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Previous Article | Next Article 
J Bacteriol, June 1998, p. 3003-3006, Vol. 180, No. 11
0021-9193/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
Characterization of Chemotactic Responses and
Flagella of Hyphomicrobium Strain W1-1B
Laura
Tuhela,1,
Jayne B.
Robinson,2,* and
Olli H.
Tuovinen1
Department of Microbiology, The Ohio State
University, Columbus, Ohio 43210-1292,1 and
Department of Biology, University of Dayton, Dayton, Ohio
45469-23202
Received 11 August 1997/Accepted 23 March 1998
 |
ABSTRACT |
Motile swarmer cells of Hyphomicrobium strain W1-1B
displayed positive chemotactic responses toward methylamine,
dimethylamine, and trimethylamine but did not display significant
chemotactic responses towards methanol and arginine. Electron
micrographs of negatively stained intact flagellar filaments indicated
a novel striated surface pattern. The flagella were composed of two
proteins of 39 and 41 kDa. Neither protein was a glycoprotein as
determined by Schiff's staining and by enzyme immunoassay. Protein
fingerprints visualized from silver-stained polyacrylamide gels and
Western blots of protease-digested samples indicated that the two
proteins were similar but not identical. Monoclonal antibodies prepared to the complex flagella of Rhizobium meliloti cross-reacted
with the striated flagella of Hyphomicrobium strain W1-1B;
however, these antibodies did not cross-react with smooth-surface
flagella. These results suggest that complex and striated flagella
possess homologous epitope regions.
| |
TEXT |
Hyphomicrobia are appendaged
organisms with differential life cycles. During their life
cycles, nascent swarmer cells are motile by means of a single subpolar
flagellum, and as the cells mature, they shed the flagella and form a
stalk. Virtually nothing is known about the regulation of cellular
differentiation, motility, and chemotaxis in hyphomicrobia. The purpose
of this work was to characterize the flagella and chemotactic responses
of a Hyphomicrobium strain. Chemotactic sensing of
substrate-rich zones enhances swarmer cell movement toward boundary
layers, and flagella may facilitate the subsequent attachment to
surfaces.
Bacteria and growth conditions.
Hyphomicrobium
strain W1-1B used in this study was originally isolated from
a water well sample and was identified as a
Hyphomicrobium sp. on the basis of phenotypic and
phylogenetic characterizations (11). Swarmer cells used for
chemotaxis experiments were grown in medium 337 (4),
which contains 0.5% (wt/vol) methylamine, dimethylamine, or
trimethylamine as the sole C, N, and energy source. Static cultures
were incubated at 22 ± 2°C for 72 h. The cells were
harvested by centrifugation and washed twice with 337 buffer at pH 7.0 (consisting of medium 337 minus any carbon source). Washed cells were
resuspended in 337 buffer.
Chemotaxis to methylated amines.
Chemotaxis was measured by
capillary assays in chemotaxis chambers as described by Palleroni
(7). The attractants tested were methylamine, dimethylamine,
trimethylamine, methanol, and arginine. Each assay was replicated three
to four times. All chemotaxis assays were conducted at 22 ± 2°C
for 60 min. After incubation, samples were plated onto agar-solidified
medium 337 supplemented with 0.5% methylamine by using a model DU
spiral plater (Spiral Systems, Inc., Cincinnati, Ohio). The plates were
incubated at 22 ± 2°C for 7 days before colony
counting.
When Hyphomicrobium strain W1-1B was grown on
methylamine, dimethylamine, or trimethylamine, swarmer cells were
motile and exhibited a positive chemotactic response toward the
respective substrates. Concentration-response curves which illustrate
the degrees of responses to various concentrations of the
chemoattractants are shown in Fig. 1.
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FIG. 1.
