Rol De Ompt

Biosci. Biotechnol. Biochem., 71 (2), 504–512, 2007

Role of the ompT Mutation in Stimulated Decrease in Colony-Forming Ability
Due to Intracellular Protein Aggregate Formation in Escherichia coli
Strain BL21
Bun-ichiro O NO,1; y Hiroko K IMIDUKA,1 Masashi K UBOTA,1
Kazuaki O KUNO,2 and Masayuki Y ABUTA2
1

Department of Biotechnology, Faculty of Science and Engineering, RitsumeikanUniversity,
1-1-1 Noji-higashi, Kusatsu, Shiga 525-8577, Japan
2
Biopharma Center, Daiichi Asubio Pharma Co., Ltd., 2716-1 Kurakake, Akaiwa,
Chiyoda-machi, Ohra-gun, Gunma 370-0503, Japan
Received October 2, 2006; Accepted November 18, 2006; Online Publication, February 7, 2007
[doi:10.1271/bbb.60541]

Recently we found that the cells of Escherichia coli
strain BL21 producing a fusionprotein, GST-Sup35NM,
show a much more rapid decrease in colony-forming
ability in the stationary phase than control cells. In this
study, it was found that an extract of the cells producing
GST-Sup35NM forms fibrous protein polymers containing GST-Sup35NM. In the course of the study, we
realized that strain BL21 carried the ompT mutation.
We suspected that the deficiency in OmpT protease wasresponsible for the observed phenotype. To test this, we
introduced the wild-type ompT gene into strain BL21,
and found that the transformed cells recovered the wildtype phenotype. We concluded that OmpT protease,
though known to localize on the cell surface, is involved
in protein quality control within the cell.
Key words:

yeast prion [PSIþ ]; colony-forming ability
of E. coli; proteinpolymer formation;
OmpT protease; protein quality control

Prions, mammalian pathogens that cause disorders of
the central nervous system, are proteins encoded by the
Prn gene. They have at least two distinct conformations.
One is pathogenic, and the other is not.1) The pathogenic
form has long been known to construct amyloid-like
rods and later was found to be rich in the -sheet
structure inthe molecule.2,3) It is now known that there
are several proteins that persist in the cell by means
of conformational alterations similarly to mammalian
prions. Of these, the best known is the [PSIþ ] factor
encoded by the Saccharomyces cerevisiae SUP35
gene.4–6) [PSIþ ] is not only an attractive research object
but also a convenient and useful model experimental
y

system of mammalianprions.
The monomeric form of the gene product of SUP35 is
eRF3, a termination factor.7) This protein consists of
three functional domains: the C domain (C-terminal 432
amino acids) has eRF3 functions, the N domain (Nterminal 123 amino acids) manifests conformational
alteration and polymerization, and the M domain
(middle 130 amino acids) appears to act as a linker of
the N and Cdomains.8–10) In the course of studying
[PSIþ ], we constructed a gene encoding a fusion protein,
GST-Sup35NM, in which Sup35NM was fused to the Cterminus of GST (glutathione S-transferase), and found
that Escherichia coli strain BL21 producing this fusion
protein grew normally to the stationary phase, but
decreased in colony-forming ability in the stationary
phase much faster than the control cellsdid.11) We
became aware that E. coli strain BL21 carried the ompT
mutation. We guessed that a deficiency of OmpT
protease in this strain might be responsible for the
observed rapid decrease in colony-forming ability. We
carried out experiments to test this speculation. Here we
present our results and also describe some additional
work which we have done to characterize proteins
produced by OmpTprotease digestion of GSTSup35NM.

Materials and Methods
Strains and culture conditions. The E. coli strains
used in this study are listed in Table 1. LB medium (1%
bacto-tryptone, 0.5% yeast extract, and 0.5% NaCl,
pH 7.2) was used to grow the E. coli strains.12) In case
of necessity, LB medium was added with 50 mg/ml

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