Bacterial Secretion Systems Research Articles

Inhibition of Xanthomonas growth by bioactive volatiles from Pseudomonas sp. triggers remarkable changes in the phytopathogen transcriptome

Volatile organic compounds (VOCs) produced by microorganisms may have a noteworthy role in the control of plant pathogens. Xanthomonas are a well-studied group of phytobacteria that cause diverse diseases in economically important crops worldwide. Key species that infect sugarcane are X. albilineans (Xab) and X. axonopodis pv. vasculorum (Xav). Here, we investigated VOC-producing bacteria with antagonistic effects against Xab and Xav. We demonstrated that VOCs produced by Pseudomonas sp. V5-S-D11 was able to abolish the growth of these pathogens. A set of 32 VOCs was identified in the volatilome of V5-S-D11, with 10 showing a concentration-dependent inhibitory effect on both phytobacteria. Among them, dimethyl disulfide (DMDS), a volatile sulfur compound, has the potential to be biotechnologically explored in agriculture since it can improve plant growth and induce systemic resistance against plant pathogens. Interestingly, transcriptomic analysis of Xab treated with DMDS revealed several up-regulated metabolic pathways such as a two-component system, flagellar assembly, chemotaxis, and a bacterial secretion system. Although the ethanol (ETOH) used as DMDS solvent did not inhibit XXab growth, it triggered a similar up-regulation of some genes, indicating that this phytopathogen can deal with ETOH better than DMDS. Overall, this study explores the wide role of VOCs in the interactions with bacteria. Moreover, our results indicate that VOCs from Pseudomonas sp. may represent a novel biotechnological strategy to counteract diseases caused by Xanthomonas species and can be further exploited for sustainable approaches in agriculture.

Reconciling plant and microbial ecological strategies to elucidate cover crop effects on soil carbon and nitrogen cycling

Abstract Plant economics, the way plants allocate and utilize resources, affect multiple soil processes through interactions with root and associated microbial communities. However, the interplay between plant economics and microbial ecological strategies remains poorly understood, which is crucial for integrated manipulation of plant‐ and microbe‐mediated functions in mitigating climate change and sustaining soil health. We used a field experiment with 11 cover crop species grown monocultures in the same base soil to test whether microbial ecological strategies are associated with plant economic strategies and if their interactions are linked to soil functions. A principal component analysis (PCA) was performed on root and leaf traits to identify the loadings of cover crop species on the plant trait space. Metagenomic analysis of rhizosphere microbial communities was conducted to infer their ecological strategies based on genetically encoded community‐aggregated traits. We found a synchronous relationship between the conservation gradient of plant economic strategies and the trade‐offs in microbial ecological strategies. Conservative plant strategists, such as Lolium multiflorum, Triticum turgidum and Brassica juncea, fostered microbial communities characterized by high growth yield potentials (Y‐strategies). This included increased microbial carbon fixation pathways, citrate cycle, ribosome and valine, leucine and isoleucine biosynthesis. As a result, microbial metabolic efficiency improved, shown by higher microbial biomass carbon content and a lower metabolic quotient (qCO2), led to enhanced soil organic carbon accumulation. In comparison, acquisitive plants like Astragalus sinicus, Vicia villosa, Trifolium incarnatum and Medicago sativa stimulated microbial resource‐acquisition strategies (A‐strategies). This included enhanced bacterial chemotaxis, secretion systems, biotin metabolism and cell motility pathways, which in turn increased soil exoenzyme activity and accelerated soil nitrogen mineralization. Consequently, these species enhanced soil nitrogen availability and had substantial feedbacks on subsequent main crop productivity. Synthesis. This study demonstrates how plant economic strategies influence the balance between different microbial ecological strategies, specifically the trade‐offs in Y‐ and A‐strategies. These interactions exert control over carbon and nitrogen dynamics in the soil ecosystem. The findings provide insights for implementing nature‐based solutions to improve agroecosystem management practices.

