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Previous Article | Next Article 
Applied and Environmental Microbiology, November 2001, p. 5233-5239, Vol. 67, No. 11
0099-2240/01/$04.00+0 DOI: 10.1128/AEM.67.11.5233-5239.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
An Altered Pseudomonas Diversity Is
Recovered from Soil by Using Nutrient-Poor
Pseudomonas-Selective Soil Extract Media
Nina
Aagot,1,2
Ole
Nybroe,2
Preben
Nielsen,3 and
Kaare
Johnsen1,*
Geological Survey of Denmark and Greenland,
DK-2400 Copenhagen NV,1 Section of
Genetics and Microbiology, Department of Ecology, The Royal Veterinary
and Agricultural University, DK-1871
Frederiksberg,2 and Novozymes A/S,
DK-2880 Bagsvaerd,3 Denmark
Received 24 April 2001/Accepted 30 August 2001
 |
ABSTRACT |
We designed five Pseudomonas-selective soil
extract NAA media containing the selective properties of trimethoprim
and sodium lauroyl sarcosine and 0 to 100% of the amount of Casamino
Acids used in the classical Pseudomonas-selective
Gould's S1 medium. All of the isolates were confirmed to be
Pseudomonas by a Pseudomonas-specific OprF antibody and a Pseudomonas-specific PCR targeting
16S ribosomal DNA. The Pseudomonas isolates were
characterized by classical physiological tests, repetitive extragenic
palindromic-PCR, Fourier transform infrared spectroscopy, and carbon
source utilization patterns. Several of these analyses showed that the
amount of Casamino Acids significantly influenced the diversity of the
recovered Pseudomonas isolates. Furthermore, the data
suggested that specific Pseudomonas subpopulations were
represented on the nutrient-poor media. The NAA 1:100 medium,
containing ca. 15 mg of organic carbon per liter, consistently gave
significantly higher Pseudomonas CFU counts than
Gould's S1 when tested on four Danish soils. NAA 1:100 may, therefore,
be a better medium than Gould's S1 for enumeration and isolation of
Pseudomonas from the low-nutrient soil environment.
| |
INTRODUCTION |
Methods for examining
bacterial numbers and diversity (26) can essentially be
divided into culture-dependent and culture-independent ones. The major
disadvantage of culturing is that one obtains only the organisms that
can grow on a particular medium under the selected growth conditions.
Although these may represent only a fraction of the total organisms in
a given sample, it is nevertheless important to obtain pure cultures of
interesting strains for further characterization or for use in
biotechnology. Many authors have compared plate counts on different
media (e.g., see references 6 and 20), but only a few have
studied the effect of medium composition on the diversity represented
by the isolates (15, 30). It has been demonstrated,
though, that different media giving comparable plate counts can select
for different bacterial types, leading to different estimates of
diversity for the same soil (30).
The genus Pseudomonas includes several species of
environmental interest, such as plant growth promoters
(24), plant pathogens (28), and xenobiotic
degraders (8, 13, 27). Due to the wide distribution of
Pseudomonas in the environment and the ease by which these
bacteria can be cultured, the genus today constitutes one of the
best-studied bacterial groups. Nevertheless, new Pseudomonas species are described after application of new media or isolation procedures (1, 13). For isolation, media rich in
nutrients, such as King's B (16) and Gould's S1
(7) agar, are traditionally used, but to our knowledge no
authors have studied the impact of nutrient level in growth media on
Pseudomonas numbers and diversity.
To identify and characterize bacteria, a number of different phenotypic
and genotypic methods are available, each permitting a certain level of
taxonomic classification (33). Traditionally, classification of bacteria has been conducted by using phenotypic tests, and the core of Pseudomonas taxonomy has been based
on the ability of the isolates to utilize a variety of carbon compounds as the sole sources of carbon and energy (25). Nutritional
characteristics still provide a good basis for characterization of
isolates, but the analysis is time-consuming and should be combined
with genotypic methods, especially when treating closely related
bacteria. Repetitive extragenic palindromic (REP)-PCR (5)
uses the conserved REP sequences originally found in Escherichia
coli as primers for PCRs (34) to distinguish between
bacteria at the subspecies level. Fourier transform infrared
spectroscopy (FT-IR) is a physical technique which establishes specific
spectral fingerprints of intact bacteria (21). FT-IR
probes the total composition of a given organism, as spectra are
influenced by the content of DNA, RNA, protein, membrane, and cell wall
components. Consequently, FT-IR spectra are growth-stage dependent
(21). Isolates can be identified using spectral data
libraries or classified according to constructed phenograms or on the
basis of multivariate statistical analysis. Characterization of
environmentally isolated Pseudomonas strains by REP-PCR and
FT-IR has previously been shown to correspond well (15).
