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INTRODUCTION |
Behavioral change in response to
alterations in the quality and quantity of light in the environment is
a ubiquitous trait among motile photosynthetic bacteria. Three distinct
types of responses to light have been described in the literature
(14, 19, 36, 37). The scotophobic response (fear of
darkness) is characterized by a tumbling, stop, or reversal that occurs when a swimming bacterium experiences a temporal, or spatial, step down
in light intensity. Photokinesis describes an alteration in the rate of
motility caused by differences in light intensity. A phototactic
response, which has been studied most extensively in algae and
cyanobacteria, involves an oriented movement of a cell toward or away
from a light source (19). An important distinction is that
the direction of irradiation is not relevant to scotophobic or
photokinetic responses, whereas it is a critical determinant in
phototaxis. Thus, phototactic organisms are uniquely capable of
migrating towards a light source, irrespective of whether they are
going up or down a gradient of light intensity (37).
The various photosensory behaviors exhibited by anoxygenic
photosynthetic bacteria have been studied mainly by physiological and
biochemical tests, with little supporting genetic data (3, 4, 8,
9, 13, 16, 27, 38). The few genetic tests that have been
undertaken have demonstrated that mutations which functionally impair
the photosystem also disrupt the ability of cells to respond to light
(3, 20). This indicates that a product of photosynthesis,
such as the generation of proton motive force or photosynthesis-driven
electron transfer, is most likely the signal that controls photosensory
behavior, rather than direct absorption of light by a
chromophore-containing receptor. This conclusion is supported by recent
physiological studies which have shown that specific inhibitors of
cyclic photosynthesis-driven electron transport inhibit photosensory
behavior in Rhodobacter sphaeroides (13, 16) and
Rhodospirillum centenum (38). By using a
site-directed mutational approach, we have shown that the scotophobic
and phototactic responses of the purple nonsulfur photosynthetic
bacterium R. centenum involve components of the chemotaxis
phosphorylation cascade (25, 26). This suggests that a
sensor of photosynthetic activity may have features similar to that of
chemoreceptors. However, which component of the photosynthesis electron
transfer chain is being sensed and what is actually sensing alterations
in electron transfer are unknown.
To identify components responsible for prokaryotic behavioral
responses to light, it is essential that techniques be developed for the isolation of mutants that are specifically defective in photosensory behavior. One of the reasons why screens for photosensory mutants have not been developed is the inherent difficulty of assaying
for photosensory behavior. Until recently, screening for such mutants
involved the onerous task of microscopically assaying individual cells
from liquid-grown cultures for a response to a step up or down in light
intensity. Since statistically meaningful results require that multiple
cells be assayed, this "brute force" approach is infeasible. A
significant advance in the isolation of prokaryotic photosensory
mutants was recently provided by our observation that colonies of the
purple photosynthetic bacterium R. centenum are capable of
macroscopic phototactic motility (36, 37). Cells of R. centenum are dimorphic, existing in liquid medium as swim cells
bearing a single polar flagellum or as hyperflagellated swarm cells on
solid surfaces (36, 37). A unique feature of R. centenum swarming colonies is that they are capable of migrating rapidly (up to 75 mm/h) toward an infrared light source or away from a
visible light source (36, 37). This behavior allows us to
rapidly screen for mutants that are deficient in photosensory responses
by simply assaying colonies for aberrant light-directed migration. In
this study, we have utilized mini-Tn5-mediated mutagenesis to isolate numerous mutants that exhibit defects in light-directed motility. The phenotypes of specific classes of mutants provide some
unique observations on photosensory behavior, as well as on the
mechanism of swim cell to swarm cell differentiation.
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MATERIALS AND METHODS |
Bacterial strains, plasmids, and growth conditions.
Wild-type swarming strain R. centenum SW (ATCC 51521) is the
parental strain used in this study (12, 37).
Escherichia coli S17-1(
pir)/pUTmini-Tn5Sm/Sp
has been described previously (10). R. centenum
strains that were selected by transposition mutagenesis in this study
are listed in Table 1.
All of the E. coli strains used were cultured at 37°C in
Luria broth. Antibiotics were added, when appropriate, at the following concentrations: ampicillin, 150 µg/ml; spectinomycin, 50 µg/ml; gentamicin, 10 µg/ml; kanamycin, 40 µg/ml. R. centenum
strains were grown either photosynthetically in CENS medium
(44) at 37°C with illumination by 60-W tungsten Lumiline
bulbs or aerobically in PYVS or CENS medium at 37°C (37).
