Effect of Shadowing on Survival of Bacteria under Conditions Simulating the Martian Atmosphere and UV Radiation

Spacecraft-associated spores and four non-spore-forming bacterial isolates were prepared in Atacama Desert soil suspensions and tested both in solution and in a desiccated state to elucidate the shadowing effect of soil particulates on bacterial survival under simulated Martian atmospheric and UV irradiation conditions. All non-spore-forming cells that were prepared in nutrient-depleted, 0.2-μm-filtered desert soil (DSE) microcosms and desiccated for 75 days on aluminum died, whereas cells prepared similarly in 60-μm-filtered desert soil (DS) microcosms survived such conditions. Among the bacterial cells tested, Microbacterium schleiferi and Arthrobacter sp. exhibited elevated resistance to 254-nm UV irradiation (low-pressure Hg lamp), and their survival indices were comparable to those of DS- and DSE-associated Bacillus pumilus spores. Desiccated DSE-associated spores survived exposure to full Martian UV irradiation (200 to 400 nm) for 5 min and were only slightly affected by Martian atmospheric conditions in the absence of UV irradiation. Although prolonged UV irradiation (5 min to 12 h) killed substantial portions of the spores in DSE microcosms (~5- to 6-log reduction with Martian UV irradiation), dramatic survival of spores was apparent in DS-spore microcosms. The survival of soil-associated wild-type spores under Martian conditions could have repercussions for forward contamination of extraterrestrial environments, especially Mars.     

Response of terrestrial microorganisms to a simulated Martian environment.

Soil samples from Cape Canaveral were subjected to a simulated Martian environment and assayed periodically over 45 days to determine the effect of various environmental parameters on bacterial populations. The simulated environment was based on the most recent available data, prior to the Viking spacecraft, describing Martian conditions and consisted of a pressure of 7 millibars, an atmosphere of 99.9% CO2 and 0.1% O2, a freeze-thaw cycle of -65 degrees C for 16 h and 24 degrees C for 8 h, and variable moisture and nutrients. Reduced pressure had a significant effect, reducing growth under these conditions. Slight variations in gaseous composition of the simulated atmosphere had negligible effect on growth. The freeze-thaw cycle did not inhibit growth but did result in a slower rate of decline after growth had occurred. Dry samples exhibited no change during the 45-day experiment, indicating that the simulated Martian environment was not toxic to bacterial populations. Psychotrophic organisms responded more favorably to this environment than mesophiles, although both types exhibited increases of approximately 3 logs in 7 to 14 days when moisture and nutrients were available.     

Response of terrestrial microorganisms to a simulated Martian environment

Soil samples from Cape Canaveral were subjected to a simulated Martian environment and assayed periodically over 45 days to determine the effect of various environmental parameters on bacterial populations. The simulated environment was based on the most recent available data, prior to the Viking spacecraft, describing Martian conditions and consisted of a pressure of 7 millibars, an atmosphere of 99.9% CO2 and 0.1% O2, a freeze-thaw cycle of -65 degrees C for 16 h and 24 degrees C for 8 h, and variable moisture and nutrients. Reduced pressure had a significant effect, reducing growth under these conditions. Slight variations in gaseous composition of the simulated atmosphere had negligible effect on growth. The freeze-thaw cycle did not inhibit growth but did result in a slower rate of decline after growth had occurred. Dry samples exhibited no change during the 45-day experiment, indicating that the simulated Martian environment was not toxic to bacterial populations. Psychotrophic organisms responded more favorably to this environment than mesophiles, although both types exhibited increases of approximately 3 logs in 7 to 14 days when moisture and nutrients were available.     

Study of Bacillus subtilis Endospores in Soil by Use of a Modified Endospore Stain

The Schaeffer-Fulton endospore stain was modified so that it would stain Bacillus subtilis endospores in soil smears. The modified stain differentiated among dormant spores, spores undergoing activation, and spores which had germinated but had not yet shown outgrowth. These differentiations were seen for spores in soil and for pure spore preparations in the laboratory. This stain was used to show reversible B. subtilis spore activation promoted by an Ensifer adhaerens-like indigenous bacterium in soil and by pure cultures of E. adhaerens added to spores in the laboratory. Under the specific conditions in the laboratory, spore germination did not proceed beyond the activation stage, and relatively little change occurred in the numbers of both E. adhaerens and B. subtilis. This was also true in soil, although some germination with destruction of spores and vegetative cells did occur if the soil had been nutritionally enriched by preincubation with incorporated ground alfalfa.     

