Arch Microbiol (2012) 194:567–574
DOI 10.1007/s00203-012-0790-5
ORIGINAL PAPER
Phylogeny and photoheterotrophy in the acidophilic phototrophic
purple bacterium Rhodoblastus acidophilus
Megan L. Kempher · Michael T. Madigan
Received: 3 August 2011 / Revised: 3 January 2012 / Accepted: 5 January 2012 / Published online: 29 January 2012
© Springer-Verlag 2012
Abstract Norbert Pfennig isolated the Wrst acidophilic
purple bacterium over 40 years ago and named the organ-
ism Rhodopseudomonas acidophila (now Rhodoblastus
acidophilus). Since the original work of Pfennig, no sys-
tematic study has been conducted on the phylogeny and
carbon nutrition of a collection of strains of Rbl. acidophi-
lus. We have isolated six new strains of Rbl. acidophilus
from a Canadian peat bog. These strains, three of the origi-
nal Pfennig strains and two additional putative R. acidophi-
lus strains isolated several years ago in this laboratory,
were characterized as to their pigments, phylogeny, and
carbon sources supporting photoheterotrophic growth.
Phototrophic cultures were either purple or orange in color,
and the color of a particular strain was linked to phylogeny.
As for the Pfennig strains of Rbl. acidophilus, all new
strains grew photoheterotrophically at pH 5 on a variety of
organic and fatty acids. However, in addition to methanol
and ethanol, the new strains as well as the Pfennig strains
grew on several other primary alcohols, results not reported
in the original species description. Our work shows that
some phylogenetic and physiological diversity exists within
the species Rbl. acidophilus and supports the observation
that few species of acidophilic purple bacteria appear to
exist in nature.
Keywords Purple nonsulfur bacteria · Acidophiles ·
Rhodoblastus acidophilus
Communicated by Jörg Overmann.
M. L. Kempher · M. T. Madigan (&)
Department of Microbiology, Southern Illinois University,
1125 Lincoln Dr., Carbondale, IL 62901, USA
e-mail: madigan@micro.siu.edu
Introduction
Anoxygenic phototrophic purple nonsulfur bacteria
(PNSB) Xourish in a variety of illuminated anoxic aquatic
and terrestrial habitats (ImhoV 1995; Madigan and Jung
2009). All known PNSB are species of either Alpha- or
Betaproteobacteria and contain bacteriochlorophyll a (or
rarely b) and a series of carotenoids as photosynthetic pig-
ments (ImhoV 1995). PNSB have the most diverse metabo-
lism of all phototrophic bacteria in that growth can occur
phototrophically with either organic compounds or CO2 as
carbon sources, or chemotrophically by respiration or fer-
mentation (ImhoV 1995; Madigan 1988; Madigan and Gest
1979; Madigan and Jung 2009).
Many PNSB inhabit extreme environments. PNSB have
been isolated from acidic, alkaline, cold, hot, and hypersa-
line habitats (Madigan 2003; Madigan and Jung 2009).
Over 40 years ago the great German microbiologist Norbert
Pfennig Wrst isolated several strains of acidophilic PNSB
from water samples of various lakes, swamps, bogs, and
springs in Europe and the US. The strains were described as
a new species of the genus Rhodopseudomonas, Rps. acido-
phila (Pfennig 1969). Once its phylogeny was understood,
the organism was elevated to its own genus as Rhodobla-
stus (Rbl.) acidophilus (ImhoV 2001). The pH optimum for
growth of Rbl. acidophilus was pH 5.8 and the pH range
from 4.8 to 7.2 (Pfennig 1969). Although some other PNSB
can grow under slightly acidic conditions (Madigan 2003),
only Rbl. acidophilus appeared in enrichments established
at pH 5.2, suggesting that it was the major acidophilic pur-
ple bacterium in the habitats surveyed by Pfennig.
In the original description of Rbl. acidophilus, two major
diVerences were reported among strains: the color of photo-
trophic mass cultures, and the G + C base ratios of their
DNA. Phototrophic cultures of the various strains of Rbl.
