The Swimming Behavior of Selected Archaea

20 Pages·2012·0.43 MB·English

AEM Accepts, published online ahead of print on 13 January 2012
Appl. Environ. Microbiol. doi:10.1128/AEM.06723-11
Copyright © 2012, American Society for Microbiology. All Rights Reserved.
1

The Swimming Behavior of Selected Archaea
1
2 Bastian Herzog1,2, Reinhard Wirth1*
3
D
o
w
4 1 Archaea Centre, University of Regensburg, Universitätsstraße 31, 93053 n
lo
a
5 Regensburg, Germany; [email protected]; phone: d
e
d
6 +49(0)941/943-1825 f
r
o
m
7 2 Present address: LS Siedlungswasserwirtschaft, TUM, Am Coulombwall, 85748 h
t
t
p
8 Garching, Germany; [email protected]; phone: [phone] :/
/
a
e
m
9 .
a
s
m
10 *: corresponding author at .o
r
g
/

o
11 Prof. Dr. Reinhard Wirth, Universität Regensburg, Mikrobiologie – Archaea Centre n

N
o
12 Universitätsstraße 31, 93053 Regensburg, GERMANY ve
m
b
13 [email protected] e
r

1
8
,
14 2
0
1
8
15 running title: archaeal swimming behavior b
y

g
u
16 e
s
t
17 key words: Archaea; swimming; Methanocaldococcus villosus; speed
18
19
2

20 SUMMARY
21 The swimming behavior of Bacteria has been studied extensively, at least for some
22 species like Escherichia coli. In contrast alomost no data have been published for
23 Archaea in this respect. In a systematic study we asked how the archaeal model D
o
w
24 organisms Halobacterium salinarum, Methanococcus voltae, Methanococcus n
lo
25 maripaludis, Methanocaldococcus jannaschii, Methanocaldococcus villosus, a
d
e
d
26 Pyrococcus furiosus and Sulfolobus acidocaldarius swim and which swimming
f
r
o
27 behavior they exhibit. The two Euryarchaeota M. jannaschii and M. villosus were m

h
28 found to be by far the fastest organisms reported up to now, if speed is measured in ttp
:
/
/
29 bps (bodies per second). Their swimming speeds, with close to 400 and 500 bps, are a
e
m
30 much higher than that of the bacterium E. coli, or a very fast animal, like the cheetah, .a
s
m
31 with ca. 20 bps, each. In addition we observed that two different swimming modes
.
o
r
g
32 are used by some Archaea. They either swim very rapidly, in a more or less straight /

o
n
33 line, or they exhibit a slower kind of zigzag swimming behavior, if cells are in close
N
o
34 proximity to the surface of the glass capillary used for observation. We argue that v
e
m
35 such a “relocate and seek” behavior enables the organisms to stay in their natural b
e
r

36 habitat. 1
8
,

2
0
37 1
8

b
y
38 INTRODUCTION
g
u
e
s
39 The ability to move is of pivotal importance for most organisms; this is especially true t
40 for microorganisms which experience many – often dramatic – changes in their
41 natural habitat. A translocation of just 1 cm in water current translates into 10,000
42 body lengths for a bacterium of 1 µm in size (for human a corresponding
43 translocation would be close to 20 km!). Within such a distance concentrations of
3

44 potential food sources may differ dramatically in open water bodies, but also
45 temperature differences of > 50° C can occur within 1 cm, e.g. within “black smoker
46 systems”. Therefore, microorganisms able to react to such differences by moving to
47 optimal surroundings have a distinct advantage over those that are immotile.
D
o
48 Swimming in liquid media is by far the best characterized motility mechanism in w
n
49 Bacteria, but various other mechanisms of movement on surfaces have been defined lo
a
d
50 for them (see, e.g. 11, 13, 17). e
d

f
r
o
51 Rotation of flagella has been identified early on as the mechanism of swimming in the m

