Screening and Characterization of Phylloplane Bacteria Producing Vlbrio fucheri Autoinducer

Vol. 4, No. 2

Screening and Characterization of Phylloplane Bacteria

Producing

Vlbrio

fucheri

Autoinducer

ANTONIUS SUWANTO

D

-

of Biobgv, Faad@ of Science and M a t h m d k , .I&Reya Pqjqjrnan, Bogor 161U Inter Uniwmi@ Center for M e - , Bqgor Agrimhral Uniwnity, Bogor 16680

Tdqkone: 251425965, TcScfa: 62-251-621 724, 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 (pH 7.4) containing approximately 10' cdlslml of BckaicMo cod carrying Vibriofirckeri hu 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 were two main groups of phylloplane bacteria belonltiag to either Xuntkmona sp. or A b n 4 ~ 1 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~ua et al. 1994).

This

system requires a difhiile com-

poundpmdwxdbythecomxpondingtmterb,

whichcan acamrolate in

the

smrounding environment during growth.

Thie

difhiile

quonnn-mnsing

compannd

is called auto-

inducer

@qua et al. 1994).

Vtbrto jischeri autoinducer (VAI) is N-3-(oxohexa-

nuyl) 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 or-

gan,

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 cell-

density) 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 pro- dwd

a ampound

that stimulated luminescence by K ps- cheri.

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. 1 994). One implication

of

this

study,

is

the understand-

!

ing

that

certain

ban&

behavior can be performed effi-

ciently 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 wer- come the stmdul or fluctuating environmental conditions (O'Brien & Lindow 1989). Recently, it has been demon- strated that colonization and survival of Pseudomonas s y i - ngae was dependent on the density of the inoculum (Wil- son & 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 (pAK- 203) were obtained from Paul Dunlap,

Woods

Hole Oceanographic Institution,

Wood

Hole, Mass. Plasmid pAK211 carries all lux genes of Vfbrio fischeri, except for the non-polar deletion of ltucl (gene for autoinducer synthe- sis), 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 Patho- logy,

Univ.

of Illinois,

Urbana.

Xanthomonas campestris pv. glycines XP202 (=NCPPB 1 14 I),

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

Vol. 4. 1997 AUTOJNDUCER IN PHYLLOPLANE BACTERIA 39

Each individual leaf was pressed onto lO?! King me- dium 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 ~emqmded in saline solution to approximately 10"' celldml. The cell suspension was mixed propor- tionally 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

final

agar 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 physiologi-

cal assays, such as: characteristics of colonies, cell mor- phology,

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 Braun- Howland et al. (1 993).

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 com- parable to that of

the

positive control, ie. JM83 (pAK21 I), 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 comple- mentation, 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

obseIV8d. Reisolation and morphological characterizations showed that these seven isolated are belonged to either of two distinct groups (ie. WP1 and WP18) which were des- cribed in Table 1.

Based

on colony morphology and limited physio- logical characteristics, isolate WPl might be assigned in the genus Xanthomonus. In addition, FISH analyses also indicated that this isolate did not belong to Entero- bacteriaceae, 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~e the 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 facut- tative

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) de- monstrated the presence of fibrous materiril (probably polysaccharide) interconnec* among the cells of this bacterium (Fig. 1). Flourescence in situ hybridi- zation (FISH) analyses showed that this isolate did not appear to be a member of either Enteroh- teriaceae, a or

p

-

group of Proteobacteria (Waese, 1987).

P i 1. SEM of isolate WPl shuwed the pmmce of atuuiw fitnow metairrls -ni among the bactaial

Ceus. This f i h materiats rmght be polyseccharde which was mponsible for the excessive mucoid appear-

ance of the colloniea grown on 10% King's

B

medium.

strain or pathwar identification in phytobacterology. To date, pathwar or strain iduuifl@tion for most plant pathogenic backria

involve

a

timeconwrming

and labo- rioustest,

as

well

as apathogenkitynnsay~partidar

plants. The -ty

of

a rapid

but reliable d@nostic

testforthesebacteria,will~facili~boththebasic

study of pathogenicity

and

practical applications for early detection of

a

disease agent in agiicdtm.

Although this bioluminescence system

is

very

sensi-

tive,itshouldbecanductedandobservedinitsoptimal light emission. On LA

(pH

7.4), b i o h m h e s m a was

am-

venicntly examined af€er an 8-24 hr incubation at 2527°C. very early examhation or ptolonged kubation produced *negative results. Light produdon might be kept longerbyincreasingbuffer~tyofthegrowthmedia In addition, the overlay agar should be

as

thin

as

possiile (not more than 3

mm),

to allow as much oxygen supply to

thecultureunderneathitduringtheluminescenoeassay.

ACKNOmEDGEMENT

This research was

conducted

in Marine Biological Laboratory,

Woods

Hole,

Mass.,

USA. I thank Professo: h4artiu Dworkin

and

P r o f m r John Breznak for thei. support

and

encouragement

in

conducting

this

research. The cost of travel and aocornodation during

the

Summer Course was granted by Zeiss, Inc. to

AS

through Marine Biological Laboratoxy.

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