Berberine Degradation Characteristics and its Degradation Pathway by a Newly Isolated Berberine-Utilizing Agrobacterium.

Berberine (BBR) is widely used as a botanical pesticide due to its broad-spectrum antibacterial and antifungal activities. However, BBR degradation pathway in soil microorganisms, which determines its impact on soil environment, remains poorly understood. Herein, a novel BBR-degrading bacterium Agrobacterium sp. V1 was isolated and characterized. Agrobacterium sp. V1 was able to utilize BBR as the sole carbon source for cell growth, and 50 µg/mL of BBR was completely degraded within 48 h. To reveal the possible BBR degradation pathway, whole genome sequencing of Agrobacterium sp. V1 was conducted, and proteins in Agrobacterium sp. V1 were aligned with enzymes involved in BBR biosynthesis in Rhizoma Coptidis. The results indicated that more than 60% of enzymes in BBR biosynthesis pathway had orthologs in Agrobacterium sp. V1. Combined with the primary mass spectra of BBR metabolites, a novel BBR degradation pathway in this bacterium was proposed. In summary, the proposed BBR degradation pathway offered new insights into the impact of BBR to the environment and also provided a reference for studying BBR metabolism in microorganisms.

Identification of the Major Facilitator Superfamily Efflux Pump KpsrMFS in Klebsiella pneumoniae That Is Down-Regulated in the Presence of Multi-Stress Factors.

Efflux pumps play important roles in bacterial detoxification and some of them are stress-response elements that are up-regulated when the host is treated with antibiotics. However, efflux pumps that are down-regulated by stimulations are rarely discovered. Herein, we analyzed multiple transcriptome data and discovered a special (Major Facilitator Superfamily) MFS efflux pump, KpsrMFS, from Klebsiella pneumoniae, which was down-regulated when treated with antibiotics or extra carbon sources. Interestingly, overexpression of kpsrmfs resulted in halted cell growth in normal conditions, while the viable cells were rarely affected. The function of KpsrMFS was further analyzed and this efflux pump was determined to be a proton-driven transporter that can reduce the intracellular tetracycline concentration. In normal conditions, the expression of kpsrmfs was at a low level, while artificial overexpression of it led to increased endogenous reactive oxygen species (ROS) production. Moreover, by comparing the functions of adjacent genes of kpsrmfs, we further discovered another four genes that can confer similar phenotypes, indicating a special regulon that regulates cell growth. Our work provides new insights into the roles of efflux pumps and suggests a possible regulon that may regulate cell growth and endogenous ROS levels.

Role of Berberine as a Potential Efflux Pump Inhibitor against MdfA from Escherichia coli: In Vitro and In Silico Studies.

Infections by Gram-negative pathogens are usually difficult to manage due to the drug export by efflux pumps. With the evolution and horizontal transfer of efflux pumps, there is an urgent need to discover safe and effective efflux pump inhibitors. Here, we found that the natural compound berberine (BBR), a traditional medicine for intestinal infection, is an inhibitor against the major facilitator superfamily (MFS) efflux pump MdfA in Escherichia coli. The impact of BBR on MdfA was evaluated in a recombinant E. coli reporter strain. We demonstrated that low levels of BBR significantly increased intracellular ciprofloxacin concentrations and restored antibiotic susceptibility of the reporter strain. At the same time, we conducted molecular dynamics simulations to investigate the mechanisms of BBR's effect on MdfA. Our data indicated that BBR can aggregate to the periplasmic and cytoplasmic sides of MdfA in both of its inward and outward conformations. Protein rigidities were affected to different degrees. More importantly, two major driving forces for the conformational transition, salt bridges and hydrophilic interactions with water, were changed by BBR's aggregation to MdfA, which affected its conformational transition. In summary, our data provide evidence for the extended application of BBR as an efflux pump inhibitor at a clinically meaningful level. We also reveal the mechanisms and provide insights into BBR's effect on the reciprocal motion of MdfA. IMPORTANCE In this work, we evaluated the role of berberine (BBR) as an inhibitor of the MFS efflux pump MdfA from E. coli. We demonstrated that low levels of BBR significantly increased intracellular ciprofloxacin concentrations and restored antibiotic susceptibility of the reporter strain. Molecular dynamics simulations revealed the effect of BBR on the conformational transition of MdfA. Our data suggested that driving forces for MdfA's conformational transition were affected by BBR and provided evidence for BBR's extended application as an effective inhibitor of MdfA.

