ABSTRACT

The number of genes encoding β-oxidation enzymes in Cupriavidus necator H16 (synonym, Ralstonia eutropha H16) is high, but only the operons A0459-A0464 and A1526-A1531, each encoding four genes for β-oxidation enzymes, were expressed during growth with long-chain-length fatty acids (LCFAs). However, we observed that C. necator ΔA0459-A0464 ΔA1526-A1531 and C. necator H16 showed the same growth behavior during growth with decanoic acid and shorter FAs. The negative effect of the deletion of these two operons increased with an increasing chain length of the utilized FAs. Transcriptome sequencing (RNA-Seq) revealed the expression profiles of genes involved in the catabolism of medium-chain-length fatty acids (MCFAs) in C. necator H16. Operon A0459-A0464 was expressed only during growth with nonanoic acid, whereas operon A1526-A1531 was highly expressed during growth with octanoic and nonanoic acid. The gene clusters B1187-B1192 and B0751-B0759 showed a log2 fold change in expression of up to 4.29 and 4.02, respectively, during growth with octanoic acid and up to 8.82 and 5.50, respectively, with nonanoic acid compared to sodium gluconate-grown cells. Several acyl-CoA ligases catalyze the activation of MCFAs with coenzyme A (CoA), but fadD3 (A3288), involved in activation of LCFAs, was not detected. The expression profiles of C. necator strain ΔA0459-A0464 ΔA1526-A1531 showed that the growth with nonanoic acid resulted in the expression of further β-oxidation enzyme-encoding genes. Additional insights into the transport of FAs in C. necator H16 revealed the complexity and putative involvement of the DegV-like protein encoded by A0463 in the transport of odd-chain-length FAs and of siderophore biosynthesis in the transport mechanism.
IMPORTANCE Although Cupriavidus necator H16 has been used in several studies to produce polyhydroxyalkanoates from various lipids, the fatty acid metabolism is poorly understood. The β-oxidation of long-chain-length FAs has been investigated, but the tremendous number of homologous genes encoding β-oxidation enzymes hides the potential for variances in the expressed genes for catabolism of shorter FAs. The catabolism of medium-chain-length FAs and connected pathways has not been investigated yet. As more sustainable substrates such as lipids and the production of fatty acids and fatty acid derivates become more critical with the dependency on fossil-based substances, understanding the complex metabolism in this highly diverse workhorse for biotechnology, C. necator, is inevitable. For further metabolic engineering and construction of production strains, we investigated the metabolism during growth on medium-chain-length FAs by RNA-Seq.

INTRODUCTION

Fatty acid metabolism is highly conserved in bacteria, and the fundamental pathways are common. Biosynthesis and catabolism of fatty acids (FAs) are performed in cycles. While the synthesis in the model organism Escherichia coli is mainly catalyzed by fab gene-encoded enzymes, the β-oxidation of long-chain-length FAs (LCFAs) is catalyzed by the fad gene-encoded enzymes and can be performed under aerobic and anaerobic conditions (15). With every cycle of β-oxidation, one acetyl-coenzyme A (CoA) molecule is cleaved from the thereby shortened acyl-CoA chain and can be utilized in the central metabolism of the cell (2, 6).
After transport across the outer membrane of E.coli promoted by FadL, β-oxidation is the central part of the degradation of FAs (7, 8). In a combined mechanism of transport into the cytoplasm and activation, FAs are activated by membrane-bound acyl-CoA synthetases (synonym, fatty acid CoA ligases) (9). The product of one passage through the cycle is acetyl-CoA and an acyl-CoA with an aliphatic chain shortened by two carbon atoms (10). Prior to acetyl-CoA cleavage by thiolases (synonym, acetyl-CoA acyltransferases), the acyl-CoA molecule is oxidized to trans2-enoyl-CoA by acyl-CoA dehydrogenases, hydrogenated to 3-hydroxyacyl-CoA by an enoyl-CoA hydratase, and again oxidized to β-ketoacyl-CoA by 3-hydroxyacyl-CoA dehydrogenase (4). While the acyl-CoA dehydrogenase transfers electrons to flavin adenine dinucleotide (FAD), the 3-hydroxyl-CoA dehydrogenase uses NAD+ as the electron acceptor (11, 12). An enzyme complex responsible for parts of the β-oxidation cycle is formed in E. coli (12). The oxidation of even-chain-length FAs finally results in two acetyl-CoA molecules. On the other hand, odd-chain-length FA oxidation leaves propionyl-CoA and acetyl-CoA. In E. coli, propionyl-CoA is subsequently converted to succinyl-CoA via the methyl citrate cycle (13, 14). In other organisms, methylmalonyl-CoA is produced by propionyl-CoA carboxylase, and a methylmalonyl-CoA mutase converts methylmalonyl-CoA to succinyl-CoA (15, 16). FadR regulates the degradation of FAs and associated processes (17). In the presence of acyl-CoAs with a chain length of over 14, FadR is released from the operator, and repression of the fad operon is stopped (18).
The ato-genes encode enzymes involved in the degradation of short-chain-length FAs (SCFAs) compared to LCFA degradation by the Fad proteins (19). In E. coli, the presence of LCFA is necessary for efficient SCFA degradation (20). Additionally, shorter FAs (C10 and shorter) are not transported exclusively across the membrane but pass by passive diffusion or through porin channels, such as OmpF (7, 20, 21).
Cupriavidus necator (synonym, Ralstonia eutropha) is a facultative chemolithoautotrophic betaproteobacterium with a versatile metabolism (22, 23). It can grow heterotrophically with sugars or organic acids, but the interest in it mainly arose because of its ability to grow with gas mixtures containing carbon dioxide (CO2) and hydrogen (H2). CO2 is assimilated by the Calvin-Benson-Bassham (CBB) cycle (24). The versatile metabolism has been used for several biotechnological applications, and most efforts and investigations have been made toward producing polyhydroxyalkanoates (PHA) (22, 25). Poly-β-hydroxybutyrate (PHB) can be produced from several carbon sources, and large amounts accumulate in the cells (26).
Although fatty acids and several lipids are sustainable and cheap carbon sources for producing value-added products, their metabolism remains relatively unknown in C. necator H16. Fatty acid synthesis in C. necator H16 has not been investigated. However, sequencing and annotation revealed fatty acid biosynthesis genes in the genome of C. necator, and synthesis of FA is likely similar to E. coli with malonyl-CoA as the precursor and elongation of acyl-ACP by two carbons with malonyl-ACP (27, 28). Nevertheless, FA degradation has been investigated with a focus on LCFAs. The genome of C. necator H16 contains numerous genes coding enzymes for each β-oxidation reaction (2830). Three fatty acyl-CoA ligases were annotated, but other genes might express functional ligases (28, 30). The acyl-CoA ligase encoded by fadD3 and a potential ligase encoded by gene A2794 showed an increased expression on trioleate, but the deletion of fadD3 did not influence growth behavior (29). Two operons showed increased expression on trioleate, containing LCFA oleic acid. The operons A0459-A0465 and A1526-A1531 encode genes for all enzymes of a β-oxidation cycle. The deletion of both operons resulted in the absence of CFU on palm oil, crude palm kernel oil, and oleate, but the single deletion of one operon had no influence, suggesting the compensation for one another (29).
As the two β-oxidation operons responsible for the degradation of LCFAs were described over a decade ago, we recently discovered that enzymes expressed by different homologous genes are involved in medium-chain-length dicarboxylic acid (MCDCA) degradation (30). For example, neither ligase FadD3 nor the ligase encoded by gene A2794 was found in cells grown with adipic acid, but a 3-oxoadipate CoA transferase possibly catalyzes the activation with CoA (30). These results already suggest that β-oxidation of carboxylic acids in C. necator is more complex than expected, that homologous genes compensate for the loss of others, and that the involvement of different homologous genes might be chain-length dependent.
Numerous studies describe the processing of oils, fats, or FAs as carbon sources for biotechnological applications (22). In C. necator, an extracellular lipase catalyzes triacylglycerols’ hydrolysis into glycerol and the corresponding FAs in the presence of oils and fats (31). High-yield PHA production from plant oils or animal fat as the carbon source is possible with C. necator (3234). Potentially fucose-containing exopolysaccharides (EPS) are produced, which support the emulsion-based growth of C. necator (35). Although glycerol backbone utilization is slow, oils and fats represent cheap carbon sources for PHA production (36). After redirection of the carbon flow, several other substances might be produced with C. necator (37). The application of C. necator to produce FAs or production from FAs and FA derivates makes the complete understanding of the versatile FA metabolism necessary for further engineering.
In this study, we provide insights into the versatile fatty acid metabolism of C. necator H16. After observing the growth of the previously described mutants C. necator ΔA0459-A0464, C. necator ΔA1526-A1531, and C. necator ΔA0459-A0464 ΔA1526-A1531 on long- and medium-chain-length FAs, we further investigated which of the numerous homologous β-oxidation genes are expressed during the β-oxidation of medium-chain-length FAs (MCFAs) (29, 30). We previously described the results of proteomic studies regarding the dicarboxylic acid (DCA) degradation in C. necator (30). Here, we present large data sets of expression profiling via transcriptome sequencing (RNA-Seq) and describe new gene clusters involved in the β-oxidation of MCFAs. We present differences between degradation of FAs with a chain length of C10 or less and FAs with a chain length of C11 or more, as well as between odd- and even-chain-length FAs.

