Sulfur metabolism in Thiorhodaceae. V. Enzymes of sulfur metabolism inThiocapsa floridana andChromatium species

In extracts ofThiocapsa floridana strain 6311 andChromatium strains 1611, 2811 and 6412 a high specific activity of adenosine 5-phosphosulfate reductase was found. In contrast, little activity of this enzyme was found inChromatium strain D.Adenosine 5-diphosphate sulfurylase is present in extracts fromThiocapsa floridana strain 6311. The enzyme appears to be involved in the oxidation of reduced sulfur compounds to sulfate.Sulfite oxidase could not be demonstrated in any of the strains examined.     

Sulfur metabolism in Thiorhodaceae. IV. Assimilatory reduction of sulfate byThiocapsa floridana andChromatium species

Thiocapsa floridana strain 1711, andChromatium strains 1611 and 6412 can grow with molecular hydrogen replacing sulfide as the electron donor. Sulfate suffices as the sulfur source. The incorporation of radioactive sulfur from³⁵S-sulfate was measured in growing cells in which molecular hydrogen or acetate was the electron donor. In cells pre-grown in sulfide, the incorporation of radioactivity began slowly after a lag period; in contrast, cells grown in sulfate took up the marker at a faster rate and without a lag. The radioactivity appeared in protein as cysteine and methionine. No elimination of sulfide was detected during growth. Thus, the reduction of sulfate was purely assimilatory.     

Anoxygenic phototrophic bacterial community of Lake Shira (Khakassia)

The anoxygenic phototrophic bacterial community of the brackish meromictic Lake Shira (Khakassia) was investigated in August 2001, July 2002, and February–March 2003. In all the periods of investigation, the prevailing microorganisms were purple sulfur bacteria similar to Lamprocystis purpurea in morphology and pigment composition. Their highest number (3 × 10⁵ cells/ml) was recorded in July 2002 at the depth of 15 m. According to 16S rRNA gene analysis, the strain of purple sulfur bacteria isolated in 2001 and designated ShAm01 exhibited 98.6% similarity to the type strain of Thiocapsa roseopersicina and 97.1–94.4% similarity to the type strains of Tca. pendens, Tca. litoralis, and Tca. rosea. The minor microorganisms of the anoxygenic phototrophic bacterial community within the period of investigation were nonsulfur purple bacteria phylogenetically close to Rhodovulum strictum (98.3% similarity, strain ShRb01), Ahrensia kielensis (of 93.9% similarity, strain ShRb02), Rhodomicrobium vannieli (of 99.7% similarity, strain ShRmc01), and green sulfur bacteria, phylogenetically close to Chlorobium limicola (of 98.7% similarity, strain ShCl03).     

Evidence for the hybrid origin of Flaveria latifolia (Compositae)

Investigations of natural populations, cytological studies, and comparisons of herbarium specimens have been used to clarify the taxonomy of the Florida species ofFlaveria. These studies indicate thatF. latifolia is a hybrid betweenF. linearis andF. floridana. Apparently the “latifolia” plants represent stabilized populations that have become adapted to the fill material dredged from the ocean and used in road building and land development in southern Florida. The wide variation noted in populations ofF. linearis andF. floridana is attributed to introgression. The paper includes a taxonomic treatment of the six species which occur in Florida. AU taxa are diploid with 2n = 36.     

Selection for novel,acid-tolerant <Emphasis Type="Italic">Desulfovibrio</Emphasis> spp. from a closed Transbaikal mine site in a temporal pH-gradient bioreactor

Almost all the known isolates of acidophilic or acid-tolerant sulphate-reducing bacteria (SRB) belong to the spore-forming genus Desulfosporosinus in the Firmicutes. The objective of this study was to isolate acidophilic/acid-tolerant members of the genus Desulfovibrio belonging to deltaproteobacterial SRB. The sample material originated from microbial mat biomass submerged in mine water and was enriched for sulphate reducers by cultivation in anaerobic medium with lactate as an electron donor. A stirred tank bioreactor with the same medium composition was inoculated with the sulphidogenic enrichment. The bioreactor was operated with a temporal pH gradient, changing daily, from an initial pH of 7.3 to a final pH of 3.7. Among the bacteria in the bioreactor culture, Desulfovibrio was the only SRB group retrieved from the bioreactor consortium as observed by 16S rRNA-targeted denaturing gradient gel electrophoresis. Moderately acidophilic/acid-tolerant isolates belonged to Desulfovibrio aerotolerans-Desulfovibrio carbinophilus-Desulfovibrio magneticus and Desulfovibrio idahonensis-Desulfovibrio mexicanus clades within the genus Desulfovibrio. A moderately acidophilic strain, Desulfovibrio sp. VK (pH optimum 5.7) and acid-tolerant Desulfovibrio sp. ED (pH optimum 6.6) dominated in the bioreactor consortium at different time points and were isolated in pure culture.     

