Oligonucleotide Microarray for 16S rRNA Gene-Based Detection of All Recognized Lineages of Sulfate-Reducing Prokaryotes in the Environment

For cultivation-independent detection of sulfate-reducing prokaryotes (SRPs) an oligonucleotide microarray consisting of 132 16S rRNA gene-targeted oligonucleotide probes (18-mers) having hierarchical and parallel (identical) specificity for the detection of all known lineages of sulfate-reducing prokaryotes (SRP-PhyloChip) was designed and subsequently evaluated with 41 suitable pure cultures of SRPs. The applicability of SRP-PhyloChip for diversity screening of SRPs in environmental and clinical samples was tested by using samples from periodontal tooth pockets and from the chemocline of a hypersaline cyanobacterial mat from Solar Lake (Sinai, Egypt). Consistent with previous studies, SRP-PhyloChip indicated the occurrence of Desulfomicrobium spp. in the tooth pockets and the presence of Desulfonema- and Desulfomonile-like SRPs (together with other SRPs) in the chemocline of the mat. The SRP-PhyloChip results were confirmed by several DNA microarray-independent techniques, including specific PCR amplification, cloning, and sequencing of SRP 16S rRNA genes and the genes encoding the dissimilatory (bi)sulfite reductase (dsrAB).     

Molecular Analysis of the Diversity of Sulfate-Reducing and Sulfur-Oxidizing Prokaryotes in the Environment, Using aprA as Functional Marker Gene

The dissimilatory adenosine-5′-phosposulfate reductase is a key enzyme of the microbial sulfate reduction and sulfur oxidation processes. Because the alpha- and beta-subunit-encoding genes, aprBA, are highly conserved among sulfate-reducing and sulfur-oxidizing prokaryotes, they are most suitable for molecular profiling of the microbial community structure of the sulfur cycle in environment. In this study, a new aprA gene-targeting assay using a combination of PCR and denaturing gradient gel electrophoresis is presented. The screening of sulfate-reducing and sulfur-oxidizing reference strains as well as the analyses of environmental DNA from diverse habitats (e.g., microbial mats, invertebrate tissue, marine and estuarine sediments, and filtered hydrothermal water) by the new primer pair revealed an improved microbial diversity coverage and less-pronounced template-to-PCR product bias in direct comparison to those of the previously published primer set (B. Deplancke, K. R. Hristova, H. A. Oakley, V. J. McCracken, R. Aminov, R. I. Mackie, and H. R. Gaskins, Appl. Environ. Microbiol. 66:2166-2174, 2000). The concomitant molecular detection of sulfate-reducing and sulfur-oxidizing prokaryotes was confirmed. The new assay was applied in comparison with the 16S rRNA gene-based analysis to investigate the microbial diversity of the sulfur cycle in sediment, seawater, and manganese crust samples from four study sites in the area of the Lesser Antilles volcanic arc, Caribbean Sea (Caribflux project). The aprA gene-based approach revealed putative sulfur-oxidizing Alphaproteobacteria of chemolithoheterotrophic lifestyle to have been abundant in the nonhydrothermal sediment and water column. In contrast, the sulfur-based microbial community that inhabited the surface of the volcanic manganese crust was more complex, consisting predominantly of putative chemolithoautotrophic sulfur oxidizers of the Betaproteobacteria and Gammaproteobacteria.     

Evolution of a Protein-Folding Machine: Genomic and Evolutionary Analyses Reveal Three Lineages of the Archaeal hsp70(dnaK) Gene

