Microbial Multitaskers: Rhizorhabdus wittichii RW1 Reveals Shared Electron Transfer Pathways for Pollutant Degradation
- Ray Sullivan
- Aug 14
- 3 min read
Microbiologists constantly seek to understand the intricate metabolic capabilities of bacteria, particularly those involved in environmental remediation. Rhizorhabdus wittichii RW1, formerly classified as Sphingomonas, is a well-known exemplar in this field, recognized for its ability to degrade polycyclic aromatic hydrocarbons like dibenzo-p-dioxin (DD) and dibenzofuran (DF). Research from Gerben Zylstra’s Lab at Rutgers delves deeper into RW1's metabolic toolkit, revealing an overlap in its degradation machinery: the electron transfer components crucial for DD/DF breakdown are also essential for benzoate degradation.
Benzoate, a ubiquitous aromatic hydrocarbon, is found in the environment from natural sources (e.g., lignin degradation, combustion) and anthropogenic activities (e.g., food preservatives, cosmetics). Its common co-occurrence with pollutants like DD and DF makes understanding its degradation pathways, especially shared ones, highly relevant for bioremediation strategies.
At the core of DD and DF degradation in R. wittichii RW1 is a three-component angular dioxygenase called DxnA1A2. This enzyme system is notable for its flexibility, utilizing two interchangeable ferredoxins (Fdx1/Fdx3) and two interchangeable reductases (RedA1/RedA2) to facilitate electron transfer. These electron transfer genes (redA1, redA2, fdx1, fdx3) are often constitutively expressed or induced when the bacterium grows on DD and DF.
Building on the knowledge that many aromatic hydrocarbon-degrading sphingomonads share electron transfer components among various dioxygenases, researchers hypothesized that RW1's benzoate 1,2-dioxygenase might similarly share these vital DxnA1A2 components.
To test this hypothesis, the researchers employed a combination of gene identification, knockout mutagenesis, and heterologous expression experiments.
Identification of benAB: The genes encoding the benzoate oxygenase component (benAB) were identified in the RW1 genome through homology to known benzoate oxygenases. The BenAB protein subunits in RW1 are evolutionarily distinct from those found in Pseudomonas putida KT2440 and Sphingomonas yanoikuyae B1, highlighting a novel enzyme.
Knockout Mutagenesis Confirms Benzoate's Dependence:
Knockout of benA (SWIT_2634) rendered the mutant strain (RW1ΔbenA) unable to grow on benzoate as its sole carbon source, confirming benAB as the primary benzoate dioxygenase. Complementation was achieved by reintroducing the entire benABD operon, suggesting a polar effect of the initial knockout on downstream genes.
Single knockouts of redA1 or redA2 (reductase genes) did not impact benzoate growth. However, a double knockout of redA1 and redA2 resulted in a mutant that could not grow on benzoate. This growth defect was restored upon reintroduction of either redA1 or redA2.
In contrast, double knockout of the ferredoxin genes, fdx1 and fdx3, did not abolish growth on benzoate, implying the involvement of other ferredoxins in benzoate degradation.
Heterologous Expression Validates Component Sharing:
When benAB genes were expressed alone in Escherichia coli, no benzoate conversion was observed.
However, co-expression of benAB with fdx3 and redA2—the specific ferredoxin and reductase components known to be utilized by DxnA1A2—resulted in a functional benzoate dioxygenase enzyme. This recombinant system could completely convert benzoate to benzoate cis-dihydrodiol within 24 hours. This unequivocally demonstrates that the benzoate dioxygenase functions as a three-component system and shares electron transfer components with DxnA1A2. While Fdx3 was highly effective, other ferredoxins tested showed only minimal activity.
This study offers several significant insights for microbiologists working on environmental cleanup:
The direct finding that DD, DF, and benzoate degradation pathways compete for the same electron transfer components (RedA1, RedA2, and Fdx3) is noteworthy. In environments co-contaminated with these pollutants, this competition could influence the rates and efficiencies of degradation for each compound, potentially slowing down cleanup efforts.
While the sharing of electron transport components among different oxygenase systems is observed in other bacteria (e.g., Sphingomonas yanoikuyae B1, Ralstonia sp. U2, Staphylococcus aureus), the identified benzoate dioxygenase in RW1 is a novel enzyme, distinct from previously characterized ones. This highlights the bacterium's metabolic adaptability and resourcefulness.
The study notes that the benzoate oxygenase is located on the main chromosome of RW1, while redA2 and fdx3 are found on the plasmid pSWIT02, which is known to have evolved under selective pressure from DF. This 'scattered' gene arrangement is characteristic of sphingomonads and underscores their complex and adaptable catabolic pathways.
Understanding this "plasticity" in electron transfer component utilization opens avenues for future research. It prompts questions about how many other oxygenase enzymes these shared components can support, potentially paving the way for engineered bacterial strains with enhanced or expanded bioremediation capabilities. Further investigation is warranted into other potential ferredoxins that can partner with RedA1 or RedA2 for benzoate degradation, possibly novel ones.
Rhizorhabdus wittichii RW1 is a highly efficient microbial scavenger. The discovery of shared electron transfer components between its DD/DF and benzoate degradation pathways deepens our understanding of bacterial catabolism. It provides insights that can inform more effective strategies for tackling complex environmental contamination.
Ivanovski I, Eleya S, Zylstra GJ. Analysis of Benzoate 1,2-Dioxygenase Identifies Shared Electron Transfer Components With DxnA1A2 in Rhizorhabdus wittichii RW1. J Basic Microbiol. 2025 Aug;65(8):e70061. doi: 10.1002/jobm.70061. Epub 2025 May 22. PMID: 40405529; PMCID: PMC12319509.
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