Because soil is generally an oligotrophic environment, most
heterotrophic bacteria indigenous to soil lack the ability to grow on
nutrient media tested so far. The purpose of this study was to assess
whether the amount of nutrients in the form of Casamino Acids in the
isolation medium can affect the recovery of Pseudomonas isolates and their diversity.
| |
MATERIALS AND METHODS |
Soil samples and preparation of cold soil extracts.
Soil
samples were obtained from six Danish localities: a polycyclic aromatic
hydrocarbon (PAH)-contaminated site near Ringe, a PAH-contaminated
former shipyard site in Copenhagen, an agricultural area at the farm
Højbakkegaard near Taastrup, an agricultural area in Græse near
Frederikssund, a deciduous forest in Suserup Skov near Sorø, and a
larch forest at Tornebakke near Slangerup. Soil properties are shown in
Table 1. Only freshly collected soil was
used in this study. To prepare cold soil extracts for use in growth
media, the soil was air dried (20°C) and passed through a sieve (mesh
size, 2 mm). The remaining part of the soil was kept at 5°C until
used for enumeration of bacteria. The air-dried, sieved soil was then
mixed 1:1 with Milli Q (Millipore, Bedford, Mass.) water, and particles
were removed by settling for 2 h and were centrifuged (5,000 × g, 20°C, 20 min). The supernatant was filter sterilized
(pore size, 0.2 µm).
Preparation of growth media.
In order to estimate and
compare the number of Pseudomonas CFU isolated on different
agar media, plate counts were obtained from seven
Pseudomonas-selective media. These were five cold soil extract (CSE) media with various levels of Casamino Acids (NAA 1:1, NAA
1:10, NAA 1:100, NAA 1:1,000, and NAA 0), Gould's S1 (7),
and King's B (16). The total number of culturable aerobic bacteria was also recovered on two general CSE media (CSE 1:1 and CSE 0).
The five Pseudomonas-selective NAA media consisted (per
liter) of 900 ml of Winogradsky's salt solution (0.4 g of
K2HPO4, 0.13 g of
MgSO · 7H2O, 0.13 g of NaCl, 1.52 mg
of MnSO4 · H2O, and
0.5 g of NH4NO3),
1.2 g of N-lauroyl sarcosine sodium salt (Sigma, St.
Louis, Mo.), 100 ml of CSE (23), 18 g of Noble agar (Difco, Detroit, Mich.), 20 mg of trimethoprim (Sigma), 50 mg of
nystatin (Sigma), and 5 g, 500 mg, 50 mg, 5 mg, or 0 mg of Casamino Acids (Difco), respectively, corresponding to 100, 10, 1, 0.1, and 0% of the amount of Casamino Acids found in Gould's S1.
Trimethoprim and nystatin were diluted in Winogradsky's salt solution
to 0.4 and 4 mg ml
1 and were filter sterilized
(pore size, 0.2 µm) and added after the medium had been autoclaved
and cooled to approximately 50°C. It was calculated that NAA 1:1, NAA
1:10, NAA 1:100, NAA 1:1,000, and NAA 0 had an organic carbon content
of 1,431, 144, 15, 3, and 1 mg/liter, respectively, based on the
organic carbon content in the added CSE and Casamino Acids. We checked
the ability of selected pure cultures to grow on carbon compound
impurities in the sarcosine sodium salt and Noble agar. This gave rise
to pinhead-size colonies emerging only after incubation at 20°C for
10 days. The two nonselective cold-extracted soil extract media were
prepared as the NAA media, but N-lauroyl sarcosine sodium
salt and trimethoprim were omitted. CSE 1:1 contained 5 g of
Casamino Acids per liter, whereas CSE 0 did not contain Casamino Acids.
All glassware used for preparing the media were placed in an acid bath
(1 M HCl) for at least 12 h, rinsed thoroughly, and oven
sterilized (14 h, 300°C) to reduce contamination with organic compounds.
Determination of number of CFU.
