Selection of available suicide vectors for gene mutagenesis using chiA (a chitinase encoding gene) as a new reporter and primary functional analysis of chiA in Lysobacter enzymogenes strain OH11

Here, three different suicide vectors were evaluated for the possibility of performing gene mutagenesis in strain OH11 using the chiA gene (accession number: DQ888611) as a new reporter. Suicide vector pEX18GM was selected, and it was successfully applied for disruption and in-frame deletions in the chiA gene in strain OH11, which was confirmed by PCR amplification and Southern hybridization. The chiA-deletion mutant OH11-3 did not have the ability to produce chitinase on chitine selection medium. Interestingly, the chiA-deletion mutants displayed wild-type antimicrobial activity against Saccharomyces cerevisiae, Magnaporthe grisea, Phytophthora capsici, Rhizoctonia solani, Sclerotinia sclerotiorum and Pythium ultimum. Our data suggest that chitinase might not be a unique lytic enzyme in controlling S. cerevisiae, M. grisea, P. capsici, and P. ultimum. R. solani, S. sclerotiorum. Also, suicide vector pEX18GM might be explored as a potential tool for gene deletions in L. enzymogenes, which will facilitate the molecular study of mechanisms of biological control in L. enzymogenes.     

Lysobacter enzymogenes antagonizes soilborne bacteria using the type IV secretion system

Soil microbiome comprises numerous microbial species that continuously interact with each other. Among the modes of diverse interactions, cell–cell killing may play a key role in shaping the microbiome composition. Bacteria deploy various secretion systems to fend off other microorganisms and Type IV Secretion System (T4SS) in pathogenic bacteria was shown to function as a contact-dependent, inter-bacterial killing system only recently. The present study investigated the role played by T4SS in the killing behaviour of the soilborne biocontrol bacterium Lysobacter enzymogenes OH11. Results showed that L. enzymogenes OH11 genome encompasses genes encoding all the components of T4SS and effectors potentially involved in inter-bacterial killing system. Generation of knock-out mutants revealed that L. enzymogenes OH11 uses T4SS as the main contact-dependent weapon against other soilborne bacteria. The T4SS-mediated killing behaviour of L. enzymogenes OH11 decreased the antibacterial and antifungal activity of two Pseudomonas spp. but at the same time, protected carrot from infection by Pectobacterium carotovorum. Overall, this study showed for the first time the involvement of T4SS in the killing behaviour of L. enzymogenes and its impact on the multiple interactions occurring in the soil microbiome.     

Expression of Bacillus thuringiensis mosquitocidal toxin in an antimicrobial Bacillus brevis strain

Brown blotch is a common disease of many mushrooms, especially oyster mushrooms, in Korea. Recently, we isolated a promising Bacillus brevis strain which has antimicrobial activity against Pseudomonas tolaasii, a serious mushroom pathogen. In this study, in order to confer insecticidal activity, a recombinant B. brevis strain was constructed via the expression of Bacillus thuringiensis mosquitocidal crystal protein. A B. thuringiensis expression vector (pPro11A), which contained the cry11Aa gene under the control of cry1Ac promoter, was introduced into this B. brevis strain. The recombinant B. brevis strain successfully expressed and produced rhomboidal shaped Cry11A protein, although the initiation time of expression was slower than that of B. thuringiensis Cry^-B transformant. The insecticidal and antimicrobial activities of the transformant were verified using two dipteran larvae, Aedes aegypti and Culex pipiens, and four bacteria, including P. tolaasii, respectively. These results demonstrate that the introduction of B. thuringiensis insecticidal activity to antimicrobial Bacillus strains might be a useful tool to construct dual functional Bacillus strains.     

Identification of Quorum-Quenching N-Acyl Homoserine Lactonases from Bacillus Species

A range of gram-negative bacterial species use N-acyl homoserine lactone (AHL) molecules as quorum-sensing signals to regulate different biological functions, including production of virulence factors. AHL is also known as an autoinducer. An autoinducer inactivation gene, aiiA, coding for an AHL lactonase, was cloned from a bacterial isolate, Bacillus sp. strain 240B1. Here we report identification of more than 20 bacterial isolates capable of enzymatic inactivation of AHLs from different sources. Eight isolates showing strong AHL-inactivating enzyme activity were selected for a preliminary taxonomic analysis. Morphological phenotypes and 16S ribosomal DNA sequence analysis indicated that these isolates probably belong to the species Bacillus thuringiensis. Enzymatic analysis with known Bacillus strains confirmed that all of the strains of B. thuringiensis and the closely related species B. cereus and B. mycoides tested produced AHL-inactivating enzymes but B. fusiformis and B. sphaericus strains did not. Nine genes coding for AHL inactivation were cloned either by functional cloning or by a PCR procedure from selected bacterial isolates and strains. Sequence comparison of the gene products and motif analysis showed that the gene products belong to the same family of AHL lactonases.     

