Megaplasmid pHG1 Research Articles

Improving growth of Cupriavidus necator H16 on formate using adaptive laboratory evolution-informed engineering

Conversion of CO2 to value-added products presents an opportunity to reduce GHG emissions while generating revenue. Formate, which can be generated by the electrochemical reduction of CO2, has been proposed as a promising intermediate compound for microbial upgrading. Here we present progress towards improving the soil bacterium Cupriavidus necator H16, which is capable of growing on formate as its sole source of carbon and energy using the Calvin–Benson–Bassham (CBB) cycle, as a host for formate utilization. Using adaptive laboratory evolution, we generated several isolates that exhibited faster growth rates on formate. The genomes of these isolates were sequenced, and resulting mutations were systematically reintroduced by metabolic engineering, to identify those that improved growth. The metabolic impact of several mutations was investigated further using RNA-seq transcriptomics. We found that deletion of a transcriptional regulator implicated in quorum sensing, PhcA, reduced expression of several operons and led to improved growth on formate. Growth was also improved by deleting large genomic regions present on the extrachromosomal megaplasmid pHG1, particularly two hydrogenase operons and the megaplasmid CBB operon, one of two copies present in the genome. Based on these findings, we generated a rationally engineered ΔphcA and megaplasmid-deficient strain that exhibited a 24% faster maximum growth rate on formate. Moreover, this strain achieved a 7% growth rate improvement on succinate and a 19% increase on fructose, demonstrating the broad utility of microbial genome reduction. This strain has the potential to serve as an improved microbial chassis for biological conversion of formate to value-added products.

Complete Nucleotide Sequence of pHG1: A Ralstonia eutropha H16 Megaplasmid Encoding Key Enzymes of H 2-based Lithoautotrophy and Anaerobiosis

The self-transmissible megaplasmid pHG1 carries essential genetic information for the facultatively lithoautotrophic and facultatively anaerobic lifestyles of its host, the Gram-negative soil bacterium Ralstonia eutropha H16. We have determined the complete nucleotide sequence of pHG1. This megaplasmid is 452,156 bp in size and carries 429 potential genes. Groups of functionally related genes form loose clusters flanked by mobile elements. The largest functional group consists of lithoautotrophy-related genes. These include a set of 41 genes for the biosynthesis of the three previously identified hydrogenases and of a fourth, novel hydrogenase. Another large cluster carries the genetic information for denitrification. In addition to a dissimilatory nitrate reductase, both specific and global regulators were identified. Also located in the denitrification region is a set of genes for cytochrome c biosynthesis. Determinants for several enzymes involved in the mineralization of aromatic compounds were found. The genes for conjugative plasmid transfer predict that R. eutropha forms two types of pili. One of them is related to the type IV pili of pathogenic enterobacteria. pHG1 also carries an extensive “junkyard” region encompassing 17 remnants of mobile elements and 22 partial or intact genes for phage-type integrase. Among the mobile elements is a novel member of the IS 5 family, in which the transposase gene is interrupted by a group II intron.

A physical map of the megaplasmid pHG1, one of three genomic replicons in Ralstonia eutropha H16

We have used pulsed field gel electrophoresis and megabase DNA techniques to investigate the basic genomic organization of Ralstonia eutropha H16, and to construct a physical map of its indigenous megaplasmid pHG1. This Gram-negative, soil-dwelling bacterium is a facultative chemolithoautotroph and a denitrifier. In the absence of organic substrates it can grow on H 2 as its sole energy source and CO 2 as its sole source of carbon. Under anaerobic conditions it can utilize nitrate as a terminal electron acceptor, whereby dinitrogen is released. Essential genetic determinants of the enzyme systems responsible for these metabolic processes are linked to the 0.44-Mb conjugative megaplasmid pHG1. Aside from pHG1, the genome of R. eutropha H16 is comprised of two circular chromosomes measuring 4.1 and 2.9 Mb, adding up to a total genome size of 7.1 Mb. An estimated five copies of rDNA are distributed on the two chromosomes. A macrorestriction map of pHG1 was derived for the endonucleases DraI and XbaI. Hybridization studies showed that genes for anaerobic metabolism are located on all three genomic replicons.

