Loss of malic enzymes leads to metabolic imbalance and altered levels of trehalose and putrescine in the bacterium Sinorhizobium meliloti

Abstract

Background: Malic enzymes decarboxylate the tricarboxylic acid (TCA) cycle intermediate malate to the glycolytic end-product pyruvate and are well positioned to regulate metabolic flux in central carbon metabolism. Despite the wide distribution of these enzymes, their biological roles are unclear in part because the reaction catalyzed by these enzymes can be by-passed by other pathways. The N2-fixing alfalfa symbiont Sinorhizobium meliloti contains both a NAD(P)-malic enzyme (DME) and a separate NADP-malic enzyme (TME) and to help understand the role of these enzymes, we investigated growth, metabolomic, and transcriptional consequences resulting from loss of these enzymes in free-living cells.

Results: Loss of DME, TME, or both enzymes had no effect on growth with the glycolytic substrate, glucose. In contrast, the dme mutants, but not tme, grew slowly on the gluconeogenic substrate succinate and this slow growth was further reduced upon the addition of glucose. The dme mutant strains incubated with succinate accumulated trehalose and hexose sugar phosphates, secreted malate, and relative to wild-type, these cells had moderately increased transcription of genes involved in gluconeogenesis and pathways that divert metabolites away from the TCA cycle. While tme mutant cells grew at the same rate as wild-type on succinate, they accumulated the compatible solute putrescine.

Conclusions: NAD(P)-malic enzyme (DME) of S. meliloti is required for efficient metabolism of succinate via the TCA cycle. In dme mutants utilizing succinate, malate accumulates and is excreted and these cells appear to increase metabolite flow via gluconeogenesis with a resulting increase in the levels of hexose-6-phosphates and trehalose. For cells utilizing succinate, TME activity alone appeared to be insufficient to produce the levels of pyruvate required for efficient TCA cycle metabolism. Putrescine was found to accumulate in tme cells growing with succinate, and whether this is related to altered levels of NADPH requires further investigation.

Keywords: Amino acids; Catabolite repression; Fatty acids; Malic enzyme; Putrescine; Sinorhizobium; Trehalose.

Fig. 1

Relative response factors (RRF) for intracellular metabolites with significantly different RRFs (P values of < 0.05 in ANOVA) from the wild-type strain, dme, tme and dme tme double mutants. Note the different RRF scale for the three growth conditions. Metabolites were 6-phospho-sugars (likely fructose-6-phosphate (6PS1), mannose-6-phosphate (6PS2) and glucose-6-phosphate (6PS3)), putrescine and trehalose. Strains were grown in M9-Succinate, M9-Glucose, M9-Succinate plus Glucose. The trehalose RRFs were negligible in extracts for strains grown in M9-glucose. For M9-Succinate + Glucose, the cells grown in Succinate + Glucose were washed and incubated for 2 h in modified M9-Succinate. Error bars were calculated using standard deviation of the mean from three independent cultures

Fig. 2

Excretion of malate (circles) and fumarate (squares) from dme (filled) and tme (open) mutant strains. Strains grown overnight in M9-glucose plus succinate were transferred into modified M9 containing 2.5 mM phosphate and 5 mM succinate. Samples were taken 1, 2 and 3.5 h post transfer and the culture supernatants were analyzed by GC-MS (see methods). The relative response factors for fumarate and malate relative to the standard ribitol were determined. Error bars were calculated using standard deviation of the means for three experimental replicates

Fig. 3

Schematic of genes and enzymes involved in central carbon metabolism in S. meliloti. Reactions are colour coded based on whether the corresponding genes were upregulated or not

Fig. 4

Schematic of genes and enzymes involved in amino acid and fatty acid synthesis in S. meliloti. Reactions are colour coded based on whether the corresponding genes were upregulated or not

Fig. 5

Growth of wild-type strain, the dme, the tme and dme tme double mutants grown in M9-Glucose (5 mM), M9-Succinate (5 mM), or M9-Succinate (5 mM) plus Glucose (5 mM). OD₆₀₀ values were from triplicate samples. The insert in the glucose + succinate panel, shows OD₆₀₀ values between 0.2 and 0.8 over the part of the growth curves where first growth stops and the second growth resumes. Note, that because the strains entered the diauxic growth transition at quite different times, the insert graph plots the OD₆₀₀ from 3 h prior to end of first growth phase to 3 h past end of first phase, allowing all growth curves to be aligned at the end of the first growth phase. Generation times in hours were determined at culture densities between OD₆₀₀ 0.1–0.3 (prior to the temporary halt in growth in the glucose + succinate cultures) and values are the means from the triplicate cultures +/− standard deviation of the mean. Both refers to media containing both succinate and glucose as carbon source