Metabolic adaptations of Microbacterium sediminis YLB-01 in deep-sea high-pressure environments.

The deep-sea environment is an extremely difficult habitat for microorganisms to survive in due to its intense hydrostatic pressure. However, the mechanisms by which these organisms adapt to such extreme conditions remain poorly understood. In this study, we investigated the metabolic adaptations of Microbacterium sediminis YLB-01, a cold and stress-tolerant microorganism isolated from deep-sea sediments, in response to high-pressure conditions. YLB-01 cells were cultured at normal atmospheric pressure and 28 ℃ until they reached the stationary growth phase. Subsequently, the cells were exposed to either normal pressure or high pressure (30 MPa) at 4 ℃ for 7 days. Using NMR-based metabolomic and proteomic analyses of YLB-01 cells exposed to high-pressure conditions, we observed significant metabolic changes in several metabolic pathways, including amino acid, carbohydrate, and lipid metabolism. In particular, the high-pressure treatment stimulates cell division and triggers the accumulation of UDP-glucose, a critical factor in cell wall formation. This finding highlights the adaptive strategies used by YLB-01 cells to survive in the challenging high-pressure environments of the deep sea. Specifically, we discovered that YLB-01 cells regulate amino acid metabolism, promote carbohydrate metabolism, enhance cell wall synthesis, and improve cell membrane fluidity in response to high pressure. These adaptive mechanisms play essential roles in supporting the survival and growth of YLB-01 in high-pressure conditions. Our study offers valuable insights into the molecular mechanisms underlying the metabolic adaptation of deep-sea microorganisms to high-pressure environments. KEY POINTS: • NMR-based metabolomic and proteomic analyses were conducted on Microbacterium sediminis YLB-01 to investigate the significant alterations in several metabolic pathways in response to high-pressure treatment. • YLB-01 cells used adaptive strategies (such as regulated amino acid metabolism, promoted carbohydrate metabolism, enhanced cell wall synthesis, and improved cell membrane fluidity) to survive in the challenging high-pressure environment of the deep sea. • High-pressure treatment stimulated cell division and triggered the accumulation of UDP-glucose, a critical factor in cell wall formation, in Microbacterium sediminis YLB-01 cells.

Pressure-Regulated Gene Expression and Enzymatic Activity of the Two Periplasmic Nitrate Reductases in the Deep-Sea Bacterium Shewanella piezotolerans WP3.

Shewanella species are widely distributed in marine environments, from the shallow coasts to the deepest sea bottom. Most Shewanella species possess two isoforms of periplasmic nitrate reductases (NAP-α and NAP-ß) and are able to generate energy through nitrate reduction. However, the contributions of the two NAP systems to bacterial deep-sea adaptation remain unclear. In this study, we found that the deep-sea denitrifier Shewanella piezotolerans WP3 was capable of performing nitrate respiration under high hydrostatic pressure (HHP) conditions. In the wild-type strain, NAP-ß played a dominant role and was induced by both the substrate and an elevated pressure, whereas NAP-α was constitutively expressed at a relatively lower level. Genetic studies showed that each NAP system alone was sufficient to fully sustain nitrate-dependent growth and that both NAP systems exhibited substrate and pressure inducible expression patterns when the other set was absent. Biochemical assays further demonstrated that NAP-α had a higher tolerance to elevated pressure. Collectively, we report for the first time the distinct properties and contributions of the two NAP systems to nitrate reduction under different pressure conditions. The results will shed light on the mechanisms of bacterial HHP adaptation and nitrogen cycling in the deep-sea environment.

Comparative Proteomic Analysis of Light-Induced Mycelial Brown Film Formation in Lentinula edodes.

Light-induced brown film (BF) formation by the vegetative mycelium of Lentinula edodes is important for ensuring the quantity and quality of this edible mushroom. Nevertheless, the molecular mechanism underlying this phenotype is still unclear. In this study, a comparative proteomic analysis of mycelial BF formation in L. edodes was performed. Seventy-three protein spots with at least a twofold difference in abundance on two-dimensional electrophoresis (2DE) maps were observed, and 52 of them were successfully identified by matrix-assisted laser desorption/ionization tandem time-of-flight mass spectrometry (MALDI-TOF/TOF/MS). These proteins were classified into the following functional categories: small molecule metabolic processes (39%), response to oxidative stress (5%), and organic substance catabolic processes (5%), followed by oxidation-reduction processes (3%), single-organism catabolic processes (3%), positive regulation of protein complex assembly (3%), and protein metabolic processes (3%). Interestingly, four of the proteins that were upregulated in response to light exposure were nucleoside diphosphate kinases. To our knowledge, this is the first proteomic analysis of the mechanism of BF formation in L. edodes. Our data will provide a foundation for future detailed investigations of the proteins linked to BF formation.

pSW2, a Novel Low-Temperature-Inducible Gene Expression Vector Based on a Filamentous Phage of the Deep-Sea Bacterium Shewanella piezotolerans WP3.

A low-temperature-inducible protein expression vector (pSW2) based on a filamentous phage (SW1) of the deep-sea bacterium Shewanella piezotolerans WP3 was constructed. This vector replicated stably in Escherichia coli and Shewanella species, and its copy number increased at low temperatures. The pSW2 vector can be utilized as a complementation plasmid in WP3, and it can also be used for the production of complex cytochromes with multiple heme groups, which has the potential for application for metal ion recovery or bioremediation. Promoters of low-temperature-inducible genes in WP3 were fused into the vector to construct a series of vectors for enhancing protein expression at low temperature. The maximum green fluorescent protein intensity was obtained when the promoter for the hfq gene was used. The WP3/pSW2 system can efficiently produce a patatin-like protein (PLP) from a metagenomic library that tends to form inclusion bodies in E. coli. The yields of PLP in the soluble fraction were 8.3 mg/liter and 4.7 mg/liter of culture at 4°C and 20°C, respectively. Moreover, the pSW2 vector can be broadly utilized in other Shewanella species, such as S. oneidensis and S. psychrophila.

Transcriptome analysis of candidate genes and signaling pathways associated with light-induced brown film formation in Lentinula edodes.

High-throughput Illumina RNA-seq was used for deep sequencing analysis of the transcriptome of poly(A)+ RNA from mycelium grown under three different conditions: 30 days darkness (sample 118), 80 days darkness (313W), and 30 days darkness followed by 50 days in the light (313C), in order to gain insight into the molecular mechanisms underlying the process of light-induced brown film (BF) formation in the edible mushroom, Lentinula edodes. Of the three growth conditions, BF formation occurred in 313C samples only. Approximately 159.23 million reads were obtained, trimmed, and de novo assembled into 31,511 contigs with an average length of 1,746 bp and an N 50 of 2,480 bp. Based on sequence orientations determined by a BLASTX search against the NR, Swiss-Prot, COG, and KEGG databases, 24,246 (76.9 %) contigs were assigned putative descriptions. Comparison of 313C/118 and 313C/313W expression profiles revealed 3,958 and 5,651 significantly differentially expressed contigs (DECs), respectively. Annotation using the COG database revealed that candidate genes for light-induced BF formation encoded proteins linked to light reception (e.g., WC-1, WC-2, phytochrome), light signal transduction pathways (e.g., two-component phosphorelay system, mitogen-activated protein kinase pathway), and pigment formation (e.g., polyketide synthase, O-methyltransferase, laccase, P450 monooxygenase, oxidoreductase). Several DECs were validated using quantitative real-time polymerase chain reaction. Our report is the first to identify genes associated with light-induced BF formation in L. edodes and represents a valuable resource for future genomic studies on this commercially important mushroom.