A physical map of the megaplasmid pHG1, one of three genomic replicons in Ralstonia eutropha H16.

We have used pulsed field gel electrophoresis and megabase DNA techniques to investigate the basic genomic organization of Ralstonia eutropha H16, and to construct a physical map of its indigenous megaplasmid pHG1. This Gram-negative, soil-dwelling bacterium is a facultative chemolithoautotroph and a denitrifier. In the absence of organic substrates it can grow on H2 as its sole energy source and CO2 as its sole source of carbon. Under anaerobic conditions it can utilize nitrate as a terminal electron acceptor, whereby dinitrogen is released. Essential genetic determinants of the enzyme systems responsible for these metabolic processes are linked to the 0.44-Mb conjugative megaplasmid pHG1. Aside from pHG1, the genome of R. eutropha H16 is comprised of two circular chromosomes measuring 4.1 and 2.9 Mb, adding up to a total genome size of 7.1 Mb. An estimated five copies of rDNA are distributed on the two chromosomes. A macrorestriction map of pHG1 was derived for the endonucleases DraI and XbaI. Hybridization studies showed that genes for anaerobic metabolism are located on all three genomic replicons.

A novel NO-responding regulator controls the reduction of nitric oxide in Ralstonia eutropha.

Ralstonia eutropha H16 mediates the reduction of nitric oxide (NO) to nitrous oxide (N2O) with two isofunctional single component membrane-bound NO reductases (NorB1 and NorB2). This reaction is integrated into the denitrification pathway that involves the successive reduction of nitrate to dinitrogen. The norB1 gene is co-transcribed with norA1 from a sigma54 (RpoN)-dependent promoter, located upstream of norA1. With the aid of norA1'-lacZ transcriptional fusions and the generation of regulatory mutants, it was shown that norB1 gene transcription requires a functional rpoN gene and the regulator NorR, a novel member of the NtrC family of response regulators. The regulator gene maps adjacent to norAB, is divergently transcribed and present in two copies on the megaplasmid pHG1 (norR1) and the chromosome (norR2). Transcription activation by NorR responds to the availability of NO. A nitrite reductase-deficient mutant that is incapable of producing NO endogenously, showed a 70% decrease of norA1 expression. Addition of the NO-donating agent sodium nitroprusside caused induction of norA1'-lacZ transcription. Truncation of the N-terminal receiver domain of NorR1 interrupted the NO signal transduction and led to a constitutive expression of norA1'-lacZ. The results indicate that NorR controls the reductive conversion of NO in R. eutropha. This reaction is not strictly co-ordinated on the regulatory level with the other nitrogen oxide-reducing steps of the denitrification chain that are independent of NorR.

A megaplasmid-borne anaerobic ribonucleotide reductase in Alcaligenes eutrophus H16.

The conjugative 450-kb megaplasmid pHG1 is essential for the anaerobic growth of Alcaligenes eutrophus H16 in the presence of nitrate as the terminal electron acceptor. We identified two megaplasmid-borne genes (nrdD and nrdG) which are indispensable under these conditions. Sequence alignment identified significant similarity of the 76.2-kDa gene product NrdD and the 30.9-kDa gene product NrdG with anaerobic class III ribonucleotide reductases and their corresponding activases. Deletion of nrdD and nrdG in A. eutrophus abolished anaerobic growth and led to the formation of nondividing filamentous cells, a typical feature of bacteria whose DNA synthesis is blocked. Enzyme activity of NrdD-like ribonucleotide reductases is dependent on a stable radical at a glycine residue in a conserved C-terminal motif. A mutant of A. eutrophus with a G650A exchange in NrdD showed the DNA-deficient phenotype as the deletion strain, suggesting that G650 forms the glycyl radical. Analysis of transcriptional and translational fusions indicate that nrdD and nrdG are cotranscribed and that the translation efficiency of nrdD is 40-fold higher than that of nrdG. Thus, the two proteins NrdD and NrdG are not synthesized at a stoichiometric level.

