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APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Oct. 1991, p. 2873-2879

0099-2240/91/102873-07$02.00/0

Copyright C 1991, American Society for Microbiology

Vol. 57, No. 10

Effect of2-Hydroxybenzoate on the Maintenance ofNaphthalene- Degrading Pseudomonads in Seeded and Unseeded Soil

0. A. OGUNSEITAN, I. L. DELGADO, Y.-L. TSAI, AND B. H. OLSON* Environmental Analysis and Design, Program in Social Ecology, University ofCalifornia, Irvine, California 92717

Received 20 February 1991/Accepted 12 July 1991

Theadditionofspecificnontoxicinducersofcatabolicoperonstocontaminatedsitesisanapproachthatmay enhance the efficiency ofin situ biodegradation. We determined the genetic response ofsix pseudomonads to salicylate (also known as 2-hydroxybenzoate) added directly to 50 g of nonsterile soil samples. The strains, isolated from a polyaromatic hydrocarbon-contaminated soil, metabolized naphthalene as the sole source of

available carbon, and their DNA sequences show significant homology to the nahAB genes ofthe degradative

plasmid

NAH7. Duplicate nonsterilesoilcultureswere incubated forup to30 days. Experimental soilcultures

were seeded with naphthalene-degrading strains (108 CFU g-1)originally isolatedfrom the soiland amended with salicylate (16 or 160 ,ug g-). Soil samples were analyzed periodically for the population density of

heterotrophic bacteria and naphthalene degraders and for the abundance of the naphthalene-degradative genotype inthebacterialcommunity. At 160 ,ugg1, salicylatesustainedthedensityofnaphthalene degraders attheintroduced densityfor30daysinadditiontoproducingatwo-tosixfoldincreaseintheoccurrence inthe bacterial community of DNA sequences homologous to the nah operon. No change in recoverable bacterial population densities was observed when soil samples were amended with 16 ,ug of salicylate g-1, but this

concentration ofsalicylate induced a significant increase in the level ofnah-related genes in the population.

Biodegradativegenotypes are abundant in microbial com- munities that colonize polynuclear aromatic hydrocarbon

(PAH)-contaminated environments, and bacterial degrada- tion of both substituted and unsubstituted PAHs has been studied in detail (2, 5). However, the complete removal of these compounds from contaminated sites in situ has yet to be demonstrated. Many reasons have been proposed forthe inefficiency ofbioremediation insitu(3,4).Two approaches to improve environmental bioremediation have been well

demonstrated: (i)the deliberate release ofdegradative bac- teria into contaminated environments, and (ii) the nonspe- cificstimulationofautochthonous bacterialpopulationswith nitrate, phosphate, and oxygen. Our laboratory has pro- posed athirdapproach thatuses specificinducers ofgenetic operons to enhance thedegradation oftoxiccompounds (7). This approach offers two direct benefits to bioremediation:

increased specificity of the target population to be stimu-

lated,

and predictability of intermediates and end products

because known degradative pathways are stimulated.

Salicylate (also known as 2-hydroxybenzoate) has been identifiedas an intermediate inthe naphthalene degradation

pathwayand

asaninducerofboththeupper(nah)and lower

(sat)operons carried on the NAH7 plasmid forthe degrada-

tion of naphthalene and salicylate, respectively (8). This

study examines the

effect of salicylate, as an inducer of

geneticexpression, on the maintenance ofdegradative phe-

notypes and genotypes in a soil bacterial community. Nu-

cleic acid hybridization

methods developed for direct and

indirect determinations of genotype distribution in natural environmental samples were used to characterize the main- tenance ofreintroduced natural bacteria in soil.

MATERIALS AND METHODS

Soil source. The test soil was obtained from a manufac- tured-gas plant site (active between 1903 and 1916), located

* Corresponding author.

