Culturable microorganisms from the earthworm digestive tract

The cultured aerobic copiotrophic bacteria and fungi from food-free digestive tracts of Aporrectodea caliginosa, Lumbricus terrestris, and Eisenia fetida earthworms, soil (compost), and fresh earthworm excrements were investigated. The microorganisms were isolated on nutrient media and identified by sequencing the fragments of bacterial 16S rRNA and fungal 28S rRNA (D1/D2 domain) gene sequences with subsequent phylogenetic analysis. Bacteria isolated from the digestive tracts of earthworms belonged to the families Aeromonadaceae, Comamonadaceae, Enterobacteriaceae, Flavobacteriaceae, Moraxellaceae, Pseudomonadaceae, and Sphingobacteriaceae (Bacteroidetes), as well as Actinobacteria. For five strains, namely Ochrobactrum sp. 341-2 (α-Proteobacteria), Massilia sp. 557-1 (β-Proteobacteria), Sphingobacterium sp. 611-2 (Bacteroidetes), Leifsonia sp. 555-1, and a bacterium from the family Microbacteriaceae, isolate 521-1 (Actinobacteria), the similarity to known 16S rRNA sequences was 93–97%; they therefore, probably belong to new species and genera. Bacterial groups isolated from the digestive tracts of earthworms were significantly different from those isolated from soil and excrements. Some bacterial taxa occurred in different sections of A. caliginosa intestine and in intestines of different earthworm species; however, the overall composition of bacterial communities in these objects is different. Existence of bacterial groupings symbiotically associated with intestines is proposed. Among the fungi, Bjerkandera adusta and Syspastospora parasitica were isolated from the cleaned digestive tracts as light-colored, sterile mycelium, as well as Geotrichum candidum, Acremonium murorum (A. murorum var. felina), Alternaria alternata, Aspergillus candidus, A. versicolor, Cladosporium cladosporioides, Rhizomucor racemosus, Mucor hiemalis, Fusarium (F. oxysporum, Fusarium sp.), and Penicillium spp. These fungi survive for a long time in the earthworm’s digestive environment. Investigation of the functional characteristics and role in the host organism is required to confirm the symbiotic status of the microorganisms associated with the earthworm digestive tract.     

Distribution of sediment bacterial and archaeal communities in plateau freshwater lakes

Both Bacteria and Archaea might be involved in various biogeochemical processes in lacustrine sediment ecosystems. However, the factors governing the intra-lake distribution of sediment bacterial and archaeal communities in various freshwater lakes remain unclear. The present study investigated the sediment bacterial and archaeal communities in 13 freshwater lakes on the Yunnan Plateau. Quantitative PCR assay showed a large variation in bacterial and archaeal abundances. Illumina MiSeq sequencing illustrated high bacterial and archaeal diversities. Bacterial abundance was regulated by sediment total organic carbon and total nitrogen, and water depth, while nitrate nitrogen was an important determinant of bacterial diversity. Proteobacteria, Acidobacteria, Actinobacteria, Bacteroidetes, Chlorobi, Chloroflexi, Cyanobacteria, Firmicutes, Gemmatimonadetes, Nitrospirae, Planctomycetes, and Verrucomicrobia were the major components of sediment bacterial communities. Proteobacteria was the largest phylum, but its major classes and their proportions varied greatly among different lakes, affected by sediment nitrate nitrogen. In addition, both Euryarchaeota and Crenarchaeota were important members in sediment archaeal communities, while unclassified Archaea usually showed the dominance.     

Towards map-based cloning of <Emphasis Type="Italic">FB_Mfu10</Emphasis>: identification of a receptor-like kinase candidate gene underlying the <Emphasis Type="Italic">Malus fusca</Emphasis> fire blight resistance locus on linkage group 10

