Transcriptome analysis expands the potential roles of quorum sensing in biodegradation and physiological responses to microcystin.

Bacterial degradation is one of the most efficient ways to remove microcystins (MCs), the most frequently detected toxin in cyanobacterial blooms. Using Novosphingobium sp. ERW19 as a representative strain, our laboratory previously demonstrated that quorum sensing (QS), the cell density-dependent gene regulation system, positively regulates biodegradation of MCs via transcriptional activation of mlr-pathway-associated genes. Increasing evidence indicates that QS is involved in a wide spectrum of regulatory circuits, but it remains unclear which physiological processes in MC degradation besides the expression of MC-degrading genes are also subject to QS-dependent regulation. This study used transcriptome analysis to identify QS-regulated genes during degradation of MCs. A large percentage (up to 32.6%) of the genome of the MC-degrading bacterial strain Novosphingobium sp. ERW19 was significantly differentially expressed in the corresponding QS mutants. Pathway enrichment analysis of QS-regulated genes revealed that QS mainly influenced metabolism-associated pathways, particularly those related to amino acid metabolism, carbohydrate metabolism, and biodegradation and metabolism of xenobiotics. In-depth functional interpretation of QS-regulated genes indicated a variety of pathways were potentially associated with bacterial degradation or physiological responses to MCs, including genes involved in cell motility, cytochrome P450-dependent metabolism of xenobiotics, glutathione S-transferase (GST), envelope stress response, and ribosomes. Furthermore, QS may be involved in regulating the initial and final steps of the catabolic pathway of phenylacetic acid, an intermediate product of MC degradation. Collectively, this global survey of QS-regulated genes in a MC-degrading bacterial strain facilitates a deeper understanding of QS-controlled processes that may be important for bacterial degradation of MCs or may contribute to the physiological responses of bacteria to MCs.

The communities and functional profiles of virioplankton along a salinity gradient in a subtropical estuary.

Viruses are the major drivers shaping microorganismal communities, and impact marine biogeochemical cycling. They are affected by various environmental parameters, such as salinity. Although the spatiotemporal distribution and dynamics of virioplankton have been extensively studied in saline environments, few detailed studies of community structure and function of viruses along salinity gradients have been conducted. Here, we used the 16S and 18S rRNA gene amplicon and metagenomic sequencing from a subtropical estuary (Pearl River Estuary, PRE; located in Shenzhen, Guangdong Province, China) to explore how viral community composition and function vary along a salinity gradient. Results showed that the detected viruses were mainly bacteriophages. The double-stranded DNA viruses were the most abundant (especially Siphoviridae, Myoviridae, Mimiviridae, Phycodnaviridae, and Podoviridae), followed by a small number of single-stranded DNA (Circoviridae) and RNA (Retroviridae) viruses. Viral biodiversity significantly declined and community structure varied greatly along the salinity gradient. The salinity, ammonium and dissolved oxygen were dominated factors influencing the community composition of viruses. Association network analysis showed that viruses had a negative effect on multiple host taxa (prokaryotic and eukaryotic species). Metagenomic data revealed that the main viral functional potential was involved in organic matter metabolism by carbohydrate-active enzymes (CAZymes). Deeper comparative functional analyses showed that viruses in the low-salinity environment had more carbohydrate-binding module and glycosidase hydrolases activities than those under high-salinity conditions. However, an opposite pattern was observed for carbohydrate esterases. These results suggest that virus-encoded CAZyme genes may alter the bacterial metabolism in estuaries. Overall, our results demonstrate that there is a spatial heterogeneity in the composition and function of virioplankton along a salinity gradient. This study enhances our understanding of viral distribution and their contribution to regulating carbon degradation throughout environments with varying salinities in subtropical estuaries.

Comparison of Gut Microbiota Diversity and Predicted Functions Between Healthy and Diseased Captive Rana dybowskii.

