APPLIED AND ENVIRONMENTAL MICROBIOLOGY
·
2024
·Vol. 91
·期 1
·
IF 3.9
Ji Zeng,Hao Wang,Yuxi Xu,Jianying Han,Yannan Li,Shu'an Wen,Changbu Wu,Dani Li,Zheng Liu,Xiaokang Zhang,Guo-Bao Tian,Min Dong
Clostridioides difficile is an obligate anaerobic, Gram-positive bacterium that produces toxins. Despite technological progress, conducting gene expression analysis of C. difficile under different conditions continues to be labor-intensive. Therefore, there is a demand for simplified tools to investigate the transcriptional and translational regulation of C. difficile. The cell-free gene expression (CFE) system has demonstrated utility in various applications, including prototyping, protein production, and in vitro screening. In this study, we developed a C. difficile CFE system capable of in vitro transcription and translation (TX-TL) in the presence of oxygen. Through optimization of cell extract preparation and reaction systems, we increased the protein yield significantly. Furthermore, our observations indicated that this system exhibited higher protein yield using linear DNA templates than circular plasmids for in vitro expression. The prototyping capability of the C. difficile CFE system was assessed using a series of synthetic Clostridium promoters, demonstrating a good correlation between in vivo and in vitro expression. Additionally, we tested the expression of tcdB and tcdR from clinically relevant C. difficile strains using the CFE system, confirming higher toxin expression of the hypervirulent strain R20291. We believe that the CFE system can not only serve as a platform for in vitro protein synthesis and genetic part prototyping but also has the potential to be a simplified model for studying metabolic regulations in Clostridioides difficile.
研究领域:
微生物学
·
分子生物学
·
生物技术
✓ 已验证
DOI
高影响因子
APPLIED AND ENVIRONMENTAL MICROBIOLOGY
·
2024
·Vol. 91
·期 1
·
IF 3.9
Yuan-Ru Xiong,Yuan-Chun Fang,Min He,Ke-Jing Li,Lei Qi,Yang Sui,Ke Zhang,Xue-Chang Wu,Liang Meng,Ou Li,Dao-Qiong Zheng
This study explored the genomic alterations in Yarrowia lipolytica, a key yeast in industrial biotechnology, under both spontaneous and mutagen-induced conditions. Our findings reveal that spontaneous mutations occur at a rate of approximately 4 × 10−10 events per base pair per cell division, primarily manifesting as single-nucleotide variations (SNVs) and small insertions and deletions (InDels). Notably, C-to-T/G-to-A transitions and C-to-A/G-to-T transversions dominate the spontaneous SNVs, while 1 bp deletions, likely resulting from template slippage, are the most frequent InDels. Furthermore, chromosomal aneuploidy and rearrangements occur, albeit at a lower frequency. Exposure to ultraviolet (UV) light, methylmethane sulfonate (MMS), and Zeocin significantly enhances the rates of SNVs and alters their mutational spectra in distinct patterns. Notably, Zeocin-induced SNVs are predominantly T-to-A and T-to-G substitutions, often occurring within the 5′-TGT*−3′ motif (* denotes the mutated base). Additionally, Zeocin exhibits a higher potency in stimulating InDels compared to UV and MMS. Translesion DNA synthesis is implicated as the primary mechanism behind most Zeocin-induced SNVs and some InDels, whereas non-homologous end joining serves as the main pathway for Zeocin-mediated InDels. Intriguingly, the study identifies the gene YALI1_E21053g, encoding a protein kinase, as negatively associated with Zeocin resistance. Overall, our results not only deepened our knowledge about the genome evolution in Y. lipolytica but also provided reference to develop innovative strategies to harness its genetic potential.