RESULTS

Growth behavior of C. necator strains on medium- to long-chain-length FAs.

In previous studies, the numerous homologous genes for β-oxidation in C. necator H16 have been the topic of investigations (2830, 38). The genes are distributed on both chromosomes and on the megaplasmid. Nevertheless, they sometimes occur in clusters or operons. Two of these clusters have been extensively described as responsible for the β-oxidation of LCFAs (29). As we have already investigated them before, we observed that the deletion of A0459-A0464 and A1526-A1531 does not prevent growth with MCDCAs and MCFAs as the sole carbon source (30). We performed growth experiments with strains C. necator H16, C. necator ΔA0459-A0464, C. necator ΔA1526-A1531, and C. necator ΔA0459-A0464 ΔA1526-A1531 in MSM supplemented with each saturated aliphatic FA from C6 hexanoic acid to C15 pentadecanoic acid (Fig. 1).
FIG 1
FIG 1 Growth behavior of different C. necator strains with different fatty acids. (A to J) Strains C. necator H16 (cross), C. necator ΔA0459-A0464 (triangle), C. necator ΔA1526-A1531 (circle), and C. necator ΔA0459-A0464 ΔA1526-A1531 (square) were grown in mineral salts medium (MSM) with 0.1% (wt/vol) (A) hexanoic acid (C6), (B) heptanoic acid (C7), (C) octanoic acid (C8) (D) nonanoic acid (C9), (E) decanoic acid (C10), (F) undecanoic acid (C11), (G) dodecanoic acid (C12), tridecanoic acid (H) (C13), (I) tetradecanoic acid (C14), or (J) pentadecanoic acid (C15). Growth experiments were performed in triplicates, and standard deviations are indicated as error bars.
The growth behavior of all strains with C6, C8, or C10 as the sole carbon source showed no differences between the strains. The deletion of one or both operons has no influence on the growth behavior (Fig. 1A, C, E, G, and I). More differences were observed with every further addition of two carbon atoms from C10 upward. While the growth of the strains with C10 showed only slight variances, the lag phase duration increased significantly in the case of C12 and C14 (Fig. 1G and I). Still, even with LCFAs as the sole carbon source, all strains showed growth, but the maximum klett units (KU) was different between the strains on longer even-chain-length FAs.
When comparing the growth with odd-chain-length FAs to that with even-chain-length FAs with a similar chain length, a difference in the growth behavior was noticed (Fig. 1B, D, F, H, and J). Compared to the similar growth of all strains with C8 octanoic acid (OA), with C9 nonanoic acid (NA), the deletion of the operons inhibited the growth of the mutants compared to the wild type. While the single deletion of operon A1526-A1531 did not influence growth behavior with any tested odd-chain-length FAs, the deletion of A0459-A0464 impeded growth. The growth curves of the strains C. necator ΔA0459-A0464 and C. necator ΔA0459-A0464 ΔA1526-A1531 are similar with all odd-chain-length FAs, suggesting the importance of the genes of the operon. While the growth behavior differed between odd and even medium-chain-length FAs, the growth behavior of the double deletion mutant was similar from C11 to C15, as the deletion of both operons impeded the growth. However, the single deletion of A0459-A0464 caused a similar effect on growth with odd-chain-length FAs, but both operons had to be deleted in the case of even-chain-length FAs (Fig. 1).
Odd medium-chain-length FA utilization was verified with GC measurements performed with samples from the beginning of the growth experiment, the exponential growth phase, and the stationary growth phase of wild-type strain H16 and mutant strain C. necator ΔA04659-A0464 ΔA1526-A1531 grown with heptanoic or nonanoic acid as the sole carbon source (data not shown). The concentration of FAs in the medium decreased during the growth of the strains and remained at the starting level during the long lag phases of the double deletion mutant, showing that growth of C. necator is promoted by the utilization of FAs as the carbon source.

Fundamental aspects of the expression profiles.

RNA sequencing with samples of C. necator H16 and C. necator ΔA0459-A0464 ΔA1526-A1531 grown with octanoic or nonanoic acid were investigated to gain insights into the expression of β-oxidation genes during growth on medium odd- and medium even-chain-length FAs and to detect potential differences to the degradation of FAs with a chain length of C11 and above. The cells were harvested at the end of the exponential growth phase (see Fig. S1 in the supplemental material). We were able to gain insights into the wild type’s MCFA degradation, the differences in the catabolism of odd-chain- and even-chain-length FAs, and the involvement and potential subsidiaries of the operons A0459-A1526 and A1526-A1531.
RNA sequencing resulted in large data sets with only 28 to 61 genes without any read at the tested conditions and a mean number of reads ranging from 2,085 to 2,823. The principal-component analysis (PCA) plots show the variances between and in the different sample triplicates (Fig. 2). The internal variances were small, and differences induced by the utilization of different carbon sources were observed. While the deletion of both operons did not lead to significant differences in general expression profiles in the case of sodium gluconate and OA-grown cells, the variance of principal component 2 (PC2) between the profiles of wild-type strain H16 (Fig. 2, violet dots) and the double deletion mutant C. necator ΔA0459-A0464 ΔA1526-A1531 (brown dots) grown with NA is significant. This more considerable variance is in line with previously shown differences in growth behavior caused by the deletion of both operons (Fig. 1).
FIG 2
FIG 2 Principal-component analysis (PCA) score plot (rlog-transformed counts) of RNA-Seq results, which allows interpretation of the relation of the samples. Each sample is represented by one circle. H16, C. necator H16; ΔΔ, C. necator ΔA0459-A0464 ΔA1526-A1531; NaG, sodium gluconate; C8, octanoic acid (OA); C9, nonanoic acid (NA).

Expression of genes involved in carboxylic acid catabolism.

