Pseudomonas denitrificans

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1. Classification

a. Higher order taxa

Domain Bacteria

Phylum Proteobacteria

Class Gammaproteobacteria

Order Pseudomonadales

Family Pseudomonadaceae

Genus Pseudomonas

Species Group Pseudomonas Pertucinogena

Species NCBI: Taxonomy Genus species Pseudomonas denitrificans

2. Description and significance

Pseudomonas denitrificans is a polar flagellated, rod-shaped, Gram-negative, aerobic, heterotrophic bacteria species with the ability to produce vitamin B12 (1). P. denitrificans is one of the few microorganisms that can synthesize vitamin B12 under aerobic conditions (2). As the name suggests, P. denitrificans is also capable of performing denitrification as a part of nitrogen cycle, a process in which nitrate is reduced into nitrogen gas (N2) (2).

Despite the enormous knowledge known about of P. denitrificans, there is still a lot of information unknown. Its cytochrome cc’ protein, which is found in the mitochondria, is essential in the electron transport chain, but yet to be studied in depth for its relationship to similar proteins in photosynthetic bacteria (3). The evolutionary implications of conserved genes encoding for vitamin B12 production may yet reveal insights into the origin of metabolism, since the pathway is thought to have developed to support fermentation processes, but as of yet not conclusively proven (4). P. denitrificans’ medical and environmental significance, in terms of its industrial use for vitamin B12 production and potential nitrate toxicity or wastewater treatment applications, is also unavailable (3-7).

P. denitrificans is believed to be phylogenetically ancient, and so provides an opportunity for understanding metabolic evolution (4). Its supplementation of oxidative phosphorylation with denitrification provides insights into how it fulfills and maintains a niche across fluctuating O and N levels in environments (8).

P. denitrificans may also be engineered to produce other commercial compounds, such as 3-hydroxypropionic acid (9). Its denitrification abilities have critical potential in wastewater management (7). Pathologically speaking, P. denitrificans may opportunistically cause meningitis in humans (10). P. denitrificans may also colonize the intestines of fish (11).

3. Genome structure

P. denitrificans ATCC 13867 genome consists of single circular chromosome with a genome size of 5,696,307 bps with 65.2% Guanine + Cytosine content (1). Its genome has 2,567 operons and 5,059 protein-encoding genes, where 59.56% of proteins are characterized as cytoplasmic and 19.41% non-cytoplasmic, while the the remaining percentage is still unknown (1). Its genome has genes for all 20 amino acids, with 63 transfer RNAs. It also contains 1,279 ribosome-binding sites, and 816 transcription terminators (1).

In addition, P. denitrificans genome contains genes encoding 26 enzymes that are involved in the biosynthesis of vitamin B12, where the genes are divided in two different clusters on the chromosome (1). The first and second clusters encode genes that are involved in the vitamin B12 biosynthesis pathway (1). Additionally, P. denitrificans genome contains methionine synthase gene, which codes for a protein that catalyzes the synthesis of L-methionine, an essential amino acid that uses vitamin B12 as a cofactor (1).

4. Cell structure

Pseudomonas denitrificans is a Gram-negative, aerobic, rod-shaped bacteria. The optimum growth temperature for P. denitrificans is 25°C (14). P. denitrificans colonies do not fluoresce, and they acquire a smooth off-white/tan color appearance. The cell sizes ranges around 1.05 x 0.8 µm, and can be composed of up to 48% lipids, depending on the strain (14). Further information of cell structure is still not clearly known for this particular Pseudomonas, and research studies are ongoing.

5. Metabolic processes

Vitamin B12 Biosynthesis

Its vitamin B12­ production can provide nutritional services to many life forms (4). In terms of more fundamental significance, its chemotrophic use of the cytochrome cc’ heme contrasts with purple bacteria photosynthetic use of a structurally and genetically similar protein, which indicates that it exists at a critical juncture in ATP production evolution (3).

Pseudomonas denitrificans is one of the few microorganisms that can synthesize Vitamin B12 de novo under aerobic conditions (2). Vitamin B12, also known as cyanocobalamin, is an essential vitamin for the proper function of the animal nervous system1, and it is used as a coenzyme in many metabolic pathways, such as the conversion of l-methylmalonyl-coenzyme A (CoA) to succinyl-CoA, catalyzed by methylmalonyl-CoA mutase (MCM) (15). Notably, deficiency has been associated with ataxia, spasticity, muscle weakness, dementia, psychosis, and Alzheimer’s disease (4).

Because P. denitrificans is is an overproducer of Vitamin B12, it has been used in large scale industrial production, and engineered to produce even higher yields via fermentation (16). In P. denitrificans, Vitamin B12 is synthesized aerobically, requiring oxygen to promote ring-contraction, and requiring approximately 30 different enzymes (16).


Pseudomonas denitrificans can conduct anaerobic denitrification through reducing NO3- → NO2- → NO → N2O → N2 (7). Initial substrates are obtained from soil8, or polluted surface waters (7). There have also been observed instances of denitrification occurring in fish, due to nitrogen bioaccumulation (11). Reactant compounds are able to act as electron acceptors in oxidative phosphorylation and produce ATP (8).