Structural basis for synthase activation and cellulose modification in the E. coli Type II Bcs secretion system

Bacterial cellulosic polymers constitute a prevalent class of biofilm matrix exopolysaccharides that are synthesized by several types of bacterial cellulose secretion (Bcs) systems, which include conserved cyclic diguanylate (c-di-GMP)-dependent cellulose synthase modules together with diverse accessory subunits. In E. coli, the biogenesis of phosphoethanolamine (pEtN)-modified cellulose relies on the BcsRQABEFG macrocomplex, encompassing inner-membrane and cytosolic subunits, and an outer membrane porin, BcsC. Here, we use cryogenic electron microscopy to shed light on the molecular mechanisms of BcsA-dependent recruitment and stabilization of a trimeric BcsG pEtN-transferase for polymer modification, and a dimeric BcsF-dependent recruitment of an otherwise cytosolic BcsE2R2Q2 regulatory complex. We further demonstrate that BcsE, a secondary c-di-GMP sensor, can remain dinucleotide-bound and retain the essential-for-secretion BcsRQ partners onto the synthase even in the absence of direct c-di-GMP-synthase complexation, likely lowering the threshold for c-di-GMP-dependent synthase activation. Such activation-by-proxy mechanism could allow Bcs secretion system activity even in the absence of substantial intracellular c-di-GMP increase, and is reminiscent of other widespread synthase-dependent polysaccharide secretion systems where dinucleotide sensing and/or synthase stabilization are carried out by key co-polymerase subunits.

Whole Cell Luminescence-Based Screen for Inhibitors of the Bacterial Sec Machinery.

There is a pressing need for new antibiotics to combat rising resistance to those already in use. The bacterial general secretion (Sec) system has long been considered a good target for novel antimicrobials thanks to its irreplacable role in maintaining cell envelope integrity, yet the lack of a robust, high-throughput method to screen for Sec inhibition has so far hampered efforts to realize this potential. Here, we have adapted our recently developed in vitro assay for Sec activity─based on the split NanoLuc luciferase─to work at scale and in living cells. A simple counterscreen allows compounds that specifically target Sec to be distinguished from those with other effects on cellular function. As proof of principle, we have applied this assay to a library of 5000 compounds and identified a handful of moderately effective in vivo inhibitors of Sec. Although these hits are unlikely to be potent enough to use as a basis for drug development, they demonstrate the efficacy of the screen. We therefore anticipate that the methods presented here will be scalable to larger compound libraries, in the ultimate quest for Sec inhibitors with clinically relevant properties.

Multi-omics analysis of excessive nitrogen fertilizer application: Assessing environmental damage and solutions in potato farming

Potatoes (Solanum tuberosum L.) are the third largest food crop globally and are pivotal for global food security. Widespread N fertilizer waste in potato cultivation has caused diverse environmental issues. This study employed microbial metagenomic sequencing to analyze the causes behind the declining N use efficiency (NUE) and escalating greenhouse gas emissions resulting from excessive N fertilizer application. Addressing N fertilizer inefficiency through breeding has emerged as a viable solution for mitigating overuse in potato cultivation. In this study, transcriptome and metabolome analyses were applied to identify N fertilizer-responsive genes. Metagenomic sequencing revealed that excessive N fertilizer application triggered alterations in the population dynamics of 11 major bacterial phyla, consequently affecting soil microbial functions, particularly N metabolism pathways and bacterial secretion systems. Notably, the enzyme levels associated with NO3- increased, and those associated with NO and N2O increased. Furthermore, excessive N fertilizer application enhanced soil virulence factors and increased potato susceptibility to diseases. Transcriptome and metabolome sequencing revealed significant impacts of excessive N fertilizer use on lipid and amino acid metabolism pathways. Weighted gene co‑expression network analysis (WGCNA) was adopted to identify two genes associated with N fertilizer response: PGSC0003DMG400021157 and PGSC0003DMG400009544.

Potential Convergence to Accommodate Pathogenicity Determinants and Antibiotic Resistance Revealed in Salmonella Mbandaka.

