Proteomic and transcriptomic elucidation of the mutant ralstonia eutropha G+1 with regard to glucose utilization

Abstract

By taking advantage of the available genome sequence of Ralstonia eutropha H16, glucose uptake in the UV-generated glucose-utilizing mutant R. eutropha G(+)1 was investigated by transcriptomic and proteomic analyses. Data revealed clear evidence that glucose is transported by a usually N-acetylglucosamine-specific phosphotransferase system (PTS)-type transport system, which in this mutant is probably overexpressed due to a derepression of the encoding nag operon by an identified insertion mutation in gene H16_A0310 (nagR). Furthermore, a missense mutation in nagE (membrane component EIICB), which yields a substitution of an alanine by threonine in NagE and may additionally increase glucose uptake, was identified. Phosphorylation of glucose is subsequently mediated by NagF (cytosolic PTS component EIIA-HPr-EI) or glucokinase (GlK), respectively. The inability of the defined deletion mutant R. eutropha G(+)1 ΔnagFEC to utilize glucose strongly confirms this finding. In addition, secondary effects of glucose, which is now intracellularly available as a carbon source, on the metabolism of the mutant cells in the stationary growth phase occurred: intracellular glucose degradation is stimulated by the stronger expression of enzymes involved in the 2-keto-3-deoxygluconate 6-phosphate (KDPG) pathway and in subsequent reactions yielding pyruvate. The intermediate phosphoenolpyruvate (PEP) in turn supports further glucose uptake by the Nag PTS. Pyruvate is then decarboxylated by the pyruvate dehydrogenase multienzyme complex to acetyl coenzyme A (acetyl-CoA), which is directed to poly(3-hydroxybutyrate). The polyester is then synthesized to a greater extent, as also indicated by the upregulation of various enzymes of poly-β-hydroxybutyrate (PHB) metabolism. The larger amounts of NADPH required for PHB synthesis are delivered by significantly increased quantities of proton-translocating NAD(P) transhydrogenases. The current study successfully combined transcriptomic and proteomic investigations to unravel the phenotype of this hitherto-undefined glucose-utilizing mutant.

FIG. 1.

First proteome analysis. Changes in the proteomes of the wild-type strain H16 and of the mutant strain G⁺1 of R. eutropha. Samples were taken from cultures (biological experiment I). Extracted proteins were focused using pH 5 to 8 nonlinear strips. Dual-channel images were generated by employing Delta 2D software. (A) Cells of the stationary growth phase cultivated with fructose (_fru). Wild-type H16, blue spots; mutant G⁺1, orange spots. (B) Cells of the stationary growth phase after the provision of glucose (_glu) and 4 h of incubation. Wild-type H16, blue spots; mutant G⁺1, orange spots; arrows and labels mark proteins with significantly different levels of expression in comparison to those of wild-type H16 and/or mutant G⁺1 under both situations investigated (cultivation with fructose or with glucose, respectively). Detailed information about the detected proteins is compiled in Table S1 and Fig. S1 in the supplemental material.

FIG. 2.

Second and third proteome analyses. Changes in the proteomes of the wild-type H16 and the mutant G⁺1 of R. eutropha of two individual biological experiments (II and III). Samples were taken from cultures. The extracted proteins were focused using pH 5 to 8 nonlinear strips. Dual-channel images were generated with Delta 2D software. (A and B) Second proteome analysis. (A) Cells (biological experiment II) of the stationary growth phase cultivated with fructose. Wild-type H16, blue spots; mutant G⁺1, orange spots. (B) Cells (biological experiment II) of the stationary growth phase after provision of glucose and 4 h of incubation. Wild-type H16, blue spots; mutant G⁺1, orange spots. (C and D) Third proteome analysis. (C) Cells (biological experiment III) of the stationary growth phase cultivated with fructose. Wild-type H16, blue spots; mutant G⁺1, orange spots. (D) Cells (biological experiment III) of the stationary growth phase after provision of glucose and 4 h of incubation. Wild-type H16, blue spots; mutant G⁺1, orange spots; red arrows and labels mark increasingly expressed proteins of mutant G⁺1 in comparison to those of wild-type H16 for the respective situations; green arrows and labels mark proteins being increasingly expressed by wild-type H16 for the respective situations. Detailed information about detected proteins is compiled in Table S2 and Fig. S2 and S3 in the supplemental material.

FIG. 3.