Breeding for resistance against the destructive fire blight disease of apples is the most sustainable strategy to control the menace of this disease, and has become increasingly important in European apple breeding programs. Since most cultivars are susceptible, wild accessions have been explored for resistance with quantitative trait loci detected in a few wild species. Fire blight resistance of Malus fusca was described following phenotypic evaluations with a C-type strain of Erwinia amylovora, Ea222_JKI, and the detection of a major QTL on chromosome 10 (Mfu10) of this crabapple. The stability of the resistance of M. fusca and Mfu10 has been evaluated using two other strains, the highly aggressive Canadian S-type strain—Ea3049, and the avrRpt2_EA mutant—ZYRKD3-1, both of which overcome the resistance of Malus ×robusta 5, a wild species accession with an already described fire blight resistance gene. To pave the way for positional cloning of the underlying fire blight resistance gene of M. fusca, we have fine mapped the QTL region on linkage group 10 using 1888 individuals and 23 newly developed molecular markers, thus delimiting the interval of interest to 0.33 cM between markers FR39G5T7xT7y/FR24N24RP and FRMf7358424/FR46H22. Tightly linked SSR markers are suitable for marker-assisted selection in breeding programs. Furthermore, a bacterial artificial chromosome (BAC) clone spanning FB_Mfu10 region was isolated and sequenced. One putative fire blight resistance candidate gene of M. fusca was predicted on the sequence of BAC 46H22 within the resistance region that encodes B-lectin and serine/threonine kinase domains.     

Rational design and analysis of an <Emphasis Type="Italic">Escherichia coli</Emphasis> strain for high-efficiency tryptophan production

l-tryptophan (l-trp) is a precursor of various bioactive components and has great pharmaceutical interest. However, due to the requirement of several precursors and complex regulation of the pathways involved, the development of an efficient l-trp production strain is challenging. In this study, Escherichia coli (E. coli) strain KW001 was designed to overexpress the l-trp operator sequences (trpEDCBA) and 3-deoxy-D-arabinoheptulosonate-7-phosphate synthase (aroG ^fbr ). To further improve the production of l-trp, pyruvate kinase (pykF) and the phosphotransferase system HPr (ptsH) were deleted after inactivation of repression (trpR) and attenuation (attenuator) to produce strain KW006. To overcome the relatively slow growth and to increase the transport rate of glucose, strain KW018 was generated by combinatorial regulation of glucokinase (galP) and galactose permease (glk) expression. To reduce the production of acetic acid, strain KW023 was created by repressive regulation of phosphate acetyltransferase (pta) expression. In conclusion, strain KW023 efficiently produced 39.7 g/L of l-trp with a conversion rate of 16.7% and a productivity of 1.6 g/L/h in a 5 L fed-batch fermentation system.     

Stability,oviposition attraction,and larvicidal activity of binary toxin from <Emphasis Type="Italic">Bacillus sphaericus</Emphasis> expressed in <Emphasis Type="Italic">Escherichia coli</Emphasis>

Bacillus sphaericus produces a two-chain binary toxin composed of BinA (42 kDa) and BinB (51 kDa), which are deposited as parasporal crystals during sporulation. The toxin is highly active against Culex larvae and Aedes and Anopheles mosquitoes, which are the principal vectors for the transmission of malaria, yellow fever, encephalitis, and dengue. The use of B. sphaericus and Bacillus thuringiensis in mosquito control programs is limited by their sedimentation in still water. In this study, the binA and binB genes were cloned and the recombinant BinAB protein was expressed in three strains of Escherichia coli. These recombinant strains were used in a toxicity assay against Culex quinquefasciatus larvae. The highest expression level was achieved when both proteins were expressed in a single operon construct. The BinAB protein expressed in the E. coli Arctic strain showed higher larvicidal activity than either of the recombinant proteins from the E. coli Ril or pLysS strains. Furthermore, it had the highest oviposition attraction (49.1%, P?相似文献   

Different laboratory populations similar bacterial profile? The case of Glossina palpalis gambiensis

Background

Microbiota plays an important role in the biology, ecology and evolution of insects including tsetse flies. The bacterial profile of 3 Glossina palpalis gambiensis laboratory colonies was examined using 16S rRNA gene amplicon sequencing to evaluate the dynamics of the bacterial diversity within and between each G. p. gambiensis colony.