0.6 km from the ocean, in Venice, Calif. The soil samples

came from the lamp black sump of the defunct plant and

thereforecontainavarietyofPAHs includingphenanthrene,

pyrene

(benzo[deA]phenanthrene),

methylnaphthalene, and

naphthalene. The

totalconcentrations ofPAHs measured in

the dry soiland in the oil-water slurry were 7.07 ,ugg-1 and 9.4 jig ml-', respectively. The concentrations of naphtha-

lene in the soiland in the oil-water scum were 0.043 ug

g-1

and 0.247 ,ug ml-', respectively (gas chromatography-mass spectrometry analyses performed by Global Geochemistry

Corp., Canoga Park, Calif.). The water content ofthe sandy

soil was 10% at the time of sampling, and the cation exchange capacity was 0.09 meq g-1. Soil samples were

asepticallycollected in stainless steel vessels equipped with

Teflon caps and kept at 4°C until needed.

Media and growth conditions. Bacteria were isolated by

serialdilutionofthe soilinphosphate-buffered saline(PBS).

Heterotrophic bacterial population

on half-strengthplatecount

density was determined

agar(Difco,Detroit,Mich.).The

population density ofbacteria able to utilize naphthalene as

the sole carbon source was determined on minimal salts

medium (MMN) containing (wt/vol) 0.4% NaNO3, 0.015%

KH2PO4, 0.05%Na2HPO4, 0.02%

MgSO4 7H20,0.00005%

FeCl3 6H20, and 0.001% CaC12 .2H20. The medium was

solidified With purified agar (Beckton Dickinson, Cockeys-

ville, Md.) and exposed to naphthalene vapor from pure

crystals (Sigma

Chemical Co., St. Louis, Mo.). To further

confirm that

bacteria that formed colonies on minimal me-

dium with naphthalene vapor actually degraded naphtha-

lene, we inoculated the colonies into MMN broth cultures

containing 2-hydroxybenzoate

sourceofcarbon.

or

naphthalene

as the sole

Aftertheinoculatedbrothsbecame turbid,

cells were removed by centrifugation

was scanned at 220 to 420 nm.

and the

supernatant

Degradation of naphthalene

was confirmed by observing the production

with maximum absorbance at 310 nm (2).

ofa

metabolite

Broth cultures of

selected bacteria were grown in minimal medium

supple-

mented with 2.0 mM sodium succinate (MMS). Bacterial

2873

  • 2874 OGUNSEITAN ET AL.

APPL. ENVIRON. MICROBIOL.

1hwithintermittentmixing,foreven distributionofbacteria s t inthe soil. For each strain,a solution of2-hydroxybenzoate _cWells (sodiumsalt;FisherScientific,Fairview,N.J.)was added to <80
1hwithintermittentmixing,foreven distributionofbacteria
s t
inthe soil. For each strain,a
solution
of2-hydroxybenzoate
_cWells
(sodiumsalt;FisherScientific,Fairview,N.J.)was
added to
<80 kb
a duplicate set ofsoilcultures to obtain finalconcentrations
of16 or 160 ,ugper g ofsoil. A third set ofsoilcultures was
inoculated with individual bacterial strains without
droxybenzoate. An uninoculated set of nonsterile soil
2-hy-
cul-
tures was also studied that contained 0, 16, or 160 ,ug
salicylate
per g of soil.
Salicylate
solutions or sterile water
was addedtothesoilculturesina finalliquidvolume of1ml.
The soil cultures were incubated at 23°C in a
humidity-
controlled chamber (Lunaire Environmental Inc.,Williams-
port, Pa.). Soil samples (2g)were
collected after0, 1,3,5,
7, 15, and 30 days
ofincubation fordetermination ofbacte-
rial densities and at 1 and 30 days for nucleic acid extrac-
tions.
Direct extraction of bacterial DNA from soil. A
simplified
freeze-thaw method was used for
extractingbacterial
DNA
from 1.0-g subsamples
of soil (10). The
advantage
of this
method over
traditional direct-extraction
techniques
is that
onlya
small amount ofsoil
(1g)isneeded.
Briefly,bacteria
were
dislodged from soil particles by
thorough
washing
of
1.0g ofsoilthree times in0.12 M
phosphatebuffer(pH8.0).
The soil slurry was then incubated
in
a 1.5% lysozyme
solution containing0.15
M NaCl and 0.1
M EDTA (pH
8.0)
at 37°C for 3
h with occasional
agitation.
The soil
samples
were then transferred onto ice, and
a 10% sodium
dodecyl
sulfate(SDS)solution(pH8.0)
was added;thiswas followed
abcdefg h ijk
mn
o
qr s
by three freeze-thaw
cycles
in
dry
ice-ethanol (5 min) and
65°Cwater (10min)baths.
The soilwas then extracted with
FIG. 1. (A) Electrophoresis gel picture showing 1-.g aliquots of
Tris-buffered phenol.
An
aliquot
ofthe
aqueous phase
was
DNA extracted from pure cultures of P. fluorescens (lanes b to h)
and P. putida (lanes i to s),strains isolated from PAH-contaminated
soil used in this study. Lanes a and t represent plasmid size markers.
extracted once
with a 1:1 solution of
phenol
and
(chloro-
form-isoamyl
alcohol [24:1]) and once
with chloroform-
(B) Autoradiograph of hybridization of DNA molecules shown in
isoamyl
alcohol (24:1). Nucleic acids were
pelletedfrom
the
panel A to a radiolabeled nahAB probe. Lane f contains the positive
aqueous phase with an equalvolume ofcold
isopropanol at
control strain PpG7(NAH7).
-200C.