The gut microbiota plays a key role in host health, and disruptions to gut bacterial homeostasis can cause disease. However, the effect of disease on gut microbiota assembly remains unclear and gut microbiota-based predictions of health status is a promising yet poorly established field. Using Illumina high-throughput sequencing technology, we compared the gut microbiota between healthy (HA and HB) and diarrhoeic (DS) Rana dybowskii groups and analyzed the functional profiles through a phylogenetic investigation of communities by reconstruction of unobserved states (PICRUSt) analysis. In addition, we estimated the correlation between gut microbiota structures and predicted the functional compositions. The results showed significant differences in the phylogenetic diversity (Pd), Shannon, and observed richness (Sobs) indices between the DS and HB groups, with significant differences observed in the gut microbiota composition between the DS group and the HA and HB groups. Linear discriminant analysis (LDA) effect size (LEfSe) results revealed that Proteobacteria were significantly enriched in the DS group; Bacteroidetes were significantly enriched in the HA and HB groups; and Aeromonas, Citrobacter, Enterococcus, Hafnia-Obesumbacterium, Morganella, Lactococcus, Providencia, Vagococcus, and Staphylococcus were significantly enriched in the DS group. Venn diagrams revealed that there were many more unique genera in the DS group than the HA and HB groups. Among 102 sensitive species selected using the indicator method, 33 indicated a healthy status and 69 (e.g., Acinetobacter, Aeromonas, Legionella, Morganella, Proteus, Providencia, Staphylococcus, and Vagococcus) indicated a diseased status. There was a significant and positive association between the composition and functional composition of the gut microbiota, thus indicating low functional redundancy of the frog gut bacterial community. Rana dybowskii disease was associated with changes in the gut microbiota, which subsequently disrupted bacterial-mediated functions. The results of this study can aid in revealing the effect of the R. dybowskii gut microbiota on host health and provide a basis for elucidating the mechanism of the occurrence of R. dybowskii disease.

Two hierarchical LuxR-LuxI type quorum sensing systems in Novosphingobium activate microcystin degradation through transcriptional regulation of the mlr pathway.

Microcystins (MCs) are the most common cyanotoxins produced by harmful cyanobacterial blooms and pose an increasing global threat to human health and ecosystems. Microbial degradation represents an efficient and sustainable approach for the removal of MCs. Although the enzymatic pathway for biodegradation of MCs has been characterized, the regulatory mechanisms underlying the degradation processes remain unclear. Quorum sensing (QS) is a cell-density-dependent regulatory mechanism that enables bacteria to orchestrate collective behaviors. The acyl-homoserine lactone (AHL)-mediated QS system regulates the biodegradation of many organic pollutants. However, it is not known whether this QS system is involved in the degradation of MCs. This study aimed to fill this knowledge gap. In this study, the proportion of culturable AHL-producers increased significantly after enrichment of MCs, and AHL-based QS systems were present in all genome-sequenced MC-degrading strains, supporting the hypothesis that QS participates in the degradation of MCs. Two bifunctional Novosphingobium strains (with MC-degrading and AHL-producing abilities) were isolated using a novel primer pair targeting mlrA, the marker gene of mlr degradation pathway. Biochemical and genetic analysis revealed that the MC-degrading bacterium Novosphingobium sp. ERW19 encodes two hierarchical regulatory QS systems designated novR1/novI1 and novR2/novI2. Gene knockout and complementation experiments indicated that both systems were required for efficient degradation of MCs. Transcriptomic analyses revealed that the QS systems positively regulate degradation of MCs through transcriptional activation of MC-degrading genes, especially mlrA. Given that QS may be a common trait within mlr pathway-dependent MC-degrading bacterial strains and the degradation activity is directly regulated by QS, manipulation of the QS systems may be a promising strategy to control biodegradation of MCs.

Environmental and host factors shaping the gut microbiota diversity of brown frog Rana dybowskii.