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acidophilus grown in dim light were either purple-red or
orange-brown in color, and their genomic DNA G + C base
ratios varied over a wide range, from as low as 62.2 to as
high as 66.8 mol% (Pfennig 1969). The base ratio data sug-
gested to us that additional genetic and phenotypic diversity
might exist within the species Rbl. acidophilus. Our objec-
tives here were to test this hypothesis by examining the pig-
ments, carbon nutrition, and phylogeny of several strains of
Rbl. acidophilus. We report here two major conclusions:
(1) phylogenetic microdiversity exists within the biotype
Rbl. acidophilus, and the color of phototrophic cultures
of a particular strain—purple or orange—correlates with
phylogeny and (2) in addition to methanol and ethanol,
Rbl. acidophilus grows photoheterotrophically on several
primary alcohols, results not described in the original
species description (Pfennig 1969) or in later work on
alcohol catabolism by this species (Douthit and Pfennig
1976; Quayle and Pfennig 1975; Sahm et al. 1976).
Materials and methods
Bog sample
An acidic boreal peat bog sample collected in north central
Alberta (Canada) was kindly provided by Brian Benscoter
Southern Illinois University. The bog had an overstory tree
layer of black spruce with the shrubs bog cranberry and
small bog cranberry and a groundcover of the moss Sphag-
num fuscum. The sample was primarily of the Sphagnum
layer immersed in bog water and sediment and was stored
in darkness at 4°C until enrichment cultures were estab-
lished.
Enrichments and pure cultures
A slurry of the bog sample was prepared by placing 5 g of
sample into 10 ml of sterile double-distilled water; the pH
of this slurry was 3.5. The slurry was placed at 4°C in the
dark for 24 h after which serial dilutions were performed in
water containing 10 mM of the buVer 2-(N-morpho-
lino)ethanesulfonic acid (MES, Sigma St. Louis) (Wnal pH
5.5). Liquid enrichment cultures were established by inocu-
lating one ml of a 10¡3, 10¡4, or 10¡5 dilution into 10-ml
screw-capped tubes containing acidic (pH 5.3) basal
medium (Pfennig 1969) supplemented with succinate
(20 mM Wnal concentration) as sole carbon source. All
tubes were completely Wlled and placed at 30°C in darkness
for 24 h before incubating in low intensity incandescent
light (20–25 mol s¡1 m¡2) for several weeks. Pigmented
cultures were streaked on plates of the same medium incu-
bated phototrophically in Gas Pak® anoxic jars (Wnal gas
mix H2:CO2:N2) and isolated colonies picked and streaked
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Arch Microbiol (2012) 194:567–574
a second time to ensure purity. Isolations were also pursued
by spreading dilutions of the bog slurry directly on plates
containing either 20 mM succinate or 10 mM methanol as
sole carbon source and incubated phototrophically. Colo-
nies were picked and puriWed as for liquid enrichments.
In addition to new isolates, three original Pfennig strains
of Rbl. acidophilus were used in this study, DSM 137T
(strain 7050T of Pfennig 1969), DSM 142 (strain 2751 of
Pfennig 1969), and DSM 145 (strain 10050 of Quayle and
Pfennig 1975). Two other strains isolated in this laboratory
from acidic enrichment cultures in the early 1980s and
thought to be Rbl. acidophilus, strains MO and FL, were
revived from freezer stocks and also used in this work.
Absorption spectra and physiological studies
For absorption spectra, 2 ml of phototrophic culture were
centrifuged at 14 K rpm for 2 min; the supernatant was dis-
carded and the pellet resuspended in a drop of medium and
mixed. A portion of the sample was then added to a cuvette
containing 30% bovine serum albumin (Sigma, St. Louis),
mixed, and absorption spectra recorded in a Hitachi U-2000
double-beam spectrophotometer.