MomL,a Novel Marine-Derived N-Acyl Homoserine Lactonase from Muricauda olearia

Gram-negative bacteria use N-acyl homoserine lactones (AHLs) as quorum sensing (QS) signaling molecules for interspecies communication, and AHL-dependent QS is related with virulence factor production in many bacterial pathogens. Quorum quenching, the enzymatic degradation of the signaling molecule, would attenuate virulence rather than kill the pathogens, and thereby reduce the potential for evolution of drug resistance. In a previous study, we showed that Muricauda olearia Th120, belonging to the class Flavobacteriia, has strong AHL degradative activity. In this study, an AHL lactonase (designated MomL), which could degrade both short- and long-chain AHLs with or without a substitution of oxo-group at the C-3 position, was identified from Th120. Liquid chromatography-mass spectrometry analysis demonstrated that MomL functions as an AHL lactonase catalyzing AHL degradation through lactone hydrolysis. MomL is an AHL lactonase belonging to the metallo-β-lactamase superfamily that harbors an N-terminal signal peptide. The overall catalytic efficiency of MomL for C₆-HSL is ∼2.9 × 10⁵ s⁻¹ M⁻¹. Metal analysis and site-directed mutagenesis showed that, compared to AiiA, MomL has a different metal-binding capability and requires the histidine and aspartic acid residues for activity, while it shares the “HXHXDH” motif with other AHL lactonases belonging to the metallo-β-lactamase superfamily. This suggests that MomL is a representative of a novel type of secretory AHL lactonase. Furthermore, MomL significantly attenuated the virulence of Pseudomonas aeruginosa in a Caenorhabditis elegans infection model, which suggests that MomL has the potential to be used as a therapeutic agent.     

The acyl-homoserine lactone-type quorum-sensing system modulates cell motility and virulence of Erwinia chrysanthemi pv. zeae

Erwinia chrysanthemi pv. zeae is one of the Erwinia chrysanthemi pathovars that infects on both dicotyledons and monocotyledons. However, little is known about the molecular basis and regulatory mechanisms of its virulence. By using a transposon mutagenesis approach, we cloned the genes coding for an E. chrysanthemi pv. zeae synthase of acyl-homoserine lactone (AHL) quorum-sensing signals (expI_Ecz) and a cognate response regulator (expR_Ecz). Chromatography analysis showed that expI_Ecz encoded production of the AHL signal N-(3-oxo-hexanoyl)-homoserine lactone (OHHL). Null mutation of expI_Ecz in the E. chrysanthemi pv. zeae strain EC1 abolished AHL production, increased bacterial swimming and swarming motility, disabled formation of multicell aggregates, and attenuated virulence of the pathogen on potato tubers. The mutation also marginally reduced the inhibitory activity of E. chrysanthemi pv. zeae on rice seed germination. The mutant phenotypes were rescued by either exogenous addition of AHL signal or in trans expression of expI_Ecz. These data demonstrate that the AHL-type QS signal plays an essential role in modulation of E. chrysanthemi pv. zeae cell motility and the ability to form multicell aggregates and is involved in regulation of bacterial virulence.     

Negative Cross-Communication among Wheat Rhizosphere Bacteria: Effect on Antibiotic Production by the Biological Control Bacterium Pseudomonas aureofaciens 30-84

Phenazine antibiotic production in the biological control bacterium Pseudomonas aureofaciens 30-84 is regulated in part via the PhzR/PhzI N-acyl homoserine lactone (AHL) system. Previous work showed that a subpopulation of the wheat rhizosphere community positively affected phenazine gene expression in strain 30-84 via AHL signals (E. A. Pierson, D. W. Wood, J. A. Cannon, F. M. Blachere, and L. S. Pierson III, Mol. Plant-Microbe Interact. 11:1078-1084, 1998). In the present work, a second subpopulation, one that negatively affected phenazine gene expression, was identified from this rhizosphere community. Strain 30-84 grown in conditioned medium (CM) from several strains produced lower levels of phenazines (1.5- to 9.3-fold) than control when grown in CM from the strain 30-84I₁/I₂. Growth of the phzB::lacZ reporter strain 30-84Z in this CM resulted in decreased lacZ expression (4.3- to 9.2-fold) compared to growth of the control strain in CM, indicating that inhibition of phzB occurred at the level of gene expression. Preliminary chemical and biological characterizations suggested that these signals, unlike other identified negative signals, were not extractable in ethyl acetate. Introduction of extra copies of phzR and phzI, but not phzI alone, in trans into strain 30-84Z reduced the negative effect on phzB::lacZ expression. The presence of negative-signal-producing strains in a mixture with strain 30-84 reduced strain 30-84's ability to inhibit the take-all disease pathogen in vitro. Together, the results from the previous work on the positive-signal subpopulation and the present work on the negative-signal subpopulation suggest that cross-communication among members of the rhizosphere community and strain 30-84 may control secondary metabolite production and pathogen inhibition.     