colonies were

routinely counted

incubation at 23°C.

after 7 and

14 days of

Identification of bacteria. Bacterial colonies that grew on

MMN were purified and identified with the Analytical Profile Index rapid NFT system (Analytab, Plainview, N.Y.). The selected bacteriawere screened forthe presence ofplasmids (1), and genetic homology to the first two genes of the naphthalene degradation operon, nahAB [cloned from Pseu- domonas putida PpG7(NAH7), a gift from B. Ensley, Am-

gen Corp., Thousand Oaks, Calif.], was determined through colony hybridization to 32P-labeled nahAB genes. Among the strains isolated from the contaminated soil, six pseudomonads were chosen forfurther study because ofthe high intensity of DNA hybridization to the nahAB genes.

The strains were identified as P. putida VNM5, VNM11, VNM43, and VNM45 and P. fluorescens V01 and V22. These strains contain plasmids that bear molecular resem-

blance through size and hybridization relationships to the NAH7 ofP. putida PpG7(NAH7), ATCC 17485 (Fig. 1). Soilculture experimental design. Soil cultures contained 50 g of subsurface soil from the contaminated site in loosely capped cylindrical polyethylene 250-ml culture flasks (Nal- gene model; internal diameter, 6 cm; height, 10 cm). Dupli- cate sets offlasks were inoculated with individual strains to a final cell density of approximately 108 CFU of mid-log- phase cultures (grown in MMS and washed in PBS) per g of nonsterile soil. All experimental (seeded) and blank (un- seeded) soil cultures were allowed to equilibrate at 23°C for

Direct extraction of bacterial mRNA from soil. The acid

guanidine thiocyanate

isolate bacterial

extraction method (11) was used to

directlyfrom

10.0

g

ofsoil. The

transcripts

method consistsofwashing10-gsoilsamplestwicewith0.12

M phosphate

buffer

(pH

5.2)

containing

1.0%

carbonate. The soil was then

incubated, for

diethyl

1

min

pyro-

with

constant

agitation,

with a 4.0 M

solution ofguanidinethio-

sarcosyl,

was extractedonce

cyanate containing 0.025 M sodium citrate, 0.5%

and0.1M

2-mercaptoethanol.The

witha 1:1 mixture

slurry

ofphenolandchloroform-isoamylalcohol

chloroform-isoamyl

with

isopropanol

alcohol

(24:1).The

at -20°C. The

treatedwith DNase

(24:1)and once with

RNA was precipitated

pelletswere

before

dissolved in0.5% SDS and

&

gelelectrophoresis or

hybridization.