Results

The three G. p. gambiensis laboratory colonies displayed similar bacterial diversity indices and OTU distribution. Larval guts displayed a higher diversity when compared with the gastrointestinal tract of adults while no statistically significant differences were observed between testes and ovaries. Wigglesworthia and Sodalis were the most dominant taxa. In more detail, the gastrointestinal tract of adults was more enriched by Wigglesworthia while Sodalis were prominent in gonads. Interestingly, in larval guts a balanced co-existence between Wigglesworthia and Sodalis was observed. Sequences assigned to Wolbachia, Propionibacterium, and Providencia were also detected but to a much lesser degree. Clustering analysis indicated that the bacterial profile in G. p. gambiensis exhibits tissue tropism, hence distinguishing the gut bacterial profile from that present in reproductive organs.

Conclusions

Our results indicated that age, gender and the origin of the laboratory colonies did not significantly influence the formation of the bacterial profile, once these populations were kept under the same rearing conditions. Within the laboratory populations a tissue tropism was observed between the gut and gonadal bacterial profile.

     

The bacterial diversity in a Brazilian non-disturbed mangrove sediment

The bacterial diversity present in sediments of a well-preserved mangrove in Ilha do Cardoso, located in the extreme south of São Paulo State coastline, Brazil, was assessed using culture-independent molecular approaches (denaturing gradient gel electrophoresis (DGGE) and analysis of 166 sequences from a clone library). The data revealed a bacterial community dominated by Alphaproteobacteria (40.36% of clones), Gammaproteobacteria (19.28% of clones) and Acidobacteria (27.71% of clones), while minor components of the assemblage were affiliated to Betaproteobacteria, Deltaproteobacteria, Firmicutes, Actinobacteria and Bacteroidetes. The clustering and redundancy analysis (RDA) based on DGGE were used to determine factors that modulate the diversity of bacterial communities in mangroves, such as depth, seasonal fluctuations, and locations over a transect area from the sea to the land. Profiles of specific DGGE gels showed that both dominant (‘universal’ Bacteria and Alphaproteobacteria) and low-density bacterial communities (Betaproteobacteria and Actinobacteria) are responsive to shifts in environmental factors. The location within the mangrove was determinant for all fractions of the community studied, whereas season was significant for Bacteria, Alphaproteobacteria, and Betaproteobacteria and sample depth determined the diversity of Alphaproteobacteria and Actinobacteria.     

<Emphasis Type="Italic">In silico</Emphasis> characterization and Molecular modeling of double-strand break repair protein <Emphasis Type="Italic">MRE11</Emphasis> from <Emphasis Type="Italic">Phoenix dactylifera</Emphasis> v deglet nour

Background

DNA double-strand breaks (DSBs) are highly cytotoxic and mutagenic. MRE11 plays an essential role in repairing DNA by cleaving broken ends through its 3′ to 5′ exonuclease and single-stranded DNA endonuclease activities.

Methods

The present study aimed to in silico characterization and molecular modeling of MRE11 from Phoenix dactylifera L cv deglet nour (DnMRE11) by various bioinformatic approaches. To identify DnMRE11 cDNA, assembled contigs from our cDNA libraries were analysed using the Blast2GO2.8 program.

Results

The DnMRE11 protein length was 726 amino acids. The results of HUMMER show that DnMRE11 is formed by three domains: the N-terminal core domain containing the nuclease and capping domains, the C-terminal half containing the DNA binding and coiled coil region. The structure of DnMRE11 is predicted using the Swiss-Model server, which contains the nuclease and capping domains. The obtained model was verified with the structure validation programs such as ProSA and QMEAN servers for reliability. Ligand binding studies using COACH indicated the interaction of DnMRE11 protein with two Mn²⁺ ions and dAMP. The ConSurf server predicted that residues of the active site and Nbs binding site have high conservation scores between plant species.

Conclusions

A model structure of DnMRE11 was constructed and validated with various bioinformatics programs which suggested the predicted model to be satisfactory. Further validation studies were conducted by COACH analysis for active site ligand prediction, and revealed the presence of six ligands binding sites and two ligands (2 Mn²⁺ and dAMP).

