Photosynth Res (2010) 104:113–122 DOI 10.1007/s11120-010-9529-9
REVIEW
Insights into heliobacterial photosynthesis and physiology from the genome of Heliobacterium modesticaldum W. Matthew Sattley • Robert E. Blankenship
Received: 28 August 2009 / Accepted: 16 January 2010 / Published online: 4 February 2010 Ó Springer Science+Business Media B.V. 2010
Abstract The complete annotated genome sequence of Heliobacterium modesticaldum strain Ice1 provides our first glimpse into the genetic potential of the Heliobacteriaceae, a unique family of anoxygenic phototrophic bacteria. H. modesticaldum str. Ice1 is the first completely sequenced phototrophic representative of the Firmicutes, and heliobacteria are the only phototrophic members of this large bacterial phylum. The H. modesticaldum genome consists of a single 3.1-Mb circular chromosome with no plasmids. Of special interest are genomic features that lend insight to the physiology and ecology of heliobacteria, including the genetic inventory of the photosynthesis gene cluster. Genes involved in transport, photosynthesis, and central intermediary metabolism are described and catalogued. The obligately heterotrophic metabolism of heliobacteria is a key feature of the physiology and evolution of these phototrophs. The conspicuous absence of recognizable genes encoding the enzyme ATP-citrate lyase prevents autotrophic growth via the reverse citric acid cycle in heliobacteria, thus being a distinguishing differential characteristic between heliobacteria and green sulfur bacteria. The identities of electron carriers that enable energy conservation by cyclic light-driven electron transfer remain in question. Keywords Photosynthesis Anoxygenic Heliobacteria Bacteriochlorophyll Type I RC W. M. Sattley Department of Biology, MidAmerica Nazarene University, 2030 E. College Way, Olathe, KS 66062, USA R. E. Blankenship (&) Departments of Biology and Chemistry, Washington University in St. Louis, Campus Box 1137, St. Louis, MO 63130, USA e-mail:
[email protected]
Abbreviations Bchl Bacteriochlorophyll CAC Citric acid cycle CDS Protein coding sequence Chl Chlorophyll PGC Photosynthesis gene cluster PS Photosystem ORF Open reading frame RC Reaction center
Introduction The Heliobacteriaceae is a unique family of phototrophic bacteria within the phylum Firmicutes (class Clostridia, order Clostridiales). Similar to many other Firmicutes, including species of Bacillus and Clostridium, but unlike all other phototrophic bacteria, heliobacteria produce endospores, a property that appears to be universal among the group (Table 1) (Ormerod et al. 1996; Kimble-Long and Madigan 2001). Many other characteristics distinguish heliobacteria from other phototrophic bacteria. The photosynthetic pigment bacteriochlorophyll (Bchl) g is uniquely employed by heliobacteria as the primary electron donor within a type I homodimeric reaction center (RC) bound to the cytoplasmic membrane; 81-hydroxy-chlorophyll (Chl) a serves as the primary electron acceptor from the RC special pair (Fuller et al. 1985; Trost and Blankenship 1989; van de Meent et al. 1991; Amesz 1995). Each heliobacterial RC contains 30–40 Bchl g, two molecules of 81-OH-Chl a, and approximately two carotenoid pigments (van de Meent et al. 1991; Oh-oka 2007). Heliobacteria synthesize C30 carotenoid pigments instead
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Table 1 Summary of major characteristics of Heliobacterium modesticaldum str. Ice1T Source
Icelandic volcanic soil
Cell morphology
Rods or curved rods
Cell size
1 9 2.5–6.5 lm
Cell differentiation
Subterminal endospores produced
Genome
Single, circular chromosome of 3.08 Mb
G ? C content
56.0%
Total ORFs
3,138
Motility Optimal temperature
Flagellar 52°C
Phototrophic growth
Photoassimilation of pyruvate, lactate, acetate, or yeast extract
Chemotrophic growth
