As previously reported [ 2 ], we also found that yeast extract 0. It is known that many undefined carbon sources, vitamin mixtures and amino acids included, are included in yeast extract. We successfully replaced yeast extract with vitamin B 12 for supporting the growth of a different photoheterotrophic bacterium [ 9 ]. In all of the growth media of H. With approaches listed in Materials and Methods, we have estimated that the amount of pyruvate, acetate and lactate in yeast extract is negligible.
However, the inclusion of pyruvate or acetate as a defined organic carbon source, along with yeast extract, can significantly enhance growth Additional file 2 : Figure S2. Alternatively, it is possible that some amino acids in yeast extract may support the growth of H. To test this hypothesis, we grew H. Also, we didn't observe significant growth enhancement with vitamin mixtures included in casamino acids-grown cultures. Together, our studies support the idea that amino acids contribute to the growth of H. Further, we have probed the contribution of glutamate and glutamine for cell growth of H.
Glutamate can serve as a nitrogen source for H. To investigate the impact of yeast extract on metabolic pathways, we compared transcriptomic data from cultures containing PYE pyruvate and yeast extract are carbon sources and PMS pyruvate as the sole organic carbon growth media all of the growth media are described in Materials and Methods section and Table 1.
It is generally assumed that proteomic and transcriptomic data are related [ 11 ], and that higher mRNA levels normally lead to more protein production, particularly in prokaryotes with no mechanism of post-transcriptional modification. Our data show that the addition of yeast extract to the culture media has little effect on the transcriptional levels of most genes involved in carbon metabolism and other cellular functions Additional file 3 : Table S1.
It has been recognized that pyruvate is the preferred organic carbon source for heliobacteria and it can support both photoheterotrophic and chemotrophic growth [ 3 ]. Consistent with previous reports, our studies show that H.
In contrast to CO 2 -enhanced growth of Chlorobaculum Cba. The lack of autotrophic growth in H. To confirm the absence of an enzyme having ACL activity, we performed activity assays in cell-free extracts of H. The latter served as a positive control for ACL activity, which is documented in Cba. Consistent with previous reports, the activity of ACL was clearly detected in cell free extracts of Cba. Additionally, the activity of citrate synthase, catalyzing the formation of citrate from condensation of OAA and acetyl-CoA in the oxidative TCA cycle, also cannot be detected data not shown.
Alternatively, the genomic data suggest that certain non-autotrophic pathways may be available for CO 2 assimilation in H. Together, our experimental data indicate that H. Figure 2A shows that H. In contrast, no CO 2 -enhanced growth was detected using pyruvate as the defined organic carbon source Figure 2B. These studies suggest that pyruvate:ferredoxin oxidoreductase PFOR contributes to CO 2 -enhanced phototrophic growth through conversion of acetyl-CoA to pyruvate equation 1 and is one of the major pathways for CO 2 assimilation in H. In some relatives of the heliobacteria, such as Clostridium thermoaceticum and other clostridia, PFOR is linked to the carbon fixation via the reductive acetyl-CoA pathway i.
While enzymes that function in this autotrophic carbon fixation pathway are commonly found in both methanogens and acetate-producing clostridia [ 20 ], they are not found in the genome of H. Because pyruvate is a required nutrient for fermentative growth [ 21 ] and also best supports phototrophic growth of heliobacteria, the following studies of heliobacterial phototrophic and chemotrophic growth were obtained from cells grown in PYE medium. The OD of cell cultures and pyruvate consumption during phototrophic and chemotrophic growth are shown in Figure 3A , and the levels of gene expression in each growth condition are reported in Table 2.
The major results from our investigation are illustrated below. Cell growth, pyruvate consumption, and acetate production during phototrophic and chemotrophic growth. Cell growth vs. Figure 3B indicates that acetate is excreted in pyruvate-grown cultures containing 0. Since either pyruvate or acetate can support the phototrophic growth, the amount of acetate production does not increase steadily during phototrophic growth. In contrast, previous reports [ 2 , 6 ] and our studies showed that only pyruvate can support chemotrophic growth of H.
