Screening and Characterization of Phylloplane Bacteria Producing Vlbrio fucheri Autoinducer
!
Vol. 4, No. 2
Screening and Characterization of Phylloplane Bacteria
Producing Vlbriofucheri Autoinducer
ANTONIUS SUWANTO
-D
of Biobgv, Faad@ of Science and M a t h m d k , .
I
&
Reya Pqjqjrnan, Bogor 161U
Inter Uniwmi@Centerfor Me-,
Bqgor Agrimhral Uniwnity, Bogor 16680
Tdqkone: 251425965, TcScfa: 62-251-621724, ESnoiL. x d n # b @ n & . n d id
Wterima 26 November 1996IMsetujui 7 April 1997
Sixty luve blades representing 14 different plant species in the Woods Hole, Massachusetts, area were collected
for the irdatlon of phyllopl.ne bacteria employing a leaf-print method on 10% King's B medium supplemented with
100 p g h l cyclohesimldc. Colonies that appeared after 2 3 days were overlaid with Laria Agar (pH7.4) containing
approximately 10' cdlslml of BckaicMo cod carrying Vibriofirckeri h
u operon with a 250-base pair non-polar
deletkm hr W. Presumptive autoinducer-producing bacteria were isolated from the spots of biolnminescens in the
overlaid plates. These kinds of phylloplme bacteria represented approximately less than two percent of the total
Ieaf-sulrce bacteria Based on preliminary observation of cdony morphology and limited physiologkd assays, they
1
were two main groups of phylloplane bacteria belonltiag to either Xuntkmona sp. or A b n 4 ~qvhgw,
Accenlda(lty, thb method bas the potential to be developed into a rapid and reliable diqphostic test, to study the
distribution u well u relative abundmre of certain strains or p a t h n of phytloplane bacteria.
INTRODUCTION
Autoinduction is an environment sensing-system that
allow8 bacteria to monitor their own population density,
which is atso wined as "quorum sensing" by some authors
(Fa~uaet al. 1994). This system requires a difhiile compoundpmdwxdbythecomxpondingtmterb, whichcan
acamrolate in the smrounding environment during growth.
Thie difhiile quonnn-mnsing compannd is called autoinducer @qua et al. 1994).
Vtbrto jischeri autoinducer (VAI) is N-3-(oxohexanuyl) hbmoserine lactone which is required to activate
lmtairadaction of biohunincscence. VAI at a concentration
of 10 nM or approximately 40 molecules/cells is sufficient
to activate autoinduction in this bacterium. In the light organ, V: fischeri can reach a very high density concentration
as high as 10'O-lOLL
celWml. In seawater, however, K
j9*heri
is only found at approximately 10' celWml
@mtap & Greenberg 1991, Ruby & McFall-Ngai 1992).
There- fore, the autoinduction system might allow K
fischeri to discrixpinate between free-living (low celldensity) stab and host-asmsiated (high celldensity) state
(Fuqua et al. 1994).
Previously, it was thought that the autoinducer for K
fkheri was s p e c i e c , ie. other v i e s of luminous
bacteria neither responded to the pure molecule nor prodwd a ampound that stimulated luminescence by K pscheri. However* a large body of evidence showed that a
campaund similar to VAI is widely employed in many
mgdatoq systems in Pmtedwkda (Bainton et al. 1992,
Pithonen et al. 1993, Canamem & More 1993, Pearson et
al. 1994). One implication of this study, is the understanding that certain ban&
behavior can be performed efficiently only by a sufFiciently large population of bacteria.
The leaf surface is considered to be an inhospitable
envifunmcnt for microbial growth. Micfoorganisms which
inhabit leaves must be able to evolve mechanisms to wercome the stmdul or fluctuating environmental conditions
(O'Brien & Lindow 1989). Recently, it has been demonstrated that colonization and survival of Pseudomonas s y i ngae was dependent on the density of the inoculum (Wilson & Lindaw 1994). In addition, virulence determinants
of certain plant and animal-pathogenic bacteria have been
shown to be regulated by VAI (Jones et al. 1993).
This study was aimed to evaauate the potential use of
lux system o f Mbrio jscheri for screening and c h a k t a i zaticm of specific autoinducer-phyloplane
bacteria.