Fermentation of pyruvate
Reaction center
Type I homodimer of PshA core protein
Photosynthetic pigments
Bchl g and 81-OH-Chl a (both esterified with farnesol)
RC pigment ratio
Approximately 17 Bchl g/81-OH-Chl a
Carotenoids
C30 pigments, predominately 4,40 diaponeurosporene
Diazotrophy
Optimal N2-fixation at 50°C via a molybdenum-dependent group I nitrogenase
of the C40 carotenoids found in other phototrophic bacteria (Takaichi 1999). Furthermore, heliobacteria contain no auxiliary light harvesting pigments or structures (e.g., Bchl-containing chlorosomes or internal membrane lamellae). Thus, heliobacteria contain perhaps the simplest known photosynthetic apparatus. Unlike all other anaerobic anoxygenic phototrophs, heliobacteria are obligately heterotrophic, with the best growth occurring photoheterotrophically using a limited number of low molecular weight organic substrates (Madigan and Ormerod 1995). Although some heliobacteria can metabolize C3/C4 fatty acids, ethanol (?CO2), and C6 monosaccharides (e.g., glucose or fructose), photoheterotrophic growth of Heliobacterium modesticaldum is limited to the use of pyruvate, lactate, or acetate as carbon sources (Table 1) (Kimble et al. 1995; Madigan 2001). Interestingly, anaerobic growth of heliobacteria can also occur in the absence of light by the fermentation of pyruvate (Kimble et al. 1994). Light-independent fermentation by photosynthetic organisms has only been observed in heliobacteria and some purple nonsulfur bacteria. Growth by this means, as well as the ability of heliobacteria to form endospores, reinforces the phylogenetic findings that these phototrophs group within the Clostridia (Bryant and Frigaard 2006). The unique photosynthetic features, physiological characteristics, and phylogenetic affiliation that combine to differentiate heliobacteria from all other phototrophs makes this group of organisms an obvious subject for
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genomic characterization. At the time of this writing, the family Heliobacteriaceae consists of four genera—Heliobacillus, Heliobacterium, Heliophilum, and Heliorestis. The genome of one species, H. modesticaldum strain Ice1T, was recently sequenced, annotated, and described (Sattley et al. 2008), thus denoting the first major step into the study of heliobacterial genomics. While interested readers should refer to Sattley et al. (2008) for a more complete summary of the genetic inventory of this photosynthetic bacterium, the following will serve as a review of the major findings of this study, as well as a consideration of some of the unanswered questions regarding heliobacterial photosynthesis and physiology.
Materials and methods Genome sequencing and annotation Details concerning the sequencing and annotation of the H. modesticaldum str. Ice1T (=ATCC 51547T) genome are described elsewhere (Swingley et al. 2007; Sattley et al. 2008). Briefly, the genome sequence was determined from shotgun libraries using the dye terminator method in Applied Biosystems 3730xl automated sequencers, and sequence assembly was carried out using ARACHNE (Batzoglou et al. 2002). Gene annotations were assigned using AutoAnnotate and checked manually using Manatee (http://manatee.sourceforge.net). Phylogenetic analyses Sequence searches were performed using the Basic Local Alignment Search Tool (BLAST) (Altschul et al. 1990). Sequence alignments and phylogenetic trees were created and analyzed using MEGA 3.1 (Kumar et al. 2004); bootstrap values were determined from 500 replicates. Sequences used in the analyses were obtained from NCBI genome databases (http://www.ncbi.nlm.nih.gov/). Nucleotide sequence accession number The complete and annotated genome sequence of H. modesticaldum str. Ice1T can be accessed from the DDBJ/EMBL/ GenBank database using accession number CP000930.
