CRYPTOENDOLITHIC ALGAL COMMUNITIES OF THE COLORADO PLATEAU1

Eighteen taxa, including representatives of both eukaryotic and prokaryotic genera, were isolated from below the surface of eight sandstones in four semi-desert and cold temperate biomes of northern Arizona, southern Utah and Western New Mexico. The algae occurred as uniform well defined bands in light-colored sandstones and also as scattered patches in dark sandstones. The algal communities varied in generic composition, chlorophyll a content, and location within the different sandstones. Biomass, estimated by chlorophyll a content, was approximately two to twenty-fold greater than reported for cryptoendolithic algal bands in a cold desert habitat. The widespread distribution of certain algae in endolithic habitats of the Colorado Plateau and their presence in rocks at quite distant locations suggests that the endolithic habitat may he utilized by algae whenever it provides more favorable conditions than the surrounding surfaces.     

Ecophysiological Studies on the Red Alga Galdieria suiphuraria Isolated from Southwest Iceland

Abstract: The acido- and thermophilic red alga, Galdieria sulphuraria , was isolated from three volcanic areas of the Reykjanes peninsula (southwest Iceland). These sites showed pH values of 1.5 to 3, low concentrations of dissolved organic carbon, and were apparently exclusively colonized by the red alga. No other eucaryotes were observed by light or electron microscopy. The isolated Galdieria strains grew heterotrophically on various sugars and polyols. At all three sites, Galdieria occupied terrestrial habitats. Extensive endolithic growth of the alga was only observed at one site where cell layers were found as deep as 3 cm within rocks of geyserite, a soft white siliceous mineral. Light is apparently insufficient for photosynthesis >10 mm below the stone surface. It is proposed that cells deep within the rock make use of metabolites released by decaying cells in the surroundings. Cells isolated from these algal layers exhibited sugar uptake rates indicative for a low-level heterotrophic state. Therefore, the extraordinary heterotrophic capabilities of Galdieria seem to be especially important in endolithic habitats.     

Anthrax-specific "AP 50-like" phages isolated from Bacillus cereus strains

Phages exerting a specific action on Bacillus anthracis were isolated from mitomycin C-induced concentrated lysates of 5 Bacillus cereus strains producing megacin A (phospholipase A). In electron micrographs the phages closely resembled the anthrax-specific, lipid containing phage AP 50 isolated earlier from soil sample. The phages were similar to AP 50 also in their antigenic and chemical structure, host range and sensitivity to organic solvents, detergents and caesium chloride. The DNA character of AP 50 nucleic acid was shown by agarose gel electrophoresis. AP 50 and related phages seem to represent a separate group of phages acting on Bacillus strains.     

DNA base compositions of halophilic and nonhalophilicBacillus firmus strains of marine origin

The mineral salt requirements of 27 strains ofBacillus firmus were determined. Twenty-six of these strains were of marine origin and one terrestrial strain was used as a reference. Three strains demonstrated strictly halophilic behavior, i.e., they showed no growth in media prepared without sodium chloride. Seven strains were nonhalophilic. The growth of 17 strains was stimulated by the addition of sodium chloride, but the cells were able to grow without it. These results were compared with the DNA base compositions of the strains. In contrast to literature data, relationships between the DNA base ratios and the halophilic or nonhalophilic behavior of the cells could not be detected. But strains with guanine plus cytosine values above 41 mol% did grow well at 44°C, and those strains showing poor growth at this temperature had lower guanine plus cytosine percentages.     

Plasmid-mediated bacteriocin production by Shigella flexneri isolated from dysenteric diarrhoea and their transformation into Escherichia coli

AIMS: To determine the production of bacteriocin by Shigella flexneri strains, to relate their production to the presence of dysenteric diarrhoea and to asses the genetic determination of the bacteriocin. METHODS AND RESULTS: One hundred and sixteen strains of Sh. flexneri were isolated from patients with diarrhoea and 49 of them produced bacteriocin active against several Escherichia coli and abacteriocinogenic Sh. flexneri strains. The extrachromosomal DNA isolated from bacteriocinogenic Sh. flexneri strains were used as a substrate to transform E. coli HB-101 cells by means of electroporation. CONCLUSIONS: Only the Sh. flexneri strains isolated from dysenteric diarrhoea produced bacteriocin. It was demonstrated that a plasmid of approx. 3 kb was responsible for the genetic determination of these anti-bacterial substances. Significance and IMPACT OF THE STUDY: A 3-kb plasmid that harboured information for the production of bacteriocin by Sh. flexneri strains was described. The production of this bacteriocin may be related to dysenteric diarrhoea produced by these bacterial strains.     

