Molecular dating of the teleost whole genome duplication (3R) is compatible with the expectations of delayed rediploidization.

Vertebrate evolution has been punctuated by three whole genome duplication (WGD) events that have been implicated causally in phenotypic evolution, from the origin of phenotypic novelties to explosive diversification. Arguably the most dramatic of these is the 3R WGD event associated with the origin of teleost fishes which comprise more than half of all living vertebrate species. However, tests of a causal relationship between WGD and teleost diversification have proven difficult due to the challenge of establishing the timing of these phenomena. Here we show, based on molecular clock dating of concatenated gene alignments, that the 3R WGD event occurred in the early-middle Permian (286.18-267.20 Million years ago; Ma), 52.02-12.84 million years (Myr) before the divergence of crown-teleosts in the latest-Permian-earliest Late Triassic (254.36-234.16 Ma) and long before the major pulses of teleost diversification in Ostariophysi and Percomorpha (56.37-100.17 Myr and at least 139.24-183.29 Myr later, respectively). The extent of this temporal gap between putative cause and effect precludes 3R as a deterministic driver of teleost diversification. However, these age constraints remain compatible with the expectations of a prolonged rediploidization process following WGD which, through the effects of chromosome rearrangement and gene loss, remains a viable mechanism to explain the evolution of teleost novelties and diversification.

Thiamine-Mediated Microbial Interaction between Auxotrophic Rhodococcus ruber ZM07 and Prototrophic Cooperators in the Tetrahydrofuran-Degrading Microbial Community H-1.

As a crucial growth factor, thiamine can regulate functional microbial communities; however, our current understanding of its effect on bioremediation is lacking. Using metatranscriptome and 16S rRNA gene sequencing, we explored the mechanism of response of an efficient tetrahydrofuran (THF)-degrading microbial culture, designated H-1, to exogenous thiamine. Rhodococcus ruber ZM07, a strain performing the THF degradation function in H-1, is a thiamine-auxotrophic bacterium. Furthermore, thiamine affected the microbial community structure of H-1 by altering resource and niche distributions. A microbial co-occurrence network was constructed to help us identify and isolate the cooperators of strain ZM07 in the microbial community. Based on the prediction of the network, two non-THF-degrading bacteria, Hydrogenophaga intermedia ZM11 and Pigmentiphaga daeguensis ZM12, were isolated. Our results suggest that strain ZM11 is a good cooperator of ZM07, and it might be more competitive than other cooperators (e.g., ZM12) in cocultured systems. Additionally, two dominant strains in our microbial culture displayed a "seesaw" pattern, and they showed completely different responses to exogenous thiamine. The growth of the THF degrader ZM07 was spurred by additional thiamine (with an increased relative abundance and significant upregulation of most metabolic pathways), while the growth of the cooperator ZM11 was obviously suppressed under the same circumstances. This relationship was the opposite without thiamine addition. Our study reveals that exogenous thiamine can affect the interaction patterns between THF- and non-THF-degrading microorganisms and provides new insight into the effects of micronutrients on the environmental microbial community. IMPORTANCE Auxotrophic microorganisms play important roles in the biodegradation of pollutants in nature. Exploring the interspecies relationship between auxotrophic THF-degrading bacteria and other microbes is helpful for the efficient utilization of auxotrophic functional microorganisms. Herein, the thiamine-auxotrophic THF-degrading bacterium ZM07 was isolated from the microbial culture H-1, and the effect of thiamine on the structure of H-1 during THF bioremediation was studied. Thiamine may help ZM07 occupy more niches and utilize more resources, thus improving THF degradation efficiency. This research provides a new strategy to improve the THF or other xenobiotic compound biodegradation performance of auxotrophic functional microorganisms/microbial communities by artificially adding special micronutrients. Additionally, the "seesaw" relationship between the thiamine-auxotrophic strain ZM07 and its prototrophic cooperator ZM11 during THF bioremediation could be changed by exogenous thiamine. This study reveals the effect of micronutrients on microbial interactions and provides an effective way to regulate the pollutant biodegradation efficiency of microbial communities.

Thiamine-Mediated Cooperation Between Auxotrophic Rhodococcus ruber ZM07 and Escherichia coli K12 Drives Efficient Tetrahydrofuran Degradation.

Tetrahydrofuran (THF) is a universal solvent widely used in the synthesis of chemicals and pharmaceuticals. As a refractory organic contaminant, it can only be degraded by a small group of microbes. In this study, a thiamine auxotrophic THF-degrading bacterium, Rhodococcus ruber ZM07, was isolated from an enrichment culture H-1. It was cocultured with Escherichia coli K12 (which cannot degrade THF but can produce thiamine) and/or Escherichia coli K12ΔthiE (which can neither degrade THF nor produce thiamine) with or without exogenous thiamine. This study aims to understand the interaction mechanisms between ZM07 and K12. We found that K12 accounted for 30% of the total when cocultured and transferred with ZM07 in thiamine-free systems; in addition, in the three-strain (ZM07, K12, and K12ΔthiE) cocultured system without thiamine, K12ΔthiE disappeared in the 8th transfer, while K12 could still stably exist (the relative abundance remained at approximately 30%). The growth of K12 was significantly inhibited in the thiamine-rich system. Its proportion was almost below 4% after the fourth transfer in both the two-strain (ZM07 and K12) and three-strain (ZM07, K12, and K12ΔthiE) systems; K12ΔthiE's percentage was higher than K12's in the three-strain (ZM07, K12, and K12ΔthiE) cocultured system with exogenous thiamine, and both represented only a small proportion (less than 1% by the fourth transfer). The results of the coculture of K12 and K12ΔthiE in thiamine-free medium indicated that intraspecific competition between them may be one of the main reasons for the extinction of K12ΔthiE in the three-strain (ZM07, K12, and K12ΔthiE) system without exogenous thiamine. Furthermore, we found that ZM07 could cooperate with K12 through extracellular metabolites exchanges without physical contact. This study provides novel insight into how microbes cooperate and compete with one another during THF degradation.