Assays of colony phototaxis toward infrared light and away from visible
light were performed as described previously (25, 37).
Recombinant DNA techniques.
Restriction and other DNA
modification enzymes were purchased from New England Biolabs and used
in accordance with the vendor's instructions. Chromosomal DNA was
prepared by a previously described protocol (40). For
Southern analyses, the SmaI fragment including a large
portion of the R. centenum chemotaxis gene operon (25, 26) was purified from an agarose gel with a GeneClean II kit (Bio
101, Inc., Vista, Calif.) and internally labeled with DIG High Prime
labeling and detection starter kit I (Boehringer Mannheim, Indianapolis, Ind.). Hybridization was performed in accordance with the
manufacturer's instructions. The results were scanned with an Epson
ES-1200C scanner attached to a Power Mac 7600/120 running Adobe
Photoshop 3.0 (Adobe Systems Incorporated). Notice that the
spectinomycin resistance (Spr) interposon does not contain
an ApaI site, but transposition introduces two new
SmaI restriction sites, so SmaI and
ApaI digestions would reveal the presence or absence of the
interposon in the chemotaxis gene cluster or its promoter region in
these mutants. Mutants showing wild-type restriction patterns in both
digestions are free of the interposon in this region.
Conjugation and transposition of the Mini-Tn5
interposon.
Mini-Tn5
Sp was delivered to
wild-type R. centenum via conjugation with S17-1(
pir)/pUTmini-Tn5Sm/Sp. The suicide plasmid
pUTmini-Tn5Sm/Sp contains the transposase gene located in
cis outside of the transposable element, thus preventing
secondary transposition events once the plasmid is lost from the cell
(10). pUTmini-Tn5Sm/Sp also contains an
incomplete replicon from plasmid R6K, and thus its replication is
dependent on the pir gene product (
protein) that is
provided in trans on the chromosome of E. coli
strains such as S17-1( pir) or SM10 ( pir)
(42). The interposon was introduced via a membrane filter
mating procedure (5, 47, 48). Usually, 5 × 109 R. centenum cells were applied to each
filter unit (Nalgene no. 245-0045). After mating for 12 to 16 h at
room temperature or for 4 to 5 h at 42°C, cells were collected
by washing with 4 ml of phosphate-buffered saline (PBS; containing 137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, and 1.7 mM
KH2PO4 at pH 7.2) and 200-µl aliquots of
resuspended cells were spread on either CENS plates for photosynthetic
growth or PYVS plates for aerobic dark growth. Plates were supplemented
with 10 µg of spectinomycin per ml for selection of the transposition
event and 40 µg of kanamycin per ml for counterselection of the
E. coli donor (note that R. centenum is naturally
resistant to kanamycin). Spr transconjugants were
identified after 48 to 72 h of incubation.
Isolation of mutants defective in the phototactic or scotophobic
response.
In a macroscopic approach, Spr strains from
mini-Tn5 mutagenesis were screened for photosensory defects
by transferring individual colonies with sterile toothpicks to square
PYVS-0.8% agar plates containing kanamycin. Alternatively, strains
were grown in liquid medium and concentrated 10- to 20-fold, and 5 to
10 µl of cell concentrate was spotted onto assay plates. Phototaxis
assays were performed for 12 to 24 h with unilateral light sources
as previously described (26, 34, 37). In a microscopic
approach, photosensory perception was tested by inspection of the cell
motility of an individual swarming colony with a Nikon OPTIPHOT-2
microscope equipped with a 40× lens, a Sony 3CCD camera, and a Sony
Trinitron monitor. The microscope beam was intercepted with a cutoff
filter that allows light with wavelengths above 550 nm to pass through (37), and the colony was then challenged with a fourfold
step down in light intensity by suddenly inserting a neutral-density filter into the light path. In both assays, wild-type R. centenum SW served as a control.
Generation of polyclonal antisera to polar and lateral
flagella.