Phylogenetic diversity of endolithic bacteria in Bole granite rock in Xinjiang

The endolithic environment is a ubiquitous microbial habitat for microorganisms, such as lichens, Cyanobacteria and fungi, and it provides mineral nutrients and growth surfaces. In extremely environments, such as hot and cold desert, endolithic communities are often the main form of life. More recently, endolithic microbial communities have been observed inhabiting a variety of rock types ranging from hard granite to porous rocks such as basalt, dolomite, limestone, sandstone and granites. Regardless of geographic location and rock type, each of these habitats is characterized by a subsurface microclimate that prevents endolithic microorganisms growth. Photosynthesis-based endolithic microbial communities commonly inhabit the outer millimeters to centimeters of rocks exposed to the surface. The ability to fix carbon dioxide and in some cases atmospheric dinitrogen, gives the Cyanobacteria a clear competitive advantage over heterotrophic bacteria, so it is been called the main primary producer. Light quality and intensity appear to be the main determinant of the maximum depth to which growth occurs in endolithic phototrophic communities. Valleys of Fantastic Rocks in Bole is close to Alashankou Port of Xinjiang which belongs to extreme continental climate. In order to investigate the structure, composition and diversity of endolithic bacterial community in exposed granitic porphyry in the Valleys of Fantastic Rocks, environmental DNA was directly extracted from granite rock, the 16S rRNA genes were amplified from the total DNA by PCR with bacterial-specific primers, and an endolithic bacterial clone library was constructed. Positive clones were randomly selected from the library and identified by Restriction Fragment Length Polymorphism (RFLP). The unique rRNA types clones were sequenced, analysised and then constructed phylogenetic tree. In total, 129 positive clones were screened and grouped into 46 operational taxonomic unites (OTUs). The clone coverage C value was 89.15%, indicating that most of the estimated endolithic bacterial diversity was sampled. BLAST analysis indicated that 46 OTUs were divided into seven phyla (Acidobacteria, Actinobacteria, Bacteroidetes, Chloroflexi, Cyanobacteria, Planctomycetes, Proteobacteria) and five unknown groups. Cyanobacteria (43%), especially the Gp I, form the functional basis for an endolithic bacteria community which contain a wide spectrum species of chemotrophic bacteria (33%) with mainly Actinobacteria, α-Proteobacteria, Acidobacteria. Additionally, most clones that derived from the endolithic bacteria clone library showed high similarity to the sequence deposited in GenBank database with 97%–99%. Besides, 35% of the clones showed less than 97% of sequence similarity, of which 12% sequences were affiliated to genus Rubrobacter. The results suggested that endolithic bacteria in Valleys of Fantastic Rocks in Xinjiang were highly diverse in species richness, and maybe have a diversity of potential novel species and lineages.     

<Emphasis Type="Italic">Microbulbifer arenaceous</Emphasis> sp. nov., a New Endolithic Bacterium Isolated from the Inside of Red Sandstone

An endolithic bacterium, strain RSBr-1, was isolated from the inside of a piece of red sandstone from coastal areas of Scotland. RSBr-1 was Gram negative, oxidase and catalase positive, and cells were non-motile rods. Sodium was required for growth. The optimum sodium chloride concentration and pH for growth were 4% and pH 8.0, respectively. Eumelanin was produced in marine broth and in BY medium. RSBr-1 hydrolyzes chitin, esculin, gelatin, and starch, but not agar. Nitrate reduction is positive. Taxonomic characterization of this strain indicated that it belongs to the genus Microbulbifer. The difference between the aligned 16S rDNA sequences of RSBr-1 and the closest relative, M. elongata, is greater than the difference between the 16S rDNA sequences of M. hydrolyticus and M. elongata. On the basis of the phenotypic and genotypic comparison of this isolate with the other strains, RSBr-1 is proposed as a new species, Microbulbifer arenaceous, with type strain RSBr-1.     

ThermotolerantCampylobacter with no or weak catalase activity isolated from dogs

ThermotolerantCampylobacter strains isolated from dog feces were characterized by phenotypical tests, DNA base composition, and DNA-DNA-hybridization. Out of 98 strains, 63 were catalase negative or weakly reacting (CNW); they were found in diarrheic as well as in healthy dogs. The CNW strains were all nalidixic-acid sensitive, hippurate negative, and grew at 42°C but not at 25°C. Seven strains were further investigated. They were sensitive to 2,3,5-triphenyl-tetrazolium chloride, reduced nitrate, and produced H₂S in the lead acetate test but not in triple-sugar-iron agar. The mol% G+C for five CNW isolates ranged from 35.2–35.8, which is higher than reported for any thermotolerantCampylobacter species. The strains also formed a well-delimitated DNA homology group with 80% or more intragroup relatedness and about 40% related toC. coli andC. jejuni.     

