43
FEMS MicrobiologyLetters 8 (1980) 43-46
© Copyright Federation of European MicrobiologicalSocieties
Published by Elsevier/North-Holland Biomedical Press
CHEMOTAXIS OF A BIRDSFOOT TREFOIL STRAIN OF RHIZOBIUM
TO SIMPLE SUGARS *
WILLIAM W. CURRIER
Department o f Microbiology and Biochemistry, College of Agriculture, Agricultural Experiment Station, University o f Vermont,
Burlington, VT 05405, U.S.A.
Received 7 March 1980
Accepted 18 March 1980
1. Introduction
Bacteria move with intent, accumulating in areas
of favorable temperature, light level and chemicals.
Movement toward (positive chemotaxis) or away
from (negative chemotaxis) chemicals has been studied
in both Gram-negative [ 1 - 4 ] and Gram-positive
organisms [5,6]. Bacteria can sense both spatial and
temporal gradients of attractant or repellant [1,7-9].
This behavior requires that bacteria have both a simple
sensory system to monitor concentration of attractants or repellants in the environment and a simple
memory to store this information. The chemosensory
mechanism must contain a receptor which receives
the chemicals and some sort of signal device which
relays information from the chemosensors to the
flagella [ 1,10]. This information ultimately controls
the direction of rotation of the flagella.
Rhizobium spp. are also chemotactic [11-13].
Chemotaxis may play a role in the accumulation of
bacteria near plant roots [14,15]. It is known that
the numbers of an infective strain of Rhizobiurn are
selectively increased in the rhizosphere of a legume
[16,17]. Before nodulation, the surface of the root
is covered with a matrix of bacteria [ 18]. When alfalfa
grown in liquid culture is inoculated with homologous
rhizobia, many more bacteria accumulate on the
surface of the root than can be accounted for by
multiplication of the bacteria [ 19]. Since other
motile soil organisms are known to travel through the
soil solution [20,21 ] and rhizobia are known to be
* University of Vermont Agricultural Experiment Station
Journal article No. 435.
motile, this suggested that chemotaxis might play a
role in the accumulation of rhizobia in the rhizosphere.
Our earlier work showed that six strains of Rhizobium show differential chemotaxis to root exudates
[11 ]. Chemotaxis is not directly correlated with
nodulation since the bacteria are attracted to many
plants which they do not nodulate, i.e. non-legumes.
However, rhizobia are generally attracted to plants
they do nodulate [11].
A glycoprotein which attracts several species of
Rhizobiurn has been isolated from birdsfoot trefoil
root exudates [12,13]. This is the only case of a
macromolecule attracting bacteria. Different strains
of Rhizobiurn respond differently to simple sugars
and amino acids. Some are unaffected by sugars and
amino acids [11,13], while others are attracted by
both sugars and amino acids.
Gitte et al. have shown that rhizobia can be
attracted by neutral root exudate fractions, presumably containing sugars [22]. Mannose is a major
monosaccharide in birdsfoot trefoil root exudates
(unpublished results).
Chemotaxis is unusual in rhizobia in that they are
attracted by a macromolecule. Are they also unusual
in their response to sugars?
2. Materials and Methods
2. l. Bacteria
Birdsfoot trefoil Rhizobium strain 95C15 was
supplied by Dr. Joe Burton, Nitragen Sales Company,
44
3101 W. Custer Ave., Milwaukee, Wl. This strain
nodulates birdsfoot trefoil efficiently and fixes
nitrogen on this host (unpublished data). This strain
is attracted b y mM concentrations of maltose, mannose, ribose, and aspartate. Cultures were maintained
on yeast extract-mannitol agar as previously described
[11]. Liquid growth and labeling cultures were as
described [11], except that phosphate buffer (0.5 M
separately sterilized pH 7.0) was added to give a
phosphate concentration of 9.8 • 10 -3 M. Bacterial
concentration was measured as previously described.
looo
NOSE
dpm
5oo
s
2.2. Chemotaxis assay
f
Chemotaxis was measured b y counting the radioactivity in bacteria, labeled by 24-h growth on
[u-a4c]glucose, that swam into a 5/al micro-pipette
dipped into a suspension of labeled bacteria. This
labeling gave about 1% incorporation of a4C. Phosphate buffer (10 mM pH 7.0) containing 10 -4 M
EDTA was used as control. Attractant solutions were
also made up in this buffer. Five replicates were used
for each sample solution. Sugar solutions were made
at 10 -1 M and 10-fold dilutions prepared from 10 -2
to 10 -1 s M. Average disintegrations per minute (dpm)
for the five samples was compared with controls by
Student's "t" test. The ratio of the average dpm in
the sample over those in the control was reported
when the average dpm in the sample was significantly
different from that in the control at the 95% confidence level [11 ].
