Applied and Environmental Microbiology最新文献

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 Periplaneta americana 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.

Importance: 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.

Incomplete oxidation of glucose by Gluconobacter 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 Gluconobacter itself via an unknown metabolic pathway. We anticipated that 2KG reductase (2KGR) would be a key enzyme in 2KG metabolism. GLF_0478 and GLF_1777 were identified in the genome of strain CHM43, which encode proteins with 70% and 48% amino acid sequence identity, respectively, to the 2KGR of Gluconobacter oxydans strain 621H. Constructed mutant derivatives of strain CHM43 lacking GLF_0478, GLF_1777, or both were examined for their 2KG consumption ability. Strains ∆GLF_0478 and ∆GLF_1777 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 ∆GLF_0478 and ∆GLF_1777 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 Gluconobacter sp. strain CHM43. The two 2KGRs showed high activity at neutral pH and lower K_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 Gluconobacter gives insights for improvement of biological 2KG production systems.

Importance: 2-Keto-D-gluconate (2KG), a product of incomplete oxidation of glucose by acetic acid bacteria including Gluconobacter spp., is used for various purposes, including in the food industry. Gluconobacter also consumes 2KG via an unclear metabolic pathway. It is reported that Pseudomonas spp. and Cupriavidus necator phosphorylate 2KG in the first step of 2KG metabolism, but some enteric bacteria including Escherichia coli reduce 2KG. This study evaluated the 2KG consumption ability of a mutant derivative of a strain of Gluconobacter 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 Gluconobacter. The results presented here give insights that might facilitate improvement of 2KG production systems that use Gluconobacter.

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, Dipsastraea veroni, a broadcast spawning hermaphrodite reef-building coral, was subjected to settlement and juvenile growth in flow-through in situ 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 D. veroni at the egg, larva, juvenile (5 days post settlement, d p.s. and 32 d p.s.) stages. Symbiodiniaceae was dominated by the genera Cladocopium, Durusdinium, Symbiodinium, with increasing relative abundance of Durusdinium at 5 d p.s. and Symbiodinium 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 Bathyarchaeota, Candidatus_Nitrosopumilus, Candidatus_Nitrocosmicus, Nitrosarchaeum, Candidatus_Nitrosotenuis, Candidatus_Nitrosopelagicus, Cenarchaeum, Haladaptatus, Halogranum, Halolamina, and Woesearchaeales and GW2011-AR15 in juveniles. All results revealed flexible symbiotic mechanisms in D. veroni 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 Dipsastraea veroni in the field, benefiting early coral development and facilitating coral population recovery and reef conversation.

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 (spdS). 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 spdS 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 Ganoderma lucidum.

Importance: 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 G. lucidum, 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 G. lucidum and that mitochondrial respiration is essential for spermidine-enhanced heat tolerance. This study elucidated a preliminary mechanism by which spermidine enhances heat tolerance of G. lucidum and provided a new insight into the understanding of how microorganisms resist heat stress.

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 Pseudoalteromonas lipolytica EPS+ strain provides protection against steel corrosion by forming a hybrid biomineralization film. In this study, we identified that a point mutation in the AT00_08765 (wspF-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 Δ08765(707A), exhibited similar phenotypes to the EPS+ strain, including colony morphology and cellulose production. Importantly, we demonstrated that moderate swimming motility in Δ08765(707A) plays a pivotal role in the development of a protective mineralization film on the steel surface. Additionally, we found that Δ08765(707A) 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, Δ08765(707A) 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 AT00_08765 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.IMPORTANCEIn this study, we revealed that moderate swimming motility significantly influences the anticorrosion activity of marine Pseudoalteromonas. 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 Pseudoalteromonas lipolytica, potentially catalyzing the advancement of an eco-friendly microbial-driven approach to marine corrosion prevention.

Chromobacterium violaceum 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 bd that protects C. violaceum against multiple stresses. The two subunits of this cytochrome bd (CioAB) are encoded by the cioRAB operon, which also encodes a GbsR-type MarR family transcription factor (CioR). A ∆cioAB 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 ∆cioAB strain was also sensitive to zinc, hydrogen peroxide, nitric oxide, sulfide, and cyanide. Expression analysis showed that the promoter activity of the cioRAB operon and the transcript levels of the cioAB genes were increased in a ∆cioR mutant. CioR bound the promoter region of the cio operon in vitro, indicating that CioR is a direct repressor of its own operon. Expression of the cio operon increased at high cell density and was dependent on the quorum-sensing regulator CviR. As cyanide is also a signal for cio expression, and production of endogenous cyanide is known to be a quorum sensing-regulated trait in C. violaceum, we suggest that CioAB is a cyanide-insensitive terminal oxidase that allows respiration under cyanogenic growth conditions. Our findings indicate that the cytochrome bd CioAB protects C. violaceum against multiple stress agents that are potentially produced endogenously or during interactions with a host.

Importance: The terminal oxidases of bacterial respiratory chains rely on heme-copper (heme-copper oxidases) or heme (cytochrome bd) to catalyze the reduction of molecular oxygen to water. Chromobacterium violaceum is a facultative anaerobic bacterium that uses oxygen and other electron acceptors for respiration under conditions of varying oxygen availability. The C. violaceum genome encodes multiple respiratory terminal oxidases, but their role and regulation remain unexplored. Here, we demonstrate that CioAB, the single cytochrome bd from C. violaceum, protects this bacterium against multiple stressors that are inhibitors of heme-copper oxidases, including nitric oxide, sulfide, and cyanide. CioAB also confers C. violaceum resistance to iron, zinc, and hydrogen peroxide. This cytochrome bd is encoded by the cioRAB operon, which is under direct repression by the MarR-type regulator CioR. In addition, the cioRAB operon responds to quorum sensing and to cyanide, suggesting a protective mechanism of increasing CioAB in the setting of high endogenous cyanide production.

