Psychrobacter: Key Features of a Psychrotolerant Bacterium from a Kentucky Salt Spring
One of the sulfur-enriched mineral springs at Big Bone Lick State Park, Boone County, KY
One of the sulfur-enriched mineral springs at Big Bone Lick State Park, Boone County, KY (Labeled view)
Psychrobacter colony morphology on tryptic soy agar
Gram stain and cellular morphology of Psychrobacter
Expanded view of gram-stained Psychrobacter showing diplococci
Expanded view of gram-stained Psychrobacter showing diplococci (Labeled view)
Salt tolerance of Psychrobacter
Psychrobacter was first described by Juni & Heym (1986) as a collection of aerobic, psychrotolerant, Gram negative, oxidase and catalase positive isolates from fresh, frozen and irradiated food sources such as fish and meat. This group, which shows a wide range of halotolerance (2), has since expanded to include psychrotolerant and psychrophilic isolates from extreme environments such as Siberian permafrost, terrestrial and aquatic Antarctic environments, and Japanese sea ice (3,10,12). A variety of Psychrobacter species occur as occasional opportunistic pathogens such as P. sanguinis and P. phenylpyruvicus involved in bacteremia (7,13) , and P. immobilis associated with meningitis (9). A recent report also identified Psychrobacter arenosus in the blood transfusion unit and blood of the transfusion recipient (4).
The Psychrobacter isolate described here was cultured from a sulphidic salt spring in Big Bone Lick State Park (Fig. 1) with an average temperature of 16°C (range 10°-19°C, n=7). The objective of the study was to characterize halotolerant culturable isolates from the spring. Using 16S rRNA gene sequence data this isolate most closely matched Psychrobacter piscatorii from the wastewater of a Japanese fish processing plant (14) (Accession # AB453700, 99% identity, 1393 nts). The discovery of Psychrobacter was unexpected as Halobacillus and Halomonas dominate the culturable community of the spring.
On agar-based media, Psychrobacter colonies are typically non-pigmented, round, opaque and glossy (Fig. 2). This isolate grew from 2-33°C with optimal growth at 25°C. Psychrobacter are Gram-negative coccobacilli that often occur as pairs or diplococci (Fig. 3A, Fig. 3B). In a study of the biogeography of cold-adapted bacteria, Rodrigues et al. (2009) found that Psychrobacter was most abundant in samples from Antarctica and Siberian permafrost with minimal, patchy distribution in temperate and tropical samples. Environmental factors correlated with Psychrobacter abundance included pH, salinity, potassium and magnesium (11). This isolate was cultured from salt spring samples collected in February. The spring is shallow (13 cm at the deepest point) and its temperature fluctuates seasonally. Perhaps favorable spring chemistry along with cooler seasonal temperatures increased the abundance of Psychrobacter. It would be interesting to quantify Psychrobacter in the spring by real-time PCR to determine how seasonal changes affect abundance.
Figure 4 shows that the Psychrobacter isolate from Big Bone Lick State Park grows readily in artificial sea water broth supplemented with up to 10% NaCl but that growth decreases dramatically at or above 15% NaCl. This is consistent with other reports that describe Psychrobacter isolates tolerant to salt ranging from 0-12% (3).
The morphology and physiology of the isolate described here match defining characteristics of the genus Psychrobacter. While 16s rRNA gene sequence data point towards Psychrobacter piscatorii as a close relative, more detailed biochemical and genomic analysis should reveal how closely this Kentucky salt spring isolate matches Psychrobacter piscatorii.
Sampling and isolation: Water and sediment from the salt spring at Big Bone Lick State Park was collected in sterile containers, transported in a cooler to IU Southeast and then stored at 4°C. Samples were plated on marine agar within 2-3 days of collection and incubated at 25°C for 48-72 hours. Individual colonies were streaked to marine agar for isolation.
Colony morphology and salt tolerance: To observe colony morphology, Psychrobacter was streaked onto tryptic soy agar and grown overnight at 25°C. For salt tolerance, Psychrobacter was grown overnight in marine broth and then transferred to artificial sea water broth (8) supplemented with 0, 5, 10, 15, or 20% NaCl. Photography was done using a Nikon D300 camera with an AF-S Micro-Nikkor 105 mm lens.
Cell morphology: Gram staining was performed on overnight cultures of Psychrobacter grown in LB. Photomicrographs were taken using a Nikon Eclipse E200 microscope with an attached Nikon DS-Fil camera at 1000x total magnification under oil.
The genus Psychrobacter is home to a number of unique cold-adapted extremophiles, and these bacteria offer much for those interested in low temperature survival strategies. For example, the recently completed genome sequence of the Siberian permafrost isolate Psychrobacter articus 273-4 revealed a number of adaptations to cold environments including preferred use of acetate as a carbon source, changes in membrane structure, and differential use of amino acids to lower protein stability and thus increase function at low temperature (1).
Those interested in biotechnology and cold-adapted enzymes should also take note of Psychrobacter. The genome sequence of Psychrobacter sp. Strain G, which produces lipases at low temperature, was recently completed (5). Further characterization of the structure and regulation of Psychrobacter lipolytic enzymes may provide clues to engineering more stable low-temperature industrial enzymes.
Psychrobacter isolates of clinical importance provide an interesting area of study. What changes, if any, are necessary for usually cold-adapted, halotolerant bacteria to become opportunistic pathogens? Perhaps a discrete set of steps allow Psychrobacter to expand its temperature range (6). Do clinical Psychrobacter isolates harbor virulence factors not found in Psychrobacter of environmental origin? Further investigations of clinically-important Psychrobacter, especially at the genome level, may provide clues to these questions.
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