Nylon

Nucleic acid

hybridizations.

membrane discs

(Biotrans;ICN,Irvine,Calif.)were

usedforcolonylifts,and

Schuell,

Keene,

nitrocellulose membranes (Schleicher

N.H.) were

bound

used for hybridizations

to membranes

by

involving

using

nucleic acids

directly

a microfiltration

manifold

(Schleicher & Schuell). Hybridization membranes

were processed as recommended

DNA fragment

containing

vector by restriction

gel-purified

random

priming(BRL,

of radiolabeled

,g

to

by

the manufacturer. The

nahAB was excised from its

Agarose

32P

by

with EcoRI and HindlIl.

nahAB fragments were labeled with

Bethesda, Md.).The

specificactivity

equal

to

108

dpm

DNA

in 50%

DNA was greater than or

Hybridization

in allcases.

of1.0 pLgofextracted

out at

42°C

radiolabeled nahAB was carried

formamide solution, followed

high-stringency

by

autoradiography

For colony hybridizations, X-ray

washes.

was used

VOL. 57, 1991

EFFECT OF SALICYLATE ON NAPHTHALENE-DEGRADING BACTERIA

2875

A a 10 7 10 A S 10 0 5 10 1s 20 25 30 35
A
a
10
7
10
A
S
10
0
5
10
1s
20
25
30
35
Time (d)
C
o
CFU/g Soil (PCA)
10
F
a
10
7
10
0
5
10
15
20
25
30
35
Time (d)
B a 10 71 10 S 10 S 10 0 5 10 15 20 25 30
B
a
10
71
10
S
10
S
10
0
5
10
15
20
25
30
35
Time (d)
D
10
CFU/g Soil (MMN)
a
10
7
10
S
10
S
10
0
5
10
15
20
25
30
35
Time (d)

FIG. 2. Bacterial populationdensities insoilmicrocosms. Heterotrophic (A)and naphthalene-degrading (B)bacterial population densities

respectively,

inoculated

in uninoculated soil. Heterotrophic

(C) and naphthalene-degrading (D) bacteria population densities, respectively, in soil

with 108CFU ofP. putida

PpG7(NAH7) per g. Salicylate was added to the soil samples at 16 (0) or 160 (A) ,ug/g. Soil samples

were also incubated without salicylate (O).

to detect signals from colonies that contained nahAB se-

quences. For quantifying the occurrence of nahAB se-

quences in purified DNA extracted from the soils, the

radioanalytic imaging system (AMBIS, San Diego, Calif.)

was used. The counts per minute retained on DNA hybrids

correlated to the abundance ofnah homologous DNA (r =

0.91).

RESULTS

Biodegradativepotentialoftestsoil.The

bacterial population

beginning

of the

of the test soil,

totalheterotrophic

determined at the

study, was (4.47 ± 1.9) x 106 CFU g-1.

Approximately 63% of the isolated colonies were able to utilize naphthalene as the sole carbon source. Of the colo-

nies that degraded naphthalene,

DNA sequences that hybridized

29% 4.8% contained

with nahAB, suggesting a

certain degree of genetic diversity in the population of

naphthalene-degrading bacteria.

Effectofsalicylate on the survival ofindigenous and intro-

duced bacteria. Changes that occurred in bacterial densities

of representative soil cultures during incubation are illus- tratedinFig. 2 to 4. In most cases, the additionof160 ,ugof salicylate per g to soil sustained the bacterial density at 108

CFU g-'for at least 3 days. The majority ofthe sustained

bacteria

represented naphthalene-degrading subpopulations

as indicated by colony counts on MMN (Fig. 2B, 3B, and

4B).The specificstimulationofnaphthalenedegradersbegan

to decline relative to the total bacterial population by 15 days.

In

the absence of salicylate, the population density of

naphthalene-degrading bacteria declined to less than 105

CFU g-1. The density

fromtheinitial2 x

g-1

of P.

putida PpG7(NAH7)

2C and D).

declined

108CFU g-1tobetween

in the

105and 106CFU

within 30 days, except

presence of 160 ,ug of

per g of soil did not

salicylate per g ofsoil (Fig.

The addition of 16 ,g

produce

of

of salicylate

a consistent stimulatory

effect on the maintenance

naphthalene-degrading

strains (Fig. 2B) or on total cell

totalbacteriathatwere

density(Fig. 3C).The

fractionofthe

  • 2876 OGUNSEITAN ET AL.

APPL. ENVIRON. MICROBIOL.

