Planetary Protection Knowledge Gap Closure Enabling Crewed Missions to Mars.

As focus for exploration of Mars transitions from current robotic explorers to development of crewed missions, it remains important to protect the integrity of scientific investigations at Mars, as well as protect the Earth's biosphere from any potential harmful effects from returned martian material. This is the discipline of planetary protection, and the Committee on Space Research (COSPAR) maintains the consensus international policy and guidelines on how this is implemented. Based on National Aeronautics and Space Administration (NASA) and European Space Agency (ESA) studies that began in 2001, COSPAR adopted principles and guidelines for human missions to Mars in 2008. At that point, it was clear that to move from those qualitative provisions, a great deal of work and interaction with spacecraft designers would be necessary to generate meaningful quantitative recommendations that could embody the intent of the Outer Space Treaty (Article IX) in the design of such missions. Beginning in 2016, COSPAR then sponsored a multiyear interdisciplinary meeting series to address planetary protection "knowledge gaps" (KGs) with the intent of adapting and extending the current robotic mission-focused Planetary Protection Policy to support the design and implementation of crewed and hybrid exploration missions. This article describes the outcome of the interdisciplinary COSPAR meeting series, to describe and address these KGs, as well as identify potential paths to gap closure. It includes the background scientific basis for each topic area and knowledge updates since the meeting series ended. In particular, credible solutions for KG closure are described for the three topic areas of (1) microbial monitoring of spacecraft and crew health; (2) natural transport (and survival) of terrestrial microbial contamination at Mars, and (3) the technology and operation of spacecraft systems for contamination control. The article includes a KG data table on these topic areas, which is intended to be a point of departure for making future progress in developing an end-to-end planetary protection requirements implementation solution for a crewed mission to Mars. Overall, the workshop series has provided evidence of the feasibility of planetary protection implementation for a crewed Mars mission, given (1) the establishment of needed zoning, emission, transport, and survival parameters for terrestrial biological contamination and (2) the creation of an accepted risk-based compliance approach for adoption by spacefaring actors including national space agencies and commercial/nongovernment organizations.

Nutrients mediate caffeine inhibition of Escherichia coli.

The consumption of coffee and other caffeinated drinks is increasingly popular across the globe. In the United States, 90% of adults consume at least one caffeinated beverage a day. While caffeine consumption of up to 400 mg/d is not generally associated with negative effects on human health, the impact of caffeine on the gut microbiome and individual gut microbiota remains unclear. We examined the effect of caffeine on the growth rate of Escherichia coli, a bacterium commonly found in the human gut, when grown aerobically or anaerobically in nutrient-rich or minimal medium. A significant negative correlation was observed between caffeine concentration and growth rate under all conditions, suggesting that caffeine can act as an antimicrobial agent when ingested. Caffeine reduced growth rates significantly more in nutrient-poor, but not in anoxic, conditions. Given the highly variable nutrient and oxygen conditions of the gut, these results suggest a need to further explore caffeine's inhibitory effects on the gut microbiome and its relation to human health.

Report of the Joint Workshop on Induced Special Regions.

The Joint Workshop on Induced Special Regions convened scientists and planetary protection experts to assess the potential of inducing special regions through lander or rover activity. An Induced Special Region is defined as a place where the presence of the spacecraft could induce water activity and temperature to be sufficiently high and persist for long enough to plausibly harbor life. The questions the workshop participants addressed were: (1) What is a safe stand-off distance, or formula to derive a safe distance, to a purported special region? (2) Questions about RTGs (Radioisotope Thermoelectric Generator), other heat sources, and their ability to induce special regions. (3) Is it possible to have an infected area on Mars that does not contaminate the rest of Mars? The workshop participants reached a general consensus addressing the posed questions, in summary: (1) While a spacecraft on the surface of Mars may not be able to explore a special region during the prime mission, the safe stand-off distance would decrease with time because the sterilizing environment, that is the martian surface would progressively clean the exposed surfaces. However, the analysis supporting such an exploration should ensure that the risk to exposing interior portions of the spacecraft (i.e., essentially unsterilized) to the martian surface is minimized. (2) An RTG at the surface of Mars would not create a Special Region but the short-term result depends on kinetics of melting, freezing, deliquescence, and desiccation. While a buried RTG could induce a Special Region, it would not pose a long-term contamination threat to Mars, with the possible exception of a migrating RTG in an icy deposit. (3) Induced Special Regions can allow microbial replication to occur (by definition), but such replication at the surface is unlikely to globally contaminate Mars. An induced subsurface Special Region would be isolated and microbial transport away from subsurface site is highly improbable.