研究领域:
微生物学
·
遗传学
·
分子生物学
✓ 已验证
DOI
高影响因子
APPLIED AND ENVIRONMENTAL MICROBIOLOGY
·
2024
·Vol. 90
·期 9
·
IF 3.9
Wenmin Chai,Xianbing Mao,Chunfeng Li,Liancai Zhu,Zongyi He,Bochu Wang
Parasites can manipulate host behavior to facilitate parasite transmission. One such host–pathogen interaction occurs between the fungus Ophiocordyceps sinensis and the ghost moth Thitarodes xiaojinensis. O. sinensis is involved in the mummification process of infected host larvae. However, the underlying molecular and chemical mechanism for this phenomenon is unknown. We characterized the small molecules regulating host behaviors and the altered metabolites in infected and mummified host larvae. Lipid-related metabolites, such as phosphatidylcholine, were identified in infected and mummified larvae. Decreased levels of the neurotransmitter acetylcholine (ACh) and elevated choline levels were observed in the brains of both the infected and mummified larvae. The aberrant activity of acetylcholinesterase (AChE) and relative mRNA expression of ACE2 (acetylcholinesterase) may mediate the altered transformation between ACh and choline, leading to the brain dysfunction of mummified larvae. Caspofungin treatment inhibited the mummification of infected larvae and the activity of AChE. These findings indicate the importance of ACh in the mummification of host larvae after O. sinensis infection.
研究领域:
真菌学
·
神经生物学
·
代谢组学
✓ 已验证
DOI
高影响因子
APPLIED AND ENVIRONMENTAL MICROBIOLOGY
·
2024
·
IF 3.9
Xuemeng Ji,Dancai Fan,Jin Wang,Bowei Zhang,Yaozhong Hu,Huan Lv,Jing Wu,Yi Sun,Jingmin Liu,Yan Zhang,Shuo Wang
Cronobacter sakazakii poses a significant threat, particularly to neonates and infants. Despite its strong pathogenicity, understanding of C. sakazakii biofilms and their role in infections remains limited. This study investigates the roles of HmsP and c-di-GMP in biofilm formation and identifies key genetic and proteomic elements involved. Gene knockout experiments reveal that HmsP and c-di-GMP are linked to biofilm formation in C. sakazakii. Comparative proteomic profiling identifies the lysozyme inhibitor protein LprI, which is downregulated in hmsP knockouts and upregulated in c-di-GMP knockouts, as a potential biofilm formation factor. Further investigation of the lprI knockout strain shows significantly reduced biofilm formation and decreased virulence in a rat infection model. Additionally, LprI is demonstrated to bind extracellular DNA, suggesting a role in anchoring C. sakazakii within the biofilm matrix. These findings enhance our understanding of the molecular mechanisms underlying biofilm formation and virulence in C. sakazakii, offering potential targets for therapeutic intervention and food production settings.
研究领域:
微生物学
·
分子生物学
·
感染病学
✓ 已验证
DOI
PubMed
高影响因子
APPLIED AND ENVIRONMENTAL MICROBIOLOGY
·
2026
·
IF 4.2
Ken Zheng,Ai-Ping Pang,Fang-Ying Zhu,Yu-Hui Liu,Xiao-Jian Zhang,Qing Yang,Xiang-Yang Li,Jun-ping Zhou,Bo Zhang,Zhi-Qiang Liu,Yu-Guo Zheng
The microbial production of pantothenic acid (d-PA) is critically limited by feedback inhibition and low activity of the key enzyme ketopantoate hydroxymethyltransferase (KPHMT). To overcome this, rational enzyme mining based on computational prediction was established. Following sequence conservation analysis, molecular dynamics simulations, and binding free energy (ΔG) calculations, five representative native KPHMT enzymes were selected for experimental validation: EcKPHMT from Escherichia coli, CgKPHMT from Corynebacterium glutamicum, MpKPHMT from Mangrovibacter plantisponsor, EpKPHMT from Enterovibrio pacificus, and BsKPHMT from Bacillus subtilis. EpKPHMT and BsKPHMT exhibited 4.25- and 4.60-fold times that of EcKPHMT, respectively, with relieved pantoate feedback inhibition (IC50: 14.07 and 19.86 mM vs. 1.08 mM) and virtually no inhibition by d-PA. The strain expressing BsKPHMT enhanced d-PA and pantoate titers by 74.30% (3.12 g/L) and 140.0% (0.84 g/L) in shake flasks, and further increased d-PA production by 55.4% in a 5 L bioreactor, over the control. This work established a predictive framework for mining superior enzymes based on in silico prediction, offering a valuable strategy for metabolic engineering of high-value chemicals.
