Multi-omics analysis of miRNA-mediated intestinal microflora changes in crucian carp Carassius auratus infected with Rahnella aquatilis.

Infection by an emerging bacterial pathogen Rahnella aquatilis caused enteritis and septicemia in fish. However, the molecular pathogenesis of enteritis induced by R. aquatilis infection and its interacting mechanism of the intestinal microflora associated with microRNA (miRNA) immune regulation in crucian carp Carassius auratus are still unclear. In this study, C. auratus intraperitoneally injected with R. aquatilis KCL-5 was used as an experimental animal model, and the intestinal pathological changes, microflora, and differentially expressed miRNAs (DEMs) were investigated by multi-omics analysis. The significant changes in histopathological features, apoptotic cells, and enzyme activities (e.g., lysozyme (LYS), alkaline phosphatase (AKP), alanine aminotransferase (ALT), aspartate transaminase (AST), and glutathione peroxidase (GSH-Px)) in the intestine were examined after infection. Diversity and composition analysis of the intestinal microflora clearly demonstrated four dominant bacteria: Proteobacteria, Fusobacteria, Bacteroidetes, and Firmicutes. A total of 87 DEMs were significantly screened, and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses revealed that the potential target genes were mainly involved in the regulation of lipid, glutathione, cytosine, and purine metabolism, which participated in the local immune response through the intestinal immune network for IgA production, lysosome, and Toll-like receptor (TLR) pathways. Moreover, the expression levels of 11 target genes (e.g., TLR3, MyD88, NF-κB, TGF-ß, TNF-α, MHC II, IL-22, LysC, F2, F5, and C3) related to inflammation and immunity were verified by qRT-PCR detection. The correlation analysis indicated that the abundance of intestinal Firmicutes and Proteobacteria was significantly associated with the high local expression of miR-203/NF-κB, miR-129/TNF-α, and miR-205/TGF-ß. These findings will help to elucidate the molecular regulation mechanism of the intestinal microflora, inflammation, and immune response-mediated miRNA-target gene axis in cyprinid fish.

Single-cell transcriptome, phagocytic activity and immunohistochemical analysis of crucian carp (Carassius auratus) in response to Rahnella aquatilis infection.

In teleost fish, kidney is an important immune and hematopoietic organ with multiple physiological functions. However, the immune cells and cellular markers of kidney require further elucidation in crucian carp (C. auratus). Here we report on the single-cell transcriptional landscape in posterior kidney, immunohistochemical and phagocytic features of C. auratus with R. aquatilis infection. The results showed that a total of 18 cell populations were identified for the main immune cells such as monocytes/macrophages (Mo/Mφ), dendritic cells (DCs), B cells, T cells, granulocytes and hematopoietic progenitor cells (HPCs). Pseudo-time trajectory analysis was reconstructed for the immune cells using Monocle2 to obtain additional insights into their developmental lineage relationships. In the detected tissues (liver, spleen, kidney, intestine, skin, and gills) of infected fish exhibited positive immunohistochemical staining with prepared for antibody to R. aquatilis. Apoptotic cells were fluorescently demonstrated by TUNEL assay, and bacterial phagocytic activity were observed for neutrophils and Mo/Mφ cells, respectively. Moreover, a similar up-ward/down-ward expression trend of the selected immune and inflammatory genes was found in the kidney against R. aquatilis infection, which were significantly involved in TLR/NLR, ECM adhesion, phago-lysosome, apoptosis, complement and coagulation pathways. To our knowledge, this is the first report on the detailed characterization of immune cells and host-R. aquatilis interaction, which will contribute to understanding on the biology of renal immune cells and repertoire of potential markers in cyprinid fish species.

Gallic and ferulic acids suppress proteolytic activities and volatile trimethylamine production in the food-borne spoiler Rahnella aquatilis KM05.

