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Journal of Bacteriology, July 1999, p. 3981-3993, Vol. 181, No. 13
0021-9193/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
Escherichia coli Mutants Lacking All
Possible Combinations of Eight Penicillin Binding Proteins: Viability,
Characteristics, and Implications for Peptidoglycan
Synthesis
Sylvia A.
Denome,
Pamela K.
Elf,
Thomas A.
Henderson,
David E.
Nelson, and
Kevin D.
Young*
Department of Microbiology and Immunology,
School of Medicine, University of North Dakota, Grand Forks, North
Dakota 58202-9037
Received 25 February 1999/Accepted 27 April 1999
The penicillin binding proteins (PBPs) synthesize and remodel
peptidoglycan, the structural component of the bacterial cell wall.
Much is known about the biochemistry of these proteins, but little is
known about their biological roles. To better understand the
contributions these proteins make to the physiology of
Escherichia coli, we constructed 192 mutants from which
eight PBP genes were deleted in every possible combination. The genes
encoding PBPs 1a, 1b, 4, 5, 6, and 7, AmpC, and AmpH were cloned, and
from each gene an internal coding sequence was removed and replaced
with a kanamycin resistance cassette flanked by two res
sites from plasmid RP4. Deletion of individual genes was accomplished
by transferring each interrupted gene onto the chromosome of E. coli via
phage transduction and selecting for
kanamycin-resistant recombinants. Afterwards, the kanamycin resistance
cassette was removed from each mutant strain by supplying ParA
resolvase in trans, yielding a strain in which a long
segment of the original PBP gene was deleted and replaced by an 8-bp
res site. These kanamycin-sensitive mutants were used as
recipients in further rounds of replacement mutagenesis, resulting in a
set of strains lacking from one to seven PBPs. In addition, the
dacD gene was deleted from two septuple mutants, creating
strains lacking eight genes. The only deletion combinations not
produced were those lacking both PBPs 1a and 1b because such a
combination is lethal. Surprisingly, all other deletion mutants were
viable even though, at the extreme, 8 of the 12 known PBPs had been
eliminated. Furthermore, when both PBPs 2 and 3 were inactivated by the
-lactams mecillinam and aztreonam, respectively, several mutants did
not lyse but continued to grow as enlarged spheres, so that one mutant
synthesized osmotically resistant peptidoglycan when only 2 of 12 PBPs
(PBPs 1b and 1c) remained active. These results have important
implications for current models of peptidoglycan biosynthesis, for
understanding the evolution of the bacterial sacculus, and for
interpreting results derived by mutating unknown open reading frames in
genome projects. In addition, members of the set of PBP mutants will provide excellent starting points for answering fundamental questions about other aspects of cell wall metabolism.
*
Corresponding author. Mailing address: Department of
Microbiology and Immunology, School of Medicine, University of North Dakota, Grand Forks, ND 58202-9037. Phone: (701) 777-2624. Fax: (701)
777-2054. E-mail: kyoung{at}medicine.nodak.edu.

Present address: Genome Therapeutics Corp., Waltham, MA
02453-8443.

Present address: Department of Biology, University of North
Dakota, Grand Forks, ND
58201.
Journal of Bacteriology, July 1999, p. 3981-3993, Vol. 181, No. 13
0021-9193/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
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