A B Io! 0 10 10 0 a 10 10 7 7 10 10 S S
A
B
Io!
0
10
10
0
a
10
10
7
7
10
10
S
S
10
10
5
10
15
20
25
30
35
0
5
10
15
20
25
30
35
Time (d)
Time (d)
C
D
10
10
10
10
7
7
10
10
0
10
15
20
25
30
35
5
10
IS
20
25
30
35
Time (d)
Time (d)

FIG. 3. Bacterial population densities in soil microcosms that were inoculated with P. putida VNM11 (A and B) and VNM43 (C and D). Panels A and C represent bacterial population densities enumeratvd on plate count agar. Panels B and D represent population densi-

ties enumerated on MMN. Salicylate was added to the soil samples at 16 (0) or salicylate (O).

160 (A) pg/g. Soil samples were also incubated without

able to degrade naphthalene (calculated as the number of colonies enumerated on MMN plates divided by the number

ofcolonies enumerated on plate count agar plates) exhibited strain- and incubation period-dependent variation. The ad-

dition of salicylate to soil at either concentration did not significantly affect the proportion ofbacteria that expressed

the degradative phenotype. Effect ofsalicylate on the maintenance ofthe nah genotype in test soil. The proportion oftotal colonies which degraded

naphthalene and contained DNA sequences homologous to nahAB was determined by colony hybridization (approxi-

mately 250 colonies were probed for each experiment). The proportion ofthe heterotrophic population isolated on non- selective media thathybridizedto nahAB was lowerthan the fraction that exhibited phenotypic naphthalene degradation. Although the latterrepresented between 80 and 100% ofthe population during the 5- to 7-day period for all soil cultures that were amended with salicylate, the proportion of total colonies that hybridized to nahAB fluctuated between 5 and 80% (data not shown). This may be due to molecular

divergence of the nah operon in the environmental strains, butfurtherinvestigation ofDNA sequence similarities under differenthybridization stringencies willbe necessary to fully

characterize the level of divergence of the naphthalene- degradative operons in the strains found in the soil.

Effect of salicylate on the density of nahAB genes in DNA

extracted from soil. The electrophoresed nucleic acid mole-

cules extracted from soil culture samples are represented in Fig. 5A. Extensive shearing ofthe DNA did not occur, and

the linear molecules were about 23 kb. The DNA extracted from the soilsaftera 1-dayincubation contained a significant number of RNA molecules (Fig. 5A, panel 1). These RNA molecules were absent in the extractions performed after a

30-day incubation, suggesting a decline in overall metabolic activity ofthe soil organisms through the 30-day incubation period (Fig. 5A, panel 2). The intensity of autoradiographic

signalsfrom DNA-DNA hybrids obtained afterprobing with nahAB isshown inFig. SB. The densityofnah genes per unit

quantity of soil increased in response to salicylate concen-

tration (Table 1). There were three major types ofresponse.

VOL. 57, 1991

 

A

10

10

a

7

10

 
  • 0 1S

5

10

20

Time (d)

C

10

9

10

a

7

10

 
  • 0 1S

5

10

20

Time (d)

EFFECT OF SALICYLATE ON NAPHTHALENE-DEGRADING BACTERIA

2877

B S 10a 10 7 10 S 10 25 30 35 0 5 10 IS 20
B
S
10a
10
7
10
S
10
25
30
35
0
5
10
IS
20
25
30
35
Time (d)
D
a
10
7
10
10
10
4
10
25
30
35
0
5
10
20
25
30
35
15

Time (d)

FIG. 4. Bacterial population densities in soil microcosms that were inoculated with P. fluorescens V22 (A and B) and V01 (C and D). Panels A and C represent bacterial densities enumerated on plate count agar, whereas panels B and D represent densities enumerated on

MMN. Salicylate was added to the soil samples at 16 (0) or

160 (A) ,ug/g. Soil samples were also incubated without salicylate (O).

(i) The uninoculated soil showed no detectable response to the addition ofeither concentration ofsalicylate after 1 and

