Rhodococcus ruber

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Classification

Figure 1: Rhodococcus ruber in rod form as a biofilm degrading polystyrene [10]

Domain: Bacteria

Phylum: Actinobacteria

Class: Actinobacteria

Order: Corynbacteriales

Family: Nocardiaceae

Genus: Rhodococcus

Species

NCBI: [2]

Rhodococcus ruber

Description and Significance

Figure 2: Rhodococcus ruber cultured on a trypticase soy broth agar [1]

Historically, the genus Rhodococcus was first defined by William Zopf in 1891. Nocardia rubra, or what is known today as Rhodococcus ruber, was isolated from a soil sludge by William Kruse in 1896. However, there were issues with taxonomic classifications due to unsophisticated and inaccurate morphological and staining identification techniques. In 1977, Micheal Goodfellow and Grace Alderson did a complete reclassification of the genus Rhodococcus, which resulted in Norcordia rubra being amended to Rhodococcus ruber.

The term Rhodococcus is from the Greek words “rhodon” and “coccus” meaning “rose” and “grain,“ respectively. R. ruber's morphological structure matches the origins of its name: rods and cocci. R. ruber initially forms a branching mycelium that breaks into shorter rods and cocci as it transitions through different growth phases. R. ruber is characteristically a gram positive, non-motile, non-spore forming bacteria. It has diverse metabolic and nutritional capabilities depending on the strain, such as utilizing gaseous hydrocarbons, aromatic hydrocarbons, complex polymers, and steroids for carbon and energy sources.

R. ruber is unique in that it is ubiquitous in many different habitats whether that is pristine, contaminated, temperate, or extreme. It is able to persist and tolerate stressful conditions, such as limited oxygen and nutrients. Given its metabolic and habitat versatility, R. ruber is a good candidate for bioremediation and other biotechnological uses to naturally degrade pollutants or remedy contaminated ecosystems.

Genome Structure

12 genome studies have been reported for the following strains of Rhodococcus ruber: R. ruber str. YC-YT1, R. ruber str. YYL, R. ruber str. P14, R. ruber str. SD3, R. ruber str. R1, R. ruber str. DSM 43338, R. ruber str. P25, R. tuber IEGM 231, R. ruber str. OA1, R. ruber str. BKS 20-38, R. ruber str. Chol-4, and R. ruber str. NCRX 15591.

The genome of Rhodococcus ruber consists of one circular chromosome with an average size of 5.57 Mb. The genome size varies for every strain and larger genome sizes can be attributed to the presence of circular plasmids. The strains of R. ruber that contain plasmids are YC-TC1, YYL, and R1, which are known to contain additional genes that code metabolic enzymes. The average amount of protein coding genes is 4932 and the average G+C content is 70.49%. A high guanine-cytosine content is characteristic of Rhodococcus species which provides DNA stability in varying conditions.

The genome sequences of R. ruber are helpful in identifying the genes necessary for metabolic pathways and cycles that R. ruber employs including the tricarboxylic acid cycle, pentose phosphate pathway, glycolysis, and gluconeogenesis. Additionally, the beta oxidation pathway and alkane degradation pathway are utilized to break down more complex xenobiotic compounds before the classic metabolic pathways are used. The genome highlights multiple metabolic gene clusters that are used for degradation of aromatic compounds, steroids, hydrocarbons, and xenobiotic polymers that R. ruber uses for carbon and energy sources. Some strains of R. ruber contain redundant genes contribute to their versatile metabolic abilities with various nutrient sources.

Cell Structure and Metabolism

Figure 3: Rhodococcus ruber cell wall structure [1]

Rhodococcus ruber is a gram positive, aerobic bacteria that is non-motile and non-spore forming. Key features of its cell structure include its thick peptidoglycan layer, mycolic acids, cell envelope glycolipids, lipoglycans, and cell envelope lipids. The mycolic acids of R. ruber are long chain fatty acids with chain lengths ranging from C42-C48, which are much longer than the carbon chains in other Rhodococcus species. Its mycolic acids are composed of saturated and unsaturated fatty acids with tuberculostearic acid. As with all Rhodococcus species, its cell wall is made up of the carbohydrates arabinose and galactose. The cell envelope glycolipids function to fill in gaps on the cells surface that mycolic acids are not present. R. ruber's lipoglycans are polysaccharides in the lipoarabinomannan family that are anchored into the membrane. The cell envelope lipids of R. ruber have notable surfactant properties due to its low hydrophilic-lipophilic value, which allows R. ruber to attach and grow on hydrophobic substrates. Other biomolecules that contribute to R. ruber's hydrophobicity and substrate binding includes its mycolic acids, extracellular polymeric substance, and cell surface proteins.

