IntroductionSpace is a complex environment characterized by a variety of extreme conditions, such as low temperatures, intense radiation, high vacuum, and microgravity. Although space stations are shielded from most extreme conditions, microgravity is still a serious mutagenic factor1. Microgravity is a state of apparent weightlessness that occurs due to the significant reduction in physical forces exerted by gravity; it is typically characterized by a gravity value ranging from ~10−3 to 10−6 g and is accompanied by low-shear and low-turbulence conditions2. Studies have shown that spaceflight can impact astronauts’ lung tissue, cardiovascular system, and immune function3,4,5. Additionally, research has indicated that certain microbes may be introduced into the space environment by humans intentionally or inadvertently. Within enclosed spacecraft, the risk of microbial infection increases the longer astronauts spend in orbit. A variety of opportunistic pathogens, such as Klebsiella pneumoniae (K. pneumoniae), have been detected by post-spaceflight tests both in the astronauts and on the spacecraft6.K. pneumoniae, a gram-negative bacterium, is commonly found on animal mucosal surfaces and in everyday environments such as soil and water. In humans, this bacterium predominantly resides in the gastrointestinal tract but is sometimes present in the nasal and pharyngeal areas. K. pneumoniae can invade the bloodstream or other tissues through the nasal and pharyngeal areas, leading to systemic infections. Consequently, K. pneumoniae is a significant risk factor for severe community-acquired infections in clinical settings7. A prevalent opportunistic pathogen, K. pneumoniae is non-pathogenic under typical conditions; its pathogenicity is conditional upon certain factors, and pathogenic K. pneumoniae is particularly common in individuals with compromised immune systems, often presenting as invasive infections. In the space environment, the immune systems of astronauts are compromised5, increasing the risk of K. pneumoniae infection.In recent years, interest in the genetic traits and mutation mechanisms of microorganisms under microgravity conditions has increased. However, because changes for microorganisms to experience authentic space environments are limited, ground-based simulated microgravity (SMG) systems have been developed8. The Rotary Cell Culture System (RCCS), a widely utilized tool, employs a specialized culture dish—the High-Aspect Rotating Vessel (HARV)—to simulate microgravity accurately on Earth. Properly filled with liquid culture medium and rotated at precise speeds and angles, the HARV effectively mimics microgravity conditions9.Studies have shown that 1 month of spaceflight can alter the biofilm formation ability of Acinetobacter baumannii and the growth rate and drug resistance of Salmonella enteritidis10,11. Furthermore, after SMG induction, genes and proteins involved in diverse metabolic processes and biological pathways were differentially regulated in Stenotrophomonas maltophilia12. However, the study periods have been limited predominantly to 14–30 days, a timeframe that differs markedly from the typical 3–6 month mission duration for astronauts. Certain phenotypic traits of K. pneumoniae, including morphology, growth rate, and biofilm formation capacity, are reportedly altered by short-term SMG exposure13, with SMG-treated strains exhibiting the upregulation of genes associated with type 3 fimbriae14. However, it remains unclear whether these alterations persist under long term SMG conditions. Moreover, RNA sequencing (RNA-seq) has been used to identify differential expressed genes (DEGs) in pathogens under SMG versus control conditions, yet differential expressed metabolites (DEMs) have seldom been characterized13,14,15. Consequently, we cultured a clinical strain of K. pneumoniae under SMG for 56 days and employed multiple experimental techniques to investigate its phenotypic changes. RNA-seq and metabolome sequencing analyses were conducted to elucidate the potential regulatory mechanisms of DEGs and DEMs in K. pneumoniae under SMG.Based on existing research, we aimed to investigate whether the phenotypic characteristics of K. pneumoniae would continue to change under long term SMG induction, and search for genes and metabolites involved in abnormal regulation of K. pneumoniae under SMG. We aimed for our findings to serve as a valuable reference for future research and the development of safer space missions.ResultsChanges in the phenotypic characteristics of K. pneumoniae under SMGFirst, we conducted growth tests on the strains cultured under SMG and NG conditions for the 24-h period of the 56th day (Fig. 1A). The OD600 values revealed that there was a difference in the growth rate of K. pneumoniae between the two environments at 1 h, and the differences in the measured values between the two environments remained non-significant until ~12 h. Notably, between 12 and 18 h, K. pneumoniae experienced a brief growth spurt, during which the differences between the two groups were significant (p 6 μg) was utilized to construct fragment library and sequenced by PacBio.Then, after quality control of the raw reads using Trimmomatic43, ABySS44, canu45, and GapCloser46 were used successively to obtain the final assembly results. And we used ab initio prediction method to get gene models for original strain. Gene models were identified using GeneMark47. Then all gene models were blastp against Non-Redundant Protein Sequence Database (NR), Cluster of Orthologous Groups (COG), Gene Ontology (GO), and Kyoto Encyclopedia of Genes and Genomes (KEGG) to do functional annotation by blastp module. And these annotations serving as references for further analysis.RNA sequencingTotal RNA was extracted from the tissue using TRIzol® Reagent according to the manufacturer’s instructions (Invitrogen), and genomic DNA was removed using DNase I (Takara). The RNA quality was assessed with a 2100 Bioanalyzer (Agilent), and the RNA concentration was measured with an ND-2000 (NanoDrop Technologies). Only high-quality RNA samples were used to construct the sequencing library. For the RNA-seq strand-specific libraries, the TruSeq RNA Sample Preparation Kit from Illumina (San Diego, CA) was utilized, starting with 5 μg of total RNA. rRNA was removed using the RiboZero rRNA Removal Kit (Epicenter), and the RNA was subjected to fragmentation. cDNA synthesis, end repair, A-base addition, and ligation of Illumina-indexed adapters were subsequently performed following Illumina’s protocol. The libraries were then size-selected for cDNA fragments ranging from 200 to 300 bp using a 2% low-range ultra agarose gel, followed by 15 cycles of PCR amplification with Phusion DNA polymerase (NEB). After quantification via TBS380, the paired-end libraries were sequenced on an Illumina NovaSeq 6000 system (150 bp*2, Biozeron, Shanghai, China). Each group had used three samples for sequencing and subsequent transcriptome analysis.Comparative transcriptomic data analysisThe raw paired-end reads were trimmed and quality controlled by Fastp48, and clean reads were aligned against the reference genome using Bowtie 249. The gene-wise read count was determined using HTSeq-count50. DEGs were identified by calculating the expression levels of transcripts with the FPKM method. The edgeR package from R/Bioconductor51 was used to normalize the read counts and analyze differential gene expression. The DEGs between two groups were selected using the following criteria: the logarithmic of fold change (FC) was greater than 2 and the false discovery rate (FDR) should be less than 0.05. To understand the functions of the differential expressed genes, GO functional enrichment and KEGG pathway analysis were carried out by Goatools52 and KOBAS53 respectively. DEGs were significantly enriched in GO terms and metabolic pathways when their Bonferroni-corrected P-value was less than 0.05.GSEA has been extensively applied to identify underlying pathways involved in various conditions54. The samples were classified as high or low according to the expression of K. pneumoniae. NOM P-values 1 and FDR q 1, P-value y/N was satisfied, the metabolic pathway was considered enriched; when the P value of the metabolic pathway was