  • 30 days of incubation. (ii) The P. putida strains typified by

PpG7(NAH7) and VNM43 (Table 1) showed higherlevels of

nahAB density per gram of soil in response to 160 ,ug of salicylate per g of soil after a 30-day incubation. (iii) The response of the P. fluorescens strains (typified by the re- sponse of strain V22 [Table 1]) showed an increased inten-

sity ofDNA-DNA hybridization after a 1-day incubation in

response to the concentration ofsalicylate. However, at the end of a 30-day incubation, the response to salicylate was not as pronounced as in the soilcultures inoculated with the P. putida strains (Table 1). Effectofsalicylateon the accumulation ofnah transcriptsin

bacteria inoculated into soil. The presence of genetic tran-

scripts derived from nah genes was detected 30 days after three strains (VNM11, VNM43, and VNM45) were inocu-

latedintosoil.The radioactive signalsfrom the mRNA-DNA hybrids were too low in all cases to be quantified by the radioanalytic image system. Therefore, detection was done by X-ray autoradiography (Fig. SC). In the absence of

salicylate amendment, no transcripts were detectable by hybridization. In the soils amended with 16 ,ug of salicylate

per g, the occurrence of nahAB transcripts was detected

only in soil inoculated with strains V22, VNM5, VNM11, and VNM43 (Fig. SC, lane 1). In soils amended with 160 ,ug

ofsalicylateper g ofsoil,nahAB transcripts were detected in soil samples inoculated with strains V22, VNM5, VNM11,

VNM43, and VNM45. The absence ofdetectabletranscripts in soil inoculated with P. putida PpG7(NAH7) is probably

due to the low cell density of this laboratory strain after a 30-day incubation in soil (Fig. 2C and D).

DISCUSSION

The data generated by this study demonstrate two points. First, naphthalene-degrading bacteria respond positively to salicylate, a known inducer of the nah operon. Hence,

salicylate at certain concentrations can be used directly in

contaminated soil to stimulate the maintenance of genetic

activity in bacteria that have the potential to degrade naph-

thalene in situ. Second, methods now available to study the

2878 OGUNSEITAN ET AL. APPL. ENVIRON. MICROBIOL. A. BR 23kb: 1. IS D : a t
2878
OGUNSEITAN ET AL.
APPL. ENVIRON. MICROBIOL.
A.
BR
23kb:
1.
IS
D :
a
t
h
I
0.1kb:
RNA
co
a
23kb
a'
b
c
a bCde0 fghi
12
FIG. 5. (A) Electrophoresis ofnucleic acids extracted directlyfrom 1.0g ofsoil samples from the microcosms after 1
day (panel 1)and
30 days (panel 2). The absence ofRNA molecules from the extractions aftera
30-day
incubation suggests that there were lower metabolic
activitiesattheendoftheincubationperiod. Lane acontainsDNA sizemarkers (phagelambdarestrictedwithHindIII). Sampleloadingwells
are not shown. The largestDNA fragments extracted were approximately 23 kb. (B) Autoradiographofsignalsfrom nahAB
hybridizationof

1.0-,ugaliquots ofDNA extracted directly from soil microcosms inoculated with the following strains in order from columns a to h, or ito

p: PpG7(NAH7), V22, VNM45, VNM11, VNM43, VNM45, V01, and uninoculated soil. DNA was extractedfrom samplestaken after1 day (row 3, columns a to h, no salicylate added; row 3, columns ito p, 16 ,ugofsalicylateadded perg ofsoil;row 2,columns a to h, 160 ,ugof salicylateadded pergofsoiland DNA extractedaftera30-dayincubation;row 2,columns ito p,no salicylateadded to soil;row 1,columns a to h, 16 ,ugofsalicylate added perg ofsoil;row 1, columns ito p, 160 ,ugofsalicylate added perg ofsoil). (C) Autoradiograph ofsignals from mRNA-nahAB hybridsfrom 10-,ugaliquotsoftotalRNA extracteddirectlyfrom soilaftera 30-dayincubation. Lanes 1and 2represent samplesfrom soilthatwas amended with60and 160 ,ugofsalicylatepergofsoil,respectively. Bacterialinocula: row a,V22;row b,VNM11;

row c, VNM43; row d, VNM45; row e, V01. Signals were detected aftera 6-day autoradiographic exposure to X-ray film.

