A novel endolysin from an Enterococcus faecalis phage and application.

Enterococcus faecalis is the primary species detected in cases of secondary persistent infection resulting from root canal therapy failure. Due to the overuse of antibacterial agents, E. faecalis has developed resistance to these drugs, making it challenging to treat clinical diseases caused by E. faecalis infection. Therefore, there is an urgent need to explore new alternative drugs for treating E. faecalis infections. We aimed to clone and express the genes of phage endolysins, purify the recombinant proteins, and analyze their antibacterial activity, lysis profile, and ability to remove biofilm. The crude enzyme of phage endolysin pEF51 (0.715 mg/mL), derived from phage PEf771 infecting E. faecalis, exhibited superior bacterial inhibitory activity and a broader bactericidal spectrum than its parental phage PEf771. Furthermore, pEF51 demonstrated high efficacy in eliminating E. faecalis biofilm. Therapeutic results of the infected Sprague-Dawley (SD) rat model indicated that among 10 SD rats, only one developed a thoracic peritoneal abscess and splenic peritoneal abscess after 72 h of treatment with pEF51. This suggests that pEF51 could provide protection against E. faecalis infection in SD rats. Based on the 16S rDNA metagenomic data of the intestinal microbial community of SD rats, endolysin pEF51 exerted a certain influence on the diversity of intestinal microorganisms at the genus level. Thus, pEF51 may serve as a promising alternative to antibiotics in the management of E. faecalis infection.

Viral AMGs-driven pentose phosphate pathway in natural wetland.

Viruses exist anywhere on earth where there is life, and among them, virus-encoded auxiliary metabolic genes (AMGs) can maintain ecosystem balance and play a major role in the global ecosystem. Although the function of AMGs has been widely reported, the genetic diversity of AMGs in natural ecosystems is still poorly understood. Exploring the genetic diversity of viral community-wide AMGs is essential to gain insight into the complex interactions between viruses and hosts. In this article, we studied the phylogenetic tree, principal co-ordinates analysis (PCoA), α diversity, and metabolic pathways of viral auxiliary metabolism genes involved in the pentose phosphate pathway (PPP) through metagenomics, and the changes of metabolites and genes of host bacteria were further studied by using Pseudomonas mandelii SW-3 and its lytic phage based on metabolic flow and AMGs expression. We found that the viral AMGs in the Napahai plateau wetland were created by a combination of various external forces, which contributed to the rich genetic diversity, uniqueness, and differences of the virus, which promoted the reproduction of offspring and better adaptation to the environment. Overall, this study systematically describes the genetic diversity of AMGs associated with the PPP in plateau wetland ecosystems and further expands the understanding of phage-host unique interactions.

A novel holin from an Enterococcus faecalis phage and application in vitro and in vivo.

Enterococcus faecalis, a conditional pathogenic bacterium, is prevalent in the intestinal, oral, and reproductive tracts of humans and animals, causing a variety of infectious diseases. E. faecalis is the main species detected in secondary persistent infection from root canal therapy failure. Due to the abuse of antibacterial agents, E. faecalis has evolved its resistant ability. Therefore, it is difficult to treat clinical diseases infected by E. faecalis. Exploring new alternative drugs for treating E. faecalis infection is urgent. We cloned and expressed the gene of phage holin, purified the recombinant protein, and analyzed the antibacterial activity, lysis profile, and ability to remove bacterial biofilm. It showed that the crude enzyme of phage holin pEF191 exhibited superior bacterial inhibiting activity and a broader lysis host range compared to the parent phage PEf771. In addition, pEF191 demonstrated high efficacy in eliminating E. faecalis biofilm. The therapeutic results of the Sprague-Dawley (SD) rats model infected showed that pEf191 did not affect SD rats, indicating that pEF191 provided greater protection against E. faecalis infection in SD rats. Based on the 16 S rDNA data of SD rats intestinal microorganism population, holin pEF191 exhibited no impact on the diversity of intestinal microorganisms at the phylum and genus levels and improved the relative abundance of favorable bacteria. Thus, pEF191 may serve as a promising alternative to antibiotics in the management of E. faecalis infection.