Phylogenetic characterization and novelty of organic sulphur metabolizing genes of <Emphasis Type="Italic">Rhodococcus</Emphasis> spp. (Eu-32)

Rhodococcus spp. (Eu-32) has the unique ability to metabolize organic sulphur containing compounds like dibenzothiophene through an extended sulphur specific pathway (Akhtar et al., in FEMS Microbiol Lett 301:95–102, 2009). Efforts were made to isolate and characterize the presumed desulphurizing genes (dszABC) involved in the sulphur specific pathway of isolate Eu-32 by employing standard and degenerate polymerase chain reaction primers. The partial dszA gene sequence of isolate Eu-32 showed 92 % sequence identity with a putative FMNH-2 dependent monooxygenase of Rhodococcus erythropolis PR4. The dszC gene sequence showed 99 % homology with the dibenzothiophene monooxygenase desulphurizing enzyme of another Rhodococcus species. The dszB gene was not unambiguously identified. A phylogenetic analysis by maximum likelihood method of the 16S rRNA gene and deduced DszA and C amino acid sequences suggest that horizontal gene transfer events might have taken place during the evolution of desulphurizing genes of Rhodococcus spp. (Eu-32).     

Growth of Thiocapsa roseopersicina purple sulfur bacteria in darkness under anaerobic conditions]

The phototrophic sulphur bacterium. Thiocapsa roseopersicina, strain BBS, was grown under anaerobic conditions in the darkness on the medium containing glucose and thiosulphate or molecular sulphur. The assimilation of glucose is accompanied by the accumulation of small amounts of pyruvate in the medium, and the uptake of thiosulphate or molecular sulphur leads to the formation of sulphates and hydrogen sulphide.     

Succession processes in the anoxygenic phototrophic bacterial community in Lake Kislo-Sladkoe (Kandalaksha Bay,White Sea)

The community of anoxygenic phototrophic bacteria (APB) in the water column of Lake Kislo- Sladkoe (Kandalaksha Bay, White Sea), which has recently become separated from the sea, was investigated in March?April 2012, March?April 2013, and in September 2013. The lake, which was previously considered meromictic, was in fact mixed and was strongly affected by the sea. In winter the lake is sometimes washed off with seawater, and this together with the seasonal cycles of succession processes determines the succession of the community. The consequences of the mixing in autumn 2011 could be observed in the APB community as late as autumn 2013. Green-colored green sulfur bacteria (GSB) usually predominated in the chemocline. In winter 2013 stagnation resulted in turbidity of water under the ice, which was responsible for both predominance of the brown GSB forms and the changes ratio of the species of purple sulfur bacteria (PSB) in anoxic water layers. Production of anoxygenic photosynthesis in the lake was at least 240 mg C m^-2 day^-1 in September and 0–20 mg C m^–2 day^–1 in March—April, which corresponded to 40 and 69%, respectively, of oxygenic photosynthesis. Okenone-containing purple sulfur bacteria, strain TcakPS12, were isolated in 2012 from lake water. The ells of this strain form filaments of not separated cells. Strain TcakPS12 exhibited 98% similarity with the type strains of Thiocapsa pendens DSM 236 and Thiocapsa bogorovii BBS, as well as with the strains AmPS10 and TcyrPS10, which were isolated from Lake Kislo-Sladkoe in 2010.     