In most prokaryotes, FAs are first transported across the outer membrane and subsequently pass into the cytosol in a connected activation-transportation step to be catabolized in the cells. They are catabolized to acetyl-CoA in the β-oxidation cycle in the cytoplasm. The log2 fold changes of all genes potentially involved in the utilization of C6 to C10 FAs by C. necator H16 are given in Tables S1 and S2.
While the genome of C. necator does not contain any genes whose products share similarity with the transporter FadL of E. coli, expression of several other transporters and outer membrane receptors was significantly higher in the wild-type strain H16 grown with FAs compared to sodium gluconate-grown cells, and additional shifts in expression could be observed caused by the deletion of both operons A0459-A0464 and A1526-A1531 (Table S1). The expression of the putative fatty acid-binding DegV-like protein encoded by gene A0463 was significantly higher in the cells of the wild-type strain cells grown with odd-chain-length NA with a log2 fold change of −1.30 and −2.06 compared to that of cells grown with OA or sodium gluconate, respectively, but not when the expression was compared between OA- and sodium gluconate-grown cells. Most noticeably, transporters with a higher expression in FA-grown cells belonged to the ABC-type transporters, and several TonB-dependent outer membrane receptors were significantly upregulated (Table S1). ABC-type transporter-encoding genes B2037-B2041 had a log2 fold change in expression of −1.65 to −4.59 comparing FA-grown cells to sodium gluconate-grown cells and were also expressed at a high level in the double deletion mutant. Several transporter-encoding genes such as A1520-A1524 (ABC-type transporter: HAAT family), A3026-A3030 (ABC-type transporter: HAAT family), A3294-A3298 (ABC-type transporter: PepT family), or especially, B0791-B0794 (TRAP-type transporter) and B1702 (oxalate/formate antiporter) were upregulated in the double deletion mutant only when grown with NA, showing a significant influence of the deletions on the transport mechanism in the case of odd-chain-length FA supplementation.
The β-oxidation of LCFAs is mediated by the operons A0459-A0464 and A1526-A1531 (29). Cells grown with OA and NA showed a higher expression of operon A1526-A1531 (Table 1). When comparing OA with sodium gluconate-grown cells, the expression change ranged from −3.99 to −4.55, while the change for NA-grown cells ranged from −3.32 to −4.62. Operon A0459-A0464 is not expressed at a significantly higher level in OA-grown cells than in sodium gluconate-grown cells, and the expression in NA-grown cells changed between −0.79 and −2.06 compared to that in sodium gluconate-grown cells (Table 1). In particular, A1526-A1531 seems to play an essential role in the degradation of FAs with a chain length between C6 and C10, and A0459-A0464 is only expressed in odd-chain-length-grown cells.
TABLE 1
TABLE 1 Differences in the expression levels of gene clusters and operons encoding β-oxidation enzymes of C. necator strains upregulated during growth with medium-chain-length fatty acidsa
Locus tagAnnotationLog2 fold change
H16NaG/H16C8H16NaG/H16C9H16C8/H16C9H16C8/ΔΔC8H16C9/ΔΔC9
A0459Transcriptional regulator 2C TetR/AcrR-family–0.37–0.79–0.417.712.73
A0460Acyl-CoA dehydrogenase0.39–0.72–1.106.542.54
A0461Two-domain protein: 3-hydroxyacyl-CoA dehydrogenase0.18–1.20–1.386.792.51
A0462Acetyl-CoA C-acyltransferase0.01–0.90–0.917.452.25
A0463Hypothetical membrane-associated protein–0.74–2.06–1.307.452.28
A0464Enoyl-CoA hydratase/carnithine racemase–0.40–1.16–0.758.151.33
A1526Enoyl-CoA hydratase/Delta(3)-cis-delta(2)-trans-enoyl-CoA isomerase–4.25–3.770.468.123.83
A1527Bifunctional pyrazinamidase/nicotinamidase–4.36–4.44-0.077.843.65
A1528Acetyl-CoA acetyltransferase–3.99–3.320.637.963.65
A1529Phenylacetic acid degradation protein PaaI–4.08–3.930.157.873.66
A1530Acyl-CoA dehydrogenase–4.55–4.62–0.068.223.45
A1531Short-chain dehydrogenase–4.47–4.190.277.973.56
B0695Probable extracytoplasmic solute receptor–0.72–1.43–0.550.06–7.95
B0696Acyl-CoA synthetase (AMP-forming)–0.12–0.49–0.36–0.01–6.90
B0697Probable extracytoplasmic solute receptor–0.21–1.09–0.830.08–6.57
B0698Enoyl-CoA hydratase/carnithine racemase–0.15–0.35–0.190.21–6.72
B0699Acyl-CoA dehydrogenase 2C long-chain specific0.511.200.60–0.05–3.36
B0751Acyl-CoA dehydrogenase–3.88–4.03–0.130.15–4.70
B0752Acyl-CoA dehydrogenase 2C short-chain specific–2.65–3.54–0.850.13–4.57
B0753Long-chain fatty acid-CoA ligase–2.67–2.83–0.140.00–4.95
B0754Transcriptional regulator 2C LuxR-family–0.79–0.82–0.020.01–0.66
B0755Nitroreductase family–3.71–4.71–0.98–0.04–3.11
B0756Enoyl-CoA hydratase–3.43–5.50–2.040.04–3.12
B0757Dioxygenase related to 2-nitropropane dioxygenase–3.58–3.92–0.320.17–2.66
B0758Phenylacetic acid degradation protein–3.67–4.59–0.890.03–2.46
B0759Acetyl-CoA acetyltransferase–4.02–4.54–0.510.07–3.29
B1187Transcriptional regulator 2C CRP-family–4.16–8.34–4.122.905.80
B1188Enoyl-CoA hydratase/isomerase–4.14–8.82–4.613.055.58
B11893-Hydroxybutyryl-CoA dehydratase–4.27–8.33–3.973.095.54
B11903-Hydroxyisobutyrate dehydrogenase–4.29–8.19–3.823.055.85
B1191Methylmalonate-semialdehyde dehydrogenase–4.05–8.04–3.933.075.75
B1192Acyl-CoA dehydrogenase–2.37–7.30–4.722.945.05
B1331Hypothetical protein0.621.701.020.22–4.38
B1332Acyl-CoA dehydrogenase–0.650.661.33–0.42–6.94
B1333Predicted aminoglycoside phosphotransferase–0.970.601.62–1.15–8.40
B1334Short-chain dehydrogenase–1.320.331.69–1.54–8.91
B1335Acyl-CoA synthetase (AMP-forming)/AMP-acid ligase II–1.37–0.021.34–1.57–9.04
B1336Conserved hypothetical membrane spanning protein–2.67–0.601.90–0.89–8.96
B1337APC transporter 2C SSS family–1.98–0.181.79–1.19–8.54
B1338Hypothetical protein0.202.502.27–1.13–6.14
B1339Transcriptional regulator 2C LysR-family–0.071.581.650.02–1.91
B1340NADPH:quinone reductase or related Zn-dependent oxidoreductase–0.74–0.110.63–0.14–2.42
B1341Hypothetical protein–0.470.170.65–0.35–3.57
B1342Transcriptional regulator 2C LuxR-family–0.471.802.29–0.16–3.02
B1343Short-chain dehydrogenase of various substrate specificities–0.481.782.29–0.05–2.90
B1344Hypothetical protein–0.370.651.150.05–1.27
B1345Hypothetical protein–1.480.572.08–1.48–7.44
B1346Enoyl-CoA hydratase/carnithine racemase–0.791.252.09–1.27–7.48
B1347Putative peptidase 2C C56 family–1.010.261.29–1.18–7.93
B1348Hypothetical protein–0.480.871.390.02–4.22
B1349Hypothetical protein–0.54–1.89–0.950.02–3.56
B1351Hypothetical protein0.291.971.610.12–2.10
B1352Insecticidal toxin complex protein–0.431.491.94–0.14–2.71
B1353Insecticidal toxin complex protein–0.230.670.91–0.15–1.98
B1354Hypothetical protein0.531.470.89–0.12–1.22
a
Gene annotations provided by database derived from complete genome sequencing and annotation (28). Log2 fold change between designated conditions and strains. A change above/below 1.00/–1.00 is considered significant. A negative value resulted from a higher gene expression in the second given condition. H16, Cupriavidus necator H16; ΔΔ, Cupriavidus necator ΔA0459-A0464 ΔA1526-A1531; NaG, sodium gluconate; C8, octanoic acid (OA); C9, nonanoic acid (NA).
RNA-Seq results showed which genes involved in β-oxidation are expressed during growth with OA or NA and allowed comparisons to the expression in wild-type C. necator H16 grown with sodium gluconate (Fig. 3). By investigating the changes of expression between the wild-type H16 and C. necator ΔA0459-A0464 ΔA1526-A1531 grown with both OA or NA, the substituting genes for the two crucial operons for LCFA degradation were detected (Fig. 4).