Results and discussion Carbon and nitrogen metabolism and related ecology Heliobacteria are the only anaerobic anoxygenic phototrophs that have no apparent autotrophic pathway for fixing
Photosynth Res (2010) 104:113–122 molybdate sulfate
115 phosphate
Co2+
Co2+
Cd2+
Ca2+
cation
drugs H+
Mg2+
Cu2+
drugs
drugs
bacitracin
ADP
ADP
ATP
ADP
Glucose-1-phosphate
H+ diphosphate
Nonoxidative Pentose Phosphate Pathway
Glucose-6-phosphate
L-lysine, L-threonine, L-asparagine
H+ Na+
Glycolysis
CO2 acetyl-CoA
Pyruvate
Fe2+ oxaloacetate
cheABCDRWY
malate
Nonautotrophic Partial Reverse CAC
fatty acids, sterols, purines
citrate
2-oxoglutarate
fumarate
ADP ATP
L-tryptophan L-phenylalanine L-tyrosine
L-glutamine
Arginine/ Lysine
Peptides
ADP ATP
porphyrin, heme Bchl g
L-glutamate
Branched Chain AAs
ADP ATP
menaquinone
farnesyl-PP
AT
22 MCPs
chorismate
acetate
(acetyl-AMP)
isocitrate
flagellum
ADP ATP
L-methionine
isopentenyl-PP 27 flagellar and motor genes
NH4+/NH3
Glutamine erythrose-5-phosphate
L-serine, L-isoleucine, L-leucine, L-valine
CO2
L-aspartate, pyrimidines
L-arginine
L-histidine
L-glycine, L-cysteine Phosphoenolpyruvate
Fe2+
PRPP
ATP
lactate
Fe2+
chemotactic signals
ATP
ATP
ADP
ADP ATP
ADP ATP
ADP ATP
ADP ATP
ADP ATP
ADP ATP
ADP ATP
2 Pi
ADP ATP
ATP
ADP ATP
Fibronectinbinding
Polyamines
ADP succinate
succinyl-CoA
Selenoamino acid metabolism selenocysteine
ADP solute Na+
N2
L-alanine
~85 sporulation/ germination genes
NAD+
e-
N2H2
4H H2
ADP
ATP
ADP
ATP
ADP
ATP
e
2NH3
Uracil/ xanthine Na+
HPr
2H
PTS EI
ATP
-
e
Lactate/ glycolate H+ Gluconate
ADP + Pi
-
H+
NADH
H+ e-
H+ ?
?
NADH:MQ oxidoreductase
H+
? MQ pool
MQ pool
K+ Zn2+
N2H4 2H
Ferredoxin
solute Na+
Na+
Molybdenum-dependent nitrogenase complex
L-proline
?
Alanine
Nitrogen fixation
ADP
ornithine
ADP ATP
ATP
ATP
?
CO2
eCyt c553
Cyt bc complex
eH2 H+ Type-1 RC
ATP synthase
[NiFe] hydrogenase
Na+ Bile acid family
Ribose
Fig. 1 Summary diagram of predicted central metabolism (photosynthetic energy conversion and carbon/nitrogen metabolism) and transport in Heliobacterium modesticaldum. Colors indicate substrate specificity: Inorganic ions (blue), amino acids/peptides/ammonia/ amines/nucleobases (red), carbohydrates/organic acids (yellow),
virulence/drug resistance (black), cytochromes/hydrogenase (orange), RC/ATP synthase (green), NADH oxidoreductases (brown), and motility (purple). Arrows indicate direction of transport; single arrows indicate an unknown energy coupling mechanism. Question marks indicate functional uncertainties
inorganic carbon, such as carbon dioxide. Genes encoding key enzymes required for CO2-fixation via any of the known autotrophic pathways were not found in the H. modesticaldum genome, including ribulose 1,5-bisphosphate carboxylase and phosphoribulokinase (Calvin cycle), ATP-citrate lyase [reverse citric acid cycle (CAC)], carbon monoxide dehydrogenase (reductive acetyl-CoA pathway), malyl-CoA lyase (3-hydroxypropionate pathway), and 4-hydroxybutyryl-CoA dehydratase (3-hydroxypropionate/4-hydroxybutyrate pathway) (Sattley et al. 2008). The absence of genes encoding ATP-citrate lyase is consistent with biochemical studies carried out by Pickett et al. (1994) in which no citrate lyase activity was detected in Heliobacterium strain HY-3. The absence of this enzyme prevents H. modesticaldum from having a complete reverse CAC (Fig. 1), which has significant implications to the metabolic versatility of heliobacteria. Without citrate lyase, CO2-fixation via the reverse CAC cannot take place, and if not for this omission, heliobacterial carbon metabolism
would presumably have much in common with that of Chlorobium and other green sulfur bacteria, including the capacity for autotrophic growth (Buchanan and Arnon 1990). Whether genes encoding a functional citrate lyase were ever present in H. modesticaldum is an interesting question. Many nonphototrophic Firmicutes closely related to heliobacteria, such as species of Clostridium and Desulfitobacterium, contain citrate lyase, which suggests that heliobacteria may have experienced a degree of genome reduction and subsequently lost these genes. It has been hypothesized that unusually high copy numbers of rRNA and tRNA genes present in otherwise average-sized genomes may be used as an indication of gene loss (Klappenbach et al. 2000; van de Guchte et al. 2006). H. modesticaldum contains eight rRNA operons (24 total rRNA genes) and over 100 genes encoding tRNAs on a 3.1 Mb chromosome (Sattley et al. 2008), suggesting that its genome has undergone a relatively recent diminution.