Nonbioluminescent Strains of Photobacterium phosphoreum Produce the Cell-to-Cell Communication Signal N-(3-Hydroxyoctanoyl)homoserine Lactone

Bioluminescence is a common phenotype in marine bacteria, such as Vibrio and Photobacterium species, and can be quorum regulated by N-acylated homoserine lactones (AHLs). We extracted a molecule that induced a bacterial AHL monitor (Agrobacterium tumefaciens NT1 [pZLR4]) from packed cod fillets, which spoil due to growth of Photobacterium phosphoreum. Interestingly, AHLs were produced by 13 nonbioluminescent strains of P. phosphoreum isolated from the product. Of 177 strains of P. phosphoreum (including 18 isolates from this study), none of 74 bioluminescent strains elicited a reaction in the AHL monitor, whereas 48 of 103 nonbioluminescent strains did produce AHLs. AHLs were also detected in Aeromonas spp., but not in Shewanella strains. Thin-layer chromatographic profiles of cod extracts and P. phosphoreum culture supernatants identified a molecule similar in relative mobility (R_f value) and shape to N-(3-hydroxyoctanoyl)homoserine lactone, and the presence of this molecule in culture supernatants from a nonbioluminescent strain of P. phosphoreum was confirmed by high-performance liquid chromatography-positive electrospray high-resolution mass spectrometry. Bioluminescence (in a non-AHL-producing strain of P. phosphoreum) was strongly up-regulated during growth, whereas AHL production in a nonbioluminescent strain of P. phosphoreum appeared constitutive. AHLs apparently did not influence bioluminescence, as the addition of neither synthetic AHLs nor supernatants delayed or reduced this phenotype in luminescent strains of P. phosphoreum. The phenotypes of nonbioluminescent P. phosphoreum strains regulated by AHLs remains to be elucidated.     

Phaeobacter sp. Strain Y4I Utilizes Two Separate Cell-to-Cell Communication Systems To Regulate Production of the Antimicrobial Indigoidine

The marine roseobacter Phaeobacter sp. strain Y4I synthesizes the blue antimicrobial secondary metabolite indigoidine when grown in a biofilm or on agar plates. Prior studies suggested that indigoidine production may be, in part, regulated by cell-to-cell communication systems. Phaeobacter sp. strain Y4I possesses two luxR and luxI homologous N-acyl-l-homoserine lactone (AHL)-mediated cell-to-cell communication systems, designated pgaRI and phaRI. We show here that Y4I produces two dominant AHLs, the novel monounsaturated N-(3-hydroxydodecenoyl)-l-homoserine lactone (3OHC_12:1-HSL) and the relatively common N-octanoyl-l-homoserine lactone (C₈-HSL), and provide evidence that they are synthesized by PhaI and PgaI, respectively. A Tn5 insertional mutation in either genetic locus results in the abolishment (pgaR::Tn5) or reduction (phaR::Tn5) of pigment production. Motility defects and denser biofilms were also observed in these mutant backgrounds, suggesting an overlap in the functional roles of these systems. Production of the AHLs occurs at distinct points during growth on an agar surface and was determined by isotope dilution high-performance liquid chromatography–tandem mass spectrometry (ID-HPLC-MS/MS) analysis. Within 2 h of surface inoculation, only 3OHC_12:1-HSL was detected in agar extracts. As surface-attached cells became established (at ∼10 h), the concentration of 3OHC_12:1-HSL decreased, and the concentration of C₈-HSL increased rapidly over 14 h. After longer (>24-h) establishment periods, the concentrations of the two AHLs increased to and stabilized at ∼15 nM and ∼600 nM for 3OHC_12:1-HSL and C₈-HSL, respectively. In contrast, the total amount of indigoidine increased steadily from undetectable to 642 μM by 48 h. Gene expression profiles of the AHL and indigoidine synthases (pgaI, phaI, and igiD) were consistent with their metabolite profiles. These data provide evidence that pgaRI and phaRI play overlapping roles in the regulation of indigoidine biosynthesis, and it is postulated that this allows Phaeobacter sp. strain Y4I to coordinate production of indigoidine with different growth-phase-dependent physiologies.     

