Dynamics of microbial-induced oil degradation at the microscale.

Microbial-induced oil degradation (MIOD) has a wide range of applications, such as microbial enhanced oil recovery and bioremediation of oil pollution. However, our understanding of MIOD is still far from complete. Particularly, how is the dynamics of degradation process at the microscale level with a single-cell resolution remains to be disclosed. In this work, using hexadecane droplets in water as a model system, we have studied the dynamics of hexadecane degradation by different strains, including Pseudomonas aeruginosa PAO1, IMP68, O-2-2, and Dietzia sp. DQ12-45-1b, at the microscale. Based on visualization of MIOD, the dynamics of MIOD can be characterized by a three-stage process, including adhesion, adaptation, and degradation stages. Although different strains showed similar three-stage dynamics of MIOD, the effective degradation rate varied and followed an order of PAO1 > O-2-2 > IMP68 > DQ12-45-1b under aerobic conditions. Different oxygen conditions were also tested, and the dynamics of MIOD was slowed down under anaerobic conditions in comparison to under aerobic conditions. Further investigations at the degradation stage revealed that biofilms formed at the oil-water interface enhanced oil degradation, but a prerequisite for such enhanced degradation is proper stimulation of biofilm cells in the course of biofilm formation. The findings in this work provided a detailed picture on the dynamics of MIOD at the microscale and would be beneficial for better applications of MIOD.IMPORTANCEMicrobial-induced oil degradation is environmental friendly and economic and has become a promising technique in the fields of enhanced oil recovery and remediation of crude oil-polluted environments. For better applications of microbial-induced oil degradation, understanding the degradation dynamics particularly at the microscale is crucial. In this study, we investigated the degradation dynamics of hexadecane oil droplets incubated with different strains, including Pseudomonas aeruginosa PAO1, O-2-2, IMP68, and Dietzia sp. DQ12-45-1b at the microscale by employing microdroplet-based methods and bacterial tracking techniques. The findings in this study provided a detailed picture on the dynamics of microbial-induced oil degradation at the microscale, which will deepen our understandings on the biodegradation mechanisms of alkanes and shed insights for developing more effective biodegradation techniques.

The evolution of autonomy from two cooperative specialists in fluctuating environments.

From microbes to humans, organisms perform numerous tasks for their survival, including food acquisition, migration, and reproduction. A complex biological task can be performed by either an autonomous organism or by cooperation among several specialized organisms. However, it remains unclear how autonomy and cooperation evolutionarily switch. Specifically, it remains unclear whether and how cooperative specialists can repair deleted genes through direct genetic exchange, thereby regaining metabolic autonomy. Here, we address this question by experimentally evolving a mutualistic microbial consortium composed of two specialists that cooperatively degrade naphthalene. We observed that autonomous genotypes capable of performing the entire naphthalene degradation pathway evolved from two cooperative specialists and dominated the community. This evolutionary transition was driven by the horizontal gene transfer (HGT) between the two specialists. However, this evolution was exclusively observed in the fluctuating environment alternately supplied with naphthalene and pyruvate, where mutualism and competition between the two specialists alternated. The naphthalene-supplied environment exerted selective pressure that favors the expansion of autonomous genotypes. The pyruvate-supplied environment promoted the coexistence and cell density of the cooperative specialists, thereby increasing the likelihood of HGT. Using a mathematical model, we quantitatively demonstrate that environmental fluctuations facilitate the evolution of autonomy through HGT when the relative growth rate and carrying capacity of the cooperative specialists allow enhanced coexistence and higher cell density in the competitive environment. Together, our results demonstrate that cooperative specialists can repair deleted genes through a direct genetic exchange under specific conditions, thereby regaining metabolic autonomy.

Biodegradation of Crude Oil by Nitrate-Reducing, Sulfate-Reducing, and Methanogenic Microbial Communities under High-Pressure Conditions.