Isolation and characterization of Flavobacterium strains that degrade pentachlorophenol.

Bacteria able to mineralize 100 to 200 ppm of pentachlorophenol (PCP) were isolated by selective enrichment from PCP-contaminated soils from three geographic areas of Minnesota. Although differing somewhat in their responses to various biochemical and biophysical tests, all strains were assigned to the genus Flavobacterium. Five representative strains were examined in detail. All strains metabolized PCP as a sole source of carbon and energy; 73 to 83% of all carbon in the form of [U-14C]PCP was returned as 14CO2, with full liberation of chlorine as chloride. A comparison between strains in their ability to metabolize PCP showed some strains to be more efficient than others. Guanine-plus-cytosine contents of DNA ranged from 58.8 to 63.8%, and DNA/DNA hybridization studies with total DNA digests suggested substantial genetic homology between strains. All strains were shown to possess an 80- to 100-kilobase plasmid, and evidence suggested the presence of a larger plasmid (greater than 200 kilobases).     

Isolation and characterization of Flavobacterium strains that degrade pentachlorophenol

Bacteria able to mineralize 100 to 200 ppm of pentachlorophenol (PCP) were isolated by selective enrichment from PCP-contaminated soils from three geographic areas of Minnesota. Although differing somewhat in their responses to various biochemical and biophysical tests, all strains were assigned to the genus Flavobacterium. Five representative strains were examined in detail. All strains metabolized PCP as a sole source of carbon and energy; 73 to 83% of all carbon in the form of [U-14C]PCP was returned as 14CO2, with full liberation of chlorine as chloride. A comparison between strains in their ability to metabolize PCP showed some strains to be more efficient than others. Guanine-plus-cytosine contents of DNA ranged from 58.8 to 63.8%, and DNA/DNA hybridization studies with total DNA digests suggested substantial genetic homology between strains. All strains were shown to possess an 80- to 100-kilobase plasmid, and evidence suggested the presence of a larger plasmid (greater than 200 kilobases).     

Bacillus endospores isolated from granite: close molecular relationships to globally distributed Bacillus spp. from endolithic and extreme environments

As part of an ongoing effort to catalog spore-forming bacterial populations in environments conducive to interplanetary transfer by natural impacts or by human spaceflight activities, spores of Bacillus spp. were isolated and characterized from the interior of near-subsurface granite rock collected from the Santa Catalina Mountains, AZ. Granite was found to contain approximately 500 cultivable Bacillus spores and approximately 10(4) total cultivable bacteria per gram. Many of the Bacillus isolates produced a previously unreported diffusible blue fluorescent compound. Two strains of eight tested exhibited increased spore UV resistance relative to a standard Bacillus subtilis UV biodosimetry strain. Fifty-six isolates were identified by repetitive extragenic palindromic PCR (rep-PCR) and 16S rRNA gene analysis as most closely related to B. megaterium (15 isolates), B. simplex (23 isolates), B. drentensis (6 isolates), B. niacini (7 isolates), and, likely, a new species related to B. barbaricus (5 isolates). Granite isolates were very closely related to a limited number of Bacillus spp. previously found to inhabit (i) globally distributed endolithic sites such as biodeteriorated murals, stone tombs, underground caverns, and rock concretions and (ii) extreme environments such as Antarctic soils, deep sea floor sediments, and spacecraft assembly facilities. Thus, it appears that the occurrence of Bacillus spp. in endolithic or extreme environments is not accidental but that these environments create unique niches excluding most Bacillus spp. but to which a limited number of Bacillus spp. are specifically adapted.     

Heterologous transformation in Bacillus subtilis. 1. Transmission of DNA of the R1drd19 plasmid]

Bac. subtilis 168 (BD-25) cells were infected with DNA of plasmide R1drd19 isolated from E. coli strain; transformants resistant to streptomycin (500 microgram/ml) and kanamycin (40 microgram/ml) appeared with the frequency of 2.10(-6). These transformants retained resistance to the mentioned antibiotics stably. A satellite DNA peak was revealed in centrifugation in the density gradient of cesium chloride with ethidium bromide. It was possible to infect cells of Bac. subtilis 168 (BD-25) with plasmide DNA isolated from the transformants. Plasmide transduction with the aid of phages AR9 and PBSI multiplied on the transformant strains was also effected. Physico-chemical analysis of the transformed plasmide DNA was conducted; its molecular weight was determined.     