The stress chaperone protein Hsp70 (DnaK) (abbreviated DnaK) and its co-chaperones Hsp40(DnaJ) (or DnaJ) and GrpE are universal in bacteria and eukaryotes but occur only in some archaea clustered in the order 5′-grpE-dnaK-dnaJ-3′ in a locus termed Locus I. Three structural varieties of Locus I, termed Types I, II, and III, were identified, respectively, in Methanosarcinales, in Thermoplasmatales and Methanothermobacter thermoautotrophicus, and in Halobacteriales. These Locus I types corresponded to three groups identified by phylogenetic trees of archaeal DnaK proteins including the same archaeal subdivisions. These archaeal DnaK groups were not significantly interrelated, clustering instead with DnaKs from three bacterial lineages, Methanosarcinales with Firmicutes, Thermoplasmatales and M. thermoautotrophicus with Thermotoga, and Halobacteriales with Actinobacteria, suggesting that the three archaeal types of Locus I were acquired by independent events of lateral gene transfer. These associations, however, lacked strong bootstrap support and were sensitive to dataset choice and tree-reconstruction method. Structural features of dnaK loci in bacteria revealed that Methanosarcinales and Firmicutes shared a similar structure, also common to most other bacterial groups. Structural differences were observed instead in Thermotoga compared to Thermoplasmatales and M. thermoautotrophicus, and in Actinobacteria compared to Halobacteriales. It was also found that the association between the DnaK sequences from Halobacteriales and Actinobacteria likely reflects common biases in their amino acid compositions. Although the loci structural features and the DnaK trees suggested the possibility of lateral gene transfer between Firmicutes and Methanosarcinales, the similarity between the archaeal and the ancestral bacterial loci favors the more parsimonious hypothesis that all archaeal sequences originated from a unique prokaryotic ancestor. Electronic Supplementary Material Electronic Supplementary material is available for this article at and accessible for authorised users. [Reviewing Editor: Dr. Stephen Freeland]     

Application of a Novel Functional Gene Microarray to Probe the Functional Ecology of Ammonia Oxidation in Nitrifying Activated Sludge

We report on the first study trialling a newly-developed, functional gene microarray (FGA) for characterising bacterial and archaeal ammonia oxidisers in activated sludge. Mixed liquor (ML) and media biofilm samples from a full-scale integrated fixed-film activated sludge (IFAS) plant were analysed with the FGA to profile the diversity and relative abundance of ammonia-oxidising archaea and bacteria (AOA and AOB respectively). FGA analyses of AOA and AOB communities revealed ubiquitous distribution of AOA across all samples – an important finding for these newly-discovered and poorly characterised organisms. Results also revealed striking differences in the functional ecology of attached versus suspended communities within the IFAS reactor. Quantitative assessment of AOB and AOA functional gene abundance revealed a dominance of AOB in the ML and approximately equal distribution of AOA and AOB in the media-attached biofilm. Subsequent correlations of functional gene abundance data with key water quality parameters suggested an important functional role for media-attached AOB in particular for IFAS reactor nitrification performance and indicate possible functional redundancy in some IFAS ammonia oxidiser communities. Results from this investigation demonstrate the capacity of the FGA to resolve subtle ecological shifts in key microbial communities in nitrifying activated sludge and indicate its value as a tool for better understanding the linkages between the ecology and performance of these engineered systems.     

Identification of a Novel Uromodulin-Like Gene Related to Predator-Induced Bulgy Morph in Anuran Tadpoles by Functional Microarray Analysis

Tadpoles of the anuran species Rana pirica can undergo predator-specific morphological responses. Exposure to a predation threat by larvae of the salamander Hynobius retardatus results in formation of a bulgy body (bulgy morph) with a higher tail. The tadpoles revert to a normal phenotype upon removal of the larval salamander threat. Although predator-induced phenotypic plasticity is of major interest to evolutionary ecologists, the molecular and physiological mechanisms that control this response have yet to be elucidated. In a previous study, we identified various genes that are expressed in the skin of the bulgy morph. However, it proved difficult to determine which of these were key genes in the control of gene expression associated with the bulgy phenotype. Here, we show that a novel gene plays an important role in the phenotypic plasticity producing the bulgy morph. A functional microarray analysis using facial tissue samples of control and bulgy morph tadpoles identified candidate functional genes for predator-specific morphological responses. A larger functional microarray was prepared than in the previous study and used to analyze mRNAs extracted from facial and brain tissues of tadpoles from induction-reversion experiments. We found that a novel uromodulin-like gene, which we name here pirica, was up-regulated and that keratin genes were down-regulated as the period of exposure to larval salamanders increased. Pirica consists of a 1296 bp open reading frame, which is putatively translated into a protein of 432 amino acids. The protein contains a zona pellucida domain similar to that of proteins that function to control water permeability. We found that the gene was expressed in the superficial epidermis of the tadpole skin.     

