Roseobacter strain 27-4 has been isolated from a turbot larval rearing unit and is capable of reducing mortality in turbot egg yolk sac larvae. Here, we demonstrate that the supernatant of Roseobacter 27-4 is lethal to the larval pathogens Vibrio anguillarum and Vibrio splendidus in a buffer system and inhibited their growth in marine broth. Liquid chromatography (LC) with both UV spectral detection and high-resolution mass spectrometry (HR-MS) identified the known antibacterial compound thiotropocin or its closely related precursor tropodithietic acid in the bioactive fractions. Antibacterial activity correlated with the appearance of a brownish pigment and was only formed in marine broth under static growth conditions. A thick biofilm of multicellular star-shaped aggregated cells formed at the air-liquid interface under static growth conditions. Here, the bioactive compound was the base peak in the LC-UV chromatograms of the extracts where it constituted 15% of the total peak area. Aerated conditions results in 10-fold-higher cell yield, however, cultures were nonpigmented, did not produce antibacterial activity, and grew as single cells. Production of antibacterial compounds may be quorum regulated, and we identified the acylated homoserine lactone (3-hydroxy-decanoyl homoserine lactone) from cultures of Roseobacter 27-4 using LC-HR-MS. The signal molecule was primarily detected in stagnant cultures. Roseobacter 27-4 grew between 10 and 30°C but died rapidly at 37°C. Also, the antibacterial compounds was sensitive to heat and was inactivated at 37°C in less than 2 days and at 25°C in 8 days. Using Roseobacter 27-4 as a probiotic culture will require that is be established in stagnant or adhered conditions and, due to the temperature sensitivity of the active compound, constant production must be ensured.The dramatic growth in the aquaculture sector (an increment of 9 to 10% per year over the last 8 to 10 years) has emphasized the importance of fish disease control. Bacterial diseases are important constraints and may be treated with antibiotics. However, due to the risk of development and transfer of antibiotic resistance, alternative disease control measures must be implemented. Vaccines have been very successful also in fish farming; however, vaccines are not efficient at the larval stages. Several studies have demonstrated that probiotic bacteria may be used to control pathogenic organisms in fish larval rearing (17,18,32), and one of several promising candidates are bacteria of the marine Roseobacter clade.The ability of Roseobacter to inhibit other bacteria was noted already by who used Roseobacter gallaeciensis strain BS107 as a probiotic treatment of scallop larvae (32). Roseobacter strains have also been isolated from turbot larval farms, and they were selected from this environment due to their strong anti-Vibrio activity (18). These strains appeared to constitute a relatively stable community since the same DNA subtypes were isolated over several months (19). Antagonistic Roseobacter were especiall...

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The flagellum protein flagellin of Listeria monocytogenes is encoded by the flaA gene. Immediately downstream of flaA, two genes, cheY and cheA, encoding products with homology to chemotaxis proteins of other bacteria, are located. In this study we constructed deletion mutants with mutations in flaA, cheY, and cheA to elucidate their role in the biology of infection with L. monocytogenes. The ⌬cheY, ⌬cheA, and double-mutant ⌬cheYA mutants, but not ⌬flaA mutant, were motile in liquid media. However, the ⌬cheA mutant had impaired swarming and the ⌬cheY and ⌬cheYA mutants were unable to swarm on soft agar plates, suggesting that cheY and cheA genes encode proteins involved in chemotaxis. The ⌬flaA, ⌬cheY, ⌬cheA, and ⌬cheYA mutants (grown at 24°C) showed reduced association with and invasion of Caco-2 cells compared to the wild-type strain. However, spleens from intragastrically infected BALB/c and C57BL/6 mice showed larger and similar numbers of the ⌬flaA and ⌬cheYA mutants, respectively, compared to the wild-type controls. Such a discrepancy could be explained by the fact that tumor necrosis factor receptor p55 deficient mice showed dramatically exacerbated susceptibility to the wild-type but unchanged or only slightly increased levels of the ⌬flaA or ⌬cheYA mutant. In summary, we show that listerial flaA, cheY, and cheA gene products facilitate the initial contact with epithelial cells and contribute to effective invasion but that flaA could also be involved in the triggering of immune responses.