Survival of Bacillus thuringiensis Spores in Soil

Bacillus thuringiensis spores and parasporal crystals were incubated in natural soil, both in the laboratory and in nature. During the first 2 weeks, the spore count decreased by approximately 1 log. Thereafter, the number of spore CFU remained constant for at least 8 months. B. thuringiensis did not lose its ability to make the parasporal crystals during its residence in soil. Spore survival was similar for a commercial spore-crystal preparation (the insecticide) and for laboratory-grown spores. In contrast to these results, spores that were produced in situ in soil through multiplication of added vegetative cells survived for only a short time. For spore additions to soil, variations in soil pH had little effect on survival for those spores that survived the first 2 weeks of incubation. Also without effect were various pretreatments of the spores before incubation in soil or nutritional amendment or desiccation of the soil. Remoistening of a desiccated soil, however, caused a decrease in spore numbers. Spores incubated in soil in the field did not show this, but the degree of soil desiccation in nature probably never reached that for the laboratory samples. The good survival of B. thuringiensis spores after the first 2 weeks in soil seemed to be a result of their inability to germinate in soil. We found no evidence for the hypothesis that rapid germination ability for spores in soil conferred a survival advantage.     

Survival of Microorganisms in a Simulated Martian Environment: II. Moisture and Oxygen Requirements for Germination of Bacillus cereus and Bacillus subtilis var. niger Spores1

The effects of moisture and oxygen concentration on germination of Bacillus cereus and B. subtilis var. niger spores were investigated in a simulated Martian environment. Less moisture was required for germination than for vegetative growth of both organisms. A daily freeze-thaw cycle lowered moisture requirements for spore germination and vegetative growth of both organisms, as compared with a constant 35 C environment. Oxygen had a synergistic effect by lowing the moisture requirements for vegetative growth, and possibly germination, of both organisms. Oxygen was not required for spore germination of either organism, but was required for vegetative growth of B. subtilis and for sporulation of both organisms.     

Airborne basidiospores of Coprinus and Ganoderma in a Caribbean region

The present study sought to examine Coprinus and Ganoderma airborne spore counts, analysing seasonal variations, the influence of meteorological variables and intraday behaviour with a view to charting the aerobiological dynamics of both genera in Havana (Cuba) during a 2-year period (November 2010–October 2012). A Hirst-type volumetric air sampler was used as sampling methodology. The spores registered in the air were identified and counted following the model proposed by the Spanish Aerobiological Network based on two longitudinal transects along the slides. Coprinus spores outnumbered those of the Ganoderma (28,468 annual total spores vs. 1,921 spores). Moreover, both genera were recorded in the atmosphere on a large number of days over the two-year study period, with daily maximum concentrations in the rainy months. The daily maximum value for Coprinus was 880 spores/m³ (30 September 2011), whereas for Ganoderma 44 spores/m³ (4 September 2011). The analysis of the Spearman correlation coefficient showed a significant positive correlation of the airborne Coprinus and Ganoderma spores with the temperature, relative humidity and rainfall, whereas the degree of association is negative with the wind speed. Regarding the intraday behaviour, both basidiospores were more abundant in the atmosphere during the night, with a maximum peak at 5–6 a.m.     

Persistence of Nosema locustae Spores in Soil as Determined by Fluorescence Microscopy

Nosema locustae, a protozoan parasite of grasshoppers, is used as a bioinsecticide. In the present study, the persistence of N. locustae spores in soil and the interaction of these spores with the indigenous soil microflora were examined with various forms of microscopy and staining. Fluorescence microscopy was found to be better than phase-contrast or bright-field microscopy for detecting and viewing spores in soil. Fluorescein isothiocyanate was a better fluorescent stain than acridine orange or fluorescein diacetate; water-soluble aniline blue did not stain spores. The eight bright-field microscopy stains tested (phenolic erythrosin, phenolic rose bengal, malachite green, crystal violet, safranin, Congo red, methyl red, and eosin B) were not satisfactory, as spore staining characteristics were either poor or masked by overstained soil debris. A procedure was developed which allowed spores to be extracted from soil with a peptone-phosphate buffer, recovered on a membrane filter, and stained with fluorescein isothiocyanate for microscopic counting. This procedure was used to assess the persistence of N. locustae spores in field and laboratory soils. The number of N. locustae spores in a laboratory model soil system persisted at a high level for over 8 weeks when the soil was incubated at 5°C but exhibited a 1,000-fold decrease after 1 week of incubation at 27°C. Persistence was related to the temperature-dependent activity of the indigenous soil microflora, which, on the basis of microscopic observations, appeared to prey on N. locustae spores. N. locustae spores were detected in an N. locustae-treated field soil at a low level consistent with the level for laboratory soil incubated at 27°C, and they persisted at this level for over 2 months. No spores were detected on vegetation from this field or in the soil from an adjacent, nontreated control field. N. locustae-like spores were also detected in soil from nontreated fields supporting large grasshopper populations.     

