Bark compost serves as an organic fertilizer, originating from the microbial decomposition of tree bark and woody residues. An examination of 84 bacterial isolates from commercially available bark composts identified nine strains capable of degrading N-acylhomoserine lactones (AHLs), which function as signaling molecules in bacterial quorum sensing. Whole-genome sequencing of one AHL-degrading isolate, Ochrobactrum quorumnocens B44, revealed the presence of multiple AHL-degrading gene homologs, with the gene OCHQB44_21820 (aiiO) encoding the principal AHL-degrading enzyme. AiiO efficiently degraded a range of AHL molecules with acyl chain lengths of 6-10, including their 3-oxo derivatives. Contrary to previous reports suggesting that AiiO functions as an AHL acylase, hydrolyzing the amide bond of AHL, biochemical analysis in this study demonstrated that AiiO acts as an AHL lactonase, catalyzing the hydrolytic opening of the lactone ring in AHL. The purified AiiO as a maltose binding protein fusion efficiently degraded AHLs at an optimal temperature of 40 °C, maintaining stability under typical soil conditions. The expression of aiiO in the plant pathogen Pectobacterium carotovorum subsp. carotovorum causes self-degradation of biosynthesized AHL and suppresses potato tissue maceration regulated by AHL-mediated quorum sensing. Co-inoculation of B44 with P. carotovorum subsp. carotovorum attenuated soft rot symptoms in potato slices. These results showed the possibility that the application of bark compost rich in AHL-degrading bacteria enhances soil resistance to bacterial diseases.

Here, we report the complete genome sequences of two strains of Bacillus velezensis, S1022 and S1172, isolated from soil in Japan. The genome sizes of S1022 and S1172 were 4,020,394 bp with a GC content of 46.4% and 3,995,622 bp with a GC content of 46.5%, respectively.

Quorum sensing mediated by N-acylhomoserine lactones (AHLs) plays a key role in the regulation of virulence in many plant-pathogenic bacteria, and enzymatic degradation of AHLs represents a promising biocontrol strategy known as quorum quenching. The AHL lactonase gene aiiA is widely distributed within the genus Bacillus and is generally considered to be present as a single-copy gene. In this study, we show that specific strains of Bacillus thuringiensis harbor two distinct aiiA homologs. Genome analyses of environmental B. thuringiensis isolates, together with publicly available genome sequences, revealed a phylogenetically distinct aiiA homolog in addition to the canonical gene. Phylogenetic analysis classified these homologs into two groups, designated AiiA1 and AiiA2. Comparative genomic analysis indicated that aiiA2 is located within variable genomic regions, suggesting acquisition via horizontal gene transfer through mechanisms other than transposon-mediated transposition. Functional assays confirmed that both AiiA1 and AiiA2 possess AHL-degrading activity. Quantitative analyses showed that the specific activities of both enzymes increased with increasing temperature, and although AiiA2 exhibited slightly higher activity than AiiA1 across the tested temperature range, no dramatic difference in AHL-degrading activity was observed between the two enzymes. These findings highlight previously unrecognized diversity in quorum-quenching systems within B. thuringiensis and suggest that the coexistence of multiple AHL lactonases with largely comparable activities may contribute to a flexible and robust quorum-quenching capacity in plant-associated environments.

Numerous Gram-negative bacteria possess N-acyl-L-homoserine lactone (AHL)-mediated quorum-sensing (QS) systems that regulate the activation of specific genes. Burkholderia plantarii causes rice seedling blight by producing the phytotoxin tropolone. In this study, we investigated multiple AHL-type QS systems in B. plantarii MAFF 301723T and their involvement in virulence regulation. MAFF 301723 harbors three AHL-mediated QS systems, designated plaI1/plaR1, plaI2/plaR2, and plaI3/plaR3. The plaI1/plaR1 system, which produces N-octanoyl-l-homoserine lactone, is functional and essential for swarming motility. When forced expression of plaI2 induces the biosynthesis of 3-OH-C10-HSL, it was suggested that expression is rarely observed in wild-type MAFF 301723. The plaI3 gene directs the synthesis of the putative C16:2-HSL, which is a rare AHL bearing two double bonds in the hexadecanoyl chain that has not been previously reported in Burkholderia spp. The plaI3/plaR3-QS system is crucial for tropolone production. These findings suggest that multiple QS systems collectively contribute to the complex virulence regulation of B. plantarii, thereby providing new insights into the development of QS-targeted biocontrol strategies for agriculture.

