Evidence for an intermediate colony morphology of Vibrio vulnificus

Abstract

Vibrio vulnificus causes both food-borne disease and wound infections. Most V. vulnificus strains express capsular polysaccharide (CPS), which is required for the virulence of this organism. Under standard growth conditions, CPS expression is lost at a relatively high frequency (10(-3) to 10(-4)), resulting in a switch from an opaque (Op, CPS+) colony morphology to a translucent (Tr, CPS-) colony morphology. The wzb gene, which encodes a phosphatase required for CPS expression, has been proposed to be involved in this switch through a site-specific deletion of the entire gene. In an examination of five strains, we found that the frequency of wzb deletion in Tr colonies varies by strain and therefore does not account for all the Tr colonies that are seen. In addition, we have identified a third, intermediate (Int) colony morphotype, in which the colonies appear less opaque but are not fully translucent. PCR studies have demonstrated that Int colonies still contain the wzb gene, while reverse transcriptase PCR studies have shown that although Int strains retain expression of wzb, in some cases the transcription of wzb is reduced. Int strains switch to a true Tr (wzb negative) morphotype at a very high frequency (nearly 100%) under certain conditions. Finally, Int colonies, which in some cases can easily be mistaken for Tr colonies, have been observed to occasionally revert to Op, while Tr colonies containing a wzb deletion presumably are unable to revert to the encapsulated form.

FIG. 1.

Tr, Int, and Op colony morphotypes of V. vulnificus strain JY1305. The strains were grown from freezer stocks on HI agar at 22°C. (A) Single colonies of Tr, Int, and Op morphotypes after 48 h growth; (B) growth of Tr, Int, and Op strains after 24 h.

FIG. 2.

Expression of wzb in Op and Int strains of V. vulnificus. Lanes: 1, negative control; 2, positive control; 3, Env1 Op; 4, Env1 Int; 5, YJ106 Op; 6, YJ016 Int; 7, C7184o Op; 8, C7184o Int; 9, JY1701 Op; 10, JY1701 Int; 11, JY1305 Op; 12, JY1305 Int.

FIG. 3.

Expression of (A) wzb and (B) tufA in Op and Int isolates of V. vulnificus strain Env1. Total RNA was extracted directly from the colonies. Lanes: 1, negative control; 2, positive control; 3, Env1 Op; 4 to 8, Env1 Int colonies 1, 2, 3, 4, and 5, respectively.