ecological impact of environmentally introduced bacteria can also be used for comparative analyses of the perfor- mance of strains that are considered significant in environ- mental bioremediation or other field applications. Salicylateisautilizablecarbon source foravariety ofsoil microorganisms and is now shown to be able to stimulate a genetic response in a subpopulation ofbiodegradative bac- teria. By comparing the survival and genetic response of strains that were isolated and reintroduced into a contam-

inated soil with those of the prototype NAH7-containing

strain,itcan beconcluded thatnative strainspossess certain

characteristics that enable them to survive and express

better in their natural habitat than exogenous strains can. The test soil had been exposed to a variety of PAHs for a protracted period; consequently, bacteria with the ability to degradePAHs, atleastnaphthalene, were abundant (greater than60% oftherecoverable heterotrophic population) inthe soil. Without the addition ofsalicylate, the bacterial density ofthesoilstabilizedatapproximately 106CFU g-1underthe conditionsofthe30-day incubation. Addition ofsalicylateat 16 ,ug g-1 did not have a notable effect on this stable cell

density. However, when salicylate was added to soil sam-

ples at 160 ,ug g-1, the bacterial densities in both the

uninoculated soiland soilthatwas inoculated with naphtha-

lene-degrading bacteria (108 CFU g-1) increased and was maintained at between 107 and 108 CFU g-' through the

30-dayincubationperiod. The effectofsalicylateon the total

heterotrophic bacterial population was not distinguishable

from the effect on the naphthalene degraders, probably

because a high proportion ofthe natural soilpopulation was

able to degrade naphthalene and presumably responded to

salicylate induction (Fig. 2 to 4).

The effectofsalicylateon themaintenance ofnaphthalene degraders was apparent within the first5 days ofincubation

during which 80 to 100% of the heterotrophs exhibited the

ability to utilize naphthalene. In the absence of sufficient

salicylate per gram of soil, the naphthalene degradation phenotype declined from near 100% after7 days to approx- imately 20% after 30 days in soil that was inoculated with

strain PpG7. Thus, a periodic dose of salicylate could be used to maintain an active degradative phenotype in the population. The addition ofa single high dose ofsalicylate,

VOL. 57, 1991

EFFECT OF SALICYLATE ON NAPHTHALENE-DEGRADING BACTERIA

2879

TABLE 1. Effect ofsalicylate on the occurrence and maintenance ofnahAB genes in soil

Bacterium and salicylate concn

CPM/g ofsoilaon:

 

(p.g/gofsoil)

day 1

day 30

Soil only

0

68.1 ±

5.6

37.5 ± 4.2

16

41.2 ± 4.4

55.6 ± 5.1

160

67.2 ± 5.6

54.7 + 5.0

  • P. putida NAH7

0

1,050.9 ± 15.2

499.5 ± 15.2

16

4,340.5 ± 44.8

176.1 ±

9.0

160

2,377.6 ± 33.2

677.5 + 17.5

  • P. putida VNM43

0

344.8 ± 12.6

102.9 ±

6.9

16

849.4 ± 19.8

242.8 ±

10.6

160

1,392.9 ± 25.4

510.6 ±

15.4

P.fluorescens V22

0

73.7 ±

5.8

74.1 ± 5.9

16

246.5 ±

10.7

53.3 ±

5.0

160

544.9 ± 15.9

91.7 ± 6.5

a Bacterial DNA was extracted directly from 1.0-g soil samples. Then 1.0 ,ug ofthe extracted DNA was probed with radiolabeled nahAB. Radioactive

signals from DNA-DNA hybrids were quantified with the AMBIS system.

however, maintained the degradative phenotype around the

inoculum density throughout the 30-day incubation period. The nativeP.putida strainsshowed asimilarbutlessdrastic

response to salicylate compared with thatofstrain PpG7. In the unseeded soil, the lower concentration of salicylate producedahigherproportionofnaphthalene degraders. This may be because the higher concentration of salicylate also providedageneralnutrientsourcefrombreakdown products

for nondegraders. The effectofsalicylate on thefractionofthe naphthalene- degrading population that contain DNA sequences which

hybridize to nahAB was more dependent on the concentra-

tionofsalicylateadded tothe soilthanon thetype ofstrain.

The population of the nah-containing colonies fluctuated considerably in the absence of sufficient salicylate. When

salicylate was added at 160

jig

per g of soil, a distinct

increase in the proportion ofnah-containing clones became apparent after a 15 day incubation, reaching near 100% for all the strains and for the uninoculated soil.