Concentration-response curves of
Hyphomicrobium strain W1-1B. The cells were grown with
methylamine (A), dimethylamine (B), or trimethylamine (C). The
responses were determined with methylamine ( ), dimethylamine ( ),
or trimethylamine ( ) as an attractant. The values (with I bars
indicating standard deviations) represent accumulations of cells in
response to the chemoattractant. Background cell accumulations (in the
absence of attractant) varied from 5.5 × 103 ± 2.5 × 103 to 1.2 × 104 ± 3.1 × 103 cells per capillary.
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Table 1 summarizes the peak response
ratios and threshold values for all experimental conditions tested.
Methylamine-grown cells proved to be the most chemotactically
responsive. The relative chemotactic responses of methylamine-grown
cells to methylamine and dimethylamine were 43- and 39-fold over
background, indicating that these compounds are strong attractants
(Fig. 1; Table 1). Dimethylamine- and trimethylamine-grown cells
exhibited moderate chemotaxis to all three methylated amines.
Arginine and methanol failed to elicit a statistically significant
response from cells of Hyphomicrobium strain W1-1B grown on
any of the methylated amines (Table 1). This result was unexpected because Hyphomicrobium strain W1-1B is able to use arginine
as the sole source of N, and amino acids have been shown to be among the strongest chemoattractants for a variety of chemotactic bacteria (1). Methanol is an excellent source of C for hyphomicrobia, and growth on methanol results in high yields of growth. However, there
is no known methanol-specific receptor or uptake system in
microorganisms, perhaps because methanol is rapidly diffused into
cells. Hyphomicrobium strain W1-1B, like many other
chemotactic bacteria, may have evolved chemotactic responses to
compounds that can serve as both C and N sources, such as the
methylated amines.
Hyphomicrobium strain W1-1B was grown aerobically with
methanol for chemotaxis experiments, but the methanol-grown swarmer cells were not motile during any phase of growth. However, flagella could be readily isolated from these cells. The addition of various concentrations of methanol to motile, methylamine-grown cells did not
inhibit motility, indicating that methanol does not directly inhibit
motility as it does for Escherichia coli (5).
Methanol-grown swarmer cells were unaffected by the addition of culture
supernatant from motile, methylamine-grown cells, indicating that there
is no extracellular, soluble factor responsible for inducing motility in this organism.
Flagellar structure.
To obtain flagellum preparations,
Hyphomicrobium strain W1-1B was grown in 10-liter fermentor
batches (1% inoculum) containing medium 337 with 0.5% (wt/vol)
methylamine or 0.5% (vol/vol) filter-sterilized methanol as the sole
source of C and energy. The fermentors were incubated at 22 ± 2°C with aeration but no stirring for 72 h. The cells were
harvested by tangential filtration (Millipore Pellicon filter),
pelleted, and resuspended in 337 buffer. Flagella were removed by
shearing cells in a blender at the maximum speed for 15 s. Cells
were observed microscopically to ascertain the loss of motility and
then separated from the free flagella by centrifugation at 10,000 × g and 4°C for 12 min. Cell debris was removed from the
flagella by centrifugation of the supernatant at 15,000 × g and 4°C for 12 min. Flagella were pelleted by
ultracentrifugation and then resuspended in HEC buffer (containing 10 mM HEPES at pH 7.0, 10 µM EDTA, and 0.2 mM CaCl2) and
stored at 4°C. The protein contents of flagellum preparations were
determined with a Micro BCA Protein Assay Reagent kit (Pierce Chemical
Co., Inc., Rockford, Ill.), with bovine serum albumin as a standard in
the HEC buffer.
Flagellum preparations were purified by sucrose density gradient
centrifugation with a 30 to 40 to 50% step gradient and separated by
ultracentrifugation at 100,000 × g at 4°C for
18 h. The sucrose density gradients were fractionated by passing
them through a UV detector set at 280 nm. The refractive indexes of the
fractions were measured with a refractometer (Bausch & Lomb, Inc.,
Rochester, N.Y.).
