Huanying Pang

Vibrio alginolyticus is an important opportunistic pathogen in marine aquaculture, causing vibriosis in a wide range of aquatic animals. Lysine 2-hydroxyisobutyrylation (Khib) is an emerging lysine acylation modification involved in regulating metabolism, stress responses, and pathogenicity in microorganisms. However, the global Khib modification profile and its potential biological functions in V. alginolyticus have remained completely unexplored. This study aims to characterize the Khib modification profile of V. alginolyticus and to preliminarily evaluate the role of CobQ as a candidate regulator of Khib homeostasis. A Khib-modified proteome of V. alginolyticus was constructed using affinity enrichment combined with LC-MS/MS-based modified proteomics. Bioinformatics analyses (GO, COG, KEGG, subcellular localization, motif, and protein–protein interaction) explored the functions of Khib-modified proteins. Selected proteins were analyzed by immunoprecipitation combined with Western blot. A cobQ deletion mutant strain was constructed to assess the association between cobQ deletion and global Khib levels, and to evaluate selected physiological traits. A total of 15,195 Khib sites were identified in 2848 proteins, indicating widespread distribution of Khib modification. Khib-modified proteins were mainly associated with metabolic processes, genetic information processing, signal transduction, membrane biogenesis, and secretion-related pathways. Motif analysis revealed conserved amino acid patterns surrounding Khib sites. Immunoprecipitation combined with Western blot confirmed the Khib modification of TpiA and Ndk at the protein level, but did not validate the specific LC-MS/MS sites. Deletion of cobQ was associated with increased global Khib levels, suggesting that CobQ may be associated with Khib modification in vivo. In vitro assays showed no statistically significant de-Khib activity of CobQ, although a trend toward lower Khib levels was observed. Phenotypic assays showed that cobQ deletion did not significantly affect growth or extracellular protease activity, but reduced swimming motility at low agar concentrations. This study provides a comprehensive catalog of putative Khib-modified proteins of V. alginolyticus. Our findings suggest that CobQ may be associated with global Khib levels, but its direct enzymatic activity and physiological substrates remain to be established. These findings provide a foundational resource for understanding Khib-mediated regulation in V. alginolyticus and identify candidate targets for future mechanistic studies.

Although numerous studies have focused on the potential application of heterotrophic nitrification–aerobic denitrification (HNAD) bacteria in wastewater treatment, research exploring their potential in aquaculture biofloc systems remains limited. In this study, a promising HNAD strain, identified as Stutzerimonas stutzeri MJ20, was isolated from mature biofloc. This strain efficiently utilized low-cost carbon sources (e.g., glucose) and small-molecule carbon sources (e.g., sodium acetate and sodium succinate). Under conditions with glucose as the carbon source, a carbon-to-nitrogen (C/N) ratio of 15, pH 6–9, temperature 25–35 °C, salinity 0–35‰, and shaker speed of 0–150 rpm, it achieved removal rates of 95–100% for NH₄⁺-N, NO₂⁻-N, and NO₃⁻-N at initial concentrations of 100 mg/L each. Even at higher concentrations (up to 200 mg/L NH₄⁺-N and 500 mg/L for both NO₂⁻-N and NO₃⁻-N), removal rates exceeded 99%. Under mixed nitrogen sources, strain MJ20 demonstrated efficient nitrogen removal, preferentially utilizing NH₄⁺-N, with only minimal and transient accumulation of nitrite and nitrate. Genomic analysis revealed that MJ20 carries key denitrification genes, including napA, nirS, norB and nosZ, and possesses complete pathways for nitrate reduction to nitrogen gas and ammonia assimilation, although typical autotrophic nitrification genes were not detected. Combined genomic data and autotrophic culture experiments indicated that, in addition to utilizing various organic carbon sources, the strain also exhibited certain autotrophic growth capabilities. Furthermore, MJ20 showed strong flocculation ability (flocculation rate > 96% within 16 h), sensitivity to multiple common antibiotics, and no toxicity to zebrafish, demonstrating favorable biosafety. In simulated seawater aquaculture wastewater with a C/N ratio of 5, it achieved a total nitrogen removal rate exceeding 94% within 72 h. These results indicate that strain MJ20 possesses comprehensive advantages, including efficient nitrogen removal, broad carbon source adaptability, strong environmental resilience, minimal accumulation of intermediate nitrogen products, excellent flocculation ability, and high biosafety. These traits highlight its potential for application in biofloc systems and in treating aquaculture tail water with a low C/N ratio. This study provides theoretical insights and practical guidance for screening HNAD bacteria suitable for biofloc systems.

Vibrio alginolyticus is a common pathogenic bacterium and can cause diseases in aquaculture animals. Lysine lactylation (Kla) is a novel post-translational modification (PTM) that has been confirmed to play critical roles in key biological processes. However, the modification landscape and functions of Kla in V. alginolyticus remain unclear. In this study, lactylation modification profiles of the bacterial pathogen V. alginolyticus were first systematically characterized; a total of 9308 lactylation sites on 2155 proteins were successfully identified. The lactylation of cAMP receptor protein (CRP) and triosephosphate isomerase (TPI) was verified by Co-immunoprecipitation (Co-IP) and Western blot to validate the lactylome data. Bioinformatic analysis of the Kla sites revealed 32 conserved sequence motifs surrounding the modified residues. Kla proteins were mainly involved in central metabolic pathways, including glycolysis/gluconeogenesis and ribosome biogen regulators were found to contain Kla modification sites. To investigate crosstalk among lysine acylations in V. alginolyticus, we integrated Kla, lysine acetylation (Kac), and lysine succinylation (Ksuc) profiles and identified 337 co-modified proteins and 5 co-modified sites. Additionally, phylogenetic analysis of Vibrio alginolyticus CobQ based on protein sequence alignment revealed no homology to the known delactylase CobB. Combined in vitro and in vivo functional validation identified VaCobQ as a candidate delactylase with potential NAD⁺-independent activity. This study establishes a lysine lactylation landscape in V. alginolyticus, providing a resource for exploring Kla functions in bacterial metabolism and its possible connections to virulence.