To extract bacteria, 2 g of freshly sieved soil was mixed with 18 ml of Winogradsky's salt
solution and sonicated (Branson 2210; Branson Ultrasonic Corporation,
Danbury, Conn.) in a glass tube for 20 s. Tenfold-dilution series
were prepared from 1-ml aliquots of the extract, and triplicate 50-µl
samples of appropriate dilutions were spread on the relevant media. The
same aliquot was used to inoculate all media, and plates were spread
alternately to give conditions as similar as possible. We checked that
there was no difference between the diversities of colonies retrieved from replicate agar plates by isolation and characterization of 40 colonies from two Gould's S1 agar plates by REP-PCR and FT-IR (data
not shown). The plates were incubated at 20°C, and colonies were
counted regularly until no new colonies developed, the colony density
was too high, or fungal hyphae appeared on the plates. Fluorescent
colonies in UV light (302 nm) on all Pseudomonas-selective media were counted.
Selection of isolates for characterization.
Dilutions of the
Copenhagen soil were spread plated on the five NAA media. Thirty-two
isolates were randomly picked from plates representing each of the NAA
media. Colonies on NAA 1:1 were isolated on day 2, colonies on NAA 1:10
were isolated on day 6, and colonies on NAA 1:100, NAA 1:1,000, and NAA
0 were isolated on day 12. Isolates were streaked on 1% tryptic soy
agar (TSA) (Difco) solidified with 1.8% BiTek agar (Difco) to assure
purity and were incubated for 2 days (20°C). Each isolate was
subsequently transferred to 1.8-ml cryo tubes containing 0.5 ml of 1%
tryptic soy broth and incubated (20°C, ~100 rpm) for 5 to 7 days.
Thereafter, 0.5 ml of glycerol was added and the cultures were mixed
and kept at 
80°C. It was not possible to obtain living cultures
from 22 out of 160 frozen stocks, and these isolates were not analyzed further.
For all subsequent work, strains were grown on 10% TSA (30°C) if not
stated otherwise. Culture collection strains were included in the tests
mentioned below to assure specificity and to evaluate the
identification methods performed. Strains used were Pseudomonas aeruginosa DSM 50071T, Pseudomonas
fluorescens I DSM 50090T, Pseudomonas
putida A DSM 291T, and Pseudomonas
chlororaphis DSM 50083T, unless stated otherwise.
Identification by Pseudomonas-specific immunoassay
and Pseudomonas-specific PCR.
The isolates were
verified as belonging to the genus Pseudomonas by using a
Pseudomonas-specific antibody (17) and
Pseudomonas-specific PCR (14, 31). A
Pseudomonas-specific antibody directed against the OprF
surface protein was used in a colony blotting assay as described by
Kragelund et al. (17). P. fluorescens DF 57 (29) and E. coli DSM 498T
were applied to the filters as positive and negative controls, respectively.
To complement the results from the colony-blotting assay, a
Pseudomonas-specific PCR targeting 16S ribosomal DNA was
performed. Template DNA of an isolate was prepared by boiling 300 µl
of an overnight bacterial culture (10% TSA, 30°C) suspended in Milli Q water (optical density at 600 nm, 0.6) in a safe-lock Eppendorf tube
for 10 min. The tubes were immediately cooled on ice and centrifuged
(20,000 × g, 10 min, 5°C), and the supernatants were subsequently kept at 20°C until PCR was carried out essentially as
previously described (14). The assay was modified by using 25 µl of master mix per reaction and Ampli Taq Gold
polymerase (Applied Biosystems, Norwalk, Conn.). The
PSMG primer (3) in combination with
the eubacterial primer 9-27 used for PCR gives Pseudomonas-specific amplification (14). To
assure this specificity, PCR was first carried out on the five
Pseudomonas and E. coli type strains. In each PCR
run, a negative control without template DNA was included as well.
Characterization by classical biochemical tests.
To
characterize the isolates the following tests were conducted: gram
determination using the Bactident Aminopeptidase kit (Merck, Darmstadt,
Germany), oxidase (BBL Dryslide Oxidase Slides; Difco), catalase using
3% H2O2, and growth on
10% TSA at 4, 37, and 41°C.
REP-PCR analysis.