Regulation of CO2 assimilation in Ralstonia eutropha: premature transcription termination within the cbb operon.

In the facultatively chemoautotrophic bacterium Ralstonia eutropha (formerly Alcaligenes eutrophus), most genes required for CO2 assimilation via the Calvin cycle are organized within two highly homologous cbb operons located on the chromosome and on megaplasmid pHG1, respectively, of strain H16. These operons are subject to tight control exerted by a promoter upstream of the 5'-terminal cbbL gene that is regulated by the activator CbbR. The existence of subpromoters within the operons was now excluded, as determined with lacZ operon fusions to suitable cbb gene fragments in the promoter-probe vector pBK. Nevertheless, marked differential expression of the promoter-proximal ribulose-1,5-bisphosphate carboxylase-oxygenase genes cbbLS and the remaining distal genes occurs within the operons. Computer analysis revealed a potential stem-loop structure immediately downstream of cbbS that was suspected to be involved in the differential gene expression. Nuclease S1 mapping identified a major 3' end and a minor 3' end of the relatively stable cbbLS partial transcript just downstream of this structure. Moreover, operon fusions containing progressively deleted stem-loop structures showed that the structure primarily caused transcriptional termination downstream of cbbS rather than increased the segmental stability of the cbbLS transcript. Premature transcription termination thus represents an important mechanism leading to differential gene expression within the cbb operons.

Identification of cbbBc as an additional distal gene of the chromosomal cbb CO2 fixation operon from Ralstonia eutropha.

Ralstonia eutropha (formerly Alcaligenes eutrophus) strain H16 possesses two highly homologous cbb operons encoding most of the Calvin cycle enzymes. One copy of the operon is located on the chromosome, the other on the megaplasmid pHG1 of the organism. Sequence analysis of the region downstream of the presumptive 3'-terminal gene (cbbAc) of the chromosomal operon revealed the presence of an open reading frame comprising 2,274 bp. Evidence is presented that this open reading frame is an additional distal gene (designated cbbBc) of the operon. In contrast to the other genes of the operon, cbbBc is not duplicated in the plasmid-borne operon. The deduced amino acid sequence of the cbbBc product (757 residues, molecular mass 83.17 kDa) showed the highest similarity to the large catalytic subunits of various bacterial formate dehydrogenases (FDH), suggesting that cbbBc might represent a structural FDH gene of R. eutropha. However, the properties of a cbbBc mutant strain indicated that the potential gene product is not related to known FDH of the organism. Transcriptional analysis in the homologous host and heterologous expression in Escherichia coli demonstrated that cbbBc is an active gene, which apparently has no essential function in the autotrophic metabolism of R. eutropha. The gene is a novel member of cbb operons in autotrophic bacteria.

Analysis of the genes forming the distal parts of the two cbb CO2 fixation operons from Alcaligenes eutrophus.

In the facultative chemoautotroph Alcaligenes eutrophus H16, most of the genes (cbb genes) encoding enzymes of the Calvin carbon reduction cycle are organized within two highly homologous cbb operons, one located on the chromosome and the other on the megaplasmid pHG1. Nucleotide sequencing of the promoter-distal part of the operons revealed three open reading frames, designated cbbG, cbbK, and cbbA. Similarity searches in databases and heterologous expressions of the subcloned genes in Escherichia coli identified them as genes encoding the Calvin cycle enzymes glyceraldehyde-3-phosphate dehydrogenase, 3-phosphoglycerate kinase, and a class II fructose-1,6-bisphosphate aldolase, respectively. The aldolase could be grouped together with the enzymes from Rhodobacter sphaeroides and Bacillus subtilis as a new subtype of class II aldolases. A phenotypic complementation analysis with a cbb operon mutant of A. eutrophus showed that the cbbG product is essential for autotrophic growth of the organism, whereas the products of cbbK and cbbA can apparently be substituted by isoenzymes encoded elsewhere on the chromosome. No or only low constitutive promoter activity was associated with cbbK and cbbA, respectively, confirming the two genes as parts of the cbb operon. Downstream of cbbA, the very high overall nucleotide sequence identity (about 94%) prevailing throughout the two cbb operons discontinues, suggesting that cbbA is the most promoter-distal gene of the operon.