Symbiotic microbial communities are common in amphibians, and the composition of gut microbial communities varies with factors such as host phylogeny, life stage, ecology, and diet. However, little is known regarding how amphibians acquire their microbiota or how their growth, development, and environmental factors affect the diversity of their microbiotas. We sampled the gut microbiota during different developmental stages of brown frog Rana dybowskii, including tadpoles (T), frogs in metamorphosis (M), frogs just post-metamorphosis and after eating (F), juvenile frogs in summer (Js), adult frogs in summer (As), adult frogs in autumn (Aa), and hibernating frogs (Ah). We recorded data on the environmental (ambient temperature, fasting status, habitat, and season) and host (body mass and developmental period) factors. We investigated whether the gut microbiota diversity of R. dybowskii differs according to the host developmental stage via high-throughput Illumina sequencing and whether the gut microbiota diversity is affected by environmental and host factors. We found that alpha and beta diversity varied significantly during different developmental stages. The linear discriminant analysis effect size (LEfSe) analysis identified eight phyla exhibiting significant differences: Cyanobacteria (T group), Proteobacteria (M group), Fusobacteria (F group), Firmicutes (As group), Actinobacteria (Aa group), Verrucomicrobia (Aa group), Tenericutes (Aa group), and Bacteroidetes (Ah group). The Venn diagrams showed that 49 shared OTUs were present during all stages of development, whereas 10 OTUs were present in >90% of the samples. The environmental and host factors were significantly correlated with microbial community changes. Furthermore, the AIC-based model results suggested that development was the only variable that needed inclusion in the redundancy analysis (RDA) to explain the variance in taxa. These results have broad implications for our understanding of gut microbiota development and its associations with amphibian development and environmental factors.

Opportunistic bacteria use quorum sensing to disturb coral symbiotic communities and mediate the occurrence of coral bleaching.

Coral associated microorganisms, especially some opportunistic pathogens can utilize quorum-sensing (QS) signals to affect population structure and host health. However, direct evidence about the link between coral bleaching and dysbiotic microbiomes under QS regulation was lacking. Here, using 11 opportunistic bacteria and their QS products (AHLs, acyl-homoserine-lactones), we exposed Pocillopora damicornis to three different treatments: test groups (A and B: mixture of AHLs-producing bacteria and cocktail of AHLs signals respectively); control groups (C and D: group A and B with furanone added respectively); and a blank control (group E: only seawater) for 21 days. The results showed that remarkable bleaching phenomenon was observed in groups A and B. The operational taxonomic units-sequencing analysis shown that the bacterial network interactions and communities composition were significantly changed, becoming especially enhanced in the relative abundances of Vibrio, Edwardsiella, Enterobacter, Pseudomonas, and Aeromonas. Interestingly, the control groups (C and D) were found to have a limited influence upon host microbial composition and reduced bleaching susceptibility of P. damicornis. These results indicate bleaching's initiation and progression may be caused by opportunistic bacteria of resident microbes in a process under regulation by AHLs. These findings add a new dimension to our understanding of the complexity of bleaching mechanisms from a chemoecological perspective.

Functional profiles of phycospheric microorganisms during a marine dinoflagellate bloom.

Harmful algal blooms (HABs) are an ecological concern but relatively few studies have investigated the functional potential of bacterioplankton over a complete algal bloom cycle, which is critical for determining their contribution to the fate of algal blooms. To address this point, we carried out a time-series metagenomic analysis of the functional features of microbial communities at three different Gymnodinium catenatum bloom stages (pre-, peak-, and post-bloom). Different microbial composition were observed during the blooming stages. The environmental parameters and correlation networks co-contribute to microbial variability, and the former explained 38.4% of total variations of the bacterioplankton community composition. Functionally, a range of pathways involved in carbon, nitrogen, phosphorus and sulfur cycling were significantly different during the various HAB stages. Genes associated with carbohydrate-active enzymes, denitrification, and iron oxidation were enriched at the pre-bloom stage; genes involved in reductive citrate cycle for carbon fixation, carbon degradation, nitrification and phosphate transport were enhanced at the peak stage; and relative gene abundance related to sulfur oxidation, vitamin synthesis, and iron transport and storage was increased at the post-bloom stage. The ecological linkage analysis has shown that microbial functional potential especially the C/P/Fe metabolism were significantly linked to the fate of the algal blooms. Taken together, our results demonstrated that microorganisms displayed successional patterns not only at the community level, but also in the metabolic potential on HAB's progression. This work contributes to a growing understanding of microbial structural elasticity and functional plasticity and shed light on the potential mechanisms of microbial-mediated HAB trajectory.

