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July 7, 2019  |  

‘Candidatus Tenderia electrophaga’, an uncultivated electroautotroph from a biocathode enrichment.

Biocathode communities are of interest for a variety of applications, including electrosynthesis, bioremediation, and biosensors, yet much remains to be understood about the biological processes that occur to enable these communities to grow. One major difficulty in understanding these communities is that the critical autotrophic organisms are difficult to cultivate. An uncultivated, electroautotrophic bacterium previously identified as an uncultivated member of the family Chromatiaceae appears to be a key organism in an autotrophic biocathode microbial community. Metagenomic, metaproteomic and metatranscriptomic characterization of this community indicates that there is likely a single organism that utilizes electrons from the cathode to fix CO2, yet this organism has not been obtained in pure culture. Fluorescence in situ hybridization reveals that the organism grows as rod-shaped cells approximately 1.8 × 0.6 µm, and forms large clumps on the cathode. The genomic DNA G+C content was 59.2 mol%. Here we identify the key features of this organism and propose ‘Candidatus Tenderia electrophaga’, within the Gammaproteobacteria on the basis of low nucleotide and predicted protein sequence identity to known members of the orders Chromatiales and Thiotrichales.


July 7, 2019  |  

Genomic, physiologic, and proteomic insights into metabolic versatility in Roseobacter clade bacteria isolated from deep-sea water.

Roseobacter clade bacteria are ubiquitous in marine environments and now thought to be significant contributors to carbon and sulfur cycling. However, only a few strains of roseobacters have been isolated from the deep-sea water column and have not been thoroughly investigated. Here, we present the complete genomes of phylogentically closed related Thiobacimonas profunda JLT2016 and Pelagibaca abyssi JLT2014 isolated from deep-sea water of the Southeastern Pacific. The genome sequences showed that the two deep-sea roseobacters carry genes for versatile metabolisms with functional capabilities such as ribulose bisphosphate carboxylase-mediated carbon fixation and inorganic sulfur oxidation. Physiological and biochemical analysis showed that T. profunda JLT2016 was capable of autotrophy, heterotrophy, and mixotrophy accompanied by the production of exopolysaccharide. Heterotrophic carbon fixation via anaplerotic reactions contributed minimally to bacterial biomass. Comparative proteomics experiments showed a significantly up-regulated carbon fixation and inorganic sulfur oxidation associated proteins under chemolithotrophic conditions compared to heterotrophic conditions. Collectively, rosebacters show a high metabolic flexibility, suggesting a considerable capacity for adaptation to the marine environment.


July 7, 2019  |  

Genomic studies of nitrogen-fixing rhizobial strains from Phaseolus vulgaris seeds and nodules.

Rhizobia are soil bacteria that establish symbiotic relationships with legumes and fix nitrogen in root nodules. We recently reported that several nitrogen-fixing rhizobial strains, belonging to Rhizobium phaseoli, R. trifolii, R. grahamii and Sinorhizobium americanum, were able to colonize Phaseolus vulgaris (common bean) seeds. To gain further insight into the traits that support this ability, we analyzed the genomic sequences and proteomes of R. phaseoli (CCGM1) and S. americanum (CCGM7) strains from seeds and compared them with those of the closely related strains CIAT652 and CFNEI73, respectively, isolated only from nodules.In a fine structural study of the S. americanum genomes, the chromosomes, megaplasmids and symbiotic plasmids were highly conserved and syntenic, with the exception of the smaller plasmid, which appeared unrelated. The symbiotic tract of CCGM7 appeared more disperse, possibly due to the action of transposases. The chromosomes of seed strains had less transposases and strain-specific genes. The seed strains CCGM1 and CCGM7 shared about half of their genomes with their closest strains (3353 and 3472 orthologs respectively), but a large fraction of the rest also had homology with other rhizobia. They contained 315 and 204 strain-specific genes, respectively, particularly abundant in the functions of transcription, motility, energy generation and cofactor biosynthesis. The proteomes of seed and nodule strains were obtained and showed a particular profile for each of the strains. About 82 % of the proteins in the comparisons appeared similar. Forty of the most abundant proteins in each strain were identified; these proteins in seed strains were involved in stress responses and coenzyme and cofactor biosynthesis and in the nodule strains mainly in central processes. Only 3 % of the abundant proteins had hypothetical functions.Functions that were enriched in the genomes and proteomes of seed strains possibly participate in the successful occupancy of the new niche. The genome of the strains had features possibly related to their presence in the seeds. This study helps to understand traits of rhizobia involved in seed adaptation.