BACKGROUND: Rahnella aquatilis is a recognised microbial threat that alters the sensory properties of seafood. The high frequency with which R. aquatilis is isolated from fish has prompted a search for alternative preservatives. In the present study, in vitro and fish-based ecosystem (raw salmon-based medium) approaches were used to validate the antimicrobial effects of gallic (GA) and ferulic (FA) acids against R. aquatilis KM05. The results were compared with data describing the response of KM05 to sodium benzoate. Bioinformatics data of the whole genome were used to analyse the potential for fish spoilage by KM05 in detail, and the results revealed the main physiological characteristics that underlie reduced seafood quality. RESULTS: In the KM05 genome, the most abundantly enriched Gene Ontology terms were 'metabolic process', 'organic substance metabolic process' and 'cellular process'. Through an evaluation of the Pfam annotations, 15 annotations were found to be directly involved in the proteolytic activity of KM05. Peptidase_M20 was the most abundantly represented (abundance value of 14060). Proteins representing the CutC family (abundance value of 427) indicated the potential for KM05 degradation of trimethyl-amine-N-oxide. Subinhibitory concentrations of GA and FA suppressed the proteolytic activities of KM05 both in vitro and in RS medium by an average of 33-45%. These results were confirmed by quantitative real-time PCR experiments, which also showed that the expression levels of genes involved in proteolytic activities and volatile trimethylamine production were also decreased. CONCLUSION: Phenolic compounds can be used as potential food additives for preventing quality deterioration of fish products. © 2023 Society of Chemical Industry.

Uptake of Selenite by Rahnella aquatilis HX2 Involves the Aquaporin AqpZ and Na+/H+ Antiporter NhaA.

Microbial transformation of selenite [Se(IV)] to elemental selenium nanoparticles (SeNPs) is known to be an important process for removing toxic soluble selenium (Se) oxyanions and recovery of Se from the environment as valuable nanoparticles. However, the mechanism of selenite uptake by microorganisms, the first step through which Se exerts its cellular function, remains not well studied. In this study, the effects of selenite concentration, time, pH, metabolic inhibitors, and anionic analogues on selenite uptake in Rahnella aquatilis HX2 were investigated. Selenite uptake by R. aquatilis HX2 was concentration- and time-dependent, and its transport activity was significantly dependent on pH. In addition, selenite uptake in R. aquatilis HX2 was significantly inhibited by the aquaporin inhibitor AgNO3 and sulfite (SO32-), and partially inhibited by carbonyl cyanide m-chlorophenyl hydrazone (CCCP) and 2,4-dinitrophenol (2,4-DNP) treatments. Three mutants with in-frame deletions of aqpZ, glpF, and nhaA genes were constructed. The transport assay showed that the water channel protein AqpZ, and not GlpF, was a key channel of selenite uptake by R. aquatilis HX2, and sulfite and selenite had a common uptake pathway. In addition, the Na+/H+ antiporter NhaA is also involved in selenite uptake in R. aquatilis HX2.

Disruption of the sensor kinase phoQ gene decreases acid resistance in plant growth-promoting rhizobacterium Rahnella aquatilis HX2.

AIMS: Rahnella aquatilis HX2, a promising plant growth-promoting rhizobacterium (PGPR) in the field, contains genes homologous to the PhoP/PhoQ two-component regulatory system. Although this system regulates stress response in numerous pathogens, PhoP/PhoQ characterization in a PGPR has not received in-depth exploration. METHODS AND RESULTS: The phoQ gene was mutated in strain HX2 using an in-frame deletion strategy. Compared to the wild type, the phoQ mutant exhibited increased sensitivity to acidic conditions (pH 4.0) in a chemically defined medium and in mild acidic natural soil (pH 5.7). The phoQ mutant also exhibited increased swimming motility under acidic conditions. Acid resistance was restored in the mutant by introducing the phoQ gene on a plasmid. Three acid resistance genes, add, cfa, and fur were downregulated significantly, whereas the chaperone encoding gene, dnak, was upregulated when the phoQ mutant was exposed to acid stress. CONCLUSIONS: This study suggested that the PhoP/PhoQ system positively regulates the acid resistance of R. aquatilis HX2.

Rahnella sikkimica sp. nov., a novel cold-tolerant bacterium isolated from the glacier of Sikkim Himalaya with plant growth-promoting properties.