Quorum-sensing regulation in soil pseudomonads

228 strains of soil and rhizosphere pseudomonads isolated in different geographic zones were screened, with the use of two tester systems, for the capacity to produce N-acyl-homoserine lactones (AHLs), which are autoinducers involved in quorum-sensing (QS) regulation. AHL production was found in 11.4% of the strains investigated. In five Pseudomonas chlororaphis strains shown to be active AHL producers and chosen for further study, PCR identified two QS systems that involved the phzI, phzR, csaI, and csaR genes; this finding suggests the conservative nature of these regulation systems in P. chloroaphis. Strain P. chlororaphis 449, chosen as a model object and studied in greater detail, produced three AHL species including N-butanoyl-homoserine lactone and N-hexanoyl-homoserine lactone. This strain produced three types of phenazine antibiotics, as well as siderophores and cyanide; it also exhibited antagonistic properties toward a wide spectrum of phytopathogenic fungi. The phzI and csaI genes, coding for synthases of AHLs of two types, were cloned and sequenced; mutants with knocked-out phzI and csaI genes were obtained. With the use of transposon mutagenesis and the gene substitution method, mutations were obtained in the global expression regulator genes gacS, coding for the GacA-GacS regulation system kinase, and rpoS, coding for the sigma S subunit of RNA polymerase. The effect of these mutations on the AHL synthesis and on the regulation of various metabolic processes in P. chlororaphis was studied.     

The quorum sensing molecule N-acyl homoserine lactone produced by Acinetobacter baumannii displays antibacterial and anticancer properties

Secretory N-acyl homoserine lactones (AHLs) mediate quorum sensing (QS) in bacteria. AHLs are shown to be inhibitory for an unrelated group of bacteria and might mimic host signalling elements, thereby subverting the regulatory events in host cells. This study investigated the AHL produced by Acinetobacter baumannii and analysed its effect on other bacterial species and mammalian cells. Chemically characterized AHL had an m/z value of 325 with a molecular formula C₁₈H₃₁NO₄ and showed its inhibitory potential against Staphylococcus aureus. Molecular docking studies identified D-alanine-D-alanine synthetase A, a cell wall synthesizing enzyme of S. aureus having a strong binding affinity towards AHL. Electron microscopy showed the disruption and sloughing off of the S. aureus cell wall when treated with AHL. In vitro experiments revealed that this bacteriostatic AHL showed time-dependent activity and induced apoptosis in cancer cell lines. This compound could be a potential structural backbone for constructing new AHL analogues against S. aureus. The findings emphasize the need to re-evaluate all previously characterized AHLs for any additional new biological functions other than QS.     

Type IV pilus biogenesis genes and their roles in biofilm formation in the biological control agent Lysobacter enzymogenes OH11

Type IV pilus (T4P) is widespread in bacteria, yet its biogenesis mechanism and functionality is only partially elucidated in a limited number of bacterial species. Here, by using strain OH11 as the model organism, we reported the identification of 26 T4P structural or functional component (SFC) proteins in the Gram-negative Lysobacter enzymogenes, which is a biocontrol agent potentially exploiting T4P-mediated twitching motility for antifungal activity. Twenty such SFC coding genes were individually knocked-out in-frame to create a T4P SFC deletion library. By using combined phenotypic and genetic approaches, we found that 14 such SFCs, which were expressed from four operons, were essential for twitching motility. These SFCs included the minor pilins (PilE_i, PilX_i, PilV_i, and FimT_i), the anti-retraction protein PilY1_i, the platform protein PilC, the extension/extraction ATPases (PilB, PilT, and PilU), and the PilMNOPQ complex. Among these, mutation of pilT or pilU caused a hyper piliation, while the remaining 12 SFCs were indispensable for pilus formation. Ten (FimT_i, PilY1_i, PilB, PilT, PilU, and the PilMNOPQ complex) of the 14 SFC proteins, as well as PilA, were further shown to play a key role in L. enzymogenes biofilm formation. Overall, our results provide the first report to dissect the genetic basis of T4P biogenesis and its role in biofilm formation in L. enzymogenes in detail, which can serve as an alternative platform for studying T4P biogenesis and its antifungal function.