DNA-DNA Homology Between Lactic Streptococci and Their Temperate and Lytic Phages

Temperate phages were induced from Streptococcus cremoris R₁, BK₅, and 134. DNA from the three induced phages was shown to be homologous with prophage DNA in the bacterial chromosomes of their lysogenic hosts by the Southern blot hybridization technique. ³²P-labeled DNA from 11 lytic phages which had been isolated on cheese starters was similarly hybridized with DNA from 36 strains of lactic streptococci. No significant homology was detected between the phage and bacterial DNA. Phages and lactic streptococci used included phages isolated in a recently opened cheese plant and all the starter strains used in the plant since it commenced operation. The three temperate phages were compared by DNA-DNA hybridizations with 25 lytic phages isolated on cheese starters. Little or no homology was found between DNA from the temperate and lytic phages. In contrast, temperate phages showed a partial relationship with one another. Temperate phage DNA also showed partial homology with DNA from a number of strains of lactic streptococci, many of which have been shown to be lysogenic. This suggests that many temperate phages in lactic streptococci may be related to one another and therefore may be homoimmune with one another. These findings indicate that the release of temperate phages from starter cells currently in use is unlikely to be the predominant source of lytic phages in cheese plants.     

Aerobic vinyl chloride metabolism in Mycobacterium aurum L1.

Mycobacterium aurum L1, capable of growth on vinyl chloride as a sole carbon and energy source, was previously isolated from soil contaminated with vinyl chloride (S. Hartmans et al., Biotechnol. Lett. 7:383-388, 1985). The initial step in vinyl chloride metabolism in strain L1 is catalyzed by alkene monooxygenase, transforming vinyl chloride into the reactive epoxide chlorooxirane. The enzyme responsible for chlorooxirane degradation appeared to be very unstable and thus hampered the characterization of the second step in vinyl chloride metabolism. Dichloroethenes are also oxidized by vinyl chloride-grown cells of strain L1, but they are not utilized as growth substrates. Three additional bacterial strains which utilize vinyl chloride as a sole carbon and energy source were isolated from environments with no known vinyl chloride contamination. The three new isolates were similar to strain L1 and were also identified as Mycobacterium aurum.     

Aerobic vinyl chloride metabolism in Mycobacterium aurum L1.

Mycobacterium aurum L1, capable of growth on vinyl chloride as a sole carbon and energy source, was previously isolated from soil contaminated with vinyl chloride (S. Hartmans et al., Biotechnol. Lett. 7:383-388, 1985). The initial step in vinyl chloride metabolism in strain L1 is catalyzed by alkene monooxygenase, transforming vinyl chloride into the reactive epoxide chlorooxirane. The enzyme responsible for chlorooxirane degradation appeared to be very unstable and thus hampered the characterization of the second step in vinyl chloride metabolism. Dichloroethenes are also oxidized by vinyl chloride-grown cells of strain L1, but they are not utilized as growth substrates. Three additional bacterial strains which utilize vinyl chloride as a sole carbon and energy source were isolated from environments with no known vinyl chloride contamination. The three new isolates were similar to strain L1 and were also identified as Mycobacterium aurum.     

The integration of donor and recipient deoxyribonucleic acid during transformation of Bacillus subtilis

1. DNA labelled with 5-bromo[(3)H]uracil was used to transform auxotrophic strains of Bacillus subtilis. 2. After various times of incubation, DNA was extracted from the transformed culture and subjected to equilibrium sedimentation in caesium chloride gradients. 3. In addition to heavy donor DNA and light recipient DNA, a component with an intermediate density was found and is believed to consist of a biological hybrid of donor and recipient. 4. The component of intermediate density was isolated and found to possess activity in transformation derived from both donor and recipient strains. 5. Denaturation of the component of intermediate density followed by centrifugation gave only one component, indicating that integration had occurred in both strands of the recipient DNA. 6. No integrated band was observed after uptake by competent cells of B. subtilis of heavy DNA prepared from Escherichia coli.     

A procedure for large-scale plasmid isolation without using ultracentrifugation.