The current study describes a novel species with the strain name ERMR1:05T isolated from the forefield soil of East Rathong Glacier in West Sikkim Himalaya (India). The isolate was facultatively anaerobic, gram-stain negative, non-spore-forming, rod-shaped, and oxidase negative. Whole-genome-based bacterial core gene phylogenetic analysis placed the strain in the genus Rahnella, well separated from Rouxiella spp. The digital DNA-DNA hybridisation and average nucleotide identity values between strain ERMR1:05T and other members of genus Rahnella were below the proposed thresholds for the species delineation. Based on these results, a new species, Rahnella sikkimica sp. nov., is proposed with strain ERMR1:05T (CIP 111636T, MTCC 12598T) as the type strain. The bacterium showed upregulation of cold-stress genes in cold conditions. Additionally, the genome analysis of the bacterium showed the presence of plant growth-promotion factors suggesting its role in crop improvement in cold hilly regions.

Antarctic Rahnella inusitata: A Producer of Cold-Stable ß-Galactosidase Enzymes.

There has been a recent increase in the exploration of cold-active ß-galactosidases, as it offers new alternatives for the dairy industry, mainly in response to the current needs of lactose-intolerant consumers. Since extremophilic microbial compounds might have unique physical and chemical properties, this research aimed to study the capacity of Antarctic bacterial strains to produce cold-active ß-galactosidases. A screening revealed 81 out of 304 strains with ß-galactosidase activity. The strain Se8.10.12 showed the highest enzymatic activity. Morphological, biochemical, and molecular characterization based on whole-genome sequencing confirmed it as the first Rahnella inusitata isolate from the Antarctic, which retained 41-62% of its ß-galactosidase activity in the cold (4 °C-15 °C). Three ß-galactosidases genes were found in the R. inusitata genome, which belong to the glycoside hydrolase families GH2 (LacZ and EbgA) and GH42 (BglY). Based on molecular docking, some of these enzymes exhibited higher lactose predicted affinity than the commercial control enzyme from Aspergillus oryzae. Hence, this work reports a new Rahnella inusitata strain from the Antarctic continent as a prominent cold-active ß-galactosidase producer.

A bacterial endophyte exploits chemotropism of a fungal pathogen for plant colonization.

Soil-inhabiting fungal pathogens use chemical signals released by roots to direct hyphal growth towards the host plant. Whether other soil microorganisms exploit this capacity for their own benefit is currently unknown. Here we show that the endophytic rhizobacterium Rahnella aquatilis locates hyphae of the root-infecting fungal pathogen Fusarium oxysporum through pH-mediated chemotaxis and uses them as highways to efficiently access and colonize plant roots. Secretion of gluconic acid (GlcA) by R. aquatilis in the rhizosphere leads to acidification and counteracts F. oxysporum-induced alkalinisation, a known virulence mechanism, thereby preventing fungal infection. Genetic abrogation or biochemical inhibition of GlcA-mediated acidification abolished biocontrol activity of R. aquatilis and restored fungal infection. These findings reveal a new way by which bacterial endophytes hijack hyphae of a fungal pathogen in the soil to gain preferential access to plant roots, thereby protecting the host from infection.

First report on the characterization of pathogenic Rahnella aquatilis KCL-5 from crucian carp: Revealed by genomic and proteomic analyses.

Rahnella aquatilis is an important pathogen of several aquatic organisms and is found widely distributed in the freshwater, soil, fish and human clinical samples. Our previously published study reported a novel pathogenic R. aquatilis strain KCL-5 to crucian carp (Carassius auratus). To further investigate the characteristics and pathogenesis caused by R. aquatilis, we here report on the pathological changes, bacterial genomic and proteomic analyses of strain KCL-5. Significantly pathological changes in liver, intestine, spleen and gills were observed in infected fish. The genome consists of one circular chromosome 5,062,299 bp with 52.02% GC content and two plasmids (506,827 bp, 52.16%; 173,433 bp, 50.00%) and predicted 5,653 genes, 77 tRNAs and 22 rRNAs. Some virulence factors were characterized, including outer membrane protein, haemolysin, RTX toxin, chemotaxis and T3SS secretion system. Antimicrobial resistance genes such as EmrAB-TolC, MexABC-OpmB and RosAB efflux pump were found in strain KCL-5. KEGG analysis showed that mainly functional modules were ABC transporters, biosynthesis of amino acids, two-component system, quorum sensing, flagellum assembly and chemotaxis, in which most of them were identified by using 2-DE/MS analyses. To our knowledge, this was first report on the molecular characteristics of R. aquatilis by multi-omics approaches, which will provide insights into the pathogenic mechanism of R. aquatilis infection in fish.

