Microbes have been an extremely important part of human history for thousands of years. They assist us in numerous daily functions and allow for life on Earth to run smoothly. One of their most important functions is in the production of food products, especially in those that are fermented. Fermented food products have been a staple in human diets due to their unique tastes, decreased perishability, and possible health benefits. An example of a popular fermented food product is daemi-jeot. This is a salt- fermented dish prepared commonly in Korea from Konosirus punctatus, a species of fish known as the dotted gizzard shad. This fish falls within the same family as sardines and anchovies (1). To make this traditional Korean dish, the fish is heavily salted and left to ferment. Like many other food products that undergo fermentation, daemi-jeot is able to be made due to the microbial interactions within its environment. However, bacterial populations found within this product are still being understood and discovered. Kushneria konosiri is the proposed name of a novel bacterial strain, strain X49T, discovered within this Korean fermented fish product (3). Kushneria konosiri is a halophilic, Gram-negative, and oval or rod-shaped organism. It is motile and contains a single flagellum that branches out from the cell. Metabolically, it is obligately aerobic. The optimum growth conditions for this bacterial strain are at 15–25 degrees Celsius, pH 5.0–7.0 and in the presence of 11–19 % (w/v) NaCl (3). Within its environment, its halophilic properties characteristic of members within its family (4) allow this microbe to survive and outcompete other microorganisms by withstanding the high salt concentrations surrounding it during the preparation of daemi-jeot. This is due to its development of several functional genes and its ability to perform carotenoid biosynthesis (2). With these adaptations this particular strain can survive in environments with salt concentrations up to 26% w/v NaCl (3). This bacterium thrives within this environment and could be a contributor to the fermentation process of daemi-jeot as it produces acids through a majority of its metabolic processes (3).
Higher Order Taxa
Eubacteria (Kingdom); Bacteria (Domain); Proteobacteria (Phylum); Gammaproteobacteria (Class); Oceanospirillales (Order); Halomonadaceae (Family); Kushneria (Genus)
Kushneria konosiri, type strain X49T (Accession GU198748)
Kushneria konosiri is a Halomonadaceae that belongs to the genus Kushneria. This genus was discovered previously as a reclassification of a cluster of species from the genus Halomonas (5). To discover Kushneria konosiri, researchers isolated genomic DNA using previous methodology (6) and used 16S and 23S rRNA gene sequencing methods in order to determine the genetic relatedness of this bacterial strain. A phylogenetic tree containing this organism was constructed using the neighbor joining method. It was used by researchers to compare 16S rRNA sequences of this species with others in the Kushneria genus. In the same study, a separate tree was constructed using 23S rRNA sequences but it revealed similar information to the results of 16S rRNA (2). This bacterium is most closely related to the species Kushneria marislavi a halophilic bacteria discovered from a water sample in the Yellow Sea (7). Through average-nucleotide identity tests, their relatedness level was found to be just below 90% (3). This indicated that this strain was in fact a novel species within the Kushneria genus.
This bacterium was isolated from the liquid portion of the Korean fermented seafood product daemi-jeot. This food product is a variation of the popular Korean dish jeotgal. Jeotgal is a popular Korean dish consumed either as a condiment or as a seasoning to other dishes. In general, all forms of jeotgal are made by heavily salting a particular type of seafood such as fish, shellfish, and squid and can be characterized by their main ingredients (8). Daemi-jeot in particular comes from the adductor muscle, stomach, and intestines of the dotted gizzard shad (9). The liquid environment of Kushneria konosiri was isolated from was not tested extensively for abiotic physical and chemical factors as it came from a homemade food product bought at a market on the coast of South Korea (3). However, as this type of dish is relatively common in Korean cuisine and is prepared in a traditional manner (8), some assumptions can be made about these factors. Since daemi-jeot is fermented using solar salt rather than traditional purified salt we can assume that within this environment there are a significant amount of micronutrients and minerals such as K, Mg, and Ca (10). The NaCl concentration is also likely significantly high within this environment with as some forms of jeotgal are prepared in environments around 25% w/v NaCl (8, 11). During the fermentation process occurring within a common jeotgal environment there is an overall decrease in pH consisting of a drop in pH initially followed by a small increase after some time (12). Oxygen concentration and temperature are also unknown for this particular environment but as this product commonly is left to sit out at the market (Fig. 2) we can assume that it was exposed to atmospheric levels of oxygen and varying temperatures that were likely mild at the time as this sample was collected in April (2).