Enhanced internal ionic interaction of MFS efflux pump MdfA contributes to its elevated antibiotic export.

Infections caused by Gram-negative pathogens are difficult to manage due to their antibiotic resistance. Efflux pumps, which transport intracellular toxins out of the cytoplasm, play an important role in the detoxification of bacteria when treated with antibiotics. The major facilitator superfamily (MFS) is a kind of widely distributed efflux pumps and can actively export clinically important antibiotics such as ciprofloxacin, while the role of internal ionic interactions in regulating drug export remains poorly understood. Herein we used a representative MFS efflux pump MdfA to investigate the impact of internal ionic interactions on the antibiotic resistance of E. coli. First, we identified the internal salt bridges of MdfA and searched their natural variants across all the sequenced E. coli isolates. By constructing these variants, we discovered that extending the salt bridge on the cytoplasmic side (E136D) conferred an elevated antibiotic resistance level of E. coli, and the level was further enhanced by combining it with an artificial mutation K346R. By analyzing the trajectories of MdfA's variants in molecular dynamics (MD) simulations, we revealed that ionic interaction strengths on the two sides were proportionally enhanced, while the protein flexibility was not affected. Moreover, enhanced interactions resulted in a larger surface for MdfA's protonation, suggesting a higher possibility for its activation. Collectively, our data revealed the importance of internal interactions on the drug export of MdfA, offering insights for the development of novel inhibitors against MFS efflux pumps.

Tetrahydroisoquinoline N-methyltransferase from Methylotenera Is an Essential Enzyme for the Biodegradation of Berberine in Soil Water.

Berberine (BBR), a Chinese herbal medicine used in intestinal infection, has been applied as a botanical pesticide in the prevention of fungal disease in recent years. However, its degradation in the environment remains poorly understood. Here, we investigated BBR's degradation in soil water from different sources accompanied by its effect on bacterial diversity. Our results indicated that BBR was only degraded in soil water, while it was stable in tap water, river water and aquaculture water. Bacterial amplicon results of these samples suggested that the degradation of BBR was closely related to the enrichment of Methylotenera. To reveal this special relationship, we used bioinformatics tools to make alignments between the whole genome of Methylotenera and the pathway of BBR's degradation. An ortholog of Tetrahydroisoquinoline N-methyltransferase from plant was discovered only in Methylotenera that catalyzed a crucial step in BBR's degradation pathway. In summary, our work indicated that Methylotenera was an essential bacterial genus in the degradation of BBR in the environment because of its Tetrahydroisoquinoline N-methyltransferase. This study provided new insights into BBR's degradation in the environment, laying foundations for its application as a botanical pesticide.

Discovering interrelated natural mutations of efflux pump KmrA from Klebsiella pneumoniae that confer increased multidrug resistance.

Klebsiella pneumoniae is a notorious pathogen that can cause multiorgan infections, which is difficult to treat mainly due to the widely distributed efflux pumps. Our previous research discovered the upregulation of efflux pump KmrA conferred enhanced antibiotic resistance, while the export mechanism and its natural mutations across K. pneumoniae isolates remain unclear. Herein, we analyzed the natural mutations of KmrA across 830 K. pneumoniae genomes to discover interrelated amino-acid substitutions (simultaneously occurred substitutions) that increase drug export. We identified two variants that contain triple amino-acid substitutions near the periplasmic side and then confirmed their roles in enhancing multidrug resistance of recombinant K. pneumoniae strains. Molecular dynamics simulations were conducted to illustrate the reason for their promoted export efficiencies. Our data indicated the triple substitutions resulted in KmrA's both stronger hydrophilic interaction with water and hydrophobic interaction with membrane. Moreover, these substitutions promoted the flexibilities of KmrA that could facilitate the conformational switch. In parallel, stronger ionic interactions (salt bridges) at cytoplasmic side also suggested the higher possibilities for the reciprocal movements. Collectively we demonstrated the potential risk of the interrelated natural mutations in efflux pump to antibiotic resistance of K. pneumoniae and provided insights into the mechanism of the enhanced drug export.