Adaptations to marine versus terrestrial low temperature environments as revealed by comparative genomic analyses of the genus Psychrobacter.

While cold-adapted bacteria isolated from marine or terrestrial low temperature environments share many similarities, cold-adapted bacteria from terrestrial environments usually grow over a broader range of temperatures suggesting different constraints of these two low temperature environments. The diversity of habitats from which Psychrobacter have been isolated (e.g. cold marine environments, frozen soils, permafrost and humans) provides a unique opportunity to examine habitat specific adaptations while reducing phylogenetic effects. Here, comparative genomic analyses of 26 strains of Psychrobacter revealed several clusters with characteristics that correlated with habitat. Marine and terrestrial Psychrobacter have amino acid composition typical of psychrophiles (e.g. fewer proline and lysine, more acidic) when compared to Psychrobacter strains associated with warm hosts, and have many potentially cold-adapted core genes (e.g. ClpX, DsbC, GroEL/GroES and MutS2). Marine and terrestrial Psychrobacter share many genes (e.g. FadB) not found in warm host Psychrobacter, which had their own distinct gene content (e.g. collagenase-like protease). Furthermore, terrestrial Psychrobacter were differentiated from marine Psychrobacter by the use of different cold adaptations and more hydrophobic and aliphatic proteins. These data suggest that terrestrial and marine Psychrobacter evolved from a mesophilic ancestor and are accumulating adaptations for low temperatures as well as for their respective habitats.

Conserved genomic and amino acid traits of cold adaptation in subzero-growing Arctic permafrost bacteria.

Permafrost accounts for 27% of all soil ecosystems and harbors diverse microbial communities. Our understanding of microorganisms in permafrost, their activities and adaptations, remains limited. Using five subzero-growing (cryophilic) permafrost bacteria, we examined features of cold adaptation through comparative genomic analyses with mesophilic relatives. The cryophiles possess genes associated with cold adaptation, including cold shock proteins, RNA helicases, and oxidative stress and carotenoid synthesis enzymes. Higher abundances of genes associated with compatible solutes were observed, important for osmoregulation in permafrost brine veins. Most cryophiles in our study have higher transposase copy numbers than mesophiles. We investigated amino acid (AA) modifications in the cryophiles favoring increased protein flexibility at cold temperatures. Although overall there were few differences with the mesophiles, we found evidence of cold adaptation, with significant differences in proline, serine, glycine and aromaticity, in several cryophiles. The use of cold/hot AA ratios of >1, used in previous studies to indicate cold adaptation, was found to be inadequate on its own. Comparing the average of all cryophiles to all mesophiles, we found that overall cryophiles had a higher ratio of cold adapted proteins for serine (more serine), and to a lesser extent, proline and acidic residues (fewer prolines/acidic residues).

Improved High-Quality Draft Genome Sequence of the Eurypsychrophile Rhodotorula sp. JG1b, Isolated from Permafrost in the Hyperarid Upper-Elevation McMurdo Dry Valleys, Antarctica.

Here, we report the draft genome sequence of Rhodotorula sp. strain JG1b, a yeast that was isolated from ice-cemented permafrost in the upper-elevation McMurdo Dry Valleys, Antarctica. The sequenced genome size is 19.39 Mb, consisting of 156 scaffolds and containing a total of 5,625 predicted genes. This is the first known cold-adapted Rhodotorula sp. sequenced to date.

Cold adaptive traits revealed by comparative genomic analysis of the eurypsychrophile Rhodococcus sp. JG3 isolated from high elevation McMurdo Dry Valley permafrost, Antarctica.

The permafrost soils of the high elevation McMurdo Dry Valleys are the most cold, desiccating and oligotrophic on Earth. Rhodococcus sp. JG3 is one of very few bacterial isolates from Antarctic Dry Valley permafrost, and displays subzero growth down to -5°C. To understand how Rhodococcus sp. JG3 is able to survive extreme permafrost conditions and be metabolically active at subzero temperatures, we sequenced its genome and compared it to the genomes of 14 mesophilic rhodococci. Rhodococcus sp. JG3 possessed a higher copy number of genes for general stress response, UV protection and protection from cold shock, osmotic stress and oxidative stress. We characterized genome wide molecular adaptations to cold, and identified genes that had amino acid compositions favourable for increased flexibility and functionality at low temperatures. Rhodococcus sp. JG3 possesses multiple complimentary strategies which may enable its survival in some of the harshest permafrost on Earth.

