The involvement of nicotinamide adenine nucleotides (NAD⁺, NADH) in the regulation of glycolysis in Lactococcus lactis was investigated by using¹³C and ³¹P NMR to monitor in vivothe kinetics of the pools of NAD⁺, NADH, ATP, inorganic phosphate (P_i), glycolytic intermediates, and end products derived from a pulse of glucose. Nicotinic acid specifically labeled on carbon 5 was synthesized and used in the growth medium as a precursor of pyridine nucleotides to allow for in vivo detection of¹³C-labeled NAD⁺ and NADH. The capacity ofL. lactis MG1363 to regenerate NAD⁺ was manipulated either by turning on NADH oxidase activity or by knocking out the gene encoding lactate dehydrogenase (LDH). An LDH⁻deficient strain was constructed by double crossover. Upon supply of glucose, NAD⁺ was constant and maximal (∼5 mm) in the parent strain (MG1363) but decreased abruptly in the LDH⁻ strain both under aerobic and anaerobic conditions. NADH in MG1363 was always below the detection limit as long as glucose was available. The rate of glucose consumption under anaerobic conditions was 7-fold lower in the LDH⁻ strain and NADH reached high levels (2.5 mm), reflecting severe limitation in regenerating NAD⁺. However, under aerobic conditions the glycolytic flux was nearly as high as in MG1363 despite the accumulation of NADH up to 1.5 mm. Glyceraldehyde-3-phosphate dehydrogenase was able to support a high flux even in the presence of NADH concentrations much higher than those of the parent strain. We interpret the data as showing that the glycolytic flux in wild type L. lactis is not primarily controlled at the level of glyceraldehyde-3-phosphate dehydrogenase by NADH. The ATP/ADP/P_i content could play an important role.

Secondary transmembrane transport carriers fall into families and superfamilies allowing prediction of structure and function. Here we describe hundreds of sequenced homologues that belong to six families within a novel superfamily, the bile/arsenite/riboflavin transporter (BART) superfamily, of transport systems and putative signalling proteins. Functional data for members of three of these families are available, and they transport bile salts and other organic anions, the bile acid:Na⁺ symporter (BASS) family, inorganic anions such as arsenite and antimonite, the arsenical resistance‐3 (Acr3) family, and the riboflavin transporter (RFT) family. The first two of these families, as well as one more family with no functionally characterized members, exhibit a probable 10 transmembrane spanner (TMS) topology that arose from a tandemly duplicated 5 TMS unit. Members of the RFT family have a 5 TMS topology, and are homologous to each of the repeat units in the 10 TMS proteins. The other two families [sensor histidine kinase (SHK) and kinase/phosphatase/synthetase/hydrolase (KPSH)] have a single 5 TMS unit preceded by an N‐terminal TMS and followed by a hydrophilic sensor histidine kinase domain (the SHK family) or catalytic domains resembling sensor kinase, phosphatase, cyclic di‐GMP synthetase and cyclic di‐GMP hydrolase catalytic domains, as well as various noncatalytic domains (the KPSH family). Because functional data are not available for members of the SHK and KPSH families, it is not known if the transporter domains retain transport activity or have evolved exclusive functions in molecular reception and signal transmission. This report presents characteristics of a unique protein superfamily and provides guides for future studies concerning structural, functional and mechanistic properties of its constituent members.