FIG 3
FIG 3 Log2 fold changes of β-oxidation-related genes in C. necator H16 between sodium gluconate- and medium-chain-length fatty acid-grown cells. The log2 fold change in expression between C. necator H16 grown on sodium gluconate and octanoic acid (blue) and the log2 fold change in expression between C. necator H16 grown on sodium gluconate and nonanoic acid (red) are shown. Genes with a log2 fold change below −1.00 are significantly upregulated in medium-chain-length fatty acid-grown cells (dotted lines). All potential genes encoded in C. necator for the five reactions of the β-oxidation are displayed. (A) All putatively acyl-CoA synthetase/ligase-encoding genes; (B) all putatively acyl-CoA dehydrogenase-encoding genes; (C) all putatively enoyl-CoA hydratase-encoding genes; (D) all putatively 3-hydroxyacyl-CoA dehydrogenase-encoding genes; and (E) all putatively thiolase/acetyl-CoA acetyltransferase-encoding genes.
FIG 4
FIG 4 Log2 fold changes of β-oxidation-related genes encoded in C. necator H16 and C. necator ΔA0459-A0464 ΔA1526-A1531 grown with medium-chain-length fatty acids. The log2 fold change in expression between C. necator H16 and C. necator ΔA0459-A0464 ΔA1526-A1531 grown with octanoic acid (green) and the log2 fold change in expression between C. necator H16 and C. necator ΔA0459-A0464 ΔA1526-A1531 grown with nonanoic acid (yellow) are shown. Genes with a log2 fold change below −1.00 are significantly more upregulated in the medium-chain-length fatty acid-grown strain, C. necator ΔA0459-A0464 ΔA1526-ΔA1526, than in the wild type on the same fatty acid (dotted lines). All potential genes in C. necator for the five reactions of the β-oxidation are displayed. (A) All putatively acyl-CoA synthetase/ligase-encoding genes; (B) all putatively acyl-CoA dehydrogenase-encoding genes; (C) all putatively enoyl-CoA hydratase-encoding genes; (D) all putatively 3-hydroxyacyl-CoA dehydrogenase-encoding genes; and (E) all putatively thiolase/acetyl-CoA acetyltransferase-encoding genes.
The three genes A3288, PHG398, and PHG399 were annotated as fatty acid acyl-CoA ligase/synthetase encoding but were not upregulated in the presence of MCFAs (Fig. 3A). However, other putatively acyl-CoA ligase-encoding genes were upregulated in the wild type and the double deletion mutant. Genes A1519, A2794, and B0753 were significantly more strongly expressed in cells grown with OA or NA than in sodium gluconate-grown cells. While gene A2252 was only expressed in the presence of OA but not of NA, genes A1519, B0696, B0753, and B1335 were expressed at a noticeably higher level in the mutant than in the wild type during growth with NA (Fig. 4A).
Aside from the described operons A0459-A0464 and A1526-A1531, other gene clusters containing several β-oxidation enzymes encoding genes such as B0751-B0759 or B1187-B1192 were upregulated on both FAs in the wild type compared to sodium gluconate (Table 1). While the expression of B0751-B0759 was recognizably higher in the wild type and increased further in the double deletion mutant with NA, the expression of cluster B1187-B1192 was induced in the wild type by the presence of MCFAs, but the deletion of A0459-A0464 and A1526-A1531 lowered the expression of B1187-B1192. However, its expression was still significantly higher comparing the double deletion mutant grown with sodium gluconate to growth with the respective MCFA. In contrast, the extensive gene clusters B1331-B1354 and B0695-B0699 were highly expressed in the deletion mutant in C9 and slightly higher in the double deletion mutant grown on C8 (Table 1).
Expression profiles emphasized the involvement of many genes in cyclic β-oxidation. Several putatively acyl-CoA dehydrogenase-encoding genes were detected (Table S2, Fig. 3B). In addition to acyl-CoA dehydrogenases A1530, B0699, B0752-B0753, B1192, and B1332, which are part of the previously mentioned operons, genes A1067-A1068, B0580, B0975, B1694, and B1696 stood out by higher expression in cells grown with both FAs. In contrast, acyl-CoA dehydrogenase-encoding B0638 was only upregulated in C. necator ΔA0459-A0464 ΔA1526-A1531 grown with NA (Fig. 4B). When examining the involved enoyl-CoA hydratases in the wild-type strain H16, genes A1526, B0756, and B1198 stood out with log2 fold changes in the range of −4.25/−3.77, −3.43/−5.50, and −4.14/−8.82, respectively during growth with OA/NA compared to growth with sodium gluconate (Table S2, Fig. 3C). While B1188 was not expressed in the mutant strain, genes B1346 and B0698 had a −7.48 and −6.72 log2 fold expression difference, respectively, comparing cells of the wild type and the mutant grown with NA (Fig. 4C). Most genes annotated as potential 3-hydroxyacyl-CoA dehydrogenases had a significant change in expression levels (Fig. 3D). While gene A1531 was highly expressed in wild-type cells grown with both FAs, B1652 was upregulated only in OA-grown cells. In NA-grown wild-type cells, A1102 and A0282 encoding 3-hydroxyacyl-CoA dehydrogenases are potential alternatives. The dehydrogenase showed an even more increased expression in the double deletion mutant than in the wild type (Fig. 4D). The final cleavage of acetyl-CoA from the acyl-CoA chain might be catalyzed by enzymes encoded by highly expressed A1445, A1528, A1887, A2148, or B0759 (Fig. 3E). Besides deleted A1528, these genes were even more expressed in the mutant strain compared to the wild-type strain (Fig. 4E).
The final cycle of β-oxidation of odd-chain-length NA results in propionyl-CoA. Propionyl-CoA can either be used to synthesize odd-chain-length FAs or is converted to succinyl-CoA in the methyl citrate cycle to be transferred to the central carbon metabolism. The gene cluster A1904-A1909 encoding enzymes of the methyl citrate cycle was upregulated significantly in cells grown with NA compared to expression in cells grown with OA (Table S3). The log2 fold change ranged from −3.90 to −5.71. Thus, we can conclude that there is a highly active methyl citrate cycle to convert propionyl-CoA (Fig. S2). However, the potential propionate catabolism activator encoded by A1904 was not upregulated under any condition. Expression profiles in the deletion mutant grown with NA reveal a potential alternative pathway by carboxylation shown by significantly increased expression of propionyl-CoA carboxylase and methyl-malonyl-CoA mutase (Table S3). Additionally, methylmalonate semialdehyde dehydrogenase-encoding genes A3664 and B1191 were significantly upregulated during growth on odd-chain-length NA. However, this enzyme class is naturally active to catalyze the synthesis of propionyl-CoA (39).
Numerous transcriptional regulators showed altering expression profiles depending on the strain and on the carbon source (Table S1). Some of the regulator-encoding genes with the highest increase in expression during cultivation with OA or NA instead of sodium gluconate were B1158-B1161, B1975-B1978, B0116, B1161, and B1187. In strain C. necator ΔA0459-A0464 ΔA1526-A1531 compared to wild-type H16, the expression of A1177 and A1666 was higher with OA as the carbon source and the expression of B0821 and B2445 was higher with NA as the carbon source. The transcriptional regulator encoded by B1497 was significantly more expressed in the deletion mutant on FAs than in C. necator H16 (Table S1).

