Edwin Minkley

Evidence that DNA helicase I and oriT site-specific nicking are both functions of the F TraI protein.

Site-specific and strand-specific nicking at the origin of transfer (oriT) of the F sex factor is the initial step in conjugal DNA metabolism. Then, DNA helicase I, the product of the traI gene, processively unwinds the plasmid from the nick site to generate the single strand of DNA that is transferred to the recipient. The nick at oriT is produced by the combined action of two Tra proteins, TraY and TraZ. The traZ gene was never precisely mapped, as no available point mutation uniquely affected TraZ-dependent oriT nicking. With several new mutations, we have demonstrated that TraZ activity is dependent upon traI DNA sequences. The simplest interpretation of this finding is that the F TraI protein is bifunctional, with DNA unwinding and site-specific DNA nicking activities.

Life in the Atacama: Searching for life with rovers (science overview)

[1] The Life in the Atacama project investigated the regional distribution of life and habitats in the Atacama Desert of Chile. We sought to create biogeologic maps through survey traverses across the desert using a rover carrying biologic and geologic instruments. Elements of our science approach were to: Perform ecological transects from the relatively wet coastal range to the arid core of the desert; use converging evidence from science instruments to reach conclusions about microbial abundance; and develop and test exploration strategies adapted to the search of scattered surface and shallow subsurface microbial oases. Understanding the ability of science teams to detect and characterize microbial life signatures remotely using a rover became central to the project. Traverses were accomplished using an autonomous rover in a method that is technologically relevant to Mars exploration. We present an overview of the results of the 2003, 2004, and 2005 field investigations. They include: The confirmed identification of microbial habitats in daylight by detecting fluorescence signals from chlorophyll and dye probes; the characterization of geology by imaging and spectral measurement; the mapping of life along transects; the characterization of environmental conditions; the development of mapping techniques including homogeneous biological scoring and predictive models of habitat location; the development of exploration strategies adapted to the search for life with an autonomous rover capable of up to 10 km of daily traverse; and the autonomous detection of life by the rover as it interprets observations on-the-fly and decides which targets to pursue with further analysis.

Robotic ecological mapping: Habitats and the search for life in the Atacama Desert

[1] As part of the three-year ‘Life in the Atacama’ (LITA) project, plant and microbial abundance were mapped within three sites in the Atacama Desert, Chile, using an automated robotic rover. On-board fluorescence imaging of six biological signatures (e.g., chlorophyll, DNA, proteins) was used to assess abundance, based on a percent positive sample rating system and standardized robotic ecological transects. The percent positive rating system scored each sample based on the measured signal strength (0 for no signal to 2 for strong signal) for each biological signature relative to the total rating possible. The 2005 field experiment results show that percent positive ratings varied significantly across Site D (coastal site with fog), with patchy zones of high abundance correlated with orbital and microscale habitat types (heaved surface crust and gravel bars); alluvial fan habitats generally had lower abundance. Non-random multi-scale biological patchiness also characterized interior desert Sites E and F, with relatively high abundance associated with (paleo)aqueous habitats such as playas. Localized variables, including topography, played an important, albeit complex, role in microbial spatial distribution. Site D biosignature trends correlated with culturable soil bacteria, with MPN ranging from 10-1000 CFU/g-soil, and chlorophyll ratings accurately mapped lichen/moss abundance (Site D) and higher plant (Site F) distributions. Climate also affected biological patchiness, with significant correlation shown between abundance and (rover) air relative humidity, while lichen patterns were linked to the presence of fog. Rover biological mapping results across sites parallel longitudinal W-E wet/dry/wet Atacama climate trends. Overall, the study highlights the success of targeting of aqueousassociated habitats identifiable from orbital geology and mineralogy. The LITA experience also suggests the terrestrial study of life and its distribution, particularly the fields of landscape ecology and ecohydrology, hold critical lessons for the search for life on other planets. Their applications to robotic sampling strategies on Mars should be further exploited.

Overproduction, purification and characterization of the F traT protein

SummaryA lambda transducing phage (EDλ110) which carries the sex factor F surface exclusion genes, traS and traT, was characterized by both genetic and physiochemical techniques. The transducing segment consists of 5.2 kilobases of F tra DNA, and carries the carboxy-terminal onehalf of the upstream traG gene, as well as traS, traT, and the adjacent downstream gene traD. These tra proteins could be identified in infected UV-irradiated cells, and the major part of their synthesis was found to occur from the phages late promoter pR under Q control.Lysogens for EDλ110 were induced and found to greatly overproduce the traT gene product (TraTp), an outer membrane protein normally found in about 20,000 copies per cell, to levels which exceeded the major outer membrane proteins. This led to the development of a simple purification procedure for TraTp, the most important step of which was the construction of an appropriate ompB derivative to eliminate the major outer membrane porin proteins, which have several physical properties in common with TraTp.Purified TraTp was added to mixtures of donor and recipient cells and found to inhibit mating. The specificity of this assay was demonstrated by using an R100-1 donor, which responds to a heterologous surface exclusion system, and by using an altered TraTp containing a missense amino acid substitution.A mechanism by which TraTp mediates surface exclusion is proposed.