The complete genome sequence of the algicidal bacterium Bacillus subtilis strain JA and the use of quorum sensing to evaluate its antialgal ability.

We describe the isolation of Bacillus subtilis strain JA and demonstrate that this bacterium exhibited strong algicidal effects on the algae Alexandrium minutum with an inhibition rate exceeding 80 % within 48 h. B. subtilis JA significantly reduced the photosynthetic efficiency of A. minutum and caused extensive morphological damage to the algae. Genomic analysis of B. subtilis JA demonstrated that a putative AI-2 type quorum sensing (QS) gene (LuxS) is present in its genome cluster, which is regulate pheromone biosynthesis. Interestingly, the exogenous addition of a QS-oligopeptide (ComX-pheromone) improved the algicidal efficiency of B. subtilis JA, thus indicating that the algicidal activity of this bacterium is potentially regulated by QS. Collectively, our data describe a potential antialgal bacterium and speculated that its behavior can be modulated by QS signal. B. subtilis JA may therefore represent a valuable tool for the development of novel chemical-ecological methods with which to control harmful algae.

Temporal Variability of Virioplankton during a Gymnodinium catenatum Algal Bloom.

Viruses are key biogeochemical engines in the regulation of the dynamics of phytoplankton. However, there has been little research on viral communities in relation to algal blooms. Using the virMine tool, we analyzed viral information from metagenomic data of field dinoflagellate (Gymnodinium catenatum) blooms at different stages. Species identification indicated that phages were the main species. Unifrac analysis showed clear temporal patterns in virioplankton dynamics. The viral community was dominated by Siphoviridae, Podoviridae, and Myoviridae throughout the whole bloom cycle. However, some changes were observed at different phases of the bloom; the relatively abundant Siphoviridae and Myoviridae dominated at pre-bloom and peak bloom stages, while at the post-bloom stage, the members of Phycodnaviridae and Microviridae were more abundant. Temperature and nutrients were the main contributors to the dynamic structure of the viral community. Some obvious correlations were found between dominant viral species and host biomass. Functional analysis indicated some functional genes had dramatic response in algal-associated viral assemblages, especially the CAZyme encoding genes. This work expands the existing knowledge of algal-associated viruses by characterizing viral composition and function across a complete algal bloom cycle. Our data provide supporting evidence that viruses participate in dinoflagellate bloom dynamics under natural conditions.

Effects of Seasonal Hibernation on the Similarities Between the Skin Microbiota and Gut Microbiota of an Amphibian (Rana dybowskii).

Both the gut and skin microbiotas have important functions for amphibians. The gut microbiota plays an important role in both the health and evolution of the host species, whereas the role of skin microbiota in disease resistance is particularly important for amphibians. Many studies have examined the effects of environmental factors on the skin and gut microbiotas, but no study has yet explored the similarities between the skin and gut microbiotas. In this study, the gut and skin microbiotas of Rana dybowskii in summer and winter were investigated via high-throughput Illumina sequencing. The results showed that the alpha diversity of gut and skin microbiotas decreased significantly from summer to winter. In both seasons, the microbial composition and structure differed significantly between the gut and skin, and the similarities between these microbiotas differed between seasons. The pairwise distances between the gut and skin microbiotas were greater in winter than in summer. The ratio of core OTUs and shared OTUs to the sum of the OTUs in the gut and skin microbiotas in summer was significantly higher than that in winter. The similarities between the gut and skin microbiotas are important for understanding amphibian ecology and life history.

Comparison of the gut microbiota of Rana amurensis and Rana dybowskii under natural winter fasting conditions.