Salmonella species are causal pathogens instrumental in human food-borne diseases. The pandemic survey related to multidrug resistant (MDR) Salmonella genomics enables the prevention and control of their dissemination. Currently, serotype Mbandaka is notorious as a multiple host-adapted non-typhoid Salmonella. However, its epidemic and MDR properties are still obscure, especially its genetic determinants accounting for virulence and MD resistance. Here, we aim to characterize the genetic features of a strain SMEH pertaining to Salmonella Mbandaka (S. Mbandaka), isolated from the patient's hydropericardium, using cell infections, a mouse model, antibiotic susceptibility test and comparative genomics. The antibiotic susceptibility testing showed that it could tolerate four antibiotics, including chloramphenicol, tetracycline, fisiopen and doxycycline by Kirby-Bauer (K-B) testing interpreted according to the Clinical and Laboratory Standards Institute (CLSI). Both the reproducibility in RAW 264.7 macrophages and invasion ability to infect HeLa cells with strain SMEH were higher than those of S. Typhimurium strain 14028S. In contrast, its attenuated virulence was determined in the survival assay using a mouse model. As a result, the candidate genetic determinants responsible for antimicrobial resistance, colonization/adaptability and their transferability were comparatively investigated, such as bacterial secretion systems and pathogenicity islands (SPI-1, SPI-2 and SPI-6). Moreover, collective efforts were made to reveal a potential role of the plasmid architectures in S. Mbandaka as the genetic reservoir to transfer or accommodate drug-resistance genes. Our findings highlight the essentiality of antibiotic resistance and risk assessment in S. Mbandaka. In addition, genomic surveillance is an efficient method to detect pathogens and monitor drug resistance. The genetic determinants accounting for virulence and antimicrobial resistance underscore the increasing clinical challenge of emerging MDR Mbandaka isolates, and provide insights into their prevention and treatment.

A coordinated attack by a bacterial secretion system and a small molecule drives prey specificity

Vibrio species are recognized for their role in food- and water-borne diseases in humans, fish, and aquatic invertebrates. We screened bacterial strains isolated from raw food shrimp for those that are bactericidal to Vibrio strains. Here we identify and characterize Aeromonas dhakensis strain A603 which shows robust bactericidal activity specifically towards Vibrio and related taxa but less potency toward other Gram-negative species. Using the A603 genome and genetic analysis, we show that two antibacterial mechanisms account for its vibriocidal activity -- a highly potent Type Six Secretion System (T6SS) and biosynthesis of a vibriocidal phenazine-like small molecule, named here as Ad-Phen. Further analysis indicates coregulation between Ad-Phen and a pore-forming T6SS effector TseC, which potentiates V. cholerae to killing by Ad-Phen.

BolA-like protein (IbaG) promotes biofilm formation and pathogenicity of Vibrio parahaemolyticus.

Vibrio parahaemolyticus is a gram-negative halophilic bacterium widespread in temperate and tropical coastal waters; it is considered to be the most frequent cause of Vibrio-associated gastroenteritis in many countries. BolA-like proteins, which reportedly affect various growth and metabolic processes including flagellar synthesis in bacteria, are widely conserved from prokaryotes to eukaryotes. However, the effects exerted by BolA-like proteins on V. parahaemolyticus remain unclear, and thus require further investigation. In this study, our purpose was to investigate the role played by BolA-like protein (IbaG) in the pathogenicity of V. parahaemolyticus. We used homologous recombination to obtain the deletion strain ΔibaG and investigated the biological role of BolA family protein IbaG in V. parahaemolyticus. Our results showed that IbaG is a bacterial transcription factor that negatively modulates swimming capacity. Furthermore, overexpressing IbaG enhanced the capabilities of V. parahaemolyticus for swarming and biofilm formation. In addition, inactivation of ibaG in V. parahaemolyticus SH112 impaired its capacity for colonizing the heart, liver, spleen, and kidneys, and reduced visceral tissue damage, thereby leading to diminished virulence, compared with the wild-type strain. Finally, RNA-sequencing revealed 53 upregulated and 71 downregulated genes in the deletion strain ΔibaG. KEGG enrichment analysis showed that the two-component system, quorum sensing, bacterial secretion system, and numerous amino acid metabolism pathways had been altered due to the inactivation of ibaG. The results of this study indicated that IbaG exerts a considerable effect on gene regulation, motility, biofilm formation, and pathogenicity of V. parahaemolyticus. To the best of our knowledge, this is the first systematic study on the role played by IbaG in V. parahaemolyticus infections. Thus, our findings may lead to a better understanding of the metabolic processes involved in bacterial infections and provide a basis for the prevention and control of such infections.