Response in Soil of Cupriavidus necator and Other Copper-Resistant Bacterial Predators of Bacteria to Addition of Water, Soluble Nutrients, Various Bacterial Species, or Bacillus thuringiensis Spores and Crystals

Soil was incubated with various species of bacteria, Bacillus subtilis, or Bacillus thuringiensis spores and crystals. These were added to serve as potential prey for indigenous, copper-resistant, nonobligate bacterial predators of bacteria in the soil. Alternatively, the soil was incubated with soluble nutrients or water only to cause potential indigenous prey cells to multiply so the predator cells would multiply. All of these incubation procedures caused excessive multiplication of some gram-negative bacteria in soil. Even greater multiplication, however, often occurred for certain copper-resistant bacterial predators of bacteria that made up a part of the gram-negative response. Incubation of the soil with copper per se did not give these responses. In most cases, the copper-resistant bacteria that responded were Cupriavidus necator, bacterial predator L-2, or previously unknown bacteria that resembled them. As was the case for C. necator and L-2, these new bacteria did not use glucose, had white colonies, produced copper-related growth initiation factor (GIF), and attacked B. thuringiensis spores on laboratory media. The results were different, however, when B. thuringiensis spores and crystals per se were added to the soil. The copper-resistant bacterial response in the soil did not, to any extent, include C. necator-like bacteria. Instead, the main copper-resistant bacterial predators that developed had yellow colonies and did not resemble C. necator or L-2 in other ways. They were not seen before, and they did not develop on the addition of B. subtilis spores to soil. Apparently, they could not produce a C. necator-like GIF. Nevertheless, they did respond very quickly to B. thuringiensis spores and crystals in soil, as if a GIF of some sort were involved. These results suggest that, under various conditions of soil incubation, gram-negative bacterial predators of bacteria multiply and that several copper-resistant types among them can be detected, counted, and isolated by plating dilutions of the soil onto media containing excess copper.     

Dry-heat resistance of selected psychrophiles.

The dry-heat resistance characteristics of spores of psychrophilic organisms isolated from soil samples from the Viking spacecraft assembly areas at Cape Kennedy Space Flight Center, Cape Canaveral, Fla., were studied. Spore suspensions were produced, and dry-heat D values were determined for the microorganisms that demonstrated growth or survival under a simulated Martian environment. The dry-heat tests were carried out by using the planchet-boat-hot plate system at 110 and 125 degrees C with an ambient relative humidity of 50% at 22 degrees C. The spores evaluated had a relatively low resistance to dry heat. D(110 degrees C) values ranged from 7.5 to 122 min, whereas the D(123 degrees C) values ranged from less than 1.0 to 9.8 min.     

Dry-heat resistance of selected psychrophiles.

The dry-heat resistance characteristics of spores of psychrophilic organisms isolated from soil samples from the Viking spacecraft assembly areas at Cape Kennedy Space Flight Center, Cape Canaveral, Fla., were studied. Spore suspensions were produced, and dry-heat D values were determined for the microorganisms that demonstrated growth or survival under a simulated Martian environment. The dry-heat tests were carried out by using the planchet-boat-hot plate system at 110 and 125 degrees C with an ambient relative humidity of 50% at 22 degrees C. The spores evaluated had a relatively low resistance to dry heat. D(110 degrees C) values ranged from 7.5 to 122 min, whereas the D(123 degrees C) values ranged from less than 1.0 to 9.8 min.     

