Affiliation:
1. Department of Microbiology, University of Georgia, Athens, Georgia, USA
2. Department of Microbiology and Plant Biology, University of Oklahoma, Norman, Oklahoma, USA
Abstract
ABSTRACT
Libraries of defined mutants are valuable research tools but necessarily lack gene knockouts that are lethal under the conditions used in library construction. In this study, we augmented a
Vibrio fischeri
mutant library generated on a rich medium (LBS, which contains [per liter] 10 g of tryptone, 5 g of yeast extract, 20 g of NaCl, and 50 mM Tris [pH 7.5]) by selecting transposon insertion mutants on supplemented LBS and screening for those unable to grow on LBS. We isolated strains with insertions in
alr
,
glr
(
murI
),
glmS
, several heme biosynthesis genes, and
ftsA
, as well as a mutant disrupted 14 bp upstream of
ftsQ
. Mutants with insertions in
ftsA
or upstream of
ftsQ
were recovered by addition of Mg
2+
to LBS, but their cell morphology and motility were affected. The
ftsA
mutant was more strongly affected and formed cells or chains of cells that appeared to wind back on themselves helically. Growth of mutants with insertions in
glmS
,
alr
, or
glr
was recovered with
N
-acetylglucosamine (NAG),
d
-alanine, or
d
-glutamate, respectively. We hypothesized that NAG,
d
-alanine, or
d
-glutamate might be available to
V. fischeri
in the
Euprymna scolopes
light organ; however, none of these mutants colonized the host effectively. In contrast,
hemA
and
hemL
mutants, which are auxotrophic for δ-aminolevulinate (ALA), colonized at wild-type levels, although mutants later in the heme biosynthetic pathway were severely impaired or unable to colonize. Our findings parallel observations that legume hosts provide
Bradyrhizobium
symbionts with ALA, but they contrast with virulence phenotypes of
hemA
mutants in some pathogens. The results further inform our understanding of the symbiotic light organ environment.
IMPORTANCE
By supplementing a rich yeast-based medium, we were able to recover
V. fischeri
mutants with insertions in conditionally essential genes, and further characterization of these mutants provided new insights into this bacterium's symbiotic environment. Most notably, we show evidence that the squid host can provide
V. fischeri
with enough ALA to support its growth in the light organ, paralleling the finding that legumes provide
Bradyrhizobium
ALA in symbiotic nodules. Taken together, our results show how a simple method of augmenting already rich media can expand the reach and utility of defined mutant libraries.
Funder
National Science Foundation
DOD | USAF | AFMC | Air Force Office of Scientific Research
Publisher
American Society for Microbiology
Subject
Ecology,Applied Microbiology and Biotechnology,Food Science,Biotechnology
Cited by
8 articles.
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