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Journal of Bacteriology, August 2004, p . 5547-5550, Vol . 186,
No . 16
Erwinia chrysanthemi O Antigen Is Required for Betaine Osmoprotection in
High-Salt Media
Thierry Touzé, Renan Goude, Sylvie Georgeault, Carlos Blanco, and
Sylvie Bonnassie*
Osmorégulation chez les bactéries, CNRS UMR 6026, Campus de Beaulieu,
Université de Rennes I, 35042 Rennes, France
Received 5 February 2004/ Accepted 17 May 2004
Cellular components necessary for osmoprotection are poorly known . In
this study we show that O antigen is specifically required for the
effectiveness of betaines as osmoprotectants for Erwinia
chrysanthemi in saline media . The phenotype is correlated with
the inability of rfb mutant strains to maintain a high
accumulation level of betaines in hypersaline media .
All microorganisms have to adapt to fluctuations in the osmolarity of
their environment . In response to elevated medium osmolarity,
bacteria first accumulate potassium and glutamate (6), which
are rapidly replaced by few organic solutes, called osmoprotectants
(4) . In the enterobacteria, this family of molecules
includes betaines like glycine betaine (GB) and imino acids (proline,
pipecolate, etc.) (4, 7,
13) . Enterobacteria like Escherichia coli
or Erwinia chrysanthemi adopt a similar response to hyperosmotic
stress regardless of the nature of the osmoprotectant available
in the medium, and these molecules are accumulated in the cell
proportionally to the osmotic stress applied via two systems, ProP
and ProU (2-4, 8,
28) .
All osmoprotectants are considered to be functionally equivalent .
Nevertheless, Gutierrez and Csonka (9) have shown that adk
mutations affect GB but not proline osmoprotection in Salmonella
enterica serovar Typhimurium, and a similar phenotype was also
found in a gltBD fnr mutant of E . coli (21) .
Moreover, an E . chrysanthemi bspA mutant grew poorly in the
presence of salt and betaines (but not proline) (24) .
These studies underline differences between the mechanisms of action
of GB and proline during ionic stress and show that osmoprotection is
a complex mechanism that could not be summarized by osmoprotectant
accumulation . To gain insight into these mechanisms, we selected
mutants whose growth was no longer restored by GB in high-salt
medium .
E . chrysanthemi A1828 (11) was subjected to
mutagenesis with transposon Tn5-B21 (23) .
Two mutants, named W91 and W96, formed colonies on M63 basal medium
supplemented or not with 0.5 M NaCl but did not grow on plates
containing 0.5 M NaCl and 1 mM GB .
The growth of A1828, W91, and W96 was analyzed in M63 medium
containing increasing NaCl concentrations in the presence or absence
of 1 mM GB . W91 and W96 do not exhibit increased osmosensitivity
(Fig . 1A to C) . The addition of GB improved the growth of all
strains on 0.3 M NaCl medium (Fig . 1A and B) . With more
stringent constraints (Fig . 1C), GB restored the
W91 and W96 growth rate in the first stages of exponential growth,
but a premature cessation of growth was observed that was more
drastic as the NaCl concentration increased .
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FIG . 1 . Effect of GB, as a function of medium osmolarity, on the growth
of A1828 and W91 cells cultivated in high-salt media . Wild-type strain
A1828 (squares) and mutant strain W91 (circles) were grown on M63 medium
(A) alone or containing 0.3 (B) or 0.5 (C, D) M NaCl in the absence
(open symbols) or in the presence (filled symbols) of 1 mM GB . The
influence of the carbon source concentration was analyzed (D) by growing
the cells on M63-0.5 M NaCl-1 mM GB medium containing 10 (small
symbols), 20 (medium symbols), or 30 (large symbols) mM glucose . Similar
results were obtained with strain W96 in place of strain W91 . OD,
optical density.
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Increasing the glucose concentration enhanced the growth yield of
strain A1828 but not that of strain W91 (Fig . 1D) . Therefore,
the phenotype did not result from carbon source depletion .
When the medium osmolarity was increased with nonionic osmotic
agents like 0.8 M sucrose (1,100 osmol/kg of H2O), the growth
of W91 and W96 was identical to that of parental strain A1828 .
Similar results were obtained when GB was added to the medium at 0.8
M, acting both as an osmotic agent (1,100 osmol/kg of H2O)
and as an osmoprotectant . Thus, GB is not toxic in media of high
osmolarity . In contrast, in the presence of 0.5 M KCl, K2SO4,
or sodium glutamate GB failed again to act as a potent
osmoprotectant . These results clearly show that the GB– phenotype is
associated with a concomitant effect of GB and high salt
concentrations .
Among the various osmoprotectants effective on E . chrysanthemi
under hyperosmotic conditions (7), pipecolate, ectoine,
and proline improved the growth of W91 and W96, whereas
dimethylsulfonioacetate and dimethylsulfoniopropionate, like GB, were
unable to alleviate the inhibitory effect of a high salt
concentration on their growth yields .
The levels of intracellular GB and pipecolate contents of strains
A1828 and W91 were analyzed (8) in media of increasing
osmolarity (from 0.3 to 0.5 M NaCl) containing 1 mM [14C]GB
or [14C]pipecolate . In the wild-type strain, the GB
content increased proportionally with the medium osmolarity, yielding
1,000 ± 96, 1,300 ± 127, and 1,400 ± 41 nmol mg of dry weight (DW)–1
at 0.3, 0.4, and 0.5 M NaCl, respectively . In contrast, the
level of GB accumulated by the W91 strain remained constant at 900 ±
24 nmol mg of DW–1 regardless of the medium osmolarity .