Carbon capture, utilization, and storage (CCUS) is an important component in many national net-zero strategies, and ensuring that CO2 can be safely and economically stored in geological systems is critical. Recent discoveries have shown that microbial processes (e.g., methanogenesis) can modify fluid composition and fluid dynamics within the storage reservoir. Oil reservoirs are under high pressure, but the influence of pressure on the petroleum microbial community has been previously overlooked. To better understand microbial community dynamics in deep oil reservoirs, we designed an experiment to examine the effect of high pressure (12 megapascals [MPa], 60 °C) on nitrate-reducing, sulfate-reducing, and methanogenic enrichment cultures. Cultures were exposed to these conditions for 90 d and compared with a control exposed to atmospheric pressure (0.1 MPa, 60 °C). The degradation characteristic oil compounds were confirmed by thin-layer analysis of oil SARA (saturates, aromatics, resins, and asphaltenes) family component rods. We found that the asphaltene component in crude oil was biodegraded under high pressure, but the concentration of asphaltenes increased under atmospheric pressure. Gas chromatography analyses of saturates showed that short-chain saturates (C8-C12) were biodegraded under high and atmospheric pressure, especially in the methanogenic enrichment culture under high pressure (the ratio of change was -81%), resulting in an increased relative abundance of medium- and long-chain saturates. In the nitrate-reducing and sulfate-reducing enrichment cultures, long-chain saturates (C22-C32) were biodegraded in cultures exposed to high-pressure and anaerobic conditions, with a ratio of change of -8.0% and -2.3%, respectively. However, the relative proportion of long-chain saturates (C22-C32) increased under atmospheric pressure. Gas Chromatography Mass Spectrometry analyses of aromatics showed that several naphthalene series compounds (naphthalene, C1-naphthalene, and C2-naphthalene) were biodegraded in the sulfate-reducing enrichment under both atmospheric pressure and high pressure. Our study has discerned the linkages between the biodegradation characteristics of crude oil and pressures, which is important for the future application of bioenergy with CCUS (bio-CCUS).

Global diversity and ecological functions of viruses inhabiting oil reservoirs.

Oil reservoirs, being one of the significant subsurface repositories of energy and carbon, host diverse microbial communities affecting energy production and carbon emissions. Viruses play crucial roles in the ecology of microbiomes, however, their distribution and ecological significance in oil reservoirs remain undetermined. Here, we assemble a catalogue encompassing viral and prokaryotic genomes sourced from oil reservoirs. The catalogue comprises 7229 prokaryotic genomes and 3,886 viral Operational Taxonomic Units (vOTUs) from 182 oil reservoir metagenomes. The results show that viruses are widely distributed in oil reservoirs, and 85% vOTUs in oil reservoir are detected in less than 10% of the samples, highlighting the heterogeneous nature of viral communities within oil reservoirs. Through combined microcosm enrichment experiments and bioinformatics analysis, we validate the ecological roles of viruses in regulating the community structure of sulfate reducing microorganisms, primarily through a virulent lifestyle. Taken together, this study uncovers a rich diversity of viruses and their ecological functions within oil reservoirs, offering a comprehensive understanding of the role of viral communities in the biogeochemical cycles of the deep biosphere.

[Element relationship and extension path of clinical evidence knowledge map with Chinese patent medicine].

This study aims to construct the element relationship and extension path of clinical evidence knowledge map with Chinese patent medicine, providing basic technical support for the formation and transformation of the evidence chain of Chinese patent medicine and providing collection, induction, and summary schemes for massive and disorganized clinical data. Based on the elements of evidence-based PICOS, the conventional construction methods of knowledge graph were collected and summarized. Firstly, the data entities related to Chinese patent medicine were classified, and entity linking was performed(disambiguation). Secondly, the study associated and classified the attribute information of the data entity. Finally, the logical relationship between entities was constructed, and then the element relationship and extension path of the knowledge map conforming to the characteristics of clinical evidence of Chinese patent medicine were summarized. The construction of the clinical evidence knowledge map of Chinese patent medicine was mainly based on process design and logical structure, and the element relationship of the knowledge map was expressed according to the PICOS principle and evidence level. The extension path crossed three levels(model layer, data layer application, and new evidence application), and the study gradually explored the path from disease, core evaluation indicators, Chinese patent medicine, core prescriptions, syndrome and treatment rules, and medical case comparison(evolution law) to new drug research and development. In this study, the top-level design of the construction of the clinical evidence knowledge map of Chinese patent medicine has been clarified, but it still needs the joint efforts of interdisciplinary disciplines. With the continuous improvement of the map construction technology in line with the characteristics of TCM, the study can provide necessary basic technical support and reference for the development of the TCM discipline.

