Previous Article | Next Article 
Applied and Environmental Microbiology, November 2000, p. 5043-5052, Vol. 66, No. 11
0099-2240/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
Phylogenetic Analysis of and Oligonucleotide Probe Development
for Eikelboom Type 021N Filamentous Bacteria Isolated from Bulking
Activated Sludge
Takahiro
Kanagawa,1,*
Yoichi
Kamagata,1
Shinobu
Aruga,1
Tetsuro
Kohno,2
Matthias
Horn,3 and
Michael
Wagner3
National Institute of Bioscience and Human-Technology,
Agency of Industrial Science and Technology, Tsukuba
305-8566,1 and Department of Civil
and Environmental Engineering, Yamanashi University, Kofu
400-0016,2 Japan and Lehrstuhl
für Mikrobiologie, Technische Universität München,
D-85350 Freising, Germany3
Received 8 May 2000/Accepted 21 August 2000
 |
ABSTRACT |
Fifteen filamentous strains, morphologically classified as
Eikelboom type 021N bacteria, were isolated from bulking activated sludges. Based on comparative 16S ribosomal DNA (rDNA) sequence analysis, all strains form a monophyletic cluster together with all
recognized Thiothrix species (88.3 to 98.7% 16S rDNA
sequence similarity) within the gamma-subclass of
Proteobacteria. The investigated Eikelboom type 021N
isolates were subdivided into three distinct groups (I to III)
demonstrating a previously unrecognized genetic diversity hidden behind
the uniform morphology of the filaments. For in situ detection of these
bacteria, 16S rRNA-targeted oligonucleotide probes specific for the
entire Eikelboom type 021N-Thiothrix cluster and the
Eikelboom type 021N groups I, II, and III, respectively, were designed,
evaluated, and successfully applied in activated sludge.
| |
INTRODUCTION |
Activated sludge systems are used
worldwide for wastewater treatment. One of the major operational
problems of these systems is the excessive growth of filamentous
bacteria (7, 14, 32). This causes poor settlement of
activated sludge flocs, a problem commonly referred to as bulking. On
the other hand, it has been suggested that a certain number of
filamentous bacteria are required for proper floc formation
(27). Thus, depending upon their identity and abundance,
filamentous bacteria can be either beneficial or detrimental for
efficient separation of the treated wastewater from the biomass in the
settling tanks. Therefore, the ability to unambiguously identify
filamentous bacteria is crucial for proper control of wastewater
treatment systems. Since cultivation of most filamentous bacteria is
difficult and time-consuming, their classification is traditionally
achieved by microscopic observation of morphological traits and simple
staining reactions of the filaments within activated sludge using the
keys proposed by Eikelboom (6). Since the majority of
filamentous bacteria in activated sludge are morphologically different
from previously identified bacteria, these filamentous bacteria were
provisionally classified by type numbers (6). Keeping in
mind that the morphology of bacteria can vary significantly depending
upon the environmental conditions, more-reliable tools for in situ
identification of filamentous bacteria are urgently required.
Eikelboom type 021N bacteria have frequently been described as
causative agents for sludge bulking (7, 16, 22, 25, 28, 32, 35,
36). Recently, an oligonucleotide probe for in situ detection of
Eikelboom type 021N bacteria has been designed by Wagner et al.
(31) and used for in situ detection of this microorganism
within bulking sludges (22, 24). Their in situ hybridization
results suggest that Eikelboom type 021N bacteria comprise a variety of
phylogenetically and physiologically different microorganisms.
Additionally, a recent report also indicated that bacteria of the
Eikelboom type 021N morphotype encompass several genotypically
different microorganisms (12).
In this study, we analyzed the phylogenetic characteristics of 15 strains of Eikelboom type 021N bacteria, most of which have been newly
isolated in the course of the present study. These strains could be
divided into three phylogenetic groups, and we subsequently designed
oligonucleotide probes specific for each group and an additional probe
which targets a 16S rRNA signature sequence of all 15 Eikelboom type
021N isolates and the recognized Thiothrix species
(T. eikelboomii, T. nivea, T. unzii, T. fructosivorans, and T. defluvii).
| |
MATERIALS AND METHODS |
Bacterial strains.
All Eikelboom type 021N isolates analyzed
in this study were recovered from bulking activated sludge of different
wastewater treatment plants (WWTPs) listed in Table
1. Four of the studied Eikelboom type
021N strains, KR-A, T1-4, T2-1, and SNR-3, were isolated previously by
Kohno (16). Eikelboom type 021N strain AP3 (T. eikelboomii, ATCC 49788) (11, 12, 34) was purchased from the American Type Culture Collection. In addition, 10 Eikelboom type 021N strains were isolated from WWTPs in Japan in the course of
this study.
View this table:
[in this window]
[in a new window]
|
TABLE 1.
Origins of Eikelboom type 021N strains used in this study
and GenBank accession numbers of 16S rDNA sequences determined in
this study
|
|
Paraformaldehyde-fixed cells of
Legionella pneumophila were
kindly provided by Dorothee Grimm (Würzburg,
Germany).
Culture media.
Medium IAM used for the isolation of
Eikelboom type 021N bacteria contained 0.15 g of sodium acetate,
0.15 g of glucose, 5 ml of vitamins mixture, 10 ml of salts
solution A, and 1 ml of phosphate solution A per liter. Vitamins
mixture contained 20 mg of calcium pantothenate, 20 mg of nicotinic
acid, 1 mg of biotin, 1 mg of cyanocobalamin, 1 mg of folic acid, 20 mg
of pyridoxine hydrochloride, 20 mg of p-aminobenzoic acid,
20 mg of inositol, 20 mg of thiamine hydrochloride, and 20 mg of
riboflavin per liter. Salts solution A contained 10 g of
(NH4)2SO4, 5.0 g of KCl,
5.0 g of MgSO4 · 7H2O, 2.0 g
of CaCl2 · 2H2O, 2.0 g of
CaCO3 and 0.05 g of Fe3Cl · 6H2O per liter. Phosphate solution A was prepared with
124 g of Na2HPO4 and 15.4 g of
NaH2PO4 per liter, and the pH was adjusted to
7.2 with 1 N HCl. Medium EGGC, which was used for the maintenance of
Eikelboom type 021N isolates, contained 0.3 g of sodium acetate,
0.3 g of Casamino Acids, 0.6 g of glucose, 10 ml of salts
solution A, 5 ml of vitamins mixture, and 2 ml of phosphate solution A
per liter. For the preparation of medium EGGC, sodium acetate and
Casamino Acids were dissolved in distilled water and mixed with salts
solution A, and the pH was adjusted to 7.2 with 1 N HCl. This mixture
was autoclaved separately, and sterile-filtered solutions of the
remaining components were added. For agar plates and slants, media were
solidified by addition of 15 g of Bacto Agar (Difco) per liter.