Analyses of the Recycling Receptor, FcRn, in Live Cells Reveal Novel Pathways for Lysosomal Delivery

Lysosomes play a central role in the degradation of proteins and other macromolecules. The mechanisms by which receptors are transferred to lysosomes for constitutive degradation are poorly understood. We have analyzed the processes that lead to the lysosomal delivery of the Fc receptor, FcRn. These studies provide support for a novel pathway for receptor delivery. Specifically, unlike other receptors that enter intraluminal vesicles in late endosomes, FcRn is transferred from the limiting membrane of such endosomes to lysosomes, and is rapidly internalized into the lysosomal lumen. By contrast, LAMP-1 persists on the limiting membrane. Receptor transfer is mediated by tubular extensions from late endosomes to lysosomes, or by interactions of the two participating organelles in kiss-and-linger-like processes, whereas full fusion is rarely observed. The persistence of FcRn on the late endosomal limiting membrane, together with selective transfer to lysosomes, allows this receptor to undergo recycling or degradation. Consequently, late endosomes have functional plasticity, consistent with the presence of the Rab5 GTPase in discrete domains on these compartments.     

Comparative Secretome Analyses of Primary Murine White and Brown Adipocytes Reveal Novel Adipokines

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Highlights

• •First comparative proteomic analysis of white and brown adipocyte secretomes.
• •100 novel adipokines differentially secreted from white versus brown adipocytes.
• •Functionally enriched protein class changes in white and brown adipocytes.
• •200 novel, NE-responsive adipokines from brown adipocytes.

     

Gene Discovery and Functional Analyses in the Model Plant Arabidopsis

The present mini-review describes newer methods and strategies, including transposon and T-DNA insertions, TILLING, Deleteagene, and RNA interference, to functionally analyze genes of interest in the model plant Arabidopsis. The relative advantages and disadvantages of the systems are also discussed.     

Functional Gene Polymorphism to Reveal Species History: The Case of the CRTISO Gene in Cultivated Carrots

Background

Carrot is a vegetable cultivated worldwide for the consumption of its root. Historical data indicate that root colour has been differentially selected over time and according to geographical areas. Root pigmentation depends on the relative proportion of different carotenoids for the white, yellow, orange and red types but only internally for the purple one. The genetic control for root carotenoid content might be partially associated with carotenoid biosynthetic genes. Carotenoid isomerase (CRTISO) has emerged as a regulatory step in the carotenoid biosynthesis pathway and could be a good candidate to show how a metabolic pathway gene reflects a species genetic history.

Methodology/Principal Findings

In this study, the nucleotide polymorphism and the linkage disequilibrium among the complete CRTISO sequence, and the deviation from neutral expectation were analysed by considering population subdivision revealed with 17 microsatellite markers. A sample of 39 accessions, which represented different geographical origins and root colours, was used. Cultivated carrot was divided into two genetic groups: one from Middle East and Asia (Eastern group), and another one mainly from Europe (Western group). The Western and Eastern genetic groups were suggested to be differentially affected by selection: a signature of balancing selection was detected within the first group whereas the second one showed no selection. A focus on orange-rooted carrots revealed that cultivars cultivated in Asia were mainly assigned to the Western group but showed CRTISO haplotypes common to Eastern carrots.

Conclusion

The carotenoid pathway CRTISO gene data proved to be complementary to neutral markers in order to bring critical insight in the cultivated carrot history. We confirmed the occurrence of two migration events since domestication. Our results showed a European background in material from Japan and Central Asia. While confirming the introduction of European carrots in Japanese resources, the history of Central Asia material remains unclear.     