While P. denitrificans- specific ecological impacts have not been extensively studied, it is believed that P. denitrificans may be engineered to aid in wastewater management (7, 11, 18).Through introducing great numbers into contaminated ponds and lakes, NO3- consumption may ward off eutrophication and help regulate nitrogen and oxygen levels (7, 11, 18). However, its ability to act as an opportunistic pathogen and cause meningitis in humans potentially limits this opportunity (10).

3-Hydroxypropionic Acid Synthesis

Pseudomonas denitrificans produces 3-hydroxypropionic acid under aerobic conditions, due to B12 -dependant enzymes (9). P. denitrificans was engineered into a recombinant strain during the development of 3-HP from glycerol by overexpressing dhaB, a gene that encodes glycerol dehydratase (19). The two major pathways for the biological assimilation of 3-HP are: (1) the oxidative pathway 3-hydroxypropionate dehydrogenase (HpdH) and (2) reductive pathway (methyl)malonate-semialdehyde dehydrogenase (MmsA). Furthermore, 3-HP inducible systems in P. denitrificans are promising in the development of gene expression systems (19).

6. Ecology

Pseudomonas denitrificans is present in a variety of habitats, including soils and surface waters (7, 8, 11). As a chemoorganoheterotroph, it is generally considered a decomposer (8). However, there have been observed instances of it infecting tropical freshwater fish species and humans, where it behaves like an opportunistic pathogen (10, 11).

In its terrestrial areas, particularly agricultural ones, its significance in the nitrogen cycle factors into overall NO3-, O, C, and micronutrient availability (20). Its presence can thus factor into manipulating crop yield and reducing nitrogenous greenhouse gas production (20). It may also be manipulated to induce carbonate mineral precipitation through denitrification, which would aid in improving water flow and subsequent nutrient uptake for plants (21).

Like other pseudomonads, it is found in the rhizosphere of plants, using different carbonaceous materials as a nutrient source (21). When added to seedlings, P. denitrificans induces structural and biochemical changes in cell wall structure that leads to heightened plant resilience against common pests and pathogens (23). Notably, P. denitrificans also acts as an antagonist against the plant fungus Vertilcillum lateritum through the production of anti-fungal metabolites, causing wheat and corn that would be otherwise affected to experience a shoot and root length increase of 30-45% (22).

In terms of aquatic environments, P. denitrificans inhabits a variety of waters, living off of sediment in surface waters (11), wastewaters (7), and also bottom lake sediment (8). As such, it is particularly active in potentially eutrophic waters (7). Since it is able to combine its denitrifying processes with its electron transport chain (8, 24), it is able to sustain life at high and low oxygen levels (8). It is able to contribute to these environments through nitrogen cycling, and so poses as a valuable potential player in wastewater management (18).

7. Pathology

There has been one documented case of Pseudomonas denitrificans implicated in human disease, in 1982. The bacteria was associated with a secondary infection of meningitis, and localized to the central nervous system of an elderly, ill patient (10). It was found to be ampicillin resistant, and developed ticarcillin and carbenicillin resistance 5 days after discovery. The patient, severely ill with systemic lupus erythematosus and chronic leg ulcers, died in 12 days (10). Since this was a singular occurrence, its clinical significance has not been corroborated with other sources or evaluated.

There are recorded cases of P. denitrificans infection in fish, particularly those exposed to wastewater. Due to its denitrification abilities, the bacteria will colonize the intestines of species that have enough nitrogenous compounds for it to sustain itself from (11). While they are susceptible to antibiotics once within hosts, free-living bacteria have been recorded as increasingly resistant (11).

8. Current Research

Current research involving Pseudomonas denitrificans focuses on its use for industrial production of vitamin B12. Challenges that are being overcome include devising an optimal growth medium, and creating systems for precise genome manipulation (2). To simplify the production process, it is being studied to engineer E. coli for large scale production of vitamin B12 (2).

There is also interest in using Pseudomonas denitrificans to reduce nitrate levels in groundwater and wastewater (18). Research focuses on which growth conditions are optimal to induce P. denitrificans’ denitrification pathway, as well as which are inexpensive enough to combat the large scales pollution and eutrophication occur in. Particularly, ethanol has been explored as a viable organic carbon source (18).

9. References

1. Ainala, S. K., Somasundar, A., & Park, S. (2013). Complete Genome Sequence of Pseudomonas denitrificans ATCC 13867. Genome Announcements, 1(3), e00257–13.

2. Fang, H., Kang, J., & Zhang, D. (2017). Microbial production of vitamin B12: a review and future perspectives. Microbial Cell Factories, 16, 15.

3. Cusanovich M.A., Tedro S.M., and Kamen M.D. (1970). Pseudomonas denitrificans cytochrome cc’. Archives of Biochemistry and Biophysics, 141(2), 557-570.

4. Martens J.H., Barg H., Warren M.J., and Jahn D. (2002). Microbial production of vitamin B12. Applied Microbiology Biotechnology, 58, 275- 285.

