Applied and Environmental Microbiology最新文献

{"title":"Assembly and engineering of BioBricks to develop an efficient NADH regeneration system.","authors":"Feng Cheng, Cheng-Jiao Wang, Xiao-Xiao Gong, Ke-Xiang Sun, Xi-Hang Liang, Ya-Ping Xue, Yu-Guo Zheng","doi":"10.1128/aem.01041-24","DOIUrl":"10.1128/aem.01041-24","url":null,"abstract":"<p><p>The cofactor regeneration system plays a crucial role in redox biocatalysis for organic synthesis and the pharmaceutical industry. The alcohol dehydrogenase (ADH)-based regeneration system offers a promising solution for the <i>in situ</i> regeneration of NAD(P)H. However, its widespread use is hindered by low activity and poor expression of ADH in <i>Escherichia coli</i>. Herein, the BioBricks (promoter, ribosome binding site [RBS], functional gene, and terminator) were assembled and engineered to constitute an efficient NADH regeneration system. The semi-rational design was employed to enhance the catalytic efficiency of <i>Gst</i>ADH (an ADH from <i>Geobacillus stearothermophilus</i>), resulting in a beneficial <i>Gst</i>ADH variant with a 2.1-fold increase in catalytic efficiency. Furthermore, the RBS optimization was used to increase the expression of ADH genes, leading to the identification of an RBS with a 3.2-fold increased translation rate. Using this developed system, the NADH generating velocity reached more than 2 s^-1 even toward 0.1 mM NAD⁺, indicating that it is the most promising NADH regeneration so far. Finally, the engineered system was utilized for the asymmetric biosynthesis of l-phosphinothricin (a chiral herbicide), with a high yield (>95%).</p><p><strong>Importance: </strong>The alcohol dehydrogenase (ADH)-based coenzyme regeneration system serves as a useful tool in redox biocatalysis. This system effectively replenishes NAD(P)H by utilizing isopropanol as a substrate, with the added advantage of easily separable acetone as a by-product. Previous studies focused on discovering new <i>adh</i> genes and engineering the ADH protein for higher catalytic efficiency, neglecting the optimization of other gene components. In this study, a remarkably efficient NADH regeneration system was developed using BioBricks assembly for system initialization. The ADH engineering was used to enhance catalytic efficiency, and RBS optimization for elevated ADH expression, which resulted in not only a 2.1-fold increase in catalytic efficiency but also a 3.2-fold increase in translation rate. Together, these improvements resulted in an overall 6.7-fold enhancement in performance. This system finds application in a wide range of NADH-dependent biocatalysis processes and is particularly advantageous for the biosynthesis of fine chemicals.</p>","PeriodicalId":8002,"journal":{"name":"Applied and Environmental Microbiology","volume":" ","pages":"e0104124"},"PeriodicalIF":3.9,"publicationDate":"2025-01-31","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11784351/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142806079","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Single-cell phenotyping of extracellular electron transfer via microdroplet encapsulation.","authors":"Gina Partipilo, Emily K Bowman, Emma J Palmer, Yang Gao, Rodney S Ridley, Hal S Alper, Benjamin K Keitz","doi":"10.1128/aem.02465-24","DOIUrl":"10.1128/aem.02465-24","url":null,"abstract":"<p><p>Electroactive organisms contribute to metal cycling, pollutant removal, and other redox-driven environmental processes via extracellular electron transfer (EET). Unfortunately, developing genotype-phenotype relationships for electroactive organisms is challenging because EET is necessarily removed from the cell of origin. Microdroplet emulsions, which encapsulate individual cells in aqueous droplets, have been used to study a variety of extracellular phenotypes but have not been applied to investigate EET. Here, we describe the development of a microdroplet emulsion system to sort and enrich EET-capable organisms from complex populations. We validated our system