The isolates were characterized by REP-PCR
with primers targeting repetitive extragenic palindromic elements
(34). Template DNA was prepared as described above. PCR
was carried out as previously described (13). The PCR was
modified by using 2- to 4-µl template DNA suspension and 9.3 or 11.3 µl of double-distilled water per reaction mixture. Amplified PCR
fragments were separated on 3% (wt/vol) LE agarose gels (20 by
20 cm) (Promega, Madison, Wis.). In each PCR run, a negative control
without template DNA was included. Isolates showing similar band
patterns were grouped manually. Electrophoresis was carried out again,
with isolates belonging to the same REP-PCR group loaded next to each
other to check similarity. Strains still giving faint or no band
patterns after three trials were considered nontypeable.
FT-IR.
For FT-IR analysis, isolates from NAA 1:1, NAA 1:10,
NAA 1:100, and NAA 1:1,000 were incubated overnight (30°C) on Luria
broth (LB) agar supplemented with (per liter) 5 g of soluble
starch, 10 ml of 1 M KPO4 (pH 7.0), and 8 ml of
50% glucose. Isolates from medium NAA 0, however, were not able
to grow on LB agar and were grown on 10% TSA instead. A loopful of
cells was used to inoculate tubes with 10 ml of TY broth, which
contains (per liter) 20 g of tryptone, 5 g of yeast extract,
0.7 ml of 1% FeCl2 · 4H2O, 0.1 ml of 1%
Mn2Cl · 4H2O, 1.5 ml
of 1% MgSO4 · 7H2O
(pH 7.3). Tubes were incubated (30°C, 250 rpm) overnight until
the cells were in the late log phase. FT-IR and cluster analyses were
carried out on all isolates as previously described (15),
but phenograms were based on 901 to 699, 1,200 to 900, and 3,001 to
2,799 parts cm1 of the first derivative
of the FT-IR spectrum. Clusters appearing in all three phenograms were
considered groups.
Characterization by carbon source utilization patterns.
We
tested the ability of the isolates to grow on a minimal medium
(13) supplemented with 31 different carbon compounds on microtiter plates. The following carbon compounds were selected on the
basis of Pseudomonas nutritional properties given by
Bergey's Manual of Systematic Bacteriology
(25) and were used at a concentration of 1 mg
ml1: 
-alanine, adipic acid, capric acid,
citric acid, D-galactose, D-gluconic acid, D-glucose,
D-maltose, D-mannitol,
D-mannose, D-sorbitol,
D-trehalose, fumaric acid, geraniol, glycerol,
glycolic acid, L-arabinose,
L-leucine, L-malic acid,
L-phenylalanine,
L-trypthophan, L-valine,
meso-inositol, mucic acid,
N-acetyl-D-glucosamine, nicotinic acid, oxalate, phenylacetate, pyruvic acid, starch, and succinic acid.
Two days prior to inoculation isolates from freeze cultures were
streaked on 10% TSA (30°C). On the day of inoculation a sterile cotton stick was used to transfer bacteria to a glass tube with minimal
medium and the bacterial concentration was adjusted to an optical
density at 600 nm of 0.3. Each microtiter plate well was inoculated
with 180 µl of medium and 20 µl of the bacterial suspension. The
plates were read immediately using a 650-nm-wavelength filter in a
microtiter plate reader (Thermo max; Molecular Devices, Sunnyvale,
Calif.). The plates were then incubated at 30°C for 43 h and
read again. The experiment was run in triplicate. Absorbances of the
wells without carbon compound were subtracted before data was subject
to statistical analysis.
Statistics.
All cell counts were subject to t
test. Groupings based on classical tests, REP-PCR, and FT-IR were
statistically evaluated using the 
2 test or
Fisher's Exact test when data were in 2-by-2 contingency tables. In
cases where more than 20% of the expected values in 2 test were less than 5, groups of media
and/or groups of isolates were pooled to increase the number of
expected values. Statistics for cell counts and groups were performed
in Sigma Stat 2.0 for Windows (SPSS Inc., Chicago, Ill.). Data from the
carbon utilization tests were further subject to pattern recognition
analysis by means of disjoint principal components analysis (PCA) using
the classification method SIMCA (35) in MATLAB
PLS_Toolbox 2.0E Version 5.3 (Eigenvector Research, Inc., Manson,
Wash.). Carbon utilization data were mean centered before analysis. In
brief, the isolation media were considered separate classes and were modeled by their own principal component (PCA) models. P
values below 0.05 were considered statistically significant.
| |
RESULTS |
Viable cell counts.
