Ana Paula Chung

Deinococcus geothermalis sp. nov. and Deinococcus murrayi sp. nov., two extremely radiation-resistant and slightly thermophilic species from hot springs

Strains of Deinococcus geothermalis sp. nov. were isolated from the hot spring and runoff at Agnano, Naples, Italy, and from the hot spring at São Pedro do Sul in central Portugal, while strains of Deinococcus murrayi sp. nov. were isolated from the hot springs at São Pedro do Sul, São Gemil, and Alcafache in central Portugal. The strains of D. geothermalis and D. murrayi produce orange-pigmented colonies and have an optimum growth temperature of about 45 to 50 degrees C. The type strains of the two new species are extremely gamma radiation resistant. The fatty acids of these new species are primarily branched-chain fatty acids. The two new species can be distinguished from each other by the lower pH range of D. geothermalis than of D. murrayi, by their fatty acid compositions, and by several biochemical parameters, including the ability of D. geothermalis to grow in minimal medium without yeast extract. 16S rRNA gene sequencing also showed that the isolates constitute two species and that these species are distinct from the other species of the genus Deinococcus. The type strain of D. geothermalis is AG-3a (= DSM 11300), and the type strain of D. murrayi is ALT-1b (= DSM 11303).

Bacterial Diversity in a Nonsaline Alkaline Environment: Heterotrophic Aerobic Populations

ABSTRACT Heterotrophic populations were isolated and characterized from an alkaline groundwater environment generated by active serpentinization, which results in a Ca(OH)2-enriched, extremely diluted groundwater with pH 11.4. One hundred eighty-five strains were isolated in different media at different pH values during two sampling periods. To assess the degree of diversity present in the environment and to select representative strains for further characterization of the populations, we screened the isolates by using random amplified polymorphic DNA-PCR profiles and grouped them based on similarities determined by fatty acid methyl ester analysis. Phenotypic characterization, determinations of G+C content, phylogenetic analyses by direct sequencing of 16S rRNA genes, and determinations of pH tolerance were performed with the selected isolates. Although 38 different populations were identified and characterized, the vast majority of the isolates were gram positive with high G+C contents and were affiliated with three distinct groups, namely, strains closely related to the species Dietzia natrolimnae (32% of the isolates), to Frigoribacterium/Clavibacter lineages (29% of the isolates), and to the type strain of Microbacterium kitamiense (20% of the isolates). Other isolates were phylogenetically related to strains of the genera Agrococcus, Leifsonia, Kytococcus, Janibacter, Kocuria, Rothia, Nesterenkonia, Citrococcus, Micrococcus, Actinomyces, Rhodococcus, Bacillus, and Staphylococcus. Only five isolates were gram negative: one was related to the Sphingobacteria lineage and the other four were related to the α-Proteobacteria lineage. Despite the pH of the environment, the vast majority of the populations were alkali tolerant, and only two strains were able to grow at pH 11.

The Chromate-Inducible chrBACF Operon from the Transposable Element TnOtChr Confers Resistance to Chromium(VI) and Superoxide

Large-scale industrial use of chromium(VI) has resulted in widespread contamination with carcinogenic chromium(VI). The abilities of microorganisms to survive in these environments and to detoxify chromate require the presence of specific resistance systems. Here we report identification of the transposon-located (TnOtChr) chromate resistance genes from the highly tolerant strain Ochrobactrum tritici 5bvl1 surviving chromate concentrations of >50 mM. The 7,189-bp-long TnOtChr of the mixed Tn21/Tn3 transposon subfamily contains a group of chrB, chrA, chrC, and chrF genes situated between divergently transcribed resolvase and transposase genes. The chrB and chrA genes, but not chrF or chrC, were essential for establishment of high resistance in chromium-sensitive O. tritici. The chr promoter was strongly induced by chromate or dichromate, but it was completely unresponsive to Cr(III), oxidants, sulfate, or other oxyanions. Plasmid reporter experiments identified ChrB as a chromate-sensing regulator of chr expression. Induction of the chr operon suppressed accumulation of cellular Cr through the activity of a chromate efflux pump encoded by chrA. Expression of chrB, chrC, or chrF in an Escherichia coli sodA sodB double mutant restored its aerobic growth in minimal medium and conferred resistance to superoxide-generating agents menadione and paraquat. Nitroblue tetrazolium staining on native gels showed that ChrC protein had superoxide dismutase activity. TnOtChr appears to represent a mobile genetic system for the distribution of the chromate-regulated resistance operon. The presence of three genes protecting against superoxide toxicity should provide an additional survival advantage to TnOtChr-containing cells in the environments with multiple redox-active contaminants.