Photoheterotrophy was tested by adding single organic
substrates to 10-ml screw-capped tubes of basal medium
supplemented with both NaHCO3 and sodium ascorbate
(10 mM and 0.05% Wnal concentrations, respectively)
[Control experiments showed that ascorbate, added as a
reducing agent, was neither toxic nor used as a carbon
source by Rbl. acidophilus]. Culture tubes were completely
Wlled and incubated at 30°C in darkness for 24 h before
phototrophic incubation (40–50 mol s¡1 m¡2). When test-
ing alcohols as carbon sources, replicate sets of tubes were
established at pH 5.5 and pH 6.8. Photoautotrophy was
tested in half-Wlled tubes of medium lacking an organic car-
bon source and incubated at 30°C in an anoxic jar for
14 days. The lower pH limit for growth was determined in
MES-supplemented media containing succinate as carbon
source and adjusted to pH 4.0, 4.5, or 5.0. In all growth
experiments, growth was scored as the average OD540 of
triplicate cultures following 7 days incubation.
Molecular methods
Genomic DNA was isolated using the Wizard® Genomic
PuriWcation Kit (Promega, Madison, WI). AmpliWcation of
16S rRNA genes was done using standard methods and the
nearly complete (1,409 nucleotide) sequences aligned with
CLUSTAL X (Larkin et al. 2007). Sequences were analyzed
using BLASTN (Altschul et al. 1997) and neighbor-joining
trees constructed using PHYLIP version 3.69 (Felsenstein
1989). SEQBOOT was utilized to calculate bootstrap values
based on 1,000 replications.
Arch Microbiol (2012) 194:567–574
Results
New strains of Rhodoblastus acidophilus
Phototrophic liquid and spread-plate enrichment cultures
(pH 5.3) inoculated with dilutions of Canadian peat bog
slurry yielded orange-brown liquid/colonies after 3–4
weeks of incubation at 30°C. Microscopic examination
revealed motile, rod-shaped budding cells, often grouped
into rosettes (Fig. 1a) as originally described for Rhodo-
pseudomonas acidophila (Pfennig 1969). Pure cultures
were then pursued and strains originating from liquid
enrichment or from direct plating were obtained. The bog
strains (Fig. 1b), along with three of the original Pfennig
strains and two other strains previously isolated in this lab-
oratory, were studied further. The sources and enrichment
details of these strains are summarized in Table 1.
Absorption properties
Phototrophic liquid cultures of all of the bog isolates, two
of the Pfennig isolates, and strain MO were orange in color
when grown in dim light, while cultures of strain FL were
purple, similar to that of strain 7050T (DSM 137T), the type
strain of Rhodoblastus acidophilus (Fig. 1c). Typical
absorption spectra for an orange- and a purple-colored
strain are shown in Fig. 2. All strains showed major peaks
in the near infrared near 860 and 805 nm from absorption
by light-harvesting (LH) II photocomplexes. In all cases,
the long wavelength absorption maximum lay above
860 nm in orange-colored strains and below 860 nm in pur-
ple-colored strains (Fig. 2; Table 1). Absorption from
carotenoids occurred near 525, 490, 465, and 420 nm, indi-
cating that “spirilloxanthin series” carotenoids (Schmidt
1971, 1978; Takaichi 1999) predominated. Notably, how-
ever, the ratio of carotenoid peaks varied between orange-
colored and purple-colored strains, and the latter lacked the
465-nm component (Fig. 2). Absorption maxima of LH II
Fig. 1 Rbl. acidophilus. Phase photomicrographs of a cell rosettes of strain MO and b cells of Canadian bog strain CBP1, showing typical bud-
ding morphology in both cultures. c Phototrophic liquid cultures of purple (top) and orange (bottom) strains of Rbl. acidophilus
569
photocomplexes and of carotenoids from the diVerent
strains are summarized in Table 1.
Phylogeny
The phylogeny of all eleven strains was determined by
comparing nearly complete 16S rRNA gene sequences; by
such criteria, all strains could be considered a single spe-
cies, with their closest established relative being Rbl. aci-
dophilus strain 7050 (Fig. 3). All six Canadian bog strains
as well as strain MO, isolated in Southern Illinois (USA),
had identical 16S rRNA gene sequences. All orange-col-
ored strains formed a cluster, and within the cluster, strain
DSM 142 was the most divergent (Fig. 3). Notably, the two
purple-colored strains formed a clade, distinct from the
cluster of orange-colored strains (Fig. 3). All 11 strains
were closely related to Rhodoblastus sphagnicola, a PNSB
that shares many phenotypic properties with Rbl. acidophi-
lus (Kulichevskaya et al. 2006). Beyond this, however, the
strains of Rbl. acidophilus were more closely related to sev-
eral chemotrophic bacteria than to other species of photo-
trophic purple bacteria; the next most closely related PNSB
was Rhodopseudomonas palustris (Fig. 3).