Herein, we report the nearly complete genome sequences of Actinobacteriota Kitasatospora sp. CMC57 and Streptomyces sp. CMC78, which were isolated from agricultural soils in Japan.

Here, we report the draft genome sequences of two strains of Pseudomonas fluorescens, MAFF 301597 and MAFF 301598, isolated from strawberry in Japan. The genome sizes of MAFF 301597 and MAFF 301598 were 6,627,466 bp with a G+C content of 60.0% and 6,449,710 bp with a G+C content of 60.1%, respectively.

Azorhizobium caulinodans is a nitrogen-fixing bacterium that forms stem and root nodules on Sesbania rostrata. All tested A. caulinodans strains exhibited degradation activity against the quorum-sensing signaling compounds, N-acyl-l-homoserine lactones (AHL). The AHL-degrading gene homolog, attM, was identified in the genome sequences of A. caulinodans MAFF 210031T and other A. caulinodans strains. Recombinant AttM functions as an AHL lactonase that hydrolyzes the lactone bond of AHL and retains its stable activity at environmental temperatures. The AHL-degrading activity of the attM-deletion mutant was completely diminished, which revealed that AHL degradation by MAFF 210031 was dependent on attM.

Tomato pith necrosis (TPN) is a common disease worldwide. In this study, we investigated the relationship between acylhomoserine lactone (AHL)-mediated quorum sensing and pathogenicity in 21 TPN isolates, which were classified into three groups: group 1 (Pseudomonas corrugata), group 2 (Pseudomonas mediterranea), and group 3 (novel taxonomic group). All strains produced N-hexanoyl-l-homoserine lactone as the dominant AHL molecule. Phylogenetic analyses revealed high conservation of quorum-sensing genes among the three groups, with nearly identical sequences and arrangements. Whole-genome sequencing of the group 3 strain MAFF 730105 revealed the presence of a type III secretion system gene cluster, similar to well-known plant pathogens. Although quorum sensing influences virulence, its impact is limited, and some virulence factors may be constitutive or regulated independently. These results suggest that quorum sensing contributes to TPN symptoms; however, additional factors and regulatory mechanisms are involved in the pathogenicity of TPN isolates.

Burkholderia gladioli produces a yellow-pigmented toxin called toxoflavin, and causes disease on a variety of plants. Previous studies have suggested that the pathogenicity of B. gladioli is regulated by an N-acyl-l-homoserine lactone (AHL)-mediated quorum sensing (QS) system. In this study, complete genome sequencing revealed that B. gladioli pv. gladioli MAFF 302385 possesses two types of AHL synthase and AHL receptor gene pairs: glaI1/glaR1 and glaI2/glaR2. Disruption of QS genes revealed that the glaI1/glaR1 QS system regulated swarming motility, biofilm formation, and colony formation via N-octanoyl-l-homoserine lactone. Although Escherichia coli harboring glaI2 produced N-(3-hydroxyoctanoyl)-l-homoserine lactone and N-(3-hydroxydecanoyl)-l-homoserine lactone, the expression of glaI2 was not confirmed in MAFF 302385 cells. We also found that toxoflavin production was regulated by the glaI1/glaR1 QS system in liquid medium, but not on agar medium. When pathogenicity tests were performed on gladiolus leaves, the wild-type and QS mutants showed a similar level of disease. Our results demonstrated that only the glaI1/glaR1-mediated QS system is active in MAFF 302385, but major virulence factors, especially toxoflavin, are not completely dependent on the QS system.

Many plant pathogenic bacteria regulate the expression of virulence factors via N-acylhomoserine lactone (AHL), a quorum-sensing signaling compound. When numerous spore-forming bacteria were isolated from a natural environment, Priestia megaterium was the dominant species, and some P. megaterium strains exhibited AHL-degrading activity. The results of a HPLC ana-lysis of AHL degradation products demonstrated that P. megaterium degraded AHL by AHL lactonase, which hydrolyzes the lactone ring of AHL. The novel AHL lactonase gene, aiiB, was found in the whole genome sequence of AHL-degrading P. megaterium. The relationship between the presence of aiiB and AHL-degrading activity in P. megaterium strains revealed that P. megaterium may be classified into three AHL degradation groups: Group 1 (with AHL-degrading activity and aiiB), Group 2 (with neither AHL-degrading activity nor aiiB), and Group 3 (without AHL-degrading activity, but with aiiB). A comparative genome ana-lysis suggested that aiiB was obtained or missed by a non-transpositional event during the process of evolution in P. megaterium. The amino acid sequences of AiiB in Group 1 and 3 strains were almost identical, and Escherichia coli harboring aiiB from Groups 1 and 3 exhibited high AHL-degrading activity. Although the AHL-degrading activity of Group 3 strains was markedly weaker than that of Group 1 strains, they degraded AHL in a long-term incubation. Based on the present results, Group 1 and 3 strains, the genomes of which contain aiiB, may reduce potato maceration activity under the control of AHL-mediated quorum sensing in P. carotovorum subsp. carotovorum NBRC 12380.

