Growth characteristics of G. daltonii strain FRC-32
G. daltonii FRC-32 can grow with various aromatic compounds including benzoate, toluene, benzene, m-, o-, and p-cresol. Toluene and benzene were toxic when amended at a high concentration (2 mM). Therefore, these aromatic hydrocarbons were supplied in a hydrophobic carrier phase with 2,2,4,4,6,8,8-Heptamethylnonane (HMN) to allow continuous supply with low concentrations (1 mM) because the direct addition to the culture was known to be inhibitory for growth (54). Also, cultures were tested for growth on toluene and benzene without HMN as a carrier in order to test if G. daltonii utilizes HMN as substrate. Good growth with toluene and benzene without HMN was observed (data not shown).
Various electron acceptors such as fumarate, nitrate and ferric citrate were tested for G. daltonii cultivation. G. daltonii was grown using 1 mM nitrate as an electron acceptor. Higher concentrations, up to 5 mM, were tested. However, these were all toxic and inhibitory to growth. Ferric citrate was tested as an electron acceptor at either 60 mM or 30 mM. Growth was observed when benzoate was used as the substrate (data not shown). However, significant growth was not observed when toluene or benzene were provided to the culture with ferric citrate as the electron acceptor. This is possibly due to the stressful nature of the combination of aromatic hydrocarbons and ferric citrate. G. daltonii culture was able to only grow using one of these at a time. Molecular genetic experiements for the present study were performed on cultures grown with 10 mM fumarate as an electron acceptor.
After establishing the growth conditions, G. daltonii FRC-32 was adapted to the respective aromatic substrates by subsequent transfer for at least five passages. A detailed growth
curve of the adapted cultures was then monitored by measuring increases in optical density, substrate loss and product formation in order to determine optimal time points for harvesting cultures. At mid-log phase cells were harvested for further genomic and metabolite analyses, so that misleading results in the gene expression study originating from growth phase effects were avoided.
These cultures grew to an average OD600 of 0.1-0.3. This growth required 7-60 days
depending upon the substrate provided. While G. daltonii is able to utilize multiple electron acceptors, it did not correspond with equal energy output. For example, more energy, and thus more rapid growth, was accomplished when this organism was grown utilizing nitrate as a sole electron acceptor than Fe(III) or even fumarate.
Genomic analysis of anaerobic aromatic compound degradation genes
In order to identify which regions of the G. daltonii genome might code for enzymes involved in anaerobic degradation of aromatic compounds, particularly benzene, genes within the reported aromatic compound-degrading gene island of G. metallireducens (55) were compared to genes within G. daltonii using BLAST analysis. As observed in the G. metallireducens genome (Fig. 9A), many genes predicted to play a role in aromatic compound degradation clustered into a large aromatic degrading gene island from Geob_0095 to Geob_0255 in the G. daltonii genome (Fig. 9B). This island spans a region of 165 genes and roughly 194 kbp. While most of the genome has a GC content of 53.5%, this region has a GC content of 56.3%. In addition to the main aromatic degrading gene island, both organisms have smaller gene clusters at other locations in the genome. The smaller cluster containing genes
Geob_2420-Geob_2451 in G. daltonii and has a GC content of 56.65% and spans a region of 32 genes and approximately 39 kbp.
Figure 9: Locations of predicted aromatic compound degradation genes in Geobacter species. Genes predicted to play a role in aromatic compound degradation in G. metallireducens and G. daltonii were mapped with black bars indicating presence of genes associated with aromatic compound degradation.
Because anaerobic benzene oxidation is such a unique characteristic of G. daltonii, its genome was scanned for genes that shared no homology to other anaerobic aromatic- degrading bacteria. Once identified, these genes were grouped into functional blocks (Fig. 10) based on physical proximity within the genome, and functions were predicted if possible based on homology and existing annotation (Table 2).
Figure 10: Location of “unique” genes in the G. daltonii genome. Genes that are predicted to have “hypothetical” or “unknown” function that are proximal to one another.
Table 2. Predicted function of the unique genesa
a
All of the G. daltonii proteins from submitted annotation were blasted against other Geobacteraceae species for which complete genome sequences were available and annotated. Genes which were present in G. daltonii, but not found in any other Geobacteraceae species were considered as unique. The algorithm was BlastP, and the e-value cut-off score was 1e-20.
