Xiao-Yan You

Alicyclobacillus ferrooxydans sp. nov., a ferrous-oxidizing bacterium from solfataric soil

A novel mesophilic and acidophilic Gram-positive bacterium, designated strain TC-34(T), was isolated from solfataric soil. Strain TC-34(T) grew aerobically at 17-40 degrees C and pH 2.0-6.0, and optimally at 28 degrees C and pH 3.0. Analysis based on 16S rRNA gene sequences showed that strain TC-34(T) was phylogenetically related to members of the genus Alicyclobacillus, with the highest similarity (94.8 %) to Alicyclobacillus pomorum. Strain TC-34(T) showed a range of phenotypic characteristics that differentiated it from recognized Alicyclobacillus species, including growth temperature, assimilation of carbon sources and production of acids from a range of compounds. Strain TC-34(T) was able to oxidize ferrous iron and its growth was correlated with the oxidation of Fe(2+) in culture medium. omega-Alicyclic fatty acids were not detected. On the basis of these results, it was concluded that strain TC-34(T) represents a novel species of the genus Alicyclobacillus, for which the name Alicyclobacillus ferrooxydans (type strain TC-34(T)=JCM 15090(T)=CGMCC 1.6357(T)) is proposed.

Alicyclobacillus aeris sp nov., a novel ferrous- and sulfur-oxidizing bacterium isolated from a copper mine

A novel mesophilic, acidophilic, endospore-forming bacterium, designated strain ZJ-6(T), was isolated from Zi-Jin copper mine in Inner Mongolia, China. Cells of strain ZJ-6(T) were rod-shaped, stained Gram-positive or were Gram-variable, and grew aerobically at 25-35 degrees C (optimum, 30 degrees C) and pH 2.0-6.0 (optimum, pH 3.5). 16S rRNA gene sequence analysis showed that strain ZJ-6(T) was related phylogenetically to members of the genus Alicyclobacillus, with 16S rRNA gene sequence similarities of 89.5-94.2 %. Cells contained MK-7 as the major quinone and the DNA G+C content was 51.2 mol%. Strain ZJ-6(T) possessed a number of phenotypic characteristics that differentiated it from recognized Alicyclobacillus species, including its growth temperature, assimilation of various carbon sources, production of acids from a range of compounds, and the ability to grow chemoautotrophically using ferrous iron, elemental sulfur and tetrathionate as electron donors. The predominant cellular fatty acids of strain ZJ-6(T) were anteiso-C(15 : 0) (67.1 %), iso-C(16 : 0) (7.7 %) and anteiso-C(17 : 0) (7.4 %); omega-alicyclic fatty acids were not found. On the basis of these results, it is concluded that strain ZJ-6(T) represents a novel species within the genus Alicyclobacillus, for which the name Alicyclobacillus aeris sp. nov. is proposed; the type strain is ZJ-6(T) (=CGMCC 1.7072(T)=NBRC 104953(T)).

Complete Genome Sequence of Metallosphaera cuprina, a Metal Sulfide-Oxidizing Archaeon from a Hot Spring

The genome of the metal sulfide-oxidizing, thermoacidophilic strain Metallosphaera cuprina Ar-4 has been completely sequenced and annotated. Originally isolated from a sulfuric hot spring, strain Ar-4 grows optimally at 65°C and a pH of 3.5. The M. cuprina genome has a 1,840,348-bp circular chromosome (2,029 open reading frames [ORFs]) and is 16% smaller than the previously sequenced Metallosphaera sedula genome. Compared to the M. sedula genome, there are no counterpart genes in the M. cuprina genome for about 480 ORFs in the M. sedula genome, of which 243 ORFs are annotated as hypothetical protein genes. Still, there are 233 ORFs uniquely occurring in M. cuprina. Genome annotation supports that M. cuprina lives a facultative life on CO(2) and organics and obtains energy from oxidation of sulfidic ores and reduced inorganic sulfuric compounds.