Improved-high-quality draft genome sequence of Rhodococcus sp. JG-3, a eurypsychrophilic Actinobacteria from Antarctic Dry Valley permafrost.

The actinobacterium Rhodococcus sp. JG-3 is an aerobic, eurypsychrophilic, soil bacterium isolated from permafrost in the hyper arid Upper Dry Valleys of Antarctica. It is yellow pigmented, gram positive, moderately halotolerant and capable of growth from 30 °C down to at least -5 °C. The 5.28 Mb high-quality-draft genome is arranged into 6 scaffolds, containing 9 contigs and 4998 protein coding genes, with 64 % GC content. Increasing the availability of genome sequences from cold-adapted species is crucial to gaining a better understanding of the molecular traits of cold adaptation in microbes.

Genome Sequence of Methanosarcina soligelidi SMA-21, Isolated from Siberian Permafrost-Affected Soil.

Here, we announce the genome sequence of Methanosarcina soligelidi SMA-21, an anaerobic methanogenic archaeon that was previously isolated from Siberian permafrost-affected soil. The sequencing of strain SMA-21 yielded a 4.06-Mb genome with 41.5% G+C content, containing a total of 2,647 open reading frames.

A new analysis of Mars "Special Regions": findings of the second MEPAG Special Regions Science Analysis Group (SR-SAG2).

A committee of the Mars Exploration Program Analysis Group (MEPAG) has reviewed and updated the description of Special Regions on Mars as places where terrestrial organisms might replicate (per the COSPAR Planetary Protection Policy). This review and update was conducted by an international team (SR-SAG2) drawn from both the biological science and Mars exploration communities, focused on understanding when and where Special Regions could occur. The study applied recently available data about martian environments and about terrestrial organisms, building on a previous analysis of Mars Special Regions (2006) undertaken by a similar team. Since then, a new body of highly relevant information has been generated from the Mars Reconnaissance Orbiter (launched in 2005) and Phoenix (2007) and data from Mars Express and the twin Mars Exploration Rovers (all 2003). Results have also been gleaned from the Mars Science Laboratory (launched in 2011). In addition to Mars data, there is a considerable body of new data regarding the known environmental limits to life on Earth-including the potential for terrestrial microbial life to survive and replicate under martian environmental conditions. The SR-SAG2 analysis has included an examination of new Mars models relevant to natural environmental variation in water activity and temperature; a review and reconsideration of the current parameters used to define Special Regions; and updated maps and descriptions of the martian environments recommended for treatment as "Uncertain" or "Special" as natural features or those potentially formed by the influence of future landed spacecraft. Significant changes in our knowledge of the capabilities of terrestrial organisms and the existence of possibly habitable martian environments have led to a new appreciation of where Mars Special Regions may be identified and protected. The SR-SAG also considered the impact of Special Regions on potential future human missions to Mars, both as locations of potential resources and as places that should not be inadvertently contaminated by human activity.

Molecular characterization of bacteria from permafrost of the Taylor Valley, Antarctica.

While bacterial communities from McMurdo Dry Valley soils have been studied using molecular techniques, data from permafrost are particularly scarce given the logistical difficulties of sampling. This study examined the molecular diversity and culturability of bacteria in permafrost from the Taylor Valley (TV), Antarctica. A 16S rRNA gene clone library was constructed to assess bacterial diversity, while a clone library of the RNA polymerase beta subunit (rpoB) gene was constructed to examine amino acid composition of an essential protein-coding gene. The 16S rRNA gene clone library was dominated by Acidobacteria from Gp6 and Gemmatimonadetes. The rpoB gene clone library (created with primers designed in this study) was also dominated by Acidobacteria. The ability of sequence analyses to garner additional information about organisms represented by TV sequences was explored. Specifically, optimum growth temperature was estimated from the stem GC content of the 16S rRNA gene, while potential cold adaptations within translated rpoB sequences were assessed. These analyses were benchmarked using known psychrophiles and mesophiles. Bioinformatic analyses suggested that many TV sequences could represent organisms capable of activity at low temperatures. Plate counts confirmed that c. 10(3) cells per gram permafrost remained viable and were culturable, while laboratory respiration assays demonstrated that microbial activity occurred at -5 °C and peaked at 15 °C.

Microbial metabolism in ice and brine at -5°C.