One of the largest segments, segment 1 is located close to the large aromatic-degrading gene island close to the start of the genome. At 55.9% GC, this region has a different GC content from the rest of the genome. This region contains multiple glycosylases and glycosyl transferases as well as dehydratases and cyclases. Because of these observations, this region is predicted to encode genes involved in biosynthesis pathways. Also unique to this region is that it is flanked by insertion sequences which could be used for genetic movement. This bares interest as it has been speculated that large mobile genetic elements, sometimes called ICE regions, are the mechanism by which the ability to degrade aromatics spread from one organism to another. An example of this is Tn4371, a 55 kbp region that confers the ability to degrade biphenyls to its host (56). Another region in G. daltonii with characteristics similar to ICE/integrated transposons is segment 7. This region has genes for DNA recombination as well as competence proteins and a recombinase. Segment 15 is an anomaly in that most of the genes in this region seem to code for a toxin/antidote system. This block also has a vastly different GC content from the rest of the genome at 48.7%. Finally, segment 18 is a region with mostly hypothetical proteins. However, it is flanked by inverted repeats and transposons as seen with ICEs. None of the regions of unique genes fell within close proximity to either of the aromatic degrading gene islands identified.
The large aromatic-degrading gene cluster from Geob_0095-Geob_0253 includes genes for utilization of phenol, which is a proposed intermediate in anaerobic oxidation of benzene. The first step in the anaerobic degradation of phenol is the addition of a phosphate group from ATP by the enzyme phenylphosphate synthase (pps). Phenylphosphate is carboxylated with CO2
the carboxyl group by an unknown enzyme, and the hydroxyl group of 4-hydroxybenzoyl-CoA is eliminated by reduction to form benzoyl CoA (57). The remainder of the pathway consists of reduction and opening of the aromatic ring, catalyzed by the bam gene products, beta-oxidation to acetyl-CoA, and oxidation to CO2 through the TCA cycle. Expression levels of pps and ppc
genes have been examined to detect activation of this pathway within anaerobic phenol- degrading organisms and could be used to determine whether they are involved in degradation of benzene by G. daltonii (58).
The smaller aromatic-degrading gene cluster from Geob_2420-Geob_2451 contains genes involved in toluene oxidation, another proposed route for anaerobic benzene degradation. The first step in anaerobic toluene degradation is the enzymatic addition of fumarate to toluene’s
methyl group, resulting in the formation of R-benzylsuccinate (59). The enzyme benzylsuccinate synthase is a duplex of three subunits (, β and γ), forming a heterohexamer (60); it is activated by an accessory protein, BssD, which generates a glycyl radical within the 98 kDa alpha subunit (61). Bss must be maintained in strictly anaerobic conditions as O2 will cause irreversible
cleavage of the protein (62). Subsequent steps are catalyzed by the bbs (beta oxidation of benzylsuccinate) gene products (Fig. 11A). Coenzyme A is transferred from succinyl-CoA to the alpha-carboxyl group of R-benzylsuccinate to form R-benzylsuccinyl-CoA. This compound is oxidized to E-2-benzylidenesuccinyl-CoA, hydrated to [2-hydroxy(phenyl)methyl]succinyl- CoA, oxidized to benzoylsuccinyl-CoA, and cleaved thiolytically to succinyl-CoA plus benzoyl- CoA. Benzoyl-CoA undergoes reduction and opening of the aromatic ring, catalyzed by the bam
gene products, followed by beta oxidation and entry into the TCA cycle as acetyl-CoA which is finally converted to CO2 (63). Upregulation of bss or bbs genes during anaerobic degradation of
benzene could be an evidence for use of the toluene pathway during anaerobic benzene degradation.
Figure 11: bbs and bss gene clusters in G. daltonii. Organization of anaerobic toluene
oxidation genes within the genome of G. daltonii. White arrows indicate genes without predicted aromatic compound degradation functions according to NCBI annotations. Yellow arrows indicate genes predicted to be responsible for Bss formation and activation. Green arrows indicate similar predicted sigma 54-dependent transcriptional regulators. Spaces within arrows indicate predicted non-coding regions between genes and/or operons.
Of particular interest is the observation that G. daltonii has two adjacent operons encoding glycl radical-like genes (Fig. 11B). No other anaerobic toluene-oxidizing prokaryotes at present have been identified which code two copies of Bss. Also, it is interesting that these copies lie within completely independent operons, each with its own divergently transcribed gene encoding a transcriptional regulator and with different genes downstream. These copies only share 76% amino acid identity to one another. The bbs genes, which are responsible for the conversion of benzylsuccinate to benzoyl-CoA, are present only once within the genome.
However, the bam genes, predicted to encode electron transfer proteins associated with benzoyl- CoA reductase, are present in two copies each, organized into two operons. One of these operons is headed by a single bamB gene (encoding benzoyl-CoA reductase proper), and the other by two paralogs of bamB. Between the two operons are seven genes, of which Geob_0225 encodes a transporter of the major facilitator superfamily with homology to a benzoate transporter of Acinetobacter sp. strain ADP1 (64). Another of the intervening genes, Geob_0224, encodes a homolog of 2,3-dihydroxybenzoate adenylyltransferase from Bacillus subtilis (1, 27), which may function as 2,3-dihydroxybenzoate-coenzyme A ligase in G. daltonii. If indeed G. daltonii is capable of degrading 2,3-dihydroxybenzoate, the pair of BamB- paralogous proteins and their associated electron transfer proteins may function in the reductive elimination of hydroxyl groups from 2,3-dihydroxybenzoyl-CoA, converting it to benzoyl-CoA for further degradation.