Structural and Functional Dynamics of Sulfate-Reducing Populations in Bacterial Biofilms

We describe the combined application of microsensors and molecular techniques to investigate the development of sulfate reduction and of sulfate-reducing bacterial populations in an aerobic bacterial biofilm. Microsensor measurements for oxygen showed that anaerobic zones developed in the biofilm within 1 week and that oxygen was depleted in the top 200 to 400 μm during all stages of biofilm development. Sulfate reduction was first detected after 6 weeks of growth, although favorable conditions for growth of sulfate-reducing bacteria (SRB) were present from the first week. In situ hybridization with a 16S rRNA probe for SRB revealed that sulfate reducers were present in high numbers (approximately 10⁸ SRB/ml) in all stages of development, both in the oxic and anoxic zones of the biofilm. Denaturing gradient gel electrophoresis (DGGE) showed that the genetic diversity of the microbial community increased during the development of the biofilm. Hybridization analysis of the DGGE profiles with taxon-specific oligonucleotide probes showed that Desulfobulbus and Desulfovibrio were the main sulfate-reducing bacteria in all biofilm samples as well as in the bulk activated sludge. However, different Desulfobulbus and Desulfovibrio species were found in the 6th and 8th weeks of incubation, respectively, coinciding with the development of sulfate reduction. Our data indicate that not all SRB detected by molecular analysis were sulfidogenically active in the biofilm.     

紫花针茅WRKY53基因的克隆、序列分析及功能鉴定

植物WRKY转录因子家族在叶片衰老中起着重要的调节作用,其成员WRKY53基因与叶片衰老密切相关。本文利用紫花针茅(Stipa purpurea Griseb.)的一条EST序列,克隆获得Sp WRKY53基因的全长CDS序列,其开放阅读框为1443 bp,编码480个氨基酸,蛋白质分子量为50.9 k D,理论等电点为7.64。序列比对结果和系统进化分析表明,Sp WRKY53基因有2个WRKY序列,与已报道的小麦(Triticum aestivem L.)的WRKY53基因结构相似。亚细胞定位结果显示该蛋白定位于细胞核上。紫花针茅WRKY53基因的过表达以及表达分析表明,该基因能够加速植物叶片衰老并在多种非生物胁迫下上调表达。本研究为WRKY53基因在牧草分子育种中的进一步研究奠定了基础。     

Culturable Diversity and Community Fatty Acid Profiling of Sulfate-Reducing Fluidized-Bed Reactors Treating Acidic,Metal-Containing Wastewater

Cultivable strains were identified from sulfate-reducing fluidized-bed reactors (FBR) treating acidic metal-containing wastewater. The FBR-communities were further characterized using culture-independent phenotypic markers, phospholipid fatty acid (PLFA) profiling. After morphological screening of 128 bacterial strains and partial sequencing of 55 strains, 17 distinct phylogenetic types were identified and characterized further. A total of 14 and 6 different bacterial strains were isolated from ethanol- and lactate-fed FBRs, respectively. Sequencing of the 16S rRNA gene showed that these strains were affiliated with members of the δ-Proteobacteria, Firmicutes and Bacteroidetes The strains were affiliated with members of the genera Desulfovibrio, Desulfotomaculum, Desulfobulbus, Desulfitobacterium, Clostridium, Caloramator, Oxobacter, and Bacteroides. Many of the strains were only distantly related to previously described species and, thus, may represent novel species or genera. A number of the strains were not detected in previously employed molecular analyses of the FBR communities, and the major component of each FBR as identified in the molecular analyses were not retrieved as cultures in this study. Most of the SRB, and two of the non-SRB utilized ethanol and lactate as a source of carbon and energy, but none of the isolates grew on acetate, an intermediate in the oxidation of ethanol and lactate. PLFA analysis revealed that the FBR community members contained large amounts of saturated fatty acids. Although the PLFA analysis showed some signatures consistent with sulfate-reducing communities, it did not show any substantial difference in the microbial communities between the reactors, an outcome that was quite contrary to the culture-independent phylogenetic analyses.