July 7, 2019  |  

Genomes of Candidatus Wolbachia bourtzisii wDacA and Candidatus Wolbachia pipientis wDacB from the cochineal insect Dactylopius coccus (Hemiptera: Dactylopiidae).

Dactylopius species, known as cochineal insects, are the source of the carminic acid dye used worldwide. The presence of two Wolbachia strains in Dactylopius coccus from Mexico was revealed by PCR amplification of wsp and sequencing of 16S rRNA genes. A metagenome analysis recovered the genome sequences of Candidatus Wolbachia bourtzisii wDacA (supergroup A) and Candidatus Wolbachia pipientis wDacB (supergroup B). Genome read coverage, as well as 16S rRNA clone sequencing, revealed that wDacB was more abundant than wDacA. The strains shared similar predicted metabolic capabilities that are common to Wolbachia, including riboflavin, ubiquinone, and heme biosynthesis, but lacked other vitamin and cofactor biosynthesis as well as glycolysis, the oxidative pentose phosphate pathway, and sugar uptake systems. A complete tricarboxylic acid cycle and gluconeogenesis were predicted as well as limited amino acid biosynthesis. Uptake and catabolism of proline were evidenced in Dactylopius Wolbachia strains. Both strains possessed WO-like phage regions and type I and type IV secretion systems. Several efflux systems found suggested the existence of metal toxicity within their host. Besides already described putative virulence factors like ankyrin domain proteins, VlrC homologs, and patatin-like proteins, putative novel virulence factors related to those found in intracellular pathogens like Legionella and Mycobacterium are highlighted for the first time in Wolbachia Candidate genes identified in other Wolbachia that are likely involved in cytoplasmic incompatibility were found in wDacB but not in wDacA. Copyright © 2016 Ramírez-Puebla et al.


July 7, 2019  |  

Borneol dehydrogenase from Pseudomonas sp. strain TCU-HL1 catalyzes the oxidation of (+)-borneol and its isomers to camphor.

Most plant-produced monoterpenes can be degraded by soil microorganisms. Borneol is a plant terpene that is widely used in traditional Chinese medicine. Neither microbial borneol dehydrogenase (BDH) nor a microbial borneol degradation pathway has been reported previously. One borneol-degrading strain, Pseudomonas sp. strain TCU-HL1, was isolated by our group. Its genome was sequenced and annotated. The genome of TCU-HL1 consists of a 6.2-Mbp circular chromosome and one circular plasmid, pTHL1 (12.6 kbp). Our results suggest that borneol is first converted into camphor by BDH in TCU-HL1 and is further decomposed through a camphor degradation pathway. The recombinant BDH was produced in the form of inclusion bodies. The apparent Km values of refolded recombinant BDH for (+)-borneol and (-)-borneol were 0.20 ± 0.01 and 0.16 ± 0.01 mM, respectively, and the kcat values for (+)-borneol and (-)-borneol were 0.75 ± 0.01 and 0.53 ± 0.01 s(-1), respectively. Two plant BDH genes have been reported previously. The kcat and kcat/Km values of lavender BDH are about 1,800-fold and 500-fold lower, respectively, than those of TCU-HL1 BDH.The degradation of borneol in a soil microorganism through a camphor degradation pathway is reported in this study. We also report a microbial borneol dehydrogenase. The kcat and kcat/Km values of lavender BDH are about 1,800-fold and 500-fold lower, respectively, than those of TCU-HL1 BDH. The indigenous borneol- and camphor-degrading strain isolated, Pseudomonas sp. strain TCU-HL1, reminds us of the time 100 years ago when Taiwan was the major producer of natural camphor in the world. Copyright © 2016, American Society for Microbiology. All Rights Reserved.