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If reductive evolution leading to a loss of the ability to grow autotrophically has occurred in H. modesticaldum, the obvious question is why? It would seem that the capacity for autotrophic growth would provide a significant selective advantage over obligate heterotrophy resulting from the loss of citrate lyase. However, if organic carbon sources were rarely in short supply, then genes allowing for autotrophic growth would become expendable, and this would be especially the case for bacteria capable of forming endospores, like heliobacteria. Such a scenario may occur in paddy fields, where heliobacteria are widespread (Stevenson et al. 1997). Here, a mutualistic relationship may exist in which rhizosphere-associated heliobacteria obtain organic carbon from rice plants in exchange for fixed nitrogen (Madigan 2006). Although this situation may accurately describe the ecology of mesophilic heliobacteria, H. modesticaldum is indigenous to hot springs and surrounding volcanic soils, which are often nutrient-deficient and contain little or no vegetation. It is likely that a loss of heliobacterial autotrophy (if indeed these bacteria ever were autotrophic) in this environment would be closely linked to a mutualistic association with unrelated photoautotrophic bacteria in hot spring microbial mats. In exchange for organic carbon, H. modesticaldum could be an important source of reactive nitrogen for these mats since nitrogen fixation is extremely rare in thermophilic anoxyphototrophic bacteria; indeed, the only other known thermophilic anoxyphototroph that can fix N2 at temperatures above 50°C is Chlorobaculum tepidum (Wahlund and Madigan 1993; Kimble et al. 1995). Genes allowing for nitrogen fixation in H. modesticaldum are contained within the nif regulon and are listed in Table 2. Unlike Heliobacterium gestii, which contains the alternative nitrogenase gene anfH (Kimble and Madigan 1992a; Enkh-Amgalan et al. 2005), H. modesticaldum contains only a molybdenum-dependent group I nitrogenase (Sattley et al. 2008). The list of defined organic substrates that support growth of H. modesticaldum is limited to only pyruvate, acetate, and lactate (Kimble et al. 1995). When acetate is used as carbon source, reduced ferredoxin is necessary to convert acetylCoA (from acetate) to pyruvate (Pickett et al. 1994; Sattley et al. 2008). Heliobacteria presumably cannot oxidize acetate completely to CO2 since a gene encoding carbon monoxide dehydrogenase was not identified in the H. modesticaldum genome, and heliobacterial growth on acetate requires the addition of CO2 itself (as NaHCO3) to culture media (Madigan 2006). Therefore, the required reduced ferredoxin is apparently not generated from reducing equivalents liberated from acetate oxidation but instead is supplied from other reactions in the cell. Although the pentose phosphate pathway is often a source of reducing power in many cells, genes corresponding to the oxidative
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branch of the pathway, in which a series of reactions converts glucose-6-phosphate to ribulose-5-phosphate, were not identified in the H. modesticaldum genome (Sattley et al. 2008). Thus, the NADPH produced by these reactions is apparently not available to this bacterium. One potential source of reducing equivalents may come from biochemical reactions of amino acid metabolism. Twenty-one amino acids, including selenocysteine, can be synthesized from enzymes encoded in the H. modesticaldum genome, and some of these biosynthetic pathways produce reducing equivalents as a byproduct. One example of this is the pathway leading to the biosynthesis of L-histidine, which is derived from phosphoribosyl pyrophosphate (PRPP), a product of the nonoxidative reactions of the pentose phosphate pathway. During the latter steps of histidine biosynthesis, the deamination of glutamate converts imidazole acetol 3-P to L-histidinol-P, which is then dephosphorylated to L-histidinol. Subsequent oxidation of L-histidinol