Whole genome analysis of Pseudomonas mandelii SW-3 and the insights into low-temperature adaptation.

Pseudomonas mandelii SW-3, isolated from the Napahai plateau wetland, can survive in cold environments. The mechanisms underlying the survival of bacteria in low temperatures and high altitudes are not yet fully understood. In this study, the whole genome of SW-3 was sequenced to identify the genomic features that may contribute to survival in cold environments. The results showed that the genome size of strain SW-3 was 6,538,059 bp with a GC content of 59%. A total of 67 tRNAs, a 34,110 bp prophage sequence, and a large number of metabolic genes were found. Based on 16S rRNA gene phylogeny and average nucleotide identity analysis among P. mandelii, SW-3 was identified as a strain belonging to P. mandelii. In addition, we clarified the mechanisms by which SW-3 survived in a cold environment, providing a basis for further investigation of host-phage interaction. P. mandelii SW-3 showed stress resistance mechanisms, including glycogen and trehalose metabolic pathways, and antisense transcriptional silencing. Furthermore, cold shock proteins and glucose 6-phosphate dehydrogenase may play pivotal roles in facilitating adaptation to cold environmental conditions. The genome-wide analysis provided us with a deeper understanding of the cold-adapted bacterium.

Diversity and composition of active and total bacteria in rhizospheric soil in response to continuous cropping years of Panax notoginseng.

The synchronous research and analysis of total and active soil microbial communities can provide insight into how these communities are impacted by continuous cropping years and pathogen infection. The diversity of total and active bacteria in rhizospheric soil of 2-year-old and 3-year-old healthy and diseased Panax notoginseng can comprehensively reveal the bacterial response characteristics in continuous cropping practice. The results showed that 4916 operational taxonomic units (OTUs) were found in the rhizospheric soil bacterial community of P. notoginseng at the DNA level, but only 2773 OTUs were found at the RNA level. The rhizospheric environment had significant effects on the active and bacterial communities, as indicated by the number of OTUs, Shannon, Chao1, Faith's phylogenetic diversity (Faith's PD), and Simpson's diversity indexes. The DNA level can better show the difference in diversity level before and after infection with root rot. The bacterial Chao1 and Faith's PD diversity indexes of 2-year-old root rot-diseased P. notoginseng rhizospheric soil (D2) were higher than that of healthy plants, while the bacterial Shannon diversity index of 3-year-old root rot-diseased P. notoginseng rhizospheric soil (D3) was the lowest in the total bacteria. Principal coordinate analysis (PCoA) illustrated that the total bacterial species composition changed markedly after root rot disease. There were significant differences in the composition of active bacterial species between the 2-year and 3-year rhizospheres. In conclusion, the total and active edaphic rhizospheric bacterial communities could provide important opportunities to understand the responses of bacteria to continuous cropping of P. notoginseng. Differential responses of total and active edaphic rhizosphere bacterial communities to continuous cropping of Panax notoginseng.

Diversity and function of viral AMGs associated with DNA biosynthesis in the Napahai plateau wetland.

Viruses play an important role in microbial community structure and biodiversity by lysing host cells, and can also affect host metabolic pathways by expressing auxiliary metabolic genes (AMGs). As a unique low-latitude, high-altitude seasonal plateau wetland in China, Napahai has high research value. However, studies on the genetic diversity of AMGs and viruses associated with DNA biosynthesis have not been reported. Based on metagenomics, with the phylogenetic tree, PCoA, and α diversity analysis, we found that three DNA biosynthesis-related viral AMGs (cobS, mazG, and purM) in the Napahai plateau wetland were rich in genetic diversity, uniqueness, and differences compared with other habitats and host sources. Through the KEGG metabolic pathway and metabolic flow analysis of Pseudomonas mandelii (SW-3) and phage (VSW-3), the AMGs (cobS, mazG, and purM) genes of the three related viruses involved in DNA biosynthesis were upregulated and their expression increased significantly. In general, we systematically described the genetic diversity of AMGs associated with DNA biosynthesis in plateau wetland ecosystems and clarified the contribution of viral AMGs in the Napahai plateau wetland to DNA biosynthesis, as well as the changes of metabolites and genes. It further expands the understanding of phage-host interactions, which is of great significance for further revealing the role of viral AMGs in the biological evolution and biogeochemical cycle of wetland ecosystems.