Opposite motion of the Central Helices of efflux pump KmrA is important for its export efficiency.

Efflux pump of Major Facilitator Superfamily (MFS) is widely distributed in bacteria, while its role in regulating antibiotic resistance of nosocomial pathogen Klebsiella pneumoniae remains unclear. Herein we analyzed the effect of amino acid substitution of MFS efflux pump KmrA on its export efficiency via molecular biology and molecular dynamics (MD). After searching across the 804 sequenced K. pneumoniae isolates, we identified four major variants of KmrA, while one of them KmrA-A was demonstrated an inactive one in MIC and ethidium bromide efflux assays. Subsequently, MD simulations of KmrA and its variants were conducted and the opposite motion of the central helices were observed for the active variants, while it was not found for KmrA-A. To further identify the importance of the opposite motion to the conformational transition, we calculated their differences in volume of binding pocket, salt bridge and hydrophilic interaction with water based on the rocker-switch model. Our results indicated that the opposite motion of KmrA conferred a larger binding pocket and stronger hydrogen bond with water at inward-facing conformation. An unusual substitution S374A of KmrA-A disrupted the normal motion of central helices by enhancing hydrophobic interactions between them, resulting into the altered positions and strengths of salt bridge, which was deduced to affect the conformational transition. Overall our data provided detailed information on the regular of KmrA's moving trajectory, demonstrating the importance of opposite motion of central helices to KmrA's export efficiency.

Saprophytic Bacillus Accelerates the Release of Effective Components in Agarwood by Degrading Cellulose.

The value of Agarwood increases with time due to the gradual release of its major components, but the mechanism behind this remains unclear. Herein we reveal that the potential driving force of this process is the degradation of cellulose in Agarwood by its saprophytic Bacillus subtilis. We selected 10-year-old Agarwood from different places and then isolated the saprophytic bacteria. We confirmed these bacteria from different sources are all Bacillus and confirmed they can degrade cellulose, and the highest cellulase activity reached 0.22 U/mL. By co-cultivation of the bacterium and Agarwood powder, we found that three of the strains could release the effective components of Agarwood, while they had little effect in increasing the same components in living Aquilaria sinensis. Finally, we demonstrated that these saprophytic Bacillus subtilis have similar effects on Zanthoxylum bungeanum Maxim and Dalbergiaod orifera T. Chen, but not on Illicium verum Hook. f, Cinnamomum cassia Presl and Phellodendron chinense Schneid. In conclusion, our experiment revealed that the saprophytic Bacillus release the effective components of Agarwood by degrading cellulose, and we provide a promising way to accelerate this process by using this bacterial agent.

Molecular Basis of the Reinforced Effect of Berberine against Cutinase from Colletotrichum capsisi by Supplying Sodium Stearate as Dispersant.

Berberine (BBR) is a promising botanical pesticide that can reduce the enzyme activity of secreted cutinase from fungal pathogens. However, only less than 15% of total activity was prohibited. Herein we researched BBR's self-aggregation in water via molecular dynamics simulations, and further investigated the effect of dispersant on blocking the aggregation together with the impact on cutinase. Strong hydrophobic interactions were found between adjacent BBR molecules, and these molecules formed clustered conformations at different BBR concentrations. Interestingly, one of the tested dispersants, sodium stearate (ST), is able to insert into BBR clusters and form stable interaction until the end of simulation, resulting in decreased hydrophobic strength in the BBR-ST cluster. More importantly, supply of ST with BBR resulted in BBR's reinforced hydrophobic interactions between BBR and the catalytic center of cutinase, which led to the inactivated mode of cutinase. Finally, wet experiments demonstrated that combined application of BBR and ST indeed resulted in a synergy-like effect on reducing the activity of cutinase. Overall, our findings revealed the mechanism of the reinforced effect of BBR against cutinase when supplying ST as dispersant, suggesting an undiscovered role of ST in enhancing the efficiency of this botanical pesticide.

Molecular Dynamics Investigation of MFS Efflux Pump MdfA Reveals an Intermediate State between Its Inward and Outward Conformations.

