Major

Microbiology

Advisor

Nelson, David

Advisor Department

Cell and Molecular Biology

Date

5-2013

Keywords

Flavobacterium columnare, columnaris disease, chondroitin AC lyase, complementation, csl operon

Abstract

Analysis of the chondroitinase operon of Flavobacterium columnare

Erin Sorlien

Major

Cell and Molecular Biology, Chemistry

Advisor

Dr. David R. Nelson

Date

May 2, 2013

Keywords

Flavobacterium columnare, columnaris disease, chondroitin AC lyase, complementation, csl operon

Abstract
Flavobacterium columnare, an opportunistic bacterial pathogen of fish, is the causative agent of columnaris disease (CD). The bacterium is a Gram-negative rod that exhibits gliding motility and avidly forms biofilms. CD affects both wild and cultured freshwater fish, and continues to cause large economic losses to the fish farming industry. According to an investigation conducted by the National Animal Health Monitoring System, CD caused losses of food-size fish in 39.0% of the US channel catfish operations, establishing CD as the most prevalent disease among catfish farms in 2010. Estimates of mortality of the disease vary significantly; however, it is clear the disease is of great economic importance. Infections have been reported in other cultured fish including tilapia, Atlantic salmon, Arctic char and rainbow trout.

The disease presents as lesions on the exterior of the fish, specifically on the fins, gills and body surface. Virulence factors proposed in F. columnare include the ability to adhere to surfaces, extracellular proteases, and chondroitin AC lyase. The evidence for the role of these proposed virulence factors is primarily based on the clinical signs of the disease and is merely suggestive. During CD outbreaks bacterial isolates have been collected from moribund fish that do not exhibit the characteristic external lesions. Characterization of the genetic basis of the virulence of this important pathogen is key to describing and accurately understanding how F. columnare generates infection in the host.
The specific virulence mechanisms that this bacterium uses to infect its host are not well studied or understood. Chondroitin AC lyase (chondroitinase) is one of the proposed virulence factors that may enhance the invasion of the pathogen. Chondroitinase functions by degrading chondroitin-containing structural components (i.e. cartilage) of the host tissues. The cslA gene encodes chondroitinase. When a mutation is made in wild type F. columnare (strain Fc2) to the cslA gene, the chondroitin AC lyase activity decreases by 98%, (unpublished data, Perreira, Staroscik and Nelson). When the cslA mutation is complemented ectopically by a wild type cslA gene on a plasmid approximately 10% of chondroitinase activity is restored (unpublished data, Perreira, Staroscik and Nelson).
It is hypothesized that the complemented mutant strain does not show full restoration of the chondroitinase activity because the cslA gene is the first gene of an operon. An operon acts as a unit of transcription, and functions in a coordinated manner to produce a single mRNA molecule which codes for more than one protein with related metabolic functions. The chondroitinase (csl) operon has been shown to contain 6 open reading frames (or genes), cslA-F. The insertion mutation in cslA disrupts the ability to transcribe downstream genes of the operon, called a pleiotropic effect. Analysis of the genes downstream of cslA suggests that they modify the enzyme chondroitin AC lyase, the gene product of cslA. In order to restore full activity of chondroitin AC lyase these downstream genes (cslB-F) must also be complemented. My investigation examines whether complementation of the complete csl operon in the cslA mutant is necessary to restore full chondroitinase activity. This project requires the construction of a genetic complementation vector, transformation into E. coli followed by conjugal mating into the F. columnare cslA mutant strain. The production of this new strain is then tested for chondroitinase activity and compared to levels seen in the wild type and the cslA mutant.