Revealing viral diversity in the Napahai plateau wetland based on metagenomics.

We focused on exploring the diversity of viruses in the Napahai plateau wetland, a unique ecosystem located in Yunnan, China. While viruses in marine environments have been extensively studied for their influence on microbial metabolism and biogeochemical cycles, little is known about their composition and function in plateau wetlands. Metagenomic analysis was employed to investigate the viral diversity and biogeochemical impacts in the Napahai wetland. It revealed that the Caudoviricetes and Malgrandaviricetes class level was the most abundant viral category based on phylogenetic analysis. Additionally, a gene-sharing network highlighted the presence of numerous unexplored viruses and demonstrated their unique characteristics and significant variation within the viral community of the Napahai wetland. Furthermore, the study identified the auxiliary metabolic genes (AMGs). AMGs provide phages with additional functions, such as protection against host degradation and involvement in metabolic pathways, such as the pentose phosphate pathway and DNA biosynthesis. The viruses in the Napahai wetland were found to influence carbon, nitrogen, sulfur, and amino acid metabolism, indirectly contributing to biogeochemical cycling through these AMGs. Overall, the research sheds light on the diverse and unique viral communities in the Napahai plateau wetland and emphasizes the significant roles of viruses in microbial ecology. The findings contribute to a deeper understanding of the characteristics and ecological functions of viral communities in plateau wetland ecosystems.

Dopamine alters phage morphology to exert an anti-infection effect.

Antiviral drug development is important for human health, and the emergence of novel COVID-19 variants has seriously affected human lives and safety. A bacteriophage-a bacterial virus with a small and simple structure-is an ideal experimental candidate for studying the interactions between viruses and their hosts. In this study, the effects and mechanisms of catecholamines on phages were explored, and dopamine (DA) was found to have general and efficient anti-infection effects. A clear dose-dependent effect was observed when different phages were treated with DA, with higher DA concentrations exhibiting stronger anti-phage activity. The half maximal inhibitory concentration values of DA for vB-EcoS-IME167, T4 Phage, and VMY22 were determined as 0.26, 0.12, and 0.73 mg mL-1, respectively. The anti-phage effect of DA increased with treatment duration. In addition, the anti-infection activities of DA against vB-EcoS-IME167, T4 Phage, and VMY22 were increased by 105, 104, and 104 folds compared to that of the control. This ability of DA was observed only in phages and not in the host bacteria. Morphological changes of phages were observed under transmission electron microscopy following their treatment with DA, and considerable changes in adsorption were confirmed via quantitative reverse transcription polymerase chain reaction. These results suggest that the anti-phage effect of DA is primarily due to the destruction of the external structure of the phage. This study, to the best of our knowledge, is the first to report the universal anti-phage infection effect of dopamine, which provides novel information regarding DA and forms a basis for further research and development of antiviral drugs. Moreover, it provides a new perspective for the research about the defense and counter-defense of bacteria and bacteriophages.

Isolation, characterization, and genomic analysis of a novel bacteriophage MA9V-1 infecting Chryseobacterium indologenes: a pathogen of Panax notoginseng root rot.

