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INTERNATIONAL
JOURNAL OF SYSTEMATIC
BACTERIOLOGY,
July 1988, p. 303-315
0020-7713/88/030303-13$02.00/0
Copyright 0 1988, International Union of Microbiological Societies
Vol. 38, No. 3
Pedomicrobium americanum sp. nov. and Pedomicrobium
australicum sp. nov. from Aquatic Habitats, Pedomicrobium gen.
emend. and Pedomicrobium ferrugineum sp. emend. -t
RAINER GEBERS” A N D MARITA BEESE
Institut fur Allgemeine Mikrobiologie, Universitat Kiel, Biologiezentrum, 0-2300 Kiel, Federal Republic of Germany
Five new strains of budding bacteria of the genus Pedornicrobiurn were isolated from freshwater habitats.
These strains formed two groups, both of which fitted the description of the genus Pedomicrobium, but their
characteristics did not match the description of any existing species. Pedornicrobiurn americanum is proposed
for strains IFAM G-1381 (= ATCC 43612), IFAM BA-868 (= ATCC 43613), and IFAM BA-869 (= ATCC
43615), with strain IFAM G-1381 as the type strain. Strains IFAM ST-1306 (= ATCC 43611) and IFAM
WD-1355 (= 43614) are named Pedomicrobium australicum, with strain IFAM ST-1306 as the type strain. The
descriptions of the genus Pedomicrobiurn and of the type species Pedomicrobium ferrugineum are emended.
Since the first pedomicrobia were discovered in podzolic
soils by Aristovskaya (1, 2), these hyphal, budding bacteria
have been observed in other types of soil (18, 19) and in
aquatic habitats (16,17, 21). In 1974, iron-depositing strains
were isolated from podzolic soils in northern Germany, and
a manganese-depositing strain was isolated from a quartzite
rock pool in France (8). Subsequently, these strains were
identified as Pedomicrobium ferrugineum and Pedomicrobium manganicum, and an emended description was given
by Gebers (7). Further investigations demonstrated the
genetic relatedness between these Pedomicrobium strains
and their relationship to other genera of hyphal, budding
bacteria (9-12, 20). In these studies, five isolates from
aquatic habitats in North America and Australia (12) proved
to be two new Pedomicrobium species. Their descriptions
and taxonomy are presented here. The two new species also
were compared with “Pedomicrobium podsolicum,” which
was described by Aristovskaya in 1963 (2) but was omitted
from the 1980 Approved Lists of Bacterial Names (23).
MATERIALS AND METHODS
Strains and cultivation. All of the bacterial strains were
supplied by the Culture Collection of the Institut fur Allgemeine Mikrobiologie (IFAM), Kiel, Federal Republic of
Germany (Tables 1 and 2). The origins, isolation, and
cultivation of these strains have been described previously
(5, 7, 12). PYVM medium for strains IFAM BA-869 and
IFAM ST-1306* (T = type strain) contained (per liter) 0.25 g
of peptone (Difco Laboratories, Detroit, Mich.), 0.25 g of
yeast extract (Difco), 10 ml of a vitamin solution (24), 20 ml
of Hutner basal salts (4),and 10 mM DL-malate; the final pH
was 7.5.
Growth characteristics and physiology. Humic gel agar (7)
contained (per liter) 5 g (wet weight) of humic gel (i.e., fulvic
acid iron sesquioxide complexes) and 20 g of Bacto-Agar
(Difco); the final pH was 7.2 to 7.5. Fe(II1) was detected by
the Prussian blue reaction. Mn(1V) was identified with the
benzidine reagent (6). Catalase, cytochrome oxidase, gelatin
liquefaction, and fermentation of glucose were detected as
described by Skerman (22). The growth medium used for
these tests was Pedomicrobium standard medium (PSM) (7)
solidified with 18 g of Bacto-Agar per liter and supplemented
* Corresponding author.
? Dedicated to Peter Hirsch on the occasion of his 60th birthday.
with 2 g of starch per liter or 4 g of gelatin per liter. Nitrate
reduction was demonstrated in PSM to which 10 mM KNO,
and 1.7 g of Bacto-Agar per liter were added. The presence
of nitrite was detected semiquantitatively with Merckoquant
nitrite test sticks (E. Merck AG, Darmstadt, Federal Republic of Germany). Hemolysis was tested on PSM agar supplemented with 100 ml of sheep blood per liter. Amino acid
utilization was determined by comparisons of quantitative
analyses of fresh PSM or PYVM medium and of the same
spent medium after 6 days of growth. Bacterial cells were
removed by centrifugation, and the supernatant was dehydrated in vacuo. The pellets were dissolved in 4 N HCl and
hydrolyzed at 100°C for 16 h. The hydrolysates were dried in
vacuo and were then dissolved in 0.2 N citrate buffer. These
preparations were then run through an amino acid analyzer
(Multichrom B; Beckman Instruments, Inc., Fullerton,
Calif.). All cultures were incubated aerobically in the dark at
30°C.
Stimulation or inhibition of growth by carbon sources and
growth dependence on vitamins, sodium chloride, temperature, pH, or buffer were determined in liquid cultures.
Cultures were pregrown in PSM or PYVM medium. In the
logarithmic growth phase, 1 volume of culture was transferred to 100 volumes of the test medium and incubated.