     

Epiphytic bacterial communities of the alga <Emphasis Type="Italic">Fucus vesiculosus</Emphasis> in oil-contaminated water areas of the Barents Sea

Taxonomic compositions of epiphytic bacterial communities in water areas differing in levels of oil pollution were revealed. In total, 82 bacterial genera belonging to 16 classes and 11 phyla were detected. All detected representatives of epiphytic bacterial communities belonged to the phyla Actinobacteria, Bacteroidetes, Planctomycetes, Proteobacteria, Verrucomicrobia, Acidobacteria, Cyanobacteria, Firmicutes, and Fusobacteria and candidate division TM7. The ratio of the phyla in the communities varied depending on the levels of oil pollution. New data on taxonomic composition of uncultivated epiphytic bacterial communities of Fucus vesiculosus were obtained.     

Complete genome sequence and pathogenic genes analysis of <Emphasis Type="Italic">Pectobacterium atroseptica</Emphasis> JG10-08

Pectobacterium atroseptica is known as a rod-shaped gram-negative bacterial pathogen associated with the blackleg of potato. P. atroseptica has been widely identified as the predominant agent causing tuber rot in temperate regions, a disease that leads to severe economic losses to potato industry. In this study, we provide the complete genome sequence of P. atroseptica JG10-08, which revealed that P. atroseptica strain JG10-08 carries a single 5,004,926 bp chromosome with 51.15% G+C content and harbors 4252 predicted coding genes. Phylogenetic analysis based on the genome sequences showed a close evolutionary relationship between P. atroseptica and Pectobacterium wasabiae. We discovered total 168 genes were potentially related to pathogenesis including 9 strain-specific genes encoding toxins on the genome of JG10-08. Further comparison with other species in Pectobacterium revealed a better understanding of pathogenic factors, especially secretion systems in P. atroseptica JG10-08. Collectively, the results of this research provide a solid foundation for discovering the underlying pathogenic mechanisms of P. atroseptica and offer the information to develop more effective strategies against blackleg of potatoes.     

Identification of D-amino acid dehydrogenase as an upstream regulator of the autoinduction of a putative acyltransferase in <Emphasis Type="Italic">Corynebacterium glutamicum</Emphasis>

Expression of a putative acyltransferase encoded by NCgl- 0350 of Corynebacterium glutamicum is induced by cell-free culture fluids obtained from stationary-phase growth of both C. glutamicum and Pseudomonas aeruginosa, providing evidence for interspecies communication. Here, we further confirmed that such communication occurs by showing that acyltransferase expression is induced by culture fluid obtained from diverse Gram-negative and -positive bacterial strains, including Escherichia coli, Salmonella Typhimurium, Bacillus subtilis, Staphylococcus aureus, Mycobacterium sp. strain JC1, and Mycobacterium smegmatis. A homologous acyltransferase encoded by PA5238 of P. aeruginosa was also induced by fluids obtained from P. aeruginosa as well as other bacterial strains, as observed for NCgl0350 of C. glutamicum. Because C. glutamicum is difficult to study using molecular approaches, the homologous gene PA5238 of P. aeruginosa was used to identify PA5309 as an upstream regulator of expression. A homologous D-amino acid dehydrogenase encoded by NCgl- 2909 of C. glutamicum was cloned based on amino acid similarity to PA5309, and its role in the regulation of NCgl0350 expression was confirmed. Moreover, NCgl2909 played positive roles in growth of C. glutamicum. Thus, we identified a D-amino acid dehydrogenase as an upstream regulator of the autoinduction of a putative acyltransferase in C. glutamicum.     

Diversity of endophytic fungal and bacterial communities in <Emphasis Type="Italic">Ilex paraguariensis</Emphasis> grown under field conditions

The composition and diversity of the endophytic community associated with yerba mate (Ilex paraguariensis) was investigated using culture-depending methods. Fungi were identified based on their micromorphological characteristics and internal transcribed spacer rDNA sequence analysis; for bacteria 16S rDNA sequence analysis was used. Fungal and bacterial diversity did not show significant differences between organ age. The highest fungal diversity was registered during fall season and the lowest in winter. Bacterial diversity was higher in stems and increased from summer to winter, in contrast with leaves, which decreased. The most frequently isolated fungus was Fusarium, followed by Colletotrichum; they were both present in all the sampling seasons and organ types assayed. Actinobacteria represented 57.5 % of all bacterial isolates. The most dominant bacterial taxa were Curtobacterium and Microbacterium. Other bacteria frequently found were Methylobacterium, Sphingomonas, Herbiconiux and Bacillus. Nitrogen fixation and phosphate solubilization activity, ACC deaminase production and antagonism against plant fungal pathogens were assayed in endophytic bacterial strains. In the case of fungi, strains of Trichoderma, Penicillium and Aspergillus were assayed for antagonism against pathogenic Fusarium sp. All microbial isolates assayed showed at least one growth promoting activity. Strains of Bacillus, Pantoea, Curtobacterium, Methylobacterium, Brevundimonas and Paenibacillus had at least two growth-promoting activities, and Bacillus, Paenibacillus and the three endophytic fungi showed high antagonistic activity against Fusarium sp. In this work we have made a wide study of the culturable endophytic community within yerba mate plants and found that several microbial isolates could be considered as potential inoculants useful for improving yerba mate production.     