The gene cluster Geob_2442-Geob_2451 was named Bss (benzylsuccinate synthase) while the gene cluster Geob_2433-Geob_2441 was named Grl (glycyl radical-like). These clusters were compared to each other as well as the Bss cluster in G. metallireducens. The organization of the Grl cluster most similarly resembles the Bss cluster found in G. metallireducens. Both clusters first encode a gene (Geob_2433/Gmet_1533) for a flavin-binding oxidoreductase. However, in lieu of the Gmet_1535 gene of the G. metallireducens Bss cluster that encodes an outer membrane protein very similar to the toluene transporters of Ralstonia pickettii and Pseudomonas putida (65), the Geob_2434 gene in Grl encodes an outer membrane protein closely related to those encoded by Geob_0136/Gmet_2107 in the phenol degradation gene clusters and Geob_0117/Gmet_2127 in the p-cresol degradation gene clusters (66). According to the information, it can be hypothesized that one or both benzylsuccinate synthase-
like complexes of G. daltonii might be involved in degradation of a hydroxyl-substituted toluene such as o-cresol or m-cresol as a substrate. Assuming that the remaining enzymes of the toluene degradation pathway (which act on the methylsuccinate moiety) can accommodate a hydroxylated aromatic ring, the end product would be 2-hydroxybenzoyl-CoA or 3- hydroxybenzoyl-CoA, plausible substrates for the BamB-paralogous reductase described above. Other than this gene, all genes in Grl and the G. metallireducens Bss cluster are predicted to be the same, encoding a metal-binding protein of the VWFA superfamily with high-sequence- identity homologs in Thauera aromatica, Aromatoleum aromaticum and deltaproteobacterial strain NaphS2, an ATPase homologous to the putative chaperone BssE (TutH) required for toluene metabolism by T. aromatica (67, 68). The benzylsuccinate synthase enzyme and its activator, BssD follows. All of these Bss operons are divergently transcribed with genes encoding sigma-54-dependent transcriptional regulators, which probably act as activators for these operons. Similarly to Grl and the G. metallireducens Bss operon, in G. daltonii Bss begins with BssCAB and BssD. However, the remaining Bss genes have homologs in G. metallireducens that are located outside the toluene degradation gene cluster.
The Bss cluster in G. metallireducens is adjacent to genes Gmet_1543 to Gmet_1556, which correspond to genes Geob_2569 to Geob_2552 of G. daltonii, separated from the Bss cluster by an island of 121 genes (Geob_2452 to Geob_2550) and roughly 102 kbp. This island is very interesting because it encodes multiple sets of proteins that resemble assembly factors for a vanadium-iron nitrogenase, yet the structural genes for this type of nitrogenase are not present in the G. daltonii genome. G. daltonii, like other Geobacter species, possesses ancestral genes for a molybdenum-iron nitrogenase and its assembly factors, Geob_2573 to Geob_2591. The assembly factors encoded by this island in G. daltonii may act in pathways analogous to the
synthesis of the molybdenum-iron cofactor of nitrogenase (69), but the end products may be various iron-sulfur-metal clusters within enzymes that catalyze difficult reactions other than nitrogen fixation. The location of the island next to Bss suggests that these reactions might serve to activate recalcitrant aromatics such as benzene.
For the first step in iron-sulfur-metal cluster synthesis, extraction of sulfur atoms from cysteine, the island encodes two cysteine desulfurases (Geob_2507, Geob_2526) and the rest of the genome encodes two more (Geob_1980, Geob_3049). The enzyme that assembles the sulfur atoms into iron-sulfur clusters is encoded by Geob_3050 outside the island. The third step, the merging of two 4Fe-4S clusters with a ninth sulfur atom and an unidentified small atom X, is carried out by NifB (Geob_2584) for the nitrogenase cofactor and presumably by three similar enzymes encoded by the island (Geob_2468, Geob_2497, Geob_2529). In many species, NifB includes an iron-sulfur cluster-binding oxidoreductase domain of the radical SAM type, which in
Geobacter species is found as a separate protein (Geob_2591); thus, a plausible source of the ninth sulfur atom is S-adenosylmethionine (SAM), which may also donate its methyl group to become atom X. A very similar radical SAM domain protein encoded by Geob_2456 in the island may function together with the three NifB-like enzymes. The island encodes several supernumerary proteins for assimilation of sulfur into both cysteine and SAM: periplasmic sulfate-binding protein (Geob_2453, redundant with Geob_1651), cysteine synthase (Geob_2459 and Geob_2530, redundant with Geob_1403), and O-acetyl-L-homoserine sulfhydrylase (Geob_2488, Geob_2499, redundant with Geob_0796, Geob_1890). There is also a gene for selenide, water dikinase (Geob_2508, redundant with Geob_1420), which could be an indication that selenium is a component of some of the iron-sulfur clusters synthesized by the island.
