A., Kikuchi, H., Ikeo, K., and Gojobori, T. “Comparative Complete Genome Sequence Analysis of the Amino Acid Replacements Responsible for the Thermostability of Corynebacterium efficiens”. Genome Research. 13:1572-1579, 2003. <http://www.genome.org/cgi/content/full/13/7/1572>

8. Marri, P., Hao, W., Golding, G.B. “The role of laterally transferred genes in adaptive evolution”. BMC Evolutionary Biology 2007, 7(Suppl 1):S8. 8 Feb 2007. <http://www.biomedcentral.com/1471-2148/7/S1/S8>

9. Kusumoto, I. “Industrial production of L-Glutamine”. J Nutr. 2001 Sep;131(9 Suppl):2552S-5S. Ajinomoto Co., Inc., Kawasaki, Japan. <http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=11533312&ordinalpos=12&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum>

10. Walter B, Hänssler E, Kalinowski J, Burkovski. “Nitrogen metabolism and nitrogen control in corynebacteria: variations of a common theme”. Journal of Molecular Microbioly and Biotechnology. 2007;12(1-2):131-8. <http://content.karger.com/ProdukteDB/produkte.asp?Aktion=ShowFulltext&ArtikelNr=96468&Ausgabe=232311&ProduktNr=228391>

11. Baumbach, J., Rahmann, S., Tauch, A. “CoryneRegNet: An Integrative Bioinformatics Platform for the Analysis of Transcription Factors and Regulatory Networks”. Bielefeld University,Germany. <http://www.eccb06.org/new_pages/program/accepted_sw.html>

12. Lexico Publishing Group, LLC. “bioinformatics”. 2007. <www.dictionary.com>

14. Shirai T, Nakato A, Izutani N, Nagahisa K, Shioya S, Kimura E, Kawarabayasi Y, Yamagishi A, Gojobori T, Shimizu H. “Comparative study of flux redistribution of metabolic pathway in glutamate production by two coryneform bacteria”. Center for Information Biology and DNA Data Bank of Japan, National Institute of Genetics. Metabolic Engineering. 2005 Mar;7(2):59-69. <http://www.ncbi.nlm.nih.gov/sites/entrez>

Edited by Chris Bunag, student of Rachel Larsen

Edited by KLB