Sequencing and expression of two arsenic resistance operons with different functions in the highly arsenic-resistant strain Ochrobactrum tritici SCII24T

BackgroundArsenic (As) is a natural metalloid, widely used in anthropogenic activities, that can exist in different oxidation states. Throughout the world, there are several environments contaminated with high amounts of arsenic where many organisms can survive. The most stable arsenical species are arsenate and arsenite that can be subject to chemically and microbiologically oxidation, reduction and methylation reactions. Organisms surviving in arsenic contaminated environments can have a diversity of mechanisms to resist to the harmful effects of arsenical compounds.ResultsThe highly metal resistant Ochrobactrum tritici SCII24 was able to grow in media with arsenite (50 mM), arsenate (up to 200 mM) and antimonite (10 mM). This strain contains two arsenic and antimony resistance operons (ars 1 and ars 2), which were cloned and sequenced. Sequence analysis indicated that ars 1 operon contains five genes encoding the following proteins: ArsR, ArsD, ArsA, CBS-domain-containing protein and ArsB. The ars 2 operon is composed of six genes that encode two other ArsR, two ArsC (belonging to different families of arsenate reductases), one ACR3 and one ArsH-like protein. The involvement of ars operons in arsenic resistance was confirmed by cloning both of them in an Escherichia coli ars-mutant. The ars 1 operon conferred resistance to arsenite and antimonite on E. coli cells, whereas the ars 2 operon was also responsible for resistance to arsenite and arsenate. Although arsH was not required for arsenate resistance, this gene seems to be important to confer high levels of arsenite resistance. None of ars 1 genes were detected in the other type strains of genus Ochrobactrum, but sequences homologous with ars 2 operon were identified in some strains.ConclusionA new strategy for bacterial arsenic resistance is described in this work. Two operons involved in arsenic resistance, one giving resistance to arsenite and antimonite and the other giving resistance to arsenate were found in the same bacterial strain.

Alicyclobacillus hesperidum sp. nov. and a related genomic species from solfataric soils of São Miguel in the Azores.

Several acidophilic, slightly thermophilic or thermophilic Gram-positive isolates were recovered from solfataric soil at Furnas on the Island of São Miguel in the Azores. Phylogenetic analysis of the 16S rRNA gene sequence showed that these organisms represented two novel species of the genus Alicyclobacillus. Strains FR-11T and FR-1b had an optimum growth temperature of about 50 degrees C, whereas strains FR-3 and FR-6T had an optimum growth temperature of about 60 degrees C. Biochemical, physiological and chemotaxonomic characteristics did not distinguish isolates FR-3 and FR-6T from the type strain of Alicyclobacillus acidocaldarius; however, strains FR-11T and FR-1b could be easily distinguished from the type strain of Alicyclobacillus acidoterrestris by the carbon source assimilation pattern and the fatty acid composition. On the basis of the phylogenetic analysis, physiological and biochemical characteristics, and fatty acid composition the name Alicyclobacillus hesperidum is proposed for the species represented by strains FR-11T and FR-1b; a formal name for the new genomic species represented by strains FR-3 and FR-6T is not proposed at this time.