MATERIAL AND METHODS
Ekcherichia coli JM83 (pAK211) and JM83 (pAK203) were obtained from Paul Dunlap, Woods Hole
Oceanographic Institution, Wood Hole, Mass. Plasmid
pAK211 carries all lux genes of Vfbriofischeri, except for
the non-polar deletion of ltucl (gene for autoinducer synthesis), while pAK203 carries functional intact lux1 gene.
Pseudomonas syringae pv. tabaci strain 2024, .Erwinia
amylovora, and Xanthomonas campestris pv. glycines 8Ra
were obtained from Paul D. Shaw, Dept. of Plant Pathology, Univ. of Illinois, Urbana. Xanthomonas campestris
pv. glycines XP202 (=NCPPB 114I), X. campestris pv.
phaseoli strain XP34, XP28, and XP20 were obtained from
Steven E. Lindow, Dept. of Environment Science, Policy
and Management, Univ.of California, Berkeley.
Phylloplane of bacteria were isolated fKrm surface of
leaf-blades collected from around Woods Hole area. Leaves
collected belonged to the following species of trees or
shrubs: Rhododendron, Black Beech, Mint plant, Maple,
roses, Acacia, Alnus, Yellow Lily, White Lily, Morning
Glory (violet), as well as four kinds of grasses belong to
Graminea.
Vol. 4. 1997
AUTOJNDUCER IN PHYLLOPLANE BACTERIA
Each individual leaf was pressed onto lO?! King medium B agar (2 g Protease Peptone; 15 ml glycerol; 0.15 g
m 4 ; 0.15 g MgSO4.7%O; 20 g Bacto agar; 1L
distilled water; pH was adjusted to 7.4 by adding 1 N
NaOH) suplemented with 100 pglml of cycloheximide.
These leaf-print plates were incubated at 2528°C for 2-3
days. Three leaf blades were used for each different plant
species.
For the werlay method, E. coli JM83 (pAK211) was
grown in Luria Bertani broth (pH 7.4) supplemented with
35 glml of chloramphenicol, at 2528°C for 20-24 hr. Cells
were harvested by centrifugation at 5000 rpm for 5 min.
After washing in saline solution (0.9% NaCl), the cell
pellet was ~emqmdedin saline solution to approximately
10"' celldml. The cell suspension was mixed proportionally with liquid LA (5 g NaC1; 5 g yeast extract; 10 g
tqptme; 15 g agar; 1 L distilled water; pH 7.4; 50°C) to
yield a final concentration of approximately lo9 cells/ml
and a finalagar cxmcenttation at 1-1.2%. This mixture (4-5
ml per plate) was quickly poured onto laaf-print plates to
cwer all of the collonies. Spots of bioluminescence were
observed in the dark room after an 8-18 hr incubation. The
pntativc autoinducer colonies were qmified by streaking
each of them, side by side, with E. coli JM83 (pAK211) on
LA plates.
The autoinducer-producing strains were characterized
further,employing limited morphological and physiological assays, such as: characteristics of colonies, cell morphology, Gram-stain,catalase, oxidase activity, ability to
grow anaembically, as well as the ability to metabolize
certain kinds of sugars (Smibert & Krieg 1994).
Scanning Electron Microscopy (SEM) was conducted
as described by Behmlander & Dworkin (1991) and
Watson et al. (1980). Fluorescence in-situ hybridization
(FISH)was essentially performed as described by BraunHowland et al. (1993).
RESULTS
The density of bacteria that occupied the leaf surface
was determined to be approximately 22-300 celldg of fhsh
leaf. AU of the phylloplane isolates could be classified into
1-7 distinct groups depending on colony morphology (ie.
color, appcamok, elevation, margin, and texture) on 1 W
King's Medium B agar.
From approximately 80 individual colonies, it was
found that isolates WP1 (WP = Woods Hole Phylloplane),
WP18, and WP19 generated bioluminescence signals comparable to that of the positive control, ie. JM83 (pAK21I),
which was streaked besides JM83 (pAK203). Isolates
WP8, WP14, and WP 17 yielded a relatively weak signal of
bioluminwce. The rest of the colonies did not show any
bioluminescence when they were streaked besides JM83
(pAK211). Isolates WP20U did not produce lux1 complementation, despite its identical colony morphology to WP1.