Schematic representation of the nag operon of R. eutropha H16 with adjacent genes, determination of promoter sequences by the method described by Reese (43), and an indication of the mutations observed with mutant G⁺1. Upper part, black arrows mark sequences that indicate what are most likely to be functional promoters, due to their adequate locations upstream of the gene. Gray arrows mark putative promoter sequences inside structural genes within the nag operon, which are unlikely to act as promoters due to their location. Genes and gene products are as follows: H16_A0310, transcriptional regulator, GntR family; nagF, protein N (pi)-phosphohistidine-sugar phosphotransferase II ABC; nagE, protein N (pi)-phosphohistidine-sugar phosphotransferase II ABC (N-acetylglucosamine specific), N-acetylglucosamine specific; nagC, outer-membrane protein (porin); nagA, N-acetylglucosamine-6-phosphate deacetylase; nagB, glucosamine-6-phosphate deaminase; zwf1, glucose-6-phosphate 1-dehydrogenase; A0317, predicted ATPase with chaperone activity/magnesium chelatase, subunit ChlI. Lower part, mutations of mutant G⁺1 observed by sequence analysis. (i) Gene H16_A0310. Pairwise alignment of partial nucleotide sequence of gene A0310 from wild-type H16 and mutant G⁺1 shows the observed insertion of a 19-bp fragment located a distance of 96 bp from the start codon and localized directly upstream (black bold letters, white background). By frameshift, this mutation causes on the protein level a nonsense amino acid sequence starting from aa 34 (corresponding codon, CGC; shaded in gray); after aa 91, translation terminates due to the occurrence of a stop codon (TGA, shaded gray in the alignment), obviously leading to a truncated and nonfunctional protein (H16_A0310 wild-type protein, 240 aa). (ii) Furthermore, a point mutation changing the first base of codon 153 of nagE from A to G was identified; it causes an alanine to be replaced by a threonine.

FIG. 4.

Quantification of proteins encoded by genes of the nag operon based on image fusions of 2D PAGE gels of the three proteome analyses presented in this study (Fig. 1 and 2). Spot quantities are given as % volume (representing the relative portion of an individual spot of the total protein present on the respective average fusion image). Quantification was done with Delta 2D software. Prefixes I, II, and II indicate the biological experiment/proteome analysis in which the protein was identified. Suffixes (a, b, and others) indicate isoforms of the same protein species present in the gels from the respective proteome analysis.

FIG. 5.

Schematic presentation of Nag PTS and parts of the central metabolism of R. eutropha H16 that are relevant for this study. The reddish gene abbreviations (small font, italics) indicate genes being upregulated in mutant G⁺1 as reported by transcriptome experiments. The reddish protein abbreviations (large font) indicate proteins being increasingly synthesized in mutant G⁺1 in comparison to wild-type H16, as revealed by proteome analyses. The same applies to the greenish gene/protein abbreviations, but these indicate downregulation or reduced synthesis by mutant G⁺1 in comparison to wild-type H16. Bluish letters are common abbreviations of trivial names of the respective enzymes. Nag PTS components are as follows: nagC (outer-membrane protein (porin), nagE (sugar phosphotransferase system, membrane component EIICB), nagF (sugar phosphotransferase system, cytosolic component EIIA-HPr-EI). KDPG pathway and subsequent reactions leading to pyruvate are as follows: G6PD/Zwf1 (glucose-6-phosphate 1-dehydrogenase), 6PGL (6-phosphogluconolactonase), EDD/Edd1 (phosphogluconate dehydratase), EDA (2-keto-3-deoxygluconate-6-phosphate aldolase), G3PD/CbbG1/cbbG1 (glyceraldehyde-3-phosphate dehydrogenase), PGM (phosphoglycerate mutase), ENO (enolase), PK (pyruvate kinase), PPS (phosphoenolpyruvate kinase). PDHC (pyruvate dehydrogenase multienzyme complex) includes pdhA1 (pyruvate dehydrogenase/decarboxylase [E1 of PDHC]), pdhB (dihydrolipoamide acetyltransferase [E2 of PDHC]), pdhL (dihydrolipoamide dehydrogenase [E3 of PDHC]). Involved in TCC and PHB metabolism are cisY (citrate synthase), PhaA (acetyl-CoA acetyltransferase), PhaB1 (acetoacetyl-CoA reductase), PhaC (PHB synthase), PhaP1/phaP1 (phasin 1), phaP3 (phasin 3), PhaP4/phaP4 (phasin 4). NAD(P)-transhydrogenases include PntAa3,2,1/pntAa2,3 [NAD(P) transhydrogenase subunits alpha 1 of gene regions 1 to 3], pntAb1,2,3 [NAD(P) transhydrogenase subunits alpha 2 of gene regions 1 to 3], and pntB3 [NAD(P) transhydrogenase subunit beta of gene region 3].