to L-histidine by histidinol dehydrogenase (HM1_3051) reduces two molecules of NAD? to NADH. Through reactions of the nonoxidative pentose phosphate pathway, PRPP is ultimately synthesized from glucose 6-phosphate, and genes encoding enzymes to carry out these reactions were identified in H. modesticaldum. However, PRPP synthesis through this mechanism requires the consumption of one molecule of reduced ferredoxin (from the production of pyruvate from acetyl-CoA) and one molecule of NADH (from the production of glyceraldehyde 3-phosphate from 1,3-bisphosphoglycerate). To circumvent this expenditure of reducing power, PRPP can also be synthesized from ribose imported into the cell through an ABC transporter, as shown in Fig. 1. Ribose is phosphorylated to ribose 5-P by ribokinase (HM1_2416), followed by the conversion of ribose 5-P to PRPP via ribose-phosphate diphosphokinase (HM1_0727). Therefore, assuming ribose is available, which would not be unlikely if heliobacteria are closely associated with primary producers in situ, then the pathway of L-histidine biosynthesis through a ribose precursor would provide one means of generating reducing power for the cell. An alternative source of reducing equivalents that could generate reduced ferredoxin may come from the oxidation of amino acids transported into the cell. A large number of amino acid ABC transporter paralogs are present in the H. modesticaldum genome, and specifically, several copies of a glutamine transporter were identified (Fig. 1). Glutamine can serve as a nitrogen source for all strains of H. modesticaldum (Kimble et al. 1995), and it is converted to glutamate and ammonia (with concomitant production of ATP) via glutamine synthetase (HM1_2353). Genes encoding glutamate synthase were also identified in the genome, and this enzyme could subsequently convert two molecules of glutamate to 2-oxoglutarate and glutamine,
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Table 2 Summary of protein coding sequences (CDSs) predicted to play important physiological roles in Heliobacterium modesticaldum Locus
Symbol
Predicted function
HM1_0289
fnr
Ferredoxin:NADP? reductase
HM1_1851, 0369
trxAB
Thioredoxin and thioredoxin reductase
bchXYZ
Chlorophyllide reductase, subunits Y, Z, and X
Photosynthesis and electron transport
HM1_0434, 0435 HM1_0654, 0655, 0659
Rubredoxin and rubrerythrin
HM1_0682, 0683, 0684
bchDHI
Magnesium chelatase, subunits H, D, and I
HM1_0685, 0686, 0687
bchBNL
Protochlorophyllide reductase, subunits B, N, and L
HM1_0688
bchE
Mg-protoporphyrin IX monomethyl ester oxidative cyclase
HM1_0689
bchM
Mg-protoporphyrin O-methyltransferase
HM1_0690
pshA
RC core polypeptide, PshA
HM1_0692 HM1_0693
bchG bchJ
Bacteriochlorophyll synthetase Bacteriochlorophyll biosynthesis protein, BchJ
HM1_0696, 0697, 0698, 0699
petADBC
Cytochrome bc complex diheme, subunit IV, cyt b6, and Rieske [2Fe-2S] subunits
HM1_0701
petJ
18 kDa cytochrome c553, PetJ
HM1_1028, 1029
nuoEFG
NADH:quinone oxidoreductase, chain EF (gene fusion) and chain G
HM1_1461
fd1
4Fe–4S ferredoxin
HM1_1462
fd2/pshB
RC-associated [4Fe-4S] ferredoxin, PshB
HM1_1803, 1802
cydA/cydB
Cytochrome ubiquinol oxidase, subunits I and II
HM1_2196–2206
nuoA–D, H–N
NADH:quinone oxidoreductase, chain A–D and chain H–N
Nitrogen fixation HM1_0858
nifV
Homocitrate synthase
HM1_0859
nifB
Dinitrogenase cofactor biosynthesis protein, NifB
HM1_0860
fdxB
Nif-specific ferredoxin III
HM1_0861
nifX
Dinitrogenase Fe/Mo cofactor
HM1_0862, 0863
nifNE
Dinitrogenase Fe/Mo cofactor biosynthesis proteins NifN and NifE
HM1_0865, 0864
nifDK
Dinitrogenase Fe/Mo protein, alpha and beta subunits
HM1_0866 HM1_0867, 0868
nifH nifI2I1
Dinitrogenase iron protein, NifH
HM1_0869
orf1
nif regulatory protein
HM1_0891, 0892
etfAB
Electron transfer flavoprotein, alpha and beta subunits
Nitrogen PII regulatory proteins
Carbon metabolism HM1_0076
pykA
Pyruvate kinase
HM1_0078
pfkA
6-Phosphofructokinase
HM1_0080, 0081
accAD
Acetyl-coenzyme A carboxylase alpha and beta subunits
HM1_0105
aco
Aconitate hydratase
HM1_0110
glgA