It would be interesting to see the degree of similarity of
lhw twa isolates (WP1 and WPZOU) at the genomic level.
Although the werlay assay was performed for all of
the leaf-print isolates, only seven luminescence spots were
39
obseIV8d. Reisolation and morphological characterizations
showed that these seven isolated are belonged to either of
two distinct groups (ie. WP1 and WP18) which were described in Table 1.
Based on colony morphology and limited physiological characteristics, isolate WPl might be assigned in
the genus Xanthomonus. In addition, FISH analyses also
indicated that this isolate did not belong to Enterobacteriaceae, where the genus of Envinia belongs to.
Therefore, the identity of the WP1 isolate needs further
c M c a t i o n employing more rigoroy physiology or
genetic analyses.
Isolate WP18 seems to be a strain of Pseudomonas
syringae, due to its obligate aerobic metabolism and its
negative result in the oxidase test. It is interesting to note
that from all plant pathogenic bacteria, included in this
study, only P. syringae pv. tabaci yielded compound that
could substi~ethe K fischerf autoinducer. Envinia
amylovora yielded only a weak bioluminescence signal in
this lux assay.
Isolates WP14 and WP20U showed similar colony
morphology to that of WPl. These isolates were also facuttative anaerobes. However, the lux assay indicated that
these isolates did not produce the K fischeri autoinducer.
All Bacillus species obtained from leaf surface in this
experiment were not able to wmplement the K fischeri
autoinducer.
DISCUSSION
Phylloplane bacteria, which can substitute the K
fischeri autoinducer (VAI), were present in low abundance
on a healthy leaf surfkx, and these can be assigned to only
two distinct p u p s as described above. Therefore, this
method might be very useful for the development of a rapid
Table 1. Two main groups, of bacteria, obtained with the overlay
method.
Isolates
Charaderistics
-
WPl
Yellow with cupious of slime on 10% King's B,
bright yellow on LA (pH 7.4), gram-negative, catalase
positive, oxidase negative, rods, motile, facultative
anaerdbe. Scanning electron microscopy (SEM) demonstrated the presence of fibrous materiril (probably
polysaccharide) interconnec*
among the cells of
this bacterium (Fig. 1). Flourescence in situ hybridization (FISH) analyses showed that this isolate did
not appear to be a member of either Enterohteriaceae, a or p - group of Proteobacteria (Waese,
1987).
WP18 White, rather mucoid on 10% Kmg's B or LA (pH
7.4), gram negative, catalase positive, oxidase
negative, rods, motile, obligate aerobe. SEM did not
show fibrous materials on the cell surface as in WPI.
FISH analyses showed identical results as in isolate
WPl.
Braun-Howland, E.B., P A Vesdo & S.A. NierdckiBauer. 1993. Use of a simpli6ed d
l blot t d m i q ~ ~
and 16s rRNAdinxted probes for idedicatiun of
common envhmental isolates. J. Appl. Ihviron.
Microbiol. 59: 3219-3224.
Canamero, M.M. & M.L More. 1993. AutoMwa
~ s t r a i n S f r o m ~
Course in MiDiversity, Marlm Biological
Labol;lsory, Woods Hole. (Unpublished).
Dunlap, P.V. & E.P. G n c n b v g 1991. Role of
htmwlluJar chemical communication in the Vibrio
fischeri-mmwatsid fish symbiosis, p. 219-253. In.
M. Dworkin (ed.), M i d i a l Cell-Cell intc~acti0)ts.
Washhgkm D.C.: American Soddy for M i M o g g .
hqm, w.c., ac. Winrar & P. G1994.
QU0nrmsensingin~:thcLuxR-LuxI-of
P i 1. SEM of isolate WPl shuwed the pmmce of atuuiw
cell dcnsity-ll%?po*
d
rcgulaton. J.
fitnow metairrls -ni
among the bactaial
Bocteriol, 176: 269-275.
Ceus. This f i h materiats rmght be polyseccharde
Jones, S., B. Yu, N.J. Brinton, M. Birdsall, B.W.
which was mponsible for the excessive mucoid appearBycraB, SR Chb.bra, IW.R Cox, P. W y , P.J.
ance of the colloniea grown on 10% King's B medium.
Reevu, S. Stephem, MIL Wilmn, G.P.C. Sahmond,
G.S.A.B.