Polar flagella were isolated from a 2-liter culture of
early-log-phase R. centenum cells grown photosynthetically
in CENS medium. Lateral flagella were isolated from swarm cells by
gently suspending cell paste from 30 0.8% agar PYVS square (9 by 9 cm) plates in liquid PYVS medium with a glass rod. The flagella were then
harvested as described previously (43). Partially purified and concentrated flagellar samples were then mixed with sodium dodecyl
sulfate (SDS) loading buffer and boiled for 5 min before being loaded
onto a preparative 12% denaturing polyacrylamide gel
(SDS-polyacrylamide gel electrophoresis). After electrophoresis, the
gels were stained with ice-cold 0.5 M KCl, the predominant bands were
excised from the gels, and the gel slices were crushed between two
glass plates and stored at 
70°C. Immunization and sampling of
antisera from rabbits were subsequently performed by a commercial
facility (Cocalico Biologicals, Reamstown, Pa.). Polyclonal
antisera and preimmune sera were tested by immunoblot assay (see below)
with samples from wild-type and mutant R. centenum strains
that are unable to synthesize flagella as determined by a
flagellar stain kit (Carr-Scarborough Microbiologicals, Decatur, Ga.). The polyclonal antiserum raised against lateral flagella cross-reacts with the polar flagellum. However, the antiserum against
the polar flagellum does not cross-react with the lateral flagella and
also displays a very high background in an immunoblot assay.
Consequently, lateral flagellar antiserum was used to detect both
flagellar types in the immunoblots.
Immunoblot assays.
Immunoblot (Western blot) assays were
performed as previously described (21) with polyclonal
anti-lateral-flagellum serum. Swarm cell flagellar samples were
collected from cells that were grown on 0.8% agar plates and then
gently rinsed off the agar surface with 5 ml of PBS buffer. For both
swim and swarm cell samples, about 5 ml of cells between 50 and 80 Klett photometric units (red filter no. 66) were centrifuged and the
cell pellet was resuspended in 500 µl of PBS buffer supplemented with
25 mM EDTA-1 mM phenylmethylsulfonyl fluoride and then vortexed for 2 min. The cells were pelleted by centrifugation, and sheared flagella
that were present in the supernatant were subjected to standard
procedures for SDS-gel electrophoresis and Western transfer to
nitrocellulose membranes (Schleicher & Schuell, Keene, N.H.). After the
transfer, membranes were blocked with 5% (wt/vol) nonfat dry milk in
PBS buffer plus 0.1% (vol/vol) Tween 20 for 1 h at 24°C. The
primary antiserum was added to the blocking mixture at dilutions of
1:10,000 to 1:20,000 and incubation proceeded for an additional 2 h at 24°C; afterwards, the membranes were washed three times in
PBS-Tween buffer and then incubated with a 1:10,000 dilution of a goat
anti-rabbit horseradish peroxidase-conjugated secondary antibody
(Amersham) in PBS-Tween buffer for 1 h at 24°C. Finally, the
membrane was washed three times in PBS-Tween buffer and the results
were visualized by development with an ECL kit (Amersham).
Chemotaxis capillary assay.
Chemotaxis measurement was
performed as previously described (26), with a few
modifications. In addition to pyruvate and acetate, we determined that
serine also serves as a strong chemoattractant for R. centenum. Therefore, 10 mM serine in CTX buffer (26) was used for all assays. Also, nonmotile strain ZJJ8-7 (Table 1), which
still synthesizes a polar flagellum in broth, was used as a
"background" control for subtraction of numbers of cells that
adhered to glass capillary tube nonspecifically. Assays were carried
out for 1 h at room temperature instead of at 42°C. The response
to serine is similarly reported as the ratio of the number of cells
entering a 1-µl capillary tube with serine to the number of cells
entering the tube with buffer only (26).
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RESULTS |
Mini-Tn5 transposon mutagenesis of R. centenum swarm cells.
Mini-Tn5 transposition
mutagenesis was used to study loci involved in light-directed
colony motility. We chose a mini-Tn5 transposon since it
exhibits a relatively high transposition frequency in diverse
gram-negative bacteria and integrates into the host chromosome with
little sequence specificity (7). Interrupting alleles with
an antibiotic resistance marker also facilitates cloning and sequence
characterization of genes by direct selection of the transposon. We
used a derivative of mini-Tn5 that contains the omega
spectinomycin interposon (Sp) cassette, since earlier experiments (24) indicated that spontaneous Spr
occurs infrequently in R. centenum, and it had
previously been useful in the isolation of photosynthesis mutants of
R. centenum (46). Southern blot analysis of a
number of strains that were derived from independent transposition
events have indicated that the transposon integrated as a single event
in random locations (24, 39).