Photoheterotrophic growth
As originally described, Rbl. acidophilus uses a variety of
organic and fatty acids as carbon sources as well as the
alcohols ethanol and methanol for photoheterotrophic
growth (Pfennig 1969; Quayle and Pfennig 1975; Sahm
et al. 1976). Substrates that supported photoheterotrophic
growth of the new isolates are compared with the original
Pfennig data in Table 2.
Besides organic and fatty acids, all strains of Rbl. aci-
dophilus grew well photoheterotrophically on several alco-
hols, including propanol, pentanol, and hexanol. Propanol
was a particularly good growth substrate and supported better
growth than did methanol or ethanol (Table 2). Glycerol was
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Table 1 Isolates of Rhodoblastus acidophilus and enrichment and pigment details

Strain GenBank Source (GPS coordinates Sample Enrichment Absorption maxima (nm)
accession of sample site) dilution protocol
numbera LHII-region Carotenoid-region

Purple-colored strains
DSM 137T M34128 Crystal Lake, Illinois (Pfennig) – Liquid 859, 805 525, 493, –, 420
FL JN408837 Forked Lake, New York – Liquid 856, 805 526, 493, –, –
(43°55⬘N; 74°33⬘W)
Orange-colored strains
DSM 142 JN408836 Forest pond, Germany (Pfennig) – Liquid 865, 805 526, 492, 464, 421
DSM 145 JN408835 Cranberry bog, USA (Pfennig) – Liquid 864, 804 527, 493, 465, 422
MO JN408838 Strip mine pond, Illinois – Liquid 861, 803 525, 490, 464, 421
(37°48⬘N; 88°33⬘W)
CBP1 JN418218 Alberta (CA) peat bog 10¡3 Plating 861, 803 524, 490, 464, 422
(55°59⬘N; 115°11⬘W)
CBP2 JN399238 Alberta (CA) peat bog 10¡4 Plating 861, 803 524, 490, 463, 421
CBP3 JN399239 Alberta (CA) peat bog 10¡5 Plating 863, 803 525, 490, 463, –
CBP8 JN399240 Alberta (CA) peat bog 10¡5 Platingb 862, 803 524, 490, 463, 420
CBL2 JN399236 Alberta (CA) peat bog 10¡3 Liquid 862, 803 524, 490, 463, –
CBL5 JN399237 Alberta (CA) peat bog 10¡4 Liquid 862, 803 525, 490, 463, 424
a
GenBank accession numbers for organisms shown in the phylogenetic tree (Fig. 3). Accession numbers for other organisms in Fig. 3 are as fol-
lows: Rhodopila globiformis M59066, Rhodopseudomonas palustris D25312, Rhodoblastus sphagnicola AM040096, Methylosinus acidophilus
DQ076754, Beijerinckia derxii AJ563934, and Methylocapsa acidiphila NR028923
b
Enriched on 10 mM methanol as carbon source. All other CBP and CBL strains enriched on 20 mM succinate

Fig. 2 Absorption spectra of in-

tact cells of a purple and an or-
ange strain of Rbl. acidophilus
grown under low-light condi-
tions

used by only one strain, while butanol and alcohols longer
than C6 did not support growth of any strain. Strain FL was
the only strain unable to grow on methanol, and methanol
was a better substrate for the new isolates than for the Pfen-
nig strains (Table 2). Growth on alcohols was tested at
both pH 5.5 and 6.8, and in contrast to previous results
that showed Rbl. acidophilus to grow better on methanol
near neutral pH (Quayle and Pfennig 1975), in our hands,
photoheterotrophic growth on any alcohol (including
methanol) was the same at both acidic and neutral pH (data
not shown).