Steward & P. WillLMI 1993. 'Ib lux
strain or pathwar identification in phytobacterology.
antoinduoer mguhs the probdon of exocaryme
To date, pathwar or strain iduuifl@tion for most plant
virulence dcttrminants in Envhia c m t o y o m md
pathogenic backria involve a timeconwrming and laboPseudomonar omrgfnosa EMBO J. 12: 247702482.
rioustest, as well as apathogenkitynnsay~partidar
O'Brien, RD. & Sk L1.darr. 1989. Effect of plant
plants. The -y
t
of a rapid but reliable d@nostic
speaesaademmanmemalconditionon~
testforthesebacteria,will~facili~boththebasic
popdation sizes o f P s e u h n m mngae and a
study of pathogenicity and practical applications for early
bact&a. PhytqWblogy. Tk 619627.
detection of a disease agent in agiicdtm.
Although this bioluminescence system is very sensi- Pearwon, PJ., KM. Gray, L. Pu#dor, KD. Trkcr,A.
Eberhard, B.E
I#emki & P. Grcmkq
tive,itshouldbecanductedandobservedinitsoptimal
1994. Stnlcture of thc auWh&mr mpbd tbr
light emission. On LA (pH7.4), b i o h m h e s m a was amexpredon of Pseudomona rrcnginosa vimlmcc
venicntly examined af€er an 8-24 hr incubation at 2527°C.
genes. A.oc. Natl. Acad Sci. USA 91: 197-201.
very early examhation or ptolonged kubation produced
Pirhmen, NJ., D. Fkgo,R HegEhrLeharo & ET. Prlva,
*negative
results. Light produdon might be kept
1993. A small d@bsible signal molecule is laspolusiblc
longerbyincreasingbuffer~tyofthegrowthmedia
for the global control of videncc and emawpe
In addition, the overlay agar should be as thin as possiile
production in the plant pathogen Envinia carutovoru
(not more than 3 mm), to allow as much oxygen supply to
EMBO J. 12: 2467-2476.
thecultureunderneathitduringtheluminescenoeassay.
Ruby, E.G. Bt, l4l.J. McFall-Ngsi. 1992. A squid that
ACKNOmEDGEMENT
glows at night: dmlopmcnt of an animal-bacmid
mutualism. J Bocteriol. 171: 48654870.
This research was conducted in Marine Biological
Smibert, RM. & N.R
1994. Phenotypic
Laboratory, Woods Hole, Mass., USA. I thank Professo:
c
o
n
,
p.
607654.
In
P.
Gerhdt, RG.E.
h4artiu Dworkin and P r o f m r John Breznak for thei.
M
u
r
r
a
y
,
W
.
Wood
&
N.R
Krieg
(ed.).Methodsfor
support and encouragement in conducting this research.
General and Molecular Bacteriology. WWashingtan
The cost of travel and aocornodation during the Summer
D.C.:American Society for Microbiolo~.
Course was granted by
Zeiss, Inc. to AS through
Watson, LP., A.E. M c b & B.R Meme& 1980.
Marine Biological Laboratoxy.
Preparation of Microbiological specimens for
REFERENCES
Scanning Elecfron Microscopy. Chicago: Scanniog
ElM-i
Inc., AMF O'Hare.
Bainton, N.J., B.W. Bycott, S.R Chhabn, P. Stead, L.
Wilson, M. & S.E. Lindm. 1994. Inocuhnn densityGledhUl, P.J. Hill, C.E.D. Rces, M.K Winson,
clependent mortality and colonization of the phyllosG.P.C. Salmond, G.S.A.B. Steward & P. Williams.
by Pseudomonas syringae. Appl. Environ.
phere
1992. A general role for the lux autoinducer in bacteMicrobiol.
60: 2232-2237.
rial cell s
i
m
:Control af antiiotic synthcsls in
Wocre,
C.R
1987.
Bacterial E?volution. Micmbiol. Rev.
h i n i a . Gene 116: 87-91.
51:221-271.
Bchmlander, RM. & M. Dworkin. 1991. Extracellular
fibrils and contact-mediakd cell htmactions in
Myxococcus xanthus. J. Bacterial. 173: 7810-7821.
a
~
Vol. 4, No. 2
Screening and Characterization of Phylloplane Bacteria
Producing Vlbriofucheri Autoinducer
ANTONIUS SUWANTO
-D
of Biobgv, Faad@ of Science and M a t h m d k , .
