RESUMEN
Flavobacterium johnsoniae forms biofilms in low nutrient conditions. Protein secretion and cell motility may have roles in biofilm formation. The F. johnsoniae type IX secretion system (T9SS) is important for both secretion and motility. To determine the roles of each process in biofilm formation, mutants defective in secretion, in motility, or in both processes were tested for their effects on biofilm production using a crystal violet microplate assay. All mutants that lacked both motility and T9SS-mediated secretion failed to produce biofilms. A porV deletion mutant, which was severely defective for secretion, but was competent for motility, also produced negligible biofilm. In contrast, mutants that retained secretion but had defects in gliding formed biofilms. An sprB mutant that is severely but incompletely defective in gliding motility but retains a fully functional T9SS was similar to the wild type in biofilm formation. Mutants with truncations of the gldJ gene that compromise motility but not secretion showed partial reduction in biofilm formation compared to wild type. Unlike the sprB mutant, these gldJ truncation mutants were essentially nonmotile. The results show that a functional T9SS is required for biofilm formation. Gliding motility, while not required for biofilm formation, also appears to contribute to formation of a robust biofilm.
RESUMEN
Flavobacterium columnare, a member of the phylum Bacteroidetes, causes columnaris disease in wild and aquaculture-reared freshwater fish. The mechanisms responsible for columnaris disease are not known. Many members of the phylum Bacteroidetes use type IX secretion systems (T9SSs) to secrete enzymes, adhesins, and proteins involved in gliding motility. The F. columnare genome has all of the genes needed to encode a T9SS. gldN, which encodes a core component of the T9SS, was deleted in wild-type strains of F. columnare The F. columnare ΔgldN mutants were deficient in the secretion of several extracellular proteins and lacked gliding motility. The ΔgldN mutants exhibited reduced virulence in zebrafish, channel catfish, and rainbow trout, and complementation restored virulence. PorV is required for the secretion of a subset of proteins targeted to the T9SS. An F. columnare ΔporV mutant retained gliding motility but exhibited reduced virulence. Cell-free spent media from exponentially growing cultures of wild-type and complemented strains caused rapid mortality, but spent media from ΔgldN and ΔporV mutants did not, suggesting that soluble toxins are secreted by the T9SS.IMPORTANCE Columnaris disease, caused by F. columnare, is a major problem for freshwater aquaculture. Little is known regarding the virulence factors produced by F. columnare, and control measures are limited. Analysis of targeted gene deletion mutants revealed the importance of the type IX protein secretion system (T9SS) and of secreted toxins in F. columnare virulence. T9SSs are common in members of the phylum Bacteroidetes and likely contribute to the virulence of other animal and human pathogens.
Asunto(s)
Proteínas Bacterianas/metabolismo , Sistemas de Secreción Bacterianos/metabolismo , Enfermedades de los Peces/microbiología , Infecciones por Flavobacteriaceae/veterinaria , Flavobacterium/metabolismo , Flavobacterium/patogenicidad , Animales , Proteínas Bacterianas/genética , Sistemas de Secreción Bacterianos/genética , Infecciones por Flavobacteriaceae/microbiología , Flavobacterium/genética , Ictaluridae/microbiología , Oncorhynchus mykiss/microbiología , Virulencia , Pez Cebra/microbiologíaRESUMEN
The 6.10-Mb genome sequence of the aerobic chitin-digesting gliding bacterium Flavobacterium johnsoniae (phylum Bacteroidetes) is presented. F. johnsoniae is a model organism for studies of bacteroidete gliding motility, gene regulation, and biochemistry. The mechanism of F. johnsoniae gliding is novel, and genome analysis confirms that it does not involve well-studied motility organelles, such as flagella or type IV pili. The motility machinery is composed of Gld proteins in the cell envelope that are thought to comprise the "motor" and SprB, which is thought to function as a cell surface adhesin that is propelled by the motor. Analysis of the genome identified genes related to sprB that may encode alternative adhesins used for movement over different surfaces. Comparative genome analysis revealed that some of the gld and spr genes are found in nongliding bacteroidetes and may encode components of a novel protein secretion system. F. johnsoniae digests proteins, and 125 predicted peptidases were identified. F. johnsoniae also digests numerous polysaccharides, and 138 glycoside hydrolases, 9 polysaccharide lyases, and 17 carbohydrate esterases were predicted. The unexpected ability of F. johnsoniae to digest hemicelluloses, such as xylans, mannans, and xyloglucans, was predicted based on the genome analysis and confirmed experimentally. Numerous predicted cell surface proteins related to Bacteroides thetaiotaomicron SusC and SusD, which are likely involved in binding of oligosaccharides and transport across the outer membrane, were also identified. Genes required for synthesis of the novel outer membrane flexirubin pigments were identified by a combination of genome analysis and genetic experiments. Genes predicted to encode components of a multienzyme nonribosomal peptide synthetase were identified, as were novel aspects of gene regulation. The availability of techniques for genetic manipulation allows rapid exploration of the features identified for the polysaccharide-digesting gliding bacteroidete F. johnsoniae.