Medium TA, which was used for the sulfur oxidation test, contained
0.082 g of sodium acetate, 10 ml of salts solution T, 5 ml of vitamins
mixture, 4 ml of phosphate solution T, and 7.5 ml of freshly prepared
thiosulfate solution (100 g of
Na2S2O3 · 5H2O
per liter) per liter. Salts solution T contained 16 g of
NH4NO3, 5.0 g of MgSO4
· 7H2O, 2.0 g of CaCO3, 0.05 g of
Fe3Cl · 6H2O, and 8.4 g of
NaHCO3 per liter. Phosphate solution T contained 66 g
of K2HPO4 and 16.6 g of
KH2PO4 per liter (pH 7.2). For the preparation
of medium TA, sodium acetate was dissolved in distilled water and mixed
with salts solution T, and the pH was adjusted to 7.2 with 1 N HCl.
This mixture was autoclaved separately, and the remaining components
were added.
Isolation of Eikelboom type 021N strains from activated
sludge.
Filamentous bacteria were collected from the activated
sludge samples using a bundle of 5 nichrome wires (0.5 by 50 mm), each bent at the end to form a hook, and transferred into sterile distilled water. Following homogenization of this suspension by a blender for 5 min (Auto Cell Master CM-200; Iuchi, Osaka, Japan), filamentous bacteria were again collected using a bundle of 12 stainless steel thin
wires (0.15 by 40 mm), each bent at the end to form a hook, and
transferred into sterile distilled water (16). This
suspension was diluted to a concentration of 500 to 3,000 bacterial
filaments or cells per ml, and 0.1-ml aliquots were spread on IAM
plates and incubated for 3 weeks at 25 or 30°C. Fingerprint-like
colonies (characteristic of Eikelboom type 021N bacteria [16,
34]) were examined under a phase-contrast microscope. Only
isolates that were classified as Eikelboom type 021N bacteria by the
Eikelboom keys (6, 7) were selected for further analysis.
Eikelboom type 021N isolates were streaked on IAM agar plates
repeatedly for further purification and maintained on EGGC agar plates
or slants.
Morphological characteristics and staining.
Eikelboom type
021N isolates grown on EGGC agar for 3 to 5 days at 30°C were
morphologically characterized by phase-contrast microscopy and several
staining procedures. Gram staining was performed with a FAVOR-G SET-S
kit (Nissui Pharmaceutical Co., Ltd., Tokyo, Japan). Neisser stain and
poly-
-hydroxybutyrate (PHB) stain techniques were performed as
described by Jenkins (13).
Quinone analysis.
Cells grown in EGGC liquid medium were
harvested by centrifugation and stored at 
20°C. Quinones were
extracted from 0.5 to 1 g (wet weight) of cells with
chloroform-methanol (2:1, vol/vol), purified by column chromatography
using Sep-Pak Cartridges (Waters Co.), and analyzed by a reverse-phase
high-performance liquid chromatography (HPLC) (29). The HPLC
system was equipped with a Beckman 126 Solvent Module, a Zorbax ODS
column (4.6 [inner diameter] by 250 mm) (Du Pont Co.) in a column
oven (30°C), a Beckman 168 diode array detector, and a Beckman System
Gold for data analysis. A mixture of methanol-isopropyl ether (9:2,
vol/vol) was used as eluent at a flow rate of 1 ml/min (10).
Oxidation of reduced sulfur.
Cells grown on EGGC agar plates
for 5 days at 30°C were inoculated in 5 ml of TA medium in sterile
test tubes (17 by 160 mm) and shaken at 200 rpm at 30°C for 12 days.
Observation of sulfur granules was performed on cultures after
incubation for 1 day and 12 days. The concentrations of thiosulfate and
sulfate in the cultures at day 0 and day 12 were determined by an HPLC
system (Shimadzu LC-6A) equipped with a CDD-6A detector (Shimadzu) and a column (4.5 by 150 mm) of Shim-pack IC-A3 (Shimadzu). HPLC analysis was performed at 40°C by using 8 mM p-hydroxybenzoic acid
and 3.2 mM bis-tris as the eluent at a flow rate of 1.2 ml/min. The injection volume of the samples was 20 µl.
Sequencing of 16S rDNA and phylogenetic analysis.
Cells
grown on EGGC agar plates were suspended in sterilized distilled water
and stored at 20°C. Crude lysates of the stored cells were prepared
by proteinase K (20 U/ml) digestion (20 min, 60°C), heat treatment
(95°C for 10 min), and centrifugation (16,000 × g, 5 min) (9). Almost complete DNA coding for 16S rRNA (rDNA) was amplified by PCR directly from the crude lysates using the bacterial consensus primers Eu8f (Escherichia coli positions
8 to 27) and Eu1492r (E. coli positions 1510 to 1492)
(33). PCR was performed using a PCR thermal cycler MP
(Takara Shuzo Co., Kyoto, Japan) with an initial denaturation at 95°C
for 9 min followed by 35 cycles of denaturation at 95°C for 1 min,
primer annealing at 50°C for 1 min, and extension at 72°C for 2 min. PCR products were extracted with chloroform-isoamyl alcohol (24:1,
vol/vol), purified by MicroSpin S-400 HR columns (Amersham Pharmacia
Biotech, Uppsala, Sweden), and directly sequenced with an ABI 377 automated DNA sequencer (Perkin-Elmer Applied Biosystems) using
sequencing primers which correspond to positions 536 to 518, 821 to
803, 1111 to 1093, 1406 to 1389, and 1094 to 1112 of the E. coli 16S rRNA. The obtained 16S rDNA sequences were subjected
to BLAST search (http://www.ncbi.nlm.nih.gov/BLAST/) to determine 16S
rDNA similarities with sequences deposited in GenBank, EMBL, and GSDB databases. The retrieved sequences were aligned by using the CLUSTAL W
program, version 1.6 (30). Subsequently, the alignment was refined by visual inspection. Evolutionary distances were calculated by
using the program MEGA, version 1.0 (17). Phylogenetic trees were constructed based on the distance matrix data obtained with the
neighbor-joining method (26). Robustness of the tree
topology was evaluated by (i) bootstrap resampling analysis
(8) with 100 replicates and (ii) applying maximum-parsimony
and maximum-likelihood methods using the ARB program package
(encompassing more than 16,000 published and unpublished homologous
small subunit rDNA primary structures; Strunk et al., unpublished data
[program available at http://www.mikro.biologie.tu-muenchen.de]). The
composition of the data sets varied with respect to the reference
sequences and the alignment positions included. Variability of the
individual alignment positions was determined using the respective tool
of the ARB package and was used as the criterion to remove or include variable positions for phylogenetic analyses.