High immigration rates critical for establishing emigration-driven diversity in microbial communities.

Unraveling the mechanisms governing the diversity of ecological communities is a central goal in ecology. Although microbial dispersal constitutes an important ecological process, the effect of dispersal on microbial diversity is poorly understood. Here, we sought to fill this gap by combining a generalized Lotka-Volterra model with experimental investigations. Our model showed that emigration increases the diversity of the community when the immigration rate crosses a defined threshold, which we identified as Ineutral. We also found that at high immigration rates, emigration weakens the relative abundance of fast-growing species and thus enhances the mass effect and increases the diversity. We experimentally confirmed this finding using co-cultures of 20 bacterial strains isolated from the soil. Our model further showed that Ineutral decreases with the increase of species pool size, growth rate, and interspecies interaction. Our work deepens the understanding of the effects of dispersal on the diversity of natural communities.

The trade-off between individual metabolic specialization and versatility determines the metabolic efficiency of microbial communities.

In microbial systems, a metabolic pathway can be either completed by one autonomous population or distributed among a consortium performing metabolic division of labor (MDOL). MDOL facilitates the system's function by reducing the metabolic burden; however, it may hinder the function by reducing the exchange efficiency of metabolic intermediates among individuals. As a result, the function of a community is influenced by the trade-offs between the metabolic specialization and versatility of individuals. To experimentally test this hypothesis, we deconstructed the naphthalene degradation pathway into four steps and introduced them individually or combinatorically into different strains with varying levels of metabolic specialization. Using these strains, we engineered 1,456 synthetic consortia and found that 74 consortia exhibited higher degradation function than both the autonomous population and rigorous MDOL consortium. Quantitative modeling provides general strategies for identifying the most effective MDOL configuration. Our study provides critical insights into the engineering of high-performance microbial systems.

Roles of sulfate-reducing bacteria in sustaining the diversity and stability of marine bacterial community.

Microbes play central roles in ocean food webs and global biogeochemical processes. Yet, the information available regarding the highly diverse bacterial communities in these systems is not comprehensive. Here we investigated the diversity, assembly process, and species coexistence frequency of bacterial communities in seawater and sediment across ∼600 km of the eastern Chinese marginal seas using 16S rRNA gene amplicon sequencing. Our analyses showed that compared with seawater, bacterial communities in sediment possessed higher diversity and experienced tight phylogenetic distribution. Neutral model analysis showed that the relative contribution of stochastic processes to the assembly process of bacterial communities in sediment was lower than that in seawater. Functional prediction results showed that sulfate-reducing bacteria (SRB) were enriched in the core bacterial sub-communities. The bacterial diversities of both sediment and seawater were positively associated with the relative abundance of SRB. Co-occurrence analysis showed that bacteria in seawater exhibited a more complex interaction network and closer co-occurrence relationships than those in sediment. The SRB of seawater were centrally located in the network and played an essential role in sustaining the complex network. In addition, further analysis indicated that the SRB of seawater helped maintain the high stability of the bacterial network. Overall, this study provided further comprehensive information regarding the characteristics of bacterial communities in the ocean, and provides new insights into keystone taxa and their roles in sustaining microbial diversity and stability in ocean.

Assembly of Microbial Communities Engaging in Metabolic Division of Labor in a Diffusion-Limited Environment Is Governed by Metabolic Flux.

Metabolic division of labor (MDOL) represents one of the most commonly occurring interactions within natural microbial communities. Specifically, in a variety of MDOL systems engaged in hydrocarbon degradation, a sequential degradation is performed by several members with final products that are necessary for the growth of each member. In these MDOL systems, each strain catalyzes one or more specific reactions of a multistep metabolic pathway, whose end products are then allocated among the participants. While the benefit allocation is independent of metabolic flux in well-mixed environments, it remains unclear how the benefits are allocated when diffusion is limited. Here, we investigated how MDOL communities assemble in a diffusion-limited environment, by combining mathematical modeling with experimental inquiry using a synthetic consortium engaged in MDOL. Our model analysis in a diffusion-limited environment showed that, when the growth of all populations in the community relies on the final product that can only be produced by the last population, a diffusion gradient of the final products may create a bias favoring the member producing the final products, resulting in a higher relative abundance of the final product producer. Moreover, such asymmetric allocation of the final products is enhanced by both the lower diffusion rate and the higher metabolic flux (i.e., the higher yields of the final products) in the MDOL. Our results show that in a diffusively confined environment, metabolic flux constitutes a determining factor in the assembly of the MDOL community. Together, our findings are critical for a better understanding of how resource-sharing microbial communities are established and should assist in designing such communities for improved biomanufacturing and bioremediation.