Rahnella aceris sp. nov., isolated from sap drawn from Acer pictum.

A Gram-reaction-negative, facultatively anaerobic bacterium, designated SAP-19T, was isolated from sap extracted from Acer pictum in Mt. Halla in Jeju, Republic of Korea and its taxonomic statue was investigated by a polyphasic approach including genome- and 16S rRNA gene-based phylogenetic analyses. Cells were motile, short rods and showed growth at 20-30 °C, pH 4-9 and 0-6% (w/v) NaCl. The whole genome- and 16S rRNA gene-based phylogenetic analyses exhibited that strain SAP-19T belongs to the genus Rahnella and forms a tight cluster with Rahnella aquatilis. The isolate shared average nucleotide identity of 92.7% and 16S rRNA gene sequence similarity of 99.6% with the type strain of Rahnella aquatilis. The polar lipids contained phosphatidylethanolamine, an unidentified aminophospholipid and an unidentified lipid. The major isoprenoid quinone was Q-8. The predominant fatty acids were C16:0 and C17:0cyclo. The G + C content of the genome was 52.3%. The low average nucleotide identity (92.7%) and digital DNA relatedness (48.6%) values between the isolate and the most closely related strain showed that the isolate can be considered a different genospecies. On the basis of combined data obtained in this study, strain SAP-19T (= KACC 21744T = NBRC 114407T) represents a novel species of the genus Rahnella, for which the name Rahnella aceris sp. nov. is proposed.

The small RNA chaperone Hfq is a critical regulator for bacterial biosynthesis of selenium nanoparticles and motility in Rahnella aquatilis.

The RNA chaperone, Hfq, is a global post-transcriptional regulator that plays an important role in regulating pleiotropic functions, such as cell growth and motility, stress tolerance, and virulence to host, in many Gram-negative bacteria. This study examined the functional roles of Hfq in Rahnella aquatilis HX2, a plant beneficial, selenium nanoparticles (SeNPs)-producing soil bacterium. A mutant HX2∆hfq with an in-frame deletion within the hfq gene in R. aquatilis HX2 was constructed and tested for various phenotypic features. Bacterial growth, motility, selenite reduction, and SeNPs production were compared between the mutant, the wild-type, and the complementation strain. The hfq gene deletion delayed the growth of strain HX2, with a lower bacterial population during the stationary phase, and significantly impaired the swimming motility of the bacterium, showing a smaller motility ring on the plate. The hfq mutation also dramatically declined microbial-induced reduction of selenite and SeNPs production in HX2, which was independent of cell growth. The introduction of a trans-expressed hfq gene into HX2∆hfq for complementation completely restored impacted phenotypes. In addition, reverse transcription real-time quantitative PCR (RT-qPCR) analysis revealed that the expression of ten genes involved in bacterial growth and survival, motility and chemotaxis, and selenite or seleno-compound metabolism were influenced by Hfq loss-of-function by at least two-fold. Six genes including two involved in SeNPs production were positively regulated by hfq, while other four genes were negatively regulated. Homolog search suggested that the rprA gene might encode a small RNA regulated by Hfq in R. aquatilis HX2. Overall, the present study provides novel information about the function of Hfq and the regulation of bacterial biosynthesis of SeNPs.

The complete genome sequence of Rahnella aquatilis ZF7 reveals potential beneficial properties and stress tolerance capabilities.

