Probing the core metabolism of Cereibacter sphaeroides by transposon mutagenesis

Abstract

During phototrophic growth, Cereibacter sphaeroides can use several carbon substrates that are central carbon intermediates (e.g., succinate and L-malate) or that require only a few steps to enter central carbon metabolism (e.g., acetate and D-malate). In addition, with light as the energy source, the carbon substrate provided will function as a carbon source for cell carbon synthesis only. Therefore, C. sphaeroides is ideally suited to understand the changes necessary to switch between different carbon sources and, consequently, to redirect carbon flow in central carbon metabolism. This study describes C. sphaeroides transposon mutants that have lost the ability to use one or more of the organic carbon sources 3-hydroxypropionate, acetate, L-malate, propionate/HCO₃^-, butyrate/HCO₃, L-lactate, D-lactate, D-malate, and L-glutamate. Pyruvate carboxylase and pyruvate dehydrogenase were confirmed to connect the precursor metabolite pools of pyruvate and oxaloacetate or acetyl-CoA, respectively, as was the ethylmalonyl-CoA pathway connecting acetyl-CoA and oxaloacetate pools. Transposon and in-frame deletion mutants suggest that 3-hydroxypropionate is oxidized to CO₂ and acetyl-CoA, involving a malonate semialdehyde dehydrogenase. The presence of this oxidative route makes pyruvate dehydrogenase dispensable during 3-hydroxypropionatedependent growth. Therefore, acetyl-CoA represents a second entry point into central carbon metabolism for 3-hydroxypropionate besides succinyl-CoA, and it is proposed that the simultaneous functioning of the two routes minimizes transiently produced CO₂/HCO₃^-. Another significant outcome of this study is the identification of genes encoding a L-glutamate TRipartite ATP-independent transporter, which was characterized biochemically 30 years ago.IMPORTANCESeveral aspects of the process of carbon assimilation, defined as the conversion of a carbon source into cell carbon, are conserved throughout life. For example, common building blocks give rise to proteins and nucleic acids, and the carbon for building blocks, cofactors, and secondary metabolites is derived from common precursor metabolites such as acetyl-CoA, pyruvate, or oxaloacetate. Using carbon substrates that require only one or a few steps to enter central carbon metabolism facilitates insights into the changes that occur to accommodate growth with different carbon substrates. In this study, transposon mutants that affect carbon flow in the core metabolism of Cereibacter sphaeroides were identified. Apparent redundancies of pathways can be explained by the need to maintain overall redox balance.

Keywords: 3-hydroxypropionate metabolism; central carbon metabolism; pyruvate carboxylase; pyruvate dehydrogenase; secondary L-glutamate TAXI transport system.

Fig 1

The core metabolism of C. sphaeroides. Entry points into central carbon metabolism for different carbon substrates (filled boxes) are indicated, based on genome annotation or experimental results. Precursor metabolites are highlighted in white boxes and are central carbon intermediates that provide carbon for biosynthesis pathways. Filled black dots indicate known central carbon intermediates. Oxidants and reductants are not considered; however, the requirement at each step can be inferred by the change in the total oxidation state of the carbon. The broken arrow for the conversion of butyrate indicates that the entry point into central carbon metabolism is not known. Genetic evidence for a so-called oxidative route (dotted line) for the conversion of 3-hydroxypropionate to acetyl-CoA and CO₂ via malonate semialdehyde is presented in this study. A so-called reductive route from 3-hydroxypropionate and bicarbonate to succinyl-CoA has been described previously (5, 6). For C. sphaeroides, there are two possibilities to connect C4- to C3-pools: either from oxaloacetate to phosphoenolpyruvate (via PEP carboxykinase) or from L-malate to pyruvate (via malic enzyme). PHB, polyhydroxybutyrate; CBB cycle, Calvin Benson-Bassham cycle.

Fig 2

Map of transposon insertion sites at two genomic loci on chromosome 1 (accession number CP000143) encoding enzymes of the methylmalonyl-CoA pathway that converts propionyl-CoA and bicarbonate to succinyl-CoA. On plates, all C. sphaeroides transposon mutants were propionate/HCO₃^– negative, butyrate/HCO₃^– negative, and 3-hydroxypropionate compromised/negative on plates incubated either aerobically in the dark or anaerobically in the light. Mutants for which the transposon insertion mapped to meaB or mcm were also acetate negative, whereas all other mutants were only acetate compromised. Compromised growth is defined as significantly less growth compared to the growth of other mutants on the same plate. Flags mark the insertion site for each mutant, and the arrow indicates the transcriptional direction of the kanamycin resistance marker gene that is part of the transposon. PccB (accession number WP_002719342) and PccA are subunits of the propionyl-CoA carboxylase, and Mcm represents the methylmalonyl-CoA mutase. PccR is a ScfR-type transcriptional regulator controlling expression of pccB and possible downstream genes (9). MeaB (accession number WP_011337157) is required for methylmalonyl-CoA mutase maturation (14).

Fig 3

Transposon insertion sites for two mutants that had acetate-compromised growth on aerobically incubated plates but showed wild-type growth with succinate. Flags mark the insertion site for both mutants, and the arrow indicates the transcriptional direction of the kanamycin resistance marker gene that is part of the transposon. The gene interrupted (rsp_0653) likely encodes an UDP-glucose-6-dehydrogenase (accession number ABA79827.1). Downstream genes encode a possible UDP-glucose-4-epimerase, a protein related to inositol monophosphate phosphatase, and a secreted lipoprotein likely acting as a transglycosylase. Upstream genes encode transport systems.