<Emphasis Type="Italic">Dankookia rubra</Emphasis> gen. nov., sp. nov., an alphaproteobacterium isolated from sediment of a shallow stream

WS-10^T–a Gram-negative, non-motile, and aerobic bacterial strain–was isolated from the sediment of a shallow stream in Korea. The optimum ranges of temperature and pH for growth were 20–40°C (optimum 28°C) and pH 6.0–8.0 (optimum pH 7.0), respectively. The DNA G+C content of strain WS-10^T was 72.7 mol%. The major fatty acids (>5%) were summed feature 8 (C_18:1ω7c), summed feature 3 (C_16:1ω7c and/or C_16:1ω6c), C_16:0, and C_18:1 2-OH. The major polar lipids consisted of phosphatidylcholine, phosphatidylglycerol, phosphatidylethanolamine, and unidentified aminolipids. Q-10 was the predominant respiratory quinone. The highest similarities in the 16S rRNA gene sequence were shown with Paracraurococcus ruber (95.3%), Belnapia soli (95.3%), B. moabensis (95.1%), and B. rosea (95.0%). A phylogenetic analysis based on 16S rRNA gene sequence comparisons showed that strain WS-10T formed a distinct line within a clade containing the genera Paracraurococcus, Craurococcus, and Belnapia in the family Acetobacteraceae. On the basis of polyphasic evidence, strain WS-10^T represents a novel species of a new genus in the family Acetobacteraceae, for which the name Dankookia rubra gen. nov., sp. nov. is proposed. The type strain of the type species is WS-10^T (= KACC 18533^T = JCM 30602^T).     

Taxonomic specificity of the sensitivity to the <Emphasis Type="Italic">Wickerhamomyces bovis</Emphasis> fungistatic mycocin

Wickerhamomyces bovis type strain was found to secrete a mycocin with a fungistatic effect at pH from 3.5 to 6.0. The peak of its activity occurred at pH 5.0 in the presence of 3% NaCl. Yeast species sensitive to this mycocin were located within the family Wickerhamomycetaceae and belonged to phylogenetically related genera Ambrosiozyma, Nakazawaea, Ogataea, and Peterozyma.     

Enhanced pyruvate production in <Emphasis Type="Italic">Candida glabrata</Emphasis> by overexpressing the <Emphasis Type="Italic">CgAMD1</Emphasis> gene to improve acid tolerance

Objectives

To enhance acid tolerance of Candida glabrata for pyruvate production by engineering AMP metabolism.

Results

The physiological function of AMP deaminase in AMP metabolism from C. glabrata was investigated by deleting or overexpresseing the corresponding gene, CgAMD1. At pH 4, CgAMD1 overexpression resulted in 59 and 51% increases in biomass and cell viability compared to those of wild type strain, respectively. In addition, the intracellular ATP level of strain Cgamd1Δ/CgAMD1 was down-regulated by 22%, which led to a 94% increase in pyruvate production. Further, various strengths of CgAMD1 expression cassettes were designed, thus resulting in a 59% increase in pyruvate production at pH 4. Strain Cgamd1Δ/CgAMD1 _(H) was grown in a 30 l batch bioreactor at pH 4, and pyruvate reached 46.1 g/l.

Conclusion

CgAMD1 overexpression plays an active role in improving acid tolerance and pyruvate fermentation performance of C. glabrata at pH 4.

     

<Emphasis Type="Italic">Spirosoma luteolum</Emphasis> sp. nov. isolated from water

A novel Gram-negative and rod-shaped bacterial strain, designated as 16F6E^T, was isolated from a water sample. Cells were yellowish in color and catalase- and oxidase-positive. The strain grew at 10–37°C (optimum at 25°C) but not at 4 and 42°C, and pH 5–7 (optimum at pH 7). It showed moderate resistance to gamma-ray irradiation. Comparative phylogenetic analysis showed that strain 16F6E^T belonged to the family Cytophagaceae of the class Cytophagia. Furthermore, this isolate showed relatively low 16S rRNA gene sequence similarities (90.7–93.1%) to the members of the genus Spirosoma. The major fatty acids were summed feature 3 (C_16:1 ω7c/C_16:1 ω6c), C_16:1 ω5c, C_16:0 N alcohol, and C_16:0. The polar lipid profile indicated presence of phosphatidylethanolamine, unknown aminophospholipids, an unknown amino lipid, unknown phospholipids, and unknown polar lipids. The predominant isoprenoid quinone was MK-7. The genomic DNA G+C content of strain 16F6E^T was 56.5 mol%. Phenotypic, phylogenetic, and chemotaxonomic properties indicated that isolate 16F6E^T represents a novel species within the genus Spirosoma, for which the name Spirosoma luteolum sp. nov. is proposed. The type strain is 16F6E^T (=KCTC 52199^T =JCM 31411^T).     