Again from cultures in the logarithmic growth phase, cell
suspensions were inoculated 1:100 (vol/vol) into fresh test
medium. At the end of the log phase of growth, turbidity,
flocculation, growth on glass walls, and cell morphology
were noted. The cultures were checked for purity by streaking onto PSM and nutrient agar (Difco). For protein determinations 4.5-ml portions of cell suspensions were hydrolyzed in 0.1 N NaOH at 60°C for 90 min. Protein
concentrations were determined in triplicate with the BioRad protein assay (Bio-Rad Laboratories, Munich, Federal
Republic of Germany). The reference protein used was
albumin. The growth of P. ferrugineum IFAM S-1290T in
modified PSM (containing 0.1% yeast extract) supplemented
with various concentrations of acetate, growth in modified
PSM (pH 8.0) supplemented with various concentrations of
HEPES (N-2-hydroxyethylpiperazine-N’-2-ethanesulfon~c
acid) buffer (Sigma Chemie, Taufkirchen, Federal Republic
of Germany), and growth in PSM supplemented with 10 mM
phenol, 10 mM benzoic acid, or 10 mM toluene were
followed optically by using a Klett-Summerson colorimeter.
The generation times in PSM or PYVM medium were
303
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304
INT. J. SYST.BACTERIOL.
GEBERS AND BEESE
TABLE 1. Levels of DN A-DNA homology among Pedornicrobiurn and selected Hyphornicrobium strains
% Homology with labeled DNA from strain:'
Source of unlabeled DNA"
Taxon
P . ferrugineurn
P. arnericanurn
P . australicurn
P. rnanganicurn
Hyphomicrobium
IFAM
strain no.
ATCC
strain no.
IFAM
S-12907'
IFAM
G-1381T
IFAM
BA-869
S-1290T
Q-1197
T-1130
F-1225
BA-869
G-1381T
BA-868
ST-1306T
WD-1355
E-1129=
MC-750
T-854
33119T
33117
33120
33122
43615
43612T
43613
43611*
43614
33121T
27500
100
99'
97'
104'
22
16
17
18
28
82
100
91
25
25
10
14
13
11
14
100
100
94
26
29
7
2 (4)
2
18
18
17
13'
7"(4)J
5
IFAM
ST-1306T
IFAM
E-1129T
14
12
9
31
6
8
8
8
8
100
2 (5)
1
11
5
10
13
20
28
100
87
3
(5)
IFAM, Institut fur Allgemeine Mikrobiologie, Kiel, Federal Republic of Germany; ATCC, American Type Culture Collection, Rockville, Md.
'Mean of at least two reactions corrected for the background values obtained with the self-reassociation controls. The self-reassociation values obtained by
hybridization with unrelated Escherichia coli K-12 DNA were as follows: strain IFAM S-1290T, 6.3%; strain IFAM G-1381T, 1.7%; strain IFAM BA-869, 1.3%;
strain IFAM ST-1306T, 4.7%; strain IFAM E-1129T, 1.7%.
Data from reference 11.
The homology values in parentheses are the values for reciprocal reactions and are included for comparison (10).
determined by following the optical densities of 150-ml
cultures colorimetrically at 620 to 640 nm (Lange, Berlin,
Federal Republic of Germany) for 161 h. Generation times
were calculated from semilogarithmic graphs of optical density versus time.
Antibiotic susceptibility was tested in liquid PSM or
PYVM medium supplemented with 1, 10, or 100 pg of
antibiotic per ml. Ampicillin, penicillin G, cycloserine, polymyxin B, neomycin, chloramphenicol, and streptomycin
were obtained from Serva, Heidelberg, Federal Republic of
Germany, cephalothin was obtained from Sigma Chemie,
and sulfanilamide was obtained from E. Merck AG. Inhibition of growth was calculated from the protein concentrations of triplicate 10-ml test cultures in relation to the protein
concentrations of cultures in the same medium without
inhibitors.
If not stated otherwise, chemicals and vitamins were
obtained from E. Merck AG; calcium panthothenate came
from Sigma Chemie.
Morphology. Cell morphology and fine structure were
investigated by transmission electron microscopy, using a
Philips model EM-300 microscope. Pictures were taken with
type 4489 electron microscope film (Eastman Kodak Co.,
Rochester, N.Y .). Negative staining, platinum-carbon shadowing, and thin-section preparation were performed as described previously (7, 8).
DNA-DNA hybridization. The methods used for cell wall
disintegration and deoxyribonucleic acid (DNA) extraction
and purification have been described previously (12). Shearing of DNA and radioactive labeling were carried out as
described by Gebers et al. (10, 11). DNA reassociation
procedures and S1 nuclease treatment were performed as
TABLE 2. Levels of DNA-DNA homology of P. ferrugineurn IFAM S-1290Twith other hyphal, budding bacteria
Source of unlabeled DNA"
Taxon
IFAM
strain no.
ATCC
strain no.
S-1290T
NQ-521gr
CO-582
1-551
EA-617
WH-563
MEV-533gr
KB-677
CO-558
zv-580
SW-808
SCH-1315
1300
ST-1307
PS-72gT
SCH-1325T
SW-814
LE-670T
117gT
33119T
27483
27492
27489
% Homology with
labeled DNA from
strain IFAM S-1290Tb
~~
P . ferrugineurn
Hyphomicrobium spp.
"Genus F"
"Genus T"
Hyphornonas polyrnorpha
Hyphomonas oceanitis
Hyphornonas sp.
Hyphomonas neptunium
Rhodornicrobiurn vannielii DSM 162T
27488
27498
27491
100
6
5
4
4
3
3
3
3
2
0
33881T
33879T
15444T
17100T
6
4
3
3
3
2
0
0
DSM, Deutsche Sammlung von Mikroorganismen, Gottingen, Federal Republic of Germany.
Mean of at least two reactions corrected for the background values obtained with the self-reassociation controls. The self-reassociation value obtained by
hybridization with unrelated E. coli K-12 DNA was 6.3%.
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VOL.38, 1988
PEDOMICROBIUM SPP.