Cu/Zn-superoxide dismutase (Cu/Zn-SOD) is an antioxidant enzyme widely present across species; however, the structural diversity and physiological roles of Cu/Zn-SOD are yet to be fully uncovered. Here, we show a unique type of Cu/Zn-SOD from Deinococcus radiodurans (DrSOD) with an additional β-propeller domain. Our structural analysis of DrSOD revealed a typical bacterial Cu/Zn-SOD domain, binding both a copper and zinc ion, alongside a six-bladed β-propeller domain coordinating a calcium ion. DrSOD was indeed expressed in D. radiodurans, but its deletion did not lead to any noticeable changes in resistance to DNA-damaging stresses, a characteristic trait of D. radiodurans. Despite this, the Cu/Zn-SOD domain retained superoxide dismutase activity, and the β-propeller domain was found to exhibit a lactonase activity specifically for hydrolyzing 2-coumaranone. Taken together, while the precise physiological role of DrSOD needs to be further investigated, our findings here reveal a unique multi-functional enzyme architecture, expanding the known structural diversity of Cu/Zn-SODs.

A spatiotemporal understanding of gene function requires the precise control of gene expression in each cell. Here, we use an infrared laser-evoked gene operator (IR-LEGO) system to induce gene expression at the single-cell level in the moss Physcomitrium patens by heating a living cell with an IR laser and thereby activating the heat shock response. We identify the laser irradiation conditions that provide higher inducibility with lower invasiveness by changing the laser power and irradiation duration. Furthermore, we quantitatively characterize the induction profile of the heat shock response using a heat-induced fluorescence reporter system after the IR laser irradiation of single cells under different conditions. Our data indicate that IR laser irradiation with long duration leads to higher inducibility according to increase in the laser power but not vice versa, and that the higher laser power even without conferring apparent damage to the cells decelerates and/or delayed gene induction. We define the temporal shift in expression as a function of onset and duration according to laser power and irradiation duration. This study contributes to the versatile application of IR-LEGO in plants and improves our understanding of heat shock-induced gene expression.

Some strains of nonpathogenic Allorhizobium vitis can control crown gall disease in grapevines caused by pathogenic A. vitis and are considered candidates for biocontrol agents. Many plant pathogenic bacteria regulate the expression of their virulence genes via quorum sensing using N-acylhomoserine lactone (AHL) as a signaling compound. The eight nonpathogenic A. vitis strains used in this study showed AHL-degrading activity. The complete genome sequence of A. vitis MAFF 212306 contained three AHL lactonase gene homologs. When these genes were cloned and transformed into Escherichia coli DH5α, E. coli harboring the aiiV gene (RvVAR031_27660) showed AHL-degrading activity. The aiiV coding region was successfully amplified by polymerase chain reaction from the genomes of all eight strains of nonpathogenic A. vitis. Purified His-tagged AiiV exhibited AHL lactonase activity by hydrolyzing the lactone ring of AHL. AiiV had an optimal temperature of approximately 30 °C; however, its thermostability decreased above 40 °C. When the AiiV-expressing plasmid was transformed into Pectobacterium carotovorum subsp. carotovorum NBRC 3830, AHL production by NBRC 3830 decreased below the detection limit, and its maceration activity, which was controlled by quorum sensing, almost disappeared. These results suggest the potential use of AHL-degrading nonpathogenic A. vitis for the inhibition of crown gall disease in grapevines and other plant diseases controlled by quorum sensing.