Rahnella aquatilis ZF7 is a plant beneficial strain isolated from Sakura tree soil with potential for biocontrol. Here, we present the complete genome sequence of R. aquatilis ZF7, which consists of one 4.49 Mb circular chromosome and a 54-kb plasmid named pRAZF7. Phylogenetic analyses revealed that R. aquatilis ZF7 is much similar to the strains Rahnella sp. Y9602 and R. aquatilis HX2 than others evaluated. In this study, multiple genes encoding functions that likely contribute to plant growth promotion, biocontrol and stress tolerance were identified by comparative genome analyses, including IAA production, phosphate solubilization, antibiotic resistance and formation of Se nanoparticles (SeNPs). In addition, these functions were also confirmed by in vitro experiments. Considering its ability to form SeNPs, strain R. aquatilis ZF7 will contribute to nano-agriculture. Overall, the features of R. aquatilis ZF7 make it a high potential and competitive strain in biocontrol, and the genome data will help further studies on the mechanisms of plant growth promotion and biocontrol.

Rahnella spp. are commonly isolated from onion (Allium cepa) bulbs and are weakly pathogenic.

AIMS: Bacterial decays of onion bulbs have serious economic consequences for growers, but the aetiologies of these diseases are often unclear. We aimed to determine the role of Rahnella, which we commonly isolated from bulbs in the United States and Norway, in onion disease. METHODS AND RESULTS: Isolated bacteria were identified by sequencing of housekeeping genes and/or fatty acid methyl ester analysis. A subset of Rahnella spp. strains was also assessed by multilocus sequence analysis (MLSA); most onion strains belonged to two clades that appear closely related to R. aquatilis. All tested strains from both countries caused mild symptoms in onion bulbs but not leaves. Polymerase chain reaction primers were designed and tested against strains from known species of Rahnella. Amplicons were produced from strains of R. aquatilis, R. victoriana, R. variigena, R. inusitata and R. bruchi, and from one of the two strains of R. woolbedingensis. CONCLUSIONS: Based on binational testing, strains of Rahnella are commonly associated with onions, and they are capable of causing mild symptoms in bulbs. SIGNIFICANCE AND IMPACT OF THE STUDY: While Rahnella strains are commonly found within field-grown onions and they are able to cause mild symptoms, the economic impact of Rahnella-associated symptoms remains unclear.

Identification of atpD as an optimal reference gene to explore antibiotic resistance and stress tolerance in Rahnella aquatilis.

AIMS: Rahnella aquatilis is a Gram-negative bacterial species with potential for agricultural and industrial applications, as well as a human pathogen. This study aims to identify an optimal reference gene to explore antibiotic resistance and stress tolerance in R. aquatilis using reverse-transcription quantitative polymerase chain reaction (RT-qPCR). METHODS AND RESULTS: Expression levels of 14 housekeeping genes in R. aquatilis were estimated by RT-qPCR under six different conditions: exponential phase, stationary phase, acid, salinity, antibiotic and oxidative stresses. BestKeeper and the ΔCt method were used to evaluate the stability of each gene. The atpD gene was stably expressed in all conditions, thus was selected and validated as an optimal reference gene. Transcript levels of 17 putative ampicillin-resistance genes in R. aquatilis strain HX2 were evaluated using the proposed RT-qPCR. Six genes encoding efflux transporters and ß-lactamase were overexpressed after ampicillin treatment. Additionally, the expression of seven putative stress response genes in strain HX2 was assessed, and five genes were up-regulated by respective stress treatments. CONCLUSIONS: The atpD gene has been identified as an optimal reference gene for expression analysis of R. aquatilis responses to abiotic stresses by RT-qPCR. SIGNIFICANCE AND IMPACT OF THE STUDY: The proposed RT-qPCR is suitable for gene expression analysis in R. aquatilis, thus useful for studying antimicrobial resistance and stress tolerance in this bacterium and others closely related.

Genomic analysis of bacteria in the Acute Oak Decline pathobiome.