305
TABLE 3. Conditions used for DNA-DNA hybridization reactions
Labeled DNA
from strain:
IFAM
IFAM
IFAM
IFAM
IFAM
S-1290T
G-1381T
BA-869
ST-1306T
E-1129T
T,, temp
in o.17
NaCl (oc)
Radioactive
95.5
95.0
95.1
95.4
95.2
3H
'I
'*'I
*''I
'1
Sp act after
labeling
(cp;~~,) of
Sp act for Amt of labeled
hybridization DNA per
(cpm/pg of hybridization
DNA)
reaction (pg)
Ratio of
labeled DNA
to unlabeled
DNA
19,348
2.40 X lo6
7.44 x lo6
2.99 x lo6
2.73 x lo6
19,348 0.12 or 0.19
2.40 X lo6
0.10
0.77 x lo6
0.10
2.99 x lo6
0.10
0.79 x lo6
0.10
1:1,250 or 1:790
1:1,500
1:1,500
1:1,500
1:1,500
HL:g-
Iwa&a;gn
temp
(u)
rC)
65
70
70
10
70
~~~1~~~
16
20
20
20
20
1,250'
100d
50d
100"
SO@
Incubation Filter
temp K) type
51
56
56
56
56
GFIC'
BA83'
BA83'
BA83'
BA83'
Amersham Buchler, Braunschweig, Federal Republic of Germany.
Miles Laboratories, Inc., Elkhart, Ind.
Whatman, Maidstone, Kent, United Kingdom.
Bethesda Research Laboratories, Karlsruhe, Federal Republic of Germany
Schleicher & Schiill, Dassel, Federal Republic of Germany.
Sigma Chernie, Taufkirchen, Federal Republic of Germany.
described previously (10). The specific conditions used for
hybridization reactions are shown in Table 3.
same time. Mature buds either separate from the hyphae as
uniflagellated swarmers or remain attached. Buds may arise
in an intercalary fashion by localized hyphal swelling. Occasionally, direct budding of mother cells or division of
single mother cells is observed (1, 3).
(ii) Cellular structure and composition. Cells stain gram
negative, although older cells may stain gram variable.
Extracellular polymers stain with ruthenium red. Oxidized
iron compounds or manganese compounds or both are
deposited primarily on mother cells and also on hyphae.
Intracytoplasmic membranes are observed in cells of manganese-depositing species. Up to three granules of poly-phydroxybutyric acid are stored per cell. Small, dense granules stainable with Loefffer methylene blue suggest the
presence of polyphosphates. Liquid cultures grown in the
dark and in synthetic light may be yellowish white and
reddish orange, respectively.
(iii) Colonial morphology. Two colony types may develop
on solid media. Type 1 colonies are round and convex and
have entire or rhizoid edges; they are yellowish red to dark
brown, sometimes with concentric rings. These colonies
have a soft consistency. Type 2 colonies are round and flat;
they pit the agar so that their surfaces remain near the agar
level. Their edges are entire or rhizoid. Type 2 colonies are
yellowish red to dark brown, and the cells in the center of the
colonies are often lysed, giving a granular appearance to the
colonies. These colonies have a cartilaginous consistency
and may be removed from the agar intact.
After restreaking on solid media, type 1 or 2 colonies give
rise to both types of colonies. With periodic transfers to
fresh medium type 1 colonies tend to predominate.
RESULTS AND DISCUSSION
The description of the genus Pedomicrobium (7) was
based on six strains of P . ferrugineum and one strain of P .
manganicum. Five new strains from aquatic habitats exhibit
the major characteristics of the genus; i.e., the cells form
threadlike outgrowths (hyphae), multiply by budding, and
produce extracellular polymers that serve as matrices for
deposition of iron oxides or manganese oxides or both.
However, these strains are unlike the previously described
species P . ferrugineum and P , manganicum, as well as " P .
podsolicum" (2, 3 ) and "P.manganicum subsp. sacchalinicum" (18, 19).
Our data (see below) allow emendation of the genus
description, an emended description of the type species, P .
ferrugineum, and descriptions of two new species.
Pedomicrobium Aristovskaya 1961 (Gebers 1981) genus
emend. (i) Morphology. Cells are spherical, oval, tetrahedral,
or rod, pear, bean, or spindle shaped. Up to five or more
prosthecae (i.e., cellular outgrowths [hyphae] of constant
diameter) are formed per cell; these are 0.15 to 0.3 pm in
diameter and vary in length with cultural conditions. At least
one hypha originates laterally; other hyphae may appear
polarly or subpolarly. True branching of the hyphae occurs.
Multiplication is primarily by budding at the hyphal tips,
where local swellings form young buds which elongate
perpendicularly to the hyphal axis and enlarge up to the size
of a mother cell. Hyphal insertion of the buds is lateral in all
cases. Up to three buds per mother cell may occur at the
TABLE 4. Growth in PYVM medium or in PSM with or without buffers added"
Buffer added to broth
shift
PH
None
10 mM HEPES + NaOH
35 mM Tris + HCl'
None
6.618.7
7.017.5
7.017.7
7.518.5
P . austrcllicum strains
P . nmerictinum strains
P . ferrugineum
IFAM s-1290T
IFAM G-1381T
Relative
growth
Relative
growth
(%)
(%I
100
71
52
67
100
44
57
100
6.618.8
1.017.5
7.018.0
7.518.9
IFAM BA-868
gt:
6.118.5
1.017.2
6.817.0
7.518.5
Relative
growth
(%I
100
18
3
52
IFAM BA-869h
gt:
Relative
growth
IFAM ST-130bTh
PH
shift
97
100
49
90
IFAM WD-1355
i:t
Relative
gr,ooth
6.718.3
7.017.1
6.817.0
7.5J8.5
50
8
2
100
(%I
(%I
6.918.8
6.818.6
6.917.4
7.619.0
Relative
growth
6.918.9
6.818.3
6.917.1
7.619.0
loo
53
23
97
Growth was calculated individually for each strain relative to the culture yielding maximum protein (= 100%). pH shift, pH of medium shortly after
inoculation/pH after 6 to 14 days.