Genome Mining and Predictive Functional Profiling of Acidophilic Rhizobacterium <Emphasis Type="Italic">Pseudomonas fluorescens</Emphasis> Pt14

Pseudomonas fluorescens Pt14 is a non-pathogenic and acidophilic bacterium isolated from acidic soil (pH 4.65). Genome sequencing of strain Pt14 was performed using Single Molecule Real Time (SMRT) sequencing to get insights into unique existence of this strain in acidic environment. Complete genome sequence of this strain revealed a chromosome of 5,841,722 bp having 5354 CDSs and 88 RNAs. Phylogenomic reconstruction based on 16S rRNA gene, Average Nucleotide Identity (ANI) values and marker proteins revealed that strain Pt14 shared a common clade with P. fluorescens strain A506 and strain SS101. ANI value of strain Pt14 in relation to strain A506 was found 99.23% demonstrating a very close sub-species association at genome level. Further, orthology determination among these three phylogenetic neighbors revealed 4726 core proteins. Functional analysis elucidated significantly higher abundance of sulphur metabolism (>1×) which could be one of the reasons for the survival of strain Pt14 under acidic conditions (pH 4.65). Acidophilic bacteria have capability to oxidize sulphur into sulphuric acid which in turn can make the soil acidic and genome-wide analysis of P. fluorescens Pt14 demonstrated that this strain contributes towards making the soil acidic.     

Functional analysis of arabinofuranosidases and a xylanase of <Emphasis Type="Italic">Corynebacterium alkanolyticum</Emphasis> for arabinoxylan utilization in <Emphasis Type="Italic">Corynebacterium glutamicum</Emphasis>

Xylooligosaccharides (XOSs) and arabinoxylooligosaccharides (AXOSs) are major oligosaccharides derived from arabinoxylan. In our previous report, Corynebacterium glutamicum was engineered to utilize XOSs by introducing Corynebacterium alkanolyticum xyloside transporter and β-xylosidase. However, this strain was unable to consume AXOSs due to the absence of α-l-arabinofuranosidase activity. In this study, to confer AXOS utilization ability on C. glutamicum, two putative arabinofuranosidase genes (abf51A and abf51B) were isolated from C. alkanolyticum by the combination of degenerate PCR and genome walking methods. Recombinant Abf51A and Abf51B heterologously expressed in Escherichia coli showed arabinofuranosidase activities toward 4-nitrophenyl-α-l-arabinofuranoside with k _cat values of 150 and 63, respectively, with optimum at pH 6.0 to 6.5. However, Abf51A showed only a slight activity toward AXOSs and was more susceptible to product inhibition by arabinose and xylose than Abf51B. Introduction of abf51B gene into the C. glutamicum XOS-utilizing strain enabled it to utilize AXOSs as well as XOSs. The xylI gene encoding a putative xylanase was found upstream of the C. alkanolyticum xyloside transporter genes. A signal peptide was predicted at the N-terminus of the xylI-encoding polypeptide, which indicated XylI was a secreted protein. Recombinant mature XylI protein heterologously expressed in E. coli showed a xylanase activity toward xylans from various plant sources with optimum at pH 6.5, and C. glutamicum recombinant strain expressing native XylI released xylose, xylobiose, xylotriose, and arabino-xylobiose from arabinoxylan. Finally, introduction of the xylI gene into the C. glutamicum AXOS-utilizing strain enabled it to directly utilize arabinoxylan.     

<Emphasis Type="Italic">Sphingorhabdus buctiana</Emphasis> sp. nov., isolated from fresh water,and reclassification of <Emphasis Type="Italic">Sphingopyxis contaminans</Emphasis> as <Emphasis Type="Italic">Sphingorhabdus contaminans</Emphasis> comb. nov.

