Motility in the marine environment: an adaptive response to patchy nutrient distributions?
Dana E. Hunt Microbial Diversity Summer Course 2004 Final Project Report
Abstract:
While the ocean is generally assumed to be well-mixed, from the vantage point of bacteria the ocean may in fact be a mosaic of evolving microenvironments. The ability of bacteria to sense and respond to these nutrient gradients may influence bacterial productivity and confer selective advantage to motile organisms. The phycosphere surrounding photosynthesizing phytoplankton is thought to contain enhanced levels of carbon and nitrogen containing substrates that can be used by heterotrophic bacteria for growth. This study establishes that cyanobacterial form strain-specific associations with heterotrophs over many years of co-culture, suggesting interactions that may be mirrored in the natural environment. Media in which cyanobacteria had been cultured resulted in positive taxis in six of the seven environmental isolates tested, suggesting that even the potentially low levels of nutrients released . by cyanobacteria can induce taxis. Additionally, seawater isolates display a tactic response to a variety of monomers that make up cyanobacterial exopolysaccharide and the amino acid leucine but not serine or glutamine. The strongest tactic response was evident in response to glucose, with severe retardation of spreading on soft agar plates. Further study of the chemotactic response to glucose suggests that marine bacteria, reflecting their oligotrophic environment, may be able to sense glucose concentrations down to 1012 M, several orders of magnitude lower than that detectable by E. coil. The fact that large numbers of isolatable marine bacteria are motile and chemotactic suggests that clustering around nutrient point sources (including phytoplankton) play a large role in bacterial ecology and productivity
Introduction:
What is the significance of motility in the marine environment? A large fraction of marine isolates have been found to be motile, but direct observation of seawater suggests a small fraction of total cell populations are motile. Flagellar motility is energetically expensive for organisms in low nutrient environments requiring greater than 10% of available resources, thus it seems logical that bacterial should derive a specific energetic benefit from this process. Theoretically, motility could confer two main advantages enhancing flux by reducing the thickness of the diffusive boundary layer or moving organisms towards sources of higher nutrient levels (Purcell 1977). Modeling has suggested that the most likely explanation is that this motility is used to respond to transient chemoeffectors. Photosynthetic phytoplankton are thought to release significant amounts of nutrients into the marine environment through exudation, representing a potentially significant nutrient point source. Bell and Mitchell (1972) first suggested the importance of the “phycosphere”, the region surrounding a photosynthesizing alga analogous to the rhizosphere of plants containing high levels of photoassimilated carbon. The compounds released by algae are thought to consist largely of polysaccharides (up to 80%) and may also contain other labile compounds including amino acids and vitamins (Myklestad 1995). Interactions between phytoplankton-heterotrophs may be highly specific with algal products serving as both bacterial attractants and repellents (Bell et al. 1972); and specific bacterial groups co-culturing with algae (Schafer et al. 2002). Modeling suggests that up to 20% of the chemotactic bacteria could be clustered around phytoplankton cells (Bowen et al. 1993); and chemotactic tracking of motile algal cells has been observed ex-situ (Barbara et al. 2003). Yet, a mesocosm experiment yielded no evidence of bacterial clustering around algae (Müller-Niklas et al. 1996). Thus while no direct evidence has established the importance of bacterial clustering in response to cyanobacterial exudates evidence suggests this would be an adaptive technique in oligotrophic aquatic ecosystems.
The components released by algae have been observed to vary between species and exudation is thought to increase during nitrogen and phosphorous limitation (Myklestad 1995). Previous study of bacterial isolates revealed that bacteria co-occurring with Microcystis aeruginosa were better more likely to exhibit positive taxis towards Microcystis cells than isolates from a site not colonized by these cyanobacteria (Casamatta et al. 2000), suggesting that specific associations form between heterotrophic and photosynthetic bacteria. The association between an Anabaena sp. and its heterocyst epibiont has been previously studied in the course (Anderson, 2003), although chemotaxis toward Anabaena cells was not observed.
The compounds released by algal cells have been extensively characterized; there has been to my knowledge no comparable study of the production of labile compounds by marine cyanobacteria, although the composition of exopolysaccharide for many organisms is known. A large fraction of marine bacterial isolates are motile, the only logical explanation for this energetically expensive activity is that bacteria derive a substantial benefit from being able to respond to spatially variable nutrient conditions. This study will attempt to quantify the importance of this chemotactic response and perhaps better inform our models of productivity in marine ecosystems.
