Differential anaerobic oxidation of benzoate in Geotalea daltonii FRC-32

Abstract

The efficient carbon source utilization in dynamic environments, including anoxic subsurface contaminated by aromatic compounds, is a challenge for anaerobic bacteria such as Geotalea daltonii strain FRC-32. The aim of this study was to elucidate the metabolic pathways employed by G. daltonii FRC-32 during anaerobic benzoate oxidation in the presence of acetate, a key intermediate in anaerobic organic matter degradation, to predict carbon source transport and utilization strategies. Simultaneous carbon source oxidation and monoauxic growth were observed in G. daltonii FRC-32 cultures grown on 1 mM benzoate + 5 mM acetate, 1 mM benzoate + 2 mM acetate, and 2 mM acetate spiked with 1 mM benzoate. Sequential carbon source oxidation and diauxic growth were observed only in cultures grown on 5 mM acetate spiked with 1 mM benzoate. Benzoate accumulation in G. daltonii FRC-32 whole cell lysates indicated that intracellular benzoate transport occurred during benzoate oxidation in the presence of acetate. Expression analyses of putative benzoate transporter BenK and protein-ligand binding affinity prediction suggested BenK's specificity for transporting benzoate. Relative expression levels for the gene benK, encoding BenK, and the genes bamNOPQ, involved in the benzoyl-CoA pathway, were significantly higher in cultures grown on both benzoate and acetate than in cultures grown on acetate as sole carbon source, indicating that intracellular benzoate accumulation facilitated the regulation of bamNOPQ. Our results demonstrated that G. daltonii FRC-32 can perform differential benzoate oxidation in the presence of acetate, by either simultaneous or sequential carbon source oxidation, which indicated the metabolic plasticity of G. daltonii FRC-32 in response to varying carbon source availability.IMPORTANCEThe contamination of anaerobic subsurface environments by crude oil derivatives including aromatic compounds is a global concern due to the persistence and toxicity of these pollutants. Anaerobic bacteria play a crucial role in the degradation of aromatic hydrocarbons under anoxic conditions; however, the potential mechanisms involved in metabolic regulation of aromatic degradation pathways are not well understood. This study contributed to elucidating how G. daltonii strain FRC-32 efficiently utilizes benzoate as a carbon source in the presence of acetate. Findings of intracellular benzoate accumulation and regulation of key genes associated with benzoate oxidation contributed to the understanding of G. daltonii FRC-32's aromatic degradation pathways, provided significant insights into potential mechanisms that modulate anaerobic benzoate oxidation in the presence of the energetically favorable carbon source acetate, and indicated metabolic strategies of G. daltonii FRC-32 in response to dynamic environmental conditions.

Keywords: Geotalea daltonii; acetate; benzoate; carbon source availability; differential anaerobic degradation; metabolic strategies.

Fig 1

Growth characteristics and carbon source oxidation in G. daltonii FRC-32 cultures grown on various carbon sources. (A) Monoauxic growth and simultaneous carbon source oxidation in cultures on 1 mM benzoate + 2 mM acetate. (B) Monoauxic growth and simultaneous carbon source oxidation in cultures on 2 mM acetate cultures spiked with 1 mM benzoate. (C) Monoauxic growth and simultaneous carbon source oxidation in cultures on 1 mM benzoate + 5 mM acetate. (D) Diauxic growth and sequential carbon source oxidation in cultures on 5 mM acetate cultures spiked with 1 mM benzoate. Arrows indicate the addition of benzoate after 1-day incubation. The results represent the means ± standard errors of triplicate OD₆₀₀ or IC determinations of each sample obtained from triplicate cultures.

Fig 2

Benzoate accumulation in whole cell lysates of G. daltonii FRC-32 cultures grown on various carbon sources. (A) Cell lysate obtained from cultures grown on 1 mM benzoate + 2 mM acetate. (B) Cell lysate obtained from cultures grown on 2 mM acetate spiked with 1 mM benzoate. (C) Cell lysate obtained from cultures grown on 1 mM benzoate + 5 mM acetate. (D) Cell lysate obtained from cultures grown on 5 mM acetate spiked with 1 mM benzoate. Arrows indicate the addition of benzoate after 1-day incubation. The results represent the means ± standard errors of triplicate IC determinations of each sample obtained from triplicate cultures.

Fig 3

G. daltonii FRC-32’s growth was enhanced in cultures grown on 1 mM benzoate spiked with 2 mM acetate. (A) Growth patterns of G. Daltonii. (B) Benzoate oxidation in cultures grown on 1 mM benzoate as a sole carbon source or on 1 mM benzoate spiked with 2 mM acetate. Arrows indicate the addition of acetate after 1-day incubation. The results represent the means ± standard errors of triplicate OD₆₀₀ or IC determinations of each sample obtained from triplicate cultures (***P > 0.0005 as determined by student’s t-test). Significant difference compared to cell density during growth on 1 mM benzoate is indicated by asterisks.