Environments of other species within the Kushneria Genus
The genus Kushneria is relatively novel and small, containing only 9 classified species (13). In general, Kushneria species are located in hypersaline environments. Species have been located in areas such as leaves of the black mangrove in Puerto Rico (14), the Yellow Sea in Korea (7), salt mines in Pakistan (15), and even within traditional Chinese cured meat (6). All members of this genus are also aerobic so they must be in the presence of oxygen to grow (13). Within their environment, they are not known to form spores (5).
Significance to the Environment
Since this bacterium is obligately aerobic and heterotrophic, it cycles nutrients through aerobic cellular respiration, obtaining its source of carbon for respiration by consuming organic materials in its environment (16). The organic materials are most likely the sugars and fats present in within the fish. While it goes through aerobic cellular respiration, being within a high-sodium environment, Kushneria konosiri is not able to form a typical proton gradient and must establish transmembrane gradients with other cations, likely sodium in its environment, and pumps them in using a modified version of ATP synthase to phosphorylate ADP (16). As this species is catalase-positive (3), through its process of aerobic cellular respiration this bacterium uses catalase to convert toxic hydrogen peroxide produced through its electron transport chain into water and oxygen gas (16). While there is an understanding of the general metabolic processes this bacterium can perform, not much is known about the exact significance that Kushneria konosiri has on the environment of daemi-jeot. While it may have some influence on the process of fermentation through the production of acid in its metabolic processes, Kushneria konosiri is not known to perform any fermentation and is also not able to reduce nitrate (3). With that being said, however, some significance of this bacterium results in its ability to outcompete other microorganisms for nutrients and space within this hypersaline environment. There is not much known about how human induced changes to its environment impact the development, evolution, and spread of Kushneria konosiri. However, increased global production of plastic products has had a negative impact in marine environments and thus could indirectly harm this bacterium. Recently, Konosirus punctatus have been shown to have high levels of microplastic pollution within their gut as a result of increased plastic pollution within their ecosystem (17). While the effects of increased microplastic contamination on food product microbial environments has not been studied extensively, previous studies have indicated adverse effects on normal physiological processes within marine microorganisms due to microplastic exposure (18). In the future, microplastics could have similar harmful effects on organisms present within seafood-based products as it has been proposed that microplastics can have bioaccumulation effects within the food chain (19).
Ecological Lifestyle and Interactions
Kushneria konosiri is a free-living bacterium within its environment (2). It is also an obligate aerobe and a heterotroph. While the specific lifestyle and interactions of this bacterium were not studied extensively, Kushneria konosiri likely interacts with a very wide variety of microorganisms as traditionally there is not much purification done with the fish products in jeotgal. Along with this, as with most multicellular organisms, a wide variety of pathogens and other microorganisms are found to coexist within the gut and surrounding tissues of the dotted gizzard shad. However, after the addition of solar salt to the fish many of those microorganisms likely died due to increased osmotic pressure. For the most part, we can assume that Kushneria konosiri interacts with halotolerant/halophilic microorganisms. The particular microbial population for the specific environment that Kushneria konosiri was isolated from, daemi-jeot, has not been studied extensively. However, many other types of jeotgal have been studied and allow us to make assumptions about that environment. Generally, through the process of fermentation in jeotgal products succession evens occur within the environment (8). This is likely due to the changes in salinity and pH during fermentation. Bacteria are not believed to be major contributors to proteolysis within jeotgal, but many are known to be the driving forces for the ripening, distinct flavoring, and for the decrease in pathogenic microbes within the environment (8, 20). The most common microorganisms present in jeotgal products are Bacillus, Brevibacterium, Flavobacterium, Micrococcus, Pediococcus, Pseudomonas, and Staphylococcus (8).
Along with those bacteria, Halobacterium, and Halomonas are also common as they are resistant to high levels of salt (8). Lactobacillus species have been shown to have antimicrobial activity to pathogens (8). In a study monitoring a jeotgal product containing anchovy, a close relative to Konosirus punctas, during its fermentation process it was shown that as fermentation goes along there is a general decrease in the population of microbes within the sample. Along with that researchers found that during the middle stage of fermentation, Pediococcus and yeasts dominated and were assumed to be the main fermenters of the fish product (21). It has also been shown that a succession from Proteobacteria to Firmicutes is a common occurrence within jeotgal products (8). However, with that being said, there is an extremely wide diversity of microbes present within each distinct jeotgal product. The main seafood base and spices added to each are unique and likely are the main reason for that large diversity. While we can assume that some microbes are present and have general functions, it is also known that there are a large number of microbes that have not been identified yet that are present in jeotgal products (8). Thus, the exact microbial interactions and populations present in daemi-jeot require more extensive research in order to be fully understood.
