Summary of information about the Trichomonas vaginalis N-acetylneuraminate lyase (sialic acid lyase, NanA), and related genes, presented by Fiona at the December 7^th meeting.

As noted in previous emails to the group, Patrick had noticed that the T. vaginalis gene within the tree Audrey presented at the last meeting seemed to be a legitimate T. vaginalis gene, though it shared very unusually high similarity with a corresponding Haemophilu influenzae gene. Fiona identified that there were other members of the Pasteurellaceae (other than H. influenzae) that contained genes that were also highly similar to the T. vaginalis gene (based on the “unfinished microbial genomes database”). The similarity is such that it was proposed an analysis of this gene be written up as a short Note.

Fiona presented a summary of information about the enzyme N-acetylneuraminate lyase (a.k.a. sialic acid lyase, NanA), the phylogeny, and other information resulting from previous discussions between Patrick, Sally, Audrey, and Fiona:

• Sialic acid lyase: cleaves free sialic acid into acylmannosamines and pyruvate
• Of sialic acids, N-acetylneuraminate is the most common. (Sialic acid: protective terminal components of many glycoproteins and glycolipids, especially of higher animals, that play a role in the recognition of self.
• Function of the sialic acid lyase, NanA, in bacteria: for nutrition and detoxification of sialic acid in the host environment ("the valve to dissipate excess sialic acid")
• NanA in mammals: prevents sialic acid recycling
• NanA in industry: reverse reaction used for sialic acid synthesis

• In bacteria: this lyase enzyme, which is cytoplasmic, may be coupled with a permease and an extracellular sialidase. Note: sialidases are involved in cleavage of sialic acid from a larger macromolecule, while sialic acid lyase only cleaves free sialic acid into subcomponents.

Functional studies of bacterial and Trichomonas sialic acid lyase (there is a wealth of information!):

· Escherichia coli: Enzymatic activity demonstrated, Crystal structure obtained.

· Clostridium perfringens: Enzymatic activity demonstrated.

· Haemophilus influenzae: Enzymatic activity demonstrated and has > 3 fold activity verses the E. coli enzyme. Protein crystals obtained.

· Pasteurella haemolytica: Enzyme activity detected (increased synthesis at 37^oC) but gene not yet reported (Barrallo et al., FEBS Lett 1999 445:325-328). Other Pasteurella also have this enzyme (as well as a sialidase).

· Trichomonas vaginalis: Gene cloned and enzymatic activity demonstrated. Sialic acid demonstrated to be exposed on the Trichomonas surface through lectin binding experiments (Filho et al., Microbios 1992 71:55-64).

Info from the paper that most discusses the issue of horizontal transfer involving the H. influenzae or T. vaginalis genes (while reporting a C. perfringens gene): Traving et al. Glycoconjugate J 1997 14:821-830:

• High similarity between lyases in C. perfringens, H. influenzae, and T. vaginalis suggest these enzymes are younger than sialidases. (While we don’t agree with this statement, it is notable that the sialidases do not share the same phylogeny as the lyases, being present in different organisms with different levels of similarity).
• E.R. Vimr (personal commun. in paper) postulates that there are two distinct lineages in the evolution of sialic acid lyases. The high similarity between H. influenzae-T. vaginalis enzyme and H. influenzae-C. perfringens enzyme lead to the assumption that horizontal transfer has occurred (note: they do not indicate in which direction the horizontal transfer occurred).
• They refer to the high G+C content of the lyase gene (30.4%) relative to the 24-27% for the chromosome and suggest this is an indication that the lyase has been horizontally transferred from an organism with a higher mol% G+C.

However, additional analyses do not support this last statement:

Data obtained from the Codon Usage Database:

C. perfringens G+C for 147 genes: 30.98%

C. perfringens G+C for nanA: 30.45%

In other words, our analysis does not support that the G+C of the C. perfringens gene is atypical for this organism.

However… the G+C seems different for T. vaginalis nanA (though based on comparison with an average of only 47 genes):

T. vaginalis G+C for 47 genes: 48.41%

T. vaginalis G+C for nanA: 42.11%

Other nanA examined from Escherichia coli, Haemophilus influenzae, and Pasteurella multocida all have G+C similar to their average (around a 1% maximum difference between G+C of the gene verses the average).