R. ruber is an aerobic chemoorganotroph, meaning they use organic compounds as their carbon and energy sources through oxidative metabolic pathways. It is able to degrade gaseous hydrocarbons, aliphatic hydrocarbons, aromatic hydrocarbons, and xenobiotic substances, such as crude oil and petroleum. Additionally, certain strains of R. ruber have unique metabolic abilities, such as R. ruber str. Chol-4 and R. ruber str. C208. R. ruber str. Chol-4 is able to use steroids, including cholesterol, testosterone, and phytosterols, as their carbon source. In the interest of biotechnological applications, the metabolic activity of R. ruber str. Chol-4 can be used for steroid transformations to active pharmaceuticals. The other strain with unique metabolic abilities is R. ruber str. C208. This strain is able to use inert polymers, such as polyethylene and polystyrene, as its sole carbon source. C208 utilizes beta oxidation and alkane degradation pathways to break down these polymers. Additionally, it has lactase-coding genes that promote biotic oxidation. Laccases are phenol oxidases that belong to a group of multi-copper enzymes. Studies have shown that the addition of copper in polyethylene cultures improved laccase's oxidizing activity, and resulted in increased degradation. R. ruber str. C208 has important implications regarding bioremediation efforts.

Ecological Impacts

Figure 4: Biofilm formation of Rhodococcus ruber on polyethylene. A) Attachment to surface B)Formation of monolayer and exopolymeric matrix C)Formation of multilayer microcolony D) Mature biofilm "mushroom" shape [13]

R. ruber will form a biofilm

This has a higher metabolic activity than if it was suspended and the solid surface serves as a substrate for R. ruber, so carbon availability is greater in a biofilm

The colonization of R. ruber to PE is nonspecific

Adheres to solid surface of PE, becomes differentiated from planktonic state, initiates formation of 3D structures of microcolonies, then further organized into multicellular mushroom-like structures that last for 60 + days

Carbon starvation enhanced the hydrophobic interactions, biofilm development, which improved biodegradation of PE

References

[1]| Alvarez, H. M. Biology of Rhodococcus; Springer, 2010; Vol. 16.

[2] | Bala, M.; Kumar, S.; Raghava, G. P. S.; Mayilraj, S. Draft Genome Sequence of Rhodococcus ruber Strain BKS 20-38. Genome Announcements 2013, 1.

[3] | Farkas, C.; Donoso, R. A.; Melis-Arcos, F.; Gárate-Castro, C.; Pérez-Pantoja, D. Complete Genome Sequence of Rhodococcus ruber R1, a Novel Strain Showing a Broad Catabolic Potential toward Lignin-Derived Aromatics. Microbiology Resource Announcements 2020, 9.

[4] | Finnerty, W. R. The Biology and Genetics of the Genus Rhodococcus. Annual Review of Microbiology 1992, 46, 193–218.

[5] | Gravouil, K.; Ferru-Clément, R.; Colas, S.; Helye, R.; Kadri, L.; Bourdeau, L.; Moumen, B.; Mercier, A.; Ferreira, T. Transcriptomics and Lipidomics of the Environmental Strain Rhodococcus ruber Point out Consumption Pathways and Potential Metabolic Bottlenecks for Polyethylene Degradation. Environmental Science & Technology 2017, 51, 5172–5181.

[6] | Guevara, G.; Lopez, M. C.; Alonso, S.; Perera, J.; Navarro-Llorens, J. M. New insights into the genome of Rhodococcus ruber strain Chol-4. BMC Genomics 2019, 20.

[7] | Heras, L. F. D. L.; Fernández, E. G.; Llorens, J. M. N.; Perera, J.; Drzyzga, O. Morphological, Physiological, and Molecular Characterization of a Newly Isolated Steroid-Degrading Actinomycete, Identified as Rhodococcus ruber Strain Chol-4. Current Microbiology 2009, 59, 548–553.

[8] | Krivoruchko, A.; Kuyukina, M.; Ivshina, I. Advanced Rhodococcus Biocatalysts for Environmental Biotechnologies. Catalysts 2019, 9.

[9] | Montazer, Z.; Najafi, M. B. H.; Levin, D. B. Challenges with Verifying Microbial Degradation of Polyethylene. Polymers 2020, 12.

[10] | Mor, R.; Sivan, A. Biofilm formation and partial biodegradation of polystyrene by the actinomycete Rhodococcus ruber. Biodegradation 2008, 19, 851–858.

[11] Rhodococcus ruber (ID 11562) https://www.ncbi.nlm.nih.gov/genome/?term=txid1830[Organism:noexp] (accessed 2020).

[12 | Santo, M.; Weitsman, R.; Sivan, A. The role of the copper-binding enzyme – laccase – in the biodegradation of polyethylene by the actinomycete Rhodococcus ruber. International Biodeterioration & Biodegradation 2013, 84, 204–210.

[13] | Sivan, A.; Szanto, M.; Pavlov, V. Biofilm development of the polyethylene-degrading bacterium Rhodococcus ruber. Applied Microbiology and Biotechnology 2006, 72, 346–352.

Author

Page authored by Hannah von Werder, student of Prof. Jay Lennon at Indiana University.