The cbb operons of the facultative chemoautotroph Alcaligenes eutrophus encode phosphoglycolate phosphatase

The two highly homologous cbb operons of Alcaligenes eutrophus H16 that are located on the chromosome and on megaplasmid pHG1 contain genes encoding several enzymes of the Calvin carbon reduction cycle. Sequence analysis of a region from the promoter-distal part revealed two open reading frames, designated cbbT and cbbZ, at equivalent positions within the operons. Comparisons with known sequences suggested cbbT to encode transketolase (TK; EC 2.2.1.1) as an additional enzyme of the cycle. No significant overall sequence similarities were observed for cbbZ. Although both regions exhibited very high nucleotide identities, 93% (cbbZ) and 96% (cbbT), only the chromosomally encoded genes were heterologously expressed to high levels in Escherichia coli. The molecular masses of the observed gene products, CbbT (74 kDa) and CbbZ (24 kDa), correlated well with the values calculated on the basis of the sequence information. TK activities were strongly elevated in E. coli clones expressing cbbT, confirming the identity of the gene. Strains of E. coli harboring the chromosomal cbbZ gene showed high levels of activity of 2-phosphoglycolate phosphatase (PGP; EC 3.1.3.18), a key enzyme of glycolate metabolism in autotrophic organisms that is not present in wild-type E. coli. Derepression of the cbb operons during autotrophic growth resulted in considerably increased levels of TK activity and the appearance of PGP activity in A. eutrophus, although the pHG1-encoded cbbZ gene was apparently not expressed. To our knowledge, this study represents the first cloning and sequencing of a PGP gene from any organism.

Analysis of a pleiotropic gene region involved in formation of catalytically active hydrogenases in Alcaligenes eutrophus H16.

In Alcaligenes eutrophus H16 a pleiotropic DNA-region is involved in formation of catalytically active hydrogenases. This region lies within the hydrogenase gene cluster of megaplasmid pHG1. Nucleotide sequence determination revealed five open reading frames with significant amino acid homology to the products of the hyp operon of Escherichia coli and other hydrogenase-related gene products of diverse organisms. Mutants of A. eutrophus H16 carrying Tn5 insertions in two genes (hypB and hypD) lacked catalytic activity of both soluble (SH) and membrane-bound (MBH) hydrogenase. Immunological analysis showed that the mutants contained SH- and MBH-specific antigen. Growing the cells in the presence of 63Ni2+ yielded significantly lower nickel accumulation rates of the mutant strains compared to the wild-type. Analysis of partially purified SH showed only traces of nickel in the mutant protein suggesting that the gene products of the pleiotropic region are involved in the supply and/or incorporation of nickel into the two hydrogenases of A. eutrophus.

A gene complex coding for the membrane-bound hydrogenase of Alcaligenes eutrophus H16

One of the key enzymes in the chemolithoautotrophic metabolism of Alcaligenes eutrophus H16 is a dimeric, membrane-associated hydrogenase. The genetic determinants of this enzyme are located on the endogenous megaplasmid pHG1 (G. Eberz, C. Hogrefe, C. Kortlüke, A. Kamienski, and B. Friedrich, J. Bacteriol. 168:636-641, 1986). Complementation studies showed that the information required for the formation of active membrane-bound hydrogenase occupies more than 7.5 kb of megaplasmid DNA. We cloned and sequenced this region and identified the genes encoding the two hydrogenase subunits (hoxK and hoxG). The nucleotide sequence contains nine additional closely spaced open reading frames. Immunoelectron microscopy showed that the gene product of one of these open reading frames (hoxM) is involved in the process leading to the attachment of hydrogenase to the membrane. Other open reading frames may encode additional processing functions and components of a hydrogenase-linked electron transport chain. Analysis of Tn5-B21-mediated transcriptional fusions provided evidence that the structural genes and accessory functions belong to at least three coordinately regulated transcriptional units.