Materials and Methods:
Cyanobacteria:
Cyanobacterial cultures were obtained from John Waterbury (Woods Hole Oceanographic
Institution) : axenic Crocosphaera WH8501 and non-axenic Crocosphaera strains E12 and 1051. No attempt has been made to purify the non-axenic strains from their co-existing heterotrophic organisms although they have been in culture for many years. Cyanobacteria were grown in a Sargasso-seawater based medium containing no combined nitrogen so that cells would grow diazotrophically with iron as the limiting growth factor. Cyanobacterial cultures were incubated on a 14 hour light 10 hour dark cycle at 28°C. the plates Isolates autoclaving added from (per identification isolation, from water; was Heterotrophic towards capillary exudate Seawater days capillary selection
Chemotactic sequenced Strains noted). vitamins Crude Swarm in States galactose, Swarm 75Omls cells substrate supplemented Chemotaxis
SWC
sequencing
obtained
liter:
of
which
the was
from
(Microbial
Chemical chemotactic 5g
were
plates: plates
growth.
were
of
produced
Swarm exudates,
or
end
tube tube
mix)
for
the isolates:
compared
tryptone;
measured
Na2HPO4
leucine,
seawater,
in
glucose
strain
they
2g
assay:
bacteria
streaked
of
obtained
by 16S order
of
assays:
were were and
bacterial
plates.
supplemented
of
primer.
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plates
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While were Corp),
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incubated
serine,
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assays 3g
MBL
minimal plated made
to after organism.
4.8g;
2
25Omls
Crocosphaera. that
several
identify
The obtained
were and
yeast
the
from
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represent
isolates
isolates
L-glutamine,
24-48
pier
with
are
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onto were
after plates
2004 then
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non-axenic
extract;
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DI, in
with
motile
times
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Handbook)).
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1
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was
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for arabinose, that NH4C1
obtained
using previously).
directly
of
1
E12-2, on
environment using
L
ml scored
into
agar
seawater.
and
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media
SWC
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and
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from method
1051.
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The
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compared primers the
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After Strains a metals
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7H20 of
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750m1s 1
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492R
in
0.5g;
isolates
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and inoculated amplified
1
a
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named
The
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the in
the
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trace
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was
at
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NaC1
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Sigma
of interest:
contents
first
seawater; was
Woods after
several
cellobiose
attempted placed
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after
(per
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51
and
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set
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of
D
partially
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in
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post
used
of
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(United
were
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motile
as
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media
MA
for
imi
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as
the
the
DI , • Pseudoalteromonas tubes the media the were culture media remaining motility and appears glucose Heterotrophic Results over-representation grown actively on associations Interestingly, medium of incubation strain organisms, 30 were Seawater (10mM aliquot 1 000x the the minutes. common closest plating grown visibly represent 1051 cyanobacterial and and heterotrophic as in A Isolates them Motile was potassium minimal likely motile buffer was complex appropriate and isolates: set isolate the short BLAST at pH nine of pelleted without with The microbial checked using placed of 30°C. the the that isolates cyanobacterial cells Discussion: and 7) in organisms were isolates incubation of media. contents nine the most three the contents medium, they phosphate, spent the plated hit were in sp. in bacteria at a obtained different for cultures marine for combined isolates the obtained to ten a weeds 5000 closely times are Md from sterile cyanobacterial chemotaxis Partial motility identify grown on “capillary of that were of using times: 213, it rpm the co-existing environment. SWC with each products. capillaries generally are appears from 1 from cyanobacterial are related glass 00um from most to 1 capillary nitrogen with for cyanobacteria 6S making the using both Vibrio, subsequent of late media. coastal strain supematant” sequences 5 capillary assays. non-axenic obtained the closely Na a that to minutes. log fast minimum 40x obtained culture In containing over a non-axenic EDTA source, use Psezidoalteromonas, tube E12 Pseudomonas the order phase Colonies seawater Although growing phase The related of cultures many pelleting tube vibrios isolates. were was were medium. The as cyanobacterial supplemented to cyanobacterial using in unless chemoeffector isolates. of contrast, at and confirm either spent SWC years most cell to on were off 99% Crocosphaera obtained least all are suggests culture-based Halomonas incubated for complex Cultures the these pellet of sp. motile cyanobacterial directly similar Considering media identity. of a enumerated The 5 the then that MBL nitrogen culturing minutes The organisms for was and with observed that products species cultures: these diluted with in of medium obtained via a specificity in pier plated Alteromonas the washed number sp. Of interest strains selective 25g/L the BLAST source. methods at shaking bacterial perhaps using after that the ocean using obtained 2,500rpm. cell in or medium. one cultures also and from isolates motility NaCI was the in first or 24 of culture both of BLAST, judging pressure could to fix motility theoretically at a involving these cyanobacteria by hours “contaminants” obtained these the blank diluted 30C. for sp. as nitrogen were direct the analyzed medium attempt capillary isolates Table Cell solution marine organisms. of culture from is with of medium A motile in plating cultures exerted rich on motility cell 1 it sterile their the to are lists from 100- for Table 1: Characterization of direct seawater isolates including information about the closest BLAST hit and percent identity of the unedited sequence read with that identification. Exponential phase cultures were also scored for motility and cell morphology.