Meiothermus cerbereus sp. nov., a New Slightly Thermophilic Species with High Levels of 3-Hydroxy Fatty Acids

Strains of Meiothermus cerbereus sp. nov. were isolated from the hot springs within the Geysir geothermal area of Iceland. The strains of Meiothermus cerbereus produce red-orange-pigmented colonies, have an optimum growth temperature of about 55 degrees C, and have higher levels of 3-OH fatty acids than the strains of the other species of the genus Meiothermus. These strains, unlike all other strains of the species of the genus Meiothermus examined previously, required cysteine, thiosulfate, or thioglycolate for growth in liquid Thermus medium, but not in the corresponding medium solidified with agar. Several strains belonging to Meiothermus silvanus, isolated from Geysir, also required reduced sulfur compounds for growth in liquid medium, leading to the hypothesis that this requirement is not a taxonomic characteristic of the new species. The new species represented by strains GY-1T and GY-5 can be distinguished from the other species of the genus Meiothermus by biochemical characteristics, fatty acid composition, DNA-DNA reassociation values, and 16S ribosomal DNA sequence. The type strain for Meiothermus cerbereus is GY-1 (= DSM 11376).

Thermus igniterrae sp. nov. and Thermus antranikianii sp. nov., two new species from Iceland

Several yellow-pigmented isolates, with optimum growth temperatures of about 65-70 degrees C, were recovered from hot springs in Iceland. Phylogenetic analysis of the 16S rDNA and DNA-DNA reassociation values showed that these organisms represented two new species of the genus Thermus. Strains RF-4T and HN1-8 had maximum temperatures for growth below 80 degrees C, while strains HN3-7T and HN2-7, unlike all other strains of the species of the genus Thermus except those belonging to Thermus thermophilus, grew at 80 degrees C. The new isolates from Iceland could not be distinguished easily from each other or from other strains of the species of the genus Thermus by biochemical characteristics; however, strains RF-4T and HN1-8 assimilated ribitol, a characteristic which was not detected in any of the other strains examined. Moreover, the species represented by strains RF-4T and HN1-8 and the species represented by strains HN3-7T and HN2-7 could be distinguished clearly from the other species of Thermus by their fatty acid composition. Strains RF-4T and HN1-8 have the highest combined levels of iso-15:0 and iso-17:0 and the lowest levels of iso-16:0 of any of the strains of the species of Thermus, while strains HN3-7T and HN2-7 are characterized by a very low iso-15:0/iso-17:0 ratio. On the basis of the phylogenetic analysis, DNA-DNA reassociation values, physiological and biochemical characteristics and fatty acid composition, the name Thermus igniterrae sp. nov. is proposed for the species represented by strains RF-4T and HN1-8 and the name Thermus antranikianii sp. nov. is proposed for the species represented by strains HN3-7T and HN2-7.

Identification of an aox System That Requires Cytochrome c in the Highly Arsenic-Resistant Bacterium Ochrobactrum tritici SCII24

ABSTRACT Microbial biotransformations have a major impact on environments contaminated with toxic elements, including arsenic, resulting in an increasing interest in strategies responsible for how bacteria cope with arsenic. In the present work, we investigated the metabolism of this metalloid in the bacterium Ochrobactrum tritici SCII24. This heterotrophic organism contains two different ars operons and is able to oxidize arsenite to arsenate. The presence of arsenite oxidase genes in this organism was evaluated, and sequence analysis revealed structural genes for an As(III) oxidase (aoxAB), a c-type cytochrome (cytC), and molybdopterin biosynthesis (moeA). Two other genes coding for a two-component signal transduction pair (aoxRS) were also identified upstream from the previous gene cluster. The involvement of aox genes in As(III) oxidation was confirmed by functionally expressing them into O. tritici 5bvl1, a non-As(III) oxidizer. Experiments showed that the As(III) oxidation process in O. tritici requires not only the enzyme arsenite oxidase but also the cytochrome c encoded in the operon. The fundamental role of this cytochrome c, reduced in the presence of arsenite in strain SCII24 but not in an O. tritici ΔaoxB mutant, is surprising, since to date this feature has not been found in other organisms. In this strain the presence of an aox system does not seem to confer an additional arsenite resistance capability; however, it might act as part of an As(III)-detoxifying strategy. Such mechanisms may have played a crucial role in the development of early stages of life on Earth and may one day be exploited as part of a potential bioremediation strategy in toxic environments.