Development,Distribution, and Host Studies of the Fungus Macrobiotophthoira vermicola (Entomophthorales)

The life cycle and host range of Macrobiotophthora vermicola were studied. Secondary spores produced from forcibly ejected primary spores adhered to the cuticle of Cruznema tripartitum, germinated, and penetrated the cuticle within 30 minutes. New primary spores were produced within 24 hours of initial spore adhesion. In a host range study, species of Rhabditidae, Diplogasteridae, and Aphelenchoidea were hosts, but not species of Bunonematidae, Tripylidae, Cephalobida, or Tylenchina. Numbers of second-stage Meloidogyne incognita juveniles were not decreased when added to soil seeded with infected C. tripartitum. In six Tennessee soybean fields, Macrobiotophthora vermicola was the most commonly encountered nematode-destroying fungus, followed by a sterile, nonseptate fungus and Arthrobotrys conoides. Nematophagous fungi were isolated more frequently from silt loam soils than from clay soils. Addition of C. tripartitum to soil extract plates as a bait nematode did not increase isolations of nematophagous fungi.     

An experimental association between Chlorella xanthella and a streptomyces

An association in the form of a lichen-like crust (“actinolichen”) was obtained by seeding Chlorella xanthella cells and spores of a Streptomyces sp. isolated from soil on the surface of potato sections partially immersed in water at temperatures with a diurnal fluctuation of 9–19 C or 14–25 C, and with illumination continuous or during the hours of the higher temperatures. Sections of the crust showed a lichen-like organized tissue with layers of Streptomyces sp. spores, hyphae alone and associated hyphae and algae.     

Host Range and Ecology of Isolates of Pasteuria spp. from the Southeastern United States

Isolates of Pasteuria penetrans were evaluated for ecological characteristics that are important in determining their potential as biological control agents. Isolate P-20 survived without loss of its ability to attach to its host nematode in dry, moist, and wet soil and in soil wetted and dried repeatedly for 6 weeks. Some spores moved 6.4 cm (the maximum distance tested) downward in soil within 3 days with percolating water. The isolates varied greatly in their attachment to different nematode species and genera. Of five isolates tested in spore-infested soil, three (P-104, P-122, B-3) attached to two or more nematode species, whereas B-8 attached only to Meloidogyne hapla and B-I did not attach to any of the nematodes tested. In water suspensions, spores of isolate P-20 attached readily to M. arenaria but only a few spores attached to other Meloidogyne spp. Isolate P-104 attached to all Meloidogyne spp. tested but not to Pratylenchus scribneri. Isolate B-4 attached to all species of Meloidogyne and Pratylenchus tested, but the rate of attachment was relatively low. Isolate P-Z00 attached in high numbers to M. arenaria when spores were extracted from females of this nematode; when extracted from M. javanica females, fewer spores attached to M. arenaria than to M. javanica or M. incognita.     

An Assay for Hirsutella rhossiliensis Spores and the Importance of Phialides for Nematode Inoculation

A spore assay was developed to measure the relative density of spores of the nematophagous fungus Hirsutella rhossiliensis in soil. Orchard soil containing H. rhossiliensis-parasitized Criconemella xenoplax was placed in vials and incubated for 0-120 days before the addition of probe nematodes, Heterorhabditis heliothidis juveniles. After 18 hours, H. heliothidis were extracted from the soil and examined for adhering spores of H. rhossiliensis. No spores were detected when H. heliothidis were added to freshly mixed soil, but the percentage of H. heliothidis with spores increased rapidly if soil was incubated undisturbed. Because mixing soil detaches spores from phialides, the results indicate that spores must be attached to phialides to adhere to nematodes. The spore assay was compared with a plate assay that measures the population density of H. rhossiliensis-parasitized C. xenoplax. Results from the two assays were highly correlated, suggesting that spores occur in three phases: reserves in nematodes that may be converted into spores; spores on phialides and therefore capable of adhering to nematodes; and spores detached from phialides and thus incapable of adhering to nematodes.     

Invasion of Spores of the Arbuscular Mycorrhizal Fungus Gigaspora decipiens by Burkholderia spp.