Metabolic activity, but not growth, has been observed in ice at temperatures from -5°C to -32°C. To improve understanding of metabolism in ice, we simultaneously examined various aspects of metabolism ((14) C-acetate utilization, macromolecule syntheses and viability via reduction of CTC) of the glacial isolates Sporosarcina sp. B5 and Chryseobacterium sp. V3519-10 during incubation in nutrient-rich ice and brine at -5°C for 50 days. Measured rates of acetate utilization and macromolecule syntheses were high in the first 20 days suggesting adjustment to the lower temperatures and higher salt concentrations of both the liquid vein network in the ice and the brine. Following this adjustment, reproductive growth of both organisms was evident in brine, and suggested for Sporosarcina sp. B5 in ice by increases in cell numbers and biomass. Chryseobacterium sp. V3519-10 cells incubated in ice remained active. These data indicate that neither low temperature nor high salt concentrations prohibit growth in ice, but some other aspect of living within ice slows growth to within the detection limits of current methodologies. These results imply that microbial growth is plausible in natural ice systems with comparable temperatures and sufficient nutrients, such as debris-rich basal ices of glaciers and ice masses.

Microbial respiration in ice at subzero temperatures (-4°C to -33°C).

The habitability of icy environments may be limited by low temperature, low nutrient concentrations, high solute concentrations and the physical ice matrix. The basal ice of ice sheets and glaciers contains sediments that may be a source of nutrients for microbial activity. Here we quantify microbial respiration and active cell populations of Antarctic glacial isolates Paenisporosarcina sp. B5 and Chryseobacterium sp. V3519-10 in laboratory ices with abundant nutrients at temperatures from -4°C to -33°C. At all temperatures, initial high rates of metabolism were followed by lower rates suggestive of a non-reproductive metabolic state such as maintenance or dormancy. Metabolism was sustained by viable cells as quantified via culturability, CTC reduction and LIVE/DEAD staining. Respiration rates based on active cell populations did not correspond to rates representative of reproductive growth from the literature, but suggested lower levels of metabolism. Our data demonstrated that bacteria actively respired acetate in polycrystalline ice with abundant nutrients despite low temperatures and the physical ice matrix. Our results suggest that the debris-rich basal ice that exists at temperatures just below the freezing point and underlies portions of both the Greenland and Antarctic ice sheets represents a significant potential habitat for metabolically active microbial communities.

Characterization of a bacterial community from a Northeast Siberian seacoast permafrost sample.

A combination of culture-dependent and -independent techniques was used to characterize a bacterial community, examine cold adaptation of isocitrate lyase (icl) genes, and detect genes with important ecological functions in a permafrost sample from the Bykovsky Peninsula on the Laptev Sea coast of northeast Siberia. According to the 16S rRNA gene sequence, 47 of the cultured isolates were members of the phyla Firmicutes, Proteobacteria, and Actinobacteria, with 85% of the isolates belonging to the genera Arthrobacter and Planococcus. The 16S rRNA gene clone library derived from DNA from the same permafrost sample contained sequences from the same phyla plus a few from Acidobacteria, but favored the Firmicutes at the cost of the Actinobacteria. A partial sequence of the icl gene, a proposed marker for cold adaptation, was determined for 25 isolates that grew at 0 °C. Two Psychrobacter isolates contained two of the four residues shown to be important for low-temperature activity in Colwellia maris or Colwellia psychrerythreaea. The presence in the permafrost DNA of genes with ecosystem functions was determined using geochip 2.0. The highest number of genes identified was from the categories of aromatic and natural polymer degradation genes, perhaps reflecting selection for the use of tundra vegetation-produced carbon.

The genome sequence of Psychrobacter arcticus 273-4, a psychroactive Siberian permafrost bacterium, reveals mechanisms for adaptation to low-temperature growth.

Psychrobacter arcticus strain 273-4, which grows at temperatures as low as -10 degrees C, is the first cold-adapted bacterium from a terrestrial environment whose genome was sequenced. Analysis of the 2.65-Mb genome suggested that some of the strategies employed by P. arcticus 273-4 for survival under cold and stress conditions are changes in membrane composition, synthesis of cold shock proteins, and the use of acetate as an energy source. Comparative genome analysis indicated that in a significant portion of the P. arcticus proteome there is reduced use of the acidic amino acids and proline and arginine, which is consistent with increased protein flexibility at low temperatures. Differential amino acid usage occurred in all gene categories, but it was more common in gene categories essential for cell growth and reproduction, suggesting that P. arcticus evolved to grow at low temperatures. Amino acid adaptations and the gene content likely evolved in response to the long-term freezing temperatures (-10 degrees C to -12 degrees C) of the Kolyma (Siberia) permafrost soil from which this strain was isolated. Intracellular water likely does not freeze at these in situ temperatures, which allows P. arcticus to live at subzero temperatures.

