Identification of Cold-Temperature-Regulated Genes in Flavobacterium psychrophilum

Abstract

Flavobacterium psychrophilum is the etiological agent of bacterial coldwater disease (BCWD) and rainbow trout fry syndrome (RTFS). It causes disease primarily in fresh water-reared salmonids, but other fish species can also be affected. A diverse array of clinical conditions is associated with BCWD, including tail rot (peduncle disease), necrotic myositis, and cephalic osteochondritis. Degradation of connective and muscular tissues by extracellular proteases is common to all of these presentations. There are no effective vaccines to prevent BCWD or RTFS, and antibiotics are often used to prevent and control disease. To identify virulence factors that might permit development of an efficacious vaccine, cDNA suppression subtractive hybridization (SSH) was used to identify cold-regulated genes in a virulent strain of F. psychrophilum. Genes predicted to encode a two-component system sensor histidine kinase (LytS), an ATP-dependent RNA helicase, a multidrug ABC transporter permease/ATPase, an outer membrane protein/protective antigen OMA87, an M43 cytophagalysin zinc-dependent metalloprotease, a hypothetical protein, and four housekeeping genes were upregulated at 8°C versus the level of expression at 20°C. Because no F. psychrophilum gene was known to be suitable as an internal standard in reverse transcription-quantitative real-time PCR (RT-qPCR) experiments, the expression stability of nine commonly used reference genes was evaluated at 8°C and 20°C. Expression of the 16S rRNA was equivalent at both temperatures, and this gene was used in RT-qPCR experiments to verify the SSH findings. With the exception of the ATCC 49513 strain, similar patterns of gene expression were obtained with 11 other representative strains of F. psychrophilum.Bacterial cold water disease (BCWD), caused by Flavobacterium psychrophilum, occurs at low water temperatures and can cause economic losses in the aquaculture industry as a result of direct mortality or vertebral deformities that decrease the market value of fish that survive the infection (31, 35). A number of putative virulence factors of this bacterium have been identified, including extracellular proteases involved in degradation of extracellular matrix components such as elastin, fibrinogen, type IV collagen, actin, and myosin (9,37,47,48). The best studied of these, psychrophilic metalloproteases Fpp1 and Fpp2, are thought to be involved in destruction of host tissues; however, their roles have not yet been demonstrated in vivo (47,48). In a recent study, Sudheesh et al. (53) used two-dimensional acrylamide gel electrophoresis and Western blot analysis to compare a virulent strain and a nonvirulent strain of F. psychrophilum and found a thermolysin that was unique to the virulent strain. A role for the iron uptake-associated gene exbD2 has also been demonstrated. Strains lacking this gene have decreased virulence and confer a high level of protection in rainbow trout fry when they are used for vaccination (4). Other putative virulence factors of F. psychrophilum include a lipo...

Seasonal changes in microbial community structure and activity imply winter production is linked to summer hypoxia in a large lake

“…Moreover, the optimum temperature range for most Flavobacterium spp. is 20–30 °C (Bernardet & Bowman, ), although at least one isolate (the fish pathogen F. psychrophilum ) has been shown to have a series of genes up‐regulated at colder temperatures (at 8 °C, Hesami et al ., ). That said, Lake Erie may host different summer populations than the Swedish lakes studied above, as a recent study of Sandusky Bay and the Western Basin of Lake Erie provided observations similar to ours (Mou et al ., ).…”

Involvement of Two-Component System CBO0366/CBO0365 in the Cold Shock Response and Growth of Group I (Proteolytic) Clostridium botulinum ATCC 3502 at Low Temperatures

“…As with many cold-responsive TCSs reported (7,12,23,31), the stimulus sensed by the CBO0366 kinase remains to be characterized. The cold-responsive DesK of B. subtilis and Hik33 of Synechocystis sp.…”

“…TCSs in bacteria are differentially specialized to respond to a wide variety of chemical and physical stimuli, including pH, osmolarity, oxidative stress, and temperature. TCSs associated with a response to low temperature in other bacteria include the DesK/DesR in B. subtilis (1)(2)(3)6), CheA/CheY in Yersinia pseudotuberculosis (31), CorS/CorR in Pseudomonas syringae (37), and Fp1516/Fp1517 in Flavobacterium psychrophilum (23). In addition, the LisK/LisR, Lmo1173/ Lmo1172, and Lmo1061/Lmo1060 systems were linked to the cold shock response but not to long-term growth of Listeria monocytogenes at low temperature (12).…”

Complete genome sequence analysis of the fish pathogen Flavobacterium columnare provides insights into antibiotic resistance and pathogenicity related genes

“…A similar mechanism may apply for F. columnare, in that we identified a paralogous gene Pf1_0182, predicted to encode metalloprotease of the fungalysin family. A correlation between proteolytic activity and the virulence of F. psychrophilum was found in cold temperature, indicating that M43 cytophagalysin may be involved in pathogenesis through destruction of host tissues [63]. Serine proteases are enzymes that cleave peptide bonds in proteins, in which serine serves as the nucleophilic amino acid at the active site.…”