h
52 bacterial model organisms Escherichia coli and Salmonella typhimurium (5). It also ttp
:
/
/
53 has been known for a long time that switching between “runs” and “tumbles” is the a
e
m
54 mechanism underlying bacterial chemotaxis (16). Today, the bacterial motility system .a
s
m
55 using flagella for swimming has been studied to a great extent and very many
.
o
r
g
56 aspects are known, not only down to molecular details, but also with respect to /

o
n
57 regulation (see e.g. 3, 21, 26 for reviews, and references therein). In principle, signals
N
o
58 are sensed by arrays of chemoreceptors; a phospho-relay then transmits the signal v
e
m
59 to the flagellar motor. The sensitivity of the chemoreceptors can be modulated by b
e
r

60 their methylation, resulting in the possibility of adaptation to stimuli. The presence of 1
8
,

2
61 chemoattractants leads to longer lasting runs (= linear movement) by 0
1
8
62 counterclockwise rotation of the peritrichously located flagella, which themselves are
b
y

63 combined in one bundle. Runs, typically at a speed of ca. 20 µm/s are interrupted by g
u
e
64 tumbles (= new, accidental orientation in space by disintegration of the flagellar st
65 bundle), which are induced by short clockwise rotation of flagella. E. coli, therefore,
66 reacts to attractant/repellent sources by integrating signal intensity over time, and not
67 cell length. It also was found that variations of the “standard E. coli swimming mode
68 system” exist. In the case of Rhizobium meliloti the new swimming direction is not
4

69 accomplished by tumbling; rather a short stop of flagellar rotation reorientates this
70 bacterium.
71 In the case of Archaea much less data were reported to their swimming mode. Only
72 in the case of the rod-shaped species Halobacterium salinarum (earlier H. halobium) D
o
w
73 and quadratic shaped halobacteria has the mechanism of swimming been shown, by n
lo
74 dark field microscopy, to be due to rotation of flagella (1, 2). For these Archaea a a
d
e
d
75 simple back and forth movement was observed and cells were relatively slow (2
f
r
o
76 µm/s). The movement of Methanococcus voltae has been described to consist of m

h
77 incomplete circles (90 – 270°), interrupted by short stops (K. Jarrell, personal ttp
:
/
/
78 communication and own data, not shown); it has to be mentioned, however that this a
e
m
79 holds true only if cells are observed at room temperature under aerobic conditions. .a
s
m
80 Direct measurements of the swimming speed of hyperthermophilic microorganisms
.
o
r
g
81 have not been reported up to now; the archaeum Pyrococcus furiosus for example /

o
n
82 was named “rushing fireball”, but no comment on its actual swimming speed was
N
o
83 given (10). In addition to the limiting data on their swimming speed and mode also v
e
m
84 only limited data are available for the synthesis, motor components, hook structures, b
e
r

85 interaction with the chemotaxis system, and other aspects of archaeal flagella (see 1
8
,

2
86 e.g. 8, 20 for recent reviews). One problem for studies of archaeal flagella clearly 0
1
8
87 relates to the extremophilic nature of archaeal model organisms. Although
b
y

88 halobacteria can be grown aerobically at ambient temperatures, studies of their g
u
e
89 proteins are difficult, due to their salt-dependence. Some members of the st
90 Sulfolobales are aerobic, too; in their case however, the dependence on very low pH
91 and high growth temperatures complicate studies. In the case of many
92 hyperthermophilic Archaea, their growth under anaerobic conditions at temperatures
93 above 80° C hinders analyses of their swimming mode. In addition the technique to
5

