Searching for eukaryotic life preserved in Antarctic permafrost

Fungi and yeasts isolated in pure culture from Antarctic permafrost collected at different depths in the McMurdo Dry Valleys were identified with cultural, physiological and molecular methods. Fungi belonged to the genera Penicillium, Eurotium, Cladosporium, Alternaria, Engyodonthium, Aureobasidium, Cordyceps, Rhizopus and yeasts to the genera Cryptococcus and Sporidiobolus. All the strains can be defined as mesophilic psychrotolerant. The molecular analyses revealed that these fungal genotypes do not deviate from the global gene pool of fungi commonly spreading worldwide at present, but possible ancestral strains have been found on the base of metabolic profiles.     

南极细菌

南极由于其独特的地理、气候和环境特征,形成了一个干燥、酷寒、强辐射的特殊生境,造就了极地微生物的新颖性和多样性。南极不仅是发现微生物新物种的重要资源宝库,也是发现新的药物先导化合物等活性物质的资源宝库,这使其将成为研究低温生物学的良好试验材料及新型活性物质的重要潜在来源。极地微生物的资源勘探与代谢活性产物研发,已成为国内外微生物学领域研究的热点之一[1-4]。     

Searching for bacteriocin-producing lactic acid bacteria in soil

A survey was conducted on the isolation and characterization of bacteriocin-producing lactic acid bacteria in soil. Forty-two acid-producing bacterial strains were isolated from 55 soil samples collected in Yamanashi prefecture, Japan. Investigation of antibacterial activities of isolates revealed that three isolates, Lactobacillus animalis C060203, Enterococcus durans C102901 and Leuconostoc mesenteroides subsp. mesenteroides C060204, showed antibacterial activities against the indicator strain of Lactobacillus sakei JCM 1157T. Bacteriocin from Enterococcus durans C102901 showed different characteristics from the known durancin L28-1A, produced by Enterococcus durans L28-1. Furthermore, this is the first report of a bacteriocin being produced by Lactobacillus animalis. Viewing from the species, bacteriocins from strains C102901 and C060203 showed high possibilities for the novel substances. These significant findings suggest that soil may be a common source for the isolation of novel bacteriocin-producing lactic acid bacteria.     

Agar-degrading bacteria isolated from Antarctic macroalgae

This study describes the taxonomic diversity of pigmented, agar-degrading bacteria isolated from the surface of macroalgae collected in King George Island, Antarctica. A total of 30 pigmented, agarolytic bacteria were isolated from the surface of the Antarctic macroalgae Adenocystis utricularis, Monostroma hariotii, Iridaea cordata, and Pantoneura plocamioides. Based on the 16S rRNA data, the agarolytic isolates were affiliated to the genera Algibacter, Arthrobacter, Brachybacterium, Cellulophaga, Citricoccus, Labedella, Microbacterium, Micrococcus, Salinibacterium, Sanguibacter, and Zobellia. Isolates phylogenetically related to Cellulophaga algicola showed the highest agarase activity in culture supernatants when tested at 4 and 37 °C. This is the first investigation of pigmented agar-degrading bacteria, members of microbial communities associated with Antarctic macroalgae, and the results suggest that they represent a potential source of cold-adapted agarases of possible biotechnological interest.     

Antibiotic susceptibility of faecal bacteria in Antarctic penguins

Faecal bacteria from 49 Gentoo penguins on the Antarctic Peninsula were identified by biochemical methods and sequencing, and tested for antibiotic susceptibility using agar dilution. Of the 42 Enterobacteriaceae isolates found, 39 belonged to the genus Edwardsiella. All isolates were susceptible to the 17 antibiotics tested. This implies that antibiotic selection pressure is a prerequisite to a high prevalence of antibiotic resistance, and in the absence of contact with human activities, antibiotic resistance in Enterobacteriaceae remains undetectable.     

Searching for monooxygenases and hydrolases in bacteria from an extreme environment

Microbial oxidation potentials of extremophiles recovered from Pampo Sul oil field, Campos Basin, Brazil, in pure culture or in consortia, were investigated using high-throughput screening (HTS) and multibioreactions. Camphor (1), cis-jasmone (2), 2-methyl-cyclohexanone (3), 1,2-epoxyoctane (4), phenylethyl acetate (5), phenylethyl propionate (6), and phenylethyl octanoate (7) were used to perform multibioreaction assays. Eighty-two bacterial isolates were recovered from oil and formation water samples and those presenting outstanding activities in HTS assays were identified by sequencing their 16S rRNA genes. These results revealed that most microorganisms belonged to the genus Bacillus and presented alcohol dehydrogenase, monooxygenase, epoxide hydrolase, esterase, and lipase activities.     