Glycogen synthase
HM1_0371–0373
sdhCAB
Fumarate reductase cytochrome b558, flavoprotein, and Fe–S subunits
HM1_0375, 0374
sucDC
Succinyl-coenzyme A synthetase, alpha and beta subunits
HM1_0380
oadA
Oxaloacetate decarboxylase
HM1_0464
fumB
Fumarate hydratase (fumarase)
HM1_0763
hxk
Putative hexokinase
HM1_0807 HM1_0951
porC acsA
Pyruvate:ferredoxin oxidoreductase AMP-forming acetyl-coenzyme A synthetase
HM1_1076
fbaA
Fructose-1,6-bisphosphate aldolase
HM1_1077
tal
Transaldolase
HM1_1078
glpX
Fructose-1,6-bisphosphatase
HM1_1192
pgm
Phosphoglucomutase
HM1_1222–1224
gln
Glutamine ABC transporter
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Table 2 continued Locus
Symbol
Predicted function
HM1_1268, 1269
tktNC
Transketolase
HM1_1300
pgi
Glucose-6-phosphate isomerase
HM1_1311
gapA
Glyceraldehyde-3-phosphate dehydrogenase
HM1_1312
pgk
Phosphoglycerate kinase
HM1_1313
tpiA
Triosephosphate isomerase
HM1_1315
gpmI
2,3-Bisphosphoglycerate-independent phosphoglycerate mutase
HM1_1316
eno
Enolase
HM1_1471
icd
Isocitrate dehydrogenase, NADP-dependent
HM1_1472
mdh
Malate dehydrogenase, NAD-dependent
HM1_2140
rpe
Ribulose-5-phosphate 3-epimerase
HM1_2157
ackA
Acetate kinase
HM1_2353
glnA
Type I glutamine synthetase
HM1_2416–2420 HM1_2461
rbsABCDK ppdK
Ribose ABC transporter and ribokinase Pyruvate-phosphate dikinase Alcohol dehydrogenase
HM1_2632
adhE
HM1_2757
ldh
L-Lactate
HM1_2762
korC
2-Oxoglutarate:ferredoxin oxidoreductase
HM1_2773
pckA
Phosphoenolpyruvate carboxykinase
dehydrogenase
Genes of physiological importance encoded in the H. modesticaldum genome not shown in the table include ATP synthase (atpABCDEFGH) and [NiFe] hydrogenase (including two copies of structural genes hupSLC with one copy of accessory/maturation genes hupD and hypABCDEF)
while at the same time producing NADPH that could be used for acetate photoassimilation. Therefore, the importance of glutamine to H. modesticaldum and other heliobacteria may extend beyond its use as a source of nitrogen. Although growth of H. modesticaldum using acetate as carbon source and ammonia as nitrogen source in the absence of amino acids has been reported (Kimble et al. 1995), the availability of glutamine and possibly other amino acids may significantly stimulate heliobacterial growth on acetate. An additional question concerning growth of H. modesticaldum on acetate is whether diazotrophic growth is possible when using this substrate. All heliobacteria have been shown to fix dinitrogen, even in the absence of light if pyruvate is available (Kimble and Madigan 1992b). However, to our knowledge, data from nitrogen fixation experiments in heliobacteria growing on acetate as carbon source have not been reported. Considering the steep energetic and reducing power demands of nitrogen fixation (8 electrons and 16 ATP per molecule of N2 reduced to 2NH3), it is questionable as to whether sufficient activated carrier molecules can be supplied for this process to occur under such conditions. Photosynthesis and electron transfer The genetic inventories of the photosynthesis gene clusters (PGCs) from two heliobacterial species, Heliobacillus mobilis and H. modesticaldum, were previously described by
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Xiong et al. (1998) and Sattley et al. (2008), respectively. Genes encoding proteins directly involved in Bchl g biosynthesis and electron transfer in the H. modesticaldum PGC are included in Table 2. A phylogenetic protein sequence analysis using bacteriochlorophyll/chlorophyll synthetase (BchG/ChlG) shows a predominately radial evolutionary divergence in a number of clearly resolved phyla of prokaryotic and eukaryotic photosynthetic organisms (Fig. 2). Heliobacterial BchG biosynthetic gene sequences show a particularly clear divergence from those of other phototrophs, suggesting the occurrence of a distant gene duplication event. No gene predicted to encode a divinyl reductase was identified from the genome data. Although bchJ is present in the PGC of H. modesticaldum (Table 2), this gene was recently shown not to encode