Multidrug resistance poses a major challenge to antibiotic therapy. A principal cause of antibiotic resistance is through active export by efflux pumps embedded in the bacterial membrane. Major facilitator superfamily (MFS) efflux pumps constitute a major group of transporters, which are often related to quinolone resistance in clinical settings. Although a rocker-switch model is proposed for description of their conformational transitions, detailed changes in this process remain poorly understood. Here we used MdfA from E. coli as a representative MFS efflux pump to investigate factors that can affect its conformational transition in silico. Molecular dynamics (MD) simulations of MdfA's inward and outward conformations revealed an intermediate state between these two conformations. By comparison of the subtle differences between the intermediate state and the average state, we indicated that conformational transition from outward to inward was initiated by protonation of the periplasmic side. Subsequently, hydrophilic interaction of the periplasmic side with water was promoted and the regional structure of helix 1 was altered to favor this process. As the hydrophobic interaction between MdfA and membrane was also increased, energy was concentrated and stored for the opposite transition. In parallel, salt bridges at the cytoplasmic side were altered to lower probabilities to facilitate the entrance of substrate. In summary, we described the total and local changes during MdfA's conformational transition, providing insights for the development of potential inhibitors.

Hormesis Effect of Berberine against Klebsiella pneumoniae Is Mediated by Up-Regulation of the Efflux Pump KmrA.

Berberine (BBR) is an effective drug for human intestinal inflammation by preventing intestinal adhesion of bacterial pathogens, while its antibacterial activity is ineffective. Although the antimicrobial mechanisms of BBR are intensively studied at high concentrations, the response of pathogens to its low concentrations remains poorly understood. Here we demonstrated that low concentrations of BBR (3 and 6 µg/mL) conferred by hormesis accelerated cell growth of an important Gram-negative pathogen, Klebsiella pneumoniae, in vitro, while higher concentrations (25 and 50 µg/mL) resulted in the opposite. Transcriptome analysis of K. pneumoniae revealed the up-regulated expression of the KmrA efflux pump and further confirmed it was hypersensitive to BBR stress. Strikingly, when cultivated in tetracycline, the growth-promoting effect of BBR became more significant, while this effect was reversed in the presence of the efflux pump inhibitor cyanide-m-chlorophenylhydrazone. The hormesis was also found in Enterobacter cloacae and Acinetobacter baumannii. More importantly, the presence of BBR at low concentrations resulted in higher minimal inhibitory concentrations of efflux-related antibiotics such as rifampicin and azithromycin. Overall, our data demonstrated the hormesis of BBR and revealed the potential risk of its applications against Gram-negative pathogens at low concentrations.

[Pathway design and key enzyme analysis of diosgenin biosynthesis].

As a naturally occurring steroid sapogenin, diosgenin acts as the precursor of hundreds of steroid medicines, and thereby has important medicinal value. Currently, industrial production of diosgenin relies primarily on chemical extraction from plant materials. Clearly, this strategy shows drawbacks of excessive reliance on plant materials and farmland as well as environment pollution. Due to development of metabolic engineering and synthetic biology, bio-production of diosgenin has garnered plenty of attention. Although the biosynthetic pathways of diosgenin have not been completely identified, in this review, we outline the identified biosynthetic pathways and key enzymes. In particular, we suggest heterologous biosynthesis of diosgenin in Saccharomyces cerevisiae. Overall, this review aims to provide valuable insights for future complete biosynthesis of diosgenin.

Molecular dynamics investigation of the interaction between Colletotrichum capsici cutinase and berberine suggested a mechanism for reduced enzyme activity.

Berberine is a promising botanical pesticide against fungal plant pathogens. However, whether berberine inhibits the invasion of fungal pathogen across plant surface remains unclear. Here we demonstrated that the enzyme activities of purified cutinase from fungal pathogen Colletotrichum capsici were partially inhibited in presence of berberine toward different substrates. Molecular dynamics simulation results suggested the rigidity of cutinase was decreased with berberine added into the system. Interestingly, aggregations of berberine to the catalytic center of cutinase were observed, and stronger hydrophobic interactions were detected between key residue His 208 and berberine with concentrations of berberine increased. More importantly, this hydrophobic interaction conferred conformational change of the imidazole ring of His 208, which swung out of the catalytic center to an inactive mode. In summary, we provided the molecular mechanism of the effect of berberine on cutinase from C. capsici.