Burkholderia species are bacterial soil inhabitants that are capable of interacting with a variety of eukaryotes, in some cases occupying intracellular habitats. Pathogenic and nonpathogenic Burkholderia spp., including B. vietnamiensis, B. cepacia, and B. pseudomallei, were grown on germinating spores of the arbuscular mycorrhizal fungus Gigaspora decipiens. Spore lysis assays revealed that all Burkholderia spp. tested were able to colonize the interior of G. decipiens spores. Amplification of specific DNA sequences and transmission electron microscopy confirmed the intracellular presence of B. vietnamiensis. Twelve percent of all spores were invaded by B. vietnamiensis, with an average of 1.5 × 10⁶ CFU recovered from individual infected spores. Of those spores inoculated with B. pseudomallei, 7% were invaded, with an average of 5.5 × 10⁵ CFU recovered from individual infected spores. Scanning electron and fluorescence microscopy provided insights into the morphology of surfaces of spores and hyphae of G. decipiens and the attachment of bacteria. Burkholderia spp. colonized both hyphae and spores, attaching to surfaces in either an end-on or side-on fashion. Adherence of Burkholderia spp. to eukaryotic surfaces also involved the formation of numerous fibrillar structures.     

Analysis of bacterial communities associated with spores of Gigaspora margarita and Gigaspora rosea

The spores of arbuscular mycorrhizal fungi (AMF) form a unique microhabitat that is suitable for the colonization by many species of bacteria. The aim of the current study was to analyze the bacterial communities associated with the surface of spores of the AMF species Gigaspora margarita MAFF 520054 and Gigaspora rosea JP1. The two AMF species were propagated with tobacco (Nicotiana tabacum) grown in a mixture of sand and soil. In another experiment, G. margarita was propagated with tobacco or alfalfa (Medicago sativa) grown in vermiculite or a mixture of sand and soil. The bacterial community composition of the new-formed spores and sand/soil substrate was analyzed using PCR of 16S rDNA fragments and denaturing gradient gel electrophoresis (DGGE). Clustering analysis revealed that the bacterial communities on the surface of G. margarita spores was different form that in the substrate or on the surface of the G. rosea spores, and both the host plant and the substrate could influence the composition of spore-associated bacterial populations of the G. margarita. Sequence analysis of the major DGGE bands of G. margarita spore samples revealed that most of the bacterial sequences were affiliated with the phyla Proteobacteria (Azospirillum, Azovibrio, Polyangium, Ramlibacter, Rubrivivax, Sphingomonas, and Rhizobium) and Actinobacteria (Streptomyces, Amycolatopsis, and Pseudonocardia).     

Histone H2B subtypes are dispensable during the yeast cell cycle

Saccharomyces cerevisiae contains two histone H2B protein subtypes, H2B1 and H2B2, which differ at 4 of 130 amino acids. We describe experiments that test whether both histone H2B subtypes are required for the completion of any stage in the yeast life cycle. Frameshift mutations were introduced into cloned copies of the H2B1 and H2B2 genes. These altered genes were integrated into the yeast genome by transformation and replaced the wild-type genes through recombination. We thus obtained strains that lacked functional H2B1 or H2B2 proteins. These mutant strains survive as haploids and homozygous diploids. During vegetative growth, they divide at the same rate as wild-type cells and are able to mate, sporulate and germinate. The h2b1^? cells grew more slowly after germination than h2b2^? or wild-type spores, but otherwise the mutants were indistinguishable from each other or from wild-type cells. We also attempted to make a strain that was mutant in both genes for H2B. We examined spores derived from a diploid that is heterozygous for both histone mutations. The two genes assort independently, so we expect one in four spores to be h2b1^?h2b2^?. Of 61 spore colonies examined, none was mutant at both loci. Our results indicate that the double mutant can germinate and bud once but cannot grow further. Since the yeast life cycle can be completed in the absence of either but not both histone H2B subtypes, we conclude that neither protein has a unique essential function.     

Comparing two methods for quantifying soil-borne Entomophaga maimaiga resting spores

To improve usability of methods for quantifying environmentally persistent entomophthoralean resting spores in soil, we modified and tested two methods using resting spores (azygospores) of the gypsy moth pathogen Entomophaga maimaiga. Both methods were effective for recovering resting spores at concentrations >100 resting spores/g dry soil. While a modification of a method originally described by Weseloh and Andreadis (2002) recovered more resting spores than a modified method based on Percoll density gradients, the ability to estimate true densities from counts was similar for both methods. Regression equations are provided for predicting true resting spore densities from counts, with R² values for both methods ?0.90.