Kai Lou

Endogenous bacteria are important for maintaining the health and other ecologically relevant functions of cotton plants. However, little is known about the community structures and diversity of endophytic bacteria in cotton plants. In our study, we used the Illumina amplicon sequencing technology to study the endophytic bacteria found in cotton root tissue in Xinjiang, China. A total of 60.84 × 10⁶ effective sequences of the 16S rRNA gene in the V5–V6 variable region revealed a large number of operational taxonomic units (OTUs), namely 81–338 OTUs, at a cut-off level of 3% and a sequencing depth of 50 000 sequences. Among the 23 classes identified, Gammaproteobacteria was the dominant group, followed by Alphaproteobacteria, Actinobacteria, and Bacillus. The diversity of endogenous bacteria differed at different growth periods, with the most OTUs detected in seedlings (654), followed by the budding stage (381), flowering stage (350), and flocking stage (351). A total of 217 OTUs were common to all four stages. Pantoea tags were more common to the Shihezi region, whereas Erwinia labels were more common to the Hami region. These results suggest that the dynamics of endophytic bacterial communities are affected by plant growth stage. This highlights the relevance of microbial diversity studies in improving our understanding of endophyte communities.

A Gram-stain-negative, oval-shaped and light pink pigmented bacterium, designated XAAS-2(T), was isolated from rhizosphere soil of Tamarix ramosissima. The sole respiratory quinone of the type strain XAAS-2(T) was MK-7, and the principal cellular fatty acids were summed feature 4 (iso-C-17:1 vertical bar and/or anteiso-C-17:1 B) and iso-C-15:0. The major polar lipids were phosphatidylethanolamine and two unidentified lipids. 16S rRNA gene sequence analysis indicated that strain XAAS-2(T) belonged to the genus Pontibacter within the family Cytophagaceae with sequence similarities of 93.9-97.1% to other type species of the genus Pontibacter and to Pontibacter xinjiangensis CCTCC AB 207200(T) as the closest neighbour. The DNA G+C content of strain XAAS-2(T) was 50.6 mol%. The level of DNA-DNA relatedness of XAAS-2(T) and P. xinjiangensis CCTCC AB 207200(T) was 47.5% (SD=3.27). Phenotypic and genotypic data suggested that strain XAAS-2(T) represents a novel species of the genus Pontibacter, for which the name Pontibacter brevis sp. nov. is proposed, with the type strain XAAS-2(T) (=CCTCC AB 2016135(T) = JCM 31443(T)).

This study details the introduction of a gfp marker into an endophytic bacterial strain (Achromobacter marplatensis strain 17, isolated from sugar beet) to monitor its colonization of sugar beet (Beta. vulgaris L.). Stability of the plasmid encoding the gfp was confirmed in vitro for at least 72 h of bacterial growth and after the colonization of tissues, under nonselective conditions. The colonization was observed using fluorescence microscopy and enumeration of culturable endophytes in inoculated sugar beet plants that grew for 10 or 20 days. gfp-Expressing strains were re-isolated from the inner tissues of surface-sterilized roots and stems of inoculated plants, and the survival of the Achromobacter marplatensis 17:gfp strain in plants 20 days after inoculation, even in the absence of selective pressure, suggests that it is good colonizer. These results also suggest that this strain could be a useful tool for the delivery of enzymes or other proteins into plants. In addition, the study highlights that sugar beet plants can be used effectively for detailed in vitro studies on the interactions between A. marplatensis strain 17 and its host, particularly if a gfp-tagged strain of the pathogen is used.