The UK's native oak is under serious threat from Acute Oak Decline (AOD). Stem tissue necrosis is a primary symptom of AOD and several bacteria are associated with necrotic lesions. Two members of the lesion pathobiome, Brenneria goodwinii and Gibbsiella quercinecans, have been identified as causative agents of tissue necrosis. However, additional bacteria including Lonsdalea britannica and Rahnella species have been detected in the lesion microbiome, but their role in tissue degradation is unclear. Consequently, information on potential genome-encoded mechanisms for tissue necrosis is critical to understand the role and mechanisms used by bacterial members of the lesion pathobiome in the aetiology of AOD. Here, the whole genomes of bacteria isolated from AOD-affected trees were sequenced, annotated and compared against canonical bacterial phytopathogens and non-pathogenic symbionts. Using orthologous gene inference methods, shared virulence genes that retain the same function were identified. Furthermore, functional annotation of phytopathogenic virulence genes demonstrated that all studied members of the AOD lesion microbiota possessed genes associated with phytopathogens. However, the genome of B. goodwinii was the most characteristic of a necrogenic phytopathogen, corroborating previous pathological and metatranscriptomic studies that implicate it as the key causal agent of AOD lesions. Furthermore, we investigated the genome sequences of other AOD lesion microbiota to understand the potential ability of microbes to cause disease or contribute to pathogenic potential of organisms isolated from this complex pathobiome. The role of these members remains uncertain but some such as G. quercinecans may contribute to tissue necrosis through the release of necrotizing enzymes and may help more dangerous pathogens activate and realize their pathogenic potential or they may contribute as secondary/opportunistic pathogens with the potential to act as accessory species for B. goodwinii. We demonstrate that in combination with ecological data, whole genome sequencing provides key insights into the pathogenic potential of bacterial species whether they be phytopathogens, part-contributors or stimulators of the pathobiome.

Disruption of acdS gene reduces plant growth promotion activity and maize saline stress resistance by Rahnella aquatilis HX2.

Rahnella aquatilis HX2 was isolated from Beijing vineyard soil and used as a plant growth-promoting rhizobacterium in the field. Previous studies have shown that it has a broad in vitro antimicrobial spectrum and could inhibit a variety of plant pathogenic bacteria and fungi. In this study, a gene, acdS, encoding 1-aminocyclopropane-1-carboxylic acid-deaminase was disrupted by in-frame deletion in the HX2 strain. Compared to the wild-type, the acdS-mutant had higher rates of nitrogen fixation, reduced indole-3-acetic acid production, lowered efficacy as a biological control agent against the grape crown gall pathogen Agrobacterium vitis. Under saline stress conditions, plant height, above-ground fresh weight, root fresh weight of corn plants were increased by treatment with HX2 but this increase was compromised by the disruption of acdS gene. Our data confirmed the function of HX2 on plant growth promoting and demonstrated that acdS gene plays a major role in its PGPR activities.

The arbuscular mycorrhizal fungus Rhizophagus irregularis MUCL 43194 induces the gene expression of citrate synthase in the tricarboxylic acid cycle of the phosphate-solubilizing bacterium Rahnella aquatilis HX2.

An increasing number of studies have demonstrated that arbuscular mycorrhizal fungi can cooperate with other soil microorganisms, e.g., bacteria, which develop near or on the surface of the extraradical hyphae where they perform multiple functions. However, the mechanisms involved in this privileged relationship are still poorly known. In the present study, we investigated how the arbuscular mycorrhizal fungus Rhizophagus irregularis MUCL 43194 influences the three pace-making enzymes (i.e., citrate synthase, isocitrate dehydrogenase, and α-oxoglutarate dehydrogenase) of the tricarboxylic acid (TCA) cycle in the phosphate-solubilizing bacterium Rahnella aquatilis HX2. The study was conducted under strict in vitro culture conditions and analysis made at the transcriptional level. Results showed that R. irregularis induced the expression of the gene-encoding citrate synthase (gltA), the pace-making enzyme involved in the first step of the TCA cycle, in R. aquatilis at all time points of observation (i.e., 1, 6, 12, 24, 48, and 72 h). The expression of the gene-encoding isocitrate dehydrogenase (icd) significantly decreased at 6, 12, 24, 48, and 72 h and the expression of the gene-encoding α-oxoglutarate dehydrogenase E1 component (kgdhc) significantly increased at 1, 6, and 48 h. The above results suggested that R. irregularis may influence the level of adenosine triphosphate production in R. aquatilis and thus the metabolism of the bacterium by stimulating the expression of gltA involved in the TCA cycle. Our results suggest a fine-tuned dialog between R. irregularis MUCL 43194 and R. aquatilis HX2 and emphasize the complexity of the interactions that might take place at the hyphal surface of arbuscular mycorrhizal fungi hosting communities of microbes.

