Download Utilization of Amino Acids and Lack of Diazotrophy in

Journal of General Microbwlogy (1986), 132, 2469-2473.
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2469
Utilization of Amino Acids and Lack of Diazotrophy in the Thermophilic
Anoxygenic Phototroph ChloroJlexus aurantiacus
By G H A N S H Y A M D . H E D A A N D M I C H A E L T . M A D I G A N *
Department of Microbiology, Southern Illinois University, Carbondale, Illinois 62901, USA
(Received 8 April 1986)
Four strains of the thermophilic phototrophic green bacterium Chlorofiexus aurantiacus were
tested for nitrogenase activity under a variety of nutritional conditions consistent with
nitrogenase derepression in anoxygenic photosynthetic bacteria. Although all strains of C.
aurantiacus could grow with various amino acids as sole nitrogen sources, no growth on N2 was
observed; acetylene reduction was undetectable in cells grown under any nutritional conditions.
The inability of Chloroflexusto fix N2 is discussed in connection with its thermophilic character.
INTRODUCTION
The ability to fix N2 is widespread among phototrophic bacteria. Since the discovery of this
important metabolic process in photosynthetic bacteria (Gest & Kamen, 1949; Kamen & Gest,
1949) representatives of all major groups have been shown to be diazotrophic (Yoch, 1978;
Madigan et al., 1984; Heda & Madigan, 1986).
The hot-spring phototroph Chlorofiexus aurantiacus has characteristics typical of both green
and purple bacteria. The light-harvesting bacteriochlorophyllof C. aurantiacus and its location
in chlorosomes (Pierson & Castenholz, 1974a, 6 ) are major characteristics shared with green
sulphur bacteria. The photosynthetic reaction centre (Pierson & Thornber, 1983), electron
transport components (Bruce et al., 1982), facultative metabolism (Pierson & Castenholz,
1974a, 6 ) and nutritional versatility (Madigan et al., 1974) of C. aurantiacus, on the other hand,
are typical of purple nonsulphur bacteria. Considering the physiological/biochemicalaffinities
between Chlorofiexusand purple and green bacteria, both groups of which are diazotrophic, one
would predict that Chlorofiexus would fix N2. Because of its unusual combination of properties
and its thermophilic character we have tested this hypothesis and describe herein aspects of
nitrogen metabolism in Chlorofiexus and our unsuccessful attempts to elicit N,-fixation in
several strains of this organism.
METHODS
Bacterial strains. Chloroflexus aurantiacus strains OK-70-fl (ATCC 29365), J-10-fl (ATCC 29366), Y-400-fl
(ATCC 29364) and 396-1 (ATCC 29363) were obtained from Dr Thomas D. Brock, University of Wisconsin,
Madison, USA, in 1976. Strains were stored at -80°C in growth medium containing 10% (v/v) glycerol.
Chlorobium Iimicola forma thwsulfatophilum strain 8327 was grown on glutamate as sole nitrogen source as
described by Heda & Madigan (1986).
Growth media and growth conditions. Strains of Chloroflexus were grown on medium D (Castenholz, 1969) or
medium D-N (Jackson & Castenholz, 1975)adjusted to pH 8 and supplemented with 0405% (w/v) yeast extract,
0.2% (w/v) sodium acetate, a metabolizable nitrogen source at the concentration specified in Table 1 and the
following vitamins (per 1of medium): nicotinic acid, 1 mg;paminobenzoic acid and biotin, 100 pg each. Sulphide
and bicarbonate were added to final concentrations of 0.05% (w/v) and 0.1 % (w/v) respectively, as described
previously (Heda & Madigan, 1986).
0001-3189 0 1986 SGM
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G . D. H E D A A N D M. T. M A D I G A N
Cultures were grown photosynthetically in completely filled 17 ml screw cap tubes or 250ml sidearm
Erlenmeyer flasks containing 100 ml medium and either high-purity-grade argon or N2/C02(99 : 1) in the head
space. Small scale cultures requiring a gas headspace were grown in 70 ml serum vials containing 20 ml medium
and stoppered with sleeve-type rubber stoppers. Cultures were illuminated with 1500 1x (incandescent lamps) and
incubation temperatures were maintained at 46 _+ 2 "C.