Chryseobacterium indologenes is one of the primary causative agents of root rot of Panax notoginseng, which significantly affected plant growth and caused economic losses. With the increasing incidence of antibiotic-resistant bacterial phytopathogens, phage therapy has been garnered renewed attention in treating pathogenic bacteria. However, the therapeutic potential of phage therapy on root rot of P. notoginseng has not been evaluated. In this study, we isolated a novel lytic phage MA9V-1 infecting C. indologenes MA9 from sewage and monitored the formation of clear and round plaques with a diameter of approximately 0.5-1.5 mm. Phage MA9V-1 exhibited rapid absorption (>75% in 8 min), a latency period of 20 min, and a burst size of 10 particles per cell. Transmission electron microscopy indicated that the phage MA9V-1 is a new myovirus hosting C. indologenes MA9. Sequencing of phage genomes revealed that phage MA9V-1 contained a linear double-stranded DNA genome of 213,507 bp with 263 predicted open reading frames, including phage structure, host lysing, and DNA polymerase/helicase but no genes of tRNA, virulence, and antibiotic resistance. Our proteomic tree and genomic analysis revealed that phage MA9V-1 shares identity with Sphingomonas phage PAU and Tenacibaculum phage PTm1; however, they also showed apparent differences. Further systemic evaluation using phage therapy experiments on P. notoginseng suggested that phage MA9V-1 can be a potential candidate for effectively controlling C. indologenes MA9 infection. Thus, we have presented a novel approach to solving root rot in P. notoginseng.

[Effect of viral infection on host cell metabolism: a review].

As specialized intracellular parasite, viruses have no ability to metabolize independently, so they completely depend on the metabolic mechanism of host cells. Viruses use the energy and precursors provided by the metabolic network of the host cells to drive their replication, assembly and release. Namely, viruses hijack the host cells metabolism to achieve their own replication and proliferation. In addition, viruses can also affect host cell metabolism by the expression of auxiliary metabolic genes (AMGs), affecting carbon, nitrogen, phosphorus, and sulfur cycles, and participate in microbial-driven biogeochemical cycling. This review summarizes the effect of viral infection on the host's core metabolic pathway from four aspects: cellular glucose metabolism, glutamine metabolism, fatty acid metabolism, and viral AMGs on host metabolism. It may facilitate in-depth understanding of virus-host interactions, and provide a theoretical basis for the treatment of viral diseases through metabolic intervention.

Function and distribution of nitrogen-cycling microbial communities in the Napahai plateau wetland.

Nitrogen is an essential component of living organisms and a major nutrient that limits life on Earth. Until now, freely available nitrogen mainly comes from atmospheric nitrogen, but most organisms rely on bioavailable forms of nitrogen, which depends on the complex network of microorganisms with a wide variety of metabolic functions. Microbial-mediated nitrogen cycling contributes to the biogeochemical cycling of wetlands, but its specific microbial abundance, composition, and distribution need to be studied. Based on the metagenomic data, we described the composition and functional characteristics of microbial nitrogen cycle-related genes in the Napahai plateau wetland. Six nitrogen cycling pathways existed, such as dissimilatory nitrate reduction, denitrification, nitrogen fixation, nitrification, anammox, and nitrate assimilation. Most genes related to the nitrogen cycling in this region come from bacteria, mainly from Proteobacteria and Acidobacteria. Habitat types and nitrogen cycle-related genes largely explained the relative abundance of total nitrogen pathways. Phylogenetic trees were constructed based on nitrogen cycle-related genes from different habitats and sources, combined with PCoA analysis, most of them clustered separately, indicating richness and uniqueness. Some microbial groups seemed to be special or general in the nitrogen cycling. In conclusion, it suggested that microorganisms regulated the N cycling process, and may lead to N loss throughout the wetland, thus providing a basis for further elucidation of the microbial regulation of N cycling processes and the Earth's elemental cycles.

Comprehensive Response of Rhodosporidium kratochvilovae to Glucose Starvation: A Transcriptomics-Based Analysis.

Microorganisms adopt diverse mechanisms to adapt to fluctuations of nutrients. Glucose is the preferred carbon and energy source for yeast. Yeast cells have developed many strategies to protect themselves from the negative impact of glucose starvation. Studies have indicated a significant increase of carotenoids in red yeast under glucose starvation. However, their regulatory mechanism is still unclear. In this study, we investigated the regulatory mechanism of carotenoid biosynthesis in Rhodosporidium kratochvilovae YM25235 under glucose starvation. More intracellular reactive oxygen species (ROS) was produced when glucose was exhausted. Enzymatic and non-enzymatic (mainly carotenoids) antioxidant systems in YM25235 were induced to protect cells from ROS-related damage. Transcriptome analysis revealed massive gene expression rearrangement in YM25235 under glucose starvation, leading to alterations in alternative carbon metabolic pathways. Some potential pathways for acetyl-CoA and then carotenoid biosynthesis, including fatty acid ß-oxidation, amino acid metabolism, and pyruvate metabolism, were significantly enriched in KEGG analysis. Overexpression of the fatty acyl-CoA oxidase gene (RkACOX2), the first key rate-limiting enzyme of peroxisomal fatty acid ß-oxidation, demonstrated that fatty acid ß-oxidation could increase the acetyl-CoA and carotenoid concentration in YM25235. These findings contribute to a better understanding of the overall response of red yeast to glucose starvation and the regulatory mechanisms governing carotenoid biosynthesis under glucose starvation.