Metabolic Checkpoint Aldehyde Dehydrogenases Are Important for Diverting ß-Oxidation into 1-Butanol Biosynthesis from Kitchen Waste Oil in Pseudomonas aeruginosa.

1-Butanol (1-BD) is a promising fuel additive which can be biosynthesized via reversed ß-oxidation pathway in bacteria. However, heterologous reversed ß-oxidation pathway is a carbon chain prolongation process with several genes overexpressed in most of bacterial hosts, leading to low titer of 1-BD and high cost for production. Here we displayed a forward ß-oxidation pathway for 1-BD production in a kitchen waste oil (KWO) degrading Pseudomonas aeruginosa PA-3, and we proved that aldehyde dehydrogenase (ALDH) is a checkpoint for diverting metabolic flux into 1-BD biosynthesis. With nitrogen source supplied, titer of 1-BD was increased accompanied with 12 ALDH coding genes transcriptionally promoted to different degrees. At the same time, binding energies of these ALDHs with different length of acyl-CoAs in ß-oxidation were calculated to identify their specificities. Based on the above information, ALDH deletions were conducted. We certified that deletion of ALDH8 and ALDH9 led to significant decreased titers of 1-BD. Finally, these two ALDHs were separately overexpressed in PA-3, and titer of 1-BD was promoted to 1.36 g/L at 72 h in shake flask. Totally in this work, we provided a forward ß-oxidation pathway for 1-BD production from KWO, and the roles of ALDHs were confirmed.

Overview of Alternaria alternata Membrane Proteins.

Alternaria species are mainly saprophytic fungi, but some pathotypes of Alternaria alternata infect economically important plants including cereal crops, vegetables and fruits. Specially, A. alternata generates toxins which contaminate food and feed. To date, management of A. alternata relies primarily on fungicides. However, the control efficacy in most cases is below expectation due to ubiquity of A. alternata and resistance to fungicides. To mitigate resistance and develop long-lasting fungicides, uncovering multiple rather than single target is a prerequisite. Membrane proteins are potential targets of fungicides owing to wide participation in myriad biochemical events especially material transport, signal transduction and pathogenicity. However, so far, little is known about the distribution and molecular structure of A. alternata membrane proteins (AAMPs). Herein we summarize AAMPs by data mining and subsequent structure prediction. We also outline the state-of-the-art research advances of AAMPs especially those closely related to pathogenicity. Overall, this review aims to portray a picture of AAMPs and provide valuable insights for future development of highly efficient fungicides towards A. alternata or beyond.

Development of orthogonal T7 expression system in Klebsiella pneumoniae.

Most expression systems are tailored for model organisms rather than nonmodel organisms. However, heterologous gene expression in model organisms constrains the innate advantages of original strain carrying gene of interest. In this study, T7 expression system was developed in nonmodel bacterium Klebsiella pneumoniae for production of chemicals. First, we engineered a recombinant K. pneumoniae strain harboring two vectors. One vector was used to express T7 RNA polymerase (T7 RNAP) which would drive the expression of egfp in the other vector. This recombinant strain demonstrated 15.73-fold of fluorescence relative to wild-type K. pneumoniae and showed similar level of fluorescence to recombinant Escherichia coli overexpressing egfp. When egfp was replaced by puuC, an endogenous aldehyde dehydrogenase catalyzing 3-hydroxypropionic acid (3-HP) biosynthesis in K. pneumoniae, the recombinant strain coexpressing T7 RNAP and PuuC showed high-level PuuC expression. In shake-flask cultivation, this recombinant strain produced 1.72 g/L 3-HP in 24 hr, which was 3.24 times that of wild-type K. pneumoniae (0.53 g/L). To mitigate plasmid burden, the vector expressing T7 RNAP was eliminated, but the T7 RNAP expression cassette was integrated into K. pneumoniae genome. The resulting strain harboring only PuuC expression vector produced 2.44 g/L 3-HP in 24 hr under shake-flask conditions, which was 1.46 times that of the strain harboring both T7 RNAP and PuuC expression vectors. In bioreactor cultivation, this strain generated 67.59 g/L 3-HP and did not show significantly halted growth. Overall, these results indicate that the engineered T7 expression system functioned efficiently in K. pneumoniae. This study provides a paradigm for the development of T7 expression system in prokaryotes.