We screened for mutants that were defective in photosensory behavior by
using two approaches (Fig. 1). With a
macroscopic approach, we assayed colonies for defects in the ability to
move toward or away from light. As discussed in detail below, this assay yielded photosensory mutants, as well as mutants exhibiting altered swarm cell behavior, such as those that migrated slower or
faster or showed irregular swarm colony morphology. In a separate assay, which we term the microscopic approach, we directly observed the motility responses of individual cells in swarm colonies by using a
microscope equipped with a 40× objective lens. As discussed above,
when liquid-grown photosynthetic bacteria encounter a sudden reduction
in light intensity, they exhibit a characteristic scotophobic response
observed as a reversal, stop, or tumble. Microscopic observation of
R. centenum swarm cell colonies on an agar plate indicated
that virtually all of the cells in the colony are highly motile and
that they also exhibit a scotophobic response. Specifically, when swarm
cells were challenged with a step down in light intensity, we observed
a consistent 3- to 5-s freeze of all cell movement in the colony,
followed by resumption of movement at a lower rate of motility. We
interpret the light-induced pause of motility to be the result of a
tumbling response. This response is followed by resumption of smooth
swimming at a lower speed, a photokinetic effect. Thus, by direct
microscopic inspection of swarm cell responses to alterations in light,
we are able to screen for mutants altered in photosensory behavior, as
well as for mutants that display a general defect in swarm cell
motility. The microscopic approach proved to be less labor intensive
than direct observation of colony migration across an agar surface, and
thus, the former assay was used for most of our screens. Overall,
approximately 1,300 colonies were assayed for defects in
light-driven motility by the macroscopic approach and
approximately 22,000 colonies were assayed by the microscopic approach.
The frequency of transposition events that produced some form of
aberrant response to light was a surprisingly high 1 in 20 assayed
colonies. Assuming that R. centenum has a typical genome size of ~4,000 genes, this frequency indicates that as many as 200 genes may be involved in the swarm cell response to light. Although
this appears to be a large number of loci, the results below
demonstrate that disruption of any number of genes involved in swim
cell to swarm cell differentiation, synthesis of a functional bacterial
photosystem, signal transduction events, or light perception results in
observable defects in photosensory behavior (Fig. 1 and Table 1 show
the categories of mutants that have been obtained in our screens).
Consequently, the challenge is not to obtain mutants that do not
respond to light but to place them correctly into appropriate
categories. Below are detailed phenotypic descriptions of mutants that
have been obtained.
Photosynthesis mutants.
One category of mutants that was
isolated early in our study included mutants with disrupted
photosynthetic growth capability. Like most nonsulfur purple
photosynthetic bacteria, R. centenum is capable of
heterotrophic growth aerobically in the dark, as well as photosynthetic
growth under anaerobic conditions. However, this species also exhibits
the unusual capability of synthesizing a functional photosystem, as
well as exhibiting photosensory behavior, when growing aerobically as a
heterotroph (46). To facilitate genetic manipulation, our
initial screens involved selection for Spr transconjugants
under heterotrophic conditions prior to assaying for defects in
photosensory behavior. Under these conditions, a large fraction of
transconjugants that were observed to be defective in photosensing
actually had mutations that disrupted photosynthetic growth
capabilities (Table 1). Included were numerous pigment biosynthesis
mutants that are deficient in the synthesis of bacteriochlorophyll (strain ZJPM1) and mutants that fail to synthesize a functional reaction center (B800) (strain ZJA4-28). Sequence analysis indicates that the photosynthesis-defective mutant ZJB9-3 has a Tn5
insertion in the pet operon, which codes for structural
polypeptides of the cytochrome bc1 complex
(24). This indicates that both the infrared- and
visible-light responses measure some component of photosynthesis-driven
electron transport. To reduce the background frequency of
photosynthesis mutants, our later screens for photosensory mutants
incorporated strict anaerobic photosynthetic growth conditions during
drug selection of the transposon.
Flagellar-biosynthesis mutants.
A second large class of
mutants was those that are defective in solid-surface motility, which
we have termed SFr mutants for surface-frozen mutants. To our surprise,
about 1 in every 25 transconjugants exhibited no solid-surface
motility, implying that a large number of genes are involved in this
process. SFr mutants could potentially have such diverse functional
defects as the inability to induce swarm cell differentiation,
disruption in lateral flagellar synthesis or assembly, a defect in
lateral flagellar rotation, or a defect in the production of a surface
lubricant.