This accumulation defect was not observed when 1 mM [14C]pipecolate
was used as an osmoprotectant, since the levels reached at 0.5 M NaCl
were 1,800 ± 200 and 1,900 ± 290 nmol mg of DW–1 for the
wild type and the mutant, respectively . On the other hand, the [14C]GB
uptake of cells of the wild-type and mutant strains cultivated at 0.5
M NaCl did not exhibit any significant differences (25 ± 3 and
17 ± 4 nmol min–1 mg of DW–1, respectively) . In
conclusion, the mutants exhibit a defect in GB accumulation at high
salt concentrations that cannot be assigned to an alteration of the
uptake systems .
To identify the mutations, the genomic regions flanking the
inserted transposon in both mutants were cloned and sequenced (see
Fig . 2 for details) (accession number
AF503594) . These nucleotide sequences were aligned to the entire
nucleotide sequence of the E . chrysanthemi 3937 genome (https://asap.ahabs.wisc.edu/annotation) .
This analysis revealed that the transposon in strains W91 and
W96 is inserted into two different and contiguous genes (wzt
and wzm, respectively) belonging to a locus of eight open reading
frames with the same transcriptional orientation (Fig . 2A) .
Searches of the databases have shown considerable homologies
with the O-antigen export and biosynthesis pathway genes (Fig .
2B) . The gene nomenclature was chosen on the basis of those
amino acid sequence homologies and in accordance with the nomenclature
proposed by Reeves et al . (19) relative to bacterial
polysaccharide synthesis .
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FIG . 2 . Genetic organization of the E . chrysanthemi rfb locus .
(A) Genetic organization of the E . chrysanthemi rfb locus,
restriction map, and subcloning of the mutated region . The Tn5-B21
transposon is represented at the top, and the line below shows the
mutated chromosomal region; the insertion points of the transposon in
strains W91 and W96 are indicated by vertical arrows . The genes are
represented by horizontal arrows, the direction of which indicates the
transcriptional direction . Putative transcriptional terminators are
indicated by
 .
Two additional mutations created by cassette insertion into the wbeA
and gmd sequences, resulting in strains W54 and W141,
respectively, are indicated by vertical arrows . The last three lines
show the inserts of the plasmids carrying the cloned chromosomal
junctions from mutants W91 and W96 . (B) Putative pathway for the
biosynthesis and assembly of E . chrysanthemi O antigen based on
sequence homologies.
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The lipopolysaccharides (LPSs) of both the wild-type and mutant
strains were extracted and separated by sodium dodecyl sulfate-13.5%
polyacrylamide gel electrophoresis and visualized by silver staining
(10, 27) . wzm and wzt
mutants lacked the O-antigenic structure but were not altered in the
electrophoretic mobility of the core component (data not shown) .
Moreover, these mutants are resistant to bacteriophage
 EC2
(EC2r),
which is known to adsorb to the O antigen of E . chrysanthemi (22) .
Strains W54 (wbeA::-Ampr)
and W141 (gmd::'uidA-Kanr) were constructed,
and they showed the same phenotype as W91 or W96 regarding the use of
GB as an osmoprotectant and O-antigen production . These results
suggest that the entire rfb locus is implicated in the GB–
phenotype .
EDTA is known to provoke a release of LPS by chelating divalent
cations that are necessary to stabilize the LPS structure (18) .
The wild-type strain was grown in M63-0.5 M NaCl-1 mM GB medium
supplemented or not with EDTA at a concentration of 50 or 100 µM, and
proline in the place of GB served as a control . The results presented
in Fig . 3 show that the addition of 50 µM EDTA had
no effect on A1828 growth in hyperosmotic medium plus GB or proline .
In contrast, GB, but not proline, osmoprotection was impaired in the
presence of 100 µM EDTA, as observed for the rfb mutant
strains grown in the same medium without EDTA . Thus, O antigen is
directly implicated in the GB– phenotype .
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FIG . 3 . Effect of EDTA on the osmoprotective efficiency of GB and
proline for E . chrysanthemi wild-type strain A1828 in high-salt
medium . Strain A1828 was grown on M63-0.5 M NaCl medium containing 1 mM
GB (open symbols) or 1 mM proline (filled symbols) in the absence
(circles) or in the presence (triangles) of EDTA at 50 (A) or 100 (B)
µM . OD, optical density.
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Several studies have revealed a significant role for the O antigen in
environmental stress adaptation (1, 16,
17) . LPS plays an essential role in outer membrane
integrity, and its implication in the secretion, assembly, or folding
of surface proteins was clearly demonstrated in vivo and in vitro (5,
20, 26) . The E . chrysanthemi
rfb mutants have a failure in the control of the internal GB pool
under salt constraint, most likely because of a defect in the efflux
systems, as the measured influx activities were correct . Osmotic
adaptation involves numerous membrane proteins, whose function has
not yet been elucidated (14, 15);
therefore it is possible that, as for IcsA of Shigella flexneri
(25) and Tcp of Vibrio cholerae O1 (12),
O antigen influences the activity of proteins involved in the control
of the internal GB pool .
This work was supported by grants from the Centre National de la
Recherche Scientifique and the Ministčre de l'Education Nationale .
We are grateful to C . Monnier, M . C . Savary, and M . Uguet for
excellent technical assistance .
* Corresponding author . Mailing address: Osmorégulation chez
les bactéries, CNRS UMR 6026, Université de Rennes I, Campus de Beaulieu, Av . du
Général Leclerc, 35042 Rennes, France . Phone: 33-223236141 . Fax: 33-223236775 .
E-mail:
sylvie.bonnassie@univ-rennes1.fr .
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