using the model electrogen <i>Shewanella oneidensis</i> and described the tooling of a benchtop microfluidic system for oxygen-limited conditions. We demonstrated the enrichment of strains exhibiting electroactive phenotypes from mixed wild-type and EET-deficient populations. As a proof-of-concept application, we collected samples from iron sedimentation in Town Lake (Austin, TX) and subjected them to microdroplet enrichment. We measured an increase in electroactive organisms in the sorted population that was distinct compared to a population growing in bulk culture with Fe(III) as the sole electron acceptor. Finally, two bacterial species not previously shown to be EET-capable, <i>Cronobacter sakazakii</i> and <i>Vagococcus fessus</i>, were further cultured and characterized for electroactivity. Our results demonstrate the utility of microdroplet emulsions for isolating and identifying EET-capable bacteria.IMPORTANCEThis work outlines a new high-throughput method for identifying electroactive bacteria from mixed populations. Electroactive bacteria play key roles in iron trafficking, soil remediation, and pollutant degradation. Many existing methods for identifying electroactive bacteria are coupled to microbial growth and fitness-as a result, the contributions from weak or poor-growing electrogens are often muted. However, extracellular electron transfer (EET) has historically been difficult to study in high-throughput in a mixed population since extracellular reduction is challenging to trace back to the parent cell and there are no suitable fluorescent readouts for EET. Our method circumvents these challenges by utilizing an aqueous microdroplet emulsion wherein a single cell is statistically isolated in a pico- to nano-liter-sized droplet. Then, via fluorescence obtained from copper reduction, the mixed population can be fluorescently sorted and gated by performance. Utilizing our technique, we characterize two previously unrecognized weak electrogens <i>Vagococcus fessus</i> and <i>Cronobacter sakazakii</i>.</p>","PeriodicalId":8002,"journal":{"name":"Applied and Environmental Microbiology","volume":" ","pages":"e0246524"},"PeriodicalIF":3.9,"publicationDate":"2025-01-31","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11784080/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142977346","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Microbiome metabolic capacity is buffered against phylotype losses by functional redundancy.","authors":"Kayla Cross, Noelle Beckman, Benjamin Jahnes, Zakee L Sabree","doi":"10.1128/aem.02368-24","DOIUrl":"https://doi.org/10.1128/aem.02368-24","url":null,"abstract":"<p><p>Many animals contain a species-rich and diverse gut microbiota that likely contributes to several host-supportive services that include diet processing and nutrient provisioning. Loss of microbiome taxa and their associated metabolic functions as result of perturbations may result in loss of microbiome-level services and reduction of metabolic capacity. If metabolic functions are shared by multiple taxa (i.e., functional redundancy), including deeply divergent lineages, then the impact of taxon/function losses may be dampened. We examined to what degree alterations in phylotype diversity impact microbiome-level metabolic capacity. Feeding two nutritionally imbalanced diets to omnivorous <i>Periplaneta americana</i> over 8 weeks reduced the diversity of their phylotype-rich gut microbiomes by ~25% based on 16S rRNA gene amplicon sequencing, yet PICRUSt2-inferred metabolic pathway richness was largely unaffected due to their being polyphyletic. We concluded that the nonlinearity between taxon and metabolic functional losses is due to microbiome members sharing many well-characterized metabolic functions, with lineages remaining after perturbation potentially being capable of preventing microbiome \"service outages\" due to functional redundancy.