Improving Rhamnolipids Biosynthesis in Pseudomonas sp. L01 through Atmospheric and Room-Temperature Plasma (ARTP) Mutagenesis.

Biosurfactants have significant applications in various industries, including microbial-enhanced oil recovery (MEOR). While the state-of-the-art genetic approaches can generate high-yield strains for biosurfactant production in fermenters, there remains a critical challenge in enhancing biosurfactant-producing strains for use in natural environments with minimal ecological risks. The objectives of this work are enhancing the strain's capacity for rhamnolipids production and exploring the genetic mechanisms for its improvement. In this study, we employed atmospheric and room-temperature plasma (ARTP) mutagenesis to enhance the biosynthesis of rhamnolipids in Pseudomonas sp. L01, a biosurfactant-producing strain isolated from petroleum-contaminated soil. Following ARTP treatment, we identified 13 high-yield mutants, with the highest yield of 3.45 ± 0.09 g/L, representing a 2.7-fold increase compared to the parent strain. To determine the genetic mechanisms behind the enhanced rhamnolipids biosynthesis, we sequenced the genomes of the strain L01 and five high-yield mutants. A comparative genomic analysis suggested that mutations in genes related to the synthesis of lipopolysaccharides (LPS) and the transport of rhamnolipids may contribute to the improved biosynthesis. To the best of our knowledge, this is the first instance of utilizing the ARTP approach to improve rhamnolipid production in Pseudomonas strains. Our study provides valuable insights into the enhancement of biosurfactant-producing strains and the regulatory mechanisms of rhamnolipids biosynthesis.

Volatile compounds-induced environmental pH shifts mediate interspecific bacterial interactions over long-distance.

Microorganisms interact with each other through exchanging metabolites as well as signals molecules. This exchange typically depends on metabolites or signaling molecules diffusing in aqueous media within spatially connected habitats, and it only occurs within a short range. However, different microorganisms frequently live in spatially discontinuous micro-habitats without the connections of aqueous media. How microorganisms populating such spatially discontinuous micro-habitats interact remains poorly understood. Here, we show that a bacterial strain, Corynebacterium glutamicum ATCC13032T, produces high amounts of ammonia in its local habitat, which disperses over long distances (up to 12 cm) through the air. As a result, the pH of another spatially disconnected habitat increases, thus inducing the growth of an acid-sensitive strain (Glycocaulis alkaliphilus 6B-8T). We also show that ammonia-mediated long-distance interactions can be commonly found in various bacterial communities. In conclusion, our work demonstrates that bacteria growing in spatially discontinuous micro-habitats can interact with each other through gaseous diffusion of volatile compounds. This finding expands our understanding of the spatial scale at which bacteria can interact and provides new insights into how spatially separated species are connected to maximizing community-level commensalism.

Predicting microbial community compositions in wastewater treatment plants using artificial neural networks.