Whole genome analysis and cold adaptation strategies of Pseudomonas sivasensis W-6 isolated from the Napahai plateau wetland.

Microbial communities of wetlands play key roles in the earth's ecology and stability. To elucidate the cold adaptation mechanisms of bacteria in plateau wetlands, we conducted comparative genomic analyses of Pseudomonas sivasensis and closely related lineages. The genome of P. sivasensis W-6, a cold-adapted bacterium isolated from the Napahai plateau wetland, was sequenced and analyzed. The genome length was 6,109,123 bp with a G+C content of 59.5%. Gene prediction yielded 5360 protein-coding sequences, 70 tRNAs, 24 gene islands, and 2 CRISPR sequences. The isolate contained evidence of horizontal gene transfer events during its evolution. Two prophages were predicted and indicated that W-6 was a lysogen. The cold adaptation of the W-6 strain showed psychrophilic rather than psychrotrophic characteristics. Cold-adapted bacterium W-6 can utilize glycogen and trehalose as resources, associated with carbohydrate-active enzymes, and survive in a low-temperature environment. In addition, the cold-adapted mechanisms of the W-6 included membrane fluidity by changing the unsaturated fatty acid profile, the two-component regulatory systems, anti-sense transcription, the role played by rpsU genes in the translation process, etc. The genome-wide analysis of W-6 provided a deeper understanding of cold-adapted strategies of bacteria in environments. We elucidated the adaptive mechanism of the psychrophilic W-6 strain for survival in a cold environment, which provided a basis for further study on host-phage coevolution.

Microbial-driven carbon fixation in natural wetland.

With the development of global industrialization, carbon neutrality has become an issue that we must be paid attention to. Microorganisms not only have an important impact on the carbon chemical cycle between the Earth's biosphere and biogeography but also play a key role in maintaining the global organic carbon balance. Wetlands are the main reservoir of organic carbon in the mainland of China, and wetland carbon sinks are indispensable for China to achieve the goal of "dual carbon," and China has taken the consolidation and improvement of wetland carbon sink capacity as an important part of the carbon peaking action plan. As a unique low-latitude, high-altitude seasonal plateau wetland in China, Napahai shows high research value. However, the role of microbes in maintaining dissolved organic carbon balance in this area has not been reported. In the study, six carbon fixation genes, accA, aclB, acsA, acsB, cbbL, and rbcL, were analyzed based on metagenomics to elucidate the rich genetic diversity, uniqueness and differences in the Napahai plateau wetland. It was found that the microbial diversity in the Napahai plateau wetland was different from other habitats. In addition, the aclB gene, a rare taxon with high genetic diversity and rich species in the Napahai plateau wetland, played a key role in the microbial metabolic pathway. Finally, the construction of a metabolic pathway through the Kyoto encyclopedia for genes and genomes revealed the contribution of microbes to carbon fixation and the role of microbes in maintaining the organic carbon balance of the Napahai plateau wetland.

Genetic diversity of virus auxiliary metabolism genes associated with phosphorus metabolism in Napahai plateau wetland.