</p><p><strong>Importance: </strong>Diet can affect gut microbiome taxonomic composition and diversity, but its impacts on community-level functional capabilities are less clear. Host health and fitness are increasingly being linked to microbiome composition and further modeling of the relationship between microbiome taxonomic and metabolic functional capability is needed to inform these linkages. Invertebrate animal models like the omnivorous American cockroach are ideal for this inquiry because they are amenable to various diets and provide high replicates per treatment at low costs and thus enabling rigorous statistical analyses and hypothesis testing. Microbiome taxonomic composition is diet-labile and diversity was reduced after feeding on unbalanced diets (i.e., post-treatment), but the predicted functional capacities of the post-treatment microbiomes were less affected likely due to the resilience of several abundant taxa surviving the perturbation as well as many metabolic functions being shared by several taxa. These results suggest that both taxonomic and functional profiles should be considered when attempting to infer how perturbations are altering gut microbiome services and possible host outcomes.</p>","PeriodicalId":8002,"journal":{"name":"Applied and Environmental Microbiology","volume":" ","pages":"e0236824"},"PeriodicalIF":3.9,"publicationDate":"2025-01-30","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143063287","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Two NADPH-dependent 2-ketogluconate reductases involved in 2-ketogluconate assimilation in <i>Gluconobacter</i> sp. strain CHM43.","authors":"Sakura Nakashima, Minenosuke Matsutani, Naoya Kataoka, Osao Adachi, Riku Yamashita, Kazunobu Matsushita, Uraiwan Tippayasak, Gunjana Theeragool, Toshiharu Yakushi","doi":"10.1128/aem.02501-24","DOIUrl":"https://doi.org/10.1128/aem.02501-24","url":null,"abstract":"<p><p>Incomplete oxidation of glucose by <i>Gluconobacter</i> sp. strain CHM43 produces gluconic acid and then 2- or 5-ketogluconic acid. Although 2-keto-D-gluconate (2KG) is a valuable compound, it is sometimes consumed by <i>Gluconobacter</i> itself via an unknown metabolic pathway. We anticipated that 2KG reductase (2KGR) would be a key enzyme in 2KG metabolism. <i>GLF_0478</i> and <i>GLF_1777</i> were identified in the genome of strain CHM43, which encode proteins with 70% and 48% amino acid sequence identity, respectively, to the 2KGR of <i>Gluconobacter oxydans</i> strain 621H. Constructed mutant derivatives of strain CHM43 lacking <i>GLF_0478</i>, <i>GLF_1777</i>, or both were examined for their 2KG consumption ability. Strains ∆<i>GLF_0478</i> and ∆<i>GLF_1777</i> consumed 2KG like the parental strain. However, the double-deletion (∆∆) strain did not consume 2KG at all, although it produced 2KG like the parental strain. Strains ∆<i>GLF_0478</i> and ∆<i>GLF_1777</i> each showed decreased 2KGR activity compared with the parental strain, and strain ΔΔ lost 2KGR activity. These results suggest that reduction of 2KG catalyzed by GLF_0478 and GLF_1777 is the committed step in 2KG metabolism in <i>Gluconobacter</i> sp. strain CHM43. The two 2KGRs showed high activity at neutral pH and lower <i>K</i>_M values for NADPH than NADH. Results of induction experiments suggest that GLF_0478 is constitutively expressed at a low level but induced by 2KG, and GLF_1777 is also inducible by 2KG but repressed in the absence of an inducer. Our study that characterizes the key genes for 2KG consumption in <i>Gluconobacter</i> gives insights for improvement of biological 2KG production systems.</p><p><strong>Importance: </strong>2-Keto-D-gluconate (2KG), a product of incomplete oxidation of glucose by acetic acid bacteria including <i>Gluconobacter</i> spp., is used for various purposes, including in the food industry. <i>Gluconobacter</i> also consumes 2KG via an unclear metabolic pathway. It is reported that <i>Pseudomonas</i> spp. and <i>Cupriavidus necator</i> phosphorylate 2KG in the first step of 2KG metabolism, but some enteric bacteria including <i>Escherichia coli</i> reduce 2KG. This study evaluated the 2KG consumption ability of a mutant derivative of a strain of <i>Gluconobacter</i> that lacks two putative 2KGR-encoding genes. The mutant strain did not consume 2KG at all; the two 2KGRs were each found to catalyze 2KG reduction. It is concluded that reduction of 2KG is the committed step in 2KG metabolism in <i>Gluconobacter</i>. The results presented here give insights that might facilitate improvement of 2KG production systems that use <i>Gluconobacter</i>.