Determination of DNA base composition.
Cells grown on EGGC
agar plates were suspended in potassium phosphate buffer and stored at
20°C. Crude lysates were prepared from the stored cells as
described above. DNA was purified from crude lysates by the method of
Marmur (21). Purified DNA was subsequently hydrolyzed by P1
nuclease (GC kit; Yamasa Shoyu Co., Choshi, Japan) followed by alkaline
phosphatase (from E. coli) (Wako Pure Chemicals Industry,
Ltd., Osaka, Japan) as described by Kamagata and Mikami
(15). The G+C contents were determined by a reversed-phase
HPLC (Shimadzu SCL-6B) using a CLC-ODS column (6.0 by 150 mm;
Shimadzu). Separation was achieved at 40°C by using 5% methanol in
10 mM phosphate buffer (pH 3.5) as the mobile phase at a flow rate of 1 ml/min. Each deoxyribonucleoside was detected by a UV-visible light
spectrophotometric detector (SPD-6AV; Shimadzu) determining
A270. An equimolar mixture of
deoxyribonucleosides (GC kit; Yamasa Shoyu Co.) was used as the standard.
Oligonucleotide probes and fluorescence in situ
hybridization.
The following oligonucleotide probes were used for
in situ hybridization of pure cultures of Eikelboom type 021N strains: (i) EUB 338 (5'-GCTGCCTCCCGTAGGAGT-3'), targeting most but
not all members of the domain Bacteria (2, 5);
(ii) 21N (5'-TCCCTCTCCCAAATTCTA-3'), previously designed to
specifically target a signature region on the 16S rRNA of the
Eikelboom type 021N isolate II-26 (31); and (iii) TNI
(5'-CTCCTCTCCCACATTCTA-3'), targeting the 16S rRNA of
T. nivea (31). In addition, the following
oligonucleotide probes (probe designations according to Alm et al.
[1]) were designed in this study using the probe
design-probe match tools of the ARB software package: (i) G1B
(S-*-021Ng1-1029-a-A-18), specific for the group I isolates of
Eikelboom type 021N; (ii) G2M (S-*-021Ng2-842-a-A-18), specific for the
group II isolates of Eikelboom type 021N; (iii) G3M
(S-*-021Ng3-996-a-A-18), specific for the group III isolates of
Eikelboom type 021N; and (iv) G123T (S-G-Thioth-697-a-A-18), specific
for the 15 Eikelboom type 021N isolates (group I, II, and III) and the
recognized Thiothrix species, T. eikelboomii,
T. nivea, T. unzii, T. fructosivorans,
and T. defluvii. In order to ensure probe specificity, all
available 16S and 23S rDNA sequences included in the ARB database were
checked for the presence of the probe target sites. Oligonucleotides
were synthesized and directly 5' labeled with
5(6)-carboxyfluorescein-N-hydroxysuccinimideester (FLUOS),
the hydrophilic sulfoindocyanine fluorescent dyes Cy3 and Cy5, or
tetramethylrhodamine-5-isothiocyanate. For in situ hybridization
activated sludge samples from 24 WWTPs in Japan and Europe as well as
the Eikelboom type 021N isolates grown on medium EGGC for 3 days at
30°C were fixed with paraformaldehyde and hybridized as described
elsewhere (3). To enhance the specificity of probe G123T it
was used together with an equimolar amount of the unlabeled competitor
oligonucleotide G123T-C (5'-CCTTCCGATCTCTACGCA-3') in all
experiments. Slides were examined using an Olympus AX80 fluorescence
microscope equipped with an IPLab-spectrum image analyzing system
(Signal Analysis) and a color charge-coupled device camera (Hamamatsu
Photonics, Hamamatsu, Japan) or a Zeiss confocal laser scanning
microscope LSM510. Optimal hybridization stringency was determined for
each probe by quantification of filament fluorescence resulting from
different formamide concentrations in the hybridization buffer using
the equipment and procedures described previously (5). For
screening of the activated sludge samples the newly designed probes
(labeled with different fluorescent dyes) were applied together in
different combinations under stringent hybridization conditions.
Simultaneous hybridization with probes requiring different stringency
was realized by a successive hybridization procedure (31).
Nucleotide accession numbers.
The 16S rDNA sequences
determined in this study have been deposited in DDBJ under accession
numbers AB042532 through AB042545 and AB042819.
| |
RESULTS |
Phylogenetic analysis of 16S rRNA sequences.
For the 15 Eikelboom type 021N isolates, including T. eikelboomii AP3,
near full-length 16S rRNA gene fragments (~1,420 bp) were
successfully amplified and directly sequenced. Subsequent comparative 16S rDNA sequence analysis revealed that the investigated Eikelboom type 021N isolates are affiliated with the gamma-subclass of
Proteobacteria and show the highest 16S rDNA sequence
similarities with members of the genus Thiothrix.
Consistently, the 15 Eikelboom type 021N isolates shared the
characteristic deletion in helix 18 of the 16S rRNA secondary
structure (corresponding to E. coli positions 455 to 477)
previously reported by Howarth et al. (12) for members of
the Eikelboom type 021N-Thiothrix group. Phylogenetic analysis revealed that the retrieved 16S rDNA sequences could be placed
into three distinct groups (Fig. 1), each
comprising highly similar sequences (99.6 to 100% for group I, 98.5 to
100% for group II, and 99.9 to 100% for group III). Independent of the data set and treeing method used, the group I isolates B3-1, B4-1,
B2-7, SCM-A, B5-1, B2-8, and OS-F formed a monophyletic lineage which
is clearly separated from all recognized Thiothrix species
and the other Eikelboom type 021N isolates investigated in this study.