5. Xia, W., Peng, W., Chen, W., and Li, K. 2015. Interactive performances of betaine on the metabolic processes of Pseudomonas denitrificans. Journal of Industrial Microbiology & Biotechnology, 42(2), 273-278.

6. Rodionov D.A., Vitreschaki A.G., Mironov A.A, and Gelfand M.S. 2003. Comparative genomics of vitamin B12 metabolism and regulation in prokaryotes. The Journal of Biological Chemistry, 278, 41148- 41159.

7. Parvanova-Mancheva T. & Beschkov V. (2009). Microbial denitrification by immobilized bacteria Pseudomonas denitrificans stimulated by constant electrical field. Biochemical Engineering Journal, 44(2-3), 208-213.

8. Koike I. & Hattori A. 1975. Energy yield of denitrification: an estimate from growth yield in continuous cultures of Pseudomonas denitrificans under nitrate-, nitrite-, and nitrous oxide-limited conditions. Microbiology, 88, 11-19

9. Zhou, S., Ashok, S., Ko, Y., Kim D., and Park S. (2014). Development of a deletion mutant of Pseudomonas denitrificans that does not degrade 3-hydroxypropionic acid. Applied Microbiology and Biotechnology, 98(10), 4389-4398.

10. Fischer, R. A., Doern, G.V., Cheeseman, S.H. (1981). Pseudomonas denitrificans meningitis. Journal of Clinical Microbiology, 13(5), 1004-1006.

11. Patra, S., Das, T.K., Ghosh, S.C., Sarkar, D., & Jana, B.B. (2010). Cadmium tolerance and antibiotic resistance of Pseudomonas sp. Isolated from water, sludge and fish raised in waste-water fed tropical ponds. Indian Journal of Experimental Biology, 48, 383-393.

12. Anzai, Y., Kim, H., Park, J. Y., Wakabayashi, H., & Oyaizu, H. (2000). Phylogenetic affiliation of the pseudomonads based on 16S rRNA sequence. International Journal Of Systematic And Evolutionary Microbiology, 50(4), 1563-1589. doi:10.1099/00207713-50-4-1563

13. Tran, P. N., Savka, M. A., & Gan, H. M. (2017). In-silico Taxonomic Classification of 373 Genomes Reveals Species Misidentification and New Genospecies within the Genus Pseudomonas. Frontiers in Microbiology, 8, 1296.

14. Environment and Climate Change Canada. “Final Screening Assessment.” Environment and Climate Change Canada - Evaluating Existing Substances - Final Screening Assessment for Pseudomonas Sp. ATCC 13867, 27 May 2016,

15. Lago, B. D., & Demain, A. L. (1969). Alternate Requirement for Vitamin B12 or Methionine in Mutants of Pseudomonas denitrificans, a Vitamin B12-producing Bacterium. Journal of Bacteriology, 99(1), 347–349.

16. Spalla, C., Grein, A., Garofano, L., & Ferni, G. (1989). Microbial Production of Vitamin B12. Biotechnology of Vitamins, Pigments and Growth Factors, 257-284. doi:10.1007/978-94-009-1111-6_15

17. Payne, W.J. (1973). Reduction of nitrogenous oxides by microorganisms. Bacteriological Reviews, 37(4), 409-452.

18. Nilsson, I., & Ohlson, S. (1982). Columnar denitrification of water by immobilized Pseudomonas denitrificans cells. European Journal of Applied Microbiology and Biotechnology,14(2), 86-90.

19. Catherine, Christy, et al. “Production of 3‐Hydroxypropionic Acid from Glycerol by Recombinant Pseudomonas Denitrificans.” Biotechnology and Bioengineering, Wiley-Blackwell, 5 July 2013,

20. Richardson, D., Felgate, H., Watmough, N., Thomson, A., & Baggs, E. (2009). Mitigating release of the potent greenhouse gas N2O from the nitrogen cycle—could enzymatic regulation hold the key? Trends in Biotechnology, 27(7), 388-397.

21. Hamdan, N., Kavazanjian, E., Rittmann, B.E., & Karatas, I. (2015). Carbonate mineral precipitation for soil improvement through microbial denitrification. Geomicrobiology Journal, 34(2), 139-146.

22. Egamberdiyeva, D. (2005) Characterization of Pseudomonas Species Isolated from the Rhizosphere of Plants Grown in Serozem Soil, Semi-Arid Region of Uzbekistan. TheScientificWorldJOURNAL, 5, 501-509.

23. Ramamoorthy, V., Viswanathan, R., Raguchander, T., Prakasam, V., Samiyappan, R. (2001) Induction of systemic resistance by plant growth promoting rhizobacteria in crop plants against pests and disease. Crop Protection, 20, 1-11.

24. Hassan, J., Qu, Z., Bergaust, L.L., & Bakken, L.R. (2016). Transient accumulation of NO2- and N2O during denitrification explained by assuming cell diversification by stochastic transcription of denitrification genes. PLoS Computational Biology, 12(1), e1004621,