We performed detailed analysis of approximately 160 SFr mutants from
our collection to categorize potential defects (Table 1). The mutants
were assayed for motility when grown in liquid medium and examined for
the presence or absence of polar and lateral flagella by Western blot
analysis with a polyclonal antiserum that cross-reacts with both
flagellar types (Fig. 2). We observed that, similar to Vibrio parahaemolyticus (1, 31,
32), wild-type R. centenum cells synthesize different
flagellar types. Specifically, wild-type swim cells synthesize a polar
flagellum comprising a 58-kDa flagellin subunit (Fig. 2A, lane 1),
whereas wild-type swarm cells synthesize the same polar flagellum, as
well as a large number of additional lateral flagella comprising 32-kDa flagellin subunits (Fig. 2B, lane 1). Thus, synthesis of the polar flagellum is constitutive, irrespective of the presence or absence of
surface-induced lateral flagella. The various mutants that are
defective in surface motility can be grouped into the following six
classes. One class (46 isolates), represented by Western blot analysis
of strains ZJH3-9 and ZJH3-26 (Fig. 2, lanes 2 and 3, respectively),
shows the normal polar-flagellum pattern in liquid medium and
agar-solidified medium, as well as normal surface-induced lateral-flagellum synthesis. Motility is observed in liquid medium, indicating normal rotation of the polar flagellum, and yet this class
of mutants is completely defective in solid-surface motility. A loss of
surface motility can arise as a consequence of a defect in
lateral-flagellar rotation or synthesis of surface surfactants such as
exopolysaccharides (18, 45) or serrawettin-like substances (28).
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FIG. 2.
Western blot analyses of the flagellar types from
nonswarming mutants. (A) Flagellar samples collected from cells grown
in liquid medium. (B) Flagellar samples collected from cells grown on
0.8% agar plates. Lanes: 1, wild-type R. centenum; 2, ZJH3-9; 3, ZJH3-26; 4, ZJF6-27; 5, ZJH8-3; 6, YB280; 7, ZJP246; 8, ZJG8-11. Protein molecular size markers are labelled on the left.
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The second class of mutants (23 isolates), represented by mutant ZJG9-9
(Table 1), synthesizes quantitatively fewer lateral flagella than do
wild-type cells, indicating that the mutants may harbor defects in the
induction of lateral-flagellar synthesis (24).
The third class (35 isolates), represented by mutants ZJP246 and
ZJG8-11 (Fig. 2, lanes 7 and 8, respectively; Table 1), fail to
synthesize a polar flagellum under our tested condition. Interestingly,
these mutants still exhibit normal surface-dependent induction of
lateral-flagellum synthesis, indicating that induction of
lateral-flagellum synthesis does not involve the presence of a polar
flagellum. Normal regulation of lateral-flagellum synthesis in these
mutants is distinctly different from that reported for polar-flagellum
mutants of V. parahaemolyticus, which exhibit a phenotype of
constitutive synthesis of lateral flagella (31). Despite the
fact that these mutants still induce lateral-flagellum synthesis,
polar-flagellum mutants exhibit a defect in surface motility.
The fourth class of mutants, represented by three isolates, ZJJ8-7,
ZJN608, and ZJP1310 (Table 1), synthesizes a polar flagellum that is
defective in rotation (synthesis of a polar flagellum was determined by
Western blot and flagellar-stain analyses, and lack of rotation was
determined by the observed lack of motility of liquid-grown swim
cells). As in the case of cells which lack synthesis of a polar
flagellum, these mutants are also defective in surface motility.
The fifth class of mutants (35 isolates), represented by strains
ZJF6-27 and ZJH8-3 (Fig. 2, lanes 4 and 5; Table 1), fails to
synthesize either lateral or polar flagella. This phenotype indicates
that the polar- and lateral-flagellum types must have some common
regulatory or structural components.
The sixth class (18 isolates), represented by mutant YB280 (Fig. 2,
lane 6; Table 1), has normal polar-flagellum synthesis and normal
liquid motility but no surface-induced lateral-flagellum synthesis. This class either has mutations that disrupt structural components of the lateral flagellum or defects in regulatory circuits that are responsible for surface-induced lateral-flagellum synthesis.