Identification of novel toxins associated with the extracellular contractile injection system using machine learning

Secretion systems play a crucial role in microbe-microbe or host-microbe interactions. Among these systems, the extracellular contractile injection system (eCIS) is a unique bacterial and archaeal extracellular secretion system that injects protein toxins into target organisms. However, the specific proteins that eCISs inject into target cells and their functions remain largely unknown. Here, we developed a machine learning classifier to identify eCIS-associated toxins (EATs). The classifier combines genetic and biochemical features to identify EATs. We also developed a score for the eCIS N-terminal signal peptide to predict EAT loading. Using the classifier we classified 2,194 genes from 950 genomes as putative EATs. We validated four new EATs, EAT14-17, showing toxicity in bacterial and eukaryotic cells, and identified residues of their respective active sites that are critical for toxicity. Finally, we show that EAT14 inhibits mitogenic signaling in human cells. Our study provides insights into the diversity and functions of EATs and demonstrates machine learning capability of identifying novel toxins. The toxins can be employed in various applications dependently or independently of eCIS.

Diverse bacterial consortia: key drivers of rhizosoil fertility modulating microbiome functions, plant physiology, nutrition, and soybean grain yield

Soybean cultivation in tropical regions relies on symbioses with nitrogen-fixing Bradyrhizobium and plant growth-promoting bacteria (PGPBs), reducing environmental impacts of N fertilizers and pesticides. We evaluate the effects of soybean inoculation with different bacterial consortia combined with PGPBs or microbial secondary metabolites (MSMs) on rhizosoil chemistry, plant physiology, plant nutrition, grain yield, and rhizosphere microbial functions under field conditions over three growing seasons with four treatments: standard inoculation of Bradyrhizobium japonicum and Bradyrhizobium diazoefficiens consortium (SI); SI plus foliar spraying with Bacillus subtilis (SI + Bs); SI plus foliar spraying with Azospirillum brasilense (SI + Az); and SI plus seed application of MSMs enriched in lipo-chitooligosaccharides extracted from B. diazoefficiens and Rhizobium tropici (SI + MSM). Rhizosphere microbial composition, diversity, and function was assessed by metagenomics. The relationships between rhizosoil chemistry, plant nutrition, grain yield, and the abundance of microbial taxa and functions were determined by generalized joint attribute modeling. The bacterial consortia had the most significant impact on rhizosphere soil fertility, which in turn affected the bacterial community, plant physiology, nutrient availability, and production. Cluster analysis identified microbial groups and functions correlated with shifts in rhizosoil chemistry and plant nutrition. Bacterial consortia positively modulated specific genera and functional pathways involved in biosynthesis of plant secondary metabolites, amino acids, lipopolysaccharides, photosynthesis, bacterial secretion systems, and sulfur metabolism. The effects of the bacterial consortia on the soybean holobiont, particularly the rhizomicrobiome and rhizosoil fertility, highlight the importance of selecting appropriate consortia for desired outcomes. These findings have implications for microbial-based agricultural practices that enhance crop productivity, quality, and sustainability.Graphical

Transcriptome Analysis Reveals Cross-Talk between the Flagellar Transcriptional Hierarchy and Secretion System in Plesiomonas shigelloides.

Plesiomonas shigelloides, a Gram-negative bacillus, is the only member of the Enterobacteriaceae family able to produce polar and lateral flagella and cause gastrointestinal and extraintestinal illnesses in humans. The flagellar transcriptional hierarchy of P. shigelloides is currently unknown. In this study, we identified FlaK, FlaM, FliA, and FliAL as the four regulators responsible for polar and lateral flagellar regulation in P. shigelloides. To determine the flagellar transcription hierarchy of P. shigelloides, the transcriptomes of the WT and ΔflaK, ΔflaM, ΔfliA, and ΔfliAL were carried out for comparison in this study. Quantitative Real-Time Polymerase Chain Reaction (qRT-PCR) and luminescence screening assays were used to validate the RNA-seq results, and the Electrophoretic Mobility Shift Assay (EMSA) results revealed that FlaK can directly bind to the promoters of fliK, fliE, flhA, and cheY, while the FlaM protein can bind directly to the promoters of flgO, flgT, and flgA. Meanwhile, we also observed type VI secretion system (T6SS) and type II secretion system 2 (T2SS-2) genes downregulated in the transcriptome profiles, and the killing assay revealed lower killing abilities for ΔflaK, ΔflaM, ΔfliA, and ΔfliAL compared to the WT, indicating that there was a cross-talk between the flagellar hierarchy system and bacterial secretion system. Invasion assays also showed that ΔflaK, ΔflaM, ΔfliA, and ΔfliAL were less effective in infecting Caco-2 cells than the WT. Additionally, we also found that the loss of flagellar regulators causes the differential expression of some of the physiological metabolic genes of P. shigelloides. Overall, this study aims to reveal the transcriptional hierarchy that controls flagellar gene expression in P. shigelloides, as well as the cross-talk between motility, virulence, and physiological and metabolic activity, laying the groundwork for future research into P. shigelloides' coordinated survival in the natural environment and the mechanisms that infect the host.

