37NISR Research GRANT
2008 Research Grant

Elucidation of Genes Relevant to Microaerobic Growth ofCorynebacterium glutamicum
Masato IKEDA
Department of Bioscience and Biotechnology, Faculty of Agriculture, Shinshu University

Research aims

Results

For most aerobic industrial microorganisms, oxygen is avery important factor for growth and production. Especially, Corynebacterium glutamicum strains that produce
amino acids require a large quantity of oxygen for efficient
production. Under oxygen limitation, production strains
usually accumulate undesirable organic acids, which results
in damaged fermentation with decreased production yields.
For this reason, considerable effort and cost have been devoted to maintenance of high oxygen supply. If we can develop so-called ‘low-O2 adapted strains’ that produce
amino acids effectively even under oxygen limitation, a significant benefit would likely arise industrially. However,there is no such technology at present. We previously compared the oxygen-requiring properties among typical aerobic bacteria and found that C. glutamicum can grow up to
relatively low concentrations of oxygen.1) On the basis of
this finding, it is likely that C. glutamicum possesses somefunction for adaptation to limited oxygen availability. In
this study, we attempted to screen for the relevant genes
from the C. glutamicum genome by genetic complementation of mutants that lost the ability to grow under low oxygen concentrations.

Screening for oxygen-high-requiring mutants. Our preliminary studies revealed that the lower limit of oxygen
concentrations where C. glutamicum can form colonies on
BY agar plates was around 0.5% O2.1) Mutagenized cell libraries of C. glutamicum wild-type strain WT-96 werescreened for mutants that lost the ability to grow at 0.5% O2
by replica plating. These mutants could form colonies
under atmospheric conditions (21% O2), but most of themshowed no growth at 0.5% O2 and even at 6% O2. Figure 1
showed the growth of five typical mutants, designated OX3, OX-96, OX-109, OX-112, and OX-119, under different
oxygen concentrations.

Methods
The wild-type strain of C. glutamicum used in this study
is strain WT-96, a single-colony derivative of C. glutamicum ATCC 31833. Complete medium BY and minimalmedium MM were used for investigation of growth dependency on different oxygen concentrations in C. glutamicum.
For the growth test on BY agar plates under different oxygen concentrations, cells were incubated at 30ºC under theindicated oxygen concentrations in a sealed 2.5-liter box,
using simple culture systems, Anaero Pack (Mitsubishi Gas
Chemical Inc., Tokyo, Japan). For static culture in MM liquid medium, a 0.05-ml amount of the seed culture grown
aerobically at 30ºC in BY medium for 12 h was inoculated
into 5 ml of medium in a test tube and cultivated at 30ºC.
Mutagenized cell libraries of strain WT-96 was prepared byincubating cells at 30ºC for 30 min in 50 mM Tris-maleate
buffer (pH 6.0) containing N-methyl-N-nitro-N-nitrosoguanidine (400 m g/ml). The genomic library of strain WT96 was constructed by ligating its chromosomal DNA digested with BamHI, SalI, or EcoRI to BamHI- or SalI-digested pCS299P or EcoRI-digested pCSEK20.

Cloning of DNA fragments that complement oxygenhigh-requiring phenotype. By the shotgun cloning method
using those five mutants as recipients, we obtained sevenplasmids that enabled those mutants to grow at 0.5% or 6%
O2. These plasmids include pEco1.9 carrying a 1.9-kb
EcoRI DNA fragment that complemented mutant OX-3,
pBam2.1 carrying a 2.1-kb BamHI DNA fragment thatcomplemented mutant OX-96, pEco3.2 carrying a 3.2-kb
EcoRI DNA fragment that complemented mutant OX-109,
pEco1.1 carrying a 1.1-kb EcoRI DNA fragment that complemented mutant OX-112, and pBam1.8, pBam3.2, andpSal2.5 carrying a 1.8-kb BamHI DNA fragment, a 3.2-kb
BamHI DNA fragment, and a 2.5-kb SalI DNA fragment,
respectively, that complemented mutant OX-119. As shownin Fig. 1, it is obvious that each plasmid substantially restored growth of its corresponding mutant under low oxygen conditions (6% and/or 0.5% O2), indicating that thecloned DNA fragments are involved in the phenotype. We
also conducted similar shotgun cloning using other ten oxygen-high-requiring mutants as recipients. However, newlycloned DNA fragments were identical to either of the DNA
fragments above.
Identification of the genes responsible for the complementation. Sequencing and genome database analyses2) ofthe insert DNAs in the seven plasmids revealed the presence of one intact gene Cgl0807 in plasmid pEco1.9, two
intact genes Cgl1101 and Cgl1102 in plasmid pBam2.1,
two intact genes Cgl2859 and Cgl2861 in plasmid pEco3.2,
one intact gene Cgl0600 in plasmid pEco1.1, one intact
gene Cgl1427 in plasmid pBam1.8, two intact genes
Cgl2857 and Cgl2858 in plasmid pBam3.2, and two intact