Strains IFAM BA-869 and IFAM ST-13MT were grown in PYVM medium.
Tris, Tris( hydrox ymet hy1)aminomethane.
(I
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306
INT.
GEBERS AND BEESE
J.
SYST.
BACTERIOL.
1
1501
h
.-cnc
c
3
-Y
b
c
Q
Y
L.
.
I
2
=8
I
8
*-
b
%
260
5'
0
m
40
I 0
300
1
600
1
50
I 0
time(h)
0
I
5'
0
I
I
100
200
I
time (h)
FIG. 1. Growth of P . ferrugineurn IFAM S-1290T in modified
PSM (pH 8) supplemented with HEPES buffer. Symbols: A, no
HEPES added, pH 8.7 at the end of growth; A,50 mM HEPES, pH
8.5; B, 100 mM HEPES, pH 7.9. 0, 200 mM HEPES, pH 7.9.
(iv) Physiological properties. Cells are microaerophilic to
aerobic and heterotrophic. Slow, poor growth occurs on 0.1
to 1%fulvic acid iron sesquioxide complexes as sole carbon
and nitrogen sources; good growth occurs with 10 mM
acetate, 10 mM malate, 10 mM succinate, or 10 mM gluconate as the carbon source and a nitrogen source such as
FIG. 2. Growth of P . ferrugineum IFAM S-1290T in PSM supplemented with aromatic compounds. Symbols: x , no supplement,
pH 8.7 at the end of growth; 0 , l O mM phenol added; pH 8.6; 0 , l O
mM benzoic acid, pH 8.9; B, 10 mM toluene, pH 8.8.
yeast extract, peptone, Casamino Acids (Difco), or Soytone
(Difco) at a concentration of 0.05%.
Vitamin mixtures stimulate growth; lack of vitamins produces pleomorphic cells. Good growth occurs in the presence of 0.1% NaC1; no growth, but survival, occurs in the
presence of 1or 2.5% NaCl; 5% NaCl is bactericidal for the
species that have been described (7).
Gelatin and starch are not hydrolyzed. Neither acid nor
gas is produced anaerobically from glucose. Anaerobic
growth is not observed in stab cultures. Catalase is pro-
TABLE 5. Growth on carbon sources"
Growth index for:
Carbon source
(10 mM)'
P . umericunum strains
P. ferrugineurn
IFAM S-1290T
IFAM G-1381'
IFAM BA-868
21
100
24
100
17
100
15
Oi
16
30
33
13
43
7i
21
102
36
17
12i
29
41
8i
28
P . uustrulicum strains
IFAM BA-869
IFAM ST-1306T
IFAM WD-1355
49
100
1%
42i
44i
51
109
56
63
20i
74
156
89
34
NT
1Oi
32i
52
NT
6
100
24
Oi
18
15
25
4
Oi
7
23
30
4
12
Oi
11
56
2i
18
~
ControlC
Acetate
Caproate
Citrate
Ethanol
Formate
Gluconate
D-Glucose
Glycerol
D-Lactate
Lactose
DL-Malate
D-Mannitol
Methanol
Phenol
Propanol
Pyruvate
D-Ribose
Succinate
a
44
3i
1Oi
14i
18
1li
Oi
8i
15i
22
24
19
Oi
8i
70
lli
37
5id
Oi
5i
30
35
6i
30
20
7i
23
21
21
1Oi
8i
60
5i
66
61
100
14i
92
44i
75
84
4%
69
23i
41i
121
52i
37i
NT'
58
39i
48i
108
A growth index was calculated from protein measurements individually for each strain as a percentage of the growth in medium to which acetate was added.
' Carbon sources were added to PYVM medium (for strains IFAM BA-869 and IFAM ST-13MT) or to PSM without acetate (for strains IFAM S-12!NT, IFAM
G-1381T, IFAM WD-1355, and IFAM BA-868).
No additional carbon source was added to the basic media.
i, Inhibition of growth compared with control.
' NT, Not tested.
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VOL. 38, 1988
PEDOMICROBIUM SPP.
307
TABLE 6. Utilization of amino acids"
% Remaining after 6 days of growth
Amino acid
DL-Alanine
Ammonia
L-Arginine
L-Aspartic acid
L-Glutamic acid
Glycine
L-Histidine
L-Isoleucine
L-Leucine
L-Lysine
L-Pheny lalanine
L-Proline
L-Serine
L-Threonine
L-Tyrosine
D-Valine
P . americanum strains
P . austrulicum strains
P.
ferrugineum
IFAM S-1290T
IFAM G-1381T
IFAM BA-868
IFAM BA-869
IFAM ST-1306T
IFAM WD-1355
18
62
50'
36
24
46
44
22
18
54
27
56
35
39
55
13
14
57
26'
34
21
47
40
19
17
50
14
99
32
38
41
10
21
67
37'
36
25
52
71
22
20
51
22
42
36
41
78'
13
64
68
62
82
32
49
22
42
82
75
86
36
72
57
57
ND
56
23
67
84'
42
45
47
75
106
88
102
92
65
73
71
56
91
55
69
56
49
19
28
29
37
55
30
76
59
53
ND'
25
Avg
35
62
58
39
36
39
50
47
43
66
37
68
49
50
58
35
" Quantitative amino acid analysis of fresh PSM or PYVM medium (strains IFAM BA-869 and IFAM ST-1306T) compared with the same spent medium. The
amino acid concentrations in the fresh media were taken t o be 100%.