An expedient procedure for large-scale plasmid isolation from Escherichia coli strains without using ultracentrifugation or special setups or reagents is described. The protocol, which utilizes a modified alkaline extraction procedure as well as differential precipitations by isopropanol and lithium chloride, is simple and rapid and yet produces plasmid DNA with a yield of about 2 mg/liter culture. The isolated plasmids consisted of mostly monomeric and dimeric covalently closed circular DNA. The plasmids could be digested by various restriction endonucleases and were compatible with gene cloning, transfection-gene expression, and viral production.     

Isolation and Characterization of the Fertility Factor P of Vibrio cholerae

Vibrio cholerae strains with the transmissible fertility factor P contained a supercoiled circular deoxyribonucleic acid (DNA) component amounting to between 2 and 6% of the total DNA obtained from the cells. Such a component was not observed in V. cholerae strains lacking the fertility factor. This supercoiled circular DNA was isolated from P(+) cells, and the molecular weight was determined by sedimentation velocity experiments and electron microscopy to be approximately 80 million daltons. These supercoiled circular DNA molecules, which have a guanine plus cytosine (G + C) composition of 42%, were concluded to be the extrachromosomal P factor. It was calculated that there is approximately one copy of the P factor per chromosome. A small amount of supercoiled circular DNA was occasionally isolated from the P(-) strains of V. cholerae. The function of this component, which has a molecular weight of 40 million daltons, is not known. The molecules found in the P(-) strains were readily distinguished from the P(+) circular molecules by their smaller molecular weight and different G + C composition.     

Metagenomic analysis of the microbial community associated with the coral Porites astreoides

The coral holobiont is a dynamic assemblage of the coral animal, zooxanthellae, endolithic algae and fungi, Bacteria,Archaea and viruses. Zooxanthellae and some Bacteria form relatively stable and species-specific associations with corals. Other associations are less specific; coral-associated Archaea differ from those in the water column, but the same archaeal species may be found on different coral species. It has been hypothesized that the coral animal can adapt to differing ecological niches by 'switching' its microbial associates. In the case of corals and zooxanthellae, this has been termed adaptive bleaching and it has important implications for carbon cycling within the coral holobiont and ultimately the survival of coral reefs. However, the roles of other components of the coral holobiont are essentially unknown. To better understand these other coral associates, a fractionation procedure was used to separate the microbes, mitochondria and viruses from the coral animal cells and zooxanthellae. The resulting metagenomic DNA was sequenced using pyrosequencing. Fungi, Bacteria and phage were the most commonly identified organisms in the metagenome. Three of the four fungal phyla were represented, including a wide diversity of fungal genes involved in carbon and nitrogen metabolism, suggesting that the endolithic community is more important than previously appreciated. In particular, the data suggested that endolithic fungi could be converting nitrate and nitrite to ammonia, which would enable fixed nitrogen to cycle within the coral holobiont. The most prominent bacterial groups were Proteobacteria (68%), Firmicutes (10%), Cyanobacteria (7%) and Actinobacteria (6%). Functionally, the bacterial community was primarily heterotrophic and included a number of pathways for the degradation of aromatic compounds, the most abundant being the homogentisate pathway. The most abundant phage family was the ssDNA Microphage and most of the eukaryotic viruses were most closely related to those known to infect aquatic organisms. This study provides a metabolic and taxonomic snapshot of microbes associated with the reef-building coral Porites astreoides and presents a basis for understanding how coral-microbial interactions structure the holobiont and coral reefs.     

DNA restriction patterns and DNA-DNA solution hybridization studies of Frankia isolates from Myrica pennsylvanica (bayberry)

Sixteen Frankia strains were isolated from Myrica pennsylvanica (bayberry) root nodules collected at diverse sites in New Jersey. Restriction pattern analysis of total genomic DNA was used to group the isolates into gel groups, and the genetic relatedness among the isolates was evaluated by DNA-DNA solution hybridization studies. Restriction pattern analysis provided a distinctive reproducible fingerprint for each isolate. Isolates fell into nine separate groups (strain types). More than one strain type was isolated from most sites. Isolates from two different gel groups were found in 3 of 10 nodules examined. Of the 16 isolates, 10 contained extrachromosomal DNA. Six different extrachromosomal DNA banding patterns were found. Genomically similar isolates carried related, but different, banding patterns. DNA hybridization studies indicated that isolates from a single plant species can be minimally related as determined by total genome homology. Homology ranged from 12 to 99%. Highly divergent strains were isolated from the same plant and found to cohabit the same nodule. Thus, this study demonstrated that Frankia strains which infect the same host plant are not only phenotypically different but also genetically diverse.