Asunto(s)
ADN Bacteriano/genética , Flavobacterium/genética , Genoma Bacteriano , Análisis de Secuencia , Adhesinas Bacterianas/genética , Proteínas Bacterianas/genética , ADN Bacteriano/química , Esterasas/genética , Glicósido Hidrolasas/genética , Locomoción/genética , Modelos Biológicos , Datos de Secuencia Molecular , Péptido Hidrolasas/genética , Polisacárido Liasas/genética , Polisacáridos/metabolismo , Proteínas/metabolismoRESUMEN
BACKGROUND: Flavobacterium columnare is the causative agent of columnaris disease, a disease affecting many freshwater fish species. Methods for the genetic manipulation for some of the species within the Bacteroidetes, including members of the genus Flavobacterium, have been described, but these methods were not adapted to work with F. columnare. RESULTS: As a first step toward developing a robust set of genetic tools for F. columnare, a protocol was developed to introduce the E. coli - Flavobacterium shuttle vector pCP29 into F. columnare strain C#2 by conjugal mating at an efficiency of 1.5 x 10(-3) antibiotic-resistant transconjugants per recipient cell. Eight of eleven F. columnare strains tested were able to receive pCP29 using the protocol. pCP29 contains the cfxA and ermF genes, conferring both cefoxitin and erythromycin resistance to recipient cells. Selection for pCP29 introduction into F. columnare was dependent on cfxA, as ermF was found not to provide strong resistance to erythromycin. This is in contrast to other Flavobacterium species where ermF-based erythromycin resistance is strong. The green fluorescent protein gene (gfp) was introduced into F. columnare strains under the control of two different native Flavobacterium promoters, demonstrating the potential of this reporter system for the study of gene expression. The transposon Tn4351 was successfully introduced into F. columnare, but the method was dependent on selecting for erythromycin resistance. To work, low concentrations of antibiotic (1 microg ml(-1)) were used, and high levels of background growth occurred. These results demonstrate that Tn4351 functions in F. columnare but that it is not an effective mutagenesis tool due to its dependence on erythromycin selection. Attempts to generate mutants via homologous recombination met with limited success, suggesting that RecA dependent homologous recombination is rare in F. columnare. CONCLUSION: The conjugation protocol developed as part of this study represents a significant first step towards the development of a robust set of genetic tools for the manipulation of F. columnare. The availability of this protocol will facilitate studies aimed at developing a deeper understanding of the virulence mechanisms of this important pathogen.
Asunto(s)
Flavobacterium/clasificación , Flavobacterium/genética , Ingeniería Genética/métodos , Animales , Conjugación Genética , Elementos Transponibles de ADN , Escherichia coli/genética , Enfermedades de los Peces/microbiología , Infecciones por Flavobacteriaceae/microbiología , Infecciones por Flavobacteriaceae/veterinaria , Vectores Genéticos , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Mutagénesis Insercional , Plásmidos , Recombinación GenéticaRESUMEN
Flavobacterium columnare is a serious pathogen in a wide range of fish species. F. johnsoniae is an opportunistic pathogen of certain fish. Both are gliding bacteria. These species were tested for their ability to infect the zebra fish Danio rerio. Both injection and bath infection methods were tested. The results indicate that F. johnsoniae is not an effective pathogen in D. rerio, but that F. columnare is an effective pathogen. F. johnsoniae did not cause increased death rates following bath infection, but did cause increased death rates following injection, with an LD50 (mean lethal dose) of approximately 3 x 10(10) cfu (colony-forming units). Non-motile mutants of F. johnsoniae produced a similar LD50. F. columnare caused increased death rates following both injection and bath infections. There was considerable strain variation in LD50, with the most lethal strain tested producing an LD50 of 3.2 x 10(6) cfu injected and 1.1 x 10(6) cfu ml(-1) in bath experiments, including skin damage. The LD50 of F. columnare in zebra fish without skin damage was > 1 x 10(8), indicating an important effect of skin damage.
Asunto(s)
Enfermedades de los Peces/microbiología , Infecciones por Flavobacteriaceae/veterinaria , Flavobacterium/patogenicidad , Pez Cebra/microbiología , Animales , Susceptibilidad a Enfermedades , Enfermedades de los Peces/transmisión , Infecciones por Flavobacteriaceae/microbiología , Infecciones por Flavobacteriaceae/transmisión , Flavobacterium/crecimiento & desarrollo , Dosificación Letal Mediana , ARN Ribosómico 16S/genética , Análisis de SupervivenciaRESUMEN
Flavobacterium johnsoniae moves rapidly over surfaces by a process known as gliding motility. The mechanism of this form of motility is not known. Four genes that are required for F. johnsoniae gliding motility, gldA, gldB, gldD, and ftsX, have recently been described. GldA is similar to the ATP-hydrolyzing components of ATP binding cassette (ABC) transporters. Tn4351 mutagenesis was used to identify two additional genes, gldF and gldG, that are required for cell movement. gldF and gldG appear to constitute an operon, and a Tn4351 insertion in gldF was polar on gldG. pMK314, which carries the entire gldFG region, restored motility to each of the gldF and gldG mutants. pMK321, which expresses GldG but not GldF, restored motility to each of the gldG mutants but did not complement the gldF mutant. GldF has six putative membrane-spanning segments and is similar in sequence to channel-forming components of ABC transporters. GldG is similar to putative accessory proteins of ABC transporters. It has two apparent membrane-spanning helices, one near the amino terminus and one near the carboxy terminus, and a large intervening loop that is predicted to reside in the periplasm. GldF and GldG are involved in membrane localization of GldA, suggesting that GldA, GldF, and GldG may interact to form a transporter. F. johnsoniae gldA is not closely linked to gldFG, but the gldA, gldF, and gldG homologs of the distantly related gliding bacterium Cytophaga hutchinsonii are arranged in what appears to be an operon. The exact roles of F. johnsoniae GldA, GldF, and GldG in gliding are not known. Sequence similarities of GldA to components of other ABC transporters suggest that the Gld transporter may be involved in export of some material to the periplasm, outer membrane, or beyond.