1 have found it important to clean the test tubes
used for the chemotaxis assay with concentrated
H2SO4 or 1 N HCI before use followed by 5 ×
water and 5 × phosphate buffer washes. Without
this treatment some batches of tubes gave high ( 3 4×) control values. Perhaps this indicates a chemorepellent is present in the tubes.
3. R e s u l t s and D i s c u s s i o n
The counts accumulating in a capillary filled with
buffer or 10 -3 M mannose increased in proportion to
the bacterial concentration over a range of 1.1 • 10 v
to 3.4 - 108 cells/ml (Fig. 1). Escherichia coli and a
motile Streptococcus gave a maximum accumulation
plateau at densities of 1 - 4 - 107 cells/ml or above
/ ~ s ~
BUFFER
1
5
2
108 Bacteria/ml
Fig. 1. Effect of density of bacterial suspension on motility
and chemotaxis toward 10 -3 M mannose. Samples incubated
for 1 h at 27°C. Bacteria were suspended in 10 -2 M phosphate, 10 -4 M EDTA (pH 7.0).
[6,23]. Bacillus subtilis showed a similar plateau at
densities of 5 • l 0 s cells/ml or above [5].
Accumulation o f bacteria in the capillary was
directly proportional to time o f incubation with both
10 -3 M mannose and buffer control for 90 rain. Accumulation to mannose peaked after 3 - 4 h, then fell
over the next 5 h from one-half to one-third o f the
peak value. This might be the result of metabolism of
mannose b y the bacteria and hence destruction o f the
gradient of attractant. Inclusion of 10 -2 M glucose in
both the bacterial suspension and the capillary had no
significant effect on chemotaxis for the first 3 h. However, the drop from 3 to 8 h incubation was not seen.
Hence, rhizobia, like E. coli, do not require an energy
source for chemotaxis [23]. Addition of high levels
of glucose prevents metabolism of the attractant
(mannose). Glucose is not a chemo-attractant for any
Rhizobiurn strain that has been tested [12,13]
(Table 1).
The accumulation of bacteria in the capillary was
highest when bacteria from early log phase were used.
Effects other than chemotaxis are not probable
because (a) an attractant must be present in the
capillary to get increased accumulation ; (b) some
45
TABLE 1
,500!
Sugar taxis of birdsfoot trefoil Rhizobium
Mannose
Glucose
Fructose
Rhamnose
Galactose
Ribose
Arabinose
Xylose
Deoxyribose
Maltose
Sucrose
Peak concentration (M)
Threshold (M)
10 .3
N.R.
10 -1
N.R.
10 -1
10 -3
N.R.
N.R.
N.R.
10 -1
10 -1
10-12
10-1 b
10-1
10-6
40C
3OO
dpm
2OO
lOb
T
Ok-//
o
10-4
10-1
a No chemo-attraction was seen with any concentration of
the sugar tested.
b Bacteria responded only to 10 -1 M attractant, hence
10 -1 M was taken as the threshold concentration.
compounds have no attractive effect; and (c) inclusion of mannose in both the capillary and the bacterial
suspension causes accumulation approximately equal
to that when buffer is used in both capillary and
bacterial suspension.
Motility, as measured by the accumulation of bacteria in a capillary containing only buffer, was greatest
at pHs of 7.0 or 7.5 in 10 mM phosphate buffer (Fig.
2). At a pH above 8.0 in 10 mM Tris-HC1 or 10 mM
20C
dpm~oc_ ~ B u f f e r
61o 615 ~o 715
81o
pH
Fig. 2. Effect of pH of 10 mM phosphate buffer on motility
and chemotaxis toward 10 .3 M mannose. Disintegrations per
minute normalized to equal dpm in the bacterial suspensions
at different pHs. Incubation was for 1 h at 27°C.
I
i
lo -8
I
i
lo -6
i
1
lo -4
I
i
L
lo -z
Monnose Concentrotion (M)
Fig. 3. Concentration response curve for mannose. Assay conditions as described in Fig. 1.
borate buffers, motility was greatly reduced and
significant chemotaxis was not observed.