The nearest neighbor of the group I isolates is T. eikelboomii (94.0 to 94.3% 16S rDNA sequence similarity). The group II isolates KR-A, T1-4, T2-1, and COM-A always clustered together
with T. eikelboomii (which was consequently included in
group II in this study), with 98.5 to 98.7% 16S rDNA sequence similarity, and the group III isolates SNR-3, EJ1M-B, and EJ2M-B, formed a stable association with T. defluvii (96.8 to 97.0%
16S rDNA sequence similarity). The other Thiothrix species,
T. nivea, T. unzii, T. ramosa, and
T. fructosivorans, formed a fourth monophyletic grouping,
which was supported by all treeing methods (Fig. 1).
View larger version (18K):
[in this window]
[in a new window]
|
FIG. 1.
Phylogenetic tree derived from 16S rDNA sequences
showing the position of the Eikelboom type 021N strains. The tree was
calculated using the neighbor-joining method. The bar represents 1%
estimated sequence divergence. Numbers at nodes represent percentage
bootstrap values. The GenBank accession numbers of the reference
strains used in the phylogenetic analysis are as follows: T. defluvii, AF127020; T. fructosivorans I, L79963;
T. fructosivorans Q, L79962; T. unzii, L79961;
T. ramosa, U32940; T. nivea JP2, L40993;
Leucothrix mucor, X87277.
|
|
G+C contents of DNA and quinones.
The determined G+C moles
percent of the DNA of all Eikelboom type 021N isolates were within a
very narrow range (Table 2). Those of
groups I, II, and III were 43.9 to 44.7, 44.1 to 46.1, and 44.0 to
44.4, respectively. All Eikelboom type 021N isolates contained an
eight-isoprene ubiquinone as the major quinone.
Morphological characteristics.
The colonies of the
investigated Eikelboom type 021N isolates were colorless and showed the
fingerprint-like structure that is characteristic of Eikelboom type
021N bacteria (16, 34). The filaments grown on EGGC agar
plates were very long (more than 0.5 mm), unbranched, sheathless,
nonmotile, slightly curled, and multicellular (Fig.
2). Single cells within
the filaments were not uniform in size and showed a barrel-like to
ovoid shape, and the septa between individual cells were clearly
visible. Overall, filaments of the majority of the investigated
Eikelboom type 021N isolates were highly similar, and thus
differentiation between the respective isolates of the three different
phylogenetic groups by morphological characteristics is not possible
(Table 2). However, it should be noted that two isolates of the
Eikelboom type 021N group II significantly differed in their morphology
from all the other isolates. Particularly, individual cells of
Eikelboom type 021N isolate AP3 (T. eikelboomii) were highly
diverse in shape and size (1.2 to 8.0 µm in width and 1.0 to 5.0 µm
in length) while the Eikelboom type 021N isolate COM-A showed an
exceptional length variation of individual cells (1.0 to 8.0 µm) but
did not vary extensively in width.
View larger version (247K):
[in this window]
[in a new window]
|
FIG. 2.
Phase-contrast photomicrographs of Eikelboom type 021N
strains in pure culture. Two strains of each Eikelboom type 021N group
and additional two strains, COM-A and AP3, which significantly differed
in morphology, are shown. The bar in photo A indicates 10 µm and
magnification was ×1,000 for all photos. (A) Group I strain B3-1; (B)
group I strain OS-F; (C) group II strain KR-A; (D) group II strain
T1-4; (E) group II strain COM-A; (F) group II strain AP3
(characteristic part is shown); (G) group III strain SNR-3; (H) group
III strain EJ2M-B.
|
|
Staining characteristics.
Gram and Neisser staining was
negative for all Eikelboom type 021N isolates. However, differences
between the groups within the investigated Eikelboom type 021N isolates
could be demonstrated using PHB staining. While distinct PHB granules
were observed in the majority of the cells of the Eikelboom type 021N
isolates belonging to groups I and III, cells of the group II isolates were stained completely and no individual granules were visible.
Oxidation of reduced sulfur.
No sulfur granules were detected
in the cells grown on IAM agar medium or on EGGC agar medium. However,
when the cells were incubated with 3 mM
Na2S2O3, high numbers of sulfur
granules were observed inside and outside of the cells of the group I
and II isolates after 1 day and 12 days of incubation. On the other
hand, only a few sulfur granules were present in the cells of the group III isolates after 1 day and no sulfur granules were present after 12 days. A significant amount of sulfuric acid was produced by the group I
and II isolates, whereas there was no production or only slight
production of this compound by the group III isolates.
Fluorescence in situ hybridization.
Previously, Wagner et al.
(31) designed the 16S rRNA-targeted probes 21N and TNI
for specific in situ detection of Eikelboom type 021N bacteria and
T. nivea in environmental samples, respectively. In the
present study, we reevaluated the specificity and sensitivity of these
probes in the light of the new 16S rRNA sequences obtained from the
15 Eikelboom type 021N isolates and the recently published 16S rRNA
sequences of additional Thiothrix species (12).
Interestingly, only the 16S rRNA of the group II isolate T1-4 is
fully complementary to probe 21N, while all the other Eikelboom type
021N isolates possess one or two mismatches with this probe (Table
3). Consequently, in situ hybridization
of isolate T1-4 with probe 21N yielded a strong signal, while very weak
or no hybridization signals could be observed for other group II, group
I, and group III isolates (Table 4).
T. eikelboomii and T. defluvii show a single
mismatch with probe 21N, while all other described Thiothrix
species have three or more mismatches with this probe and will thus not
be detected with this probe. The 16S rRNA sequences of all
Eikelboom type 021N isolates included in this study showed two or three mismatches with probe TNI (Table 3). While all group I isolates shared
one centrally located strong C-C mismatch and one distal weak T-G
mismatch with probe TNI, all group II isolates (except for isolate
T1-4, which has three mismatches with probe TNI) and all group III
isolates exhibited two distal mismatches (a weak T-G and a strong C-A
mismatch) with the 5' end of probe TNI (Table 3). Considering that a
mismatch at the 5' or 3' end of an oligonucleotide probe is less
destabilizing for the probe-target hybrid compared to a centrally
located mismatch, hybridization with probe TNI yielded the expected
results: No signal could be observed for group I isolate OS-F and group
II isolate T1-4, while strong signals were detectable for group II
isolate KR-A and group III isolate SNR-3 (Table 4).