Disinfectant polyhexamethylene guanidine triggered simultaneous efflux pump antibiotic- and metal-resistance genes propagation during sludge anaerobic digestion

The environmental transmission of antibiotic resistance genes (ARGs) and metal resistance genes (MRGs) exerted devastating threats to global public health, and their interactions with other emerging contaminants (ECs) have raised increasing concern. This study investigated that the abundances of ARGs and MRGs with the predominant type of efflux pump were simultaneously increased (8.4–59.1%) by disinfectant polyhexamethylene guanidine (PHMG) during waste activated sludge (WAS) anaerobic digestion. The aggregation of the same microorganisms (i.e., Hymenobacter and Comamonas) and different host bacteria (i.e., Azoarcus and Thauera) were occurred upon exposure to PHMG, thereby increasing the co-selection and propagation of MRGs and ARGs by vertical gene transfer. Moreover, PHMG enhanced the process of horizontal gene transfer (HGT), facilitating their co-transmission by the same mobile genetic elements (20.2–223.7%). Additionally, PHMG up-regulated the expression of critical genes (i.e., glnB, trpG and gspM) associated with the HGT of ARGs and MRGs (i.e., two-component regulatory system and quorum sensing) and exocytosis system (i.e., bacterial secretion system). Structural equation model analysis further verified that the key driver for the simultaneous enrichment of ARGs and MRGs under PHMG stress was microbial community structure. The study gives new insights into the aggravated environmental risks and mechanisms of ECs in sludge digestion system, providing guidance for subsequent regulation and control of ECs.

Parachlorometaxylenol stress caused multidrug-type antibiotic resistance genes proliferation via simultaneously reshaping microbial community and interfering metabolic traits during wastewater treatment process

Despite biological wastewater treatment processes (e.g., sequencing batch reactors (SBR)) being able to reduce the dissemination of antibiotic resistance genes (ARGs), the variation of ARGs under exogenous pollutant stress is an open question. This work investigated the impacts of para-chloro-meta-xylenol (PCMX, typical antibacterial contaminants) on ARGs spread in long-term SBR operation. Although the SBR process inherently decreased ARGs abundance, the presence of PCMX substantially amplified both the prevalence (mainly multidrug) and abundance of total ARGs (1.17-fold of the control). Further analysis demonstrated that PCMX disintegrated sludge structures as well as increased membrane permeability, facilitating the release of mobile genetic elements and subsequent horizontal transfer of ARGs. In addition, PCMX selectively enriched potential ARG hosts, notably Nitrospira and Candidatus Accumulibacter, which predominantly served as multidrug ARG hosts. Concurrently, the self-adaptive functions of ARGs hosts in the PCMX-exposed SBR system were activated via quorum sensing, two-component regulatory system, ATP-binding cassette transporters, and bacterial secretion system. The upregulation of these metabolic pathways also contributed to the dissemination of ARGs.

Comparative transcriptome reveals importance of export apparatus subunit (ascR) in type III secretion system and its roles on biological properties, gene expression profiles, virulence and colonization of Aeromonas veronii

Aeromonas veronii, an opportunistic pathogen, is known to cause serious infections across various species. In our previous study, we discovered that A. veronii GL2 exhibited a virulence up to ten times greater than that of FO1. To ascertain the factors contributing to the disparity in virulence between the two strains, we conducted a comparative transcriptome analysis. This analysis reveals a significant upregulation (P < 0.05) of the ascR gene in GL2 compared with FO1. Additionally, six differentially expressed genes (DEGs) were identified within the “Bacterial secretion system” pathway (map03070), with ascR being an essential component of type III secretion system (T3SS). AscR, considered as SctR family export apparatus subunit within the T3SS, has ambiguous roles in the biological properties, gene expression profiles, virulence and colonization of A. veronii. Therefore, we constructed a mutant strain (ΔascR) by homologous recombination. Comparative analysis with the wide-type GL2 reveals no significant differences in terms of colony morphology, growth curve, hemolytic activity and protease activity. However, significant reductions (P < 0.01) were observed in the abilities of biofilm formation and swimming mobility. No remarkable difference was noted in the lengths of flagella. The LD50 value of ΔascR was to be 5.15 times higher than that of GL2. Interestingly, the mRNA expression of ascC, ascD, ascJ and ascI genes in the T3SS, and mshB, mshE, mshK and mshP genes in the MSHA type pili were significantly upregulated (P < 0.05) in ΔascR, potentially due to transcriptional compensation. Further analysis of enzymatic biomarkers revealed that ΔascR might not destruct the recognition of innate immune response in host remarkably, but the colonization levels of A.veronii were significantly suppressed (P < 0.01) in ΔascR group. In conclusion, the ascR gene may be a key determinant in regulating the virulence of A. veronii, and the destruction of the T3SS caused by ascR deficiency results in these notable changes.