Although the influence of this bacterium on the jeotgal environment is relatively unknown, Kushneria bacteria have been shown to have influence on the fermentation of other food products. Bacteria of this genus were shown to be a core microbe of a Chinese food product called Panxian ham (22). In a research study on Panxian ham, bacteria of the Kushneria genus were found to be associated with the production of organic acids such as succinic and malic acids. Succinic acid is important in producing sour and umami tastes in that ham product (22). Similar to jeotgal, Panxian ham was shown to have multiple succession events that occur during its fermentation process. Proteobacteria dominated the raw and post salting stages and ultimately Firmicutes ended up dominating the resting and ripening stages of this product. The population of Kushneria bacteria was found to be largely increased during the final stages of ripening for this product due to increased salt content (22). Due to the similarities in the stages of fermentation for these products, we can assume the Kushneria konosiri also grew in abundance during this period for daemi-jeot,
Significance to Humans
While the specific influence of Kushneria konosiri on society is generally unknown, the environment it was discovered in has a rich history surrounding it. As stated previously, daemi-jeot is a type of the food product known as jeotgal. This fermented food product was created thousands of years ago during the Chosun Dynasty and is a staple in the diets of many Koreans today (8). Similar to other fermented foods, it was created in an attempt to prolong the shelf life of perishable food products. Through the creation of jeotgal, people were able to prevent deterioration of highly perishable fish and shellfish. Along with a prevention of early spoilage, jeotgal is enjoyed by many due to its enhanced flavor profile that is brought about during its fermentation process. Although it typically has a high sodium content, jeotgal is also known to be of high nutritional value and can contribute to human health by helping our appetite, protecting the liver, aiding in digestion, and by giving us beneficial microbes (8). Some forms of jeotgal have also been shown to have anticancer, antioxidant, antidiabetics, and immune system boosting activities (8). Along with the health benefits of jeotgal, by researching and identifying novel microorganisms like Kushneria konosiri, we can have a better understanding of fermented food products. This will allow us to better regulate the fermentation process to make healthier, higher quality, and better tasting products in the future.
Not much is known about the influence that Kushneria konosiri or other members of its genus have had on human society. This is likely due to the relatively recent classification and isolation of this bacterium and its genus. Kushneria konosiri in particular was proposed as a novel species roughly 4 years ago and its genome was sequenced only 3 years ago. Although research has not specifically been done on its practical applications, genome sequencing of Kushneria konosiri has revealed a possibility for development in biotechnological applications surrounding hypersaline tolerance (2). Previously, applications of halophilic microbes have been shown for both industrial and environmental uses. For example, some halophilic microbes and their enzymes are used to catalyze processes in high salt environments. The production of certain organic compounds by halophilic microbes can be useful in the biotechnology industry (23). Kushneria konosiri could have commercial potential as it is known to possess ectoine biosynthesis-related genes (2). Ectoine is a commonly used osmotic solute that can protect unstable enzymes and act as a molecular chaperone. It has also been used within the cosmetics industry as an additive to moisturizers to help those suffering from aged, dry, or irritated skin (23). Ecotine has also been shown to protect the skin from the effects of UVA radiation (24) so a wide variety of applications could be developed through a better understanding of its biosynthesis process.
Cell Structure, Metabolism & Life Cycle
Morphology and optimum growth conditions
Kushneria konosiri is a Gram-negative, oval or rod-shaped bacterium with cells that typically have a size of 0.5-1.0 X 1.2-3.2 μm (3). Additionally, this bacterium has a single polar flagellum which attaches to the cell on either end by anchoring into the double membrane of the cell envelope via its basal body structure (16). The presence of the flagellum within this bacterium is likely useful for motility in its wet environment (16). From a macroscopic view, Kushneria konosiri forms orange circular colonies when grown on marine agar medium (2). While Kushneria konosiri has the same general characteristics as other species within its genus, it was differentiated from other species via genetic, phylogenetic and biochemical analysis (3). Its combination of physiological and biochemical characteristics differ from members of its genus along with its major fatty acids (3). These differences were likely acquired as an adaptation to the fermented fish environment Kushneria konosiri developed in. In terms of growth, Kushneria konosiri can grow in a wide range of conditions that likely assist in its survival in the dynamic environment it is a part of. It is able to grow in temperatures between 10-37 degrees Celsius with optimum growth occurring from 15-25 degrees Celsius (3). This wider range of temperatures is essential in the market environment that this microorganism is exposed to.