divinyl reductase in green sulfur bacteria (Chew and Bryant 2007). Therefore, a pathway for Bchl g biosynthesis was proposed in which a series of reductions converted divinyl protochlorophyllide a to 8vinyl bacteriochlorophyllide a through the activity of the bchLNBXYZ gene products (Sattley et al. 2008). An isomerase that could convert the 81-vinyl group to an ethylidene group would produce bacteriochlorophyllide g, and following the addition of farnesol by BchG, the mature Bchl g would be synthesized (Sattley et al. 2008). Genes encoding farnesol biosynthesis via a complete nonmevalonate pathway are present in the H. modesticaldum genome (Sattley et al. 2008).
Photosynth Res (2010) 104:113–122
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Fig. 2 Phylogeny of bacteriochlorophyll/chlorophyll synthetases (BchG/ChlG) from Heliobacterium modesticaldum and selected photosynthetic species. The scale bar indicates the number of substitutions per site. (Included as supplemental material in Sattley et al. 2008)
Although this putative pathway effectively describes the biosynthesis of the primary electron donor in the RC, genes involved in the biosynthesis of the pigment that functions as the primary electron acceptor, 81-OH-Chl a, are unknown. The synthesis of 81-OH-Chl a from a Bchl g precursor could occur under the proper conditions. The spontaneous isomerization of Bchl g to a form of Chl a in the presence of light and oxygen is well known, and the transition can be readily observed as the pigment changes from a brown-olive color to emerald green (Beer-Romero et al. 1988). Following this abiotic oxidation, the formation of 81-OH-Chl a could then be achieved by the conversion of the C-8 ethylidene group to a hydroxyethyl group on ring B of the porphyrin tetrapyrrole. However, if 81-OHChl a is formed from Bchl g, oxidation of the latter would presumably need to be catalyzed enzymatically in vivo since heliobacteria are strictly anaerobic and quickly lose viability upon exposure to oxygen and light (Madigan 2006). Consequently, additional molecular and/or biochemical studies are necessary to identify genes encoding enzymes involved in 81-OH-Chl a biosynthesis in heliobacteria. Phylogenetic analyses of components of pigment biosynthesis, such as that shown in Fig. 2, are useful for elucidating the early evolution of photosynthesis since all lineages of phototrophic organisms can be represented in a linear fashion (Blankenship 1992; Xiong et al. 2000). However, phylogenetic trees constructed comparing RC core polypeptides have also been shown to be useful tools for visualizing the natural history of photosynthesis (Sadekar et al. 2006). An analysis showing a RC core polypeptide phylogeny for heliobacteria and other
selected phototrophs containing type I RCs is shown in Fig. 3. Protein sequences used in the alignment include PscA (Chlorobi and Acidobacteria), PsaA (Cyanobacteria and phototrophic eukaryotes), and PshA (heliobacteria). The tree shows a loose clustering of type I RC core polypeptides from anoxygenic versus oxygenic phototrophs, indicating the distant separation of these two phototrophic groups. Among anoxygenic phototrophic bacteria, the heliobacterial clade is tightly clustered and far removed from that of PscA-containing Chlorobi and the aerobic photoheterotroph Candidatus Chloracidobacterium thermophilum. Genomic features concerning electron transfer and photophosphorylation in H. modesticaldum were recently shown in detail (see Fig. 6 from Sattley et al. 2008). Although much work over the past 25 years has contributed to our current understanding of heliobacterial electron transfer (Nuijs et al. 1985; Smit et al. 1987; Vos et al. 1989; Fischer 1990; Trost et al. 1992; Kleinherenbrink et al. 1994; Lin et al. 1995; Nitschke et al. 1995; Kramer et al. 1997; Oh-oka et al. 2002; Heinnickel et al. 2006, 2007), some important questions remain. Until recently, the nature and identity of the complex that transfers electrons from NADH to the menaquinone pool was unknown. However, with the putative identification of 14 genes (nuoA-N) encoding NADH:quinone oxidoreductase, it is likely that electrons enter the transport chain through this complex, though this has not been confirmed biochemically (Sattley et al. 2008). The movement of electrons through the cytochrome bc complex (including the Rieske [2Fe–2S] subunit PetC) and cytochrome c553 (PetJ) to RC pigments is becoming