Plants harbor complex and variable microbial communities. Using molecular-based techniques targeting the 16S rRNA gene, we studied the developmental stages and geographical location diversity of endophytic archaea in two locations (Shihezi and Changji) and four periods (the seedling growth, rosette formation, tuber growth and sucrose accumulation sampling periods) in the north slope of Tianshan Mountain, China. Community structure of mixed sample from 60 sugar beet plants was examined using PCR-based 454 pyrosequencing and terminal restriction fragment length polymorphism (T-RFLP). In total, 5290 archaea 16S rRNA sequences were obtained from all sugar beet samples. The most abundant archaea groups in all sugar beet were Methanococci, the miscellaneous Crenarchaeotic Group and Thermoplasmata. There was a marked difference in diversity of endophytic archaea in sugar beet for different growth periods. The greatest number of Operational T-RFLP Units (OTUs) was detected during sucrose accumulation (298) and rosette formation (282). Endophytic archaea diversity was reduced during seedling growth (128 OTUs) and tuber growth (55 OTUs). Nine OTUs were common to all four periods of growth. There were more OTUs in Shihezi than in Changji. Clustering analysis and principal component analysis of T-RFLP data revealed distinct shifts in endophytic archaea community profiles that corresponded to plant growth stage rather than geographical location. The dynamics of endophytic archaea communities were influenced by plant growth stage. To our knowledge, this is the first report that archaea has been identified as endophytes associated with sugar beet by the culture-independent approach. The results suggest that the diversity of endophytic archaea is abundant in sugar beet.

Our aim was to know response of spring bacteria and metabolic characteristics of sensitive bacteria to felt earthquake. Water samples were collected from January 31 to December 31, 2012, during which period 5 felt earthquakes occurred and the epicenter was 100 kilometers away from the No. 10 Spring in Urumqi. We monitored the spring bacterial activities and function diversity changes from No. 10 Spring in Urumqi during the pre- and post-earthquake stages by using plate culture counting methods and BIOLOG GEN III bacteria plate. The spring bacterial numbers presented stochastic dynamic changes through the year. The culturable bacteria numbers and average well color development (AWCD ) of carbon source utilization of bacterial community were higher after the earthquake. Besides, there were some correlations with magnitude and epicenter distance of earthquake. The main carbon source utilization types of sensitive bacteria group for felt earthquake were sugar alcohol at the No. 10 Spring. The results indicated that the BIOLOG GEN III plate can be used for spring bacterial metabolism diversity research. Culturable bacteria numbers and carbon source utilization of bacterial communities showed some reflecting earthquake law.

As bacteria-originated crude violacein, a natural indolocarbazole product, consists of violacein and deoxyviolacein, and can potentially be a new type of natural antibiotics, the reconstruction of an effective metabolic pathway for crude violacein (violacein and deoxyviolacein mixture) synthesis directly from glucose in Escherichia coli was of importance for developing industrial production process. Strains with a multivariate module for varied tryptophan productivities were firstly generated by combinatorial knockout of trpR/tnaA/pheA genes and overexpression of two key genes trpE fbr /trpD from the upstream tryptophan metabolic pathway. Then, the gene cluster of violacein biosynthetic pathway was introduced downstream of the generated tryptophan pathway. After combination of these two pathways, maximum crude violacein production directly from glucose by E. coli B2/pED + pVio was realized with a titer of 0.6 ± 0.01 g L−1 in flask culture, which was four fold higher than that of the control without the tryptophan pathway up-regulation. In a 5-L bioreactor batch fermentation with glucose as the carbon source, the recombinant E. coli B2/pED + pVio exhibited a crude violacein titer of 1.75 g L−1 and a productivity of 36 mg L−1 h−1, which was the highest titer and productivity reported so far under the similar culture conditions without tryptophan addition. Metabolic pathway analysis using 13C labeling illustrated that the up-regulated tryptophan supply enhanced tryptophan metabolism from glucose, whereas the introduction of violacein pathway drew more carbon flux from glucose to tryptophan, thereby contributing to the effective production of crude violacein in the engineered E. coli cell factory.