✓ 已验证
产品关联
DOI
PubMed
高影响因子
APPLIED AND ENVIRONMENTAL MICROBIOLOGY
·
2026
·
IF 4.2
Jiaxin Zhang,Libing Cai,Lin Wang,Lianhui Zhang,Nan Meng,Andong Chen,Qiao Ma
The organic booster biocide DCOIT (4,5-dichloro-2-n-octyl-4-isothiazolin-3-one) is widely used in marine antifouling systems, yet its ecological impacts on sediment microbiomes remain poorly understood. Here, we integrated absolute quantitative 16S rRNA gene sequencing, metagenomics, and enzyme activity assays to examine microbial responses to DCOIT exposure (0–50 μg/g sediment) over 30 days. DCOIT induced oxidative stress and bioenergetic impairment, accompanied by reduced microbial activity and inhibition of key enzyme-mediated processes involved in organic matter turnover and nitrogen transformation. Absolute quantification revealed a compensatory increase in total microbial abundance by Day 30, despite persistent diversity loss and community restructuring. Metagenomic analysis showed that DCOIT disturbed functional potentials related to carbon and nitrogen cycling. Kordiimonas, Aliikangiella, and Neptuniibacter emerged as potential contributors to nitrogen transformation, whereas Marinobacter was more closely associated with potential DCOIT transformation. DCOIT exposure also enriched adaptive traits, including chemotaxis, motility, quorum sensing, and biofilm regulation, and was accompanied by increased multidrug efflux systems and heavy metal resistance determinants. Our findings provide novel insights into the ecotoxicological risks of isothiazolinone biocides and highlight the potential for DCOIT to undermine sediment ecosystem functions and microbial habitat health. Given its extensive application, this study emphasizes the need to consider the microbial ecological consequences of DCOIT accumulation in seafloor environments.
✓ 已验证
产品关联
DOI
PubMed
高影响因子
APPLIED AND ENVIRONMENTAL MICROBIOLOGY
·
2026
·
IF 4.2
Haiqin Meng,Shun Pan,Zening Chai,Lixiang Zhou,Tianyu Zheng,Hanyu Zhang,He Fu,Lei Zou,Dongqi Liu,Jingcheng Dai,Dazhong Yan,Hong-Jun Chao
The catabolism of the priority environmental pollutant 2,4-xylenol requires bacteria to oxidize its ortho-methyl group. Its exact biochemical mechanism is uncharacterized until now. In this study, we resolve key metabolic bottlenecks in Pseudomonas putida NCIMB 9866. We demonstrate that functionally redundant Rieske oxygenase systems ensure the robust ortho-methyl oxidation of 2,4-xylenol. On the para-methyl oxidation branch, PchA2 acts as the main aldehyde dehydrogenase. It shows significantly higher catalytic efficiency than the previously reported PchA. Additionally, we characterized PmmA1B1, a newly identified three-component Rieske oxygenase system responsible for ortho-methyl oxidation, which is supported by two redundant isoenzymes (PmmA2B2 and PmmA3B3), by recruiting the shared endogenous ferredoxin Orf05169. The PmmA1B1-Orf05169 system catalyzes the successive oxidation of the ortho-methyl group of 4-hydroxy-3-methylbenzoate to a carboxyl group, yielding the high-value pharmaceutical precursor 4-hydroxyisophthalate. Structural and kinetic data revealed that the active site architecture of PmmA1 enables efficient successive oxidation, distinguishing it from isoenzymes PmmA2B2 and PmmA3B3 that, when coupled with Orf05169, catalyze only incomplete oxidation. Overall, these results complete the 2,4-xylenol catabolic pathway by defining the enzymes required for both para- and ortho-methyl oxidation. They also reveal functional redundancy as an adaptive feature of microbial aromatic degradation and establish Rieske oxygenases as biocatalysts for converting methylated aromatic pollutants into value-added chemicals.