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Fig. 3 Phylogeny of type I reaction center core polypeptides from selected species of green sulfur bacteria and acidobacteria (PscA), heliobacteria (PshA), and cyanobacteria (PsaA). The scale bar indicates the number of substitutions per site. (Included as supplemental material in Sattley et al. 2008)
increasingly well characterized through recent biochemical and genomic studies (Ducluzeau et al. 2008; Sattley et al. 2008). However, electron transfer beyond the primary electron acceptor (81-OH-Chl a) within the RC is uncertain. Despite the structural and phylogenetic connection between the heliobacterial RC and Photosystem I of oxygenic phototrophs (Trost et al. 1992; Madigan 2006), conflicting experimental results have lead to the questionable existence of a secondary quinone acceptor in the heliobacteria. In addition, Kramer et al. (1997) proposed the cyclic transfer of electrons from the heliobacterial RC to the cytochrome bc complex via the menaquinone pool. However, the mechanism by which this takes place has not been unequivocally confirmed. The resolution of these questions remains a major focus of research on heliobacterial RC photochemistry. Electrons within the RC core polypeptide homodimer PshA are eventually shuttled to an FX-like [4Fe–4S] cluster before subsequent donation to the terminal Fe/S dicluster FA/FB within PshB, a [4Fe–4S]-binding ferredoxin that associates with PshA on the cytoplasmic side of the membrane (Kleinherenbrink et al. 1994; Heinnickel and Golbeck 2007; Oh-oka 2007; Jagannathan and Golbeck 2008). The pshA gene (HM1_0690) is contained within the heliobacterial PGC, while pshB (HM1_1462) is situated adjacent to a second [4Fe–4S]-binding ferredoxin (HM1_1461) on a distant region of the chromosome. This second ferredoxin presumably supplies reducing equivalents for cytoplasmic reactions primarily focused on carbon assimilation and nitrogen fixation (Fig. 1) (Sattley et al. 2008). Although initially not detected, a homolog for a
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gene encoding ferredoxin:NADP? reductase (FNR) (HM1_0289) has now been identified in the H. modesticaldum genome, thus providing for the ferredoxin-dependent reduction of NADP?. Similar to the open question of cyclic electron transfer from the heliobacterial RC, the possibility of direct electron transfer from reduced cytoplasmic ferredoxin to the cytochrome bc complex is unconfirmed. Final remarks Obtaining the genome sequence of H. modesticaldum str. Ice1 is but the first step into heliobacterial genomics. The sequencing of a second heliobacterial genome has recently been funded and thus more heliobacterial genomes should be available within the next few years. The new study will focus on an alkaliphilic heliobacterial species of the genus Heliorestis. It is expected that a comparison of such a genome with that from H. modesticaldum will provide valuable information concerning cellular processes in contrasting extremophilic heliobacteria, including those related to carbon and nitrogen metabolism, photophosphorylation, nutrient transport, and sporulation. Acknowledgments The authors thank Michael T. Madigan, Mark Heinnickel, and Joseph (Kuo-Hsiang) Tang for kindly participating in insightful discussions concerning the physiology and ecology of heliobacteria. Genome sequencing of Heliobacterium modesticaldum strain Ice1T was supported by the National Science Foundation Phototrophic Prokaryotes Sequencing Project (grant 0412824). We also gratefully acknowledge continuing support from the Exobiology Program from NASA.
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