We investigated the primary successions of soil enzyme activity and heterotrophic microbial communities at the forefields of the Tianshan Mountains No. 1 Glacier by investigating soil microbial processes (microbial biomass and nitrogen mineralization), enzyme activity and community-level physiological profiling. Soils deglaciated between 1959 and 2008 (0, 5, 17, 31 and 44 years) were collected. Soils >1,500 years in age were used as a reference (alpine meadow soils). Soil enzyme activity and carbon-source utilization ability significantly increased with successional time. Amino-acid utilization rates were relatively higher in early, unvegetated soils (0 and 5 years), but carbohydrate utilization was higher in later stages (from 31 years to the reference soil). Discriminant analysis, including data on microbial processes and soil enzyme activities, revealed that newly exposed soils (0–5 years) and older soils (17–44 years) were well-separated from each other and obviously different from the reference soil. Correlation analysis revealed that soil organic carbon, was the primary factor influencing soil enzyme activity and heterotrophic microbial community succession. Redundancy analysis suggested that soil pH and available P were also affect microbial activity to a considerable degree. Our results indicated that glacier foreland soils have continued to develop over 44 years and soils were significantly affected by the geographic location of the glacier and the local topography. Soil enzyme activities and heterotrophic microbial communities were also significantly influenced by these variables.

The endophytic bacterial diversity of drunken horse grass (DHG) (Achnatherum inebrians) growing in native habitat station in Xinjiang Province, China was analyzed by 16S rDNA cloning, amplified ribosomal DNA restriction analysis (ARDRA), and sequence homology comparison. To effectively exclude the interference of chloroplast DNA and mitochondrial DNA of DHG, a pair of bacterial PCR primers was selected to specifically amplify bacterial 16S rDNA sequences directly from DHG root tissues. Among 249 positive clones in the 16S rDNA library of endophytes, 57 OTUs were identified based on the similarity of the ARDRA banding profiles. Sequence analysis revealed diverse phyla of bacteria in the 16S rDNA library, which consisted of alpha, beta, gamma, delta, and epsilon subclasses of the Proteobacteria, Flavobacteria, Actinobacteria, Sphingobacteria, and uncultured bacteria. The dominant group was Proteobacteria, whie the most numerous genus was Bacillus. More than 8.00% of the total clones showed a high similarity to uncultured bacteria, suggesting that nonculturable bacteria make a significant part of DHG endophytic bacterial community. To our knowledge, this is the first report that endophytic bacteria associated with DHG are revealed by the culture-independent approach. The results suggest that the diversity of endophytic bacteria is abundant in DHG.

Plants harbors complex and variable microbial communities. Endophytic bacteria play an important function and potential role more effectively in developing sustainable systems of crop production. To examine how endophytic bacteria in sugar beet (Beta vulgaris L.) vary across both host growth period and location, PCR-based Illumina was applied to revealed the diversity and stability of endophytic bacteria in sugar beet on the north slope of Tianshan mountain, China. A total of 60.84 M effective sequences of 16S rRNA gene V3 region were obtained from sugar beet samples. These sequences revealed huge amount of operational taxonomic units (OTUs) in sugar beet, that is, 19–121 OTUs in a beet sample, at 3 % cutoff level and sequencing depth of 30,000 sequences. We identified 13 classes from the resulting 449,585 sequences. Alphaproteobacteria were the dominant class in all sugar beets, followed by Acidobacteria, Gemmatimonadetes and Actinobacteria. A marked difference in the diversity of endophytic bacteria in sugar beet for different growth periods was evident. The greatest number of OTUs was detected during rossette formation (109 OTUs) and tuber growth (146 OTUs). Endophytic bacteria diversity was reduced during seedling growth (66 OTUs) and sucrose accumulation (95 OTUs). Forty-three OTUs were common to all four periods. There were more tags of Alphaproteobacteria and Gammaproteobacteria in Shihezi than in Changji. The dynamics of endophytic bacteria communities were influenced by plant genotype and plant growth stage. To the best of our knowledge, this study is the first application of PCR-based Illumina pyrosequencing to characterize and compare multiple sugar beet samples.