Signal transduction (che) mutants.
From our
screen, we isolated 17 mutants, represented in Table 1 by strain
ZJP1177, that exhibit normal photosynthetic growth capabilities, as
well as normal induction of lateral flagella on a solid surface.
Microscopic analysis indicated that swarm cells of this mutant class
are actively motile on an agar surface; however, unlike wild-type
cells, they fail to exhibit the characteristic freeze of movement when
given a step down in light intensity. Swim cells from a majority of
these mutants displayed a biased smooth-swimming pattern and also
failed to exhibit the scotophobic tumbling response when challenged
with a step down in light intensity. Macroscopically, colonies of these
mutants also failed to migrate toward or away from light. The
phenotypes manifested by these mutants are virtually identical to those
of smooth-swimming chemotaxis mutants that we previously described
(26). Southern blot analysis using a che gene
probe subsequently demonstrated that 12 of the 17 isolates contained a
mini-Tn5 insertion in the che gene cluster (Fig.
3, mutants ZJP1177, ZJP1315, ZJA8-25,
ZJC2-35, ZJC2-33, ZJC4-20, ZJE6-29, ZJE7-17, ZJI3-1, ZJJ5-26, ZJJ6-5,
and ZJJ8-2). The remaining five isolates, which do not contain a
mini-Tn5 insertion in the che gene cluster, are
discussed in the section below.
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FIG. 3.
Southern blot analysis of genomic DNAs from scotophobic
and phototactic mutants by probing with an SmaI fragment of
the R. centenum chemotaxis gene cluster. (A) Diagram of the
che gene cluster and flanking sequences along with relevant
restriction sites. (B) Genomic DNA samples digested with
ApaI. (C) Genomic DNA samples digested with SmaI.
Lengths of DNA markers and fragments are marked in kilobase pairs on
the left. W.T., wild-type R. centenum sample. ZJP1177,
ZJP1315, ZJA8-25, ZJC2-35, ZJC2-33, ZJC4-20, ZJE6-29, ZJE7-17, ZJF6-4,
ZJF7-13, ZJH7-24, ZJI3-1, ZJJ5-26, ZJJ6-5, ZJO3-35, YB300-3, and ZJJ8-2
are samples from the corresponding mutant strains. The loading order in
panel C is identical to that in panel B.
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Photoperception mutants.
As discussed above, five isolates
(Fig. 3, strains ZJF6-4, ZJF7-13, ZJF7-24, ZJO3-35, and YB300-3) were
obtained which exhibited defects in the scotophobic response when
challenged with a step down in infrared light intensity similar to that
observed with che mutants (Table 1). One important
observation provided by Southern analysis is that these mutants contain
an intact che operon, which was characterized by us
previously (25, 26). Furthermore, chemotaxis to serine (Fig.
4) or pyruvate (data not shown) indicated
that, with the exception of YB300-3, which has a general growth defect,
these mutants are not defective in chemotaxis per se, compared to the
che gene cluster deletion mutant (25). As such,
each of the mutations potentially disrupts components of the light
perception and/or light signal transduction pathway or down regulates
the functions of these components. The results of light-driven colony
motility assays of these five mutants (Fig. 5) demonstrated that mutants ZJF6-4 and
ZJO3-35 are defective in both infrared- and visible-light phototactic
responses; mutant ZJF7-13 displays no infrared-light response;
mutant ZJH7-24 has attenuated infrared- and visible-light
responses (particularly the infrared-light response); and mutant
YB300-3 has a diminished visible-light response and a completely
defective infrared-light response.
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FIG. 4.
Chemotaxis capillary assays for photoperception mutants.
The vertical axis represents the relative ratio (see Materials and
Methods). Columns: a, wild-type R. centenum strain; b,
ZJF6-4; c, ZJF7-13; d, ZJH7-24; e, ZJO3-35; f, R. centenum
che mutant (26).
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FIG. 5.
Phototactic colony migration assays of isolated mutants.
(A) Positive swarm colony phototaxis toward an infrared-light source.
(B) Negative phototaxis away from a visible-light source. Lanes: w.t.,
wild-type R. centenum; 1, ZJF6-4; 2, ZJF7-13; 3, ZJH7-24; 4, ZJO3-35; 5, YB300-3; 6, che mutant.