Growth meusurements. Growth was measured in a Klett-Summerson photometer fitted with a no. 66 (red) filter.
Because cells of Chiorojexus tend to form thick clumps, tube cultures were vortexed extensively immediately
before each reading. Photometer readings were standardized to cell protein (Lowry method) using bovine serum
albumin as standard.
Nitrogenuse assay. Nitrogenase (acetylene reduction) activity was tested in intact cells of Chloroflexus at 4548 "C except where indicated. Chiorobiurn nitrogenase was assayed at 30 "C in thiosulphate-supplemented cell
suspensions. The assay protocol was as described previously (Heda & Madigan, 1986).
RESULTS A N D DISCUSSION
Nitrogen nutrition of C . aurantiacus
Initially, the range of nitrogenous compounds that would support growth as sole nitrogen
source was examined. Medium D (Castenholz, 1969) contains NO, as sole nitrogen source but
NO, did not serve as a nitrogen source for Chloroflexus (Table 1). However, NHI, alanine,
aspartate, glutamate, glutamine, glycine and serine, when added to medium D, supported good
growth of all strains (Table 1). Tyrosine and valine were utilized by all strains, but growth yields
were lower on these substrates than on glutamate or aspartate. Certain other amino acids were
used by one or more strains, but proline, adenine and urea were not metabolized by any of the
Chlorofexus strains tested.
Initial attempts to grow ChloroJlexus strains J-10-fl and OK-70-fl on N, were unsuccessful.
These strains were then grown on limiting (0.3 mM) NH,+in flasks containing either N2 or Ar as
gas phase. As shown in Fig. 1 for strain J-10-fl, no evidence of growth on N2 was obtained; cell
densities were nearly the same under Ar or N,. A control flask containing a nonlimiting (3 mM)
NH,+concentration allowed growth to much higher cell densities (Fig. 1). These results suggest
that N2 does not serve as a nitrogen source for growth of strain J-10-fl; similar results were
obtained with strain OK-70-fl.
Nitrogenase activity
Growth on amino acids as nitrogen sources by nonsulphur purple bacteria (Gest & Kamen,
1960; Yoch, 1978) and green sulphur bacteria (Heda & Madigan, 1986) is associated with
nitrogenase derepression. Therefore, strains of Chlorofexus were grown in similar fashion and
tested for acetylene reduction. Chlorobium limicola strain 8327 was used as a positive control.
None of the Chlorofexus isolates reduced acetylene when grown on glutamate, aspartate or
limiting NHI as nitrogen source, while nitrogenase activity was readily detected in glutamategrown cells of Chlorobium [mean activity 5 pmol ethylene h-l (mg cell protein-')]. To ensure that
the Chloroflexuscultures were not failing to derepress because of a nutritional deficiency, cells of
strain J-10-fl and OK-70-fl were grown on 3 mwglutamate, 3 mmaspartate or limiting NH,+
(0.3 mM) in medium D with the following supplements in separate experiments: 10 mMpyruvate, 10- and 100-fold excess molybdenum, in a gas phase of H2/N2(50 :50, medium free of
sulphide) and in the presence of 0.2% (w/v) yeast extract (with and without sulphide). To ensure
that NO: was not repressing nitrogenase synthesis in Chlorofexus, as it does in the
cyanobacterium Anabaena CA (Bottomley et al., 1979), cells were grown on 3 mwglutamate or
0.3 w - N H , +in Nosfree medium (D-N). To test the effect of lowered growth temperature on
nitrogenase expression, the low-temperature Chlorofexus strain 396-1 (Bauld & Brock, 1973)
was grown at 39 "C in medium D or D-N on glutamate, aspartate or limiting NH,+as nitrogen
sources with and without 10mM-pyruvate and tested for acetylene reduction at the same
temperature. None of the above conditions yielded cells capable of reducing acetylene.