Chemotaxis was seen at temperatures between
17°C and 35°C. No significant chemotaxis was seen
at 4°C, although the bacteria were still motile.
Trefoil rhizobia were attracted by mannose at concentrations of 10 -1 to 10 -9 M (Fig. 3). The chemoattraction seems to be bimodal with a sharp peak at
10 -3 M and a broader peak from 10 -s to 10 -8 M. This
biphasic response was seen in three repetitions on
separate days. This may reflect two different chemosensors. On different days the threshold varied by an
order of magnitude. From such concentration response
curves, the peak response and threshold for each compound tested were determined [5] (Table 1). Three
general classes can be seen: strong attractants (mannose, ribose, maltose), with thresholds of 10 -3 M or
less; weak attractants (fructose, galactose, sucrose)
with thresholds of 10 -1 M;and non-attractants
(glucose, rhamnose, arabinose, xylose, deoxyribose).
Of the strong attractants, both ribose and mannose
showed a decline in accumulation at concentrations
above 10 -3 M.
In this work I have shown that rhizobial chemotaxis is similar to chemotaxis in other bacteria in that
it occurs over a wide range of bacterial concentrations
and temperatures [5,6,23]. Like chemotaxis in a
motile Streptococcus, it is confined to a narrow range
of pHs around neutrality [6]. Chemotaxis in rhizobia
does not require an exogenous energy source, as is the
46
case in •: coli [23]. Unlike c h e m o t a x i s in E. coli, B.
subtilis and Streptococcus, high c o n c e n t r a t i o n s o f
rhizobia do not lead to a plateau in bacterial accumulation [5,6].
C o n c e n t r a t i o n response curves for m a n n o s e were
bimodal, unlike those for o t h e r bacteria, or for o t h e r
attractants in rhizobia. Finally, rhizobia remain the
only bacteria attracted by a m a c r o m o l e c u l e , trefoil
c h e m o t a c t i n [12].
References
[1] Adler, J. (1975) J. Gen. Microbiol. 74, 77 91.
[2] Aswad, D.W. and Koshland Jr., D.E. (1975) J. Mol.
Biol. 97,207 223.
[3] Macnab, R.M. and Ornston, M.K. (1977) J. Mol. Biol.
112, 1 30.
[4] Springer, M.S., Goy, M.F. and Adler, J. (1977) Proc.
Natl. Acad. Sci. USA 72, 3312-3316.
[5] Ordal, G.W. and Gibson, K.J. (1977) J. Bacteriol. 129,
151 155.
[6] Van der Drift, C., Duiverman, J., Bexkens, H. and
Krijen, A. (1975) J. Bacteriol. 124, 1142-1147.
[7] Berg, H.C. and Brown, D.A. (1972) Nature 234,500
504.
[8] Macnab, R. and Koshland Jr., D.E. (1972) Proc. Natl.
Acad. Sci. USA 69, 2504-2512.
[9] Tsang, N., Macnab, R. and Koshland Jr., D.E. (1973)
Science 181, 60- 63.
[10] Lursen, S.H., Reader, R.W., Kent, E.N., Yso, W.W. and
Adler, J. (1974) Nature 249, 74-77.
[11 ] Currier, W.W. and Strobel, G.A. (l 976) Plant Physiol.
57,820 823.
[12] Currier, W.W. and Strobel, G.A. (1977) Science 196,
434 -436.
[13] Currier, W.W. and Strobel, G.A. (1977) FEMS Microbiol. Lett. 1,243 246.
[14] Rovira, A.D. (1965) Annu. Rev. Microbiol. 19, 241
266.
[15] Wilson, J.K. (1930) Soil Science 30,289- 296.
[16] Purchase, tt.F. and Nutman, P.S. (1957) Ann. Bot. 21,
439 454.
[171 Rovira, A.D. (1961) Austral. J. Agric. Res. 12, 77 83.
[181 Dart, P.J. and Mercer, F.W. (1964) Arch. Mikrobiol. 47,
344 378.
[19] Muns, D.N. (1968) Plant and Soil 28, 129 146.
[20] Griffin, D.M. and Quail, G. (1968) Austral. J. Biol. Sci.
21,579-582.
[21] Hamdi, Y.A. (1971) Soil Biol. Biochem. 3, 121 126.
[22] Gitte, R., Rai, P. and Patil, R. (1978) Plant and Soil
50, 533-566.
[23] Adler, J. (1973) J. Gen. Microbiol. 74, 77 91.