There is not a distinct temperature at which daemi-jeot is stored or prepared so temperatures can vary among samples. Additionally, it can survive in a pH range of 4.5-8.5 with optimum growth at pH 5.0-7.0. The ability to survive within a wide range of pH is especially important for Kushneria konosiri. The process of fermentation brings about a rapid change in pH within the environment and it is essential that the microorganisms present in this environment are able to adapt to these changes. Finally, and perhaps most interestingly for this bacterium, it is able to grow in NaCl concentrations of 0-26% w/v with optimum growth between 11-19% (3). This high salt tolerance is not only necessary for survival but also a defining characteristic for this bacterium and members of its genus. Optimal growth pertaining to the abiotic factors of UV and oxygen concentration were not measured for this bacterium. However, the presence of carotenoids within this bacterium likely allows it to have some levels of resistance to UV radiation (25).
Antibiotic resistance and metabolic abilities
Antibiotic resistance information for this particular bacterium is not mentioned in the literature describing it or within The Comprehensive Antibiotic Resistance Database (CARD). While antibiotic resistance information is not currently available for this bacterium, other members of its genus were found to be susceptible to certain antibiotics such as penicillin, chloramphenicol, vancomycin, novobiocin, polymyxin B, sulfamethazole/trimethoprim, and rifampicin (5). As these species are in the same genus, we can infer that there is a somewhat similar resistance profile with Kushneria konosiri, but due to its cell structure and unknown inhibition methods this may be different. It is unknown how well the resistance of this bacterium would be to chloramphenicol a, but we can assume that if the drug was able to enter this bacterium it would be susceptible to it as this antibiotic targets the 50S subunit of the ribosome (26). Rifampicin would likely not have a bactericidal effect on this bacteria but rather a bacteriostatic effect (26). Sulfamethazole/trimethoprim and polymyxin B would likely be able to attack this bacterium as these antibiotics are effective against many Gram-negative bacteria (27, 28). In contrast to the other member of its genus, we may not see this bacterium to be resistant to penicillin as it is usually limited in effectiveness on Gram-negative bacteria(29). This is the same case with vancomycin and novobiocin as these antibiotics are typically effective only against Gram-positive bacteria (26, 30).
In terms of metabolism, the exact sources of carbon from the environment that Kushneria konosiri uses for growth are unknown and more research must be done in order to understand its overall metabolic processes. While it is unknown, based on the information known about this bacterium and its environment it is likely that the carbon sources are sugars present from the remaining food left the stomach and intestines of the dotted gizzard shad, the remains of dead microorganisms that had shriveled up due to increased salt concentrations, and possibly the broken-down muscle and other tissues produced during the fermentation process of daemi-jeot. Being a heterotroph, Kushneria konosiri finds its sources of energy from its surrounding environment. Though much is not known about its exact metabolism, this bacterium is known to be able to use various molecules such as D-Glucose, L-arabinose, D-mannose, maltose, potassium gluconate, malate and trisodium citrate as substrates. It is also able to hydrolyze aesculin to produce glucose and esculetin (3). Along with that, Kushneria konosiri contains numerous enzymes (alkaline phosphatase, acid phosphatase, naphthol-AS- BI-phosphohydrolase) that catalyze the hydrolysis of organic phosphate molecules in its environment (3).
Genome Structure, Content, and/or Gene Expression
The genome of Kushneria konosiri is has a length of 3,584,631 base pairs with a 59.1% GC content. Its genome consists of a single circular chromosome. Of its 3347 genes, the majority are used for protein coding while the rest are RNA genes (2). Researchers found that 6.04% of genes are responsible for energy production and conversion, 1.66% are for defense mechanisms, 5.98% are for the biogenesis of the cell envelope. Along with that, a large portion of genes are dedicated to the transport and metabolism of various molecules such as amino acids and carbohydrates (2).