Percent Morphology! Closest BLAST hit identity Motile comments Capillary supernatant isolates SWC1 Vibrio sp. MD 208 98% yes short rods SWC2 Vibrio sp. MD 208 98% yes short rods SWC3 Photobacterium damselae subsp. Piscicida 99% slow short rods SWC4 V.nereis (ATCC 259 17T) 99% yes smaller, short rods long chains of rods SWC5 Vibrio pomeroi 98% yes snake-like swimming Pseudoalteromonas haloplanktis subsp. SWC6 tetraodonis 99% maybe? longer rods SWC7 Vibrio splendidus strain 636 100% short rods and long SWC8 Vibrio splendidus 99% darting filaments SWC9 Vibrio splendidus 98% fast tiny, darting motility SWC1O V.natriegens (ATCC 14048T) 97% fast tiny, run-reverse
SWC1 1 Vibrio alginolyticus strain NRL-SS41 98% slow small rods
Direct Seawater Plating Isolates Spi Pseudoalteromonas citrea 99% yes rods Sp2 Vibrio tasmanius 100% yes short rods Sp3 Pseudoalteromonas sp. SKA2O 98% fast short rods Sp4 Vibrio splendidus strain 636 100% Sp5 Pseudoalteromonas sp. TEl-i 16S 99% slow rods Sp7 Pseudoalteromonas sp. SM9913 99% wiggly rods Sp8 Pseudoalteromonas sp. SKA32 99% yes rods Sp9 Pseudoalteromonas sp. ANG.ro2 99% SplO Vibriotasmaniensis 100% slow rods
Spi 1 Alteromonas sp 99% Sp12 Pseudoalteromonas sp. TEl-i 99% yes rods
Swarm Plate Assay:
The swarm plate assay was used as a rapid method of assessing the chemotactic response of organisms to various potential growth substrates compared to growth on seawater —based medium supplemented with minerals and vitamins. Potential chemoeffectors were chosen based on their presence as monomers making up the exopolysaccharide of many cyanobacteria (glucose, galactose, rhamnose, arabinose, glucuronic acid) (Nicolaus et al. 1999; Giroldo et al. 2003). Chemotaxis was also tested for several amino acids (serine, glutamine, and leucine). Chemotaxis swarm plates supplemented with 100jiM of the component of interest were scored based on the diameter of the spreading circle of bacteria through the medium compared to the blank. A chemotactic response was observed when spreading of the bacteria was significantly decreased in the presence of a chemoattractant. For some substrates- i.e. glucose there was almost no migration through the agar plates for any of the isolates, suggesting that glucose is a strong chemoeffector. For other substrates, notably serine and glutamate the spreading of the cultures appeared to be enhanced with a diameter up to 175% of the control value, suggesting that there may be an interesting response to these substrates; however, leucine, the other amino acid tested, appeared to generate a positive taxis response in many isolates. It may be possible that this represents a negative taxis as it is been noted that natural bacterial populations are negatively tactic towards serine {Barbara, 2003 #173}. This observation was further investigated using a capillary assay with no observed neative or positive taxis in response to serine for two 0b0 separate isolates over a range of 10 to 1 M serine. Thus perhaps the presence of serine served to activate motility in these organisms as it did not appear to be a good substrate for growth, although this hypothesis warrants further testing. Other compounds such as cellobiose that in general made good growth substrates did not appear to induce a chemotactic response, which is interesting as cellobiose is a dimer of glucose. Galactose which is chemically identical to glucose but has a different arrangement of ring substituents appeared to induce only weak chemotactic responses. Several of these observations warrant further testing using a more sophisticated method such as the capillary assay.
Table 2: Swarm Plate assay results: after 24-48 hours the plates were scored based on the extent of growth on various added substrates (100jiM) compared to growth on the control plate (seawater and agar). The check mark reflects definite growth, a check minus was difficult to discern and a minus reflected no observable growth. The chemotaxis was scored based on the diameter of the motile bacteria- a culture less than half the diameter of the control was scored as +; +1-reflects a diameter between half and 75% of the diameter of the control; while a — reflected a growth diameter greater than 75% of the control. oapltlarY
sp7 sp8
ap9 splO spli
splZ
direct
SWC1 SWC2
SWC3
SWC6 SWC5
SWC7 swcs
swc9 SWCIO SWC12
Isolates
1051-9 105110
1051-Il Chemotaxis
exopolysaccharide range table iron-limited medium tactic organisms replicate between statistical chemo cyanobacterial
seawater
assay
In
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while - --
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A arabiflOsO SWC10(rep) 4.94 spi 1.24 spi (rep) 2.32 6-E12 3.45 sp5 1.04 SWC8 2.29 SWC12 6.38 SWC4 9.24
Additionally, since glucose appears to be such a strong chemoattractant for all of the cells
studied, the capillary assay was performed with glucose concentrations ranging from 102 to 10.12 M. The ratio of colony forming units for isolate SWC1Ofor this range of glucose concentrations compared to the control is plotted in Figure 1. The colonies for the 10 M glucose concentration were too dense to enumerate, but appeared to be more abundant than those on the 10 M glucose plate, suggesting that cells may be able to recognize very high levels of glucose. The other interesting result is the low concentrations at which chemotaxis appears to be able to sense a glucose gradient. The receptors of E. coil cannot detect glucose below 5* 910M, while this vibrio isolate appears to exhibit taxis to glucose down to 2M10’ levels. More sensitive receptors may be a distinct advantage in the marine environment where nutrient levels are significantly lower than the gut environment experienced by E. coil.