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Fig. 1. Growth on agar plates under different oxygen concentrations. Appropriate dilutions (ca. 103/ml) of cultures of C. glutamicum wild-type strain WT96, its oxygen-high-requiring mutants, and their transformants were spread onto BY agar plates and cultured at 30ºC for two days under indicated oxygen
concentrations. Growth levels are signified as follows: , good growth; , weak growth; /, marginal growth; -, no growth.

genes Cgl2857 and Cgl2859 in plasmid pSal2.5. As for thethree plasmids, pBam2.1, pEco3.2, and pBam3.2, which
carried two genes on each insert, further subcloning experiments identified that Cgl1102, Cgl2859, and Cgl2857, respectively, were responsible for the complementation.
Taken together, six genes in total were identified as thegenes involved in the oxygen-high-requiring phenotypes:(i) Cgl0600 encoding a putative RNA polymerase s 24 subunit, (ii) Cgl0807 encoding a putative siderophore-interacting protein involved in iron transport, (iii) Cgl1102 encoding a putative ferredoxin involved in electron transfer, (iv)Cgl1427 encoding a putative cytidylate kinase involved innucleic-acid biosynthesis, and (v) Cgl2857 and (vi)Cgl2859 both encoding putative membrane proteins.3) Noneof those are known for their relations to adaptability to low
oxygen conditions in C. glutamicum. In addition, putative
membrane proteins encoded by Cgl2857 and Cgl2859 remain to be assigned even a tentative function. Since orthologs of the two genes could not be found in other microorganisms except C. glutamicum R and Corynebacterium efficiens, the function might be unique to so-called‘glutamic-acid bacteria’.

the five different mutants and examined their cross-complementation abilities in oxygen-limiting static culture with
MM medium. As for plasmids to introduce the four genes
Cgl0807, Cgl1102, Cgl0600, and Cgl1427, we usedpEco1.9, pBam2.1d, pEco1.1, and pBam1.8, respectively,
all carrying single genes alone. As for the other two genes
Cgl2857 and Cgl2859 locating next to each other on thegenome, we used plasmid pSal2.5 carrying the gene cluster,because the two gene products, putative membrane proteins, likely play a single role by forming a complex. The
three plasmids pEco1.1, pBam1.8, and pSal2.5 could moreor less restore the growth defects of not only the corresponding mutants but any other mutants, indicating cross-complementation abilities of the four genes (Cgl0600, Cgl1427,Cgl2857, and Cgl2859).3) The other two plasmids pEco1.9
and pBam2.1d showed cross-complementation with respect
to the two corresponding mutants OX-3 and OX-96, butshowed no substantial cross-complementation toward otherthree mutants, indicating the limited cross-complementation abilities of the two genes (Cgl0807 and Cgl1102).3)
The similar results were obtained on solid plates under different oxygen concentrations.

Cross-complementation of different mutants with eachgene. The fact that the two separate genes Cgl1427 andCgl2857 were obtained by functional complementation of asingle mutant OX-119 (Fig. 1) has suggested the crosscomplementation ability of the gene(s). To verify this possibility for the six genes all, we introduced five plasmids into

Conclusion
Mutagenized cell libraries of Corynebacterium glutamicum were screened for mutants that lost the ability to growunder low oxygen concentrations. The resulting oxygenhigh-requiring mutants were used to clone wild-type DNA

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fragments that could complement the phenotype. Sequencing and subcloning analyses showed that the following six
genes were responsible for the complementation: (i)Cgl0600 encoding a putative RNA polymerase s 24 subunit,(ii) Cgl0807 encoding a putative siderophore-interacting
protein involved in iron transport, (iii) Cgl1102 encoding aputative ferredoxin involved in electron transfer, (iv)
Cgl1427 encoding a putative cytidylate kinase involved innucleic-acid biosynthesis, and (v) Cgl2857 and (vi)Cgl2859 both encoding putative membrane proteins. Someof these genes showed cross-complementation of the mutants in oxygen-limiting static culture, suggesting applicability of those genes to improved growth and production
under oxygen limitation.

References
1)

Takeno, S., Ohnishi, J., Komatsu, T., Masaki, T., Sen, K., and Ikeda,
M. Anaerobic growth and potential for amino acid production by nitrate respiration in Corynebacterium glutamicum. Appl. Microbiol.
Biotechnol. 75, 1173–1182 (2007)
2) Ikeda, M., and Nakagawa, S. The Corynebacterium glutamicum
genome: features and impacts on biotechnological process. Appl. Microbiol. Biotechnol. 62, 99–109 (2003)
3) Ikeda, M., Baba, M., Tsukamoto, N., Komatsu, T., Mitsuhashi, S., andTakeno, S. Elucidation of genes relevant to the microaerobic growthof Corynebacterium glutamicum. Biosci. Biotechnol. Biochem. 73,2806–2808 (2009)