A Gram-stain-negative, strictly aerobic, non-motile and rod-shaped bacterial strain, designated T5^T, was isolated from the Chishui River in Maotai town, Guizhou Province, Southwest of China. Strain T5^T was found to grow optimally at pH 9.0 and 25 °C. The 16S rRNA gene sequence analysis indicated that strain T5^T belongs to the family Sphingomonadaceae within the phylum Proteobacteria; the strain T5^T clustered with the type strains of Sphingopyxis contaminans, Sphingorhabdus wooponensis and Sphingorhabdus rigui, with which it exhibits 16S rRNA gene sequence similarity values of 96.2–96.9%. The DNA G+C content was 58.5 mol%. The major respiratory quinone was Q-10 and the major polar lipid was phosphatidylethanolamine. The major polyamine was homospermidine and the major fatty acids were C_18:1 ω7c (37.5%) and C_16:1 ω7c (30.1%). On the basis of phylogenetic, phenotypic and genetic data, strain T5^T represents a novel species of the genus Sphingorhabdus, for which the name Sphingorhabdus buctiana sp. nov. is proposed. The type strain is T5^T (= CGMCC 1.12929^T = JCM 30114^T). It is also proposed that Sphingopyxis contaminans should be reclassified as a member of the genus Sphingorhabdus.     

<Emphasis Type="Italic">Spirosoma daeguensis</Emphasis> sp. nov., isolated from beach soil

A Gram-stain-negative, non-motile, non-spore-forming, rod-shaped, aerobic bacterium, designated 15J9-6^T, was isolated from beach soil on Jeju Island, South Korea. Strain 15J9-6^T, grew at 10–30°C (optimum growth at 25°C) and pH 7–8 (optimum growth at pH 7) on R2A, NA, and TSA agar. Phylogenetically, the strain was closely related to members of the genus Spirosoma (92.3–90.1% 16S rRNA gene sequence similarities) and showed highest sequence similarity to Spirosoma panaciterrae DSM 21099^T (92.3%). The G+C content of the genomic DNA of strain 15J9-6^T was 45.7 mol%. The strain contained phosphatidylethanolamine, two unidentified aminophospholipids, an unidentified phospholipid, and an unidentified lipid as the major polar lipids; menaquinone MK-7 as the predominant respiratory quinone and summed feature 3 (C_16:1 ω6c/C_16:1 ω7c; 30.1%), C_16:1 ω5c (23.1%), iso C_15:0 (13.3%), and C_16:0 (8.4%) as the major fatty acids which supported the affiliation of strain 15J9-6^T to the genus Spirosoma. The results of physiological and biochemical tests allowed genotypic and phenotypic differentiation of strain 15J9-6^T from recognized Spirosoma species. On the basis of its phenotypic properties and phylogenetic distinctiveness, strain 15J9-6^T represents a novel species of the genus Spirosoma, for which the name Spirosoma daeguensis sp. nov. is proposed. The type strain is 15J9-6^T (=KCTC 52036^T =JCM 31995T)     

In vitro probiotic potential of <Emphasis Type="Italic">Lactobacillus delbreuckii</Emphasis> subsp. <Emphasis Type="Italic">bulgaricus</Emphasis> F18 isolated from homemade butter

Present study was carried out to evaluate a new bacterial strain, Lactobacillus delbreuckii subsp. bulgaricus F18 as probiotic strain. L. delbreuckii subsp. bulgaricus F18 was isolated from homemade butter and identified by conventional and molecular techniques. The 16S rRNA sequence of the isolate was registered in National Centre for Biotechnology Information (NCBI) under accession number KT865224. In the present study, L. delbreuckii subsp. bulgaricus F18 exhibited highest viable counts against acid tolerance or low pH tolerance (at pH 1 after 2h of incubation), bile tolerance (conc. 1%), autoaggregation (68%), cell surface hydrophobicity against O-xylene (33.9%), antimicrobial activity against various food borne pathogens (inhibition = 100%), antibiotic sensitivity following standard test methods suggested by various research workers.     