Inhibiting biofilm formation triggered by a quorum sensing (QS) mechanism is promising in industries where biofilms cause deterioration of production performance. QS can be disrupted by degrading or scavenging the signal compounds that mediate QS, such as N-acyl-homoserine lactones (AHLs). This study investigated the effect of lactonase, an AHL-degrading enzyme, on bacterial biofilm formation; we applied lactonase QsdS, produced by Sphingopyxis sp. strain EG6, which was isolated from a cooling water system, to biofilms of Pseudomonas putida strain TS312, which was isolated from white water in a paper mill. Addition of QsdS lactonase inhibited biofilm formation by P. putida strain TS312; the magnitude of the effect was Qsds lactonase dosedependent, decreasing the biofilm mass by 85% at a Qsds lactonase concentration of 10 mu g/ mL. Addition of QsdS lactonase decreased AHL concentrations in the culture supernatant of P. putida strain TS312, indicating that degradation of AHLs inhibited biofilm formation by disrupting QS. Observing three-dimensional biofilms by confocal laser scanning microscopy (CLSM) and optical coherence tomography (OCT) showed that addition of QsdS lactonase decreased the secretion of alpha-polysaccharides by P. putida strain TS312.

Phytopathogenic bacteria (MAFF 302110T and MAFF 302107) were isolated from lesions on Japanese angelica trees affected by bacterial soft rot in Yamanashi Prefecture, Japan. The strains were Gram-reaction-negative, facultatively anaerobic, motile with peritrichous flagella, rod-shaped, and non-spore-forming. The genomic DNA G+C content was 51.1 mol % and the predominant cellular fatty acids included summed feature 3 (C16 : 1 ω7c and/or C16 : 1 ω6c), C16 : 0, summed feature 8 (C18 : 1 ω7c and/or C18 : 1 ω6c), summed feature 2 (comprising any combination of C12 : 0 aldehyde, an unknown fatty acid with an equivalent chain length of 10.928, C16 : 1 iso I, and C14 : 0 3OH), and C12 : 0. Phylogenetic analyses based on 16S rRNA and gyrB gene sequences, along with phylogenomic analysis utilizing whole-genome sequences, consistently placed these strains within the genus Pectobacterium. However, their phylogenetic positions did not align with any known species within the genus. Comparative studies involving average nucleotide identity and digital DNA–DNA hybridization with the closely related species indicated values below the thresholds employed for the prokaryotic species delineation (95–96 % and 70 %, respectively), with the highest values observed for Pectobacterium polonicum DPMP315T (92.10 and 47.1 %, respectively). Phenotypic characteristics, cellular fatty acid composition, and a repertoire of secretion systems could differentiate the strains from their closest relatives. The phenotypic, chemotaxonomic, and genotypic data obtained in this study show that MAFF 302110T/MAFF 302107 represent a novel species of the genus Pectobacterium, for which we propose the name Pectobacterium araliae sp. nov., designating MAFF 302110T (=ICMP 25161T) as the type strain.

Root-colonizing fluorescent pseudomonads, represented by Pseudomonas protegens, produce a number of antibiotic secondary metabolites and extracellular enzymes that contribute to the suppression of pathogens in the rhizosphere. An antibiotic exoproduct that is commonly produced by P. protegens and related strains is 2,4-diacetylphloroglucinol (DAPG). We recently screened and characterized DAPG-producing strains among ca. 3000 fluorescent pseudomonads isolated from samples collected in the field in Japan. The genome data obtained will provide insights into the characteristics of novel factor(s) contributing to the antibiotic activities of newly isolated strain(s). DAPG-producing strains were subjected to a comparative genomic analysis, and the results obtained showed the accumulation of strain-specific genomic repertoires for the biosynthesis of secondary metabolites that prevent soil-borne diseases. Exoproduct expression levels were dependent on the Gac/Rsm signal transduction pathway, indicating the conservation of this mechanism in these strains. We herein propose a number of strategies to control these useful strains by promoting this signal transduction pathway in the rhizosphere.

Many gram-negative pathogens can activate virulence factors under the control of N-acylhomoserine lactone (AHL）-mediated quorum sensing. AHL-degrading enzymes have been investigated for their application in disease control. Trichoderma is a genus of fungi inhabiting various types of soil and are widely used as biocontrol agents for plant pathogens. When the AHL-degrading activity of 33 strains belonging to Trichoderma species was investigated, most strains can degrade AHL. AHL lactonase catalyzes AHL ring opening by hydrolyzing lactone. Two model strains, Trichoderma atroviride MAFF 242473 and MAFF 242475, degrade AHL using their AHL lactonase activity and rapidly metabolize ring-opening AHL. Moreover, co-inoculation with MAFF 242473 and MAFF 242475 effectively inhibited AHL production by the plant pathogens, Pantoea ananatis and Pectobacterium carotovorum subsp. carotovorum. Our study suggested that Trichoderma might be an effective biocontrol agent to inhibit the expression of virulence factors via AHL-mediated quorum sensing.