The colony hybridization data suggest a certain degree of evolutionary divergence of the naphthalene-degradative

genes presentinthenaturaltestsoil.Thisissupportedbythe

evidence presented inFig. 1, inwhich only 7 of18 naphtha- lene degraders from the soil exhibited significant homology to nahAB (at the hybridization stringency of95%).

The limitation of the colony hybridization technique for assessing genotype maintenance, however, isthat the colo-

niesthatwere examined must beabundantenoughinthe soil to be detected by the platingtechnique and must be capable

offormingindependentcolonies. Forthisandotherreasons,

several investigators have pursued alternative methods for direct assessment of bacterial genotypes in environmental samples. The studiesthathave been conducted on thedirect extractionofnucleicacidsfromenvironmental sampleshave

been designed to characterize the microbial diversity with

respect tothe relativeabundance ofspecificgene

sequences

of

which mightbeexpected tobe amplifiedasaconsequence

populationinvasion(9).Inanattempttoquantifycommunity

response at the genetic level, DNA probes were applied to

nucleic acid molecules extracted directly from the soil cul-

tures (Fig. 5).

The addition of salicylate to soil samples that were inoc-

ulatedwithnaphthalene-degradingbacteriaclearlyenhanced

the occurrence of the genetic determinants of naphthalene

degradation in the bacterial community. Experiments to

characterize the contribution ofthe enhanced geneticpoten-

tial (abundance of genetic determinants in the population

irrespectiveofgeneticexpression) tothe rate ofnaphthalene

degradation in soil have been conducted (6). The direct

application of salicylate or other nontoxic inducers of ge- neticpathways ofbiodegradationintocontaminatedenviron-

ments may be an efficientmeans ofachievingenvironmental

bioremediation, particularly where the degradative bacteria

have evolved naturally. Furthermore, the high-density rein- oculation ofnative bacteriawhich respond to specificinduc-

ersofbiodegradation may prove betterthantheintroduction ofengineered or exogenous strains into contaminated envi-

ronments.

ACKNOWLEDGMENTS This study was supported by research grant no. 8000-25 from the environment division of the Electric Power Research Institute

(EPRI), Palo Alto, Calif.

REFERENCES

  • 1. Anderson, D. G., and L. L. McKay. 1983. Simple and rapid method for isolating large plasmid DNA from lactic strepto- cocci. Appl. Environ. Microbiol. 46:549-552.

  • 2. Cerniglia, C. E. 1984. Microbial metabolism ofpolycyclic aro- matic hydrocarbons. Adv. Appl. Microbiol. 30:31-71.

  • 3. Goldstein, R. M., L. M. Mallory, and M. Alexander. 1985. Reasons for possible failure of inoculation to enhance biodeg- radation. Appl. Environ. Microbiol. 50:977-983.

  • 4. Jain, R. K., and G. S. Sayler. 1987. Problems and potential for in situ treatment of environmental pollutants by engineered microorganisms. Microbiol. Sci. 4:59-63.

  • 5. Mueller, J. G., P. J. Chapman, and P. H. Pritchard. 1989. Actionoffluoranthene-utilizingbacterialcommunity on polycy- clic aromatic hydrocarbon components of creosote. Appl. En- viron. Microbiol. 55:3085-3090.

  • 6. Ogunseitan, 0. A., and B. H. Olson. Unpublished data.

  • 7. Olson, B. H., and R. A. Goldstein. 1988. Applying genetic ecology to environmental management. Environ. Sci. Technol. 22:370-372.

  • 8. Scheli, M. A. 1985. Transcriptional control of the nah and sal hydrocarbon degradation operons by the nahR gene product. Gene 36:301-309.

  • 9. Torsvik, V., K. Salte, R. Sorheim, and J. Goksoyr. 1990. Comparison ofphenotypic diversity and DNA heterogeneity in a populationofsoilbacteria. Appl. Environ. Microbiol. 56:776-
    781.

  • 10. Tsai, Y.-L., and B. H. Olson. 1991. Rapid method forthe direct extraction of DNA from soils and sediments. Appl. Environ. Microbiol. 57:1070-1074.

  • 11. Tsai, Y.-L., M. J. Park, and B. H.

Olson. 1991. Rapid method

for direct extraction of mRNA from seeded soils. Appl. Envi- ron. Microbiol. 57:765-768.