Metallosphaera cuprina sp. nov., an acidothermophilic, metal-mobilizing archaeon

A novel acidothermophilic archaeon, strain Ar-4(T), was isolated from a sulfuric hot spring in Tengchong, Yunnan, China. Cells of strain Ar-4(T) were Gram-staining-negative, irregular cocci and motile by means of flagella. Strain Ar-4(T) grew over a temperature range of 55-75 °C (optimum, 65 °C), a pH range of 2.5-5.5 (optimum, pH 3.5) and a NaCl concentration range of 0-1 % (w/v). The novel strain was aerobic and facultatively chemolithoautotrophic. The strain could extract metal ions from sulfidic ore. It was also able to oxidize reduced sulfur compounds. In addition, it was able to use heterogeneous organic materials for organotrophic growth. The main cellular lipids were calditoglycerocaldarchaeol (CGTE) and caldarchaeol (DGTE). The DNA G+C content of the strain was 40.2 mol%. Analysis of 16S rRNA gene sequences showed that strain Ar-4(T) was phylogenetically related to members of the genus Metallosphaera and had sequence similarities of 97.7 %, 97.0 % and 96.8 % with Metallosphaera hakonensis DSM 7519(T), Metallosphaera sedula DSM 5348(T) and Metallosphaera prunae DSM 10039(T), respectively. Strain Ar-4(T) showed DNA-DNA relatedness values of 47.5 %, 30.8 % and 29.1 % with M. hakonensis DSM 7519(T), M. sedula DSM 5348(T) and M. prunae DSM 10039(T), respectively. The differences in cell motility, the temperature and pH ranges for growth, the ability to utilize carbon sources, the DNA G+C content, and the low DNA-DNA relatedness values distinguished strain Ar-4(T) from recognized species of the genus Metallosphaera. On the basis of these results, it was concluded that strain Ar-4(T) represents a novel species of the genus Metallosphaera, for which the name Metallosphaera cuprina is proposed. The type strain is Ar-4(T) ( = JCM 15769(T) = CGMCC 1.7082(T)).

Construction and application of an expression vector from the new plasmid pLAtc1 of Acidithiobacillus caldus

In this study, a recently sequenced 9.8-kb plasmid, pLAtc1, from Acidithiobacillus caldus strain SM-1 was characterized and developed into an expression vector. The pLAtc1 backbone carried an oriV, three rep genes, five mob genes, a Nic site, and an addiction system. Multilocus sequence analysis indicated that pLAtc1 was phylogenetically more related to the IncQ-like broad host range plasmids than to other IncQ plasmids. pLAtc1 was able to replicate and reside in Gram-negative Escherichia coli, Comamonas testosteroni, but not in Gram-positive Corynebacterium glutamicum. pLAtc1 was mobilized via conjugation into E. coli BL21 and A. caldus SM-1 from E. coli S17-1. Quantitative PCR revealed seven and four copies of plasmid in A. caldus and E. coli cells, respectively. The expression vector pLAtcE was constructed from pLAtc1 by introducing a regulatable promoter (PtetH), a transcriptional terminator, a multiple cloning site, a kanamycin resistance gene, and a streptomycin resistance gene. The functionality of pLAtcE was demonstrated by expressing a gene encoding enhanced green fluorescence protein in E. coli and in A. caldus. pLAtcE was used to express α-ketoglutarate dehydrogenase (sucAB) and succinate dehydrogenase (sdhA) genes in A. caldus. The newly engineered strain that harbored sucAB and sdhA on a plasmid pLAtcE-sucA-sucB-sdhA grew better than the parent strain SM-1/pLAtcE in tetrathionate and glucose-supplemented medium and produced more acidity and resulted in a more oxidative environment. This study created a useful molecular tool for genetic manipulation of the thermoacidophilic and autotrophic A. caldus.

Resolution of carbon metabolism and sulfur-oxidation pathways of Metallosphaera cuprina Ar-4 via comparative proteomics.