The 8Fe-9S-X cluster assembled by each NifB-like protein is expected to be transferred to a scaffold protein homologous to NifEN (Geob_2581) or a heterodimer of VnfE and VnfN in the case of a vanadium-iron nitrogenase, where the iron atom at one apex of the cluster may be displaced by a different metal before the cluster is inserted into the active site of an enzyme. The island encodes four homologs of VnfE (Geob_2460, Geob_2466, Geob_2500, Geob_2539) and five homologs of VnfN (Geob_2461, Geob_2467, Geob_2491, Geob_2501, Geob_2540), suggesting that G. daltonii can synthesize at least five different clusters. Although these proteins are clearly homologs, they are not most closely related each other according to the BLAST analysis output, suggesting that their evolutionary divergence is older than their acquisition by G. daltonii. The island encodes numerous metal transporters (Geob_2474, Geob_2475, Geob_2476 for nickel uptake, Geob_2512 for magnesium uptake, Geob_2552, Geob_2553, Geob_2554, Geob_2555, Geob_2556 for a heavy metal) and efflux pumps (Geob_2462, Geob_2477, Geob_2478,Geob_2487, Geob_2562,Geob_2563,Geob_2564 for a metal), and other ABC transporters, for which the number of periplasmic substrate-binding proteins (Geob_2470, Geob_2532, Geob_2533, Geob_2534, Geob_2536) and membrane proteins (Geob_2489, Geob_2490, Geob_2493, Geob_2494, Geob_2535, Geob_2537) exceeds the number of ATP- binding proteins (Geob_2492, Geob_2524, Geob_2531), suggesting adaptability to transport different solutes. These transporters may provide the VnfE and VnfN scaffolds with a variety of trace metals to insert into iron-sulfur clusters.
Displacement of a cluster’s apical iron atom with molybdenum, which requires ATP
hydrolysis and electron transfer to molybdate, is catalyzed by the NifH protein (Geob_2574), which also transfers reducing equivalents to nitrogenase during nitrogen fixation. The island encodes two homologs of NifH (Geob_2455 and Geob_2458), which may reduce different
metals and provide the energy to insert them into iron-sulfur clusters, and may also transfer electrons to the enzymes that bind these clusters. The molybdenum atom is ligated by homocitrate, which is synthesized by the Geob_2573 gene product; the island-derived clusters may or may not contain homocitrate. Consistent with a role of these clusters in activation of benzene by methylation to toluene, the island encodes three proteins involved in methyl group transfer: methyl-Co(III) 5-hydroxybenzimidazolylcobamide-binding protein (Geob_2541), UroD-like decarboxylase/methyltransferase (Geob_2542), and methylcobamide--coenzyme M methyltransferase (Geob_2543).
An anaerobic sulfate-reducing Deltaproteobacterial strain NaphS2, which was isolated from anoxic marine sediment, can anaerobically degrade naphthalene (70). It is reasonable to predict that dearomatization of naphthalene might occur by mechanisms relevant to dearomatization of benzene. Therefore, genes that were upregulated in NaphS2 during growth on naphthalene, versus benzoate, as its substrate were compared to the G. daltonii genome in order to identify potential anaerobic benzene oxidation genes. Most of the genes which shared homology with G. daltonii were housekeeping genes related to protein translation. In addition to these, the two organisms shared genes for an ATPase and benzoate degradation genes. However, there were some genes in NaphS2 which corresponded to aromatic-degrading genes in G. daltonii. Among these were a fumarate reductase, sensor histidine kinase, and BbsF genes within the smaller aromatic-degrading gene cluster. Finally, both organisms share genes for the Clp protease. This protein has been indicated in a wide range of genetic regulation mechanisms within multiple organisms, including regulation of aromatic degradation (71).
Utilization of benzene as an electron donor by G. daltonii
G. daltonii was able to grow using benzene as a sole carbon source and an electron donor with fumarate as the electron acceptor (Fig. 12B). The growth was accompanied by the disappearance of benzene and formation of succinate, the product of fumarate reduction (Fig. 12A). In a G. daltonii culture grown on benzene and fumarate the electron equivalents recovered in succinate production measured was commensurate with the electron equivalents calculated from consumed benzene with a calculated electron recovery of 121.2%. In order to test if G. daltonii utilizes fumarate as a substrate some cultures were incubated only with fumarate but without addition of benzene. Without benzene G. daltonii did not grow and no succinate