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Applied and Environmental Microbiology, November 1998, p. 4328-4332, Vol. 64, No. 11
Institute of Applied Biochemistry, University
of Tsukuba, Tsukuba, Ibaraki 305-8572, Japan
Received 2 June 1998/Accepted 24 August 1998
Growth of Thermus thermophilus HB27 was inhibited by a
proline analog, 3,4-dehydroproline (DHP). This result suggested that the
0099-2240/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
Construction of a Proline-Producing Mutant of the
Extremely Thermophilic Eubacterium Thermus
thermophilus HB27
-glutamyl kinase (the product of the proB gene) was
inhibited by feedback inhibition in T. thermophilus.
DHP-resistant mutants were reported previously for Escherichia
coli (A. M. Dandekar and S. L. Uratsu, J. Bacteriol.
170:5943-5945, 1988) and Serratia marcescens (K. Omori, S. Suzuki, Y. Imai, and S. Komatsubara, J. Gen. Microbiol.
138:693-699, 1992), and their mutated sites in the proB
gene were identified. Comparison of the amino acid sequence of T. thermophilus -glutamyl kinase with those of E. coli and S. marcescens mutants revealed that the DHP
resistance mutations occurred in the amino acids conserved among the
three organisms. For eliminating the feedback inhibition, we first
constructed a DHP-resistant mutant, TH401, by site-directed mutagenesis
at the proB gene as reported for the proline-producing
mutant of S. marcescens. The mutant, TH401, excreted about
1 mg of L-proline per liter at 70°C after 12 h of
incubation. It was also suggested that T. thermophilus had
a proline degradation and transport pathway since it was able to grow
in minimal medium containing L-proline as sole nitrogen
source. In order to disrupt the proline degradation or transport genes,
TH401 was mutated by UV irradiation. Seven mutants unable to utilize
L-proline for their growth were isolated. One of the
mutants, TH4017, excreted about 2 mg of L-proline per liter
in minimal medium at 70°C after 12 h of incubation.
*
Corresponding author. Mailing address: Institute of
Applied Biochemistry, University of Tsukuba, Ten-nodai, Tsukuba,
Ibaraki 305-8572, Japan. Phone: 81-298-53-6782. Fax: 81-298-53-6782. E-mail: takachan{at}sakura.cc.tsukuba.ac.jp.
Applied and Environmental Microbiology, November 1998, p. 4328-4332, Vol. 64, No. 11
0099-2240/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
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