Proteomics analysis of global regulatory cascades involved in clavulanic acid production and morphological development in <Emphasis Type="Italic">Streptomyces clavuligerus</Emphasis>

The genus Streptomyces comprises bacteria that undergo a complex developmental life cycle and produce many metabolites of importance to industry and medicine. Streptomyces clavuligerus produces the β-lactamase inhibitor clavulanic acid, which is used in combination with β-lactam antibiotics to treat certain β-lactam resistant bacterial infections. Many aspects of how clavulanic acid production is globally regulated in S. clavuligerus still remains unknown. We conducted comparative proteomics analysis using the wild type strain of S. clavuligerus and two mutants (ΔbldA and ΔbldG), which are defective in global regulators and vary in their ability to produce clavulanic acid. Approximately 33.5 % of the predicted S. clavuligerus proteome was detected and 192 known or putative regulatory proteins showed statistically differential expression levels in pairwise comparisons. Interestingly, the expression of many proteins whose corresponding genes contain TTA codons (predicted to require the bldA tRNA for translation) was unaffected in the bldA mutant.     

Validation of reference genes for gene expression analysis of response to anthocyanin induction in cell cultures of <Emphasis Type="Italic">Vitis davidii</Emphasis> (Rom. Caill.) Foëx

Real-time quantitative polymerase chain reaction (RT-qPCR) is an effective method for detecting changes of gene expression in plant cell metabolic regulation. A set of 15 reference gene candidates were selected for the present study of anthocyanin biosynthesis regulation, and stability. The suitability of their expression was evaluated in eight different experimental treatments in spine grape (Vitis davidii [Rom. Caill.] Foëx.) cell cultures. The results indicated that SAND family protein (SAND) and V-type proton ATPase subunit G (VAG) were the most stable reference genes for culture duration, tubulin alpha-3/alpha-5 chain (α-tubulin) and tubulin beta-1 chain (β-tubulin) for illumination conditions, ubiquitin-conjugating enzyme E2-17 kDa (UBQ) and VAG for UVB treatment, VAG and 60S ribosomal protein L18-2 (60SRP) for temperature treatment, AP47, clathrin adaptor complex subunit mu (AP-2) and 60SRP for cinnamic acid treatment, α-tubulin and UBQ for chitosan treatment, actin and alcohol dehydrogenase 2 (ADH2) for kinetin treatment, and β-tubulin and elongation factor 1-α (EF1-α) for cell line. Finally, the reliability of the selected reference genes was confirmed by investigating the expression profiles of the target gene dihydroflavonol 4-reductase (DFR) in spine grape cell cultures. The results of the present study offer the most robust platform for the most precise and broad application of RT-qPCR to investigate gene expression associated with anthocyanin biosynthesis in spine grape cell cultures.     