Co-production of 1,3-Propanediol and 2,3-Butanediol from Waste Lard by Co-cultivation of Pseudomonas alcaligenes and Klebsiella pneumoniae.

The platform chemicals 1,3-propanediol (1,3-PD) and 2,3-butanediol (2,3-BD) are important raw materials for polyesters and biofuels. However, the biosynthesis of the compounds relies on massive consumption of glucose or glycerol, leading to the uneconomical production in industrial scale. In this work, we developed a new method for co-production of 1,3-PD and 2,3-BD from waste lard to reduce the cost in carbon source supply. A waste lard utilizing Pseudomonas alcaligenes PA-3 and a 1,3-PD producing Klebsiella pneumoniae AA405 were co-cultivated by using waste lard as the sole carbon source. In a shake flask, 1.05 g/L 1,3-PD and 0.35 g/L 2,3-BD were produced from waste lard within 24 h. The addition of nitrogen source significantly increased the relative ratio of K. pneumoniae AA405 in the medium, which further favored to the higher titers of the two products. In bioreactor, the co-cultivation system produced 5.98 g/L 1,3-PD and 4.29 g/L 2,3-BD from 100 g/L waste lard within 72 h, and the conversion rate of 1,3-PD and 2,3-BD from waste lard were 62.95% and 0.75%, respectively. In all, this is the first work on 1,3-PD and 2,3-BD production from waste triglyceride, which will favor the utilization of low-cost carbon source in industrial production of chemicals.

Expression of the TetA gene encoding TetA efflux protein in E. coli contributes to its increased bacterial resistance toward berberine.

Berberine (BBR) is a traditional Chinese medicine in various applications due to its antibacterial effect. Here we investigated the increased bacterial resistance of E. coli toward BBR. The median effective concentration (EC50) of BBR against E. coli was increased when TetA efflux protein (TEP) was introduced. Sixty-five percent of the intracellular BBR was expelled and molecular docking demonstrated the intensive interaction of TEP to BBR. Finally, the combined antibacterial experiment identified that BBR acted as an inhibitor of TEP in detoxification of tetracycline. TEP is the first discovered protein that was related to the bacterial susceptibility to BBR.

Evaluation of berberine as a natural fungicide: biodegradation and antimicrobial mechanism.

Berberine (BBR) is a traditional Chinese medicine which recently was applied as a biological pesticide. Here, we studied the antimicrobial mode of BBR and its impact on soil bacterial diversity. BBR was more effective against fungi than bacteria due to the specific interaction between BBR and glucan. Also, BBR was degraded rapidly in soil, leading to the limited effect on soil bacterial diversity. Collectively, BBR is an environment-friendly pesticide and it is promising in dealing with fungal plant diseases.

Isolation and characterization of a novel bacterium Pseudomonas aeruginosa for biofertilizer production from kitchen waste oil.

Kitchen waste oil is composed of long chain triglycerides (LCTs) that has high energy density. However, it is hard to be degraded by microbes, thereby leading to increasing levels of environmental pollution due to landfill disposition. In this study, we isolated and characterized a novel bacterium Pseudomonas aeruginosa PA-3 that could convert kitchen waste oil into biofertilizer. PA-3 could survive on trilaurin or kitchen waste oil as the sole carbon source, and 10 g L-1 trilaurin or kitchen waste oil was completely consumed within 7 days. Interestingly, the degradation products of kitchen waste oil can be used as biofertilizer in promoting cabbage growth. The plant height, leaf area and stem diameter of cabbage plants were all increased with the addition of kitchen waste oil cultivation products into the soil. Kitchen waste oil degradation products were analyzed by gas chromatography mass spectrometry (GC-MS), and short chain alcohols or fatty acids were observed to be the main products. To unravel the mechanism underlying the accelerated cabbage growth, bacterial diversity of the soil was investigated after using this biofertilizer. Results showed that agricultural probiotics accumulated with the addition of kitchen waste oil cultivation products. Finally, the whole genome of PA-3 was sequenced and analyzed, which showed the existence of a complete ß-oxidation pathway in the genome of PA-3. To our knowledge, this is the first study on kitchen waste oil degradation and re-utilization by bacteria, which provides a new method for waste source re-utilization.