July 7, 2019  |  

Neuraminidase A-exposed galactose promotes Streptococcus pneumoniae biofilm formation during colonization.

Streptococcus pneumoniae is an opportunistic pathogen that colonizes the nasopharynx. Herein we show that carbon availability is distinct between the nasopharynx and bloodstream of adult humans: glucose is absent from the nasopharynx, whereas galactose is abundant. We demonstrate that pneumococcal neuraminidase A (NanA), which cleaves terminal sialic acid residues from host glycoproteins, exposed galactose on the surface of septal epithelial cells, thereby increasing its availability during colonization. We observed that S. pneumoniae mutants deficient in NanA and ß-galactosidase A (BgaA) failed to form biofilms in vivo despite normal biofilm-forming abilities in vitro Subsequently, we observed that glucose, sucrose, and fructose were inhibitory for biofilm formation, whereas galactose, lactose, and low concentrations of sialic acid were permissive. Together these findings suggested that the genes involved in biofilm formation were under some form of carbon catabolite repression (CCR), a regulatory network in which genes involved in the uptake and metabolism of less-preferred sugars are silenced during growth with preferred sugars. Supporting this notion, we observed that a mutant deficient in pyruvate oxidase, which converts pyruvate to acetyl-phosphate under non-CCR-inducing growth conditions, was unable to form biofilms. Subsequent comparative transcriptome sequencing (RNA-seq) analyses of planktonic and biofilm-grown pneumococci showed that metabolic pathways involving the conversion of pyruvate to acetyl-phosphate and subsequently leading to fatty acid biosynthesis were consistently upregulated during diverse biofilm growth conditions. We conclude that carbon availability in the nasopharynx impacts pneumococcal biofilm formation in vivo Additionally, biofilm formation involves metabolic pathways not previously appreciated to play an important role. Copyright © 2016, American Society for Microbiology. All Rights Reserved.


July 7, 2019  |  

Expansion of lysine-rich repeats in Plasmodium proteins generates novel localisation sequences that target the periphery of the host erythrocyte.

Repetitive low-complexity sequences, mostly assumed to have no function, are common in proteins that are exported by the malaria parasite into its host erythrocyte. We identify a group of exported proteins containing short lysine-rich tandemly repeated sequences that are sufficient to localise to the erythrocyte periphery where key virulence-related modifications to the plasma membrane and the underlying cytoskeleton are known to occur. Efficiency of targeting is dependent on repeat number, indicating that novel targeting modules could evolve by expansion of short lysine-rich sequences. Indeed, expression GARP fragments from different species shows that two novel targeting sequences have arisen via the process of repeat expansion in this protein. In the protein Hyp12, the targeting function of a lysine-rich sequence is masked by a neighbouring repetitive acidic sequence, further highlighting the importance of repetitive low complexity sequences. We show that sequences capable of targeting the erythrocyte periphery are present in at least nine proteins from Plasmodium falciparum, and one from Plasmodium knowlesi. We find these sequences in proteins known to be involved in erythrocyte rigidification and cytoadhesion, as well as in previously uncharacterised exported proteins. Together, these data suggest that expansion and contraction of lysine-rich repeats could generate targeting sequences de novo as well as modulate protein targeting efficiency and function in response to selective pressure. Copyright © 2016, The American Society for Biochemistry and Molecular Biology.


July 7, 2019  |  

Unbiased identification of signal-activated transcription factors by barcoded synthetic tandem repeat promoter screening (BC-STAR-PROM).