<Emphasis Type="Italic">Flavobacterium parvum</Emphasis> sp. nov., isolated from soil polluted by sewer water

A novel Gram-stain-negative, motile by means of gliding, and short rod-shaped bacterium, designated HS916T, was isolated from soil polluted by sewer water in Cheonan-si, South Korea. Growth occurred at 10–35°C (optimum 30°C), pH 6.0–8.0 (optimum pH 7.0), and 0–1% sodium chloride (NaCl, w/v). Based on similarities of 16S rRNA gene sequences, strain HS916^T was closely related to members of the genus Flavobacterium, exhibiting the highest sequence similarities with Flavobacterium glycines Gm-149^T (96.4%), followed by F. granuli Kw05^T (96.3%), F. fluminis 3R17^T (96.3%), F. aquicola TMd3a3^T (96.2%), and F. nitratireducens N1^T (96.2%). Phylogenetic analysis based on 16S rRNA gene sequences indicated that strain HS916T was placed in a monophyletic cluster with F. nitratireducens N1^T and F. fluminis 3R17^T. The predominant fatty acids (> 5% of the total) of strain HS916^T were iso-C_15:0, anteiso-C_15:0, iso-C_15:0 3-OH, C_17:1ω6с, C_16:0 3-OH, iso-C_17:0 3-OH, and summed feature 3 (C_16:1ω7с and/or C_16:1ω6с). The major polar lipids of the strain comprised phosphatidylethanolamine, unidentified aminolipids, and five unidentified lipids. The predominant respiratory quinone and the major polyamine were menaquinone-6 (MK-6) and symhomospermidine, respectively. The DNA G + C content of strain HS916^T was 34.9 mol%. Based on polyphasic analyses, strain HS916^T represents a novel species belonging to the genus Flavobacterium, for which the name Flavobacterium parvum sp. nov. is proposed. The type strain is HS916T (= KACC 19448^T = JCM 32368^T).     

<Emphasis Type="Italic">Halomonas chromatireducens</Emphasis> sp. nov., a new denitrifying facultatively haloalkaliphilic bacterium from solonchak soil capable of aerobic chromate reduction

A heterotrophic bacterial strain AGD 8-3 capable of denitrification under extreme haloalkaline conditions was isolated from soda solonchak soils of the Kulunda steppe (Russia). The strain was classified within the genus Halomonas. According to the results of 16S rRNA gene sequencing, Halomonas axialensis, H. meridiana, and H. aquamarina are most closely related to strain AGD 8-3 (96.6% similarity). Similar to other members of the genus, the strain can grow within a wide range of salinity and pH. The strain was found to be capable of aerobic reduction of chromate and selenite on mineral media at 160 g/l salinity and pH 9.5–10. The relatively low level of phylogenetic similarity and the phenotypic characteristics supported classification of strain AGD 8-3 as a new species Halomonas chromatireducens.     

Probiotic Evaluation of Antimicrobial <Emphasis Type="Italic">Lactobacillus plantarum</Emphasis> VJC38 Isolated from the Crop of Broiler Chicken

The aim of this study was to evaluate probiotic properties of antimicrobial Lactobacillus plantarum VJC38 in vitro. L. plantarum VJC38 was isolated from the crop of broiler chicken and characterized using dnaK gene sequence. The inhibitory activities of L. plantarum VJC38 against bacterial and fungal pathogens were evaluated. Antifungal compounds secreted by the strain VJC38 were identified using Gas Chromatography and Mass Spectrometry (GC-MS). The strain was evaluated for its tolerance to low pH, resistance to bile salts, auto-aggregation, co-aggregation with pathogenic Escherichia coli, cell surface hydrophobicity, cholesterol lowering activity, β-galactosidase production, adhesion ability to Caco-2 cells, mucin degradation, hemolytic activity and biogenic amine production. Phylogenetic analysis of dnaK gene of bacterial strain VJC38 showed 99% sequence similarity to Lactobacillus plantarum var. plantarum. It showed effective inhibition against food spoiling and pathogenic organisms like Escherichia coli, Listeria monocytogenes, Staphylococcus aureus, Aspergillus niger, Penicillium expansum and Eurotium species. The antifungal compound phenol- 2,4-bis(1,1-dimethylethyl) (PD) was identified in the culture filtrate of L. plantarum VJC38 and reported to have inhibition against Aspergillus species. L. plantarum VJC38 exhibited tolerance to low pH, resistance to bile salts, bile salt hydrolase activity, auto-aggregation (87.5%), co-aggregation with Escherichia coli (55.7%), cholesterol lowering activity (64%), β-galactosidase production (1206 MU), adherence to Caco-2 cells (11%), negative for mucin degradation, hemolytic activity and biogenic amine production. L. plantarum VJC38 could be a good candidate for further investigation in vivo to elucidate its health benefits and to evaluate its technological properties as a bio-protective strain.