The effect of Astragalus polysaccharides on the repair of adverse effects in largemouth bass (Micropterus salmoides) under enrofloxacin stress

Antibiotics have important role in controlling the outbreak of bacterial diseases in fish farming, but long-term use or persistent existence of antibiotics in water can cause adverse effects on aquatic animals. In order to evaluate repairing effect of Astragalus polysaccharides (APS) on damaged intestine of fish under enrofloxacin (ENR) stress, ENR-pretreated (1.0 mg/L, 7 days) largemouth bass were randomly divided into two groups, and subsequently were fed basal diet and diet supplemented with 1.0 g/kg APS for 14 days. Intestine tissue and content were sampled on day 0 (D0), days 7 (D7), days 21 (D21_APS and D21) to detect the intestinal structure, intestinal microbiota structure, oxidative stress, non-specific immune and inflammatory response.Intestinal tissue section showed that the height, width of intestinal villi, and muscular thickness in D21_APS were significantly greater than those on D0, D7, and D21 (P < 0.05). APS-supplemented diet was significantly increased expression of tight junction protein related genes occludin, claudin-1, ZO-1 (P < 0.05). APS addition significantly improved the activities of catalase (CAT) and peroxidase (POD) and decreased superoxide dismutase (SOD) activity (P < 0.05). The activity of CAT and SOD in D21_APS and D0 were similar. The highest activities of acid phosphatase (ACP), lysozyme (LZM) and alkaline phosphatase (AKP) were found in D21_APS group. The expression of proinflammatory factors IL-1β and TNF-α in D21_APS and D21 were significantly down-regulated compared to that of D7, while anti-inflammatory factor IL-10 was up-regulated (P < 0.05). Compared with D7 group, quinolone resistance gene (qnrB) in D21 and D21_APS groups were significantly down-regulated, and in contrast to D21 group, qnrB and qnrS genes in D21_APS group were significantly down-regulated (P < 0.05).High-throughput 16SrRNA gene sequencing revealed proportion of Cyanobacteria in intestinal microbiota increased from 8.23% to 52.66%, while Firmicutes decreased from 28.54% to 2.03% after ENR treatment. In D21 group, Proteobacteria was notable decrease compared to D0, while Firmicutes increased. In D21_APS, the dominant bacteria were Proteobacteria, Fusobacteriota and Firmicutes, and Acinetobacter, Cetobacterium and Aeromonas were the predominant genera. Cluster analysis indicated that bacterial community structure in D21_APS has a similar level to D0 at phylum level. Feeding APS increased abundance of genes related to styrene, isoleucine and lysine degradation; propanoate, butanoate phenylalanine, and tryptophan metabolism; lipopolysaccharide biosynthesis; bacterial secretion system; cell motility and secretion at KEGG pathway Level 3 compared to the basal diet. Taken together, APS can repair the damage of intestinal structure and intestinal microbiota caused by ENR.