The discovery of transcription factors (TFs) controlling pathways in health and disease is of paramount interest. We designed a widely applicable method, dubbed barcorded synthetic tandem repeat promoter screening (BC-STAR-PROM), to identify signal-activated TFs without any a priori knowledge about their properties. The BC-STAR-PROM library consists of ~3000 luciferase expression vectors, each harboring a promoter (composed of six tandem repeats of synthetic random DNA) and an associated barcode of 20 base pairs (bp) within the 3′ untranslated mRNA region. Together, the promoter sequences encompass >400,000 bp of random DNA, a sequence complexity sufficient to capture most TFs. Cells transfected with the library are exposed to a signal, and the mRNAs that it encodes are counted by next-generation sequencing of the barcodes. This allows the simultaneous activity tracking of each of the ~3000 synthetic promoters in a single experiment. Here we establish proof of concept for BC-STAR-PROM by applying it to the identification of TFs induced by drugs affecting actin and tubulin cytoskeleton dynamics. BC-STAR-PROM revealed that serum response factor (SRF) is the only immediate early TF induced by both actin polymerization and microtubule depolymerization. Such changes in cytoskeleton dynamics are known to occur during the cell division cycle, and real-time bioluminescence microscopy indeed revealed cell-autonomous SRF-myocardin-related TF (MRTF) activity bouts in proliferating cells.© 2016 Gosselin et al.; Published by Cold Spring Harbor Laboratory Press.


July 7, 2019  |  

Identification and expression analysis of wheat TaGF14 genes.

The 14-3-3 gene family members play key roles in various cellular processes. However, little is known about the numbers and roles of 14-3-3 genes in wheat. The aims of this study were to identify TaGF14 numbers in wheat by searching its whole genome through blast, to study the phylogenetic relationships with other plant species and to discuss the functions of TaGF14s. The results showed that common wheat harbored 20 TaGF14 genes, located on wheat chromosome groups 2, 3, 4, and 7. Out of them, eighteen TaGF14s are non-e proteins, and two wheat TaGF14 genes, TaGF14i and TaGF14f, are e proteins. Phylogenetic analysis indicated that these genes were divided into six clusters: cluster 1 (TaGF14d, TaGF14g, TaGF14j, TaGF14h, TaGF14c, and TaGF14n); cluster 2 (TaGF14k); cluster 3 (TaGF14b, TaGF14l, TaGF14m, and TaGF14s); cluster 4 (TaGF14a, TaGF14e, and TaGF14r); cluster 5 (TaGF14i and TaGF14f); and cluster 6 (TaGF14o, TaGF14p, TaGF14q, and TaGF14t). Tissue-specific gene expressions suggested that all TaGF14s were likely constitutively expressed, except two genes, i.e., TaGF14p and TaGF14f. And the highest amount of TaGF14 transcripts were observed in developing grains at 20 days post anthesis (DPA), especially for TaGF14j and TaGF14l. After drought stress, five genes, i.e., TaGF14c, TaGF14d, TaGF14g, TaGF14h, and TaGF14j, were up-regulated expression under drought stress for both 1 and 6 h, suggesting these genes played vital role in combating against drought stress. However, all the TaGF14s were down-regulated expression under heat stress for both 1 and 6 h, indicating TaGF14s may be negatively associated with heat stress by reducing the expression to combat heat stress or through other pathways. These results suggested that cluster 1, e.g., TaGF14j, may participate in the whole wheat developing stages, e.g., grain-filling (starch biosynthesis) and may also participate in combating against drought stress. Subsequently, a homolog of TaGF14j, TaGF14-JM22, were cloned by RACE and used to validate its function. Immunoblotting results showed that TaGF14-JM22 protein, closely related to TaGF14d, TaGF14g, and TaGF14j, can interact with AGP-L, SSI, SSII, SBEIIa, and SBEIIb in developing grains, suggesting that TaGF14s located on group 4 may be involved in starch biosynthesis. Therefore, it is possible to develop starch-rich wheat cultivars by modifying TaGF14s.


July 7, 2019  |  

Oryza rufipogon Griff.