View this table:
[in this window]
[in a new window]
|
TABLE 3.
Difference alignment of the target region of the 16S
rRNA of Eikelboom type 021N isolates for the previously
designed probes 21N and TNIa
|
|
To extend the probe set for in situ detection of
Thiothrix
spp. and Eikelboom type 021N bacteria, four additional 16S
rRNA-targeted
oligonucleotide probes were designed (Table
5). Probe G1B specifically
targets
all Eikelboom type 021N group I isolates and displays
at least three
mismatches with all other available 16S rRNA sequences,
including
the Eikelboom type 021N group II and group III isolates
(Table
6). Probe G2M was designed to
specifically detect the
group II isolates (including
T. eikelboomii) and displays one
central strong C-A mismatch to the
group I isolates and at least
three mismatches with all other available
16S rRNA sequences,
including all Eikelboom type 021N group III
isolates. Probe G3M
was designed to specifically detect the group III
isolates and
displays one weak, putatively nondiscriminative G-U
mismatch with
T. defluvii, at least two mismatches to
all other available 16S
rRNA sequences, and at least five
mismatches to Eikelboom type
021N group I and II isolates. In addition,
G123T was designed
to specifically hybridize to a signature region on
the 16S rRNA
of all recognized
Thiothrix species,
including all Eikelboom type
021N isolates analyzed in this study.
Probe G123T has a relatively
weak T-G mismatch with a few other
microorganisms (mainly
Legionella sp.) which are not members
of the Eikelboom type 021N-
Thiothrix clade. Thus, to enhance
specificity this probe should only be
applied in combination with the
competitor probe G123T-C. Optimal
hybridization stringency was
determined for the four probes by
increasing the formamide
concentration in the hybridization buffer
in increments of 5 or 10% at
a constant hybridization temperature
of 46°C. Probe-conferred signals
remained at the same level following
the addition of formamide up to
30% for probe G1B, 35% for probe
G2M, 30% for probe G3M, and 40%
for probe G123T, and then decreased
rapidly (Fig.
3). Using these stringent hybridization
conditions,
the application of the group-specific probes allowed us to
specifically
discriminate between the three Eikelboom type 021N groups,
while
G123T was successfully used to visualize all Eikelboom type 021N
isolates analyzed in this study (Fig.
4).
For each of the probes
no signals were observed with the tested
nontarget organisms if
the stringent hybridization conditions were
applied (Fig.
3).
View this table:
[in this window]
[in a new window]
|
TABLE 5.
Eikelboom type 021N group specific probe sequences,
target sites, and formamide concentration in the hybridization
buffer required for specific in situ hybridization
|
|
View this table:
[in this window]
[in a new window]
|
TABLE 6.
Difference alignment of the target region of the 16S
rRNA of Eikelboom type 021N isolates for various probes
|
|
View larger version (27K):
[in this window]
[in a new window]
|
FIG. 3.
Probe dissociation curves of newly designed probes under
increasingly stringent hybridization and washing conditions. For each
data point mean fluorescence intensity values (arbitrary units
[a.u.]) of at least 100 cells of the filaments and suitable nontarget
reference organisms were determined. Error bars indicate the standard
deviation. (A) probe G1B, specific for group I; (B) probe G2M, specific
for group II; (C) probe G3M, specific for group III; (D) probe G123T,
specific for the Eikelboom Type 021N-Thiothrix cluster. For
each microorganism used, the number of mismatches (MM) with the
respective probe is given in parentheses. It should be mentioned that
the nontarget organisms with one mismatch (strain B5-1 and L. pneumophila) were slightly visible to the human eye after
nonstringent hybridization with the respective probes. However, due to
constraints of the eight-bit digital image analysis software, these
weak signals could not be recorded with the confocal microscope during
the probe dissociation curve experiments.
|
|
View larger version (24K):
[in this window]
[in a new window]
|
FIG. 4.
Whole-cell hybridization of Eikelboom type 021N strains
in pure culture with newly designed probes. Rows: I, group I strain
B5-1; II, group II strain T2-1; III, group III strain EJ1M-B. Columns:
A, Probe G1B; B, probe G2M; C, probe G3M; D, probe G123T.
|
|
Subsequently, the developed probes were used in different combinations
for an in situ diversity survey of Eikelboom type 021N
bacteria in
bulking and nonbulking activated sludge samples from
24 municipal and
industrial WWTPs in Japan and Europe (Table
7).
Eikelboom type 021N bacteria of group
I were detected in five
Japanese and one Swedish WWTP. Eikelboom type
021N bacteria of
group II could be visualized in seven Japanese and two
European
WWTPs (Fig.
5) while Eikelboom
type 021N bacteria of group III
were not found in any of the
investigated samples. No signals
were detected with the three Eikelboom
type 021N bacterial group-specific
probes in 12 sludge samples from
German WWTPs. Except for the
Swedish WWTP all filaments detected with
the group-specific probe
G1B or G2M also hybridized simultaneously with
probe G123T that
is specific for the entire Eikelboom type
021N-
Thiothrix cluster.
In the Swedish plant a minor
fraction of the filaments identified
as Eikelboom type 021N bacteria of
group II did not hybridize
with probe G123T.
View this table:
[in this window]
[in a new window]
|
TABLE 7.
Application of developed probes for in situ survey of
filamentous bacteria of the Eikelboom type 021N in activated sludge
from different WWTPs
|
|
View larger version (71K):
[in this window]
[in a new window]
|
FIG. 5.