Fatty acids up to C6 supported growth of all strains with
the exception of formate, which was used by only a few
strains, and butyrate, which did not support growth of strain
DSM 142; butyrate was an excellent growth substrate for
most other strains of Rbl. acidophilus (Table 2). In general,

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Fig. 3 Neighbor-joining 16S rRNA gene phylogenetic tree of all strains of Rbl. acidophilus used in this study. Bootstrap values based on 1,000
replications are shown at the nodes. GenBank accession numbers for all sequences in this tree are listed in Table 1
strain-to-strain diVerences in fatty acid use were more
apparent than those in organic acids or alcohols (Table 2).
Four of the new strains grew on glucose; however, none of
the other sugars tested, including hexoses, pentoses, and
disaccharides, supported growth (Table 2). All strains were
also capable of photoautotrophic growth using H2 as the
electron donor (data not shown).
The lower pH limit for growth of the isolates from this
laboratory was tested in MES-buVered succinate media
adjusted to pH 4.0, 4.5, or 5.0, and the results compared
with those of Pfennig (1969). In agreement with the latter,
none of the strains grew at a starting pH of 4.0 but all grew
to full densities in media adjusted to pH 5. In addition,
however, all strains showed noticeable growth at pH 4.5
except for strain FL, which grew to full densities at both pH
4.5 and 5.0 (data not shown).
Discussion
Although many extremophilic purple bacteria are known
(Madigan 2003; Madigan and Jung 2009), few are acido-
philic. The only purple bacteria capable of growth at pH 4.5
are Rhodoblastus acidophilus and Rhodopila globiformis
(formerly Rhodopseudomonas globiformis). The latter was
isolated from an acidic (pH 3.5) sulWde spring in Yellow-
stone and is more acidophilic than even Rbl. acidophilus
(Pfennig 1974). However, Rbl. acidophilus is likely to be the
more widely distributed of the two species in nature, as
enrichments for Rbl. acidophilus were successful from
571
samples of freshwater lakes and ponds as well as from bogs
and other acidic sites worldwide (Pfennig 1969 and this
study), whereas Rpi. globiformis has not been reported out-
side of certain acidic Yellowstone springs (Madigan et al.
2005).
The original species description of Rbl. acidophilus
reported its lower pH limit for growth to be 4.8 (Pfennig
1969). However, the lower limit is actually below this, pH
4.5, or perhaps even slightly lower. In our experiments, we
used MES-buVered media and showed that all strains grew
at pH 4.5 but that none grew at pH 4.0. As a species, Rbl.
acidophilus should thus be successful in virtually any illu-
minated acidic habitat where acidity is derived from
organic rather than mineral acids. Moreover, the observa-
tion that acidic enrichment for PNSB in the work of Pfen-
nig (1969) and herein yielded only Rbl. acidophilus
suggests that the diversity of acidophilic purple bacteria in
nature is probably low. Rhodoblastus sphagnicola, also an
acidophilic PNSB and a close relative of Rbl. acidophilus,
has a higher minimum pH for growth than does Rbl.
acidophilus and has only been reported from one habitat
(Kulichevskaya et al. 2006).
Phototrophic cells of Rbl. acidophilus contain bacterio-
chlorophyll a and carotenoids of the spirilloxanthin series;
however, when grown in dim light, cell suspensions are
either orange or purple in color, depending on the strain.
Orange-colored strains are orange whether grown at high-
or low-light intensities while purple-colored strains are pur-
ple when grown in dim light but orange when grown at high
light. The mechanism for this diVerence is well understood.