Membrane fatty acid analysis of Antarctic bacteria

Abstract Randomly selected strains of a bacterial collection of marine sea-ice bacteria from Antarctica were analyzed to obtain a profile of the membrane fatty acids. Results showed that short chain saturated and unsaturated fatty acids were more common in the psychrotrophs when compared to psychrophiles. In contrast, branched-chain fatty acids were more abundant in the psychrophiles.     

Isolation of denitrifying bacteria from hydrocarbon-contaminated Antarctic soil

In this study, we report the isolation of denitrifiers from hydrocarbon-contaminated Antarctic soils. Seventy-two isolates were obtained from soils that had received a fertilizer treatment to stimulate hydrocarbon degradation. All isolates, except one, belonged to the genus Pseudomonas. The one exception was a member of the Microbacteriaceae, which was also, coincidentally, the only isolate negative for the nirS gene. The diversity of the 16S rRNA and nosZ genes was assessed by denaturing gradient gel electrophoresis and sequencing. There was a slight correlation between the 16S rRNA and nosZ operational taxonomic units. Surprisingly, many isolates contained nosZ on plasmids and, to the best of our knowledge, this is the first report of nosZ being extra-chromosomally present in Pseudomonas spp.     

Antifungal activity of Arctic and Antarctic bacteria isolates

Various psychrotolerant bacterial strains with broad-spectrum antimicrobial potential were isolated from a number of Antarctic and Arctic samples (lake sediments, water, as well as faeces, feathers and soils collected in penguin rookeries). Seven isolates from all types of samples exhibited clear antifungal activities against the multidrug-resistant pathogenic yeast strain Candida albicans NCIM 3471. One isolate from the penguin rookery, identified by means of 16S rDNA sequencing as a strain of Enterococcus faecium, showed very strong antimycotic activity against a total of six C. albicans strains. The antibiotic activity was found after ammonium sulphate precipitation and dialysis but was sensitive to proteolytic enzymes.     

Cloning of complete genes for novel hydrolytic enzymes from Antarctic sea water bacteria by use of an improved genome walking technique

The increased demand for enzymes with new properties makes indispensable the development of easy and rapid strategies to obtain complete genes of new enzymes. Here a strategy is described which includes screening by PCR of new subtilases mediated by Consensus-Degenerate Hybrid Oligonucleotide Primers (CODEHOP) and an improved genome walking method to obtain the complete sequence of the identified genes. Existing methods of genome walking have many limitations, which make them inefficient and time consuming. We have developed an improved genome walking method with novel advances to get a simple, rapid and more efficient procedure based on cassette-ligation. Improvements consist basically in the possibility of a genomic DNA digestion with any restriction enzyme, blunting and 3' adenylation of digested DNA by Taq DNA polymerase to avoid self-circularization, followed by TA ligation of the adenine 3' overhanging end to the same unphosphorylated oligo-cassette. The efficiency of the genome walking method was demonstrated by finding the unknown ends of all gene fragments tested, previously obtained by CODEHOP-mediated PCR, including three subtilases (P4, P6 and P7), one xylanase and one lipase, from different strains of Antarctic marine bacteria.     

Antimycobacterial activity in vitro of pigments isolated from Antarctic bacteria

In this study, we describe the antimycobacterial activity of two pigments, violacein, a purple violet pigment from Janthinobacterium sp. Ant5-2 (J-PVP), and flexirubin, a yellow-orange pigment from Flavobacterium sp. Ant342 (F-YOP). These pigments were isolated from bacterial strains found in the land-locked freshwater lakes of Schirmacher Oasis, East Antarctica. The minimum inhibitory concentrations (MICs) of these pigments for avirulent and virulent mycobacteria were determined by the microplate Alamar Blue Assay (MABA) and Nitrate Reductase Assay (NRA). Results indicated that the MICs of J-PVP and F-YOP were 8.6 and 3.6 μg/ml for avirulent Mycobacterium smegmatis mc²155; 5 and 2.6 μg/ml for avirulent Mycobacterium tuberculosis mc²6230; and 34.4 and 10.8 μg/ml for virulent M. tuberculosis H₃₇Rv, respectively. J-PVP exhibited a ~15 times lower MIC for Mycobacterium sp. than previously reported for violacein pigment from Chromobacterium violaceum, while the antimycobacterial effect of F-YOP remains undocumented. Our results indicate these pigments isolated from Antarctic bacteria might be valuable lead compounds for new antimycobacterial drugs used for chemotherapy of tuberculosis.     

