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.     

Production of two phosphatases by Lysobacter enzymogenes and purification and characterization of the extracellular enzyme.

Lysobacter enzymogenes produces an extracellular phosphatase (EC. 3.1.3.1) during the stationary phase of growth. The cells also produce a cell-associated alkaline phosphatase. This enzyme is found in the particulate fraction of cell extracts and may be membrane bound. The production of both phosphatases, especially the extracellular enzyme, is reduced by inorganic phosphate. The extracellular phosphatase was purified to a specific activity of 270 U/mg primarily by chromatography on carboxymethyl cellulose and gel filtration. The enzyme is stable under normal storage conditions but is rapidly inactivated above 70 degrees. It consists of one polypeptide with an approximate molecular weight of 25,000. The pH optimum is 7.5, and the Km for p-nitrophenylphosphate is 2.2 X 10(-4) M. The enzyme degrades a number of other phosphomonoesters but at a reduced rate compared with the rate obtained with p-nitrophenylphosphate. Phosphate and arsenate inhibit the enzyme, but EDTA and other chelating agents have no effect. The lack of a metal ion requirement for activity, the lower molecular weight, the soluble nature of the enzyme, and the lower pH optimum clearly distinguish the extracellular phosphatase from the cell-associated phosphatase and from other bacterial phosphatases.     

Influence of Lysobacter enzymogenes Strain C3 on Nematodes

Chitinolytic microflora may contribute to biological control of plant-parasitic nematodes by causing decreased egg viability through degradation of egg shells. Here, the influence of Lysobacter enzymogenes strain C3 on Caenorhabditis elegans, Heterodera schachtii, Meloidogyne javanica, Pratylenchus penetrans, and Aphelenchoides fragariae is described. Exposure of C. elegans to L. enzymogenes strain C3 on agar resulted in almost complete elimination of egg production and death of 94% of hatched juveniles after 2 d. Hatch of H. schachtii eggs was about 50% on a lawn of L. enzymogenes strain C3 on agar as compared to 80% on a lawn of E. coli. Juveniles that hatched on a lawn of L. enzymogenes strain C3 on agar died due to disintegration of the cuticle and body contents. Meloidogyne javanica juveniles died after 4 d exposure to a 7-d-old chitin broth culture of L. enzymogenes strain C3. Immersion of A. fragariae, M. javanica, and P. penetrans juveniles and adults in a nutrient broth culture of L. enzymogenes strain C3 led to rapid death and disintegration of the nematodes. Upon exposure to L. enzymogenes strain C3 cultures in nutrient broth, H. schachtii juveniles were rapidly immobilized and then lysed after three days. The death and disintegration of the tested nematodes suggests that toxins and enzymes produced by this strain are active against a range of nematode species.     

Beta-lactamase of Lysobacter enzymogenes: induction, purification and characterization

Lysobacter enzymogenes produces an inducible beta-lactamase and induction with 100 micrograms ampicillin ml-1 resulted in an increase of more than 100-fold in enzyme activity. Various other beta-lactam antibiotics also served as effective inducers. The enzyme was obtained from cells by osmotic shocking to release periplasmic components and it was purified primarily by ion-exchange chromatography and PAGE. The beta-lactamase consists of one polypeptide with a molecular mass of about 28 kDa and an isoelectric point greater than 9.6. It is strongly inhibited by p-chloromercuribenzoate and clavulanic acid but not by EDTA. The enzyme readily hydrolyses several penicillins and cephalosporins, but not oxacillin or cloxacillin. The enzyme therefore belongs to group 2b of the bacterial beta-lactamases.     

Use of endoproteinase Lys-C from Lysobacter enzymogenes in protein sequence analysis

Endoproteinase Lys-C from Lysobacter enzymogenes, which is commercially available, proved to be useful in the determination of primary structures of proteins. The enzyme preferentially cleaves at the carboxyl side of lysine residues.     

Nucleotide sequence and characterization of the gene for secreted alkaline phosphatase from Lysobacter enzymogenes.

Lysobacter enzymogenes produces an alkaline phosphatase which is secreted into the medium. The gene for the enzyme (phoA) was isolated from a recombinant lambda library. It was identified within a 4.4-kb EcoRI-BamH1 fragment, and its sequence was determined by the chain termination method. The structural gene consists of an open reading frame which encodes a 539-amino-acid protein with a 29-residue signal sequence, followed by a 119-residue propeptide, the 281-residue mature phosphatase, and a 110-residue carboxy-terminal domain. The roles of the propeptide and the carboxy-terminal peptide remain to be determined. A molecular weight of 30,000 was determined for the mature enzyme from sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The amino acid sequence was compared with sequences available in the current protein data base, and a region of the sequence was found to show considerable homology with sequences in mammalian type 5 iron-containing purple acid phosphatases.     

