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Journal of Bacteriology, March 2000, p. 1731-1738, Vol. 182, No. 6
Infectious Disease Division, Massachusetts
General Hospital, Boston, Massachusetts 02114,1
and Department of Microbiology and Molecular Genetics, Harvard
Medical School, Boston, Massachusetts 021152
Received 22 September 1999/Accepted 21 December 1999
A 7.5-kbp fragment of chromosomal DNA downstream of the
Vibrio cholerae vibriobactin outer membrane receptor,
viuA, and the vibriobactin utilization gene,
viuB, was recovered from a Sau3A lambda library
of O395 chromosomal DNA. By analogy with the genetic organization of
the Escherichia coli enterobactin gene cluster, in which
the enterobactin biosynthetic and transport genes lie adjacent to the
enterobactin outer membrane receptor, fepA, and the
utilization gene, fes, the cloned DNA was examined for the ability to restore siderophore synthesis to E. coli ent
mutants. Cross-feeding studies demonstrated that an E. coli
entF mutant complemented with the cloned DNA regained the ability
to synthesize enterobactin and to grow in low-iron medium. Sequence
analysis of the cloned chromosomal DNA revealed an open reading frame
downstream of viuB which encoded a deduced protein of
greater than 2,158 amino acids, homologous to Yersinia sp.
HMWP2, Vibrio anguillarum AngR, and E. coli
EntF. A mutant with an in-frame deletion of this gene, named
vibF, was created with classical V. cholerae strain O395 by in vivo marker exchange. In cross-feeding studies, this
mutant was unable to synthesize ferric vibriobactin but was able to
utilize exogenous siderophore. Complementation of the mutant with a
cloned vibF fragment restored vibriobactin synthesis to
normal. The expression of the vibF promoter was found to be negatively regulated by iron at the transcriptional level, under the
control of the V. cholerae fur gene. Expression of
vibF was not autoregulatory and neither affected nor was
affected by the expression of irgA or viuA. The
promoter of vibF was located by primer extension and was
found to contain a dyad symmetric nucleotide sequence highly homologous
to the E. coli Fur binding consensus sequence. A footprint
of purified V. cholerae Fur on the vibF promoter, overlapping the Fur binding consensus sequence, was observed
using DNase I footprinting. The protein product of vibF is
homologous to the multifunctional nonribosomal protein synthetases and
is necessary for the biosynthesis of vibriobactin.
0021-9193/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
Vibrio cholerae VibF Is Required for Vibriobactin
Synthesis and Is a Member of the Family of Nonribosomal Peptide
Synthetases
and
*
Corresponding author. Mailing address: Infectious
Disease Division, Massachusetts General Hospital, 55 Fruit St., Boston, MA 02114. Phone: (617) 726-3818. Fax: (617) 726-7416. E-mail: jbutterton{at}partners.org.
Present address: Scriptgen Pharmaceuticals, Inc., Waltham, MA 02154.
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