For the Ringe soil, Gould's S1 agar
resulted in the lowest estimate of the Pseudomonas
population of 9.8 × 104 CFU g of
soil1, as observed by the end of the incubation
period (Fig. 1A). All NAA media except
that from NAA 1:100 yielded significantly higher plate counts than
Gould's S1, exceeding the number recovered on Gould's S1 by two- to
fourfold, within the same incubation period. The highest plate count
was obtained on NAA 1:100, although differences between NAA media were
not statistically significant. Plating on King's B agar resulted in
4.1 × 106 CFU g of
soil1, or 2.1 × 105
(± 8.9 × 104) fluorescent
Pseudomonas CFU g of soil1, as
observed in UV light. The total number of aerobic bacteria was
determined on CSE 0 and CSE 1:1, yielding 6.2 × 106 and 1.5 × 107 CFU
g of soil1 (Fig. 1B). A further comparison of
the recovery in four more soils with very different soil
characteristics showed that the number of CFU was significantly higher
on NAA 1:100 than on Gould's S1 agar in all four soils (Table
2). The Copenhagen soil harbored a
smaller Pseudomonas population than the other tested soils
(compare Table 2 and Fig. 1 and 2).
Again, the highest plate counts were obtained from NAA 1:100, although
the differences between NAA media were not statistically significant.
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FIG. 1.
The number of CFU per gram of Ringe soil on seven
Pseudomonas-selective media and two general media.
Different letters indicate significantly different
(P < 0.05) levels of CFU per gram of soil at the
end of the incubation period. (A) NAA 1:1, NAA 1:10, NAA 1:100, NAA
1:1,000, NAA 0, and Gould's S1; (B) CSE 1:1, CSE 0, and King's B
agar.
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FIG. 2.
The number of CFU per gram of Copenhagen soil on NAA
1:1, NAA 1:10, NAA 1:100, NAA 1:1,000, and NAA 0. Different letters
indicate significantly different (P < 0.05) levels
of CFU per gram of soil by the day of isolation.
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Identification of isolates by Pseudomonas-specific
immunoassay and Pseudomonas-specific PCR.
The
diversity represented on the different NAA media was compared in an
experiment with the Copenhagen soil. First, the specificity of the NAA
media for Pseudomonas was verified. All isolates were identified as Pseudomonas by a colony-blotting procedure
employing a Pseudomonas-specific OprF antibody (Table
3). Correspondingly, Pseudomonas-specific PCR and gel electrophoretic analysis of
all isolates and the culture collection strains confirmed the presence of one band of the correct size (445 bp), suggesting that all isolates
belonged to the genus Pseudomonas (Table 3).
Characterization by classical tests.
At primary isolation, the
percentage of fluorescent colonies on NAA 1:1, NAA 1:10, NAA 1:100, NAA
1:1,000, NAA 0, Gould's S1, and King's B plates was 68% ± 10%,
68% ± 37%, 0% ± 0%, 8% ± 13%, 16% ± 13%, 86% ± 23%, and
5% ± 2%, respectively. All 138 isolates were gram negative and were
oxidase and catalase positive (Table 3). In the temperature tests 99%
of the isolates were capable of growth at 4°C, 17% were capable of
growth at 37°C, and 7% were capable of growth at 41°C. The
proportion of isolates growing at the respective temperatures were not
significantly different between the media.
Grouping by REP-PCR, FT-IR, and phenotypic tests.
Using
REP-PCR, 106 isolates were typeable, among which 72 formed groups with
at least one other isolate, constituting 11 groups (Table
4). Five different REP-PCR groups and 11 unique isolates originated from NAA 1:100, which thereby represented
more REP-PCR groups than any other medium. The distribution of REP-PCR
groups was significantly different between the media below (NAA 0 and 1:1,000) and above (NAA 1:1, 1:10, and 1:100) 15 mg of carbon per
liter. Fifteen milligrams of carbon per liter is the limit below which
Kuznetsov et al. (18) designate bacteria as oligotrophic. In particular, group IV isolated from NAA 1:1,000 appears to be medium
specific, suggesting the existence of unique Pseudomonas subpopulations on the nutrient-poor media. None of the reference strains fitted into any of the REP-PCR groups, indicating that no
isolates are closely related to these strains.
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TABLE 4.