Additional upregulated genes and clusters in FA-grown cells.

RNA sequencing results revealed additional upregulated genes in cells grown with FAs as the carbon source. Several genes connected to iron uptake and iron-requiring enzymes were highly expressed in FA-grown cells (Table 2). Cluster B0115-B0119, encoding iron metabolism-associated enzymes, was significantly upregulated, by a log2 fold change of between −2.81 and −4.98 with NA or OA in comparison to sodium gluconate-grown cells. The log2 fold change of between −5.00 and −7.03 in the expression of genes B1676 to B1692, linked to iron transporting siderophore biosynthesis, was even higher, verifying the correlation of the encoded genes with the presence of MCFAs (Fig. 5). Additionally, genes PHG120 to PHG126, also putatively connected to siderophore biosynthesis, were expressed in the case of OA and NA utilization.
FIG 5
FIG 5 Log2 fold changes of the cluster B1676-B1692 putatively encoding siderophore biosynthesis-related enzymes in C. necator H16 between sodium gluconate- and medium-chain-length fatty acid-grown cells. The log2 fold change in expression between C. necator H16 grown on sodium gluconate and octanoic acid (blue) and the log2 fold change in expression between C. necator H16 grown on sodium gluconate and nonanoic acid (red) are shown. Genes with a log2 fold change below −1.00 are significantly upregulated in medium-chain-length fatty acid-grown cells.
TABLE 2
TABLE 2 Differences in the expression levels of genes encoding iron-transport-related enzymes of C. necator strains upregulated during growth on medium-chain-length fatty acidsa
Locus tagAnnotationLog2 fold change
H16NaG/H16C8H16NaG/H16C9H16C8/H16C9H16C8/ΔΔC8H16C9/ΔΔC9
B0115Conserved hypothetical protein–4.98–3.331.52–0.02–1.26
B0116Transcriptional regulator 2C ferric uptake–4.44–3.940.43–0.03–0.68
B0117Putative iron uptake protein–3.08–2.810.25–0.21–0.18
B0118Uncharacterized iron-regulated membrane protein–3.17–3.32–0.12–0.081.20
B0119Putative iron uptake protein–3.62–3.570.05–0.280.57
B1676Hypothetical membrane-spanning protein–6.55–5.001.530.050.34
B1677Iron-regulated protein–6.52–6.62–0.100.010.93
B1678Hypothetical protein–6.45–6.74–0.27–0.050.85
B1679Outer membrane receptor 2C TonB dependent–6.34–6.89–0.540.040.50
B1680Lysine/ornithine N-monooxygenase–6.39–6.160.220.180.00
B1681Siderophore biosynthesis-related protein/acetyltransferase or N-acetylase of ribosomal proteins–6.33–6.56–0.21–0.040.20
B1682ABC-type transporter 2C ATPase and permease components: Pep3E family–6.47–6.450.03–0.080.48
B1683Nonribosomal peptide synthetase modules and related proteins–6.36–6.360.00–0.190.37
B1684Putative iron transport-related membrane protein–6.42–5.420.970.27–0.22
B1685Nonribosomal peptide synthetase–6.53–5.780.73–0.040.19
B1686Nonribosomal peptide synthetase–6.39–5.760.61–0.09–0.07
B1687Nonribosomal peptide synthetase–6.52–6.430.09–0.010.34
B1688Pyoverdine biosynthesis regulatory gene–6.26–6.44–0.18–0.010.11
B1689Antibiotic/siderophore biosynthesis protein–6.12–6.100.03–0.09–0.14
B1690Thioesterase–6.55–6.68–0.13–0.11–0.05
B1691Phosphopantetheinyltransferase family protein–6.55–7.03–0.46–0.090.31
B1692Putative aminotransferase–6.51–6.360.15–0.08–0.25
B0084Ferrous iron transport protein B–2.61–3.22–0.520.010.68
B0085Ferrous iron transport protein A–2.09–1.700.37–0.080.38
B0116Transcriptional regulator 2C ferric uptake–4.44–3.940.43–0.03–0.68
B0117Putative iron uptake protein–3.08–2.810.25–0.21–0.18
B0118Uncharacterized iron-regulated membrane protein–3.17–3.32–0.12–0.081.20
B0119Putative iron uptake protein–3.62–3.570.05–0.280.57
PHG120Putative aldolase–4.49–5.02–0.490.181.06
PHG121Putative diaminopimelate decarboxylase–4.25–6.22–1.860.161.09
PHG122Putative iron transport protein–4.35–6.09–1.640.511.38
PHG123Putative efflux protein–5.13–6.34–1.120.492.00
PHG124Putative siderophore biosynthesis protein–5.15–6.61–1.390.321.75
PHG125Putative siderophore biosynthesis protein–5.40–6.65–1.180.241.95
PHG126Probable ferrisiderophore receptor protein 2C TonB dependent–4.35–5.57–1.090.011.28
a
Gene annotations provided by a database derived from complete genome sequencing and annotation (28). Log2 fold change between designated conditions and strains. A change above/below 1.00/–1.00 is considered significant. A negative value resulted from a higher gene expression in the second condition. H16, Cupriavidus necator H16; ΔΔ, Cupriavidus necator ΔA0459-A0464 ΔA1526-A1531; NaG, sodium-gluconate; C8, octanoic acid (OA); C9, nonanoic acid (NA).
Aside from iron-associated genes, genes encoding enzymes putatively involved in the metabolism of different aromatic compounds were significantly upregulated in cells grown with FAs, especially in C. necator ΔA0459-A0464 ΔA1526-A1531 grown with NA (Table S4). Other remarkable changes in expression were shown for genes A1594-A1612, potentially indicating modifications in cell wall composition during growth of the wild type with OA and the double deletion mutant with NA. Genes B0494-B0507, putatively encoding membrane, and transport-associated proteins, showed a log2 fold change of up to −9.66 when comparing strain H16 to strain ΔA0459-A464 ΔA1526-A1531, both grown with NA.
As the glyoxylate bypass is essential to provide 3- and 4-carbon intermediates during catabolism of fatty acids in C. necator H16, we compared the expression profiles under different conditions (29, 40). Our results confirm an active glyoxylate bypass during growth with MCFAs and further conversion to phosphoenolpyruvate (PEP) by malate dehydrogenase (encoded by A2634) or malic enzyme (encoded by A1002). The genes’ highest expression was observed during the growth of C. necator H16 with NA, while growth with OA or the deletion of both β-oxidation operons resulted in a lower expression, especially of isocitrate lyase A2211 (Table S5).

Construction of marker-free deletion mutant C. necator ΔB1187-B1192 and growth behavior on various fatty acids.