</p>","PeriodicalId":8002,"journal":{"name":"Applied and Environmental Microbiology","volume":" ","pages":"e0250124"},"PeriodicalIF":3.9,"publicationDate":"2025-01-29","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143057757","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Diversity and dynamics of multiple symbionts contribute to early development of broadcast spawning reef-building coral <i>Dipsastraea veroni</i>.","authors":"Minglan Guo, Lei Jiang, Guowei Zhou, Jiansheng Lian, Xiaolei Yu, Hui Huang","doi":"10.1128/aem.02359-24","DOIUrl":"https://doi.org/10.1128/aem.02359-24","url":null,"abstract":"<p><p>Sexual reproduction and recruitment enhance the genetic diversity and evolution of reef-building corals for population recovery and coral reef conservation under climate change. However, new recruits are vulnerable to physical changes and the mechanisms of symbiosis establishment remain poorly understood. Here, <i>Dipsastraea veroni</i>, a broadcast spawning hermaphrodite reef-building coral, was subjected to settlement and juvenile growth in flow-through <i>in situ</i> seawater at 27.93 ± 0.96°C. Symbiosis of Symbiodiniaceae, bacteria, and/or archaea by horizontal acquisition and/or hypothetical vertical transmission through the mucus with symbionts from the parent appears to be a heritable process of selection and adaptation in <i>D. veroni</i> at the egg, larva, juvenile (5 days post settlement, d p.s. and 32 d p.s.) stages. Symbiodiniaceae was dominated by the genera <i>Cladocopium</i>, <i>Durusdinium</i>, <i>Symbiodinium,</i> with increasing relative abundance of <i>Durusdinium</i> at 5 d p.s. and <i>Symbiodinium</i> at 32 d p.s. Mixed acquisition of the dominant phyla Pseudomonadota, Bacteroidota, Cyanobacteriota, Bacillota, Planctomycetota, and Actinomycetota in egg, larva, and/or juvenile showed a winnowing and regulated bacterial diversity and dynamics, resulting in stage-abundant orders Pseudomonadales and Bacillales in egg and Rhodobacterales, Rhodospirillales, Cyanobacteria, and Cyanobacteriales in larva and/or juvenile. The photoautotrophic Chloroflexales, Cyanobacteriales, and Chlorobiales were abundant in adults. The stable archaeal community contained predominant Crenarchaeota, Halobacterota, Nanoarchaeia Thermoplasmatota, and eight rare phyla, with increased relative abundance of the genera <i>Bathyarchaeota</i>, <i>Candidatus_Nitrosopumilus</i>, <i>Candidatus_Nitrocosmicus</i>, <i>Nitrosarchaeum</i>, <i>Candidatus_Nitrosotenuis</i>, <i>Candidatus_Nitrosopelagicus</i>, <i>Cenarchaeum</i>, <i>Haladaptatus</i>, <i>Halogranum</i>, <i>Halolamina,</i> and <i>Woesearchaeales</i> and <i>GW2011-AR15</i> in juveniles. All results revealed flexible symbiotic mechanisms in <i>D. veroni</i> during early ontogeny for coral survival and evolution.IMPORTANCEFlexible symbioses of Symbiodiniaceae, bacteria, and archaea appear to be a heritable process of selection and adaptation in <i>Dipsastraea veroni</i> in the field, benefiting early coral development and facilitating coral population recovery and reef conversation.