Viruses play important roles in ecosystems by interfering with the central metabolic pathways of the host during infection via the expression of auxiliary metabolic genes (AMGs), altering the productivity of ecosystems and thus affecting geochemical cycling. In this study, the genetic diversity of phosphorus metabolism AMGs phoH, phoU and pstS was investigated by phylogenetic analysis, PCoA analysis, and alpha diversity analysis based on metagenomic data. It was found that the majority of the sequences were unique to Napahai plateau wetland. It was shown that the genetic diversity of phoH, phoU and pstS genes was independent of both habitats and host origins. In addition, the metabolic pathway of AMGs associated with the phosphorus cycling was identified based on metagenomic data. When phosphorus is deficient, virus utilizes AMGs to affect the metabolic pathway, contributing to higher phosphorus levels in the host and facilitating virus survival, replication, and propagation in the host cell.

Mitomycin C enhanced the antitumor efficacy of Rocaglamide in colorectal cancer.

Rocaglamide (ROC), a natural phytochemical isolated from Aglaia species, is a translational inhibitor of de novo c-FLIP synthesis, which relieves the inhibition of c-FLIP dimerization with procasoase-8 and downstream activation. Unfortunately, a lot of cancer cells, especially colorectal cancer cells (CRC), exhibit marked resistance to Rocaglamide-induced cell death. Research has demonstrated that mitomycin C (MMC) has broad-spectrum anti-tumor activity that it can synergize with a wide range of clinical drugs to inhibit tumor growth. The current study investigated whether MMC combined with ROC could sensitize CRC cells with different ROC resistance to apoptosis. HCT116 and HT29, two different CRC cells, were treated with ROC and/or MMC, and the induction of apoptosis, inhibition of cell migration and invasion, arrest of cell cycle, induction of reactive oxygen species, and effects on Bcl-2 family signaling pathway were investigated. The results showed that low concentration of MMC combined with ROC significantly promoted HCT116 and HT29 cell apoptosis and inhibited cell proliferation by downregulating the expression of Bcl-2 and c-FLIP, upregulating the expression of Bax, activating the caspase cascade (involving the mitochondrial apoptosis pathway), arresting cell cycle in G1 phase, and increasing the level of reactive oxygen species (ROS). In addition, the viability and morphology of MRC-5 cells were not significantly affected by the combined treatment with ROC and MMC, indicating its safety. Therefore, it is concluded that the combination treatment of ROC and MMC is a highly effective tumor therapy and may offer a promising therapeutic strategy for the treatment of CRC.

Biogeochemical sulfur cycling of virus auxiliary metabolic genes involved in Napahai plateau wetland.

Virus plays important roles in regulating microbial community structure, horizontal gene transfer, and promoting biological evolution, also augmenting host metabolism during infection via the expression of auxiliary metabolic genes (AMGs), and thus affect biogeochemical cycling in the oceans. As the "kidney of the earth," wetlands have rich biodiversity and abundant resources. Based on metagenomic data, 10 AMGs associated with sulfur cycling, i.e., tusA, moaD, dsrE, soxA, soxB, soxC, soxD, soxX, soxY, and soxZ, were analyzed in Napahai plateau wetland. The phylogenetic trees of AMGs involved in sulfur metabolism from different habitats and host origins were constructed. Combined with principal coordinate analysis, it revealed that most AMGs associated with sulfur metabolism clustered separately, indicating the abundance and uniqueness in this region. The sulfur metabolism pathways involved by AMGs were mainly SOX systems, among which sulfur oxidation was associated with moaD and dsrE genes, while sulfur transport was related to tusA genes. It provides an insight into the biogeochemical sulfur cycling in plateau wetlands and lays the foundation for further study on the co-evolution of virus and host.

Hybrid histidine kinase HisK2301 modulates carotenoid production to counteract cold-induced oxidative stress in Rhodosporidium kratochvilovae YM25235 under low temperature.