Extracellular nucleases of Lysobacter enzymogenes: production of the enzymes and purification and characterization of an endonuclease

Lysobacter enzymogenes produced a nonspecific extracellular nuclease and an extracellular RNAase when grown in tryptone broth. Both enzyme activities appeared after the exponential growth phase of the organism. The addition of RNA to the medium specifically inhibited the production of the nuclease and the addition of phosphate prevented the synthesis of the RNAase. DNA had no effect on the enzyme production. The Lysobacter nuclease was purified 274-fold and its molecular weight was estimated to be between 22 000 and 28 000. Freshly purified nuclease showed one major protein band and one major activity band on polyacrylamide gels, whereas two major bands were seen after prolonged storage of the enzyme. The nuclease was most active at pH 8.0 and required Mg2+ or Mn2+. Little activity was obtained in the presence of Ca2+. The enzyme degraded double-stranded DNA more rapidly than single-stranded DNA or RNA and was essentially inactive with poly(A) or poly(C) as the substrate. Extensive hydrolysis of double-stranded DNA by the enzyme yielded oligodeoxyribonucleotides with terminal 5'-phosphate groups. The Lysobacter RNAase appeared to have a molecular weight approximately twice that of the nuclease and was specific for ribonucleotide polymers.     

Specific detection of Lysobacter enzymogenes (Christensen and Cook 1978) strain 3.1T8 with TaqMan® PCR

Aims: To develop a strain‐specific TaqMan^® PCR method for detecting and quantifying the biocontrol strain Lysobacter enzymogenes 3.1T8. Methods and Results: A primer–probe combination was designed on the basis of a strain‐specific sequence selected using REP‐PCR (repetitive extragenic palindromic‐polymerase chain reaction). The specificity of this combination was demonstrated by 14 other Lysobacter strains that did not react with the selected primer–probe combination. To quantify strain 3.1T8 in cucumber root samples, a calibration curve was prepared by spiking roots with a 10‐fold dilution series of the strain. Detection of the biocontrol strain 3.1T8 with this method showed that the strain survived well for 22 days on root tips as well as on older cucumber roots. Survival was higher when the strain was inoculated to younger plants. In a cucumber production system with large volumes of substrate, strain 3.1T8 was detected in high numbers on cucumber roots 3 weeks after inoculation. Conclusions: The primer–probe combination developed was strain specific, because it did not react with other strains of the same species and genus. The TaqMan^® PCR method successfully quantified the inoculated biocontrol strain on cucumber roots grown in different cropping systems. Significance and Impact of the Study: The developed TaqMan^® PCR method is a strain‐specific real‐time detection method that can be used to assess the population dynamics of L. enzymogenes strain 3.1T8 for further optimization of its biocontrol efficacy.     

Systematic optimization for production of the anti-MRSA antibiotics WAP-8294A in an engineered strain of Lysobacter enzymogenes

WAP-8294A is a group of cyclic lipodepsipeptides and considered as the first-in-class new chemical entity with potent activity against methicillin-resistant Staphylococcus aureus. One of the roadblocks in developing the WAP-8294A antibiotics is the very low yield in Lysobacter. Here, we carried out a systematic investigation of the nutritional and environmental conditions in an engineered L. enzymogenes strain for the optimal production of WAP-8294A. We developed an activity-based simple method for quick screening of various factors, which enabled us to optimize the culture conditions. With the method, we were able to improve the WAP-8294A yield by 10-fold in small-scale cultures and approximately 15-fold in scale-up fermentation. Additionally, we found the ratio of WAP-8294A2 to WAP-8294A1 in the strains could be manipulated through medium optimization. The development of a practical method for yield improvement in Lysobacter will facilitate the ongoing basic research and clinical studies to develop WAP-8294A into true therapeutics.     