BACKGROUND: Activated sludge (AS) of wastewater treatment plants (WWTPs) is one of the world's largest artificial microbial ecosystems and the microbial community of the AS system is closely related to WWTPs' performance. However, how to predict its community structure is still unclear. RESULTS: Here, we used artificial neural networks (ANN) to predict the microbial compositions of AS systems collected from WWTPs located worldwide. The predictive accuracy R21:1 of the Shannon-Wiener index reached 60.42%, and the average R21:1 of amplicon sequence variants (ASVs) appearing in at least 10% of samples and core taxa were 35.09% and 42.99%, respectively. We also found that the predictability of ASVs was significantly positively correlated with their relative abundance and occurrence frequency, but significantly negatively correlated with potential migration rate. The typical functional groups such as nitrifiers, denitrifiers, polyphosphate-accumulating organisms (PAOs), glycogen-accumulating organisms (GAOs), and filamentous organisms in AS systems could also be well recovered using ANN models, with R21:1 ranging from 32.62% to 56.81%. Furthermore, we found that whether industry wastewater source contained in inflow (IndConInf) had good predictive abilities, although its correlation with ASVs in the Mantel test analysis was weak, which suggested important factors that cannot be identified using traditional methods may be highlighted by the ANN model. CONCLUSIONS: We demonstrated that the microbial compositions and major functional groups of AS systems are predictable using our approach, and IndConInf has a significant impact on the prediction. Our results provide a better understanding of the factors affecting AS communities through the prediction of the microbial community of AS systems, which could lead to insights for improved operating parameters and control of community structure. Video Abstract.

MiR-328-3p promotes TGF-ß1-induced proliferation, migration, and inflammation of airway smooth muscle cells by regulating the PTEN/Akt pathway.

BACKGROUND: Recent studies have shown that the up-regulation of microRNA miR-328-3p expression increases seasonal allergy and asthma symptoms in children, but the specific mechanism remains unclear. Therefore, the aim of this study was to explore the role and mechanism of -miR-328-3p in transforming growth factor (TGF)-ß1-induced airway smooth muscle cells (ASMCs). METHODS: The effect of TGF-ß1 on the expression of miR-328-3p in ASMCs was examined by real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR). Cells proliferation, migration, and inflammatory factors in TGF-ß1-induced ASMCs were measured by cell counting kit-8 (CCK-8), transwell, and enzyme-linked immunosorbent assay (ELISA), respectively. Besides, TargetScan was used to predict phosphatase and tensin homolog (PTEN), the downstream target of miR-328-3p; double-luciferase reporter assay, western blot, and qRT-PCR were used to verify the targeting relationship between miR-328-3p and PTEN; western blot was also used to examine the effects of PTEN and miR-328-3p knockdown on the expression levels of PTEN, Akt, and p-Akt proteins. RESULTS: The expression of miR-328-3p was up-regulated in TGF-ß1-induced ASMCs. Knockdown of miR-328-3p significantly inhibited proliferation, migration, and inflammation of ASMCs induced by TGF-ß1 and decreased levels of tumor necrosis factor (TNF)-α and interleukin (IL)-1ß. The dual--luciferase reporter assay results confirmed that PTEN was a target gene of miR-328-3p. Moreover, inhibition of PTEN expression reversed the inhibitory effect of low miR-328-3p expression on -TGF-ß1-induced ASMC's proliferation, migration, and inflammation. In comparison to the knockdown of miR-328-3p alone, the simultaneous knockdown of miR-328-3p with PTEN decreased PTEN protein expression levels and increased p-Akt/Akt ratio in TGF-ß1-induced ASMCs. CONCLUSION: Through regulating the expression of PTEN and the activity of Akt signaling pathway, miR-328-3p promotes TGF-ß1-induced proliferation, migration, and inflammation of ASMCs.

pH and Nitrate Drive Bacterial Diversity in Oil Reservoirs at a Localized Geographic Scale.

Oil reservoirs are one of the most important deep subsurface biospheres. They are inhabited by diverse microorganisms including bacteria and archaea with diverse metabolic activities. Although recent studies have investigated the microbial communities in oil reservoirs at large geographic scales, it is still not clear how the microbial communities assemble, as the variation in the environment may be confounded with geographic distance. In this work, the microbial communities in oil reservoirs from the same oil field were identified at a localized geographic scale. We found that although the injected water contained diverse exogenous microorganisms, this had little effect on the microbial composition of the produced water. The Neutral Community Model analysis showed that both bacterial and archaeal communities are dispersal limited even at a localized scale. Further analysis showed that both pH and nitrate concentrations drive the assembly of bacterial communities, of which nitrate negatively correlated with bacterial alpha diversity and pH differences positively correlated with the dissimilarity of bacterial communities. In contrast, the physiochemical parameters had little effect on archaeal communities at the localized scale. Our results suggest that the assembly of microbial communities in oil reservoirs is scale- and taxonomy-dependent. Our work provides a comprehensive analysis of microbial communities in oil reservoirs at a localized geographic scale, which improves the understanding of the assembly of the microbial communities in oil reservoirs.