Together, these results are coherent with our investigation that no significant amount of pyruvate is included in yeast extract see "growth on yeast extract". Additionally, no lactate excretion is detected in pyruvate-grown cultures Table 3. While our results are different from the report of Heliobacterium strain HY-3 [ 18 ], the authors found more acetate being produced during chemotrophic growth Both our and their studies demonstrate that acetate can be produced from pyruvate-grown heliobacterial cultures during phototrophic and chemotrophic growth.
In contrast, the enzymatic activity of ACK and PTA can be detected in cell extracts of pyruvate-grown cultures during both phototrophic and chemotrophic growth. Relative gene expression levels during phototrophic versus chemotrophic growth.
Only representative genes responsible for carbon metabolism, nitrogen fixation and hydrogen production are shown. Together, our studies suggest that: i H. Lack of enzymatic activity of ACS and low expression of acsA in the cultures grown in darkness is consistent with the physiological evidence that acetate cannot support the chemotrophic growth of H. ATP is generated via substrate-level phosphorylation in the reaction of acetyl-phosphate being converted to acetate; and iii while no pta gene has been annotated in the genome, function of PTA is identified in H.
Also, no pox gene is annotated in the genome. The proposed acetate metabolism of H. The proposed carbon flux in H. Enzymes or pathways investigated in our report are highlighted in red. Dot line represents that the gene is missing and activity is not detected.
To extend our understanding from the physiological studies shown in Figure 3 , we monitored some key genes for carbon, nitrogen and hydrogen metabolism during phototrophic and chemotrophic growth. Compared to the photoheterotrophic growth of H. In agreement with this hypothesis, most of the genes involved in energy metabolism are down-regulated during chemotrophic growth Table 2 and Figure 4. Despite the lack of genes encoding pyruvate dehydrogenase, PFOR can be an alternative enzyme for converting pyruvate into acetyl-CoA and Fd red in pyruvate fermentation equation 1 , and Fd red can interact with FNR, known to be the last electron transporter in the light-induced electron transfer chain, to produce NADPH equation 2.
Together, the discovery of FNR activity in cell extracts indicates that the reducing power required for carbon and nitrogen metabolisms in H. The genomic information indicates that H.
A putative mechanism of BChl g biosynthesis was recently proposed [ 1 ]. The biosynthesis of photosynthetic pigments during chemotrophic growth under nitrogen fixing conditions has been observed for some species of heliobacteria, including Heliobacillus mobilis , Heliobacterium gestii and Heliobacterium chlorum [ 21 ]. Here, we would like to examine if H. Figure 6 shows the normalized absorption spectra of the intact cell cultures from phototrophic and chemotrophic growth, after cell light-scattering has been digitally subtracted from the raw data see Methods.
The absorption peaks of the unique pigment BChl g at nm and of 8 1 -OH-Chl a F at nm can be detected in Figure 6 , indicating that photosynthetic pigments can be produced by H. The expression levels of genes responsible for B Chls biosynthesis, bchY chlorophyll reductase, subunit Y , bchB protochlorophyllide reductase, subunit B , bchE anaerobic cyclase and bchG bacteriochlorophyll synthase , are fold lower in darkness than grown phototrophically Figure 4 and Table 2.
Normalized absorption spectra of whole cell cultures during phototrophic and chemotrophic growth. The cell scattering was digitally subtracted in the spectra.
Biological nitrogen assimilation i. In the energy metabolism of H. Because of the energy and reducing power demanded for nitrogen fixation, diazotrophic growth of H. Figure 7 shows diazotrophic and non-diazotrophic growth during phototrophic and chemotrophic growth, and growth of H. Table 3 indicates that a similar amount of acetate is excreted during diazotrophic and non-diazotrophic growth. Together, our studies suggest that H. Cell growth with or without nitrogen fixation in pyruvate-grown cultures during phototrophic and chemotrophic growth.