Changes in phycospheric and environmental microbes in Neoporphyra haitanensis during the cultivation cycle

Neoporphyra haitanensis is an economic seaweed widely cultivated along the coast of China, allowing 4–5 times harvests during a cultivation season. The composition and functions of algal-microbial communities are intricate, dynamic, and closely linked to the health of algae. This study utilized metagenomic and 16S ribosomal RNA (rRNA) sequencing to investigate the dynamic changes in the phycospheric microbial communities of Neoporphyra haitanensis (PMC-Nh) and bacterial communities of surrounding seawater (BC-SW) during the cultivation cycle, providing valuable reference data for monitoring and preventing diseases of cultivated seaweed. The results indicated that N. haitanensis might possess the ability to “recruit” and “select” surrounding microbial communities. Beneficial bacteria contributing to carbon, nitrogen, sulfur or phosphorus cycles, such as Proteobacteria, Firmicutes, Cyanobacteria at the class level, and Rhodobacteraceae, Saprospiraceae, Flavobacteriaceae at the family level, exhibited increased abundance. A symbiotic relationship based on resource supply formed between algae and PMC-Nh. However, potential pathogenic bacteria capable of degrading algae, such as Aquimana, Nonlabens, Alteromonas, Loktanella, also successfully colonized. The yield and quality of N. haitanensis in the 2nd and 3rd harvests are crucial for annual production and income. However, potential pathogenic bacteria and factors peaked in abundance during the 2nd harvest. At this point, the symbiotic body formed by Neoporphyra and the PMC-Nh was in a state of equilibrium on the “tip of the needle”. Biomarkers in the 2nd harvest, such as Nonlabens and Alteromonas, have been reported as algal pathogens. Moreover, pathways or factors associated with bacterial pathogenesis were significantly enriched in the algal-associated microbial community in the 2nd harvest, such as biofilm formation, flagellar assembly, bacterial secretion system pathways, and virulence factors of the Virulence Factors of Pathogenic Bacteria (VFDB) database. This may be attributed to the variable and frequently intense temperature increases in the coastal areas of the East China Sea during the 2nd harvest period in mid to late October, leading to significant shifts in microbial balance. The PMC-Nh remained relatively stable during the 3rd harvest. Until the end of cultivation, pathways related to glycometabolism and the content of glycoside hydrolases in the PMC-Nh increased, possibly due to heightened polysaccharide content and the maturation or senescence of the final stage of N. haitanensis, favoring polysaccharide exudation. Our results suggest that the 2nd harvest stage is a high-risk period for N. haitanensis cultivation, and effective cultivation management should be conducted to prevent widespread algal decay and reduce production losses.

Comparative genomic and transcriptome analyses of two Pectobacterium brasiliense strains revealed distinct virulence determinants and phenotypic features.

Potato soft rot caused by Pectobacterium spp. are devastating diseases of potato which cause severe economic losses worldwide. Pectobacterium brasiliense is considered as one of the most virulent species. However, the virulence mechanisms and pathogenicity factors of this strain have not been fully elucidated. Here, through pathogenicity screening, we identified two Pectobacterium brasiliense isolates, SM and DQ, with distinct pathogenicity levels. SM exhibits higher virulence compared to DQ in inducing aerial stem rot, blackleg and tuber soft rot. Our genomic and transcriptomic analyses revealed that SM encodes strain specific genes with regard to plant cell wall degradation and express higher level of genes associated with bacterial motility and secretion systems. Our plate assays verified higher pectinase, cellulase, and protease activities, as well as fast swimming and swarming motility in SM. Importantly, a unique endoglucanase S specific to SM was identified. Expression of this cellulase in DQ greatly enhances its virulence compared to wild type strain. Our study sheds light on possible determinants causing different pathogenicity of Pectobacterium brasiliense species with close evolutionary distance and provides new insight into the direction of genome evolution in response to host variation and environmental stimuli.

Modulation of the rat intestinal microbiota in the course of Anisakis pegreffii infection.

Anisakis are globally distributed, marine parasitic nematodes that can cause human health problems, including symptoms such as vomiting, acute diarrhea, and allergic reactions. As parasitic nematodes that primarily affect the patient's digestive tract, intestinal helminths can interact directly with the host microbiota through physical contact, chemicals, or nutrient competition. It is widely accepted that the host microbiota plays a crucial role in the regulation of immunity. Nematodes collected from the abdominal cavity of marine fish were identified by molecular biology and live worms were artificially infected in rats. Infection was determined by indirect ELISA based on rat serum and worm extraction. Feces were collected for 16S rDNA-based analysis of microbiota diversity. Molecular biology identification based on ITS sequences identified the collected nematodes as A. pegreffii. The success of the artificial infection was determined by indirect ELISA based on serum and worm extraction from artificially infected rats. Microbiota diversity analysis showed that a total of 773 ASVs were generated, and PCoA showed that the infected group was differentiated from the control group. The control group contained five characterized genera (Prevotellaceae NK3B31 group, Turicibacter, Clostridium sensu stricto 1, Candidatus Stoquefichus, Lachnospira) and the infected group contained nine characterized genera (Rodentibacter, Christensenella, Dubosiella, Streptococcus, Anaeroplasma, Lactococcus, Papillibacter, Desulfovibrio, Roseburia). Based on the Wilcoxon test, four processes were found to be significant: bacterial secretion system, bacterial invasion of epithelial cells, bacterial chemotaxis, and ABC transporters. This study is the first to analyze the diversity of the intestinal microbiota of rats infected with A. pegreffii and to determine the damage and regulation of metabolism and immunity caused by the infection in the rat gut. The findings provide a basis for further research on host-helminth-microbe correlationships.