</p>","PeriodicalId":8002,"journal":{"name":"Applied and Environmental Microbiology","volume":" ","pages":"e0235924"},"PeriodicalIF":3.9,"publicationDate":"2025-01-29","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143057755","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Spermidine enhances the heat tolerance of <i>Ganoderma lucidum</i> by promoting mitochondrial respiration driven by fatty acid β-oxidation.","authors":"Xiaofei Han, Zi Wang, Lingyan Shi, Ziyang Wei, Jiaolei Shangguan, Liang Shi, Mingwen Zhao","doi":"10.1128/aem.00979-24","DOIUrl":"https://doi.org/10.1128/aem.00979-24","url":null,"abstract":"<p><p>High temperature is an unavoidable environmental stress that generally exerts detrimental effects on organisms and has widespread effects on metabolism. Spermidine is an important member of the polyamines family and is involved in a range of abiotic stress responses in plants. Mitochondria play an essential role in cellular homeostasis and are key components of the stress response. Our results indicated that mitochondrial respiratory intensity increased by 80% in wild-type (WT) under heat stress, but the activities of key enzymes of the tricarboxylic acid (TCA) cycle and electron transport chain (ETC) were significantly reduced upon the knockdown of the spermidine synthase gene (<i>spdS</i>). Furthermore, the content of mitochondrial pyruvate decreased by 36.1%, whereas the levels of free fatty acid increased by 28.8% under heat stress. Upon <i>spdS</i> knockdown, the content of mitochondrial pyruvate was similar to that in the WT, but the medium-chain fatty acid (C6:0) decreased by 68.6%-84.2%, whereas the long-chain fatty acid (C18:2) marginally increased. Subsequent studies demonstrated that spermidine promoted the translation of long chain acyl-CoA dehydrogenase (LCAD) and mitochondrial trifunctional protein (MTP, also known as HADH), thereby enhancing fatty acid β-oxidation under heat stress. In conclusion, spermidine enhances key TCA cycle and ETC enzyme activities and is involved in heat stress-induced fatty acid β-oxidation by promoting the translation of LCAD and HADH, thereby improving the heat tolerance of <i>Ganoderma lucidum</i>.</p><p><strong>Importance: </strong>Polyamines are stress-responsive molecules that enhance the tolerance of plants to multiple abiotic stresses by regulating a variety of biological processes. Our previous research indicated that heat stress induces the the biosynthesis of polyamines and promotes the conversion of putrescine to spermidine in <i>G. lucidum</i>, but the physiological role of elevated spermidine levels is yet to be elucidated. In this study, our findings demonstrated that spermidine enhances the heat tolerance in <i>G. lucidum</i> and that mitochondrial respiration is essential for spermidine-enhanced heat tolerance. This study elucidated a preliminary mechanism by which spermidine enhances heat tolerance of <i>G. lucidum</i> and provided a new insight into the understanding of how microorganisms resist heat stress.</p>","PeriodicalId":8002,"journal":{"name":"Applied and Environmental Microbiology","volume":" ","pages":"e0097924"},"PeriodicalIF":3.9,"publicationDate":"2025-01-29","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143057756","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"A point mutation in a <i>wspF-</i>like gene in <i>Pseudoalteromonas lipolytica</i> enhances the anticorrosion activity.","authors":"Zhenshun Zeng, Dan He, Zhiying Zhao, Tianci He, Qian Li, Yuqi Wang","doi":"10.1128/aem.02154-24","DOIUrl":"https://doi.org/10.1128/aem.02154-24","url":null,"abstract":"<p><p>The protection of steel based on microbial biomineralization has emerged as a novel and eco-friendly strategy for corrosion control. However, the molecular basis of the biomineralization process in mineralization bacteria remains largely unexplored. We previously reported that <i>Pseudoalteromonas lipolytica</i> EPS+ strain provides protection against steel corrosion by forming a hybrid biomineralization film. In this study, we identified that a point mutation in the <i>AT00_08765</i> (<i>wspF-</i>like) gene, responsible for encoding a chemotaxis protein that regulates swimming motility and polysaccharide production, is linked to the observed anticorrosion activity in EPS+ strain. The engineered point mutation mutant strain, designated Δ<i>08765(707A</i>), exhibited similar phenotypes to the EPS+ strain, including colony morphology and cellulose