✓ 已验证
产品关联
DOI
高影响因子
APPLIED AND ENVIRONMENTAL MICROBIOLOGY
·
2026
·
IF 4.2
Hua Li,Fangjuan Li,Yajie Shi,Xinxin Wang,Xiaomeng Wang,Guanyu Zheng,Lixiang Zhou,Barth F. Smets
The dissemination of extracellular antibiotic resistance genes (eARGs) through horizontal gene transfer poses a serious threat to global water safety. While Escherichia coli MG1655 is known to employ intracellular nucleases to defend against foreign DNA, the potential of its cell lysate to enzymatically degrade plasmid-borne eARGs and disrupt their environmental propagation remains unexplored. This study demonstrated that E. coli MG1655 cell lysate can degrade the plasmid-borne ampR gene by 99.9% within 48 h, and the maximum first-order degradation rate constant measured by long amplicon qPCR was 0.20 h−1 at the selected dosage state. This degradation eliminated the ability of the plasmid pUC19 for transformation, reducing it to below detectable levels (<10−6 transformants/recipients). The DNase I, produced by the chromosomal endA gene, was identified as the likely nuclease driving eARG cleavage. Environmental conditions critically modulated degradation efficiency: humic acid (50 mg/L) decreased the rate by 41.3%, while divalent cations (e.g., Mg2+) increased the rate by 1.65-fold. To transmit laboratory findings into real-world application, a polyacrylamide hydrogel-immobilized lysate (PAM-cell lysate) was engineered, which outperformed free lysate in treating plasmid-spiked real wastewater, sustaining 99.9% of ampR removal across three treatment cycles. This research demonstrated that PAM-cell lysate, without genetic modification, can be a cost-effective and scalable solution for attenuating the spread of eARGs in water, advancing the development of complementary enzymatic strategies for sustainable water remediation.
✓ 已验证
DOI
PubMed
高影响因子
APPLIED AND ENVIRONMENTAL MICROBIOLOGY
·
2026
·
IF 4.2
Zhengyu Hou,Hejuan Pan,Shihan Wang,Yulin Li,Yang Yu,Pei Qiao,Haixia Wang,Weihong Zhong
Phthalate esters (PAEs) are ubiquitous in the environment, and their microbial metabolism is associated with transport proteins. In this study, multiple novel membrane proteins were identified, revealing their pivotal role in PAEs degradation. Based on whole-genome and transcriptomic analyses, approximately 20 potential PAEs transport membrane proteins were predicted to be involved in PAEs degradation by Rhodococcus sp. AH-ZY2. Among them, four protein genes (0620, 3572, 4497, and 5299) exhibited increased transcription levels (Log2F(c)>2.8) in response to di-n-octyl-phthalate (DnOP) as the sole carbon source, instead of fructose, were selected for functional verification. Gene knockout and complementation experiments showed that these four transport protein genes, belonging to the MFS family (0620, 3572, and 5299) and the ABC family (4497), respectively, exhibited the function to transport multiple PAEs into cells. Specifically, membrane protein 5299 could transport the most diverse kinds of PAEs. Molecular docking results also showed that Ser and Arg play a key role in the PAEs transport process of membrane protein 5299, and the PAEs-binding pocket size of 5299 in the transition region was larger than that of 0620, 3572, and 4497. Then, the expression of the genes encoding 0620, 3572, 5299, and 4497 was enhanced after they were cloned into the plasmid pNV18 and transformed into the strain AH-ZY2. The enhancement of 5299 expression exhibited a better improvement in PAEs degradation than the other three genes. This study provides four novel transport proteins and a new strategy for effective bioremediation of PAEs via enhanced membrane transport.
✓ 已验证
DOI
PubMed
高影响因子
APPLIED AND ENVIRONMENTAL MICROBIOLOGY
·
2026
·
IF 4.2
Pengrui Zhang,Haiyang Zhang,Xinyu Yang,Yuanli Wu,Shanshan Dai,Yanqin Ding,Shanshan Sun
Bacteria play an important role in the biodegradation of polycyclic aromatic hydrocarbons (PAHs), especially the more recalcitrant high-molecular-weight compounds like pyrene. In this study, we isolated and characterized a novel pyrene-degrading bacterium, Glutamicibacter soli ENR6, from petroleum-contaminated soil. Strain ENR6 was able to utilize various PAHs as sole carbon sources and degrade 88.8% of 50 mg L−1 pyrene and 76.8% of 100 mg L−1 pyrene within 15 days. During pyrene degradation, eight metabolites were detected, with 2,2′-diphenic acid emerging as the predominant intermediate. By day 7, 40.6% of the degraded pyrene had accumulated in the form of 2,2′-diphenic acid, which was subsequently metabolized via the phthalate pathway. To our knowledge, this is the first report identifying 2,2′-diphenic acid as an intermediate in pyrene degradation. Integrated genomic and transcriptomic analyses revealed four gene clusters (clusters I–IV) involved in pyrene degradation in strain ENR6. Reverse transcription-quantitative PCR (RT-qPCR) analysis demonstrated that these clusters were specifically induced by pyrene and its intermediates, with genes chr_3400-chr_3404 in cluster I strongly associated with 2,2′-diphenic acid degradation. Based on these findings, we propose a distinct 2,2′-diphenic acid-centered pathway for pyrene degradation in strain ENR6. This study not only broadens the bacterial repertoire available for PAH bioremediation but also provides new insights into the metabolic diversity of PAH-degrading microorganisms.