Approximate values.
ND, Not determined.
TABLE 7. Inhibition of growth by antibiotics
~~
% Inhibition of growth of"
Antibiotic
Ampicillin
Penicillin G
Cephalothin
Cycloserine
Polymyxin B
Neom y cin
Chloramphenicol
Streptomycin
Sulfanilamide
Concn
(&ml)
1
10
100
1
10
100
1
10
100
1
10
100
1
10
100
1
10
100
1
10
100
1
10
100
1
10
100
P. ferrugineum
IFAM S-1290T
P . uustrulicum strains
P . americunum strains
IFAM G-1381T
IFAM BA-868
IFAM BA-869
IFAM ST-1306'
IFAM WD-1355
19
91
95
0
0
93
0
0
77
16
89
95
80
17
74
91
10
59
90
19
45
26
43
91
23
98
97
0
41
59
59
92
33
0
17
94
18
+
+
+
3
78
93
73
73
97
4
60
68
63
90
96
75
61
91
91
69
78
95
76
75
84
71
77
95
55
89
82
81
84
74
83
79
85
76
86
85
84
43
90
92
84
88
84
85
82
74
94
98
73
69
77
90
84
5
47
88
78
83
80
85
83
76
78
98
84
77
96
95
95
81
85
82
85
82
5
3
16
0
0
3
16
20
22
48
49
47
13
13
8
15
19
33
22
65
82
3
82
87
18
81
+b
12
73
82
5
82
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
" Inhibition was evaluated by comparing the protein concentrations in cultures grown in the presence of antibiotics with the concentrations in untreated
cultures.
Higher concentrations of cephalothin, polymyxin B, and streptomycin caused a yellow color reaction during protein hydrolysis with 0.1 N NaOH. Inhibition
was estimated by visual comparison of culture turbidity. +, Strong inhibition (approximately 60 to 100%).
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308
INT. J. SYST.BACTERLOL.
GEBERS AND BEESE
FIG. 3. Electron micrograph of p . americanum IFAM G-1381*
negatively stained with 0.5% phosphotungstic acid: oval-shaped
imother cell with lateral hypha and bud. The arrows indicate extracellular polymers incrusted with iron oxides or manganese oxides or
both. Bar = 1 bm.
FIG. 5 . Electron micrograph of P. arnericanurn IFAM G-1381T
negatively stained with 0.5% phosphotungstic acid: mother cell with
polar hypha and bud. Note beginning outgrowth of second, subpolar
hypha (arrow). Bar = 1 wn.
duced. On PSM agar supplemented with sheep blood (100
mlAiter), all strains grow well but are not hemolytic.
(v) Occurrence. These organisms are widely distributed in
podzolic and other soils, freshwater habitats, iron springs,
and seawater; they are probably ubiquitous.
The DNA base composition (four species, 11 strains)
ranges from 63 to 66 mol% guanine plus cytosine (G+C) as
determined by the thermal denaturation (T,) method.
The levels of DNA-DNA homology of P . ferrugineum
IFAM S-1290Tto representative strains of other genera of
hyphal, budding bacteria are less than 7% (Table 2).
The type species is P . ferrugineum Aristovskaya 1961.
P . ferrugineum Aristovskaya 1961 emend. Cells are spherical, oval, tetrahedral, or rod or bean shaped and are 0.6 to
2 by 0.6 to 2.5 pm. At least one hypha originates laterally;
less frequently, other hyphae appear polarly or subpolarly.
Multiplication is by budding at the tips of the hyphae which
are 0.2 pm or less in diameter. Initially, buds are spherical to
oval; they are motile by a single polar to subpolar flagellum.
Occasionally, intercalary buds are observed. Deposition of
oxidized iron compounds occurs when cells are grown with
fulvic acid iron sesquioxide complexes, elemental iron powder, FeS, or iron paper clips; there is no accumulation of
manganese compounds. Depositions occur primarily on
mother cells and later on hyphae. Intracytoplasrnic membranes have not been found in ultrathin sections of strain
IFAM S-1290T.The temperature for optimum growth is 29 to
30°C; the temperature range is 18 to 43°C. The pH range for
growth is 6.6 to 8.7 (Table 4). P. ferrugineum IFAM S-1290T
grows in 50 mM HEPES-buffered PSM (Fig. 1); however,
100 and 200 mM HEPES prevent growth. Growth in unbuffered PSM at pH 6.6 gives higher protein yields than growth
in PSM buffered with 10 mM HEPES or 35 rnM tris(hydroxymethy1)aminomethane hydrochloride (Table 4). Growth in
PSM containing 10 mM HEPES is best at an initial pH of 8.1.
During growth, the pH of all cultures increases.
The generation time of strain IFAM S-1290T in PSM is 10
h at 30°C. The concentration of acetate in modified PSM
(containing 0.1% yeast extract) influences the generation
time and maximum optical density of P. ferrugineum
cultures; 20 mM acetate results in a minimum generation
time of 15.5 h and an optical density of 210 Klett units. With
30 mM acetate, the generation time is 20.5 h, and a value of
240 Klett units is reached; however, cells of the latter
cultures produce large granules of poly-P-hydroxybutyric
acid.
FIG. 4. Electron micrograph of P. americanum IFAM BA-869
negatively stained with 0.5% phosphotungstic acid: tetrahedral
mother cell with branching hypha. Bar = 1 pm.
FIG. 6. Electron micrograph of P. arnericanurn IFAM G-1381T
negatively stained with 0.5% phosphotungstic acid: separated
daughter cell in the flagellated swarmer stage. Note the subpolarly
inserted flagellum and the separation nose (arrow), where the former
mother cell hypha was attached. Bar = 1 pm.
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VOL. 38, 1988
PEDOMICROBIUM SPP.