production. Importantly, we demonstrated that moderate swimming motility in Δ<i>08765(707A</i>) plays a pivotal role in the development of a protective mineralization film on the steel surface. Additionally, we found that Δ<i>08765(707A</i>) enhances biofilm formation by rapidly forming small aggregates in the initial stage of biofilm growth. This process facilitated the assembly of more compact and larger mineralization products, effectively inhibiting steel corrosion. In addition, Δ<i>08765(707A</i>) formed a uniform mineralization film that completely covered the steel surface, preventing the formation of sheet-like steel corrosion products. Therefore, this study demonstrates that an engineering strain carrying a point mutation in the <i>AT00_08765</i> gene can significantly enhance the anticorrosion activity. This enhancement is accomplished through the formation of small bacteria-induced aggregates, followed by the development of larger mineralization products and the creation of a uniform organic-inorganic hybrid film.<b>IMPORTANCE</b>In this study, we revealed that moderate swimming motility significantly influences the anticorrosion activity of marine <i>Pseudoalteromonas</i>. Furthermore, our study demonstrated that overproduction of cellulose facilitates cell aggregation rapidly during the initial stages of biofilm formation, thereby promoting the development of larger mineralization products and the formation of a uniform organic-inorganic hybrid film on the steel surface. Our findings provide new insights into the biomineralization mechanisms in <i>Pseudoalteromonas lipolytica</i>, potentially catalyzing the advancement of an eco-friendly microbial-driven approach to marine corrosion prevention.</p>","PeriodicalId":8002,"journal":{"name":"Applied and Environmental Microbiology","volume":" ","pages":"e0215424"},"PeriodicalIF":3.9,"publicationDate":"2025-01-28","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143051403","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"A cytochrome <i>bd</i> repressed by a MarR family regulator confers resistance to metals, nitric oxide, sulfide, and cyanide in <i>Chromobacterium violaceum</i>.","authors":"Bianca B Batista, Vinicius M de Lima, W Ryan Will, Ferric C Fang, José F da Silva Neto","doi":"10.1128/aem.02360-24","DOIUrl":"10.1128/aem.02360-24","url":null,"abstract":"<p><p><i>Chromobacterium violaceum</i> is a ubiquitous environmental pathogen. Despite its remarkable adaptability, little is known about the mechanisms of stress resistance in this bacterium. Here, in a screen for iron-susceptible transposon mutants, we identified a cytochrome <i>bd</i> that protects <i>C. violaceum</i> against multiple stresses. The two subunits of this cytochrome <i>bd</i> (CioAB) are encoded by the <i>cioRAB</i> operon, which also encodes a GbsR-type MarR family transcription factor (CioR). A ∆<i>cioAB</i> mutant strain was sensitive to iron and the iron-requiring antibiotic streptonigrin and showed a decrease in siderophore production. Growth curves and survival assays revealed that the ∆<i>cioAB</i> strain was also sensitive to zinc, hydrogen peroxide, nitric oxide, sulfide, and cyanide. Expression analysis showed that the promoter activity of the <i>cioRAB</i> operon and the transcript levels of the <i>cioAB</i> genes were increased in a ∆<i>cioR</i> mutant. CioR bound the promoter region of the <i>cio</i> operon <i>in vitro</i>, indicating that CioR is a direct repressor of its own operon. Expression of the <i>cio</i> operon increased at high cell density and was dependent on the quorum-sensing regulator CviR. As cyanide is also a signal for <i>cio</i> expression, and production of endogenous cyanide is known to be a quorum sensing-regulated trait in <i>C. violaceum</i>, we suggest that CioAB is a cyanide-insensitive terminal oxidase that allows respiration under cyanogenic growth conditions. Our findings indicate that the cytochrome <i>bd</i> CioAB protects <i>C. violaceum</i> against multiple stress agents that are potentially produced endogenously or during interactions with a host.