In situ detection of Eikelboom type 021N bacteria in
activated sludge from the WWTP Großlappen (Munich, Germany). (A) An
interference contrast image of a sludge floc. (B) Fluorescence in situ
hybridization of the same microscopic field using probes G2M (labeled
with Cy3, colored in green) and G123T (labeled with Cy5, colored in
red). Yellow filaments did hybridize with both probes and were thus
identified as members of group II. Red filaments (arrow) only bound
probe G123T and are most likely members of the genus
Thiothrix. Bar, 20 µm.
|
|
| |
DISCUSSION |
The 15 isolates of filamentous bacteria analyzed in this
study showed highly similar morphological, staining, and
physiological characteristics, which are indicative of
classification as Eikelboom type 021N bacteria. Comparative 16S
rRNA sequence analysis revealed that these isolates can be
divided into three distinct evolutionary groups which, together with
recognized Thiothrix species, form a monophyletic grouping
within the gamma-subclass of the class Proteobacteria. It
should be noted that the investigated Eikelboom type 021N isolates were
clearly different from Thiothrix species described in
Bergey's Manual of Systematic Bacteriology (18) in the shape of colonies, the length of filaments, sheath formation, and sulfur granule production. Furthermore, the G+C contents of Eikelboom type 021N isolates analyzed here are much lower (43.9 to
46.1%) than the G+C contents reported for T. nivea (52%)
(19) and T. ramosa (51.0 to 52.4%)
(23). In addition, 16S rRNA sequence similarities of the
investigated Eikelboom type 021N isolates with T. nivea and
T. ramosa are lower than 92%. However, recently, Howarth et
al. (12) have proposed a change of the definition of the
genus Thiothrix and suggested the inclusion of Eikelboom type 021N strains into this genus. Consequently, Eikelboom type 021N
strain AP3 was designated as T. eikelboomii, and the newly isolated Eikelboom type 021N strain Ben 57 was classified as T. defluvii. In our study we showed a previously unrecognized
diversity within the Eikelboom type 021N strains, and these findings
underline the necessity of obtaining more phenotypic and genotypic
information of Eikelboom type 021N isolates to clarify their taxonomic position.
Based on the extended 16S rRNA data set for members of
the Eikelboom type 021N-Thiothrix group, the
specificity and sensitivity of the previously published 16S
rRNA-targeted probes TNI and 21N (31) was reevaluated.
Results demonstrated that, using the hybridization conditions
recommended by Wagner et al. (31), probe TNI also targets
the Eikelboom type 021N group II, including T. eikelboomii (except for strain T1-4), and the Eikelboom type 021N group III, including T. defluvii. Of the newly analyzed strains, only
strain T1-4 of the Eikelboom type 021N group II is targeted by probe 21N. Thus, members of the Eikelboom type 021N group I, for the first
time described in this study, cannot be detected in environmental samples by application of these probes. This might explain the findings
of Nielsen et al. (22), who used the oligonucleotide probes
21N and TNI for in situ analysis of bulking activated sludges in
industrial and domestic WWTPs dominated by filamentous bacteria morphologically identified as Eikelboom type 021N bacteria. The authors
reported that the dominant filamentous bacteria in some treatment
plants did not hybridize with probe 21N but hybridized with probe TNI
and that the dominant filamentous bacteria in other treatment plants
did not hybridize with either probe 21N or TNI. Nielsen and coworkers
thus hypothesized that the Eikelboom type 021N morphotype comprises
different species of filamentous bacteria which are dominating in
different bulking sludges. Our results confirm this hypothesis and
suggest that previous reports on sludge bulking caused by Eikelboom
type 021N bacteria (7, 13, 14, 16, 25, 32, 35, 36) were
descriptions of the same phenomenon caused by different bacterial
species which share the same morphology.
New 16S rRNA-targeted probes were designed and evaluated for three
subgroups of Eikelboom type 021N bacteria and for all recognized Eikelboom type 021N-Thiothrix species and subsequently
successfully applied in a diversity survey of Eikelboom type 021N
bacteria in various Japanese and European WWTPs. The results of the
survey showed that members of group I and group II Eikelboom type 021N bacteria do occur in WWTPs on both continents. In contrast, group III
Eikelboom type 021N bacteria could not be detected in situ in any of
the analyzed WWTPs. Future studies will have to show whether this is
caused by low in situ abundance (below the detection limit of the in
situ hybridization method) or by restricted occurrence of these
filaments in particular WWTPs. The finding of Eikelboom type 021N
bacteria which hybridize with probe G2M but not with probe G123T in one
Swedish WWTP suggests the existence of additional, not-yet-recognized
diversity of Eikelboom type 021N bacteria.
Currently we are using the developed probes to investigate (i) the in
situ physiology of the different groups of Eikelboom type 021N bacteria
by combining fluorescence in situ hybridization with
microautoradiography (20) and (ii) more generally, the links
between the different Eikelboom type 021N bacteria and bulking of
activated sludge.
| |
ACKNOWLEDGMENTS |
M.H. was supported by a grant of the Sonderforschungsbereich 411 from the Deutsche Forschungsgemeinschaft (Research Center for
Fundamental Studies of Aerobic Biological Wastewater Treatment) to M.W.
We thank Yasuyo Ashizawa and Yoko Ueda for quinone and the G+C content determination.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: National
Institute of Bioscience and Human-Technology, Agency of Industrial
Science and Technology, 1-1 Higashi, Tsukuba 305-8566, Japan. Phone:
81-298-61-6026. Fax: 81-298-61-6009. E-mail:
kanagawa{at}nibh.go.jp.
| |
REFERENCES |
| 1.
|
Alm, E. W.,
D. B. Oerther,
N. Larsen,
D. A. Stahl, and L. Raskin.
1996.
The oligonucleotide probe database.
Appl. Environ. Microbiol.
62:3557-3559[Medline].
|
| 2.
|
Amann, R. I.,
B. J. Binder,
R. J. Olson,
S. W. Chisholm,
R. Devereux, and D. A. Stahl.
1990.
Combination of 16S rRNA-targeted oligonucleotide probe with flow cytometry for analyzing mixed microbial populations.
Appl. Environ. Microbiol.
56:1919-1925[Abstract/Free Full Text].
|
| 3.
|
Amann, R. I.
1995.
In situ identification of micro-organisms by whole cell hybridization with rRNA-targeted nucleic acid probes, p. 1-15.
In
A. D. L. Akkermans, J. D. Elsas, and F. J. Bruij (ed.), Molecular microbial ecology manual, 3.3.6. Kluwer, London, United Kingdom.
|
| 4.
|
Brosius, J.,
T. L. Dull,
D. D. Sleeter, and H. F. Noller.
1981.
Gene organization and primary structure of a ribosomal operon from Escherichia coli.