✓ 已验证
产品关联
DOI
高影响因子
APPLIED AND ENVIRONMENTAL MICROBIOLOGY
·
2026
·
IF 4.2
Xiaofang Li,Sen Yang,Yuping Liu,Hua Huang,Xinshuai Zhang,Yin Zhong,Lorenz Adrian,Ping’an Peng
Reductive dehalogenases (RDases) are a family of enzymes that employ a cobamide cofactor and two iron-sulfur clusters to selectively cleave carbon-halogen bonds. The identification of yet-uncharacterized RDases is crucial for advancing our understanding of dehalogenation processes in both natural and engineered environments, particularly for bioremediation. In this study, we developed a heterologous expression system in Escherichia coli DH5α to produce a chloroform-reducing RDase (TmrA) from Dehalobacter sp. UNSWDHB, using a pTrcHisA vector without requiring an additional vector for vitamin B12 transporters. By co-expressing the pRSFDuet-BtuCEDFB plasmid, we further enhanced active TmrA production without relying on the T7 promoter system. Using this system, we achieved the first heterologous expression of an RDase (the debrominase Ros_A3X954) from a marine heterotrophic Roseobacter strain, without chemical cofactor reconstitution. Ros_A3X954 catalyzes conversion of 2,6-dibromophenol to 2-bromophenol with a specific activity of 21.8 ± 1.0 nmol s−1 mg−1. The enzyme exhibited optimal activity at pH 6.0 and demonstrated significant thermal stability by maintaining full catalytic capacity across a broad temperature range (20–50°C), a key advantage for applications in thermally fluctuating marine environments. These findings expand our understanding of RDase functional diversity in common marine microbiota and highlight the significant application potential of Roseobacter for bioremediation of bromophenol pollution.
✓ 已验证
DOI
PubMed
高影响因子
APPLIED AND ENVIRONMENTAL MICROBIOLOGY
·
2026
·
IF 4.2
Fang Nan,Huihui Song,Min Sun,Lei Cui,Zeran Bian,Jie Yin,Zipeng Lin,Yan Wang
Microbial antagonism is a fundamental ecological process that shapes community composition and maintains ecosystem balance. However, the molecular signals, such as cross-kingdom interactions between bacteria and fungi in natural environments, remain largely unexplored. Lysobacter enzymogenes is a widespread predatory bacterium that produces the antifungal secondary metabolite heat-stable antifungal factor (HSAF), which enables interactions with diverse fungi. Here, we investigated the interkingdom interactions between L. enzymogenes and the environmental fungus Candida krusei, both of which are widely distributed, to elucidate the bacterial-fungal communication. We found that the antagonistic effect of bacteria-fungi was significant under both contact and non-contact co-culture conditions, indicating the involvement of diffusible metabolites. Given that farnesol is a common quorum-sensing (QS) molecule in Candida, metabolite profiling combined with exogenous addition and biosynthesis inhibition experiments demonstrated that farnesol functions as a cross-kingdom signal regulating HSAF production and bacterial antagonism. Further mechanistic analysis of the intrinsic mechanism revealed that the two-component system (TCS) RcsB/C in L. enzymogenes can sense farnesol and activate the production of HSAF through the MarR family regulators, mediating the antagonistic pathway. In addition, our work identified the key amino acid residues in RcsC of L. enzymogenes responsible for recognizing farnesol. In summary, we report the bacterial TCS involved in farnesol sensing and reveal a novel bacterial-fungal antagonistic mechanism, in which L. enzymogenes “hijacks” a fungal QS molecule to enhance its antifungal capacity, uncovering a previously unrecognized strategy of cross-kingdom communication.
✓ 已验证
DOI
PubMed
高影响因子
危险品化学品经营许可证（带存储）