</p><p><strong>Importance: </strong>The terminal oxidases of bacterial respiratory chains rely on heme-copper (heme-copper oxidases) or heme (cytochrome <i>bd</i>) to catalyze the reduction of molecular oxygen to water. <i>Chromobacterium violaceum</i> is a facultative anaerobic bacterium that uses oxygen and other electron acceptors for respiration under conditions of varying oxygen availability. The <i>C. violaceum</i> genome encodes multiple respiratory terminal oxidases, but their role and regulation remain unexplored. Here, we demonstrate that CioAB, the single cytochrome <i>bd</i> from <i>C. violaceum</i>, protects this bacterium against multiple stressors that are inhibitors of heme-copper oxidases, including nitric oxide, sulfide, and cyanide. CioAB also confers <i>C. violaceum</i> resistance to iron, zinc, and hydrogen peroxide. This cytochrome <i>bd</i> is encoded by the <i>cioRAB</i> operon, which is under direct repression by the MarR-type regulator CioR. In addition, the <i>cioRAB</i> operon responds to quorum sensing and to cyanide, suggesting a protective mechanism of increasing CioAB in the setting of high endogenous cyanide production.</p>","PeriodicalId":8002,"journal":{"name":"Applied and Environmental Microbiology","volume":" ","pages":"e0236024"},"PeriodicalIF":3.9,"publicationDate":"2025-01-24","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143031628","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"A novel FadL family outer membrane transporter is involved in the uptake of polycyclic aromatic hydrocarbons.","authors":"Qiu Meng, Yuxuan Liang, Yinming Xu, Saiyue Li, Haiyan Huang, Yuanyou Xu, Feifei Cao, Jianhua Yin, Tingheng Zhu, Haichun Gao, Zhiliang Yu","doi":"10.1128/aem.00827-24","DOIUrl":"https://doi.org/10.1128/aem.00827-24","url":null,"abstract":"<p><p>Gram-negative bacteria play a pivotal role in the bioremediation of persistent organic pollutants, such as polycyclic aromatic hydrocarbons (PAHs). Because the outer membrane (OM) of these bacteria hinders the direct permeation of hydrophobic substances into the cells, trans-OM proteins are required for the uptake of PAHs. However, neither the characteristics of PAH transporters nor the specific transport mechanism has been well interpreted. In this study, we revealed the participation of a novel FadL family transporter, PadL, in the biodegradation of the representative PAH phenanthrene in <i>Novosphingobium pentaromativorans</i> US6-1, an efficient PAH-degrading bacterium. PadL facilitates the cross-OM transport of phenanthrene, thus upregulating the expression of the gene <i>ahdA1e</i> that is critical to the PAH catabolism. We then showed that hydrophobic amino acid residues in the substrate binding pockets of PadL are essential for the binding of PAHs, such as phenanthrene and benzo[<i>a</i>]pyrene. PadL homologs commonly exist in most of the PAH-degrading species from <i>Sphingomonas</i> and <i>Novosphingobium</i>. The characterization of PadL provided in this study holds significant potential for improving the PAH biodegradation efficiency.</p><p><strong>Importance: </strong>Persistent organic pollutants, including polycyclic aromatic hydrocarbons (PAHs), pose serious threats to human health, and biodegradation has been applied as an efficient strategy for PAH removal. However, due to the high hydrophobicity of PAHs, their uptake is hindered by the bacterial outer membrane, restraining degradation efficiency. The present study reveals the critical roles of a novel FadL family protein (PadL) in the biodegradation of PAHs. PadL specifically transports PAHs such as phenanthrene and benzo[a]pyrene and PadL homologs generally exist in PAH-degrading bacteria of Sphingomonas and Novosphingobium. Our findings fill the knowledge gap in the bacterial trans-membrane uptake process of PAHs and provide a future direction for enhancing the bacterial PAH bioremediation capacity.