309
FIG. 7. Thin section of P.arnericanurn IFAM G-1381T. Note the large granule of poly-P-hydroxybutyric acid (PHB) and the extracellular
polymers incrusted with iron oxides or manganese oxides or both (arrow), as well as the section through the branching hypha. Bar = 0.5 pm.
Good growth occurs with 10 mM fumarate or 10 mM
glutamate. Slow, poor growth occurs with 10 mM oxalate, 10
mM tartrate, 10 mM cholesterol, 10 mM aspartate, 10 mM
tryptophan, 10 mM alanine, or 0.3% paraffin. No growth
occurs with 10 mM galactose, 10 mM fructose, 10 mM
maltose, 10 mM sucrose, 10 mM phenylalanine, 10 mM
serine, or 10 mM lysine (7). The utilization of other substrates is shown in Table 5. Amino acids are utilized simultaneously as nitrogen sources (7).
The growth of strain IFAM S-1290Tin PSM supplemented
with 10 mM phenol, 10 mM benzoic acid, or 10 mM toluene
was monitored by determining optical density values. In
these cultures, the lag phase was increased compared with
cultures in pure PSM (Fig. 2). Benzoic acid and toluene
reduced the growth of strain IFAM S-1290T only to a small
extent, whereas phenol caused a strong inhibition. A second
transfer to PSM supplemented with 10 mM phenol completely prevented growth (Table 5 ) .
Good growth occurs with 0.05% yeast extract or 0.05%
peptone as a nitrogen source. Within 6 days, strain IFAM
S-1290T removes 44 to 87% of the amino acids from PSM
culture broth (Table 6). Slow, poor growth occurs with
0.05% Soytone, 0.05% Casamino Acids, 10 mM 2-aminobenzoic acid, 10 mM NaNO,, 10 mM acetamide, 10 mM urea, 10
mM N-acetylglucosamine, or 10 mM methylamine hydrochloride; and no growth occurs with 0.01 M NaNO,, 0.58 M
(4%) NaNO,, or 1.06 M (9%) NaNO, (7).
A vitamin mixture (24) or 1 pg of cyanocobalamin per liter
is required for good growth. The presence of 0.1% NaCl
stimulates growth.
The cells are positive for cytochrome oxidase and for
reduction of nitrate to nitrite.
FIG. 8. Electron micrograph of P . australicitrn IFAM ST-1306=,
Pt-C shadowed: oval mother cell with polar hypha and spindleshaped bud. Bar = 5 pm.
FIG. 9. Electron micrograph of P . austraficitm IFAM WD-1355,
Pt-C shadowed: spindle-shaped mother cell with lateral hypha and
bud. Bar = 1 pm.
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310
INT. J . SYST.BACTERIOL.
GEBERS AND BEESE
FIG. 10. Electron micrograph of P. australicum IFAM ST-1306T
negatively stained with 0.5% phosphotungstic acid: mother cell with
one lateral b p h a and one polar hypha. Note the terminal swelling of
the polar hypha. Bar = 1 pm.
FIG, 12. Electron micrograph of P . australicum IFAM ST-1306T
negatively stained with 0.5% phosphotungstic acid: mother cell with
hypha branching twice and with three buds at different developmental stages. Bar = 1 pm.
P.ferrugineum strains are not pathogenic for guinea pigs
after intraperitoneal injection of lo8 cells (7).
In addition to antibiotic susceptibilities in liquid cultures
(Table 7), cells of strain IFAM S-1290T are susceptible in
agar diffusion tests (7) to rifampin, tetracycline, gentamicin,
and nitrofurazone,
The DNA base composition of the type strain is 65 mol%
G+C (TnI method) (7, 9). The average DNA base composition of four additional strains is 65 mol% G+C (T, method).
These strains exhibit levels of DNA-DNA homology of 97 to
104% to P. ferrugineum IFAM S-1290T (11). The levels of
DNA-DNA homology to the other Pedomicrobium species
are listed in Table 1.
All strains were isolated from podzolic soils in northern
Germany (8).
The type strain is strain IFAM S-1290 (= ATCC 33119 =
DSM 1540). Other strains include strains IFAM P-1196 (=
ATCC 33116 = DSM 1541), IFAM Q-1197 (= ATCC 33117
= DSM 1542), IFAM R-1198 (= ATCC 33118 = DSM 1543),
and IFAM T-1130 (= ATCC 33120 = DSM 1544).
Pedomicrobium americanum sp. nov. Cells are oval (Fig. 3),
tetrahedral (Fig. 4), or short rod, bean, or spindle shaped and
are up to 1.3 by 1.8 km. Up to three hyphae of various
lengths originate laterally or polarly with similar frequency
(Fig. 5). True branching of the hyphae occurs (Fig. 4).
Multiplication is by budding at the hyphal tips (Fig. 3 and 5).
Occasionally, intercalary buds are observed. Mature buds
separate from the hyphae as motile swarmers with one
subpolar flagellum (Fig. 6). Direct budding or division of
single mother cells has not been observed.
The cells have gram-negative cell walls (Fig. 7). Extracellular structures appear primarily on cell surfaces opposite
the lateral hyphae (Fig. 3 and 7). These structures have been
identified as polyanionic, ruthenium red- and Alcian bluestaining polymers which form fine filaments or arrays of
particles (7, 14, 15) and provide the matrix for incrustations
of iron oxides and manganese oxides. Deposition of iron
compounds occurs when cells are grown on fulvic acid iron
sesquioxide complexes or on PSM agar containing 5 mg of
FeSO, . 7H,O per liter. Deposition of manganese(1V) oxides
occurs on PSM agar containing 1.54 mg of MnSO, . H,O per
liter. Intracytoplasmic membranes are present. Granules of
poly-P-hydroxybutyric acid appear frequently in the cytoplasm (Fig. 7). Polyphosphate granules are present.