Enrichment and characterization of a highly efficient tetrahydrofuran-degrading bacterial culture.

Tetrahydrofuran (THF) is a ubiquitous toxic and carcinogenic pollutant. Screening for pure or mixed-culture microorganisms that can efficiently degrade THF is difficult due to its chemical stability. In this study, an enrichment culture, H-1, with a stable THF-degrading ability and microbial community structure was enriched from activated sludge and could efficiently degrade 95% of 40 mM THF within 6 days. The optimal THF degradation conditions for H-1 were an initial pH of 7.0-8.0 and a temperature of 30 °C. The substrate tolerance concentration of H-1 reached 200 mM. Heavy metals tolerance concentrations of Cu2+, Cd2+ and Pb2+ of H-1 was 0.5 mM, 0.4 mM and 0.03 mM, and 4 mM Mn2+ did not significantly influence the THF degradation ratio or biomass of H-1. H-1 might be a good material for actual wastewater treatment because of its efficient THF degradation performance and ability to resist various stressful conditions. In addition, the THF-degrading efficiency of H-1 was enhanced by the addition of moderate carbon sources. High-throughput sequencing of the 16S rRNA gene showed that Rhodococcus sp. (a potential THF-degrading strain) and Hydrogenophaga sp. (a potential non-THF-degrading strain) were the dominant microorganisms in the H-1 culture. These results indicate the potential coexistence of cooperation and competition between THF-degrading bacteria and nondegrading bacteria in this enrichment culture.

Metabolite Cross-Feeding between Rhodococcus ruber YYL and Bacillus cereus MLY1 in the Biodegradation of Tetrahydrofuran under pH Stress.

Bacterial consortia are among the most basic units in the biodegradation of environmental pollutants. Pollutant-degrading strains frequently encounter different types of environmental stresses and must be able to survive with other bacteria present in the polluted environments. In this study, we proposed a noncontact interaction mode between a tetrahydrofuran (THF)-degrading strain, Rhodococcus ruber YYL, and a non-THF-degrading strain, Bacillus cereus MLY1. The metabolic interaction mechanism between strains YYL and MLY1 was explored through physiological and molecular studies and was further supported by the metabolic response profile of strain YYL, both monocultured and cocultured with strain MLY1 at the optimal pH (pH 8.3) and under pH stress (pH 7.0), through a liquid chromatography-mass spectrometry-based metabolomic analysis. The results suggested that the coculture system resists pH stress in three ways: (i) strain MLY1 utilized acid metabolites and impacted the proportion of glutamine, resulting in an elevated intracellular pH of the system; (ii) strain MLY1 had the ability to degrade intermediates, thus alleviating the product inhibition of strain YYL; and (iii) strain MLY1 produced some essential micronutrients for strain YYL to aid the growth of this strain under pH stress, while strain YYL produced THF degradation intermediates for strain MLY1 as major nutrients. In addition, a metabolite cross-feeding interaction with respect to pollutant biodegradation is discussed.IMPORTANCERhodococcus species have been discovered in diverse environmental niches and can degrade numerous recalcitrant toxic pollutants. However, the pollutant degradation efficiency of these strains is severely reduced due to the complexity of environmental conditions and limitations in the growth of the pollutant-degrading microorganism. In our study, Bacillus cereus strain MLY1 exhibited strong stress resistance to adapt to various environments and improved the THF degradation efficiency of Rhodococcus ruber YYL by a metabolic cross-feeding interaction style to relieve the pH stress. These findings suggest that metabolite cross-feeding occurred in a complementary manner, allowing a pollutant-degrading strain to collaborate with a nondegrading strain in the biodegradation of various recalcitrant compounds. The study of metabolic exchanges is crucial to elucidate mechanisms by which degrading and symbiotic bacteria interact to survive environmental stress.

Novel tetrahydrofuran (THF) degradation-associated genes and cooperation patterns of a THF-degrading microbial community as revealed by metagenomic.

Our understanding of the tetrahydrofuran (THF) degradation in complex environment is limited. The majority of THF degrading genes reported are group V soluble diiron monooxygenases and share greater than 95% homology with one another. In this study, we used sole-carbon-source incubation combined with high-throughput metagenomic sequencing to investigate this contaminant's degradation in environmental samples. We identified as-yet-uncultivated microbe from the genera Pseudonocardia and fungi Scedosporium sp. (Scedosporium sp. was successfully isolated) as THF degraders as containing THF degradation genes, while microbes from the genera Bordetella, Pandoraea and Rhodanobacter functioned as main cooperators by utilizing acidic intermediates and providing anti-acid mechanisms. Furthermore, a 9387-bp THF degradation cluster designated thmX from the as-yet-uncultivated Pseudonocardia (with 6 main ORFs and with 79-93% amino acid sequence identity with previously reported clusters) was discovered. We also found a THF-degrading related cytochrome P450 monooxygenase from the genus Scedosporium and predicted its cognate reductase for the first time. All the genes and clusters mentioned above were successfully amplified from samples and cloned into the suitable expression vectors. This study will provide novel insights for understanding of THF degradation mechanisms under acid stress conditions and mining new THF degradation genes.