94 observe flagella by direct staining with fluorescent dyes, originally developed for in
95 situ analyses of swimming E. coli (24), is not applicable to a great variety of Archaea
96 (25).
97 Here, we present the first systematic analysis of the swimming mode of selected D
o
w
98 archaeal model organisms making use of our so-called “thermo-microscope” (12). n
lo
99 We asked which swimming patterns these microorganisms exhibit under their optimal a
d
e
d
100 growth conditions and determined their swimming speeds. Remarkably, the motility of
f
r
o
101 two species of methanocaldococci is the highest relative speed of any organism on m

h
102 earth. ttp
:
/
/
a
103 e
m
.
a
s
104 MATERIALS AND METHODS m
.
o
r
g
105 Growth of Microorganisms /

o
n

N
106 The following microorganisms (species; medium; gas phase; optimal growth
o
v
e
107 temperature) were included in this study: Escherichia coli (fresh fecal isolate), LB, air, m
b
108 37° C; Halobacterium salinarum DSM 3754T, DSM-medium 97, air; 50° C; er

1
109 Methanococcus maripaludis DSM 2067T, DSM-medium 141, H2/CO2 (80/20%), 37° 8,
2
0
110 C; Methanococcus voltae DSM 1224T, MGG (4), H2/CO2 (80/20%), 37° C; 1
8

111 Methanocaldococcus jannaschii DSM 2661T, MJ (4), H2/CO2 (80/20%), 85° C; by

g
112 Methanocaldococcus villosus DSM 22612T, MGG (4), H2/CO2 (80/20%), 80° C; ue
s
t
113 Methanothermobacter thermoautotrophicus DSM 1053T, DSM-medium 119, H2/CO2
114 (80/20%), 65° C; Pyrococcus furiosus DSM3638T, ½ SME (+ 0.1 % yeast extract +
115 0.1% peptone; 10), N2/CO2 (80/20%), 95 °C; Sulfolobus acidocaldarius DSM639T,
116 DSM-medium 88, air, 70° C. Cells were grown by shaking, aerobically in Erlenmeyer
117 flasks filled to a maximum of 1/10 their nominal volume (E. coli and H. salinarum) or
6

118 semi-aerobically in Erlenmeyer flasks filled to 1/3 of their nominal volume (S.
119 acidocaldarius). The other species were grown in 120 ml serum bottles, filled with 20
120 ml medium under the gas phase given above, again with shaking. Growth was in all
121 cases close to stationary phase, a condition under which optimal motility was
D
o
122 observed. w
n
lo
123 Cells were transferred into rectangular glass capillaries, 1 x 0.1 mm interior, by a
d
e
d
124 capillary action and the capillaries closed on both ends by use of instant super glue.
f
r
o
125 Thereafter the capillaries were immediately transferred onto an electrically heated m

h
126 stage which was placed on the microscope table. Since the capillaries had a length ttp
:
/
/
127 of ca. 3 cm and observations were confined to the center of capillaries, potential toxic a
e
m
128 effects of super glue were avoided. .a
s
m
.
129 Microscopic Analyses of Swimming Behavior o
r
g
/

o
130 Analyses were done by use of our so-called “thermo-microscope” (see 12 for a n

N
131 detailed description), which in principle consists of a normal phase-contrast light o
v
e
m
132 microscope (Olympus BX50) located inside a plexiglass housing. Heating fans inside
b
e
133 this housing were used to heat the system up to ca. 55° C. To obtain higher r
1
8
,
134 temperatures the phase-contrast objectives were heated electrically as were the 2
0
1
135 glass capillaries (containing cells) located on the electrically heated stage. Objectives 8

b
y
136 and stage were equipped with temperature sensors to ensure that the desired
g
u
137 temperature, which could be raised to a maximum of 95° C, had been obtained. All es
t
138 operation elements like focus, shutters etc. were extended to the outside of the
139 plexiglass housing to allow operation of the microscope at up to 95° C. After the
140 desired temperature was reached (monitored via the heat sensors on the objectives
141 and stage), motility was recorded using a CCD camera (pco1600; PCO Company,
7