J. Mol. Biol.
148:107-127[CrossRef][Medline].
|
| 5.
|
Daims, H.,
A. Brühl,
R. Amann,
K.-H. Schleifer, and M. Wagner.
1999.
The domain-specific probe EUB338 is insufficient for the detection of all bacteria: development and evaluation of a more comprehensive probe set.
Syst. Appl. Microbiol.
22:434-444[Medline].
|
| 6.
|
Eikelboom, D. H.
1975.
Filamentous organisms observed in activated sludge.
Water Res.
9:365-388[CrossRef].
|
| 7.
|
Eikelboom, D. H., and H. J. J. van Buijsen.
1981.
Microscopic sludge investigation manual. Report A94a.
IMG-TNO, Delft, The Netherlands.
|
| 8.
|
Felsenstein, J.
1985.
Confidence limits on phylogenies: an approach using the bootstrap.
Evolution
39:783-791[CrossRef].
|
| 9.
|
Hiraishi, A.
1992.
Direct automated sequencing of 16S rRNA amplified by polymerase chain reaction from bacterial cultures without DNA purification.
Lett. Appl. Microbiol.
15:210-213[Medline].
|
| 10.
|
Hiraishi, A.,
Y. Ueda,
J. Ishihara, and T. Mori.
1996.
Comparative lipoquinone analysis of influent sewage and activated sludge by high-performance liquid chromatography and photodiode array detection.
J. Gen. Appl. Microbiol.
42:457-469[CrossRef].
|
| 11.
|
Howarth, R.,
M. Head, and R. F. Unz.
1998.
Phylogenetic assessment of five filamentous bacteria isolated from bulking activated sludges.
Water Sci. Technol.
37(4-5):303-306.
|
| 12.
|
Howarth, R.,
R. F. Unz,
E. M. Seviour,
R. J. Seviour,
L. L. Blackall,
R. W. Pickup,
J. G. Jones,
J. Yaguchi, and I. M. Head.
1999.
Phylogenetic relationships of filamentous sulfur bacteria (Thiothrix spp. and Eikelboom type 021N bacteria) isolated from wastewater-treatment plants and description of Thiothrix eikelboomii sp. nov., Thiothrix unzii sp. nov., Thiothrix fructosivorans sp. nov. and Thiothrix defluvii sp. nov.
Int. J. Syst. Bacteriol.
49:1817-1827[Abstract/Free Full Text].
|
| 13.
|
Jenkins, D.
1992.
Towards a comprehensive model of activated sludge bulking and foaming.
Water Sci. Technol.
25(6):215-230.
|
| 14.
|
Jenkins, D.,
M. G. Richard, and G. T. Daigger.
1993.
Manual on the causes and control of activated sludge bulking and forming, 2nd ed.
Lewis Publishers, Chelsea, Mich.
|
| 15.
|
Kamagata, Y., and E. Mikami.
1991.
Isolation and characterization of novel thermophilic Methanosaeta strain.
Int. J. Syst. Bacteriol.
41:191-196.
|
| 16.
|
Kohno, T.
1988.
Morphology, physiology, and nutrition of a sulfur-oxidizing filamentous organism isolated from activated sludge.
Water Sci. Technol.
20(11-12):241-247.
|
| 17.
|
Kumar, S.,
K. Tamura, and M. Nei.
1993.
MEGA; molecular evolutionary genetics analysis, version 1.0.
The Pennsylvania State University, University Park.
|
| 18.
|
Larkin, J. M.
1989.
Genus II. Thiothrix, p. 2098-2101.
In
J. P. Staley, M. P. Bryant, N. Pfennig, and J. G. Holt (ed.), Bergey's manual of systematic bacteriology, vol. 3. The Williams & Wilkins Co., Baltimore, Md.
|
| 19.
|
Larkin, J. M., and D. L. Shinabarger.
1983.
Characterization of Thiothrix nivea.
Int. J. Syst. Bacteriol.
33:841-846[Abstract/Free Full Text].
|
| 20.
|
Lee, N.,
P. H. Nielsen,
K. H. Andreasen,
S. Juretschko,
J. L. Nielsen,
K.-H. Schleifer, and M. Wagner.
1999.
Combination of fluorescent in situ hybridization and microautoradiography: a new tool for structure-function analyses in microbial ecology.
Appl. Environ. Microbiol.
65:1289-1297[Abstract/Free Full Text].
|
| 21.
|
Marmur, J.
1961.
A procedure for the isolation of deoxyribonucleic acid from microorganisms.
J. Mol. Biol.
3:208-218.
|
| 22.
|
Nielsen, P. H.,
K. Andeasen,
M. Wagner,
L. L. Blackall,
H. Lemmer, and R. J. Seviour.
1998.
Variety of type 021N in activated sludge as determined by in situ substrate uptake pattern and in situ hybridization with fluorescent rRNA targeted probes.
Water Sci. Technol.
37(4-5):423-430.
|
| 23.
|
Odintsova, E. V., and G. A. Dubinina.
1990.
Thiothrix ramosa nov. sp., a new species of colourless filamentous sulfur bacteria.
Mikrobiologii
59:637-646.
|
| 24.
|
Pernelle, J.,
E. Cotteux, and P. Duchene.
1998.
Effectiveness of oligonucleotide probes targeted against Thiothrix nivea and type 021N 16S rRNA for in situ identification and population monitoring in activated sludges.
Water Sci. Technol.
37(4-5):431-440[CrossRef].
|
| 25.
|
Richard, M. G.,
G. P. Shimizu, and D. Jenkins.
1985.
The growth physiology of the filamentous organism type 021N and its significance to activated sludge bulking.
J. Water Pollut. Control Fed.
57:1152-1161.
|
| 26.
|
Saitou, N., and M. Nei.
1987.
The neighbor joining method: a new method for reconstructing phylogenetic trees.
Mol. Biol. Evol.
4:406-425[Abstract].
|
| 27.
|
Sezgin, M.,
D. Jenkins, and D. S. Parker.
1978.
A unified theory of filamentous activated sludge bulking.
J. Water Pollut. Control Fed.
50:362-381.
|
| 28.
|
Strom, F., and D. Jenkins.
1984.
Identification and significance of filamentous microorganisms in activated sludge.
J. Water Pollut. Control Fed.
57:449-459.
|
| 29.
|
Tamaoka, K.,
Y. Katayama-Fujimura, and H. Kuraishi.
1983.