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Table 2 Carbon sources for photoheterotrophic growth of strains of Rhodoblastus acidophilusa

Substrate Strain and growthb

CBP1 CBP2 CBP3 CBP8 CBL2 CBL5 MO FL DSM 137Tc DSM 142c DSM 145c

Fatty acids
Formate ¡ ¡ + + + + + ¡ ¡ ¡ +
Acetate ++ ++ ++ ++ ++ ++ ++ ++ ++ + ++
Propionate ++ +++ +++ +++ ++ ++ ++ +++ + ++ +++
Butyrate ++++ ++++ ++++ +++ + +++ ++++ +++ + ¡ ++++
Valerate + + + + + + ++ ++ ++ + ++
Caproate ++ +++ ++++ +++ + ++ +++ +++ + + +++
Organic acids
Pyruvate ++ ++ ++ ++ ++ ++ +++ +++ +++ +++ ++++
Lactate +++ +++ +++ +++ ++ ++ +++ +++ +++ +++ +++
Malate ++ +++ +++ +++ ++ ++ + +++ +++ +++ +++
Succinate +++ +++ +++ +++ ++ ++ +++ +++ +++ +++ +++
Fumarate +++ +++ +++ +++ ++ +++ ++ ++ +++ +++ ++
Malonate + + + + + + + ¡ ¡ + ¡
Alcoholsc
Methanol ++ ++ ++ ++ ++ ++ + ¡ +d + ++
Ethanol ++ ++ ++ ++ ++ ++ ++ + ++ ++ ++
Propanol +++ ++ +++ ++ +++ ++ ++ ++ +++ +++ +++
Pentanol ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++
Hexanol + + + + + + + + + + +
Glycerol ¡ + ¡ ¡ ¡ ¡ ¡ ¡ ¡ ¡ ¡
a
All cultures were grown phototrophically in the acidic (pH 5.5) medium of Pfennig (1969) with the single carbon source as indicated. All strains
grew phototrophically on H2/CO2, and strains CBP1, 2, 3, and 8 grew weakly (OD, +) on glucose. All substrates added at 10 mM Wnal concentration
except 5 mM for valerate, caproate, malonate, pentanol, and hexanol
b
++++, OD540 >2.0; +++, OD540 1.5–2.0; ++, OD540 >0.4 and <1.5; +, OD540 0.1–0.4; ¡, OD540 <0.1. The following carbon sources were test-
ed in this study but did not support growth: benzoate (5 mM), citrate (5 mM), tartrate (5 mM), butanol (5 mM, 2 mM), heptanol (2 mM), octanol
(2 mM), benzyl alcohol (2 mM), ribose (10 mM), galactose (10 mM), sucrose (10 mM), lactose (10 mM), xylose (10 mM), fructose (10 mM), and
mannitol (5 mM)
c
Data adapted from Pfennig (1969)
d
Strain DSM 137T grew photoheterotrophically on methanol at pH 6.8 but not at pH 5.5

Rhodopin, lycopene, and spirilloxanthin are the main
carotenoids of all strains of Rbl. acidophilus; in addition,
orange-colored but not purple-colored strains contain large
amounts of rhodopin glucoside (Schmidt 1971, 1978;
Schmidt et al. 1971; Takaichi 1999). Purple-colored strains
produce large amounts of rhodopinal glucoside but only
under low-light conditions (Gardiner et al. 1993; Heinemeyer
and Schmidt 1983; Takaichi 1999). By contrast, rhodopinal
glucoside was absent from 5 of 6 low-light-grown orange-
colored strains of Rbl. acidophilus (Schmidt 1971) and is
likely the reason why orange-colored strains are never purple
in color. In place of rhodopinal glucoside, high-light-grown
purple-colored strains synthesize large amounts of rhodopin
glucoside, a major carotenoid of orange-colored strains grown
at any light intensity (Gardiner et al. 1993; Heinemeyer and
Schmidt 1983; Takaichi 1999).
In addition to carotenoids, the composition of the light-
harvesting complexes of certain strains of Rbl. acidophilus
can change with light intensity (Gardiner et al. 1993). Thus,
light intensity appears to be an important regulatory signal
for this species. No speciWc physiological traits linked to
the orange or purple phenotype of Rbl. acidophilus were
noted by Pfennig (1969). Nevertheless, it would be surpris-
ing if the distinctly diVerent colors observed in the diVerent
strains of this species (Fig. 1c) did not have some ecologi-
cal signiWcance.
