Lysobacter enzymogenes Employs Diverse Genes for Inhibiting Hypha Growth and Spore Germination of Soybean Fungal Pathogens

Lysobacter enzymogenes strain C3 (LeC3) is a potential biocontrol agent for plant diseases caused by fungi and oomycetes. Understanding the interaction between LeC3 and soybean pathogens at the molecular level could help improve its biocontrol efficacy. In this study, we obtained mutants with decreased abilities in inhibiting hypha growth of the white mold pathogen Sclerotinia sclerotiorum. Insertion sites for 50 mutants, which no longer inhibited S. sclerotiorum hypha growth in dual cultural assay, were determined and seven mutants were selected for further characterization. These seven mutants also completely lost their abilities in suppressing spore germination of Fusarium virguliforme, the causal agent of soybean sudden death syndrome. Furthermore, mutation of the seven genes, which encode diguanylate cyclase, transcriptional regulators from the TetR family, hemolysin III family channel protein, type IV secretion system VirB10 protein, phenol hydroxylase, and phosphoadenosine phosphosulfate reductase, respectively, led to reduced production or secretion of four extracellular enzymes and heat-stable antifungal factor (HSAF). These results suggest that these seven genes play important roles in L. enzymogenes in suppressing hypha growth and spore germination of fungal pathogens, probably by influencing production or secretion of extracellular enzymes and HSAF.

Inactivation ofcysLInhibits Biofilm Formation by Activating the Disulfide Stress Regulator Spx inBacillus subtilis

ABSTRACTBacillus subtilisforms biofilms in response to internal and external stimuli. I previously showed that thecysLdeletion mutant was defective in biofilm formation, but the reason for this remains unidentified. CysL is a transcriptional activator of thecysJIoperon, which encodes sulfite reductase, an enzyme involved in cysteine biosynthesis. Decreased production of sulfite reductase led to biofilm formation defects in the ΔcysLmutant. The ΔcysLmutation was suppressed by disruptingcysHoperon genes, whose products function upstream of sulfite reductase in the cysteine biosynthesis pathway, indicating that defects in cysteine biosynthesis were not a direct cause for the defective biofilm formation observed in the ΔcysLmutant. ThecysHgene encodes phosphoadenosine phosphosulfate reductase, which requires a reduced form of thioredoxin (TrxA) as an electron donor. High expression oftrxAinhibited biofilm formation in the ΔcysLmutant but not in the wild-type strain. Northern blot analysis showed thattrxAtranscription was induced in the ΔcysLmutant in a disulfide stress-induced regulator Spx-dependent manner. On the basis of these results, I propose that the ΔcysLmutation causes phosphoadenosine phosphosulfate reductase to consume large amounts of reduced thioredoxin, inducing disulfide stress and activating Spx. Thespxmutation restored biofilm formation to the ΔcysLmutant. The ΔcysLmutation reduced expression of theepsoperon, which is required for exopolysaccharide production. Moreover, overexpression of theepsoperon restored biofilm formation to the ΔcysLmutant. Taken together, these results suggest that the ΔcysLmutation activates Spx, which then inhibits biofilm formation through repression of theepsoperon.IMPORTANCEBacillus subtilishas been studied as a model organism for biofilm formation. In this study, I explored why thecysLdeletion mutant was defective in biofilm formation. I demonstrated that the ΔcysLmutation activated the disulfide stress response regulator Spx, which inhibits biofilm formation by repressing biofilm matrix genes. Homologs of Spx are highly conserved among Gram-positive bacteria with low G+C contents. In some pathogens, Spx is also reported to inhibit biofilm formation by repressing biofilm matrix genes, even though these genes and their regulation are quite different from those ofB. subtilis. Thus, the negative regulation of biofilm formation by Spx is likely to be well conserved across species and may be an appropriate target for control of biofilm formation.

Isolation of New Stenotrophomonas Bacteriophages and Genomic Characterization of Temperate Phage S1

ABSTRACT Twenty-two phages that infect Stenotrophomonas species were isolated through sewage enrichment and prophage induction. Of them, S1, S3, and S4 were selected due to their wide host ranges compared to those of the other phages. S1 and S4 are temperate siphoviruses, while S3 is a virulent myovirus. The genomes of S3 and S4, about 33 and 200 kb, were resistant to restriction digestion. The lytic cycles lasted 30 min for S3 and about 75 min for S1 and S4. The burst size for S3 was 100 virions/cell, while S1 and S4 produced about 75 virus particles/cell. The frequency of bacteriophage-insensitive host mutants, calculated by dividing the number of surviving colonies by the bacterial titer of a parallel, uninfected culture, ranged between 10−5 and 10−6 for S3 and 10−3 and 10−4 for S1 and S4. The 40,287-bp genome of S1 contains 48 open reading frames (ORFs) and 12-bp 5′ protruding cohesive ends. By using a combination of bioinformatics and experimental evidence, functions were ascribed to 21 ORFs. The morphogenetic and lysis modules are well-conserved, but no lysis-lysogeny switch or DNA replication gene clusters were recognized. Two major clusters of genes with respect to transcriptional orientation were observed. Interspersed among them were lysogenic conversion genes encoding phosphoadenosine phosphosulfate reductase and GspM, a protein involved in the general secretion system II. The attP site of S1 may be located within a gene that presents over 75% homology to a Stenotrophomonas chromosomal determinant.

Proteoglycan sulfation and storage parallels storage of basic secretory proteins in exocrine cells

Increased storage of basic proline-rich secretory proteins induced in rat parotid acinar cells by isoproterenol is accompanied by increased storage of a chondroitin sulfate-containing proteoglycan. Amino acid analysis of the purified proteoglycan and the chondroitinase digestion products reveals that the polypeptide backbone is a proline-rich protein. Most sulfation occurs in Golgi elements; however, a small fraction of the proteoglycan can be labeled by incubating isolated secretion granules with [35S]phosphoadenosine phosphosulfate ([35S]PAPS), and the amount of sulfate incorporation decreases with increased granule maturity. In vitro incorporation is sensitive to inhibitors of PAPS transport and occurs in intact granules as shown by radioautography. Both the increased production of a chondroitin sulfate proteoglycan following isoproterenol treatment and its sulfation at sites of secretory condensation and storage suggest that sulfation may aid secretory packaging by reducing the known fixed positive charge that stems from the large concentration of basic secretory proteins.

Cerebroside sulfotransferase: preparation of antibody and localization of antigen in kidney.

This immunohistochemical study describes the localization of the enzyme cerebroside sulfotransferase (phosphoadenosine phosphosulfate: galactosylceramide sulfotransferase, EC 2.8.2.11) in rat kidney. The enzyme was purified from kidney and the preparation was used to raise antibodies for immunocytochemical investigations. In the kidney, the antigen was present only on the brush border of the epithelial cells of the proximal tubules, suggesting that sulfation of glycolipids occurs in the cytoplasm and plasma membranes of these specific cells. Moreover, biochemical and immunocytochemical studies of cerebroside sulfotransferase during development indicate that catalytic activity is correlated with the appearance of enzyme protein.