Frank Sargent

Sialic acid (Sia) transporters are critical to the capacity of host-associated bacteria to utilise Sia for growth and/or cell surface modification. While N-acetyl-neuraminic acid (Neu5Ac)-specific transporters have been studied extensively, little is known on transporters dedicated to anhydro-Sia forms such as 2,7-anhydro-Neu5Ac (2,7-AN) or 2,3-dehydro-2-deoxy-Neu5Ac (Neu5Ac2en). Here, we used a Sia-transport-null strain of Escherichia coli to investigate the function of members of anhydro-Sia transporter families previously identified by computational studies. First, we showed that the transporter NanG, from the Glycoside-Pentoside-Hexuronide:cation symporter family, is a specific 2,7-AN transporter, and identified by mutagenesis a crucial functional residue within the putative substrate-binding site. We then demonstrated that NanX transporters, of the Major Facilitator Superfamily, also only transport 2,7-AN and not Neu5Ac2en nor Neu5Ac. Finally, we provided evidence that SiaX transporters, of the Sodium-Solute Symporter superfamily, are promiscuous Neu5Ac/Neu5Ac2en transporters able to acquire either substrate equally well. The characterisation of anhydro-Sia transporters expands our current understanding of prokaryotic Sia metabolism within host-associated microbial communities.

Escherichia coli is a facultative anaerobe that can grow in a variety of environmental conditions. In the complete absence of O 2 , E. coli can perform a mixed-acid fermentation that contains within it an elaborate metabolism of formic acid. In this study, we use cavity-enhanced Raman spectroscopy (CERS), FTIR, liquid Raman spectroscopy, isotopic labelling and molecular genetics to make advances in the understanding of bacterial formate and H 2 metabolism. It is shown that, under anaerobic (anoxic) conditions, formic acid is generated endogenously, excreted briefly from the cell, and then taken up again to be disproportionated to H 2 and CO 2 by formate hydrogenlyase (FHL-1). However, exogenously added D-labelled formate behaves quite differently from the endogenous formate and is taken up immediately, independently, and possibly by a different mechanism, by the cell and converted to H 2 and CO 2 . Our data support an anion-proton symport model for formic acid transport. In addition, when E. coli was grown in a micro-aerobic (micro-oxic) environment it was possible to analyse aspects of formate and O 2 respiration occurring alongside anaerobic metabolism. While cells growing under micro-aerobic conditions generated endogenous formic acid, no H 2 was produced. However, addition of exogenous formate at the outset of cell growth did induce FHL-1 biosynthesis and resulted in formate-dependent H 2 production in the presence of O 2 .