Biochemical characterization of Escherichia coli DNA helicase I

The gene product of F tral is a bifunctional protein which nicks and unwinds the F plasmid during conjugal DNA transfer. Further biochemical characterization of the Tral protein reveals that it has a second, much lower, Km for ATP hydrolysis, in addition to that previously identified. Measurement of the single‐stranded DNA‐stimulated ATPase rate indicates that there is co‐operative interaction between the enzyme monomers for maximal activity. Furthermore, 18O‐exchange experiments indicate that Tral protein hydrolyses ATP with, at most, a low‐level reversal of the hydrolytic step during each turnover.

Application of pulsed‐excitation fluorescence imager for daylight detection of sparse life in tests in the Atacama Desert

A daylight fluorescence imager was deployed on an autonomous rover, Zoe, to detect life on the surface and shallow subsurface in regions of the Atacama Desert in Chile during field tests between 2003 and 2005. In situ fluorescent measurements were acquired from naturally fluorescing biomolecules such as chlorophyll and from specific fluorescent probes sprayed on the samples, targeting each of the four biological macromolecule classes: DNA, protein, lipid, and carbohydrate. RGB context images were also acquired. Preparatory reagents were applied to enhance the dye probe penetration and fluorescence intensity of chlorophyll. Fluorescence imager data sets from 257 samples were returned to the Life in the Atacama science team. A variety of visible life forms, such as lichens, were detected, and several of the dye probes produced signals from nonphotosynthetic microorganisms.

Investigating microbial diversity and UV radiation impact at the high-altitude Lake Aguas Calientes, Chile

The High-Lakes Project is funded by the NAI and explores the highest perennial volcanic lakes on Earth in the Bolivian and Chilean Andes, including several lakes ~6,000 m elevation. These lakes represent an opportunity to study the evolution of microbial organisms in relatively shallow waters not providing substantial protection against UV radiation. Aguas Calientes (5,870 m) was investigated (November 2006) and samples of water and sediment collected at 1, 3, 5, and 10 cm depth. An Eldonet UV dosimeter positioned on the shore records UV radiation and temperature, and is logging data year round. A UV SolarLight sensor allowed acquisition of point measurements in all channels at the time of the sampling. UVA, UVB, and PAR peaks between 11:00 am and 1:00 pm reached 7.7 mW/cm2, 48.5 μW/cm2, and 511 W/m2, respectively. The chemical composition of the water sample was analyzed. DNA was extracted and DGGE analyses with bacterial and archaeal 16S fragments were performed to describe microbial diversity. Antibiotic resistances were established previously in similar environments in Argentine Andean wetlands. In order to determine these resistances in our samples, they were inoculated onto LB and R2A media and onto R2A medium containing either chloramphenicol, ampicillin or tetracycline. Bacterial was higher than archeal cell number determined by RT-PCR in all the samples, reaching maximum total values of 5x105 cell mL-1. DGGE results from these samples and Licancabur summit lake (5,916 m) samples were also compared. Eight antibiotic-resistant Gram negative strains have been isolated with distinct resistance patterns.

Dynamics of declining lake habitat in changing climate

We illustrate the impact of climate variability on lake habitat at high altitude as a proxy to the evolution of declining lakes on Mars. The geophysical environment at the summit lake of the Licancabur volcano (5970 m) in the Central Andes of Chile and Bolivia was monitored between 2002 and 2006. The lake’s environment presents analogies with Mars at a time when the planet was transitioning from a wetter to a drier, colder climate: thin atmosphere, high solar irradiance, low ozone, high daily and yearly temperature fluctuations with low averages, ice, reduced yearly precipitation, and volcanic geology. The Licancabur lake is also located in a region impacted by climate change resulting in enhanced evaporation and strong negative water balance that rapidly modifies lake habitat. Data show that although the decline is not monotical, interannual fluctuations in precipitation, water balance, major ion concentration, and pH are well marked. Microorganisms dwelling near or at the water/atmosphere interface are exposed to a solar irradiance 160% that of sea level. Maximum averaged UVB peaks at 4 W/m2 and shorter (260–270 nm) radiation is also detected at the surface. The thin and cold atmosphere generates sudden and significant inverse relationship between UV radiation and temperatures. In this cold, unstable environment the lake ecosystem is abundant. In addition to adaptation strategies, data suggest that the timing of key cycles is a critical factor in life survival and provide important information on the lake habitability and life survival potential on Mars as the climate changed.

Correction to ''The High-Lakes Project''

Nathalie A.Cabrol,EdmondA.Grin, GuillermoChong,EdwinMinkley,AndrewN. Hock,Youngseob Yu, Leslie Bebout, Erich Fleming, Donat P. Ha¨der, Cecilia Demergasso,John Gibson, Lorena Escudero, Cristina Dorador, Darlene Lim, Clayton Woosley,Robert L. Morris, Cristian Tambley, Victor Gaete, Matthieu E. Galvez,Eric Smith, Ingrid Ukstins Peate, Carlos Salazar, G. Dawidowicz, and J. Majerowicz