     

A new self-assembled inorganic-organic hybrid layered compound containing hexarhenium cluster: [MV][{Mn(CH3OH)2}{Re6Se8(CN)6}]

A new layered compound, [MV][{Mn(CH₃OH)₂}{Re₆Se₈(CN)₆}] (1) consists of a layer alternately knitted by hexarhenium cluster and Mn complex, and MV²⁺ cations (methyl viologen dication = 1,1′-dimethyl-4,4′-bipyridilium dication) reside between the layers. The title compound 1 is the first layered framework containing cyano-hexarhenium clusters with photoactive guest molecules, MV²⁺. The MV²⁺ can be partly exchanged by H₂TMB²⁺ (N,N,N′,N′-tetramethylbenzidine dication) to form a compound [H₂TMB²⁺]_x[MV²⁺]_1−x [{Mn(CH₃OH)₂}{Re₆Se₈(CN)₆}] (2) showing an electronic interaction between the layered framework and [H₂TMB]²⁺ cation.     

Expression of a bifunctional cellulase with exoglucanase and endoglucanase activities to enhance the hydrolysis ability of cellulase from a marine Aspergillus niger

Low exoglucanase and endoglucanase activities of marine Aspergillus niger cellulase decreased the hydrolyzing ability of cellulase. To increase the activity of halostable cellulase obtained from a marine A. niger, a cellulase with endoglucanase and exoglucanase activity was efficiently expressed by constructing a vector with promoter glaA. Exoglucanase and endoglucanase activities increased from 0.21 and 4.51 U/ml of the original strain to 0.89 U/ml and 15.12 U/ml of the transformant, respectively. Filter paper activity (FPA) increased by 7.1 folds from 0.63 to 4.47 U/ml. The release of glucose by hydrolysis of wheat straw with cellulase from the transformant was 1.37 folds higher than that with cellulase from the original strain under high salinity condition. Cellulase with endoglucanase and exoglucanase activities could be well expressed in marine A. niger. The cellulase from the transformant not only showed higher activity, but also retained halostability. An appreciate proportion of β-glucosidase, exoglucanase, endgolucanasein cellulase was important for hydrolyzing cellulose.     

Restraining Erwinia virulence by expression of N-acyl homoserine lactonase gene pro3A-aiiA in Bacillus thuringiensis subsp leesis

To widen the biological control function of a genetically modified Bacillus thuringiensis subsp leesis strain BMB-005, an acyl homoserine lactonase (AHL lactonase) gene aiiA transcribed by the promoter of insecticidal crystal protein coding gene cry3A, was transformed into strain BMB-005. The amount of AHL lactonase protein produced by transformant BMB821A was 2.4-fold more than that produced by BMB-005. AHL-degradation assay showed that transformant BMB821A could degrade more AHLs molecules than the original strain BMB-005. The result of Erwinia carotovora pathogenicity test showed that the parental strain BMB-005 had no restraint of Erwinia infection, but the transformants exhibited strong restraint of E. carotovora infection on potato slices and cactus stems. Insecticidal bioassay against lepidopteran Spodoptera exigua showed that both strain BMB-005 and transformant BMB821A were toxic to S. exigua. The toxicity of transformant BMB821A (LC(50) was 3.8) was a little attenuated comparing with the toxicity of the original strain BMB-005 (LC(50) was 2.9). The B. thuringiensis strain BMB-005 has high toxicity against Helicoverpa armigera, Plutella xylostella, and S. exigua. This work provided new strategy for developing genetically engineered multi-functional B. thuringiensis strain that possesses insecticidal activity together with restraint of bacterial pathogenicity.     

Biofouling inhibition in MBR by Rhodococcus sp. BH4 isolated from real MBR plant

It has been reported that an indigenous quorum quenching bacterium, Rhodococcus sp. BH4, which was isolated from a real plant of membrane bioreactor (MBR) has promising potential to control biofouling in MBR. However, little is known about quorum quenching mechanisms by the strain BH4. In this study, various characteristics of strain BH4 were investigated to elucidate its behavior in more detail in the mixed liquor of MBR. The N-acyl homoserine lactone hydrolase (AHL–lactonase) gene of strain BH4 showed a high degree of identity to qsdA in Rhodococcus erythropolis W2. The LC-ESI-MS analysis of the degradation product by strain BH4 confirmed that it inactivated AHL activity by hydrolyzing the lactone bond of AHL. It degraded a wide range of N-acyl homoserine lactones (AHLs), but there was a large difference in the degradation rate of each AHL compared to other reported AHL–lactonase-producing strains belonging to Rhodococcus genus. Its quorum quenching activity was confirmed not only in the Luria-Bertani medium, but also in the synthetic wastewater. Furthermore, the amount of strain BH4 encapsulated in the vessel as well as the material of the vessel substantially affected the quorum quenching activity of strain BH4, which provides useful information, particularly for the biofouling control in a real MBR plant from an engineering point of view.