Methanogenesis in subglacial sediments.

Methanogenic archaea have a unique role in Earth's global carbon cycle as producers of the greenhouse gas methane (CH4 ). However, despite the fact that ice covers 11% of Earth's continental landmass, evidence for methanogenic activity in subglacial environments has yet to be clearly demonstrated. Here we present genetic, biochemical and geochemical evidence indicative of an active population of methanogens associated with subglacial sediments from Robertson Glacier (RG), Canadian Rockies. Porewater CH4 was quantified in two subglacial sediment cores at concentrations of 16 and 29 ppmv. Coenzyme M (CoM), a metabolic biomarker for methanogens, was detected at a concentration of 1.3 nmol g sediment(-1) corresponding to ∼3 × 10(3) active cells g sediment(-1) . Genetic characterization of communities associated with subglacial sediments indicated the presence of several archaeal 16S rRNA and methyl CoM reductase subunit A (mcrA) gene phylotypes, all of which were affiliated with the euryarchaeal order Methanosarcinales. Further, CH4 was produced at 9-51 fmol g dry weight sediment(-1) h(-1) in enrichment cultures of RG sediments incubated at 4°C. Collectively, these findings have important implications for the global carbon cycle in light of recent estimates indicating that the Earth's subglacial biome ranges from 10(4) to 10(6) km(3) sediment.

Microbial communities in subpermafrost saline fracture water at the Lupin Au mine, Nunavut, Canada.

We report the first investigation of a deep subpermafrost microbial ecosystem, a terrestrial analog for the Martian subsurface. Our multidisciplinary team analyzed fracture water collected at 890 and 1,130 m depths beneath a 540-m-thick permafrost layer at the Lupin Au mine (Nunavut, Canada). 14C, 3H, and noble gas isotope analyses suggest that the Na-Ca-Cl, suboxic, fracture water represents a mixture of geologically ancient brine, approximately25-kyr-old, meteoric water and a minor modern talik-water component. Microbial planktonic concentrations were approximately10(3) cells mL(-1). Analysis of the 16S rRNA gene from extracted DNA and enrichment cultures revealed 42 unique operational taxonomic units in 11 genera with Desulfosporosinus, Halothiobacillus, and Pseudomonas representing the most prominent phylotypes and failed to detect Archaea. The abundance of terminally branched and midchain-branched saturated fatty acids (5 to 15 mol%) was consistent with the abundance of Gram-positive bacteria in the clone libraries. Geochemical data, the ubiquinone (UQ) abundance (3 to 11 mol%), and the presence of both aerobic and anaerobic bacteria indicated that the environment was suboxic, not anoxic. Stable sulfur isotope analyses of the fracture water detected the presence of microbial sulfate reduction, and analyses of the vein-filling pyrite indicated that it was in isotopic equilibrium with the dissolved sulfide. Free energy calculations revealed that sulfate reduction and sulfide oxidation via denitrification and not methanogenesis were the most thermodynamically viable consistent with the principal metabolisms inferred from the 16S rRNA community composition and with CH4 isotopic compositions. The sulfate-reducing bacteria most likely colonized the subsurface during the Pleistocene or earlier, whereas aerobic bacteria may have entered the fracture water networks either during deglaciation prior to permafrost formation 9,000 years ago or from the nearby talik through the hydrologic gradient created during mine dewatering. Although the absence of methanogens from this subsurface ecosystem is somewhat surprising, it may be attributable to an energy bottleneck that restricts their migration from surface permafrost deposits where they are frequently reported. These results have implications for the biological origin of CH4 on Mars.

Survivability of Psychrobacter cryohalolentis K5 under simulated martian surface conditions.