I
&
Reya Pqjqjrnan, Bogor 161U
Inter Uniwmi@Centerfor Me-,
Bqgor Agrimhral Uniwnity, Bogor 16680
Tdqkone: 251425965, TcScfa: 62-251-621724, ESnoiL. x d n # b @ n & . n d id
Wterima 26 November 1996IMsetujui 7 April 1997
Sixty luve blades representing 14 different plant species in the Woods Hole, Massachusetts, area were collected
for the irdatlon of phyllopl.ne bacteria employing a leaf-print method on 10% King's B medium supplemented with
100 p g h l cyclohesimldc. Colonies that appeared after 2 3 days were overlaid with Laria Agar (pH7.4) containing
approximately 10' cdlslml of BckaicMo cod carrying Vibriofirckeri h
u operon with a 250-base pair non-polar
deletkm hr W. Presumptive autoinducer-producing bacteria were isolated from the spots of biolnminescens in the
overlaid plates. These kinds of phylloplme bacteria represented approximately less than two percent of the total
Ieaf-sulrce bacteria Based on preliminary observation of cdony morphology and limited physiologkd assays, they
1
were two main groups of phylloplane bacteria belonltiag to either Xuntkmona sp. or A b n 4 ~qvhgw,
Accenlda(lty, thb method bas the potential to be developed into a rapid and reliable diqphostic test, to study the
distribution u well u relative abundmre of certain strains or p a t h n of phytloplane bacteria.
INTRODUCTION
Autoinduction is an environment sensing-system that
allow8 bacteria to monitor their own population density,
which is atso wined as "quorum sensing" by some authors
(Fa~uaet al. 1994). This system requires a difhiile compoundpmdwxdbythecomxpondingtmterb, whichcan
acamrolate in the smrounding environment during growth.
Thie difhiile quonnn-mnsing compannd is called autoinducer @qua et al. 1994).
Vtbrto jischeri autoinducer (VAI) is N-3-(oxohexanuyl) hbmoserine lactone which is required to activate
lmtairadaction of biohunincscence. VAI at a concentration
of 10 nM or approximately 40 molecules/cells is sufficient
to activate autoinduction in this bacterium. In the light organ, V: fischeri can reach a very high density concentration
as high as 10'O-lOLL
celWml. In seawater, however, K
j9*heri
is only found at approximately 10' celWml
@mtap & Greenberg 1991, Ruby & McFall-Ngai 1992).
There- fore, the autoinduction system might allow K
fischeri to discrixpinate between free-living (low celldensity) stab and host-asmsiated (high celldensity) state
(Fuqua et al. 1994).
Previously, it was thought that the autoinducer for K
fkheri was s p e c i e c , ie. other v i e s of luminous
bacteria neither responded to the pure molecule nor prodwd a ampound that stimulated luminescence by K pscheri. However* a large body of evidence showed that a
campaund similar to VAI is widely employed in many
mgdatoq systems in Pmtedwkda (Bainton et al. 1992,
Pithonen et al. 1993, Canamem & More 1993, Pearson et
al. 1994). One implication of this study, is the understanding that certain ban&
behavior can be performed efficiently only by a sufFiciently large population of bacteria.
The leaf surface is considered to be an inhospitable
envifunmcnt for microbial growth. Micfoorganisms which
inhabit leaves must be able to evolve mechanisms to wercome the stmdul or fluctuating environmental conditions
(O'Brien & Lindow 1989). Recently, it has been demonstrated that colonization and survival of Pseudomonas s y i ngae was dependent on the density of the inoculum (Wilson & Lindaw 1994). In addition, virulence determinants
of certain plant and animal-pathogenic bacteria have been
shown to be regulated by VAI (Jones et al. 1993).
This study was aimed to evaauate the potential use of
lux system o f Mbrio jscheri for screening and c h a k t a i zaticm of specific autoinducer-phyloplane
bacteria.
MATERIAL AND METHODS
Ekcherichia coli JM83 (pAK211) and JM83 (pAK203) were obtained from Paul Dunlap, Woods Hole
Oceanographic Institution, Wood Hole, Mass. Plasmid
pAK211 carries all lux genes of Vfbriofischeri, except for
the non-polar deletion of ltucl (gene for autoinducer synthesis), while pAK203 carries functional intact lux1 gene.