This bacterium was sequenced due to its ability to survive and adapt within hypersaline environments. It was also chosen as a part of the Agricultural Microbiome R&D program at the Korea Institute of Planning and Evaluation for Technology in Food, Agriculture, Forestry (2). A greater understanding for tolerance within this type of environment will allow for future research and development of various practical applications within the biotechnology industry. The sequencing of this genome highlights the mechanisms in which Kushneria konosiri uses to have high resistance to hypersaline environments. It is able to adapt by using functional genes. These genes allow it to develop mechanisms that increase internal osmolarity using inorganic ions and organic compounds (2). While the exact specifics of these mechanisms will be discussed in a later section, this bacterium encodes potassium uptake-related loci such as the Trk system, KUP ststem and Kdp two- component system. In addition to ion regulation, the genome also encodes ectioine and betaine biosynthesis related genes along with betaine uptake-related genes and a glycerol uptake facilitator protein (2). Its genome also encodes genes that can be used to synthesize carotenoids that act as antioxidants and additional supports to increase tolerance of high salt concentrations (2).
As discussed previously, the main feature that sets this bacterium apart from others is its hypersaline tolerance. This is similar to other halophilic microorganisms that live in Hypersaline Lakes and the Dead Sea. These microorganisms are fascinating due to their ability to withstand high osmotic pressure resulting from high concentrations of salt in their extracellular environment. In response to high osmotic pressure on a microorganism, most face potentially fatal stressors. With the process osmosis, when there is a higher ion concentration in an extracellular environment, water tends to diffuse out of a cell to reach equilibrium between the external and internal environment. When there is high osmotic pressure on microbes what essentially occurs is a decreased availability of water to cells. Without water many end up shriving up, drying out and thus dying due to a lack of available water. Another important factor for most prokaryotes is their ability to have a high turgor pressure to maintain shape and to expand cell walls during growth. High osmotic pressure reduces an organisms turgor pressure by decreasing the osmotic pressure difference between the intracellular and extracellular environment (16). While most microorganisms face fatal consequences to the stressor of high osmotic pressure, Kushneria konosiri and other halophilic microorganisms are able to withstand this pressure through internal systems of regulation. In order to maintain their high turgor pressure, halophiles will increase the concentration of solutes within the cytoplasm by either pumping them in through transport systems or by synthesizing them within the cytoplasm (16).
Though it is not completely understood, genome analysis of Kushneria konosiri shows several possible mechanisms that uniquely allow it to regulate its internal osmolarity. In Kushneria konosiri, the major solute that increases in concentration in response to high osmotic pressure is potassium. When this bacterium faces high osmotic pressure, potassium ions are pumped into the cytoplasm by the Trk-type transport systems (31), the KUP system (32) or the Kdp two-component system (33). Trk systems are low-affinity and high-capacity so they allow for potassium uptake in the environment when potassium concentration is high (31). When potassium ion concentration is low, the high-affinity Kdp two-component system may be used (33). The KUP system has medium levels of affinity and is able to function better than the Trk systems at low pH levels (32). As potassium levels within the cell increase in response to high osmotic pressure, a surplus of these ions in the cytoplasm may cause some damage to the cell. Therefore, Kushneria konosiri also requires osmoprotective compounds to withstand this stress. Within the genome of this bacterium there are two organic molecules that likely act as osmoprotectants: ectoine and betaine. They may also in some cases act as compatible solutes as well (2). In order to accumulate these molecules into the cytoplasm, Kushneria konosiri encodes genes for the biosynthesis pathways of ectoine and betaine. It also encodes genes for the uptake of betaine molecules (2). Additionally, there is the presence of a carotenoid biosynthesis pathway within this genome. Carotenoids are pigments that can act as antioxidants and can help cells tolerate salt stress. With the presence of these pigments in the bacterium, it could be hypothesized that they may act as osmoprotectants as well (2). It is important to keep in mind however that these molecular mechanisms are not completely understood yet. This is a relatively new bacterium, and its internal mechanisms will require additional research to become confirmed. Additional research on this bacterium should be conducted in the near future. Although these aspects are speculative at the moment, they can quite possibly provide great insight into the overall molecular systems needed for hypersaline tolerance. This could be useful in future applications within multiple industries. This microorganism is yet another example of the amazing things that bacteria are able to do for us.
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