Research on heterotrophic bioflocs is extensive, whereas investigations into autotrophic bioflocs remain limited. This study established an in situ autotrophic biofloc (ABF) system for intensive Pacific white shrimp (Penaeus vannamei) farming, aiming for zero water exchange and optimized water quality. A 120-day indoor experiment tested three stocking densities (300 (T1), 250 (T2), and 200 shrimp per m³ (T3)) with no water exchange. Water quality was monitored every two days, and bacterial communities were analyzed on days 10 and 70. The results indicated that ABF maturation was achieved by day 70 across all treatments, marked by three key indicators: (1) synchronous declines in nitrite and nitrate concentrations; (2) concurrent decreases in pH and total alkalinity approaching maturation; and (3) sustained high nitrogen removal efficiency (nitrite < 0.7 mg/L, ammonia < 0.6 mg/L). All density groups displayed similar patterns in both water quality dynamics and microbial community evolution. Bacterial analysis revealed that dominant genera such as Ruegeria, Bacillus, Muricauda, SM1A02, and Nitrospira played critical roles in toxic nitrogen removal, while pathogenic Klebsiella and Vibrio significantly decreased post-maturation. Heterotrophic nitrification and aerobic denitrification microorganisms (HNADMs) were identified as potentially responsible for nitrite accumulation. Nitrite accumulation was found in all groups. T2 and T3 achieved satisfactory breeding performance despite pre-maturation nitrate peaks exceeding 40 mg/L, whereas T1 suffered a low survival rate (27.47%) due to severe nitrite accumulation (>50 mg/L). A biofloc volume (BFV) of 4–8 mL/L effectively managed daily feed inputs of 75–110 g/m³. These findings lay a theoretical and technical foundation for the application of in situ ABF cultivation in intensive farming and enhance the sustainability of aquaculture.

The bacterial flagellum plays a crucial role in pathogenesis. However, the mechanism by which the flagellum interferes with host energy metabolism remains unclear. In this study, we confirmed that deletion of the fliC gene resulted in a 30% reduction in the virulence of Pseudomonas plecoglossicida against the large yellow croaker (Larimichthys crocea). Compared to the wild-type strain (WT) infection group, the ΔfliC infection group exhibited a 29.54% decrease in the number of vacuolar degeneration hepatocytes and a 50.83% higher liver glycogen content. Furthermore, infection led to decreased mitochondrial complex V activity, a reduced NAD+/NADH ratio, lower levels of reduced glutathione (GSH), and increased lipid peroxide levels; however, these metabolic perturbations were significantly ameliorated in the ΔfliC group compared to the WT group. Proteomic analysis revealed that the dysregulation of the complement cascade and core carbon metabolic pathways observed in the WT infection group liver was significantly alleviated in the ΔfliC infection group. Additionally, in the ΔfliC infection group, pro-inflammatory genes (such as Tlr5, Tnfα, and Il1β) were downregulated, while lipid metabolism-related genes (such as Acox1, Cpt1a, and Pparα) were upregulated, suggesting the suppression of the Tlr5/NF-κB immune signaling axis and enhanced fatty acid β-oxidation. Collectively, fliC may mediate the disruption of host glucose and lipid metabolism homeostasis through a Tlr5-triggered immunometabolic regulatory axis. In conclusion, this study demonstrates that bacterial flagella modulate host energy metabolism, expanding our understanding of flagellum-mediated pathogenesis.

Vibrio harveyi, a pathogenic vibrio, is ubiquitous and the most prevalent disease infecting tropical and subtropical mariculture animals in marine and estuarine environments. It presents a major risk to mariculture companies worldwide and can cause serious disease problems in aquaculture. Recent studies have shown that various pathogens employ post-translational modifications (PTMs) to regulate cellular processes. One of the major PTMs is lysine succinylation, which is widespread in eukaryotic and prokaryotic cells. Many basic biological functions of bacteria are associated with the regulation of lysine (K) succinylation (Ksuc). However, little is known about the role of lysine succinylation in V. harveyi pathogenesis. Here, we performed LC-MS/MS analysis of 1271 proteins from V. harveyi to identify 4252 Ksuc modification sites. The modification of S-ribosylhomocysteine lyase (LuxS) and transcription elongation factor GreA proteins by Ksuc was confirmed through immunoprecipitation combined with Western blot, further validating our proteomics results. Bioinformatics study revealed that the identified Ksuc proteins play roles in multiple cellular processes and vital metabolic pathways, including LuxS, biofilm exopolysaccharide biosynthesis protein EpsG, and the general secretory system (Sec systems), and are proteins that regulate bacterial virulence. Generally, this scientific study serves as the basis for additional research on the pathogenic nature of Ksuc in V. harveyi and reveals potential targets that would accelerate the manufacturing of attenuated vaccines.