Pseudomonas syringae pv. tabaci strain 2024, .Erwinia
amylovora, and Xanthomonas campestris pv. glycines 8Ra
were obtained from Paul D. Shaw, Dept. of Plant Pathology, Univ. of Illinois, Urbana. Xanthomonas campestris
pv. glycines XP202 (=NCPPB 114I), X. campestris pv.
phaseoli strain XP34, XP28, and XP20 were obtained from
Steven E. Lindow, Dept. of Environment Science, Policy
and Management, Univ.of California, Berkeley.
Phylloplane of bacteria were isolated fKrm surface of
leaf-blades collected from around Woods Hole area. Leaves
collected belonged to the following species of trees or
shrubs: Rhododendron, Black Beech, Mint plant, Maple,
roses, Acacia, Alnus, Yellow Lily, White Lily, Morning
Glory (violet), as well as four kinds of grasses belong to
Graminea.
Vol. 4. 1997
AUTOJNDUCER IN PHYLLOPLANE BACTERIA
Each individual leaf was pressed onto lO?! King medium B agar (2 g Protease Peptone; 15 ml glycerol; 0.15 g
m 4 ; 0.15 g MgSO4.7%O; 20 g Bacto agar; 1L
distilled water; pH was adjusted to 7.4 by adding 1 N
NaOH) suplemented with 100 pglml of cycloheximide.
These leaf-print plates were incubated at 2528°C for 2-3
days. Three leaf blades were used for each different plant
species.
For the werlay method, E. coli JM83 (pAK211) was
grown in Luria Bertani broth (pH 7.4) supplemented with
35 glml of chloramphenicol, at 2528°C for 20-24 hr. Cells
were harvested by centrifugation at 5000 rpm for 5 min.
After washing in saline solution (0.9% NaCl), the cell
pellet was ~emqmdedin saline solution to approximately
10"' celldml. The cell suspension was mixed proportionally with liquid LA (5 g NaC1; 5 g yeast extract; 10 g
tqptme; 15 g agar; 1 L distilled water; pH 7.4; 50°C) to
yield a final concentration of approximately lo9 cells/ml
and a finalagar cxmcenttation at 1-1.2%. This mixture (4-5
ml per plate) was quickly poured onto laaf-print plates to
cwer all of the collonies. Spots of bioluminescence were
observed in the dark room after an 8-18 hr incubation. The
pntativc autoinducer colonies were qmified by streaking
each of them, side by side, with E. coli JM83 (pAK211) on
LA plates.
The autoinducer-producing strains were characterized
further,employing limited morphological and physiological assays, such as: characteristics of colonies, cell morphology, Gram-stain,catalase, oxidase activity, ability to
grow anaembically, as well as the ability to metabolize
certain kinds of sugars (Smibert & Krieg 1994).
Scanning Electron Microscopy (SEM) was conducted
as described by Behmlander & Dworkin (1991) and
Watson et al. (1980). Fluorescence in-situ hybridization
(FISH)was essentially performed as described by BraunHowland et al. (1993).
RESULTS
The density of bacteria that occupied the leaf surface
was determined to be approximately 22-300 celldg of fhsh
leaf. AU of the phylloplane isolates could be classified into
1-7 distinct groups depending on colony morphology (ie.
color, appcamok, elevation, margin, and texture) on 1 W
King's Medium B agar.
From approximately 80 individual colonies, it was
found that isolates WP1 (WP = Woods Hole Phylloplane),
WP18, and WP19 generated bioluminescence signals comparable to that of the positive control, ie. JM83 (pAK21I),
which was streaked besides JM83 (pAK203). Isolates
WP8, WP14, and WP 17 yielded a relatively weak signal of
bioluminwce. The rest of the colonies did not show any
bioluminescence when they were streaked besides JM83
(pAK211). Isolates WP20U did not produce lux1 complementation, despite its identical colony morphology to WP1.
It would be interesting to see the degree of similarity of
lhw twa isolates (WP1 and WPZOU) at the genomic level.
Although the werlay assay was performed for all of
the leaf-print isolates, only seven luminescence spots were
39
obseIV8d. Reisolation and morphological characterizations
showed that these seven isolated are belonged to either of
two distinct groups (ie. WP1 and WP18) which were described in Table 1.