Spacecraft launched to Mars can retain viable terrestrial microorganisms on board that may survive the interplanetary transit. Such biota might compromise the search for life beyond Earth if capable of propagating on Mars. The current study explored the survivability of Psychrobacter cryohalolentis K5, a psychrotolerant microorganism obtained from a Siberian permafrost cryopeg, under simulated martian surface conditions of high ultraviolet irradiation, high desiccation, low temperature, and low atmospheric pressure. First, a desiccation experiment compared the survival of P. cryohalolentis cells embedded, or not embedded, within a medium/salt matrix (MSM) maintained at 25 degrees C for 24 h within a laminar flow hood. Results indicate that the presence of the MSM enhanced survival of the bacterial cells by 1 to 3 orders of magnitude. Second, tests were conducted in a Mars Simulation Chamber to determine the UV tolerance of the microorganism. No viable vegetative cells of P. cryohalolentis were detected after 8 h of exposure to Mars-normal conditions of 4.55 W/m(2) UVC irradiation (200-280 nm), -12.5 degrees C, 7.1 mbar, and a Mars gas mix composed of CO(2) (95.3%), N(2) (2.7%), Ar (1.6%), O(2) (0.2%), and H(2)O (0.03%). Third, an experiment was conducted within the Mars chamber in which total atmospheric opacities were simulated at tau = 0.1 (dust-free CO(2) atmosphere at 7.1 mbar), 0.5 (normal clear sky with 0.4 = dust opacity and 0.1 = CO(2)-only opacity), and 3.5 (global dust storm) to determine the survivability of P. cryohalolentis to partially shielded UVC radiation. The survivability of the bacterium increased with the level of UVC attenuation, though population levels still declined several orders of magnitude compared to UVC-absent controls over an 8 h exposure period.

Psychrobacter arcticus 273-4 uses resource efficiency and molecular motion adaptations for subzero temperature growth.

Permafrost soils are extreme environments that exert low-temperature, desiccation, and starvation stress on bacteria over thousands to millions of years. To understand how Psychrobacter arcticus 273-4 survived for >20,000 years in permafrost, transcriptome analysis was performed during growth at 22 degrees C, 17 degrees C, 0 degrees C, and -6 degrees C using a mixed-effects analysis of variance model. Genes for transcription, translation, energy production, and most biosynthetic pathways were downregulated at low temperatures. Evidence of isozyme exchange was detected over temperature for D-alanyl-D-alanine carboxypeptidases (dac1 and dac2), DEAD-box RNA helicases (csdA and Psyc_0943), and energy-efficient substrate incorporation pathways for ammonium and acetate. Specific functions were compensated by upregulation of genes at low temperature, including genes for the biosynthesis of proline, tryptophan, and methionine. RNases and peptidases were generally upregulated at low temperatures. Changes in energy metabolism, amino acid metabolism, and RNase gene expression were consistent with induction of a resource efficiency response. In contrast to results observed for other psychrophiles and mesophiles, only clpB and hsp33 were upregulated at low temperature, and there was no upregulation of other chaperones and peptidyl-prolyl isomerases. relA, csdA, and dac2 knockout mutants grew more slowly at low temperature, but a dac1 mutant grew more slowly at 17 degrees C. The combined data suggest that the basal biological machinery, including translation, transcription, and energy metabolism, is well adapted to function across the growth range of P. arcticus from -6 degrees C to 22 degrees C, and temperature compensation by gene expression was employed to address specific challenges to low-temperature growth.

Development and use of genetic system to identify genes required for efficient low-temperature growth of Psychrobacter arcticus 273-4.

We describe the development of genetic tools (electroporation, conjugation, vector for targeted gene replacement) for use in the psychrophile Psychrobacter arcticus 273-4 to test hypotheses about cold adaptation. Successful electroporation only occurred with nonstandard parameters, such as: electrocompetent cells freshly prepared from stationary-phase cultures, high field strengths (25 kV cm(-1)), long recovery times (16-24 h), and selection with low concentrations of antibiotics. Transformation frequencies were greatly affected by a methylation-dependent restriction barrier homologous to DpnI. The vector pJK100 (which was self-transmissible and contained a Pir-dependent R6K origin of replication) proved effective as a suicide plasmid that could be used to recombine mutations into the P. arcticus 273-4 genome. We used this vector for targeted replacement of dctT, the substrate-binding periplasmic subunit of a TRAP (tripartite ATP-independent periplasmic) transporter (which we have named dctTUF), as it was more highly expressed at cold temperatures. The replacement of dctT (with kan) decreased the rate of growth at low temperatures in mineral medium with glutamate, acetate, butyrate, and fumarate, but not with pyruvate suggesting that DctTUF participates in the transport of glutamate, acetate, butyrate, and fumarate at cold temperatures. This is the first report to demonstrate the creation of site-specific mutants in the genus Psychrobacter, their affect on low-temperature growth, and a substrate range for TAXI proteins of TRAP transporters.