Broad-spectrum biocontrol by Pseudomonas protegens CHA0 and other fluorescent pseudomonads is achieved through the generation of various secondary metabolites with antibiotic activities against not only other microbes but, also, nematodes and insects present in the rhizosphere. A previous metabolomic study demonstrated that intracellular low-molecular weight effectors, such as guanosine tetraphosphate and γ-aminobutyrate, function as important signals in niche adaptation by strain CHA0 to plant roots. We investigated the role of amino acids in the biocontrol trait of P. protegens Cab57 towards Pythium damping off and root rot in cucumber. Among the 11 amino acids tested, only glutamate markedly enhanced the efficacy of biocontrol. An RNA-Seq analysis revealed that glutamate upregulated the expression of a chitinase gene cluster (c21370-c21380, in which the c21370 gene was annotated as a gene encoding the chitin-binding protein cbp and the c21380 gene encoded chitinase chiC) in strain CHA0. Glutamate upregulated the expression of the regulatory small RNA rsmZ but reduced the production levels of other Gac/Rsm-regulated biocontrol factors, such as 2,4-diacetylphloroglucinol and pyoluteorin. The promoter activity of cbp and chitinase activity were characterized in detail; their activities were up-regulated in response to glutamate and their expression was under the control of GacA. Therefore, glutamate appears to be essential for biocontrol activity in which chitinase production is regulated in response to glutamate. [Formula: see text] Copyright © 2023 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Pseudomonas putida is a major species belonging to the genus Pseudomonas. Although several hundred strains of P. putida have been deposited in culture collections, they potentially differ from the genetically defined "true Pseudomonas putida" because many were classified as P. putida based on their phenotypic and metabolic characteristics. A phylogenetic ana-lysis based on the concatenated sequences of the 16S rRNA and rpoD genes revealed that 46 strains of P. putida deposited in Japanese culture collections were classified into nine operational taxonomic units (OTUs) and eleven singletons. The OTU7 strain produces N-acylhomoserine lactone as a quorum-sensing signal. One of the OTU7 strains, JCM 20066, exhibited a ppuI-rsaL-ppuR quorum-sensing system that controls biofilm formation and motility. The P. putida type strain JCM 13063T and six other strains were classified as OTU4. Classification based on the calculation of whole-genome similarity revealed that three OTU4 strains, JCM 20005, 21368, and 13061, were regarded as the same species as JCM 13063T and defined as true P. putida. When orthologous genes in the whole-genome sequences of true P. putida strains were screened, PP4_28660 from P. putida NBRC 14164T (=JCM 13063T) was present in all true P. putida genome sequences. The internal region of PP4_28660 was successfully amplified from all true P. putida strains using the specific primers designed in this study.

The search for bacteria that can be used as biocontrol agents to control crop diseases yielded a promising candidate, Sm006T, which was isolated from the rhizosphere of eggplant (Solanum melongena) growing in a field in Aichi Prefecture, Japan, in 2006. The cells were Gram-stain-negative, aerobic, non-spore-forming, rod-shaped and motile with one polar flagellum. The results of homology searches and phylogenetic analyses based on the 16S rRNA gene sequence indicated that Sm006T represents a member of the genus Pseudomonas. The genomic DNA G+C content was 66.3 mol% and the major cellular fatty acids (more than 5 % of the total fatty acids) were summed feature 8 (C18 : 1ω7c and/or C18 : 1ω6c), summed feature 3 (C16 : 1ω7c and/or C16 : 1ω6c), C16 : 0 and C12 : 0. Phylogenetic analyses using the rpoD gene sequence and phylogenomic analysis of the whole genome sequence revealed that Sm006T represents a member of the Pseudomonas resinovorans group; however, its phylogenetic position does not match that of any known species of the genus Pseudomonas. The average nucleotide identity and digital DNA-DNA hybridisation values between the strain and closely related species were lower than the thresholds for prokaryotic species delineation (95-96 and 70 %, respectively), with the highest values observed for Pseudomonas tohonis TUM18999T (92.05 and 46.3 %, respectively). Phenotypic characteristics, cellular fatty acid composition and possession of 2,4-diacetylphloroglucinol biosynthetic gene cluster could be used to differentiate the strain from its closest relatives. The phenotypic, chemotaxonomic and genotypic data obtained during this study indicated that Sm006T represents a novel species of the genus Pseudomonas, for which we propose the name Pseudomonas solani sp. nov., with Sm006T (= MAFF 212523T = ICMP 24689T) as the type strain.