Grouping by REP-PCR, the number of isolates per medium in
each group, and correspondence with growth temperatures
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All isolates were typeable by using FT-IR, and cluster analysis in
FT-IR by complete linkage, average linkage, and Ward's algorithm
yielded very similar groups (data not shown). We formed FT-IR groups
when isolates were grouped together by all three methods. Only 19 isolates did not fall into any FT-IR group (Table 5). Again, isolates from NAA 1:100
represented the highest number of groups constituting 19 FT-IR groups.
The distribution of FT-IR groups was significantly different between
media below (NAA 0 and 1:1,000) and above (NAA 1:1, 1:10 and 1:100) 15 mg of carbon per liter. The FT-IR groups F and I, originating from NAA
1:1,000 and NAA 0, respectively, were larger medium-specific groups,
again supporting the existence of specific Pseudomonas
populations on low-nutrient media. In general, there was good agreement
between the groups formed by FT-IR and REP-PCR. For instance, the
REP-PCR groups IV, V, VI, VII, and VIII were analogous to the FT-IR
groups F, H, I, J, and S. The FT-IR technique generally resulted in
formation of more and smaller groups than REP-PCR, but two FT-IR groups fitted into the same REP-PCR group on some occasions, and on one occasion two REP-PCR groups clustered in the same FT-IR group (data not
shown). The groups formed by the two typing methods coincided very well
with the classical phenotypic tests. In general, isolates sharing
REP-PCR and FT-IR groups grew at the same temperatures (Table 4).
Carbon source utilization data.
PCA on carbon utilization data
coincided well with grouping appearing after FT-IR and REP-PCR
analysis, showing that colonies appearing on the NAA 1:100 medium
represented the highest proportion of unique strains analyzed by SIMCA
(data not shown). The PCA model based on all carbon utilization rate
data explained 81% of the variation. PC1 and PC2 each explained
47 and 19% of the variation, respectively, and this variation was
largely governed by the utilization rates of D-mannitol,
D-mannose, D-sorbitol, D-trehalose,
glycerol, L-arabinose, meso-inositol, succinic
acid, L-valine, and
N-acetyl-D-glucosamine. The proportion
of isolates able to grow on selected carbon compounds is shown in Table
6. The proportions of isolates able to
grow on the remaining carbon compounds were the following: -alanine,
92%; adipic acid, 0%; capric acid, 100%; citric acid, 100%;
D-galactose, 88%;
D-gluconic acid, 99%;
D-glucose, 100%;
D-maltose, 2%; fumaric acid, 99%; geraniol,
1%; glycerol, 99%; glycolic acid, 0%;
L-leucine, 99%; L-malic
acid, 7%; L-phenylalanine, 76%;
L-valine, 96%; mucic acid, 99%; nicotinic acid,
6%; oxalate, 0%; phenylacetate, 1%; pyruvic acid, 99%; starch, 0%;
and succinic acid, 99%. The isolates from NAA 0 had a significantly
narrower carbon source utilization profile than isolates from the other
NAA media in the sense that there were proportionally fewer positive
tests for NAA media than for the isolates from other media.
| |
DISCUSSION |
Pseudomonas-specific NAA media.
The NAA
media are derived from the classical Gould's S1 Pseudomonas
isolation medium (7) and contain the selective agent trimethoprim against facultative gram-negative bacteria
(7). Yao and Moellering (36) state that
trimethoprim is an inhibitor of DNA synthesis and is active against
many gram-positive cocci and most gram-negative bacilli. Sodium lauroyl
sarcosine prevents the growth of gram-positive bacteria
(7). These selective compounds were also used by Andersen
and coworkers (2) when they constructed a medium for the
isolation of phenanthrene-degrading Pseudomonas isolates. In
Gould's S1 medium, sucrose and glycerol are included to create an
osmotic stress that selects for Pseudomonas, but apparently
we could omit these compounds in the NAA media without problems. Cold
soil extract was included, as environmental extracts can replace some
ingredients in media used for enumeration and isolation of specific
bacteria (reviewed by Lochhead and Burton [19]). Viable
cell counts on the five NAA soil extract media, which vary in amino
acid content, significantly exceed those obtained on Gould's S1 medium
(Fig. 1 and Table 2). The media are specific for
Pseudomonas, as shown by two independent molecular methods targeting the outer membrane protein OprF (17) and the 16S
ribosomal DNA (14), respectively.