We observed that cluster B1187-B1192 was highly expressed during growth with OA and NA in the wild-type H16. We constructed new marker-free deletion mutants with a deleted cluster B1187-B1192 originating from the wild-type H16 and previously generated double deletion mutant (Table 3) to verify this cluster’s involvement and further investigate the FA degradation in C. necator.
TABLE 3
TABLE 3 Bacterial strains used in this study
Bacterial strainDescriptionSouce or reference
Cupriavidus necator H16WT, GmrDSM 428
Cupriavidus necator ΔA0459-A0464Marker-free deletion of genes A0459 to A046430
Cupriavidus necator ΔA1526-A1531Marker-free deletion of genes A1526 to A153130
Cupriavidus necator ΔA0459-A0464 ΔA1526-A1531Marker-free deletion of genes A0459 to A0464 and A1526 to A153130
Cupriavidus necator ΔB1187-B1192Marker-free deletion of genes B1187 to B1192This study
Cupriavidus necator ΔA0459-A0464 ΔA1526-A1531 ΔB1187-B1192Marker-free deletion of genes A0459 to A0464, A1526 to A1531, and B1187 to B1192This study
Escherichia coli S17-1Cloning strain, F mcrA Δ(mrr-hsdRMS-mcrBC) galK Φ80lacZΔM15 ΔlacX74 recA1 araD139 Δ(ara-leu)7697 rpsL galU (Strr) endA1 nupGLife Technologies
Escherichia coli TOP10thi-1 proA hsdR17 (rK mK+) recA1; tra genes of plasmid RP4 integrated into the genome78
The additional deletion of the newly detected cluster B1187-B1192 in the double mutant ΔA0459-A0464 ΔA1526-A1531 showed the slowest growth and lowest maximum optical density on all tested FAs, while the single deletion of cluster B1187-B1192 has only a minor influence (Fig. S3). The triple deletion mutant showed no growth on C18 and C19 FAs. As previously observed, all deletion mutants showed more phenotypical changes on long-chain-length acids than on MCFAs. The deletion of B1187-B1192 did not change this pattern. However, the cluster is involved in the FA metabolism, and the deletion impeded, but not wholly prevented, the growth of C. necator on medium- and long-chain-length FAs (Fig. S3).

DISCUSSION

While the catabolism of LCFAs in C. necator H16 was elucidated previously, the catabolism of MCFAs has remained unclear so far (29). The high number of homologous genes encoding β-oxidation enzymes in the genome is unique (2830). Although the deletion of β-oxidation operons A0459-A0464 and A1526-A1531 inhibited growth on LCFAs, we observed growth with various FAs from hexanoic acid to octadecanoic acid of the wild-type strain H16 and strain C. necator ΔA0459-A0464 ΔA1526-A1531. Nevertheless, growth behavior, lag-phases, and maximum optical density differed (Fig. 1 and Fig. S2). The RNA-Seq results elucidated involved genes in the degradation of OA and NA in C. necator, and we conclude that the operons A1526-A1531, B1187-B1192, and B0751-B0759 are expressed for the β-oxidation of LCFAs, while operon A0459-A0464 is important for LCFAs and odd-chain-length FA catabolism. Activation of MCFAs leading to acyl-CoA chains is catalyzed by the enzymes encoded by A1519, A2794, B0753, and B1709. The genes encoding β-oxidation enzymes with the highest expression levels during growth with OA and NA are displayed in Fig. 6. These results suggest variances in the expressed genes for transport and catabolism of MCFAs with a chain length of C10 and below compared to LCFAs and differences regarding catabolism or transport between odd- and even-chain-length FAs.
FIG 6
FIG 6 Schematic visualization of the β-oxidation of medium-chain-length FAs in C. necator H16. The locus tags of genes with the highest expression in cells grown with medium-chain-length FAs compared to cells grown with sodium gluconate putatively encoding the displayed catalyzing enzymes are displayed. The log2 fold change of each displayed gene can be found in Table S2. CoA, coenzyme A; FAD, flavin adenine dinucleotide; TCA, tricarboxylic acid cycle.
It is plausible that MCFAs, like LCFAs, are also catabolized via β-oxidation, independently whether odd- or even-chained. The high number of homologous genes in C. necator results in a possible compensation of deleted genes involved in catabolism. In other organisms, even two different gene sets result in differences in the involved operons between short- and long-chain-length FAs (4, 19). In E. coli, the ato genes encode proteins involved in SCFA degradation, while the fad operon encodes the respective enzymes for catabolism of LCFAs (19, 41, 42). Therefore, it is highly likely that several genes in C. necator encode several functional β-oxidation enzymes. Operons A0459-A0464 and A1526-A1531 were detected during growth on trioleate (29). While A1526-A1531 is also highly expressed during growth with octanoic and nonanoic acid, operon A0459-A0464 is not expressed with octanoic acid, but expression during growth with nonanoic acid was detected. The deletion of A0459-A0464 inhibits growth with odd-chain-length FAs (<C11) (Fig. 1).
The involvement of cluster B1187-B1192 in the catabolism of MCFAs was verified by growth experiments (Fig. S3), and cluster B0751-B0759 was expressed during growth in all investigated strains. In C. necator ΔA0459-A0464 ΔA1526-A1531 grown with OA, no additional genes encoding β-oxidation enzymes were detected compared to expression in the wild type. Therefore, already expressed genes such as B0751-B0759 substitute for the prevented function of enzymes expressed by A1526-A1531. In contrast, the deletion of A0459-A0464 and A1526-A1531 requires additional genes that need to be expressed to promote growth with nonanoic acid. B0751-B0759 is even more highly expressed in the case of the double deletion and completed by expressing additional clusters such as B1331-B1337 or B0694-B0699. The necessity for additional genes during growth with nonanoic acid is one possible explanation for differences in the observed growth behavior.
The deletion of A0459-A0464 and A1526-A1531 resulted in a lowered expression of B1187-B1192 compared to the wild type. Either this cluster is responsible for encoding enzymes involved in the degradation of substrates that do not occur in the double deletion mutant, or the expression of the cluster is regulated by a regulator with dependency on a molecule that does not occur in the same concentrations in the double deletion mutant. Potentially, B1187-B1192 is highly involved in degrading medium- and short-chain-length FAs, while it is not involved in the degradation of LCFAs.
The presence of LCFAs (14 or more carbon atoms) is necessary to induce the expression of the fad operon in E. coli (4). The genome of C. necator H16 does not encode proteins with high sequence similarity to transcriptional regulators of the LCFA degradation FadR or YsiA from E. coli and Bacillus subtilis, respectively (5, 28, 43). However, the expression of the ato genes in E. coli is under the control of AtoC (19). AtoC shows sequence similarity with several sigma-54-dependent transcriptional regulators, which are not upregulated during growth with OA or NA.
While the potential ligases encoded by fadD3 (A3288) and A2794 were upregulated during growth on LCFAs, only A2794 was significantly more highly expressed in cells grown on OA and NA (29, 44). Several additional putatively ligase-encoding genes, A1519, B0753 and B1709, might activate MCFAs. In addition to previously described upregulated operons, several other genes encoding the respective β-oxidation genes are expressed during growth with OA and NA (Fig. 3 and 4). This complexity underlines the high versatility in MCFA catabolism in C. necator. Enoyl-CoA hydratases encoded by A3593-A3594 and A0179 upregulated during growth with MCFAs were also detected in the proteome of adipic and hexanoic acid-grown cells of C. necator. However, other genes putatively involved in the degradation of hexanoic acid and adipic acid were not detected. In particular, operon B0198-B0200 seems to be exclusively involved in dicarboxylic acid degradation (30).
RNA-Seq showed that operon A0459-A0464 was only expressed during growth with NA as the sole carbon source. Gene A0463 encodes a DegV-like protein with putative fatty acid-binding capabilities (29, 45, 46). Potentially, this protein has a higher affinity to bind specific FAs like other described proteins of this class, and the binding capability is influenced by characteristics of FAs (47, 48). DegV-like proteins are involved in fatty acid incorporation in Staphylococcus strains and catalyze the phosphorylation of fatty acids (47, 49). ABC-type transporters encoded by A3310 and A3661 were also detected in proteome analysis of cells grown with hexanoic and adipic acid (30). As we also detected their upregulation in octanoic and nonanoic acid-grown cells, their involvement in transportation seems likely. In C. necator, no protein similar to fatty-acid transportation enzyme FadL from E. coli was detected, but transport of short- to medium-chain-length FAs might also occur through porins or passive diffusion (21, 28, 50). Multiple outer membrane proteins and porins were upregulated during growth with OA and NA.
Siderophores are a diverse molecule class occurring in bacteria, fungi, and plants. They are involved in iron accumulation, binding of other metal ions, or even acting as signaling molecules (5156). They are transferred across the outer membrane into the periplasm by TonB-dependent receptors and various ABC transporters (57, 58), both of which were highly upregulated during the growth of C. necator with OA and NA. A TonB-dependent receptor encoded by B1489, which was strongly expressed during growth with MCFAs, shares high sequence similarity with iron receptor AleB of Cupriavidus metallidurans CH34 (59, 60). The highly upregulated cluster B1676-B1692 encodes proteins involved in the biosynthesis of cupriachelin, a siderophore produced by C. necator H16 (61). Since siderophores are produced in other Cupriavidus species as a response to aromatic stress, a connection to IGEPAL-CA630, used to allow solubilization of FAs, remains to be investigated (62). However, IGEPAL-CA630 did not influence growth with sodium gluconate and was not used as a carbon source by strain H16 (data not shown). Therefore, the incorporation without the presence of FAs is not likely. Cupriachelin contains even medium-chain-length FAs as sidechains when produced as an iron accumulation chelator in the case of iron deficiency (61). Potentially, the presence of even medium-chain-length FAs supports the extracellular production of cupriachelin, which is subsequently transported to the periplasm and cleaved, resulting in the release of FAs in the periplasm and potential further activation-coupled transportation in the cytoplasm by expressed ligases (61, 63). Naturally occurring cupriachelin contains only even-chain-length FAs, which might explain the different growth behavior.
Our results showed the tremendous growth speed of C. necator with medium- and long-chain-length FAs. While several studies, in which LCFAs containing TAGs such as animal fat, soybean oil, or waste oil were used as carbon source, targeted the optimization of PHB production, a complete understanding of the catabolism of FAs is lacking (27, 34, 64, 65). Although β-oxidation of LCFAs has been investigated, this study gives additional insights regarding the catabolism of shorter FAs and important uninvestigated topics such as transportation and regulation. The prevention of the fast degradation previously resulted in higher production of FAs in C. necator, and the detected ligases in this study provide a promising target for engineering the controlled FA degradation (29, 44, 66). Nevertheless, the possibilities of metabolic engineering are limited by the number of homologous genes and the metabolic diversity of C. necator H16. Our results show that FA catabolism is complex, and further detailed metabolomic studies might show intermediates to verify additional alternative pathways for catabolism or transport.