Besides the type strain of Rbl. acidophilus, whose 16S
rRNA gene sequence was known, the 16S sequences of two
other Pfennig isolates and the eight strains isolated in this
laboratory were determined herein. The 16S sequences of
the six Canadian bog isolates were identical regardless of
the enrichment method (liquid or plating) or dilution factor
used to obtain them. This suggests that only one cultivable
biotype of Rbl. acidophilus inhabits this bog. More surpris-
ing, however, is the fact that strain MO (Table 1), isolated
from a coal strip mine pond in Southern Illinois (located

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some 3,200 km from the Canadian bog), had a 16S rRNA
gene sequence identical to that of the bog isolates. Among
other orange-colored strains, strain DSM 142 was the most
divergent, and this is consistent with the fact that its geno-
mic DNA G + C base ratio was near the lowest for the
rather signiWcant range of G + C base ratios reported for
diVerent strains in the original species description of Rbl.
acidophilus (Pfennig 1969).
The 16S sequences of the two purple-colored strains,
isolated from habitats 1,500 km apart, were highly similar
but distinct from those of the orange-colored strains. In
fact, purple-colored strains formed a clade to the exclusion
of all orange-colored strains, suggesting that orange- and
purple-colored strains diverged from a common ancestor to
improve Wtness for growth in speciWc habitats. Analyses of
a larger number of strains would be necessary to garner
stronger support for this hypothesis, but at the very least,
our results suggest that the orange and purple phenotypes
may have phylogenetic roots.
It is not uncommon for PNSB to be closely related to
chemotrophic bacteria (ImhoV 1995), and thus it was not
surprising to Wnd that with the exception of the purple bac-
terium Rbl. sphagnicola, the closest phylogenetic relatives
of Rbl. acidophilus were nonphototrophic bacteria. Of three
chemotrophic relatives included in our phylogenetic tree,
two were methylotrophic while the other was a free-living
nitrogen-Wxing bacterium. However, despite this metabolic
diversity, these bacteria all inhabit acidic environments and
are themselves acidophilic (Bowman 2005; Dedysh et al.
2002, 2006; Kennedy 2005). Thus, acidophily rather than
speciWc energy metabolisms is the likely phylogenetic link
between these chemotrophs and Rbl. acidophilus.
Rbl. acidophilus uses a variety of organic and fatty acids
as well as methanol and ethanol as carbon sources (Pfennig
1969; Quayle and Pfennig 1975; Sahm et al. 1976). For the
most part, similar Wndings were obtained with the new iso-
lates. However, the more extensive survey done here of pri-
mary alcohols as potential carbon sources for growth of
Rbl. acidophilus indicated that in addition to methanol and
ethanol, propanol, pentanol, and hexanol are used and sup-
port good growth of this species. Indeed, the consumption
of alcohols may be signiWcant for the competitive success
of acidophilic phototrophs. Unlike organic and fatty acids—
compounds that are protonated (and thus more toxic) under
acidic conditions—alcohols are neutral molecules. It is thus
possible that alcohols are widely consumed by Rbl. acidophi-
lus in nature and may actually be prime growth substrates.
The rapid phototrophic growth of Rbl. acidophilus on metha-
nol coupled with the inability of most other PNSB to use
methanol (Douthit and Pfennig 1976) may be another example
of the diversity of alcohol catabolism by this species.
Even though PNSB grow best photoheterotrophically, most
species can also grow photoautotrophically (Madigan and Jung
573
2009). All strains of Rbl. acidophilus grew autotrophically on
H2 + CO2, indicating their potential as contributors of organic
matter in acidic natural habitats. Acidic bogs such as the Cana-
dian peat bog studied here are known to sequester huge
amounts of carbon, harboring roughly one-third of the global
reserves of soil organic carbon (Gorham 1991). Rbl. acidophi-
lus may be the major or even sole anoxygenic phototroph in
these carbon-rich anoxic environments, using H2 produced by
chemotrophic anaerobes as electron donor to drive photoauto-
trophy when usable low-molecular-weight organic compounds
become scarce.
Acknowledgments This work was supported in part by grant
0950550 from the United States National Science Foundation. We
thank Dr. Brian Benscoter (now of Florida Atlantic University) and Dr.
Dale Vitt (Southern Illinois University) for the Canadian bog sample.
We also thank Deborah O. Jung for help in preparing Figs. 1 and 2 and
formatting the tables, and for helpful comments on the manuscript.
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