</p>","PeriodicalId":8002,"journal":{"name":"Applied and Environmental Microbiology","volume":" ","pages":"e0082724"},"PeriodicalIF":3.9,"publicationDate":"2025-01-24","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143031632","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Development of a conditional plasmid for gene deletion in non-model <i>Fusobacterium nucleatum</i> strains.","authors":"Peng Zhou, Bibek G C, Chenggang Wu","doi":"10.1128/aem.01816-24","DOIUrl":"https://doi.org/10.1128/aem.01816-24","url":null,"abstract":"&lt;p&gt;&lt;p&gt;&lt;i&gt;Fusobacterium nucleatum&lt;/i&gt; is an opportunistic pathogen with four subspecies: &lt;i&gt;nucleatum&lt;/i&gt; (FNN), &lt;i&gt;vincentii&lt;/i&gt; (FNV), &lt;i&gt;polymorphum&lt;/i&gt; (FNP), and &lt;i&gt;animalis&lt;/i&gt; (FNA), each with distinct disease potentials. Research on fusobacterial pathogenesis has mainly focused on the model strain ATCC 23726 from FNN. However, this narrow focus may overlook significant behaviors of other FNN strains and those from other subspecies, given the genetic and phenotypic diversity within &lt;i&gt;F. nucleatum&lt;/i&gt;. While ATCC 23726 is highly transformable, most other &lt;i&gt;Fusobacterium&lt;/i&gt; strains exhibit low transformation efficiency, complicating traditional gene deletion methods that rely on non-replicating plasmids. To address this, we developed a conditional plasmid system in which the RepA protein, essential for replication of a pCWU6-based shuttle plasmid, is controlled by an inducible system combining an &lt;i&gt;fdx&lt;/i&gt; promoter with a theophylline-responsive riboswitch. This system allows plasmid replication in host cells upon induction and plasmid loss when the inducer is removed, forcing chromosomal integration via homologous recombination in the presence of the antibiotic thiamphenicol. We validated this approach by targeting the &lt;i&gt;galK&lt;/i&gt; gene, successfully generating mutants in FNN (ATCC 23726, CTI-2), FNP (ATCC 10953), FNA (21_1A), and the closely related species &lt;i&gt;Fusobacterium periodonticum&lt;/i&gt;. Incorporating a &lt;i&gt;sacB&lt;/i&gt; counterselection marker in this conditional plasmid enabled the deletion of the &lt;i&gt;radD&lt;/i&gt; gene in non-model strains. Interestingly, while &lt;i&gt;radD&lt;/i&gt; deletion in 23726, 10953, and 21_1A abolished coaggregation with &lt;i&gt;Actinomyces oris&lt;/i&gt;, the CTI-2 mutant retained this ability, suggesting the involvement of other unknown adhesins. This work significantly advances gene deletion in genetically recalcitrant &lt;i&gt;F. nucleatum&lt;/i&gt; strains, enhancing our understanding of this pathogen.IMPORTANCE&lt;i&gt;Fusobacterium nucleatum&lt;/i&gt; is implicated in various human diseases, including periodontal disease, preterm birth, and colorectal cancer, often linked to specific strains and reflecting the species' genetic and phenotypic diversity. Despite this diversity, most genetic research has centered on the model strain ATCC 23726, potentially missing key aspects of other strains' pathogenic potential. This study addresses a critical gap by developing a novel conditional plasmid system that enables gene deletion in genetically recalcitrant strains of &lt;i&gt;F. nucleatum&lt;/i&gt;. We successfully deleted genes in the FNN clinical strain CTI-2, the FNA strain 21_1A, and &lt;i&gt;F. periodonticum&lt;/i&gt; for the first time. Our findings, particularly the varying behavior of the &lt;i&gt;radD&lt;/i&gt; gene production in coaggregation across strains, underscore the complexity of &lt;i&gt;F. nucleatum&lt;/i&gt; and the need for broader genetic studies. This work advances our understanding of &lt;i&gt;F. nucleatum&lt;/i&gt; virulence at the strain level and provides a valuable tool for future bacterial ","PeriodicalId":8002,"journal":{"name":"Applied and Environmental Microbiology","volume":" ","pages":"e0181624"},"PeriodicalIF":3.9,"publicationDate":"2025-01-24","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143031646","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}