Oryza rufipogon, the progenitor of present-day cultivated rice, O. sativa, is one of the most studied wild species of rice. It is a perennial plant commonly found in a marsh or aquatic habitats of eastern and southern Asia. It has partial outcrossing behavior and is photoperiod sensitive. The flowering time usually ranges between September and November. It has been and is being exploited as a source of valuable genes and QTLs for yield components as well as resistance against biotic and abiotic stresses. A number of populations like chromosome segment substitution lines, backcross inbred lines, near-isogenic lines, and recombinant inbred lines have been developed from crosses between O. rufipogon and O. sativa as a prebreeding resource. These are being employed for broadening the genetic base of cultivated rice and diversify the breeder’s pool. With the advent of sequencing technologies, a number of phylogenetic studies have been conducted to reveal the evolutionary relationship of O. rufipogon with cultivated rice O. sativa. Further, transcriptomic studies characterizing the effect of various abiotic stresses have been conducted on this wild species. Role of miRNA under stress reaction has also been studied. Though the genetic, genomic, and transcriptomic resources are abundant, the proteomic resources for O. rufipogon are limited.


July 7, 2019  |  

Complete genome sequence of “Thiodictyon syntrophicum” sp. nov. strain Cad16T, a photolithoautotrophic purple sulfur bacterium isolated from the alpine meromictic Lake Cadagno.

Thiodictyon syntrophicum sp. nov. strain Cad16T is a photoautotrophic purple sulfur bacterium belonging to the family of Chromatiaceae in the class of Gammaproteobacteria. The type strain Cad16T was isolated from the chemocline of the alpine meromictic Lake Cadagno in Switzerland. Strain Cad16T represents a key species within this sulfur-driven bacterial ecosystem with respect to carbon fixation. The 7.74-Mbp genome of strain Cad16T has been sequenced and annotated. It encodes 6237 predicted protein sequences and 59 RNA sequences. Phylogenetic comparison based on 16S rRNA revealed that Thiodictyon elegans strain DSM 232T the most closely related species. Genes involved in sulfur oxidation, central carbon metabolism and transmembrane transport were found. Noteworthy, clusters of genes encoding the photosynthetic machinery and pigment biosynthesis are found on the 0.48 Mb plasmid pTs485. We provide a detailed insight into the Cad16T genome and analyze it in the context of the microbial ecosystem of Lake Cadagno.


July 7, 2019  |  

Lifestyle of Lactobacillus hordei isolated from water kefir based on genomic, proteomic and physiological characterization.

Water kefir is a traditional fermented beverage made from sucrose, water, kefir granules, dried or fresh fruits. In our water kefir granules, Lactobacillus (L.) hordei is one of the predominant lactic acid bacteria (LAB) species of this presumed symbiotic consortium. It faces abundant sucrose versus limitation of amino- and fatty acids in an acidic environment. Sequencing of the genome of L. hordei TMW 1.1822 revealed one chromosome plus three plasmids. The size of the chromosome was 2.42?Mbp with a GC content of 35% GC and 2461 predicted coding sequences. Furthermore, we identified 1474 proteins upon growth on water kefir medium. Metabolic prediction revealed all enzymes required for the glycolytic Embden-Meyerhof (EMP) and phosphoketolase (PKP) pathways. Genes encoding all enzymes involved in citrate, pyruvate and mannitol metabolism are present. Moreover, it was confirmed that L. hordei is prototrophic for 11 amino acids and auxotrophic for 6 amino acids when combining putative biosynthesis pathways for amino acids with physiological characterization. Still, for glycine, serine and methionine no sure auxotype could be determined. The OppABCDF peptide transport system is complete, and 13 genes encoding peptidases are present. The arginine deiminase system, was predicted to be complete except for carbamate kinase, thus enabling neutralization reactions via ammonium formation but no additional energy generation. Taken together our findings enable prediction of the L. hordei lifestyle in water kefir: Abundant sucrose is consumed directly via parallel EMP and PK pathways and is also extracellularly converted to dextran and fructose by a glucansucrase, leaving fructose as additional carbon source. Essential amino acids (in the form of peptides) and citrate are acquired from fruits. In the lack of FabB unsaturated fatty acids are synthesized by predicted alternative enzymes. Formation of acetoin and diacetyl as well as arginine conversion reactions enable acidification limitation. Other members of the water kefir consortium (yeasts, acetic acid bacteria) likely facilitate or support growth of L. hordei by delivering gluconate, mannitol, amino- and fatty acids and vitamins. Copyright © 2018 Elsevier B.V. All rights reserved.


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