Response of Microbial Communities on Cathode with Different Potentials in a Single-Chamber Reactor.

Microbes use both organic and inorganic compounds as electron donors, with different electronic potentials, to produce energy required for growth in environments. Conventional studies on the effects of different electron donors on microbial community has been extensively studied with a set cathode potential. However, it remains under-researched how a microbial community response to the different redox potentials in different environments. Here, we incubated a lake sediment in a single-chamber reactor equipped with three working electrodes, i.e., with potentials of - 0.29 V, - 0.05 V versus standard hydrogen electrode and open-circuit, respectively. Results reveal that the structure of bacterial communities was highly similar for all closed-circuit electrodes (- 0.29 V, - 0.05 V), while differing significantly from those on open-circuit electrodes. We also show that specific bacteria were preferentially enriched by different electrode potentials, i.e., Pseudomonas and Rhodobacter preferentially grew on - 0.05 V and - 0.29 V cathode potentials, Azospirillum and Bosea preferentially grew on - 0.05 V; while Ferrovibrio, Hydrogenophaga, Delftia, and Sphingobium preferentially grew on - 0.29 V. In addition, microorganisms selectively enriched on open-circuit electrodes possess higher connectivity and closer relationship than microorganisms selectively enriched on closed-circuit electrode.

Metabolic profiling of petroleum-degrading microbial communities incubated under high-pressure conditions.

While pressure is a significant characteristic of petroleum reservoirs, it is often overlooked in laboratory studies. To clarify the composition and metabolic properties of microbial communities under high-pressure conditions, we established methanogenic and sulfate-reducing enrichment cultures under high-pressure conditions using production water from the Jilin Oilfield in China. We utilized a metagenomics approach to analyze the microbial community after a 90-day incubation period. Under methanogenic conditions, Firmicutes, Deferribacteres, Ignavibacteriae, Thermotogae, and Nitrospirae, in association with the hydrogenotrophic methanogen Archaeoglobaceae and acetoclastic Methanosaeta, were highly represented. Genomes for Ca. Odinarchaeota and the hydrogen-dependent methylotrophic Ca. Methanosuratus were also recovered from the methanogenic culture. The sulfate-reducing community was dominated by Firmicutes, Thermotogae, Nitrospirae, Archaeoglobus, and several candidate taxa including Ca. Bipolaricaulota, Ca. Aminicenantes, and Candidate division WOR-3. These candidate taxa were key pantothenate producers for other community members. The study expands present knowledge of the metabolic roles of petroleum-degrading microbial communities under high-pressure conditions. Our results also indicate that microbial community interactions were shaped by syntrophic metabolism and the exchange of amino acids and cofactors among members. Furthermore, incubation under in situ pressure conditions has the potential to reveal the roles of microbial dark matter.

Type IV pili trigger episymbiotic association of Saccharibacteria with its bacterial host.

Recent characterization of the obligate episymbiont Saccharibacteria (TM7) belonging to the candidate phyla radiation (CPR) has expanded the extent of microbial diversity. However, the episymbiotic lifestyle of TM7 is still underexploited due to the deficiency of cultivated representatives. Here, we describe gene-targeted TM7 cultivation guided by repurposing epicPCR (emulsion, paired isolation, and concatenation PCR) to capture in situ TM7‒host associations. Using this method, we obtained a novel Saccharibacteria isolate TM7i and its host Leucobacter aridicollis J1 from Cicadae Periostracum, the castoff shell of cicada. Genomic analyses and microscopic characterizations revealed that TM7i could bind to J1 through twitching-like motility mediated by type IV pili (T4P). We further showed that the inhibition of T4P extrusion suppressed the motility and host adherence of TM7i, resulting in its reduced growth. However, the inactivation of T4P had little effect on the growth of TM7i that had already adhered to J1, suggesting the essential role of T4P in host recognition by TM7i. By capturing CPR‒host association and elaborating the T4P-dependent episymbiotic association mechanism, our studies shed light on the distinct yet widespread lifestyle of CPR bacteria.

Single-Cell-Based High-Throughput Cultivation and Functional Characterization of Biosurfactant-Producing Bacteria from Soil and Oilfield-Produced Water.

