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Flavobacterium psychrophilum is an important infectious Gram-negative bacterium causing cold-water disease (CWD) and rainbow trout fry syndrome. Outer-membrane proteins (OMPs) are key molecules with regard to the interface between the cell and its environment. Therefore, we sought to define the outer-membrane (OM) subproteome of F. psychrophilum in order to gain insight into the biology and pathogenesis of this bacterium and to identify the dominant antigens targeted by the rainbow trout (Oncorhynchus mykiss) immune system during infection. First, OMs were prepared from a cell-envelope suspension by differential Sarkosyl (sodium lauryl sarcosinate) solubility. We then isolated the OMPs and identified 36 proteins from 34 spots resolved by two-dimensional electrophoresis and LC-MS/MS. An immunoproteomic approach using antibodies from CWD-convalescent rainbow trout was then used to identify 25 immunoreactive F. psychrophilum antigens that may be relevant in pathogenesis and diagnosis. These included the previously characterized surface-exposed OMPs OmpA, OmpH/P18 and FspA, as well as newly described antigenic proteins. This study provides a number of novel candidate proteins for developing vaccine(s) against flavobacteriosis infection in aquaculture.

The stable maintenance and expression of foreign genes in mollicutes (mycoplasmas) have been difficult to achieve due to the lack of suitable vectors. In this paper we show for the first time that a replicating vector can been used to express foreign genes other than antibiotic resistance genes in Acholeplasma laidlawii. Plasmids derived from the lactococcal vector pNZ18 could introduce and maintain four different genes for many generations in A. laidlawii. One of these, encoding the dominant membrane lipoprotein spiralin from the mollicute Spiroplasma citri, was expressed; however, expression was weak, the signal peptide of spiralin was not cleaved and the protein was not covalently modified by fatty acids. This resulted in a hydrophilic character of spiralin and its cytoplasmic localization in A. laidlawii. To increase the expression of foreign genes, random A. laidlawii DNA fragments were cloned into a pNZ18-related plasmid and expression signals were selected using the Bacillus licheniformis [alpha]-amylase gene as a probe. Selection was done in Escherichia coli as well as directly in A. laidlawii. Active recombinants from E. coli were also able to express [alpha]-amylase activities and an enzyme of native size in A. laidlawii. The highest activity was obtained from a recombinant selected directly in A. laidlawii. This is the first example of a promoter sequence selected in a mollicute. Analysis of the putative promoters in seven clones revealed similar - 10 and - 35 regions, and similar spacer distances in A. laidlawii, Acholeplasma oculi, Lactococcus and E. coli. Vectors related to pNZ18 should be useful for the genetic analysis of specific A. laidlawii proteins and functions.