To observe the ultrastructures of flagella, a diluted suspension of
flagella was placed onto a Formvar, carbon-coated grid and negatively
stained with uranyl acetate. Samples were observed with a Zeiss 10 transmission electron microscope.
Electron micrographs of Hyphomicrobium strain W1-1B
flagellum preparations are shown in Fig.
2. The flagellar filaments did not appear
to have the cross-hatched surface pattern typical of complex flagella,
such as those of Rhizobium meliloti, Rhizobium lupini, and Bradyrhizobium japonicum (10).
However, the surfaces of the filaments appeared to be of a striated
type rather than of the smooth-surface type characteristic of the
surfaces of flagella of E. coli and Salmonella
spp. (2).
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FIG. 2.
Transmission electron micrograph of flagella of
Hyphomicrobium strain W1-1B following purification by
density gradient centrifugation. (A) Purified flagella; (B) flagellar
hook (arrow). Bars, 100 nm.
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To further analyze the structures of the flagella, flagellin proteins
were separated by sodium dodecyl sulfate-polyacrylamide gel
electrophoresis (SDS-PAGE). Analysis by SDS-PAGE demonstrated the
presence of two flagellin monomers, of 39 and 41 kDa, in the purified
Hyphomicrobium flagellum preparations (Fig.
3A, lane 1). At least two flagellins have
also been reported for Bdellovibrio bacteriovorus,
Halobacterium halobium, Campylobacter coli,
Caulobacter crescentus, and R. meliloti
(8). In contrast, the filaments of E. coli and
Salmonella typhimurium are made up of a single species of
flagellin.
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FIG. 3.
SDS-polyacrylamide gel (A) and Western blot (B) of
purified flagella of Hyphomicrobium strain W1-1B previously
grown with either methylamine (lane 1) or methanol (lane 2).
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The two proteins associated with the flagella of
Hyphomicrobium strain W1-1B were tested for glycosylation
with a First Choice Glycan Detection kit (Boehringer Mannheim Corp.,
Indianapolis, Ind.) to visualize antibody-glycoprotein complexes and by
Schiff's staining procedure (3). In both cases, transferrin
was used as a positive control and creatinase was used as a negative
control. Results from both the Schiff's staining and enzyme
immunoassay indicated that neither the 39- nor the 41-kDa flagellin
monomer was glycosylated (data not shown).
Western blotting was performed to examine the antigenic surface
structures of Hyphomicrobium. Proteins blotted onto
nitrocellulose membranes were exposed to monoclonal antibodies
specific to R. meliloti 7201 flagella and then visualized by
the ProtoBlot II AP System with Stabilized Substrate, Mouse (Promega
Corp., Madison, Wis.). The monoclonal antibodies were prepared at
the Monoclonal Antibody Facility at The Ohio State University
(Columbus) by using sucrose-gradient-purified flagellum preparations
from R. meliloti RMB7201 as the antigen.
Western hybridization of the two Hyphomicrobium flagellar
proteins showed that both the 39- and 41-kDa proteins were
cross-reactive with the monoclonal antibody against R. meliloti flagella (Fig. 3B). This monoclonal antibody also
cross-reacted with the flagella of Bradyrhizobium japonicum
but not with those of Agrobacterium tumefaciens,
Pseudomonas aeruginosa PAO1, or the atrazine-degrading soil
isolate M91-3 (9) (Fig. 4).
These results indicate that homology exists between the complex
flagella of R. meliloti RMB7201 and B. japonicum
and the flagella of Hyphomicrobium strain W1-1B. Because the
monoclonal antibodies were prepared against whole, intact flagella of
R. meliloti, the common antigen may be related to surface
structure.
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FIG. 4.
Western blot of flagellum preparations from several
bacteria with monoclonal antibody prepared against whole, intact
flagella from R. meliloti RMB7210. Lane 1, R. meliloti; lane 2, Hyphomicrobium strain W1-1B; lane 3, P. aeruginosa; lane 4, A. tumefaciens; lane
5, B. japonicum; lane 6, M91-3.