Based on colony morphology and limited physiological characteristics, isolate WPl might be assigned in
the genus Xanthomonus. In addition, FISH analyses also
indicated that this isolate did not belong to Enterobacteriaceae, where the genus of Envinia belongs to.
Therefore, the identity of the WP1 isolate needs further
c M c a t i o n employing more rigoroy physiology or
genetic analyses.
Isolate WP18 seems to be a strain of Pseudomonas
syringae, due to its obligate aerobic metabolism and its
negative result in the oxidase test. It is interesting to note
that from all plant pathogenic bacteria, included in this
study, only P. syringae pv. tabaci yielded compound that
could substi~ethe K fischerf autoinducer. Envinia
amylovora yielded only a weak bioluminescence signal in
this lux assay.
Isolates WP14 and WP20U showed similar colony
morphology to that of WPl. These isolates were also facuttative anaerobes. However, the lux assay indicated that
these isolates did not produce the K fischeri autoinducer.
All Bacillus species obtained from leaf surface in this
experiment were not able to wmplement the K fischeri
autoinducer.
DISCUSSION
Phylloplane bacteria, which can substitute the K
fischeri autoinducer (VAI), were present in low abundance
on a healthy leaf surfkx, and these can be assigned to only
two distinct p u p s as described above. Therefore, this
method might be very useful for the development of a rapid
Table 1. Two main groups, of bacteria, obtained with the overlay
method.
Isolates
Charaderistics
-
WPl
Yellow with cupious of slime on 10% King's B,
bright yellow on LA (pH 7.4), gram-negative, catalase
positive, oxidase negative, rods, motile, facultative
anaerdbe. Scanning electron microscopy (SEM) demonstrated the presence of fibrous materiril (probably
polysaccharide) interconnec*
among the cells of
this bacterium (Fig. 1). Flourescence in situ hybridization (FISH) analyses showed that this isolate did
not appear to be a member of either Enterohteriaceae, a or p - group of Proteobacteria (Waese,
1987).
WP18 White, rather mucoid on 10% Kmg's B or LA (pH
7.4), gram negative, catalase positive, oxidase
negative, rods, motile, obligate aerobe. SEM did not
show fibrous materials on the cell surface as in WPI.
FISH analyses showed identical results as in isolate
WPl.
Braun-Howland, E.B., P A Vesdo & S.A. NierdckiBauer. 1993. Use of a simpli6ed d
l blot t d m i q ~ ~
and 16s rRNAdinxted probes for idedicatiun of
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t
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detection of a disease agent in agiicdtm.
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Eberhard, B.E
I#emki & P. Grcmkq
tive,itshouldbecanductedandobservedinitsoptimal
1994. Stnlcture of thc auWh&mr mpbd tbr
light emission. On LA (pH7.4), b i o h m h e s m a was amexpredon of Pseudomona rrcnginosa vimlmcc
venicntly examined af€er an 8-24 hr incubation at 2527°C.
genes. A.oc. Natl. Acad Sci. USA 91: 197-201.
very early examhation or ptolonged kubation produced
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*negative
results. Light produdon might be kept
1993. A small d@bsible signal molecule is laspolusiblc
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for the global control of videncc and emawpe
In addition, the overlay agar should be as thin as possiile
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ACKNOmEDGEMENT
glows at night: dmlopmcnt of an animal-bacmid
mutualism. J Bocteriol. 171: 48654870.
This research was conducted in Marine Biological
Smibert, RM. & N.R
1994. Phenotypic
Laboratory, Woods Hole, Mass., USA. I thank Professo:
c
o
n
,
p.
607654.
In
P.
Gerhdt, RG.E.
h4artiu Dworkin and P r o f m r John Breznak for thei.
M
u
r
r
a
y
,
W
.
Wood
&
N.R
Krieg
(ed.).Methodsfor
support and encouragement in conducting this research.
General and Molecular Bacteriology. WWashingtan
The cost of travel and aocornodation during the Summer
D.C.:American Society for Microbiolo~.
Course was granted by
Zeiss, Inc. to AS through
Watson, LP., A.E. M c b & B.R Meme& 1980.
Marine Biological Laboratoxy.
Preparation of Microbiological specimens for
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