142 Kelheim, Germany). Movies of swimming microorganisms were analyzed using
143 software ImageJ (ImageJ version 1.41o with Java 1.6.0) with the add-on modules
144 particle tracker and manual tracking. For each movie the diameter of the particles to
145 be analyzed had to be defined; a certain threshold of grey values (maximal deviation
D
o
146 of 50%) was used to avoid analyses of microorganisms deviating in the z-plane by w
n
147 more than 2 µm. In some of the supplementary movies tracks which were used for lo
a
d
148 analyses are indicated by coloration. Tracks that consisted of at least five continuous e
d

f
r
149 frames were used for determination of maximum swimming speed. Data are only o
m

150 reported for measurements for which the speed calculations for the five frames varied h
t
t
p
:
151 by less than 10%. The average speed was determined by measuring the speed of at //
a
e
152 least 10 different cells (tracks) of one sample. To further eliminate experimental m
.
a
s
153 variations cells were observed in at least 5 independent experiments under the same m
.
o
154 conditions, especially growth/observation temperature. Therefore, at least 50 tracks r
g
/

o
155 were combined for determination of the average speed. For recording the swimming n

N
156 tracks of the fastest organisms a high-speed CCD camera (PCO 1200 hs) with a o
v
e
157 resolution of 100 frames per second had to be used. Calibration for swimming m
b
e
158 distances was accomplished by photographing a micrometer scale with the standard r
1
8
159 equipment used. ,
2
0
1
8
160
b
y

g
161 RESULTS AND DISCUSSION u
e
s
t
162 The species we have analyzed were selected for the following reasons: The
163 bacterium E. coli was chosen as a positive control because many data are available
164 for this standard bacterium. The archaeum M. thermoautotrophicus was chosen as a
165 negative control, because we knew from earlier analyses (23) that this species is not
8

166 motile. The other Archaea were chosen because of their known motility and the fact
167 that they represent model systems for various aspects: H. salinarum for salt-
168 dependence; M. maripaludis and M. voltae for mesophilic, genetically manipulable
169 methanogens; M. jannaschii for hyperthermophilic methanogens; M. villosus as a
D
o
170 control for the data obtained with M. jannaschii; P. furiosus for heterotrophic w
n
171 hyperthermophilic anaerobic Archaea; S. acidocaldarius for thermoacidophilic lo
a
d
172 Archaea. e
d

f
r
o
173 Control experiments (data not shown) indicated that no difference in swimming m

h
174 behavior was observed if cells were transferred into the rectangular glass capillaries ttp
:
/
/
175 under anaerobic or aerobic conditions. For ease of operation, glass capillaries a
e
m
176 therefore were filled under aerobic conditions and closed on both ends by superglue. .a
s
m
177 This procedure took less than 1 min and the medium was still anaerobic inside the
.
o
r
g
178 glass capillaries as indicated by a lack of coloration of the redox indicator resazurin /

o
n
179 used in the anaerobic media. Table 1 gives a summary of the results obtained, which
N
o
180 we interpret as follows. v
e
m
b
181 Swimming speeds e
r

1
8
,
182 We did observe an extensive range of swimming speed for the various species 2
0
1
183 analyzed, with absolute values for maximal speeds ranging from 10 to ~ 590 µm/s; 8

b
y
184 average speeds varied from 3 to ~ 380 µm/s. The average swimming speed we
g
u
185 observed for our E. coli fecal isolate did exceed with ~ 45 µm/s that reported for E. es
t
186 coli AW405 with 21 µm/s (6). It has to be emphasized that the experimental setups
187 differ markedly between these two data sets: we did analyses at 37° C, enhancing
188 swimming speed by ca. a factor of 1.5 compared with swimming at room temperature
189 (data not shown) and in rich medium, which clearly shifts runs to longer times (6; and
9