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Flagella prepared from cells grown with methanol and with methylamine
were analyzed by SDS-PAGE and with Western blots that were prepared
with the R. meliloti RMB7201 antiflagellar monoclonal antibody (Fig. 3). Comparable proteins were detected in both flagellum preparations, although the methanol-grown cells lacked motility. Interestingly, the 39-kDa protein was more abundant in the flagella of
methanol-grown cells while the 41-kDa protein was more abundant in
the flagella of methylamine-grown cells. Flagellar preparations from methanol-grown cells cross-reacted with the monoclonal antibody, indicating that the proteins were comparable to those from the flagella
of cells grown in methylamine.
The two proteins associated with Hyphomicrobium strain W1-1B
flagella were compared to each other and to flagellin proteins of
R. meliloti for degrees of similarity with a Protein
Fingerprinting System kit (Promega Corp.). Proteins were separated by
electrophoresis, and gels were processed according to the directions of
the manufacturer (Promega Corp.). Gels were stained by a modified
silver stain method (1a) or transferred to a nitrocellulose
membrane by Western blotting with a Bio-Rad transblot apparatus. The
blotted proteins were probed with the monoclonal antibody specific to
R. meliloti RMB7201 flagella.
Fingerprints of the 39- and 41-kDa flagellin proteins of
methylamine-grown Hyphomicrobium strain W1-1B and of the
flagellin protein from R. meliloti RMB7201 were prepared by
digestion with alkaline protease, endoproteinase Lys-C, and
endoproteinase Glu-C. Only the digests with Glu-C resulted in the
complete digestion of the proteins to peptide fragments, yielding
reproducible fingerprints (Fig. 5). The
fingerprints of the Hyphomicrobium flagellins as visualized
on the Western blot were more similar than were the fingerprints from
SDS-PAGE, suggesting homology between the epitope regions. However, the
fingerprints of the two Hyphomicrobium proteins were
different enough to indicate that the proteins were not identical. The
two Hyphomicrobium flagellins showed more similarity to each other than either did to R. meliloti flagellin, in spite of
the monoclonal antibody cross-reactivity.
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FIG. 5.
Endoproteinase Glu-C digestion of the two
flagellum-associated proteins of Hyphomicrobium strain W1-1B
and a flagellum preparation of R. meliloti. (A)
SDS-polyacrylamide gel; (B) Western blot. Lane 1, 41-kDa flagellin of
Hyphomicrobium; lane 2, 39-kDa flagellin of
Hyphomicrobium; lane 3, flagellum preparation of R. meliloti.
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Conclusions.
This work shows that Hyphomicrobium
strain W1-1B is chemotactic to methylated amines that can serve as both
C and N sources for this methylotrophic bacterium. Chemotactic
responses, which have not been previously described for any
Hyphomicrobium spp., were exhibited during the motile phase
of the organism's differential life cycle, Hyphomicrobium
strain W1-1B has complex, striated flagella which posses epitope
regions homologous to those of the flagella of R. meliloti.
The production of nonfunctional flagella by methanol-grown cells
suggests an unusual structure-function relationship that warrants
further studies of the regulatory mechanisms of the motility and
changes with life cycle in this methylotrophic organism.
| |
ACKNOWLEDGMENTS |
We thank W. D. Bauer, Department of Horticulture and Crop
Science, The Ohio State University, for valuable discussions and placing laboratory facilities at our disposal.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Department of
Biology, University of Dayton, 300 College Park, Dayton, OH 45469. Phone: (937) 229-2580. Fax: (937) 229-2021. E-mail:
robinson{at}neelix.udayton.edu.
Present address: Ohio Wesleyan University, Delaware, OH 43015.
| |
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J Bacteriol, June 1998, p. 3003-3006, Vol. 180, No. 11
0021-9193/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
This article has been cited by other articles:
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