Hymenobacter perfusus sp. nov., Hymenobacter flocculans sp. nov. and Hymenobacter metalli sp. nov. three new species isolated from an uranium mine waste water treatment system

Three red-pink pigmented strains, designated A1-12(T), A2-50A(T) and A2-91(T), were recovered from two different sites in a uranium mine. For all strains, the optimum growth temperature was 25°C, the optimum pH was 6.0-6.5 and the DNA G+C contents were between 60 and 63.4 mol%. The major respiratory quinone was menaquinone 7 (MK-7) and the fatty acid profiles contained iso- and anteiso-branched C15 fatty acids, summed feature 3 (16:1 ω6c and/or ω7c and/or 15:0 iso 2-OH), summed feature 4 (17:1 anteiso B and/or iso I) and the unsaturated fatty acid 16:1 ω5c as the major components. Phylogenetic analysis of the 16S rRNA gene sequences showed that these organisms represented three distinct branches within the family Flexibacteraceae most closely related to the members of the genus Hymenobacter. Strain A1-12(T) formed a distinct phylogenetic line along with H. rigui KCTC 12533(T) and they shared approximately 98.9% 16S rRNA gene sequence similarity. However, these two strains shared only 14.7% pairwise similarity in their genomic DNA. Strains A2-50A(T) and A2-91(T) formed two distinct lineages, related to the species H. soli KCTC 12607(T), sharing about 95.5% 16S rRNA gene sequence similarity between themselves, and 88.3 and 92.0% with other members of the genus Hymenobacter. Based on the phylogenetic analysis and physiological and biochemical characteristics, these isolates were considered to represent three novel species for which we propose the names Hymenobacter perfusus for strain A1-12(T) (=CIP 110166=LMG 26000), Hymenobacter flocculans for strain A2-50A(T) (=CIP 110139=LMG 25699) and Hymenobacter metalli for strain A2-91(T) (=CIP 110140=LMG 25700).

Roseomonas pecuniae sp. nov., isolated from the surface of a copper-alloy coin

Strain N75(T) was isolated from the surface of a copper-alloy 50 Euro cent coin collected from general circulation. Phylogenetic analysis of the 16S rRNA gene sequence revealed that strain N75(T) formed a distinct branch within the genus Roseomonas and placed it in the Alphaproteobacteria. Strain N75(T) showed 16S rRNA gene sequence similarities of 92.4-97.1  % to type strains of species of the genus Roseomonas. Strain N75(T) was a Gram-negative, non-spore-forming, non-motile coccoid, with an optimum growth temperature of about 30 °C; the strain did not grow at 5 or 37 °C. Strain N75(T) did not grow in medium containing NaCl. The major respiratory quinone was ubiquinone 10 (Q-10). The major fatty acids were unsaturated C₁₆:₁ω7c/C₁₆:₁ω6c and C₁₈:₁ω7c (around 70 % of the total fatty acids); the third most abundant fatty acid was the hydroxylated C₁₈:₁ 2-OH. The major polar lipids were phosphatidylcholine, phosphatidylethanolamine and an unknown aminolipid. The DNA G+C content was 72.8 mol%. On the basis of the phylogenetic analysis and physiological and biochemical characteristics, we conclude that strain N75(T) represents a novel species of the genus Roseomonas, for which we propose the name Roseomonas pecuniae sp. nov. (type strain N75(T) =LMG 25481(T) =CIP 110074(T)).