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We also obtained an interesting mutant, YB72, which exhibits a normal
scotophobic response as measured microscopically. However, light-directed colony migration was not observed (39).
Colony morphology mutants.
Another class of mutants exhibited
pleiotropic effects on swarm colony morphology. These mutants do not
appear to have defects in light perception per se but, instead, display
altered light-driven colony motility as a result of aberrant swarm cell
behavior. One frequently obtained type of morphology mutants is
hyperswarmers, represented by strain YB600-1 in Fig.
6. Hyperswarmer mutants quickly spread
across the surface of the agar medium, in contrast to the more discrete
colony formation exhibited by the wild-type strain (Fig. 6). Variations
exist among this class of mutants, with some strains forming very thin
layers of cells and others forming thick layers. Additional isolates
form ruffled edges rather than smooth edges. Another type of morphology
mutants, which we categorize as superdrivers, is represented by mutants
BR2-68 and BR2-39. BR2-68 exhibits consistently faster colony motility
in response to both visible light and infrared light, whereas BR2-39 has a reduced response to infrared light and an enhanced response to
visible light (39).
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FIG. 6.
Swarming morphology of wild-type R. centenum
versus that of hyperswarming mutant YB600-1. The plates were spotted
with equal numbers of cells and incubated in the dark for 40 h at
42°C on PYVS-0.8% agar plates. (A) Wild-type R. centenum. (B) Transposon mutant YB600-1.
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DISCUSSION |
This study demonstrates that light-directed swarm colony motility
is a very complex process involving numerous (possibly 200) genetic
loci. They include genes involved in swarm cell differentiation, synthesis of the lateral flagella, and synthesis of a functional photosystem, as well as those involved in light perception and sensory
transduction. As shown by our results, it is relatively easy to obtain
mutants that exhibit defects in light-driven swarm colony motility. The
difficulty is in properly analyzing and classifying the numerous
mutants that are obtained. Despite these hurdles, we were successful in
obtaining and characterizing a number of strains that give us a unique
perspective on the complexities involved in R. centenum
swarm cell differentiation and how these cells perceive and respond to
light signals.
Motility-defective isolates providing insights into swarm cell
differentiation.
Even though surface-dependent swarm cell
differentiation is known to occur in many species (22, 23),
little is known about the mechanisms used to sense a solid surface. The
best-understood example is V. parahaemolyticus, which has a
pattern of swarm cell differentiation very similar to that of R. centenum (31, 32). As observed with R. centenum, V. parahaemolyticus swim cells have a single,
sheathed polar flagellum, whereas swarm cells contain additional,
numerous, unsheathed lateral flagella. Genetic studies of V. parahaemolyticus have demonstrated that loss of polar-flagellum synthesis results in constitutive (liquid and solid) synthesis of
lateral flagella (31). This observation and other
experimental results have led to a model proposing that impeded
movement of the polar flagellum generates a signal that leads to
induction of lateral-flagellum synthesis. Transmission of a signal
regarding polar-flagellum rotation appears to involve the
che gene products, since che mutants of V. parahaemolyticus are deficient in the induction of
lateral-flagellum synthesis (41). Our mutational analysis of
R. centenum clearly indicates that swarm cell
differentiation involves a distinctly different mechanism. This is
evidenced by the isolation of mutants that fail to synthesize a polar
flagellum but which are still capable of undergoing normal swim cell to swarm cell differentiation when placed on a solid surface. We have also
observed that disruption of the che operon has no effect on
swarm cell differentiation (26). Thus, it appears that
R. centenum has a mechanism of sensing a solid surface that
does not involve synthesis or rotation of the polar flagellum. Indeed, some mutants in our collection that fail to synthesize lateral flagella
could potentially contain defects in a surface sensor receptor or in
transmission of a signal from a surface receptor. Additional sequence
and expression analysis of this class of mutations needs to be
undertaken to further subgroup these mutants into those that contain
defects in structural components of the lateral flagellum versus those
that contain defects in the regulation of the induction of
lateral-flagellum synthesis.