Reducing Pectobacterium virulence by expression of an N-acyl homoserine lactonase gene Plpp-aiiA in Lysobacter enzymogenes strain OH11

An N-acyl homoserine lactonase gene aiiA, transcribed by a strong and constitutive Escherichia coli promoter P_lpp (Accession No. EU723847), was transformed into Lysobacter enzymogenes strain OH11, creating strain OH11A. The N-acyl-homoserine lactone (AHL)-degradation assay showed that transformant OH11A acquired the ability to degrade AHL molecules produced by Agrobacterium tumefaciens, Pectobacterium carotovorum, Pseudomonas syringae pv. tomato strain DC3000 and Acidovorax avenae subsp. citrulli. Pathogenicity tests showed that while the parental strain OH11 did not reduce P. carotovorum infection, the transformant OH11A caused a strong reduction of Pectobacterium virulence on Chinese cabbage and cactus, whereas strain OH11A did not seem to interfere with the normal growth of this pathogen in cabbages. In antimicrobial activity assays, strain OH11A and OH11 showed similar antimicrobial activity against Phytophthora capsici and Sclerotinia sclerotiorum. This work provided a new strategy for developing genetically engineered multi-functional L. enzymogenes strains that possessed the ability to biologically control fungal pathogens and reduce bacterial pathogenicity.     

Unusual activities of the thioesterase domain for the biosynthesis of the polycyclic tetramate macrolactam HSAF in Lysobacter enzymogenes C3

HSAF is an antifungal natural product with a new mode of action. A rare bacterial iterative PKS-NRPS assembles the HSAF skeleton. The biochemical characterization of the NRPS revealed that the thioesterase (TE) domain possesses the activities of both a protease and a peptide ligase. Active site mutagenesis, circular dichroism spectra, and homology modeling of the TE structure suggested that the TE may possess uncommon features that may lead to the unusual activities. The iterative PKS-NRPS is found in all polycyclic tetramate macrolactam gene clusters, and the unusual activities of the TE may be common to this type of hybrid PKS-NRPS.     

Use of the tetrazolium salt MTT to measure cell viability effects of the bacterial antagonist Lysobacter enzymogenes on the filamentous fungus Cryphonectria parasitica

Despite substantial interest investigating bacterial mechanisms of fungal growth inhibition, there are few methods available that quantify fungal cell death during direct interactions with bacteria. Here we describe an in vitro cell suspension assay using the tetrazolium salt MTT as a viability stain to assess direct effects of the bacterial antagonist Lysobacter enzymogenes on hyphal cells of the filamentous fungus Cryphonectria parasitica. The effects of bacterial cell density, fungal age and the physiological state of fungal mycelia on fungal cell viability were evaluated. As expected, increased bacterial cell density correlated with reduced fungal cell viability over time. Bacterial effects on fungal cell viability were influenced by both age and physiological state of the fungal mycelium. Cells obtained from 1-week-old mycelia lost viability faster compared with those from 2-week-old mycelia. Likewise, hyphal cells obtained from the lower layer of the mycelial pellicle lost viability more quickly compared with cells from the upper layer of the mycelial pellicle. Fungal cell viability was compared between interactions with L. enzymogenes wildtype strain C3 and a mutant strain, DCA, which was previously demonstrated to lack in vitro antifungal activity. Addition of antibiotics eliminated contributions to MTT-formazan production by bacterial cells, but not by fungal cells, demonstrating that mutant strain DCA had lost complete capacity to reduce fungal cell viability. These results indicate this cell suspension assay can be used to quantify bacterial effects on fungal cells, thus providing a reliable method to differentiate strains during bacterial/fungal interactions.     

Two direct gene targets contribute to Clp-dependent regulation of type IV pilus-mediated twitching motility in Lysobacter enzymogenes OH11

Applied Microbiology and Biotechnology - Lysobacter enzymogenes is an agriculturally important Gram-negative bacterium that employs a multitude of antifungal mechanisms to inhibit and infect...     

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.

     

The alpha-lytic protease gene of Lysobacter enzymogenes. The nucleotide sequence predicts a large prepro-peptide with homology to pro-peptides of other chymotrypsin-like enzymes

alpha-Lytic protease is a 19.8-kDa protein secreted from the Gram-negative bacterium Lysobacter enzymogenes. We have cloned and sequenced the gene for this serine protease. The nucleotide sequence contains an open reading frame which codes for the 198-residue mature enzyme and a potential prepro-peptide, also of 198 residues. The COOH-terminal 49 residues of the pro-peptide are significantly homologous to the propeptides of Streptomyces griseus proteases A and B. We suggest that this pro-peptide region facilitates formation of the active enzyme. A region bridging the NH2-terminal pre- and pro-peptides is homologous to a maize inhibitor of serine proteases. We speculate that this region inhibits enzymatic activity of the prepro-enzyme.