Fig 4

(A) Transposon insertion sites on chromosome 1 (accession number CP000143) mapping to the dddC (rsp_2962) gene proposed to encode an acetylating malonate semialdehyde dehydrogenase from C. sphaeroides (accession number ABA79124). Based on visual inspection, all transposon mutants listed were scored as 3-hydroxypropionate-negative on plates incubated either aerobically in the dark or anaerobically in the light. Flags mark the insertion site for each mutant, and the arrow indicates the transcriptional direction of the kanamycin resistance marker gene that is part of the transposon. For the ΔdddCMA4 strain, most of the coding region of dddC was deleted, with the in-frame deletion resulting in a 19-amino acid peptide, as indicated. The gene upstream of dddC and marked in orange encodes a LysR-type transcriptional regulator that may control the expression of dddC. (B) Growth of the wild-type and ΔdddCMA strains, carrying no plasmid, an empty vector control (pBBR) or the intact dddC gene on a pBBR-derived plasmid (dddC). Growth was either anaerobically in the light (photoheterotrophically, top graph) or aerobically in the dark (respiratory growth, bottom graph) with 3-hydroxypropionate as the carbon substrate. The same symbols for strains are used for both growth conditions.

Fig 5

(A) Map of insertion sites on chromosome 2 (accession number CP000144) for transposon mutants of C. sphaeroides isolated on plates incubated aerobically with severely compromised 3-hydroxy-propionate-dependent growth. Flags mark the insertion site for each mutant, and the arrow indicates the transcriptional direction of the kanamycin resistance marker gene that is part of the transposon. For the ΔABC35KB mutant, most of the coding regions of five genes encoding an ABC transporter (orange) were deleted, resulting in a peptide consisting of 47 N-terminal amino acids of RSP_3297 (accession number ABA80904) and 44 C-terminal amino acids of RSP_3293, as indicated. For the Δgmor47KB mutant, most of the coding region of gmor was deleted, with the in-frame deletion resulting in a 62-amino acid peptide, as indicated. The gene rsp_3291, shown in dark pink, is predicted to encode a symporter, and the four genes downstream of it, shown in light pink, encode an ABC transporter. (B) Growth of the wild-type and the Δgmor47KB strains, carrying no plasmid, an empty vector control (pBBR) or the intact gmor gene on a pBBR-derived plasmid (gmor), as well as growth of the ΔABC35KB strain either anaerobically in the light (photoheterotrophically, left graph) or aerobically in the dark (respiratory growth, right) with 3-hydroxypropionate as the carbon source. Gmor (accession number ABA80899) is proposed to encode a 3-hydroxypropionate dehydrogenase.

Fig 6

Transposon insertion sites on chromosome 1 (accession number CP000143) mapping to two genes (rsp_4047 and rsp_4049) proposed to encode the alpha- (accession number ABA78645.1) and beta-subunits of the E1 component of pyruvate dehydrogenase (Pdh) from C. sphaeroides. The mutants were isolated as D-lactate, L-lactate, or L-malate-negative on plates incubated aerobically in the dark. A full range of substrates was not tested for these mutants; however, the mutants were acetate- and 3-hydroxy-propionate-positive on plates incubated aerobically in the dark. Flags mark the insertion site for each mutant, and the arrows indicate the transcriptional direction of the kanamycin resistance marker gene that is part of the transposon.

Fig 7

Transposon insertion sites on chromosome 1 (accession number CP000143) mapping to the pycA (rsp_2090) gene proposed to encode pyruvate carboxylase from C. sphaeroides (accession number ABA78245.1). All mutants listed were isolated as D-lactate-, L-lactate- D/L-lactate, or D-malate-negative, but L-malate-positive on plates incubated aerobically in the dark. Flags mark the insertion site for each mutant, and the arrows indicate the transcriptional direction of the kanamycin resistance marker gene that is part of the transposon.

Fig 8

L-Glutamate-negative or -compromised mutants. Insertion sites for transposon mutants isolated as L-glutamate negative (A) or L-glutamate compromised (C) on plates incubated aerobically in the dark. Flags mark the insertion site for each mutant, and the arrows indicate the transcriptional direction of the kanamycin resistance marker gene that is part of the transposon. (A) For the possible operon on chromosome 2 (CP000144), the first two genes (rsp_1412 and rsp_1413) encode the periplasmic-binding protein (Rsp_1412, accession number AMJ49705.1) and the membrane component (Rsp_1413, accession number: AMJ49704.1) of a TRAP transporter from C. sphaeroides, respectively. The three genes (rsp_3804-3802) downstream are likely co-transcribed with rsp_1412 and rsp_1413 because of the short spacing between the open reading frames, as indicated. (B) Phototrophic growth of the wild-type and the Δrsp_1412-1413SK7 deletion strains, carrying no plasmid, an empty vector control (pBBR) or the intact rsp_1412 and rsp_1413 genes on a pBBR-derived plasmid (rsp_1412 + 1413), with L-glutamate as the carbon source, either in the presence (top) or absence (bottom graph) of sodium ions in the medium. The genes rsp_1412 and rsp_1413 are likely to encode a sodium-dependent TRAP transport system that was previously biochemically characterized by Jacobs et al. (12). (C) Transposon insertion sites on chromosome 1 (CP000143) mapping to the rsp_0398 gene proposed to encode a glutamate dehydrogenase from C. sphaeroides (accession number ABA79572.1) for mutants that were isolated as L-glutamate compromised on plates.