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 Novosphingobium pentaromativorans US6-1, an efficient PAH-degrading bacterium. PadL facilitates the cross-OM transport of phenanthrene, thus upregulating the expression of the gene ahdA1e 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[a]pyrene. PadL homologs commonly exist in most of the PAH-degrading species from Sphingomonas and Novosphingobium. The characterization of PadL provided in this study holds significant potential for improving the PAH biodegradation efficiency.

Importance: 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.

The biosynthesis of mupirocin, a clinically significant antibiotic produced by Pseudomonas sp. NCIMB 10586, is activated by the N-acyl homoserine lactone (AHL) MupR/I quorum sensing (QS) system. However, to date, limited research has focused on the influence of global regulators such as the GacS/A two-component system (TCS) on the MupR/I QS system or mupirocin biosynthesis. In this study, we characterized the regulatory components of the Gac/Rsm transduction system in the mupirocin-producing model strain NCIMB 10586 and investigated their interconnection with the MupR/I QS circuit and subsequent mupirocin biosynthesis. The production of mupirocin was hampered by either gacS inactivation, gacA inactivation, or the double-mutant of the sRNAs ( RsmY and RsmZ). Similarly, the expressions of mupR and mupI, and AHL synthesis significantly decreased in gacS, gacA, or rsmY/Z mutants, indicating that the GacS/A system stimulates mupirocin biosynthesis via the MupR/I QS system. Five CsrA family proteins, RsmA/E/I/F/N, were found in strain NCIMB 10586, and the single and multiple mutants of rsmA/E/I/F/N showed different phenotypes with respect to mupirocin production. Our results revealed that mupirocin biosynthesis was likely to be negatively regulated by RsmA/E/I, but positively regulated by RsmF. Additionally, the RsmF protein was shown to interact with the 5' leader of mupR mRNA. In summary, the Gac/Rsm system positively regulates the biosynthesis of mupirocin mainly through the MupR/I QS system, and the model of the regulatory mechanism is proposed. The elucidation of the Gac/Rsm-MupR/I regulatory pathway could help devise ways for improving mupirocin production through genetic engineering.IMPORTANCEThe Gac/Rsm regulatory system plays a global regulatory role in bacterial physiology and metabolism, including secondary metabolism. Mupirocin is a clinically important antibiotic, produced by Pseudomonas sp. NCIMB 10586, whose biosynthesis is activated by the MupR/I quorum sensing system. Global regulators have important impacts on the gene expression of secondary metabolic gene clusters and QS genes, and the GacS/A two-component system is one of the main regulators across Pseudomonas species, which significantly influences antibiotic production. Our study presented that the expressions of QS genes and mup gene cluster were downregulated in gacS, gacA, or rsmY/Z mutants compared to the wild-type. The inactivation of rsmA/E/I/F/N in NCIMB 10586, encoding CsrA family proteins, showed different regulatory traits of mupirocin production, in which the RsmF protein could interact with the 5' UTR region of mupR mRNA. These findings provide the understanding of the regulatory role of Gac/Rsm on mupirocin biosynthesis and mupR/I QS system and lay foundations for further improving mupirocin production.

The gene mutAW encoding Trichoderma harzianum fungus mutanase (MutA, GH71 family, α-1,3-glucanase, EC 3.2.1.59) was cloned and heterologously expressed by the highly productive Penicillium verruculosum fungus. P. verruculosum MutA strain secreted crude enzyme preparations with the recombinant MutA content of 40% of the total secreted protein, and the specific activity increased 150 folds compared to that of enzyme preparation obtained by the host strain. Homogeneous MutA had molecular mass of 70 kDa and displayed maximum of the activity on mutan at pH 5.0 and 50°C, with K_m and k_cat being 1.0 g/L and 30 s^-1, respectively. At 40-50°C, the MutA was stable for at least 3 h. Glucose was the main product of long-term mutan hydrolysis. HPLC analysis of hydrolysis product of oligo-α-(1→3)-D-glucosides bearing UV-detectable N-trans-cinnamoyl residue in the aglycon clearly indicated that MutA has an endo-processive hydrolytic mode of action. It was demonstrated that MutA can destroy the polysaccharide matrix of both gram-positive and gram-negative pathogenic bacteria biofilms.

Importance: The manuscript describes the properties of a novel recombinant GH71 mutanase Mut A from Trichoderma harzianum. Gene mutAW encoding mutanase was heterologously expressed in the host strain Penicillium verruculosum B1-537 (ΔniaD). The recipient strain has a high secretory ability and allowed to obtain preparations containing the target recombinant enzyme up to 80% of the total protein pool. MutA exhibited a high activity against mutan and negligible or zero activity toward other types of glucans including α-(1→4)-, β-(1→3)-, β-(1→4)-, and β-(1→6)-glucans. By using a series of synthetic oligo-α-(1→3)-D-glucosides, we demonstrated that MutA is an endo-processive enzyme, which hydrolyzes the internal glucosidic bonds and releases glucose from the reducing end sliding into the non-reducing end. MutA recognizes tetrasaccharide as a minimal substrate and hydrolyzes it to trisaccharide and glucose. The effectiveness of the use of MutA for the destruction of clinical isolates of gram-positive and gram-negative bacteria is also described.