Composition and abundance of microbiota in the pharynx in patients with laryngeal carcinoma and vocal cord polyps

The pharynx is an important site of microbiota colonization, but the bacterial populations at this site have been relatively unexplored by culture-independent approaches. The aim of this study was to characterize the microbiota structure of the pharynx. Pyrosequencing of 16S rRNA gene libraries was used to characterize the pharyngeal microbiota using swab samples from 68 subjects with laryngeal cancer and 28 subjects with vocal cord polyps. Overall, the major phylum was Firmicutes, with Streptococcus as the predominant genus in the pharyngeal communities. Nine core operational taxonomic units detected from Streptococcus, Fusobacterium, Prevotella, Granulicatella, and Veillonella accounted for 21.3% of the total sequences detected. However, there was no difference in bacterial communities in the pharynx from patients with laryngeal cancer and vocal cord polyps. The relative abundance of Firmicutes was inversely correlated with Fusobacteria, Proteobacteria, Actinobacteria, and Bacteroidetes. The correlation was evident at the genus level, and the relative abundance of Streptococcus was inversely associated with Fusobacterium, Leptotrichia, Neisseria, Actinomyces, and Prevotella. This study presented a profile for the overall structure of the microbiota in pharyngeal swab samples. Inverse correlations were found between Streptococcus and other bacterial communities, suggesting that potential antagonism may exist among pharyngeal microbiota.     

The relation between <Emphasis Type="Italic">Blastocystis</Emphasis> and the intestinal microbiota in Swedish travellers

Background

Blastocystis sp. is a unicellular eukaryote that is commonly found in the human intestine. Its ability to cause disease is debated and a subject for ongoing research. In this study, faecal samples from 35 Swedish university students were examined through shotgun metagenomics before and after travel to the Indian peninsula or Central Africa. We aimed at assessing the impact of travel on Blastocystis carriage and seek associations between Blastocystis and the bacterial microbiota.

Results

We found a prevalence of Blastocystis of 16/35 (46%) before travel and 15/35 (43%) after travel. The two most commonly Blastocystis subtypes (STs) found were ST3 and ST4, accounting for 20 of the 31 samples positive for Blastocystis. No mixed subtype carriage was detected. All ten individuals with a typable ST before and after travel maintained their initial ST. The composition of the gut bacterial community was not significantly different between Blastocystis-carriers and non-carriers. Interestingly, the presence of Blastocystis was accompanied with higher abundances of the bacterial genera Sporolactobacillus and Candidatus Carsonella. Blastocystis carriage was positively associated with high bacterial genus richness, and negatively correlated to the Bacteroides-driven enterotype. These associations were both largely dependent on ST4 – a subtype commonly described from Europe – while the globally prevalent ST3 did not show such significant relationships.

Conclusions

The high rate of Blastocystis subtype persistence found during travel indicates that long-term carriage of Blastocystis is common. The associations between Blastocystis and the bacterial microbiota found in this study could imply a link between Blastocystis and a healthy microbiota as well as with diets high in vegetables. Whether the associations between Blastocystis and the microbiota are resulting from the presence of Blastocystis, or are a prerequisite for colonization with Blastocystis, are interesting questions for further studies.

     

Exploring the mechanism and efficient use of a durable gene-mediated resistance to bacterial blight disease in rice

Xanthomonas oryzae pv. oryzae (Xoo) causes bacterial blight, the most devastating bacterial disease of rice worldwide. The major disease resistance gene Xa3/Xa26 confers a durable resistance to Xoo with a dosage effect. However, the mechanism of Xa3/Xa26-mediated resistance remains to be elucidated. We created near-isogenic lines carrying Xa3/Xa26 with a background of indica and japonica, the two major subspecies of Asian cultivated rice. Analyzing these rice lines showed that the japonica background facilitated resistance to Xoo, which was associated with increased Xa3/Xa26 expression, compared with rice lines with an indica background. This characteristic of Xa3/Xa26 was related to the WRKY45 locus, which had higher expression with the japonica background than with the indica background. However, the two alleles of the WRKY45 locus had different expression levels, with the WRKY45-1 expression level being higher than that of WRKY45-2 for both japonica and indica backgrounds. In addition, the resistance level conferred by Xa3/Xa26 was higher in the presence of WRKY45-1 than in the presence of WRKY45-2 for both japonica and indica backgrounds. Xa3/Xa26-mediated resistance was associated with increased accumulation of jasmonic acid (JA), JA-isoleucine, and terpenoid and flavonoid phytoalexins. Exogenous JA application enhanced Xa3/Xa26-mediated resistance. These results not only provide more knowledge toward understanding the mechanism of Xa3/Xa26-mediated resistance but also offer the best choice for using Xa3/Xa26 for rice resistance improvement, specifically, a japonica background with the WRKY45-1 allele.