The inability of C. aurantiacus to grow on N2 as sole nitrogen source and the lack of acetylenereducing activity in the four strains examined are evidence that this photosynthetic bacterium is
phenotypically Nif -. This is unusual since virtually all anoxygenic phototrophic bacteria are
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Lack of diazotrophy in C . aurantiacus
247 1
Time (d)
Fig. 1. Growth of C. uuruntiucus strain J-10-fl on limiting and excess NHZ. Cells were grown
photosynthetically in 250 ml Erlenmeyer flasks containing 100 ml medium D supplemented with
0.3 ~ M - N H $ ( ON2
, headspace; 0 ,Ar headspace)or 3 m - N H $ ( O , Ar headspace). The results shown
are typical of those obtained in two separate experiments.
Table 1. Growth of strains of ChloroJexus aurantiacus on various nitrogenous compounds
All strains were grown photosynthetically (1500 lx, 46 f 2 "C) in medium D for 7 d; cultures were
scored for growth following a third transfer in the same medium. The results shown are typical of those
observed in two separate experiments. Growth responses: 3, > 300 Klett units; 2, 150-300 Klett units;
1, 50-150 Klett units; 0, no growth. (200 Klett units are equivalent to approximately 200 pg bacterial
protein ml-I.) No growth was observed with proline, urea or adenine.
Nitrogen source*
396- 1
J-10-fl
OK-70-fl
Y-400-fl
2
2
2
2
NHf
3
2
3
3
Alanine
3
0
3
0
Arginine
2
2
Aspartate
3
3
3
3
0
3
Asparagine
0
3
2
2
Cysteine
2
3
3
2
Glycine
2
2
3
Glutamate
3
2
3
3
3
Glutamine
0
0
2
Histidine
0
2
1
0
0
Isoleucine
0
0
1
0
Leucine
0
2
0
Lysine
0
1
0
1
0
Methionine
1
0
0
0
NO;
1
2
0
1
Phenylalanine
3
3
Serine
3
3
1
0
0
Threonine
3
1
0
2
0
Tryptophan
1
1
1
Tyrosine
2
1
1
2
1
Valine
* Compounds were added at the following concentrations: NHI, NO: and amino acids, 3 m;tyrosine and
adenine, 2-5 InM.
diazotrophic (Madigan et al., 1984; Yoch, 1978; Heda & Madigan, 1986). It is of course
conceivable that some specific factor is required for nitrogenase expression in Chlorojlexus.
Pyruvate, recently demonstrated to be required for N,-fixation by sulphate-reducing bacteria
(Postgate & Kent, 1985) and lowered growth temperatures, necessary for nitrogenase expression
by Klebsiella pneumoniae (Hennecke & Shanmugam, 1979), were both unsuccessful in eliciting
acetylene reduction in ChloroJexus. HZ,which is taken up by ChloroJexus in the absence of
sulphide (Drutschmann & Klemme, 1985), also did not induce putative nitrogenase activity in
Chlorojexus.
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G . D. HEDA A N D M. T . MADIGAN
The fact that ChlorocfEexus is a thermophile may explain its lack of diazotrophy. In general,
thermophilic organisms do not fix N2 (Postgate, 197l), although occasional exceptions have
been noted. For example, a close neighbour of ChlorofIexus in low-temperature algal-bacterial
mats devoid of sulphide is the cyanobacterium Mastigocladus laminosus (Brock, 1978). This
photograph has been shown to reduce acetylene at temperatures above 50 "C both in the field
(Stewart, 1970) and in culture (Stevens et af., 1985), indicating that N2 fixation per se is not
incompatible with the temperatures required for growth of Chloro_Pexus.On the other hand, the
newly described thermophilic purple sulphur bacterium Chromatiurn tepidum also appears
incapable of N2 fixation (Madigan, 1986). The latter organism is a small-celled Chromatiurn
species resembling the N,-fixing species Chromatiurn vinosum except for its growth temperature
optimum of 48 "C (Madigan, 1984), and would therefore be expected to fix N2.The inability of
both Chromatiurn tepidum and Chforo_fexusaurantiacus to fix N2suggests that diazotrophy is not
compatible with thermophily in anoxygenic phototrophs. More isolates of thermophilic
phototrophic bacteria, especially nonsulphur purple bacteria (if thermophilic representatives
exist), will be needed to answer this question definitively.
This work was supported by grant 83-CRCR-1-1308 from the Science and Education Administration of the
United States Department of Agriculture.
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