Biosurfactants are a group of surface-active compounds that can be produced by diverse microorganisms. They have been widely used in various industrial fields. Reducing production costs, improving efficiency, and collecting more diverse producing strains have become major challenges in the biosurfactant industry. These challenges could be overcome by screening for more diverse and efficient biosurfactant-producing strains. The conventional methods for the isolation and functional characterization of microorganisms are laborious and biased toward fast-growing or strongly competitive microorganisms. Here, we established a high-throughput approach of single-cell-based cultivation and functional characterization of biosurfactant-producing bacteria (SCCBB). This approach combines single-cell cultivation with the detection of optical distortions. Using this approach, we isolated 431 strains with biosurfactant production potential from petroleum-contaminated soil and oilfield-produced water. The surfactant production capabilities of the strains were subsequently validated using surface tension measurements, TLC, and CMC measurements. To investigate the industrial production potential, we optimized the production conditions of a representative glycolipids-producing strain, Pseudomonas sp. L01, using response surface methodology (RSM). Optimal conditions yielded a crude biosurfactant yield of 8.43 g/L in a flask. Our work provides a high-throughput approach to the isolation and screening of biosurfactant-producing bacteria, as well as other functional bacteria in a wide range of fields.

The Roles of the Two-Component System, MtrAB, in Response to Diverse Cell Envelope Stresses in Dietzia sp. DQ12-45-1b.

Two-component systems (TCSs) act as common regulatory systems allowing bacteria to detect and respond to multiple environmental stimuli, including cell envelope stress. The MtrAB TCS of Actinobacteria is critical for cell wall homeostasis, cell proliferation, osmoprotection, and antibiotic resistance, and thus is found to be highly conserved across this phylum. However, how precisely the MtrAB TCS regulates cellular homeostasis in response to environmental stress remains unclear. Here, we show that the MtrAB TCS plays an important role in the tolerance to different types of cell envelope stresses, including environmental stresses (i.e., oxidative stress, lysozyme, SDS, osmotic pressure, and alkaline pH stresses) and envelope-targeting antibiotics (i.e., isoniazid, ethambutol, glycopeptide, and ß-lactam antibiotics) in Dietzia sp. DQ12-45-1b. An mtrAB mutant strain exhibited slower growth compared to the wild-type strain and was characterized by abnormal cell shapes when exposed to various environmental stresses. Moreover, deletion of mtrAB resulted in decreased resistance to isoniazid, ethambutol, and ß-lactam antibiotics. Further, Cleavage under targets and tagmentation sequencing (CUT&Tag-seq) and electrophoretic mobility shift assays (EMSAs) revealed that MtrA binds the promoters of genes involved in peptidoglycan biosynthesis (ldtB, ldtA, murJ), hydrolysis (GJR88_03483, GJR88_4713), and cell division (ftsE). Together, our findings demonstrated that the MtrAB TCS is essential for the survival of Dietzia sp. DQ12-45-1b under various cell envelope stresses, primarily by controlling multiple downstream cellular pathways. Our work suggests that TCSs act as global sensors and regulators in maintaining cellular homeostasis, such as during episodes of various environmental stresses. The present study should shed light on the understanding of mechanisms for bacterial adaptivity to extreme environments. IMPORTANCE The multilayered cell envelope is the first line of bacterial defense against various extreme environments. Bacteria utilize a large number of sensing and regulatory systems to maintain cell envelope homeostasis under multiple stress conditions. The two-component system (TCS) is the main sensing and responding apparatus for environmental adaptation. The MtrAB TCS highly conserved in Actinobacteria is critical for cell wall homeostasis, cell proliferation, osmoprotection, and antibiotic resistance. However, how MtrAB works with regard to signals impacting changes to the cell envelope is not fully understood. Here, we found that in the Actinobacterium Dietzia sp. DQ12-45-1b, a TCS named MtrAB is pivotal for ensuring normal cell growth as well as maintaining proper cell morphology in response to various cell envelope stresses, namely, by regulating the expression of cell envelope-related genes. Our findings should greatly advance our understanding of the adaptive mechanisms responsible for maintaining cell integrity in times of sustained environmental shocks.