Figure 1. Plot of glucose concentrations versus the chemotactic response level
Chemotaxis of SWCIO to Glucose . > 26 16oc 11 . . 6 . . , L
1 I I I I 0 2 4 6 8 10 12 14 -log of glucose concentration
Conclusions:
This project suggests that motile cells in the marine environment may be highly sensitive to low concentration nutrient point sources, such as those produced by phytoplankton leaking photoassimilated carbon and other nutrients. Interactions between phytoplankton and . heterotrophs may be highly specific as suggested by previous research {Schäfer, 2002 #129} and • • Nicolaus, those Purcell, Myklestad, there Bowen, with Müller-Niklas, Anderson, Casamatta, Bell, Barbara, concentrations. other compounds. especially through Giroldo, Cited important Ferguson assemblages the Schafer, interesting seawater seems in cultures which observation order-of—magnitude W. may compounds, provided Works likely phytoplankton to cyanobacteria.” marine degradation Microbial communities exopolysaccharides emphasis Thalassiosira heterocyst extracellular Microbiology 1577-15 E. J. consumption of H., D., and G. these isolates phytoplankton: B., 1986, selective D., be if C. M. to Crocosphaera D. S. M. B. A. certain that for R. Another A. important test (2003) K. M. (1977). A. diatoms.” organisms G., by Abbas. A. and 82. Mitchell Ayo This Panico, glucose in D. on these are and Ecology if (1995). of marine although H. M. this advantages of J. cyanobacterial essential Stolzenbach, polysaccharides.” products.” to et an “Isolation, apparently sp result Vieira, Ecology C. of G. interesting Agis, “Life cells an et heterotrophs study Phytochemistry exudates al. Anabaena cross-talk (Bacillariophyceae).” extracellular et E. and Mitchell FEMS (1972). al. extracellular Results “Release are cyanobacteria were variations 2001). 40(1): al. from Wickstrom is in the at et (2002). amino et nutrients that cultured consistent (1999). a low 44(1): al. by The largest highly al. turbulent growth Microbiology highly from representative “Chemotactic observation 64-73. et from the (1996). between (2003). enabling (2003). sp.” Reynolds-number.” of growth While Biological acids al. are “Genetic in 79-87. culturable products extracellular “Chemical the contains polysaccharide strain-specific Science chemotactic (P, (1993). nutrient Microbial (2000). sensitive 100-nl 52(4): and making by with ocean.” suggests “Microscale cyanobacterium etc.) energetically “Bacterial “Relative is phytoplankton exudation bacteria investigation enhanced the that diversity Journal samples.” Ecology 639-647. Bulletin or of were “Simulating “Sensitivity and no members levels a heterotrophic to observed composition through Limnology the Diversity living bears added that products low response growth to increase provided tracking Total after of or released (Azam distribution make American of’satellite’ bacteria by 42(1): concentrations expensive, 143(2): further off Limnology Phycology cell combined of of feeding and the Microcystis years multiphastic responses Environment of bacterial Summer of by heterocystous the use was lysis. of and & of presence contaminants in two and 25-35. heterotrophs the by phytoplankton investigation 265-277. are bacterial deoxy Hodson motile in the of observed Journal Oceanography a off cyanobacteria, production disjunct of motility co-culture. bacteria adapted nutrient nitrogen tropical 39(6): and form Course clustering bacterioplankton of of of sugars aeruginosa algae.” of uptake marine lysed Oceanography 1981, 165(1-3): epibiont glucose of of heterotrophs, and at 1109-1115. bacterioplankton must and present to point of freshwater Physics or 2003. complex is high during of with cells. an non-axenic Fuhrman non-heterocystous FEMS that kinetics Since carbon around it bacteria 38(1): confer whether environment sources would and of Kutzing.” 155-164. special culturable in 45(1): Additionally the microbial the organic perhaps cultures sources- in in 36-5 41(7): to & be medium relation such natural algal 3-11. 1. of as it