Construction and properties of a triprotein containing the high-affinity nickel transporter of Alcaligenes eutrophus

The high-affinity nickel transporter of Alcaligenes eutrophus H16 is encoded by gene hoxN, which maps within the hydrogenase gene cluster of megaplasmid pHG1. A tripartite gene fusion was constructed, consisting of (i) the Escherichia coli lacZ gene for β-galactoxidase, (ii) a segment encoding an endoproteolytically cleavable peptide, and (iii) the A. eutrophus gene hoxN. An E. coli strain harboring this construct (plasmid pCH307) efficiently produced the corresponding triprotein upon induction. A broad-host-range derivative of pCH307 was shown to complement an A. eutrophus HoxN − mutant.

Three trans-acting regulatory functions control hydrogenase synthesis in Alcaligenes eutrophus

Random Tn5 mutagenesis of the regulatory region of megaplasmid pHG1 of Alcaligenes eutrophus led to the identification of three distinct loci designated hoxA, hoxD, and hoxE. Sequencing of the hoxA locus revealed an open reading frame which could code for a polypeptide of 482 amino acids with a molecular mass of 53.5 kDa. A protein of comparable apparent molecular mass was detected in heterologous expression studies with a plasmid-borne copy of the hoxA gene. Amino acid alignments revealed striking homologies between HoxA and the transcriptional activators NifA and NtrC of Klebsiella pneumoniae and HydG of Escherichia coli. HoxA- mutants of A. eutrophus lacked both NAD-reducing soluble hydrogenase and membrane-bound hydrogenase. In HoxA- mutants, the synthesis of beta-galactosidase from a hoxS'-'lacZ operon fusion was drastically reduced, indicating that HoxA is essential for the transcription of hydrogenase genes. Mutants defective in hoxD and hoxE also lacked the catalytic activities of the two hydrogenases; however, in contrast to HoxA- mutants, they contained immunologically detectable NAD-reducing soluble hydrogenase and membrane-bound hydrogenase proteins, although at a reduced level. The low hydrogenase content in the HoxD- and HoxE- mutants correlated with a decrease in beta-galactosidase synthesized under the direction of a hoxS'-'lacZ operon fusion. Thus, hoxD and hoxE apparently intervene both in the regulation of hydrogenase synthesis and in subsequent steps leading to the formation of catalytically active enzymes.

Cloning, nucleotide sequence, and heterologous expression of a high-affinity nickel transport gene from Alcaligenes eutrophus.

High-affinity nickel transport in Alcaligenes eutrophus H16 is mediated by a function designated hoxN. hoxN lies within the hydrogenase gene cluster of megaplasmid pHG1. An insertional mutation at the hoxN locus led to an increased nickel requirement. In this mutant (strain HF260) both autotrophic growth on hydrogen and wild-type level of urease, a nickel-containing enzyme, were dependent on high concentration of nickel in the medium. Studies with a heterologous in vivo expression system revealed that the hoxN locus encodes two proteins with Mr = 30,000 and 28,000. Only the larger polypeptide was essential for nickel transport. The hoxN locus was cloned on a 2.2-kilobase pair fragment. Nucleotide sequence analysis of the hoxN locus revealed an open reading frame with a coding capacity for a protein of 33.1 kDa. The insertion leading to the Nic- phenotype of strain HF260 maps within this open reading frame indicating that it does in fact have coding function. The deduced amino acid sequence of the hoxN gene has several features typical of a hydrophobic integral membrane protein. Alkaline phosphatase fusion proteins produced by insertion of the transposon TnphoA into hoxN gave significant levels of alkaline phosphatase activity indicating that protein HoxN contains periplasmic domains. Taken together, our results suggest that gene hoxN encodes the high-affinity nickel transporter of A. eutrophus.