Microbiome and infectivity studies reveal complex polyspecies tree disease in Acute Oak Decline.

Decline-diseases are complex and becoming increasingly problematic to tree health globally. Acute Oak Decline (AOD) is characterized by necrotic stem lesions and galleries of the bark-boring beetle, Agrilus biguttatus, and represents a serious threat to oak. Although multiple novel bacterial species and Agrilus galleries are associated with AOD lesions, the causative agent(s) are unknown. The AOD pathosystem therefore provides an ideal model for a systems-based research approach to address our hypothesis that AOD lesions are caused by a polymicrobial complex. Here we show that three bacterial species, Brenneria goodwinii, Gibbsiella quercinecans and Rahnella victoriana, are consistently abundant in the lesion microbiome and possess virulence genes used by canonical phytopathogens that are expressed in AOD lesions. Individual and polyspecies inoculations on oak logs and trees demonstrated that B. goodwinii and G. quercinecans cause tissue necrosis and, in combination with A. biguttatus, produce the diagnostic symptoms of AOD. We have proved a polybacterial cause of AOD lesions, providing new insights into polymicrobial interactions and tree disease. This work presents a novel conceptual and methodological template for adapting Koch's postulates to address the role of microbial communities in disease.

First report of Rahnella aquatilis infection in crucian carp Carassius auratus in China.

Rahnella aquatilis infection is rare in aquaculture. Here, a Gram-negative rod-shaped bacterium was isolated from diseased crucian carp Carassius auratus in Xuzhou City, Jiangsu Province, eastern China. The isolate was tentatively named strain KCL-5, and subsequently identified as R. aquatilis by biochemical properties and molecular techniques. The results showed that the isolate KCL-5 was most closely related to the type strain ATCC33071 (= DSM4594) of R. aquatilis, which shared 99.67, 96.26 and 99.58% nucleotide sequence identities for 16S rDNA, gyrB and toxin yhaV genes, respectively. Experimental challenges were conducted which demonstrated pathogenicity of the isolate in crucian carp. Antimicrobial susceptibility testing showed that the isolated strain was susceptible to piperacillin, gentamicin, kanamycin, nalidixic acid, norfloxacin, ofloxacin, azithromycin and erythromycin. To our knowledge, this is the first report on R. aquatilis infection in crucian carp, and the first evidence of pathogenicity in fish.

The replication protein of pHW126 auto-controls its expression.

pHW126 belongs to a small group of rolling circle plasmids. So far, the region mediating autonomous replication has been identified and it was shown that the rep gene is required for replication. However, the regulation of rep expression remained elusive. Here evidence is presented that expression of the replication gene rep is auto-regulated. Sequence analysis revealed a conserved stretch in the rep promoter consisting of three imperfect direct repeats (DR2.1, DR2.2 and DR2.3). Assays for promoter activity showed that these direct repeats act as an enhancer of transcriptional activity. Interestingly, the activating effect was reduced in the presence of Rep protein. Electrophoretic mobility shift assays demonstrated that the Rep protein can directly bind to direct repeats DR2.1 and DR2.3 while DR2.2 is not bound but places DR2.1 and DR2.3 in an appropriate distance. These results show that the synthesis of Rep protein is auto-regulated. In the absence of Rep protein the promoter is, due to the presence of the direct repeats acting as a transcriptional enhancer, highly active. Binding of Rep to the direct repeats reduces the transcription rate significantly. Since this regulation mechanism is independent of a specialised regulator protein it is presumably a very economic strategy.