MATERIALS AND METHODS

Growth and harvest of cells.

The different strains used in this study are listed in Table 3. The strains were grown either in liquid medium at 130 rpm in flasks with baffles or on agar plates containing 1.8% (wt/vol) agar. All E. coli strains were grown on lysogeny broth medium (LB [67]). Liquid cultures of E. coli were grown at 30°C, and plates were cultivated at 37°C. Experiments with C. necator strains were all performed at 30°C, and the strains were grown in nutrient broth (NB) or mineral salts medium (MSM [68]) with 1% (wt/vol) sodium gluconate or different carboxylic acids at different concentration as the sole carbon source. Two different photometers were used in this study to follow the growth of cells in liquid medium. A spectral photometer (Thermo Spectronic GENESYS 20 visible spectrophotometer; Conquer Scientific, San Diego, CA, USA) was used to follow the optical density (OD) at a 600-nm wavelength. The growth experiments described here were followed with a Klett-Summerson photometer (Manostat Corporation, New York, NY, USA) equipped with filter no. 54 for a wavelength of 520 to 580 nm. Depending on the culture volume, cells were harvested in 15- or 50-mL tubes at 15 to 20 min at 7,690 × g at 4°C (Universal 320R; Hettich Lab Technology, Tuttlingen, Germany) or in Eppendorf tubes for 5 min at 12,000 × g at 4°C (Eppendorf centrifuge 5424R; Eppendorf AG, Hamburg, Germany).

Isolation and modification of DNA.

Genomic DNA was isolated with the NucleoSpin tissue kit (Macherey-Nagel, Düren, Germany), and plasmids were isolated with the NucleoSpin plasmid kit (Macherey-Nagel) as recommended by the manufacturer. Phusion polymerase (Thermo Fisher Scientific, Waltham, MA, USA) was used to amplify DNA fragments with specific primer oligonucleotides (Table 4; Eurofins Genomics, Ebersberg, Germany) in the peqSTAR 2X gradient thermocycler (Peqlab, VWR, Radnor, PA, USA). For modification of DNA fragments and plasmids, restriction endonucleases and T4-ligase (Thermo Fisher Scientific) were applied. Verification of successful modification, isolation, and amplification was performed with agarose gel electrophoresis and sequencing (Eurofins Genomics).
TABLE 4
TABLE 4 Oligonucleotides used in this study
OligonucleotideSequence (5′–3′)
B1187_92_up_fw_XbaIAaaTCTagACGCTGTCCAAGAGCTACAACATGG
B1187_92_up_revacTTgccGCCTGCGCGGTTGATGC
B1187_92_dw_fwcgCagGCGGCAAGTCAGGCAGAATGCG
B1187_92_dw_rev_SacIaaagagctCAGCCGCACGCCGATATCG
B1187_92_ko_in_fwCCGAAGATCTCCTCGGTGTAGATCG
B1187_92_ko_in_revGCCTGTAGTCCCCCATCCCTTG
B1187_92_ko_ex_fwCCATCGGGTCCAAGGAAGGC
B1187_92_ko_ex_revGGAGTACTTCACCACCACGTTGTGC

RNA extraction and RNA sequencing.

The strains C. necator H16 (samples 1 to 3, 7 to 9, and 13 to 15) and C. necator ΔA0459-A0464 ΔA1526-A1531 (samples 4 to 6, 10 to 12, and 19 to 21) employed for the transcriptional analysis were grown in triplicates in 50 mL MSM with 1% (wt/vol) sodium gluconate (samples 1 to 6), 0.1% (vol/vol) octanoic acid (C8, OA) (samples 7 to 12), or 0.1% (vol/vol) nonanoic acid (C9, NA) (samples 13 to 15 and 19 to 21). The cells were harvested at the end of the exponential-growth phase. Cells were stored at −70°C prior to RNA isolation.
For further analysis, the harvested cells were suspended in 800 μL RLT buffer (RNeasy minikit, Qiagen) with β-mercaptoethanol (10 μL × mL−1), and cell lysis was performed using a laboratory ball mill. Subsequently, 400 μL RLT buffer (RNeasy minikit, Qiagen) with β-mercaptoethanol (10 μL × mL−1) and 1,200 μL 96% (vol/vol) ethanol were added. For RNA isolation, the RNeasy minikit (Qiagen) was used as recommended by the manufacturer, but instead of RW1 buffer, RWT buffer (Qiagen) was used to isolate RNAs smaller than 200 nucleotides [nt]. To determine the RNA integrity number (RIN), the isolated RNA was run on a Bioanalyzer 2100 using an RNA 6000 nano kit as recommended by the manufacturer (Agilent Technologies, Waldbronn, Germany). The remaining genomic DNA was removed by digesting it with TURBO DNase (Invitrogen, Thermo Fisher Scientific, Paisley, UK). The Pan-Prokaryozes riboPOOL kit v4 (siTOOLS Biotech, Planegg/Martinsried, Germany) was used to reduce the number of rRNA-derived sequences (samples 1 to 12), and the Ribo-Zero plus rRNA depletion kit (Illumina, Inc., San Diego, CA, USA) was used to reduce the amount of rRNA-derived sequences of samples 13 to 15 and 19 to 21. For sequencing, the strand-specific cDNA libraries were constructed with a NEBNext Ultra II directional RNA library preparation kit for Illumina and the NEBNext Multiplex Oligos for Illumina (New England Biolabs, Frankfurt am Main, Germany). To assess the quality and size of the libraries, samples were run on a Agilent Bioanalyzer 2100 using an Agilent high-sensitivity DNA kit as recommended by the manufacturer. The concentration of the libraries was determined using the Qubit double-stranded DNA (dsDNA) high-sensitivity (HS) assay kit as recommended by the manufacturer (Life Technologies GmbH, Darmstadt, Germany). Sequencing was performed on the NovaSeq 6000 instrument (Illumina, Inc.) using the NovaSeq 6000 SP reagent kit (100 cycles) and the NovaSeq XP 2-lane kit v1 (samples 1 to 12) and v1.5 (samples 13 to 15 and 19 to 21) for sequencing in the paired-end mode and running 2 × 50 cycles. Trimmomatic v0.39 (69) and a cutoff phred-33 score of 15 were used for quality filtering and removal of the remaining adaptor sequences. The mapping against the reference genomes of C. necator H16 (28) was performed with Salmon (v1.2.1 for samples 1 to 12 and v1.5.0 for samples 1 to 15 and 19 to 21) (70). The mapping backbone, a file that contains all annotated transcripts excluding rRNA genes and the whole genome of the references as decoy, was prepared with a k-mer size of 11. Decoy-aware mapping was done in selective-alignment mode with the –mimicBT2, –disableChainingHeuristic, and –recoverOrphans flags as well as sequence and position bias correction and 10,000 bootstraps. For –fldMean and –fldSD, a value of 325 and 25, respectively, was used. The quant.sf files produced by Salmon were subsequently loaded into R (v4.0.3) (71) using the tximport package (v1.18.0) (72). DESeq2 (v1.30.0) (73) was used for normalization of the reads, and fold change shrinkages were also calculated with DESeq2 and the apeglm package (v1.12.0) (74). Genes with a log2 fold change of +1/– 1 and an adjusted P (P-adjust) value of <0.05 were considered differentially expressed. For PCA analysis, the rlog function of DESeq2 with the “blind” parameter was set to FALSE, and ggplot2 (v3.3.0) was used to plot the figure (75).