Genetics of CO2fixation in the chemoautotrophAlcaligenes eutrophus

The genome of Alcaligenes eutrophus H16 contains two functional, highly homologous clusters of genes (cfx genes) for at least six Calvin cycle enzymes, with one cluster located on the chromosome and the other on megaplasmid pHG1. The genes within each cluster presumably belong to a single large operon of about 11 kilobase pairs (kb) in size. Both operons form a cfx regulon that is controlled by the chromosomally encoded regulatory gene cfxR. The product of cfxR seems to be a transcriptional activator. Occurrence of isoenzymes within the central carbon metabolism of the organism is a consequence of this unusual genetic setup.

Genetics of CO 2 fixation in the chemoautotroph Alcaligenes eutrophus

The genome of Alcaligenes eutrophus H16 contains two functional, highly homologous clusters of genes ( cfx genes) for at least six Calvin cycle enzymes, with one cluster located on the chromosome and the other on megaplasmid pHG1. The genes within each cluster presumably belong to a single large operon of about 11 kilobase pairs (kb) in size. Both operons form a cfx regulon that is controlled by the chromosomally encoded regulatory gene cfxR. The product of cfxR seems to be a transcriptional activator. Occurrence of isoenzymes within the central carbon metabolism of the organism is a consequence of this unusual genetic setup.

Cloning and expression of chromosomally and plasmid-encoded glyceraldehyde-3-phosphate dehydrogenase genes from the chemoautotroph Alcaligenes eutrophys

Hybridizations using heterologous glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene probes suggested the existence of three GAPDH genes in Alcaligenes eutrophus H16. Two of these, located on the chromosome and the megaplasmid pHG1 of the organism, respectively, mapped about 2.5 kilobase pairs (kb) downstream of the two duplicated CO 2 fixation gene clusters ( cfx genes). They were identified as GAPDH genes ( cfxG c and cfxG p ) by cloning and expression in Escherichia coli. These genes encode GAPDH isoenzymes functioning in the Calvin cycle. The third gene ( gap) is chromosomally encoded but not linked to the cfx c cluster. Its product is probably involved in heterotrophic carbon metabolism.

Cloning and expression of chromosomally and plasmid-encoded glyceraldehyde-3-phosphate dehydrogenase genes from the chemoautotrophAlcaligenes eutrophys

Hybridizations using heterologous glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene probes suggested the existence of three GAPDH genes in Alcaligenes eutrophus H16. Two of these, located on the chromosome and the megaplasmid pHG1 of the organism, respectively, mapped about 2.5 kilobase pairs (kb) downstream of the two duplicated CO2 fixation gene clusters (cfx genes). They were identified as GAPDH genes (cfxGc and cfxGp) by cloning and expression in Escherichia coli. These genes encode GAPDH isoenzymes functioning in the Calvin cycle. The third gene (gap) is chromosomally encoded but not linked to the cfxc cluster. Its product is probably involved in heterotrophic carbon metabolism.

Genetic determinants of a nickel-specific transport system are part of the plasmid-encoded hydrogenase gene cluster in Alcaligenes eutrophus.

Nickel-deficient (Nic-) mutants of Alcaligenes eutrophus requiring high levels of nickel ions for autotrophic growth with hydrogen were characterized. The Nic- mutants carried defined deletions in the hydrogenase gene cluster of the indigenous pHG megaplasmid. Nickel deficiency correlated with a low level of the nickel-containing hydrogenase activity, a slow rate of nickel transport, and reduced activity of urease. The Nic+ phenotype was restored by a cloned DNA sequence (hoxN) of a megaplasmid pHG1 DNA library of A. eutrophus H16. hoxN is part of the hydrogenase gene cluster. The nickel requirement of Nic- mutants was enhanced by increasing the concentration of magnesium. This suggests that the Nic- mutants are impaired in the nickel-specific transport system and thus depend on the second transport activity which normally mediates the uptake of magnesium.