UNLABELLED Metallosphaera cuprina is able to grow either heterotrophically on organics or autotrophically on CO2 with reduced sulfur compounds as electron donor. These traits endowed the species desirable for application in biomining. In order to obtain a global overview of physiological adaptations on the proteome level, proteomes of cytoplasmic and membrane fractions from cells grown autotrophically on CO2 plus sulfur or heterotrophically on yeast extract were compared. 169 proteins were found to change their abundance depending on growth condition. The proteins with increased abundance under autotrophic growth displayed candidate enzymes/proteins of M. cuprina for fixing CO2 through the previously identified 3-hydroxypropionate/4-hydroxybutyrate cycle and for oxidizing elemental sulfur as energy source. The main enzymes/proteins involved in semi- and non-phosphorylating Entner-Doudoroff (ED) pathway and TCA cycle were less abundant under autotrophic growth. Also some transporter proteins and proteins of amino acid metabolism changed their abundances, suggesting pivotal roles for growth under the respective conditions. BIOLOGICAL SIGNIFICANCE The described work is of great significance: For general microbiology: How do extremophile organisms use their unique metabolic capabilities in adapting to autotrophic and hetetrotrophic growth conditions? Which are important enzymes involved in the metabolic adaptation and which enzyme candidate should be investigated in more detail with microbiological/biochemical approaches? For applied microbiology: Which are the key enzymes and reaction pathways for sulfur oxidation and autotrophic growth? This knowledge should accelerate future design of improved bioleaching processes in biomining industries or bioremediation.

Structural Basis for the Thermostability of Sulfur Oxygenase Reductases

Abstract The thermostability of three sulfur oxygenase reductases (SORs) was investigated from thermoacidophilic achaea Acidianus tengchongensis (SOR AT ) and Sulfolobus tokodaii (SOR ST ) as well as the moderately thermophilic bacterium Acidithiobacillus sp. SM-1 (SOR SB ). The optimal temperatures for catalyzing sulfur oxidation were 80 °C (SOR AT ), 85 °C (SOR ST ), and 70 °C (SOR SB ), respectively. The half-lives of the three SORs at their optimal catalytic conditions were 100 min (SOR AT ), 58 min (SOR ST ), and 37 min (SOR SB ). In order to reveal the structural basis of the thermostability of these SORs, three-dimensional structural models of them were generated by homology modeling using the previously reported high-resolution X-ray structure of SOR AA (from Acidianus ambivalens ) as a template. The results suggest that thermostability was dependent on: (a) high number of the charged amino acid glutamic acid and the flexible amino acid proline, (b) low number of the thermolabile amino acid glutamine, (c) increased number of ion pairs, (d) decreased ratio of hydrophobic accessible solvent surface area (ASA) to charged ASA, and (e) increased volumes of the cavity. The number of cavities and the number of hydrogen bonds did not significantly affect the thermostability of SORs, whereas the cavity volumes increased as the thermal stability increased.

Some Physiological Properties of Three Novel Fe(II)- and Sulfur-Oxidizing Strains Affiliated to Alicyclobacillus

This study focused on the soils of sofataric region and acid mine drainage from a copper mine. Based on cultivation, 8 and 6 strains that grow on Fe(II) and sulfur compounds, respectively, were obtained from samples from these two environments. Analysis of 16S rRNA genes of the 14 strains indicated that they were affiliated to Acidithiobacillus, Alicyclobacillus, Sulfobacillus and Leptospirillum. Physiological and phylogenetic studies indicated that three strains (TC-34, TC-71 and ZJ-6) might represent three novel members of Alicyclobacillus. These strains showed 94.8-97.1% 16S rRNA gene identity to other species of Alicyclobacillus. Otherwise, strain TC-34, TC-71 and ZJ-6 showed a range of phenotypic characteristics that differentiated them from previously recognized Alicyclobacillus species, including the growth temperature, assimilation of carbon sources and production of acids from a range of compounds. Chemoautotrophic growth using Fe2+, elemental sulfur and tetrathionate as sole energy source was observed. Especially strain TC-71 was obligately dependent on Fe(II) for growth and quickly oxidized Fe2+. It is concluded that the Fe(II)-oxidizers are metabolically diverse and represent novel Alicyclobacillus species. These are proposed to take part in biogeochemical cycling of iron and sulfur in the solfataric region and could be relevant for biomining.