a. Higher order taxa
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
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).
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).
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).
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).
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