FIG. 11. Electron micrograph of P . uustralicum IFAM ST-1306T
negatively stained with 0.5% phosphotungstic acid: mother cell with
branching hypha and two buds. Bar = 1 pm.
FIG. 13. Electron micrograph of P. uustralicum IFAM ST-1306T
negatively stained with 0.5% phosphotungstic acid: hypha showing
local swellings, one of which formed an intercalary bud (arrow). Bar
= 1 pm.
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VOL.38. 1988
PEDOMICROBIUM SPP.
FIG. 14. Electron micrograph of P . australicum IFAM ST-1306T
negatively stained with 0.5% phosphotungstic acid: motile swarmer
cell with subpolarly inserted flagellum. The arrow indicates the basal
hook of the flagellum. Bar = 1 pm.
311
FIG. 16. Thin section of P . australicum IFAM ST-1306T. Note
the intracytoplasmic membranes in the transverse section of the left
cell. Bar = 0.5 pm.
FIG. 15. Thin section of P . australicum IFAM ST-1306T.Note the intracytoplasmic membranes and extracellular polymers incrusted with
iron oxides or manganese oxides or both (arrows). Bar = 0.5 pm.
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312
INT. J. SYST.BACTERIOL.
GEBERS AND BEESE
FIG. 17. Thin section of P . australicum IFAM WD-1355. Note the intracytoplasmic membranes which formed cell compartments;note the
section through the separation nose and the extracellular polymers incrusted with iron oxides or manganese oxides or both (arrows). Bar =
0.5 pm.
The optimal growth temperature is 32 to 38°C; the temperature range is 15 to 41°C. The pH range for optimal growth in
unbuffered media is 6.6 to 7.5; during growth, the pH
increases (Table 4). The optimal pH of buffered media
(containing 10 mM HEPES) is 7.6 to 8.4.
The generation times in PSM or PYVM medium are 10 to
20 h.
Stimulation or inhibition of growth by various carbon
sources differs for individual strains (Table 5). Good growth
occurs with 0.05% yeast extract or 0.025% yeast extract plus
0.025% peptone; nitrate does not serve as a sole nitrogen
source. Within 6 days, the strains of P . americanurn remove
1 to 90% of the amino acids from PSM or PYVM culture
broth (Table 6).
A vitamin mixture (24) or 1 pg of cyanocobalamin per liter
is required for growth of strains IFAM G-1381Tand IFAM
BA-868 and stimulates the growth of strain IFAM BA-869.
The presence of 0.1% NaCl stimulates the growth of strain
IFAM BA-868.
Catalase is produced by strains IFAM G-1381T and IFAM
BA-868, but not by strain IFAM BA-869. All strains are
positive for cytochrome oxidase and for reduction of nitrate
to nitrite.
The strains of P . americanum are susceptible to several
antibiotics (Table 7); only strain IFAM G-1381T is resistant
to sulfanilamide.
The DNA base compositions of strains IFAM G-1381T,
IFAM BA-868, and IFAM BA-869 are 64,65, and 64 mol%
G+C, (Tnzmethod) (12), respectively. The DNA relatedness
of the three strains is shown in Table 1.
All strains are from freshwater habitats in North America
(12); strain IFAM G-1381Twas isolated from pond water in
TABLE 8. Differential characteristics of Pedomicrobium species
Species
P . ferrugineum"
P . americanum
P . australicum
P . manganicum"
Deposition Of iron ('I1)
during growth on
humic gel agar
+
+
+
-
Deposition of manganese (IV)
during growth on PSM agar
Intracytoplasmic
membranes
present
-
-
+
+
+
+
+
+
'' Data from reference 7.
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Spindle-shaped
cells occur
Spherical
cells
occur
-
+
PEDOMICROBIUM SPP.
VOL.38, 1988
FIG. 18. Thin section of P. australicum IFAM WD-1355. Note
the invagination of the cytoplasmic membrane and the mesosomelike cell compartment. Bar = 0.2 pm.
Sapsucker Woods, Ithaca, N.Y., by E. Dale and strains
IFAM BA-868 and IFAM BA-869 were isolated from a
temporary puddle by J. A. Babinchak in Ann Arbor, Mich.
The type strain is strain IFAM G-1381 (= ATCC 43612).
Other strains include strains IFAM BA-868 (= ATCC 43613)
and IFAM BA-869 (= ATCC 43615).
Pedomicrobium australicum sp. nov. Cells are oval (Fig. 8),
tetrahedral, or short rod, bean, or spindle shaped (Fig. 9) and
313
are up to 1.2 by 1.8 bm. Up to three hyphae of varying
lengths originate laterally or polarly with similar frequency
(Fig. 10). True branching of the hyphae occurs (Fig. 11 and
12). Multiplication is by budding at the hyphal tips (Fig. 8
through 10). Up to three buds per mother cell may occur at
the same time (Fig. 11 and 12). Occasionally, intercalary
buds are formed (Fig. 13). Mature buds separate from the
mother hyphae as motile swarmers with one subpolar flagellum (Fig. 14).
The cells have a gram-negative cell wall structure (Fig. 15
and 16). Extracellular polymers appear primarily on cell
surfaces opposite the lateral hyphae (Fig. 15 and 17). Deposition of iron compounds occurs when cells are grown on
fulvic acid iron sesquioxide complexes or on PSM agar
which contains 5 mg of FeSO, . 7H,O per liter. Deposition
of manganese(1V) oxides occurs on PSM agar which contains 1.54 mg of MnSO, . H,O per liter. Intracytoplasmic
membranes appear to form compartments within the cells
(Fig. 17 and 18). Involvement of these membranes in the
oxidation or deposition of manganese remains questionable
(13). Granules of poly-P-hydroxybutyric acid and polyphosphate occur in the cytoplasm (Fig. 19).