Hybrid histidine kinases (HHKs) are major sensor proteins for fungi that contribute to stress tolerance. In the present work, we investigated the roles and mechanisms of the HHK HisK2301 in cold-adapted Rhodosporidium kratochvilovae strain YM25235. The HisK2301 deletion strain was constructed by homologous recombination method and arranged for multiple stress tests. We analysed the content of carotenoid using UV-Vis and HPLC. Relative transcript levels of genes phytoene desaturase (RKCrtI) and phytoene synthase and lycopene cyclase (RKCrtYB) were analysed by RT-qPCR. Intracellular reactive oxygen species (ROS) generation was measured using 2',7'-dichlorodihydrofluorescein diacetate (DCFH-DA). Our results clearly indicated that YM25235 produces γ-carotene, torulene, ß-carotene and torularhodin, with the latter two components strongly related to adapt to cold. HisK2301 is crucial for YM25235 adaptation to different types of stress such as cold, salt, osmotic and oxidative stress. Growth at low temperature clearly induced oxidative stress in YM25235, as more ROS accumulated at cold. During cold stress, HisK2301 is suggested to sense cold-induced ROS signals and then promote carotenoid production partially by RKCrtI and RKCrtYB to scavenge excessive ROS production. Such an inducible protective system may confer YM25235 fast response and better adaptation to cold stress. To conclude, our findings give the first insight into the effect of HisK2301 on carotenoid biosynthesis and cold-induced oxidative stress in fungi under low temperature and suggest the potential use of the cold-adapted HHK HisK2301 in industrial production of carotenoid.

Cd2+ tolerance and removal mechanisms of Serratia marcescens KMR-3.

Heavy metal contamination is a global issue, with cadmium (Cd2+) and its treatment becoming major environmental challenge that could be solved by microbial restoration, an eco-friendly technique. Serratia marcescens KMR-3 exhibits high tolerance and removal rate of Cd2+ (≤500 mg/L). Here, we aimed to explore mechanisms underlying tolerance to and removal of Cd2+ by KMR-3. Scanning electron microscopy, X-ray photoelectron spectroscopy, and Fourier transform infrared spectrometry were conducted to analyze characteristics of the KMR-3 biofilm and Cd2+ combined forms. The results revealed varying degrees of cell adhesion, membrane thickening, and shrinkage on the surface of the bacteria. The binding elements, electronic binding energy, and functional groups on the surface of the bacteria exhibited changes. Furthermore, the biofilm amount following treatment with Cd2+ was 1.5-3 times higher than that in the controls, treatment with Cd2+ substantially enhanced biofilm generation and increased Cd2+ adsorption. Cd2+ adsorption by its own secondary metabolite prodigiosin produced by KMR-3 was enhanced by 19.5 % compared with that observed without prodigiosin. Through transcriptome sequencing and RT-qPCR, we observed that Znu protein-chelating system regulated gene expression (znuA, znuB, and znuC), and the efflux mechanism of the P-type ATPase regulated the expression of genes (zntA, zntB, and zntR), which were significantly enhanced. Through the combined action of various strategies, KMR-3 demonstrated a high tolerance and removal ability of Cd2+, providing a theoretical basis to treat Cd2+ pollution.

Mechanisms underlying the inhibitory effects of Cd2+ on prodigiosin synthesis in Serratia marcescens KMR-3.

Prodigiosin (2-methyl-3-pentyl-6-methoxyprodiginine), a red-colored microbial pigment, is produced in large quantities by Serratia marcescens KMR-3. This bacterium can grow in a medium with a Cd2+ concentration of 500 mg/L, but it does not produce prodigiosin when the Cd2+ concentration in the medium is higher than 140 mg/L. Therefore, we investigated the mechanisms by which Cd2+ inhibits prodigiosin synthesis. Upon addition of Cd2+ to the medium, the expression of the prodigiosin (pig) gene cluster was significantly downregulated. Simultaneously, genes encoding proteins related to the synthesis of arginine and proline(prodigiosin precursors) were significantly downregulated, while the degradation-related genes were upregulated. Furthermore, PigF, which encodes a key enzyme involved in the synthesis of 4-methoxy-2,2'-bipyrrole-5-carboxaldehyde and PigC, which encodes a key enzyme involved in the last step of prodigiosin synthesis, were downregulated by 80% and 55%, respectively, following Cd2+ treatment. As PigC and PigF are located on the cell membrane and are involved in the final steps of prodigiosin synthesis, the cell membrane might be presumed to be the site of prodigiosin synthesis. The bacterial membrane exhibited different degrees of elongation, folding, fragmentation, and sagging after the addition of Cd2+, while likely destroying the site of prodigiosin synthesis.