When tested for several different soils,
Pseudomonas-specific counts on NAA 1:100 were at least
threefold higher than counts on Gould's S1 (Table 2), and NAA
1:100 yielded the highest number of bacterial groups within a
relatively short incubation time. Hence, although
Pseudomonas is considered an organism typical for
nutrient-rich media, the NAA 1:100 medium appears to be useful for
enumeration and isolation of Pseudomonas from the
low-nutrient soil environment. This medium contains an amino acid
concentration of 50 mg liter1. In accordance
with this fact, Olsen and Bakken (23) found higher
heterotrophic colony counts on general soil extract media containing 7 and 70 mg of nutrient mixture liter1 than on
media with 0, 700, or 7,000 mg liter1. A
negative effect of media with high nutrient concentrations on colony
formation has often been observed for the quantification of bacteria in
both soil (10, 11) and water (4). For
instance, Hattori and Hattori (9) found that dilute
nutrient broth organisms, considered oligotrophs, were severely
suppressed by 1 or 2% (wt/vol) Casamino Acids.
Kuznetsov et al. (18) classified oligotrophs as bacteria
that develop on media with an organic carbon content of about 1 to 15 mg liter1 at first cultivation. Other authors
have suggested that the term oligotrophs should be used for bacteria
which do not form colonies on high-nutrient media at the first
cultivation, though this definition is much more difficult to use
experimentally (22, 23). Using Kuznetsov's definition,
the strains isolated on NAA 1:100, NAA 1:1,000, and NAA 0 with organic
carbon contents of approximately 15, 3, and 1 mg per liter,
respectively, can be classified as oligotrophs (18). All
isolates were maintained on 1% tryptic soy broth after the first
cultivation step. However, the strains from NAA 0 could not be
recultivated on the rich LB agar, suggesting that these isolates are
nutritionally different from the isolates recovered on the other media.
We propose that the Pseudomonas strains recovered on
low-nutrient NAA media may include specific groups that can grow on
richer media at subsequent recultivation. This group is defined as
facultative oligotrophs by Ishida et al. (12).
Pseudomonas diversity.
The concentration of
Casamino Acids in the NAA media had a significant effect on the
diversity represented by the Pseudomonas isolates as shown
by both genotypic and phenotypic typing methods. The data suggest that
unique Pseudomonas groups occur on the more nutrient-poor
media. These subpopulations might occupy other ecological niches than
Pseudomonas strains obtained on the traditional
nutrient-rich Pseudomonas isolation media King's B
(16) and Gould's S1 (7) and could represent
new species.
Hence, we have shown that by varying the level of Casamino Acids in a
Pseudomonas-selective growth medium, significantly different assessments of diversity will be obtained from the same soil. Other
authors have shown that the media used for isolation play an important
role in the study of bacterial diversity. For example, Sørheim et al.
(30) demonstrated that the carbon source of a general soil
extract medium influenced the bacterial diversity markedly. Not much
literature, however, exists on comparative diversity studies of strains
isolated on eu- and oligotrophic media that are otherwise comparable.
The culture-dependent techniques may seem inadequate for addressing
bacterial diversity, since only about 1 to 5% of the total bacterial
population can be readily cultivated using the media known so far
(23) and soil most likely contains a vast number of
virtually unknown bacteria. Torsvik et al. (32) found that
the diversity of the total bacterial community estimated by
reassociation analysis of DNA isolated directly from soil was
approximately 170 times higher than the diversity represented by
cultured strains from the same soil. Even though culture-independent
DNA-based methods are powerful tools for diversity studies,
microbiologists will still be dependent on cultivation methods to
obtain pure cultures for further physiological characterization.
Hopefully, further attention to culturing methods might reduce the huge
gap between the culturable and nonculturable bacterial community and
provide a better understanding of the soil ecosystem.
| |
ACKNOWLEDGMENTS |
This work was supported by BIOPRO (Centre for biological
processes in contaminated soils and sediments) established under The
Danish Environmental Research Programme (www.biopro.dk).
We gratefully thank Per Rosenberg for excellent multivariate
statistical help.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Danish
Veterinary Laboratory, Bülowsvej 27, DK-1790 Copenhagen V,
Denmark. Phone: 45 35 30 01 00. Fax: 45 35 30 01 20. E-mail:
kjo{at}svs.dk.
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Abstract
Introduction
