J Bacteriol, February 1998, p. 634-641, Vol. 180, No. 3
0021-9193/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
Department of Microbiology and Immunology, University of Tennessee and Veterans Affairs Medical Center, Memphis, Tennessee 38163
Received 17 July 1997/Accepted 19 November 1997
Chronic pulmonary infection with Pseudomonas aeruginosa
is a common and serious problem in patients with cystic fibrosis (CF). The P. aeruginosa isolates from these patients typically
have a mucoid colony morphology due to overproduction of the
exopolysaccharide alginate, which contributes to the persistence of the
organisms in the CF lung. Most of the alginate biosynthetic genes are
clustered in the algD operon, located at 34 min on the
chromosome. Alginate biosynthesis begins with the formation of an
activated monomer, GDP-mannuronate, which is known to occur via the
products of the algA, algC, and
algD genes. Polymannuronate forms in the periplasm, but the
gene products involved in mannuronate translocation across the inner
membrane and its polymerization are not known. One locus of the operon
which remained uncharacterized was a new gene called algK
between alg44 and algE. We sequenced
algK from the mucoid CF isolate FRD1 and expressed it in
Escherichia coli, which revealed a polypeptide of the
predicted size (52 kDa). The sequence of AlgK showed an apparent signal
peptide characteristic of a lipoprotein. AlgK-PhoA fusion proteins were
constructed and shown to be active, indicating that AlgK has a
periplasmic subcellular localization. To test the phenotype of an
AlgK
mutant, the algK coding sequence was
replaced with a nonpolar gentamicin resistance cassette to avoid polar
effects on genes downstream of algK that are essential for
polymer formation. The algK
mutant was nonmucoid,
demonstrating that AlgK was required for alginate production. Also,
AlgK
mutants demonstrated a small-colony phenotype on L
agar, suggesting that the loss of AlgK also caused a growth defect. The
mutant phenotypes were complemented by a plasmid expressing
algK in trans. When the algK
mutation was placed in an algJ::Tn501
background, where algA was not expressed due to polar
transposon effects, the growth defect was not observed.
AlgK
mutants appeared to accumulate a toxic extracellular
product, and we hypothesized that this could be an unpolymerized
alginate precursor. High levels of low-molecular-weight uronic acid
were produced by the AlgK
mutant. When AlgK
culture supernatants were subjected to dialysis, high levels of uronic
acids diffused out of the dialysis sac, and no uronic acids were
detectable after extensive dialysis. In contrast, the mucoid wild-type
strain produced only polymerized uronic acids (i.e., alginate), whereas
the algK
algJ::Tn501 mutant
produced no uronic acids. Thus, the alginate pathway in an
AlgK
mutant was blocked after transport but at a step
before polymerization, suggesting that AlgK plays an important role in
the polymerization of mannuronate to alginate.
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