Rana amurensis and R. dybowskii occupy similar habitats. As temperatures decrease with the onset of winter, both species migrate to ponds for hibernation. Our goal was to determine whether different species possess different intestinal microbiota under natural winter fasting conditions. We used high-throughput Illumina sequencing of 16S rRNA gene sequences to analyse the diversity of intestinal microbes in the two species. The dominant gut bacterial phyla in both species were Bacteroidetes, Proteobacteria and Firmicutes. Linear discriminant analysis (LDA) effect size revealed significant enrichment of Proteobacteria in R. amurensis and Firmicutes in R. dybowskii. There were significant differences in the gut microbiota composition between the species. The core operational taxonomic unit numbers in R. amurensis and R. dybowskii shared by the two species were 106, 100 and 36. This study indicates that the intestinal bacterial communities of the two frog species are clearly different. Phylum-level analysis showed that R. amurensis was more abundant in Proteobacteria and Verrucomicrobia than R. dybowskii was This is the first study of the composition and diversity of the gut microbiota of these two species, providing important insights for future research on the gut microbiota and the role of these bacterial communities in frogs.

Modelling the growth of the brown frog (Rana dybowskii).

Well-controlled development leads to uniform body size and a better growth rate; therefore, the ability to determine the growth rate of frogs and their period of sexual maturity is essential for producing healthy, high-quality descendant frogs. To establish a working model that can best predict the growth performance of frogs, the present study examined the growth of one-year-old and two-year-old brown frogs (Rana dybowskii) from metamorphosis to hibernation (18 weeks) and out-hibernation to hibernation (20 weeks) under the same environmental conditions. Brown frog growth was studied and mathematically modelled using various nonlinear, linear, and polynomial functions. The model input values were statistically evaluated using parameters such as the Akaike's information criterion. The body weight/size ratio (Kwl) and Fulton's condition factor (K) were used to compare the weight and size of groups of frogs during the growth period. The results showed that the third- and fourth-order polynomial models provided the most consistent predictions of body weight for age 1 and age 2 brown frogs, respectively. Both the Gompertz and third-order polynomial models yielded similarly adequate results for the body size of age 1 brown frogs, while the Janoschek model produced a similarly adequate result for the body size of age 2 brown frogs. The Brody and Janoschek models yielded the highest and lowest estimates of asymptotic weight, respectively, for the body weights of all frogs. The Kwl value of all frogs increased from 0.40 to 3.18. The K value of age 1 frogs decreased from 23.81 to 9.45 in the first four weeks. The K value of age 2 frogs remained close to 10. Graphically, a sigmoidal trend was observed for body weight and body size with increasing age. The results of this study will be useful not only for amphibian research but also for frog farming management strategies and decisions.

Seroprevalence and risk factors of Chlamydia infection in dogs in Southwestern China.

Chlamydia spp. are obligate intracellular bacteria distributed globally, known to cause various forms of diseases in animals and humans. To date, there is limited information about the seroprevalence of Chlamydia and the risk factors associated with Chlamydia infection in dogs in the world. In the present study, a serological survey was undertaken to examine the seroprevalence and risk factors associated with dog chlamydiosis in Yunnan Province, southwestern China. A total of 591 dogs were sampled, antibodies to Chlamydia were determined by indirect hemagglutination assay (IHA). The overall seroprevalence was estimated at 17.6%. The risk factors associated with seroprevalence were determined by a multivariate logistic regression analysis. Gender and age of dogs were not significant in the logistic regression analysis (P>0.05) and left out of the final model. Type and geographical origin of dogs were considered as main risk factors associated with Chlamydia infection, stray dogs (31.37%) were more than 16 times (OR=16.167, 95% CI=6.283-41.599, P<0.01) at risk of acquiring the infection compared to the police dogs (7.62%), while pet dogs (14.41%) had a 3 times (OR=2.968, 95% CI=1.349-6.529, P=0.007) higher risk. Positive dogs were found in 5 districts of Yunnan Province with prevalence ranging from 2.56% to 31.67% except Diqing (0/56). Dogs in Kunming (20.21%) had a 9 times higher risk of being seropositive compared to dogs in Lijiang (2.56%) (OR=9.057, 95% CI=1.211-67.714, P=0.032), although no regional differences were found in other 4 administrative divisions compared to Lijiang (P>0.05). Our study revealed a widespread and high prevalence of Chlamydia infection in dogs in Yunnan Province, southwestern China, with higher exposure risk in stray dogs and distinct geographical distribution. These findings suggest the potential importance of dogs in the transmission of zoonotic Chlamydia infection, and thus Chlamydia should be taken into consideration in diagnosing dog diseases.