Analysis of bacterial menaquinone mixtures by high performance liquid chromatography.
J. Appl. Bacteriol.
54:31-36.
|
| 30.
|
Thompson, J. D.,
D. G. Higgins, and T. J. Gibson.
1994.
CLUSTAL W; improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice.
Nucleic Acids Res.
22:4673-4680[Abstract/Free Full Text].
|
| 31.
|
Wagner, M.,
R. Amann,
P. Kaempeer,
B. Assmus,
A. Hartmann,
P. Hutzler,
N. Springer, and K.-H. Schleifer.
1994.
Identification and in situ detection of Gram-negative filamentous bacteria in activated sludge.
Syst. Appl. Microbiol.
17:405-417.
|
| 32.
|
Wanner, J.
1994.
Activated sludge bulking and foaming control.
Technomic Publishing Company, Lancaster, Pa.
|
| 33.
|
Weisburg, W. G.,
S. M. Barns,
D. A. Pelletier, and D. J. Lane.
1991.
16S ribosomal DNA amplification for phylogenetic study.
J. Bacteriol.
173:697-703[Abstract/Free Full Text].
|
| 34.
|
Williams, T. M., and R. F. Unz.
1985.
Filamentous sulfur bacteria of activated sludge: Characterization of Thiothrix, Beggiatoa, and Eikelboom type 021N strains.
Appl. Environ. Microbiol.
49:887-898[Abstract/Free Full Text].
|
| 35.
|
Williams, T. M., and R. F. Unz.
1985.
Isolation and characterization of filamentous bacteria present in bulking activated sludge.
Appl. Microbiol. Biotechnol.
22:273-282.
|
| 36.
|
Ziegler, M.,
M. Lange, and W. Dott.
1990.
Isolation and morphological and cytological characterization of filamentous bacteria from bulking sludge.
Water Res.
24:1437-1451[CrossRef].
|
Applied and Environmental Microbiology, November 2000, p. 5043-5052, Vol. 66, No. 11
0099-2240/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
This article has been cited by other articles:
-
Amachi, S., Kawaguchi, N., Muramatsu, Y., Tsuchiya, S., Watanabe, Y., Shinoyama, H., Fujii, T.
(2007). Dissimilatory Iodate Reduction by Marine Pseudomonas sp. Strain SCT. Appl. Environ. Microbiol.
73: 5725-5730
[Abstract]
[Full Text]
-
Payne, M. S., Hall, M. R., Sly, L., Bourne, D. G.
(2007). Microbial Diversity within Early-Stage Cultured Panulirus ornatus Phyllosomas. Appl. Environ. Microbiol.
73: 1940-1951
[Abstract]
[Full Text]
-
Okabe, S., Odagiri, M., Ito, T., Satoh, H.
(2007). Succession of Sulfur-Oxidizing Bacteria in the Microbial Community on Corroding Concrete in Sewer Systems. Appl. Environ. Microbiol.
73: 971-980
[Abstract]
[Full Text]
-
Kragelund, C., Kong, Y., van der Waarde, J., Thelen, K., Eikelboom, D., Tandoi, V., Thomsen, T. R., Nielsen, P. H.
(2006). Ecophysiology of different filamentous Alphaproteobacteria in industrial wastewater treatment plants.. Microbiology
152: 3003-3012
[Abstract]
[Full Text]
-
Okabe, S., Ito, T., Sugita, K., Satoh, H.
(2005). Succession of Internal Sulfur Cycles and Sulfur-Oxidizing Bacterial Communities in Microaerophilic Wastewater Biofilms. Appl. Environ. Microbiol.
71: 2520-2529
[Abstract]
[Full Text]
-
Amachi, S., Mishima, Y., Shinoyama, H., Muramatsu, Y., Fujii, T.
(2005). Active Transport and Accumulation of Iodide by Newly Isolated Marine Bacteria. Appl. Environ. Microbiol.
71: 741-745
[Abstract]
[Full Text]
-
Kalanetra, K. M., Huston, S. L., Nelson, D. C.
(2004). Novel, Attached, Sulfur-Oxidizing Bacteria at Shallow Hydrothermal Vents Possess Vacuoles Not Involved in Respiratory Nitrate Accumulation. Appl. Environ. Microbiol.
70: 7487-7496
[Abstract]
[Full Text]
-
Uyeno, Y., Sekiguchi, Y., Sunaga, A., Yoshida, H., Kamagata, Y.
(2004). Sequence-Specific Cleavage of Small-Subunit (SSU) rRNA with Oligonucleotides and RNase H: a Rapid and Simple Approach to SSU rRNA-Based Quantitative Detection of Microorganisms. Appl. Environ. Microbiol.
70: 3650-3663
[Abstract]
[Full Text]
-
Gillan, D. C., Dubilier, N.
(2004). Novel Epibiotic Thiothrix Bacterium on a Marine Amphipod. Appl. Environ. Microbiol.
70: 3772-3775
[Abstract]
[Full Text]
-
Engel, A. S., Lee, N., Porter, M. L., Stern, L. A., Bennett, P. C., Wagner, M.
(2003). Filamentous "Epsilonproteobacteria" Dominate Microbial Mats from Sulfidic Cave Springs. Appl. Environ. Microbiol.
69: 5503-5511
[Abstract]
[Full Text]
-
Bjornsson, L., Hugenholtz, P., Tyson, G. W., Blackall, L. L.
(2002). Filamentous Chloroflexi (green non-sulfur bacteria) are abundant in wastewater treatment processes with biological nutrient removal. Microbiology
148: 2309-2318
[Abstract]
[Full Text]
-
Suzuki, T., Kanagawa, T., Kamagata, Y.
(2002). Identification of a Gene Essential for Sheathed Structure Formation in Sphaerotilus natans, a Filamentous Sheathed Bacterium. Appl. Environ. Microbiol.
68: 365-371
[Abstract]
[Full Text]
-
Seka, M. A., Kalogo, Y., Hammes, F., Kielemoes, J., Verstraete, W.
(2001). Chlorine-Susceptible and Chlorine-Resistant Type 021N Bacteria Occurring in Bulking Activated Sludges. Appl. Environ. Microbiol.
67: 5303-5307
[Abstract]
[Full Text]
Top
Abstract
Introduction