Analysis of global Aeromonas caviae genomes revealed that strains carrying T6SS are more common in human gastroenteritis than in environmental sources and are often phylogenetically related.

Aeromonas caviae is an emerging human enteric pathogen. However, the genomic features and virulence genes of A. caviae strains from human gastroenteritis and other sources have not been fully elucidated. Here, we conducted a genomic analysis of 565 global A. caviae strains isolated from different sources, including 261 strains isolated from faecal samples of gastroenteritis patients, of which 18 genomes were sequenced in this study. The presence of bacterial virulence genes and secretion systems in A. caviae strains from different sources was compared, and the phylogenetic relationship of A. caviae strains was assessed based on the core genome. The complete genome of A. caviae strain A20-9 isolated from a gastroenteritis patient was obtained in this study, from which 300 putative virulence factors and a T4SS-encoding plasmid, pAC, were identified. Genes encoding T4SS were also identified in a novel genomic island, ACI-1, from other T4SS-positive strains. The prevalence of T4SS was significantly lower in A. caviae strains from gastroenteritis patients than in environmental strains (3 %, P<0.0001 vs 14 %, P<0.01). Conversely, the prevalence of T6SS was significantly higher in A. caviae strains isolated from gastroenteritis patients than in environmental strains (25 %, P<0.05 vs 13 %, P<0.01). Four phylogenetic clusters were formed based on the core genome of 565 A. caviae strains, and strains carrying T6SS often showed close phylogenetic relationships. T3SS, aerolysin and thermostable cytotonic enterotoxin were absent in all 565 A. caviae strains. Our findings provide novel information on the genomic features of A. caviae and suggest that T6SS may play a role in A. caviae-induced human gastroenteritis.

Transcriptome analysis reveals the inhibitory mechanism of 3,4-Dimethoxyphenol from Streptomyces albidoflavus strain ML27 against Xanthomonas oryzae pv. oryzae

Bacterial leaf blight, caused by Xanthomonas oryzae pv. oryzae (Xoo), poses a significant threat to rice cultivation across diverse regions. Growing concerns about pesticide resistance and environmental impact underscore the urgent necessity for eco-friendly biopesticides. Here, the complete genome sequence of Streptomyces albidoflavus strain ML27 revealed substantial antimicrobial activity and secondary metabolite production potential through genome mining. 3,4-dimethoxyphenol (purity 97%) was successfully isolated from the fermentation broth of S. albidoflavus strain ML27, exhibiting broad and pronounced inhibitory effects on the growth of seven different fungi and five tested bacteria. The efficacy of 3,4-dimethoxyphenol in controlling rice bacterial leaf blight was evaluated through pot tests, demonstrating substantial therapeutic (69.39%) and protective (84.53%) effects. Application of 3,4-dimethoxyphenol to Xoo resulted in cells displayed notable surface depressions, wrinkles, distortions, or even ruptures compared to their typical morphology. Transcriptome analysis revealed significant inhibition of membrane structures, protein synthesis and secretion, bacterial secretion system, two-component system, flagellar assembly, as well as various metabolic and biosynthetic pathways by 3,4-dimethoxyphenol. Notably, the down-regulation of the type III secretion system (T3SS) expression was a pivotal finding. Furthermore, validation via quantitative real-time polymerase chain reaction (qRT-PCR) analysis confirmed significant downregulation of 10 genes related to T3SS upon 3,4-dimethoxyphenol treatment. Based on these results, it is promising to develop 3,4-dimethoxyphenol as a novel biopesticide targeting the T3SS of Xoo for controlling bacterial leaf blight in rice.