The optimal growth temperature is 29 to 32°C; the temperature range is 15 to 36°C. The pH for optimal growth in
unbuffered media is 6.9 to 7.5; during growth, the pH
increases (Table 4). The optimal pH of buffered media is 7.3
to 7.6.
The generation times in PSM or PYVM medium are 11to
26 h.
Stimulation or inhibition of growth by various carbon
sources differs between strains (Table 5). Good growth
occurs with 0.05% yeast extract or 0.025% yeast extract plus
0.025% peptone; nitrate does not serve as a sole nitrogen
source. Within 6 days, strains IFAM ST-1306T and IFAM
WD-1355 utilize 8 to 78% of the amino acids from PSM or
PYVM culture broth (Table 6). Strain IFAM WD-1355 does
not use isoleucine and lysine. Good growth of strain IFAM
WD-1355 depends on the presence of 1 pg of cyanocobalamin per liter. The presence of 0.1% NaCl stimulates the
growth of strain IFAM WD-1355. Both strains are positive
for cytochrome oxidase and for reduction of nitrate to
nitrite.
The strains are susceptible to several antibiotics (Table 7);
sulfanilamide causes only little inhibition of growth.
The DNA base compositions of strains IFAM ST-1306T
and IFAM WD-1355 are 65 and 63 mol% G + C (T, method)
(12), respectively. The DNA relatedness of the strains is
shown in Table 1.
FIG. 19. Thin section of P. australicum IFAM WD-1355. Note the transparent granule of poly-p-hydroxybutyric acid (PHB) (arrow) and
the dark, electron-dense granule of polyphosphate (PP) (arrow) within the mother cell, as well as the dark genome within the bud. Bar = 0.5
CLm.
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INT. J. SYST. BACTERIOL.
GEBERS AND BEESE
TABLE 9. Compositions of optimal nutrient media for Pedomicrobium strains
Strain
IIFAM S-1290T
IFAM G-1381T
lFAM BA-868
Carbon
source (10
mM)
lFAM BA-869
Acetate
Acetate
Acetate or
malate
Malate
IFAM ST-1306T
Malate
[FAM WD-1355
Acetate
Nitrogen source
Vitamin source"
Mineral salts
sourceb
Sodium
chloride
concn
(g/liter)
medium
pH of 10 mM
HEPESbuffered
medium
pH of
Yeast extract (0.5 g/liter)
Yeast extract (0.5 g/liter)
Yeast extract (0.5 g/liter)
Mixture (10 ml)
Mixture (10 ml)
Mixture (10 ml)
Metals 44 (1ml)
Metals 44 (1 ml)
Metals 44 (1ml)
1
0
1
6.6
6.6 or 7.5
6.7
8.1
8.4
7.7
Yeast extract (0.25 g/liter)
+ peptone (0.25 g/liter)
Yeast extract (0.25 g/liter)
+ peptone (0.25 g/liter)
Yeast extract (0.5 g/liter)
Mixture (10 ml)
HBM (20 ml)
0
6.9
7.6
Mixture (10 ml)
HBM (20 ml)
0
6.9
7.6
Cyanocobalamin
(1 Fg/liter)
Metals 44 (1 ml)
1
7.5
7.3
a The vitamin mixture (24) contained (per liter) 2 mg of D-( +)-biotin, 2 mg of folic acid, 5 mg of thiamine hydrochloride, 5 mg of calcium-D-(+)-panthothenate,
0.1 mg of cyanocobalamin, 5 mg of riboflavin, 5 mg of nicotinic acid, 5 mg of para-aminobenzoic acid, and 10 mg of pyridoxin hydrochloride.
Metals 44 was a mineral salts solution (4) containing (per liter) 10.95 g of ZnSO, . 7H20, 5 g of FeSO, . 7H20, 1.54 g of MnSO, . H,O, 392 mg of
CuSO, . 5H20, 248 mg of C O ( N O ~.)6H20,
~
177 mg of Na2B40, . 10H,O, and 2.5 g of ethylenediaminetetraaceticacid. HBM was a mineral salts solution (4)
containing (per liter) 29.7 g of MgSO, . 7H20, 3.34 g of CaCI, . 2H,O, 99 mg of FeS04 . 7H,O, 9.25 mg of (NH4)6M07024
. 4H20, 10 g of nitrilotriacetic acid, and
50 ml of metals 44 solution. The pH was adjusted with KOH to 6.8.
Both strains were isolated from a freshwater reservoir in
New South Wales, Australia, by J. T. Staley (12, 25).
The type strain is strain IFAM ST-1306 (= ATCC 43611).
The other strain which has been identified is strain IFAM
WD-1355 (= ATCC 43614).
Differentiation of Pedomicrobiurn species is based primarily on morphological characteristics (Table 8) and on DNADNA homologies (Table 1). All of the Pedomicrobiurn
strains that have been described so far have similar morphologies, biochemical capabilities, and antibiotic susceptibilities. With regard to carbon source preference, amino acid
utilization, optimal pH, and incubation temperature, each of
the strains exhibits its individual pattern, so differentiation
among species is unreliable on these bases. Suggested media
for the species are shown in Table 9.
ACKNOWLEDGMENTS
We are grateful to P. Hirsch and J. L. Johnson for advice and
fruitful discussions and to U. Wehmeyer for technical assistance.
We thank W. Liesack for performing the amino acid analyses.
Without the donation of bacterial strains by J. A. Babinchak, W. C,
Ghiorse, P. Hirsch, and J. T. Staley, this study would not have been
possible.
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