Construction of marker-free deletion mutants.

The cloning vector pJET1.2/blunt (Thermo Fisher Scientific) was used to construct a plasmid containing approximately 500-bp DNA fragments of the up- and downstream flanking regions of the targeted region of B1187-B1192. Up- and downstream fragments were combined and ligated with pJET1.2/blunt again. After digestion with endonucleases XbaI and SacI (Thermo Fisher Scientific), the combined fragment was ligated into the suicide plasmid pJQ200mp18Tc (76). The conjugal transfer of pJQ200mp18Tc::B1187-B1192updn into C. necator H16 and C. necator ΔA0459-A0464 ΔA1526-A1531 was realized using E. coli S17-1 as a transfer organism (77). The first homologous recombination resulted in heterogenotes containing the wild-type B1187-B1192 region and the plasmid up- and downstream region on the genome. After the second homologous recombination, the target region B1187-B1192 was deleted, and the resulting mutants contained a marker-free deletion (76). The constructed strains C. necator ΔB1187-1192 and C. necator ΔA0459-A0464 ΔA1526-A1531 ΔB1187-B1192 were verified by PCR and sequencing. All strains used for the construction and the constructed strains a listed in Table 3.

Growth experiments with various fatty acids as the sole carbon sources.

Newly constructed and previously constructed strains (Table 3) were tested regarding their growth behavior with fatty acids as the sole carbon source. Nonidet P-40 substitute IGEPAL-CA630 (Alfa Aesar, Ward Hill, MA, USA) was used to improve the solubility of FAs (44). It was used in low concentrations up to 2% (vol/vol) and did not impede growth, nor did it serve as a utilizable carbon source. The strains were grown in an NB preculture (20 mL in 100-mL flasks with baffles) inoculated from NB plates. The following day, a mineral salts medium (MSM) preculture (50 mL in a 300-mL flask with baffles) containing 1% (wt/vol) sodium gluconate was inoculated with 500 μL of the NB preculture. The final culture of 50 mL in 300-mL Klett flasks with baffles was inoculated to an optical density at 600 nm (OD600) of 0.3 from the MSM preculture. Subsequently, growth was followed using a Klett-Summerson photometer (Manostat Corporation). All growth experiments were conducted in triplicates. FAs were used in different concentrations regarding the respective solubility and acidity. The medium-chain-length FAs heptanoic acid (C7, enanthic acid) and octanoic acid (OA, C8, caprylic acid) were supplemented with 0.2% (vol/vol), nonanoic acid (NA, C9, pelargonic acid), decanoic acid (C10, capric acid), tetradecanoic acid (C14, myristic acid), and pentadecanoic acid (C15, pentadecylic acid) were used at a final concentration of 0.1% (wt/vol), and octadecanoic acid (C18, stearic acid) and nonadecanoic acid (C19, nondecylic acid) were used at a final concentration of 0.05% (wt/vol).

Measurement of fatty acids in culture broth by gas chromatography.

Various fatty acid concentrations were measured using gas chromatography (GC). After creating a calibration curve using pure FAs, concentrations were measured from 2 mL freeze-dried supernatant. The freeze-dried pellet was resuspended in 2 mL chloroform and 2 mL methanol-sulfuric acid mixture (85%/15%, vol/vol). Subsequently, the acidic methanolysis was performed for 4 h in a 100°C oil bath. After adding 1 mL double-distilled water (ddH2O), the sample was vortexed for 30 s. Cotton wool-stuffed tips were used for the final filtration of the sample before collecting it in a glass vial. A BP21 capillary column 50-m by 0.32-mm, 0.25-μm film (SGE, Darmstadt, Germany) was used to separate a 10-μL injection sample volume in a GC-System Serie 6890 instrument (Hewlett Packard GmbH, Dortmund, Germany) with a split ratio of 1:20 at 250°C and 86.5 kPa with hydrogen as the carrier gas. The following temperature program was applied: 120°C for 5 min, increase of 3°C per min to 180°C, increase of 10°C per min to 220°C, holding for 31 min.

Data availability.

The raw reads have been deposited in the Sequence Read Archive (SRA) under BioProject PRJNA802999 and accession numbers SRR17982167 to SRR17982184.

ACKNOWLEDGMENTS

Alexander Steinbüchel is very grateful for the support of the Rahn-Quade-Stiftung, which allowed the employment of one Ph.D. student.
We declare that we have no conflicts of interest.

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Information & Contributors

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Published In

cover image Applied and Environmental Microbiology
Applied and Environmental Microbiology
Volume 89Number 131 January 2023
eLocator: e01428-22
Editor: Ning-Yi Zhou, Shanghai Jiao Tong University
PubMed: 36541797

History

Received: 29 August 2022
Accepted: 22 November 2022
Published online: 21 December 2022

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Keywords

  1. Cupriavidus necator
  2. Ralstonia eutropha
  3. RNA sequencing
  4. medium-chain-length fatty acid catabolism
  5. β-oxidation

Contributors

Authors

Institut für Molekulare Mikrobiologie und Biotechnologie, Westfälische Wilhelms-University Münster, Münster, Germany
Genomic and Applied Microbiology & Göttingen Genomic Laboratory, Institute of Microbiology and Genetics, Georg-August University of Göttingen, Göttingen, Germany
Axel Himmelbach
Leibniz Institute of Plant Genetics and Crop Plant Research (IPK), Seeland, Germany
Genomic and Applied Microbiology & Göttingen Genomic Laboratory, Institute of Microbiology and Genetics, Georg-August University of Göttingen, Göttingen, Germany
Institut für Molekulare Mikrobiologie und Biotechnologie, Westfälische Wilhelms-University Münster, Münster, Germany
Environmental Science Department, King Abdulaziz University, Jeddah, Saudi Arabia

Editor

Ning-Yi Zhou
Editor
Shanghai Jiao Tong University

Notes

The authors declare no conflict of interest.

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