No mutants of V. parahaemolyticus missing both the polar and
lateral flagella have been isolated (29, 30, 32, 33). In
contrast, we have obtained numerous mutants of R. centenum that lack both flagellar types (Table 1). This phenotype suggests that
lateral and polar flagella in R. centenum have structural and/or regulatory factors in common (such as a commonly utilized sigma
factor). Another possibility is that lateral- and polar-flagellum genes
are cotranscribed in one or more large operons. If so, integration of
the mini-Tn5 transposon, which contains flanking
transcription termination sites, would produce polarity effects on the
synthesis of both flagellar types. At this early stage of our
structural analyses, we do know (24, 39) that at least two
structural components of the lateral flagella are not utilized by the
polar flagellum. Specifically, lateral and polar flagella have separate flagellin polypeptides, as well as different subunits of FlgI, which is
a component of the P-ring of the flagellar basal body. Future sequence
analysis of loci that are disrupted in mutants that fail to synthesize
one or both flagellar types should sort out whether or not common
subunits and/or regulatory factors are, indeed, shared.
Another surprising observation is that a number of polar-flagellum
mutants were isolated that are defective in lateral-flagellum rotation.
This suggests that these two flagellar types have motor or switch
subunits in common. An argument against common motor or switch
components is that we have isolated mutants that are defective in
lateral-flagellum rotation but have normal polar-flagellum rotation.
Thus, even more complex scenarios must be considered, such as
communication of polar-flagellum rotation to the lateral flagella. For
example, perhaps rotation of the polar flagellum is needed as a
nucleation point for the formation of a lateral-flagellar bundle. It
should also be noted that since we screened for cells that are
defective in surface motility, we do not know whether it is
possible to obtain mutants that are defective in polar-flagellum rotation but are still capable of surface motility. A separate screen
for nonmotile swim cells has to be undertaken to address this issue.
Finally, the involvement of the polar flagellum in swarm cell motility
has also been observed in V. parahaemolyticus, in which
mutations that disrupt polar-flagellum synthesis result in swarm cells
that exhibit aberrant "wobbly" surface motility (31).
This indicates that the presence of a polar flagellum is also important
for proper lateral-flagellum function in this species.
Photoperception and signal transduction.
In addition to
obtaining information on swarm cell differentiation, this study also
established that light-directed colony motility can be exploited to
obtain mutants that are specifically defective in photosensory
perception. To our knowledge, this investigation provides the first
extensive genetic screen for bacterial mutations that exhibit altered
photosensory behavior. From analysis of isolated mutants, it is evident
that part of the signal(s) for both positive and negative phototactic
responses involves light-driven photosynthetic electron transport.
Movement toward infrared light is most likely a response to increased
energy conversion caused by efficient utilization of infrared light to
drive photosynthesis (3, 13, 16, 20, 37). Movement away from
visible light is somewhat more complex, since visible light is also
utilized by the photosystem to drive photosynthesis. Thus, the negative
response must measure photosynthetic electron transport, as well as an
additional component of the light spectrum. The isolation of mutants
ZJF6-4 and ZJO3-35, which have normal chemotaxis but no visible- or
infrared-light phototaxis, indicates that the signal transduction
pathways for both responses converge. Presumably, these pathways
converge at a receptor or transducer that communicates with the
chemotaxis cascade, since a number of che gene mutations
were also obtained which affect phototaxis, as well as chemotaxis
(25, 26).
Morphology mutants.
Our ability to obtain colony morphology
mutants that are crippled in light-driven colony migration suggests
that cell-cell communication or production of a surface surfactant is a
factor in swarm motility. Multicellular cooperative behavior is
widespread in prokaryotes and is involved in processes such as fruiting
body formation in Myxococcus xanthus and swarming
differentiation. In swarming, perhaps the best-understood systems are
those of Proteus mirabilis (2, 6, 17) and
Serratia marcescens (11, 15, 35). Mutants of
these species which exhibit altered swarming patterns have been
isolated. Most of the morphology mutants that we obtained in this study
tend to form sheets of cells rapidly across the surfaces of the plates
rather than forming discrete colonies. Western blot and
flagellum-staining analyses suggest that the hyperswarmer mutants have
the normal complement of flagella per cell, so we suspect that this
phenotype could be caused by alterations in exopolysaccharide synthesis
or surface surfactant production (18, 28, 45). More
extensive macroscopic screening for colony morphology mutants can be
undertaken to address the nature and number of loci involved in
potential cell-cell communication events that are involved in
light-driven swarm colony motility.
This work was supported in part by a grant from the U.S. National
Science Foundation (IBN-9303836), as well as by a National Science
Foundation postdoctoral fellowship awarded to B.G.R.
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Abstract
Introduction