Microhabitat selection of Gyrodactylus derjavini on the body surface of rainbow trout changed markedly during a 6-week experimental infection period. Pectoral fins, pelvic fins and anal fins were the most important sites (expressed in terms of parasite density) during the initial part of the infection. In the later stages of infection, the corneal surface and tail fin became increasingly more heavily infested. Factors responsible for this dynamic site selection were investigated. The density of superficial mucous cells in the epithelium of fins and skin was weakly correlated (r = 0.23) with parasite density in the first part of the infection. This association changed into a significant negative correlation (r = -0.92) as the infection progressed and the parasite population increased. These results strongly indicate that mucous cell contents play a decisive role in gyrodactylid site selection. Lysozyme, protease, immunoglobulin (Ig), complement factor C3, enzymes, lectinbinding carbohydrates and peptides adrenocorticotropic hormone, interleukin (IL-1) and somastatin) were detected in mucus and some of these (Ig, C3, IL-1, carbohydrates) are suggested to influence the infection dynamics. Thus, some molecules in mucus are liable first to attract the gyrodactylids, but subsequently reactive substances present in increasing amounts will counteract the performance of parasites in mucous-cell-rich microhabitats. The mechanisms involved in this process are discussed.

The aim of this work was to identify genes responsible for host recognition in the lactococcal phages sk1 and bIL170 belonging to species 936. These phages have a high level of DNA identity but different host ranges. Bioinformatic analysis indicated that homologous genes, orf18 in sk1 and orf20 in bIL170, could be the receptor-binding protein (RBP) genes, since the resulting proteins were unrelated in the C-terminal part and showed homology to different groups of proteins hypothetically involved in host recognition. Consequently, chimeric bIL170 phages carrying orf18 from sk1 were generated. The recombinant phages were able to form plaques on the sk1 host Lactococcus lactis MG1614, and recombination was verified by PCR analysis directly with the plaques. A polyclonal antiserum raised against the C-terminal part of phage sk1 ORF18 was used in immunogold electron microscopy to demonstrate that ORF18 is located at the tip of the tail. Sequence analysis of corresponding proteins from other lactococcal phages belonging to species 936 showed that the N-terminal parts of the RBPs were very similar, while the C-terminal parts varied, suggesting that the C-terminal part plays a role in receptor binding. The phages investigated could be grouped into sk1-like phages (p2, fd13, jj50, and 7) and bIL170-like phages (P008, P113G, P272, and bIL66) on the basis of the homology of their RBPs to the C-terminal part of ORF18 in sk1 and ORF20 in bIL170, respectively. Interestingly, sk1-like phages bind to and infect a defined group of L. lactis subsp. cremoris strains, while bIL170-like phages bind to and infect a defined group of L. lactis subsp. lactis strains.Lactococcus lactis is the most important bacterium used for starter cultures by the dairy industry. An important problem in industrial milk fermentation is infection of the starter bacteria by bacteriophages, which leads to bacterial lysis. The consequences of phage infection are fermentation delay, alteration of product quality, and in severe cases loss of the product. All these outcomes result in considerable economic loss to dairies (5,22). Industrial phage ecology is dominated by three phage species: the predominant species 936 and species c2 and P335, which also have considerable importance (5,22).The first step in phage infection is adsorption of the phage to the host cell. Despite the fact that little information concerning the adsorption process of lactococcal phages is available, it seems to be a two-step process that starts with reversible binding to specific carbohydrates exposed on the surface of the cell wall (30, 37, 39), which is followed by, at least in species c2, irreversible binding to a protein in the cell membrane (14,30,38). Information on receptor-binding proteins (RBPs) in phages infecting gram-positive bacteria is sparse compared to the information available for phages infecting gram-negative bacteria. However, recently, Duplessis and Moineau identified the first RBP genes for the Streptococcus thermophilus phages DT1 and MD4 (11). These authors repo...

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Two putative structural genes, orf tmp (tape measure protein) and orf bpp (baseplate protein), of the temperate lactococcal phage TP901-1 were examined by introduction of specific mutations in the prophage strain Lactococcus lactic ssp. cremoris 901-1. The adsorption efficiencies of the mutated phages to the indicator strain L. lactic ssp. cremoris 3107 were determined and electron micrographs were obtained. Specific mutations in orf tmp resulted in the production of mostly phage head structures without tails and a few wild-type looking phages. Furthermore, construction of an inframe deletion or duplication of 29% in orf tmp was shown to shorten or lengthen the phage tail by approximately 30%, respectively. The orf tmp is proposed to function as a tape measure protein, TMP, important for assembly of the TP901-1 phage tail and involved in tail length determination. Specific mutations in orf bpp produced phages which were unable to adsorb to the indicator strain and electron microscopy revealed particles lacking the baseplate structure. The orf bpp is proposed to encode a highly immunogenic structural baseplate protein, BPP, important for assembly of the baseplate. Finally, an assembly pathway of the TP901-1 tail and baseplate structure is presented.

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