While the EMP pathway is annotated in the genome, no sugar-supported growth has been reported for H. It is not uncommon for microorganisms that have the EMP pathway annotated but do not use glucose and other sugars as carbon sources, and to date only one heliobacterium, Heliobacterium gestii , has been reported to grow on C6-sugars, i. Alternatively, fermentation of glucose through the EMP pathway has been reported in non-phototrophic bacteria in the phylum Firmicutes [ 26 , 27 ].
In this paper, we present the first report on the growth of H. With the support of multiple lines of experimental evidence, including physiological studies, activity assays, gene expression data, and mass spectra, our work addresses the puzzle that the EMP pathway has previously been annotated but no sugar-supported growth has been reported for H. As D-fructose and D-glucose are polar molecules, glucose, fructose or hexose transporter proteins are required to move those molecules across the cell membrane into the cells.
No known hexose transporter has been reported for H. It remains to be determined if the putative ribose transporter of H. Moreover, utilization of glucose can provide an additional path for H 2 production in H. The CO 2 -anaplerotic pathways are known to replenish the intermediates of TCA cycle, so that removal of the intermediates for synthesizing cell materials will not significantly slow down the metabolic flux through the TCA cycle. Our recent studies showed that the photoheterotrophic bacterium R. All of the genes encoding the enzymes for CO 2 -anaplerotic pathways, PEP carboxylase, PEP carboxykinase, pyruvate carboxylase and malic enzyme, have been annotated in the R.
Our studies presented here also suggest that H. One carbon C1 metabolism plays a central role in microbial metabolism and global biogeochemical cycles. Key molecules of C1 metabolism include methane, carbon monoxide, carbon dioxide, and methanol, coenzyme-bound one-carbon moieties e. Many of them e. The conference spans natural and synthetic systems under oxic and anoxic conditions at scales from molecules to ecosystems. There is an emphasis on the molecular aspects of autotrophic microbes, methanogens, methylotrophs, methanotrophs, and acetogens. Participants will hear the latest cutting-edge research on microbial C1 metabolism, the role of these reactions in biogeochemical cycles, and current approaches to engineer microbes and biochemical pathways for societal applications.
These applications include the production of biofuels and the remediation of environmental contamination. The conference program will feature talks from a mix of junior and senior investigators. Complementary perspectives will be provided by leaders in biochemistry, general microbiology, molecular biology, structural biology, microbial ecology, and systems and synthetic biology.
The meeting will also feature dynamic and interactive poster sessions. This process is an essential storage form of carbon, which can be used when light conditions are too poor to satisfy the immediate needs of the organism. A heterotroph is an organism that depends on organic matter already produced by other organisms for its nourishment. Photoheterotrophs obtain their energy from sunlight and carbon from organic material and not carbon dioxide.
Most of the well-recognized phototrophs are autotrophs, also known as photoautotrophs, and can fix carbon.
They can be contrasted with chemotrophs that obtain their energy by the oxidation of electron donors in their environments. Photoheterotrophs produce ATP through photophosphorylation but use environmentally obtained organic compounds to build structures and other bio-molecules. Photoautotrophic organisms are sometimes referred to as holophytic.
Chemoautotrophs and chemoheterotrophs make their food using chemical energy rather than solar energy.
Chemotrophs are a class of organisms that obtain their energy through the oxidation of inorganic molecules, such as iron and magnesium. The most common type of chemotrophic organisms are prokaryotic and include both bacteria and fungi.
All of these organisms require carbon to survive and reproduce. The ability of chemotrophs to produce their own organic or carbon-containing molecules differentiates these organisms into two different classifications—chemoautotrophs and chemoheterotrophs. Organismal and environmental interactions in a wetland : sources of energy and carbon for each trophic level.
Chemoautotrophs are able to synthesize their own organic molecules from the fixation of carbon dioxide. These organisms are able to produce their own source of food, or energy. The energy required for this process comes from the oxidation of inorganic molecules such as iron, sulfur or magnesium. Chemoautotrophs are able to thrive in very harsh environments, such as deep sea vents, due to their lack of dependence on outside sources of carbon other than carbon dioxide.