Searching pathogenic bacteria in the rare biosphere of the ocean

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Degradation of nonane by bacteria from Antarctic marine sediment

A microbial enrichment culture tolerant of petroleum hydrocarbons was developed from sediment collected near Casey Station, Antarctica. To select cold-adapted microbes that would degrade diesel, enrichments were cultured at 0°C during six successive transfers to fresh medium, with Special Antarctic Blend diesel (SAB) as the sole carbon source. Biodegradation of components of the SAB was then measured in microcosms inoculated with the enrichment culture. After 16 weeks, the amount of biodegradation was small, but nonane (a C9 alkane) had degraded significantly more in inoculated microcosms than in sterile controls. DNA was then extracted from the enrichment cultures and a fragment of the 16S rRNA gene was amplified for denaturing gradient gel electrophoresis. Bands were excised from the gel and, following sequencing, were found to belong to the genera Pseudomonas and Colwellia.     

Diversity and association of psychrophilic bacteria in Antarctic sea ice.

The bacterial populations associated with sea ice sampled from Antarctic coastal areas were investigated by use of a phenotypic approach and a phylogenetic approach based on genes encoding 16S rRNA (16S rDNA). The diversity of bacteria associated with sea ice was also compared with the bacterial diversity of seawater underlying sea ice. Psychrophilic (optimal growth temperature, < or = 15 degrees C; no growth occurring at 20 degrees C) bacterial diversity was found to be significantly enriched in sea ice samples possessing platelet and bottom ice diatom assemblages, with 2 to 9 distinct (average, 5.6 +/- 1.8) psychrophilic taxa isolated per sample. Substantially fewer psychrophilic isolates were recovered from ice cores with a low or negligible population of ice diatoms or from under-ice seawater samples (less than one distinct taxon isolated per sample). In addition, psychrophilic taxa that were isolated from under-ice seawater samples were in general phylogenetically distinct from psychrophilic taxa isolated from sea ice cores. The taxonomic distributions of psychrotrophic bacterial isolates (optimal growth temperature, > 20 degrees C; growth can occur at approximately 4 degrees C) isolated from sea ice cores and under-ice seawater were quite similar. Overall, bacterial isolates from Antarctic sea ice were found to belong to four phylogenetic groups, the alpha and gamma subdivisions of the Proteobacteria, the gram-positive branch, and the Flexibacter-Bacteroides-Cytophaga phylum. Most of the sea ice strains examined appeared to be novel taxa based on phylogenetic comparisons, with 45% of the strains being psychrophilic. 16S rDNA sequence analysis revealed that psychrophilic strains belonged to the genera Colwellia, Shewanella, Marinobacter, Planococcus, and novel phylogenetic lineages adjacent to Colwellia and Alteromonas and within the Flexibacter-Bacteroides-Cytophaga phylum. Psychrotrophic strains were found to be members of the genera Pseudoalteromonas, Psychrobacter, Halomonas, Pseudomonas, Hyphomonas, Sphingomonas, Arthrobacter, Planococcus, and Halobacillus. From this survey, it is proposed that ice diatom assemblages provide niches conducive to the proliferation of a diverse array of psychrophilic bacterial species.     

Antarctic desert soil bacteria exhibit high novel natural product potential,evaluated through long-read genome sequencing and comparative genomics

Actinobacteria and Proteobacteria are important producers of bioactive natural products (NP), and these phyla dominate in the arid soils of Antarctica, where metabolic adaptations influence survival under harsh conditions. Biosynthetic gene clusters (BGCs) which encode NPs, are typically long and repetitious high G + C regions difficult to sequence with short-read technologies. We sequenced 17 Antarctic soil bacteria from multi-genome libraries, employing the long-read PacBio platform, to optimize capture of BGCs and to facilitate a comprehensive analysis of their NP capacity. We report 13 complete bacterial genomes of high quality and contiguity, representing 10 different cold-adapted genera including novel species. Antarctic BGCs exhibited low similarity to known compound BGCs (av. 31%), with an abundance of terpene, non-ribosomal peptide and polyketide-encoding clusters. Comparative genome analysis was used to map BGC variation between closely related strains from geographically distant environments. Results showed the greatest biosynthetic differences to be in a psychrotolerant Streptomyces strain, as well as a rare Actinobacteria genus, Kribbella, while two other Streptomyces spp. were surprisingly similar to known genomes. Streptomyces and Kribbella BGCs were predicted to encode antitumour, antifungal, antibacterial and biosurfactant-like compounds, and the synthesis of NPs with antibacterial, antifungal and surfactant properties was confirmed through bioactivity assays.