190 own observations, data not shown). An absolute average swimming speed of 45
191 µm/s translates into a relative swimming speed of ca. 20 bps for E. coli. The
192 maximum speed of the sulfur bacterium Thiovulum majus was reported to be approx.
193 600 µm/s, but because of its size of ~ 7 µm this relates to ca. 85 bps (9). In contrast,
D
o
194 the bacterium Bdellovibrio bacteriovorus swims at approx. 100 µm/s which due to its w
n
195 small size relates to a maximum of approx. 150 bps (9). lo
a
d
e
d
196 The average absolute swimming speeds we have measured here for the various
f
r
o
197 archaeal model species are in the range of very slow swimming (3 µm/s for H. m

h
198 salinarum, corroborated by earlier data, 1) to very fast swimming (380 µm/s for M. ttp
:
/
/
199 jannaschii). There was no correlation between swimming speed and growth a
e
m
200 temperature: M. voltae grown at 37° C swims faster than P. furiosus observed at 95° .a
s
m
201 C, but slower than M. villosus grown at 80° C. The maximal swimming speed of M.
.
o
r
g
202 jannaschii with 589 µm/s is similar to that of T. majus; due to the marked difference in /

o
n
203 size between those two microorganisms their relative swimming speeds, however
N
o
204 differ drastically: ca. 390 bps vs. ca. 85 bps. Because of its smaller size M. villosus v
e
m
205 has an even higher maximal relative swimming speed of ~ 470 bps. These two b
e
r

206 archaeal species therefore possess the highest relative speed measured for any 1
8
,

2
207 organism on earth up to now (15). 0
1
8

208 Mode of swimming b
y

g
u
209 The fresh fecal isolate of E. coli was used here for comparisons and showed the es
t
210 mode of swimming reported earlier: runs were interrupted by tumbles. We noted that
211 the swimming speed (45 µm/s) and length of runs (up to 250-300 µm, and up to 8-10
212 s) of this fresh isolate exceed those reported for E. coli AW405 (21 µm/s, and ~ 1s;
213 6). This very probably is due not only to different experimental set-ups (see above),
10

214 but also to the fact that E. coli K12 strains have a “lab history” of > 50 years, a period
215 in which physiological alterations might have accumulated. We have deliberately
216 chosen to compare the fresh fecal E. coli isolate with the archaeal species tested,
217 because all of the latter were type strains, representing “wild-types”.
D
o
w
218 Very interestingly, these Archaea (Methanococcus maripaludis, Methanococcus n
lo
219 voltae, Methanocaldococcus jannaschii, Methanocaldococcus villosus, Sulfolobus a
d
e
d
220 solfataricus) exhibited a swimming behavior we observed also for P. furiosus, which
f
r
o
221 is in stark contrast to that of, for example, E. coli. This latter bacterium swims in more m

h
222 or less smooth runs, disrupted by tumbles to alter the swimming direction. The ttp
:
/
/
223 hyperthermophilic Archaea, on the other hand exhibit a swimming behavior best a
e
m
224 characterized as a “relocate and seek” strategy; for relocation they swim in more or .a
s
m
225 less straight lines, covering a (relatively) long distance due to their high swimming
.
o
r
g
226 speed – see supplementary movies. In this fast swimming mode some smaller /

o
n
227 changes in direction are observed, but not abrupt ones; thus, tumbles observed in E.
N
o
228 coli are not observed in these Archaea. For comparative reasons these v
e
m
229 supplementary movies show: swimming of E. coli (medium velocity- 2 movies, one b
e
r

230 without and one with indication of tracks used for analyses); swimming of H. 1
8
,

2
231 salinarum (slow velocity) and swimming of H. salinarum at 10-fold time lapse; 0
1
8
232 swimming of P. furiosus (fast swimming); swimming of M. voltae (fast swimming);
b
y

233 swimming of M. villosus (very high velocity); swimming of M. jannaschii (highest g
u
e
234 velocity). This type of swimming behavior was observed especially for cells swimming st
235 parallel to the observation plane in the middle of the capillary. Most of these cells,
236 therefore, did not experience a contact of their flagella with the glass capillary wall; in
237 average, they had a distance of ca. 50 µm from the glass capillary wall.