Cloning of Prevotella intermedia loci demonstrating multiple hemolytic domains

Abstract

A gene bank was created from Prevotella intermedia strain 27 chromosomal DNA, and a clone was isolated that conferred the expression of two separate modes of hemolytic activity in recombinant Escherichia coli. The original recombinant hemolytic strain (EB34) contained plasmid, pEB34, with a 5.6-kb insert from Sau 3 AI-digested P. intermedia strain 27 chromosomal DNA cloned into the Bam HI site of pUC18. EB34 and deletion subclones were tested for expression of hemolytic activity in a standard tube assay, measuring lysis of erythrocytes spectrophotometrically as a function of hemoglobin release. Cell suspensions of EB34 demonstrated a dose-dependent hemolytic activity, inhibitable by proteases, and heat treatment but not dependent on calcium ions, and not inhibitable by osmoprotectants. Cell-free lysates also demonstrated a heat inhibitable, dose dependent hemolytic activity. Sub-cloning experiments localized the hemolytic region of the insert to a 3.9-kb fragment under direction of the lac promoter. Sequence analysis of the entire insert revealed the presence of multiple open reading frames (1 to 3) in this region which correlated to different forms of hemolytic expression, such that subclones containing all open reading frames 1 to 3 demonstrated strong hemolytic phenotype on blood plates and in the tube assay. Subclones containing only ORF1 demonstrated hemolysis on plates, but not in the tube assay. Subclones containing only open reading frames 2 and 3, but not ORF1 demonstrated hemolysis in the tube assay but not on plates. Homology searches of DNA and protein databases have not revealed significant homologies with reported hemolysins or proteins in any of the open reading frames.

A Genomic Sample Sequence of the Entomopathogenic Bacterium Photorhabdus luminescens W14: Potential Implications for Virulence

“…In addition to the Tc and Rtx-like toxins discussed above, W14 also appears to contain several non-Rtx hemolysins and other classes of toxins. One of the non-Rtx hemolysins, hemocyte erythrocyte lysis protein 2 from Prevotella intermedia, is notable in that searches of DNA and protein databases have not previously revealed any significant homologies (14); the Photorhabdus homology reported here is, therefore, possibly such a match. Other classes of toxins include two ADP-ribosyltransferases and cytotoxic necrotizing factor 2, all of which are cytotoxins.…”

Genome features of Leptospira interrogans serovar Copenhageni

“…LIC12134 codes for an HlpA-related protein which was characterized as a hemolysin in Prevotella intermedia, a common oral bacterium associated with periodontitis (38). Another identified gene (LIC10325) is related to the hlyX gene which codes for a predicted hemolysin previously identified in L. borgpetersenii serovar Hardjo (Accession number AAF09252, unpublished results).…”

Novel Rhamnosyltransferase Involved in Biosynthesis of Serovar 4-Specific Glycopeptidolipid from Mycobacterium avium Complex

“…Previously, it was reported that the GPL biosynthetic gene cluster of MAC serovar 2 strains contained one gene whose amino acid sequences are similar to that of hlpA with 69% identity (14). This has been regarded as the gene not associated with GPL biosynthesis, because its amino acid sequences are similar to those of hemolytic proteins distributed in some species of bacteria (4). Thus, as shown in Fig.…”

“…Therefore, HlpA in the serovar 2 strain originally might function as a glycosyltransferase in the biosynthesis of oligosaccharides of other serovars. Most HlpA homologues are putatively categorized as hemolytic proteins because they are similar to one protein from Prevotella intermedia, which is actually proved to have hemolytic activity (4). Since the amino acid sequences of HlpA show 38% identity and 54% similarity to the above protein of P. intermedia, we predicted that HlpA also possesses hemolytic activity as an additional function.…”

“…The extracts in organic phase were separated by adding water and evaporated until dry. To remove the lipid components except for GPLs, the whole-lipid extracts were subjected to mild alkaline hydrolysis to prepare the crude GPLs as previously described (27,28 4 and charring. Purified GPLs were prepared from crude GPLs by preparative TLC on the same plates and extracted from the bands corresponding to each GPL.…”