Molecular genetic and biochemical investigations into the bacterial PTS system were inspired by the complete genome sequences of E. coli and B. subtilis which had just become available:

• In Escherichia coli EI(Ntr) was shown to phosphorylate NPr in vitro using either a [(32)P]PEP-dependent protein phosphorylation assay or a quantitative sugar phosphorylation assay. EI(Ntr) phosphorylated NPr but not HPr, whereas Enzyme I exhibited a strong preference for HPr. These two pairs of proteins (EI(Ntr)/NPr and EI/HPr) thus exhibit little cross-reactivity. The results suggest that E. coli possesses at least two distinct PTS phosphoryl transfer chains, EI(Ntr) à NPr à IIA(Ntr) and EI à HPr à IIA(sugar). The EI(Ntr)/NPr system is suggested to be involved in regulatory cross-talk between carbon and nitrogen cycle.
  
• In Bacillus subtilis HPr(Ser) kinase is the sensor in a multicomponent phosphorelay system that controls catabolite repression, sugar transport and carbon metabolism in gram-positive bacteria. The gene (ptsK) encoding this serine/threonine protein kinase was identified and the purified protein product characterized. The PtsK kinase was shown to be allosterically activated by metabolites such as fructose-1,6-bisphosphate and inhibited by inorganic phosphate. In contrast to wild-type B. subtilis, the ptsK mutant was insensitive to transcriptional regulation by catabolite repression.

Taking advantage of the increasing genomic databases, the phylogeny of transport proteins was studied.

• Tripartite ATP-independent periplasmic transporters (TRAP-T) represent a novel type of secondary active transporter (DctMQ) that functions in conjunction with an extracytoplasmic solute-binding receptor (DctP). Phylogenetic data suggest that all present day TRAP-T systems probably evolved from a single ancestral transporter with minimal shuffling of constituents between systems. DctM appears to belong to a large superfamily of transporters, the ion transporter (IT) superfamily.

• A comprehensive analysis of solute transport systems encoded within the completely sequenced genomes of 18 prokaryotic organisms was conducted. Membrane proteins are analyzed in terms of putative membrane topology, and the recognized transport systems are classified into 76 families. The mode of transport generally correlates with the primary mechanism of energy generation, and the numbers of secondary transporters relative to primary transporters are roughly proportional to the total numbers of primary H(+) and Na(+) pumps in the cell. These results provide insight into the relevance of transport to the overall physiology of prokaryotes