Fig 4

Monoauxic growth and simultaneous carbon source oxidation in G. daltonii FRC-32 cultures grown on 1 mM benzoate spiked with 2 mM acetate. Carbon source oxidation in cultures on 1 mM benzoate spiked with 2 mM acetate. Arrow indicates addition of acetate after 1-day incubation. The results represent the means ± standard errors of triplicate OD₆₀₀ or IC determinations of each sample obtained from triplicate cultures.

Fig 5

Effect of pre-cultivation conditions on growth characteristics of benzoate-oxidizing G. daltonii FRC-32 cultures. Benzoate-oxidizing cultures were inoculated from various cultures that were adapted to varying benzoate and/or acetate availability. The results represent the means ± standard errors of triplicate OD₆₀₀ determinations of each sample obtained from triplicate cultures. (*P > 0.05 as determined by student’s t-test). Significant difference compared with cell density in benzoate-oxidizing cultures inoculated with cultures grown on 2 mM acetate spiked with 1 mM benzoate or 5 mM acetate spiked with 1 mM benzoate is indicated by asterisks.

Fig 6

Structure and protein-ligand-binding affinity prediction of the putative benzoate transporter BenK in G. daltonii FRC-32. (A) In silico structure prediction of BenK shows the presence of transmembrane regions creating a central transport channel (left). Structural alignment prediction of BenK compared with P. putida CSV86’s BenK (green: G. daltonii FRC-32; blue: P. putida) (right). (B) Protein topology prediction for BenK reveals the presence of 12 transmembrane regions. (C) In silico prediction of protein-ligand binding affinity of aromatic compounds to BenK. Protein-ligand binding affinity predictions were performed in triplicates. The results are represented as the means ± standard errors (*P > 0.05; as determined by Student’s t-test). Significant difference compared to binding affinity of BenK to acetate, toluene, and benzene is indicated by an asterisk.

Fig 7

Relative expression levels of putative benzoate transporter gene benK (Geob_0193) in G. daltonii FRC-32 cultures grown on different aromatic carbon sources. Transcript levels for benK were normalized to transcript levels for housekeeping gene recA. The fold change is relative to expression levels of benK in cultures grown on acetate as a negative control. The results represent the means ± standard errors of the triplicate qRT-PCR determinations of each cDNA sample obtained from triplicate cultures (***P > 0.0005; as determined by student’s t-test). Significant difference compared with expression during growth on benzene and toluene is indicated by asterisks.

Fig 8

SDS-PAGE image showing the band indicative of putative benzoate transporter BenK in whole cell lysates of G. daltonii FRC-32 cultures grown on various aromatic compounds. Lane 1: cells grown on 5 mM acetate as the sole carbon source. Lane 2: cells grown on 1 mM benzoate as sole carbon source. Lane 3: cells grown on 1 mM toluene as the sole carbon source. Lane 4: cells grown on 1 mM benzene. Arrows indicate the location of the putative benzoate transporter BenK, ca. 43 kDa.

Fig 9

Relative expression levels of putative aromatic transporter gene benK (Geob_0193) in different growth phases of G. daltonii FRC-32 cultures grown on 5 mM acetate and spiked with 1 mM benzoate. Transcript levels for benK were normalized to transcript levels for housekeeping gene recA. The fold change is relative to expression levels of benK in cultures grown on acetate as a negative control. The results represent the means ± standard errors of the triplicate qRT-PCR determinations of each cDNA sample obtained from triplicate cultures (***P > 0.0005; as determined by student’s t-test). Significant difference compared with expression during the second early and mid-log phases is indicated by asterisks.

Fig 10

Relative expression levels of putative aromatic transporter gene benK (Geob_0193) in different growth phases of G. daltonii FRC-32 cultures grown on 1 mM benzoate + 5 mM acetate. Transcript levels for benK were normalized to transcript levels for housekeeping gene recA. The fold change is relative to expression levels of benK in cultures grown on acetate as a negative control. The results represent the means ± standard errors of the triplicate qRT-PCR determinations of each cDNA sample obtained from triplicate cultures (***P > 0.0005; as determined by student’s t-test). Significant difference compared with expression during the early and mid-log phases is indicated by asterisks.

Fig 11

Transcript levels for genes bamNOPQ in G. daltonii FRC-32 cultures grown on 1 mM benzoate + 5 mM acetate, 5 mM acetate spiked with 1 mM benzoate and 5 mM acetate. (A) Transcript levels for gene bamN, encoding thiolase. (B) and (C) Transcript levels for genes bamO and bamP, encoding the electron transfer flavoprotein subunits beta and alpha, which facilitate electron transfer for the strictly ETF-coupled benzoate degradation protein BamM (glutaryl-coenzyme A dehydrogenase). (D) Transcript levels for gene bamQ, encoding 6-hydroxycyclohex-1-ene-1-carbonyl-CoA dehydrogenase. The results represent the means ± standard errors of the triplicate qRT-PCR determinations of each cDNA sample obtained from triplicate cultures (***P > 0.0005, **P > 0.005; as determined by Student’s t-test). Significant difference compared to transcript levels during the early and late-log phases is indicated by asterisks.