Cloning of Klebsiella pneumoniae pqq genes and PQQ biosynthesis in Escherichia coli

We have cloned genes from Klebsiella pneumoniae which are required for pyrroloquinoline quinone (PQQ) biosynthesis. The cloned 6.7 kb fragment can complement several chromosomal pqq mutants. Escherichia coli strains are unable to synthesize PQQ but E. coli strains containing the cloned 6.7 kb K. pneumoniae fragment can synthesize PQQ in large amounts and E. coli pts mutants can be complemented on minimal glucose medium by this clone.     

Continued growth of Escherichia coli after stopping medium addition to a K+-limited chemostat culture

The steady-state bacterial dry wt of Escherichia coli, growing under K+-limited conditions in the chemostat, was inversely dependent on the growth rate. This phenomenon was more carefully investigated in medium-flow stop experiments. Growth did not stop immediately but continued for a time, initially at the same rate as before. The dry wt increased to a value corresponding to a steady-state growth rate near zero, independent of the initial specific growth rate. This was observed in both the wild-type strain and a mutant that lacked the high-affinity K+ uptake system. The wild-type strain maintained a low extracellular K+ concentration both in the chemostat under steady-state conditions and after stopping the medium flow. The mutant, on the other hand, maintained a much higher extracellular K+ concentration in the steady state, which decreased to much lower values after stopping the medium flow. From the increase in bacterial dry wt and the low external K+ concentration after stopping the medium flow it is concluded that the intracellular K+ is redistributed among the cells, including new cells. The growth yield on K+ was highest in the stationary growth phase of a batch culture and all steady-state cultures converged ultimately to this yield value after the medium flow had been stopped. It is proposed that the growth rate of E. coli under K+-limited conditions is determined by the intracellular K+ concentration.     

Regulation of glycerol kinase by enzyme IIIGlc of the phosphoenolpyruvate:carbohydrate phosphotransferase system.

Wild-type glycerol kinase of Escherichia coli is inhibited by both nonphosphorylated enzyme IIIGlc of the phosphoenolpyruvate:carbohydrate phosphotransferase system and fructose 1,6-diphosphate. Mutant glycerol kinase, resistant to inhibition by fructose 1,6-diphosphate, was much less sensitive to inhibition by enzyme IIIGlc. The difference between the wild-type and mutant enzymes was even greater when inhibition was measured in the presence of both enzyme IIIGlc and fructose 1,6-diphosphate. The binding of enzyme IIIGlc to glycerol kinase required the presence of the substrate glycerol.     

Role of IIIGlc of the phosphoenolpyruvate-glucose phosphotransferase system in inducer exclusion in Escherichia coli.

The phosphoenolpyruvate-D-glucose phosphotransferase system of Enterobacteriaceae is thought to regulate the synthesis and activity of a number of catabolite uptake systems, including those for maltose, lactose, and glycerol, via the phosphorylation state of one of its components, IIIGlc. We have investigated the proposal by Kornberg and co-workers (FEBS Lett. 117(Suppl.):K28-K36, 1980) that not IIIGlc, but an unknown protein, the product of the iex gene, is responsible for the exclusion of the above-mentioned compounds from the cell. The iex mutant HK738 of Escherichia coli contains normal amounts of IIIGlc as measured by specific antibodies, in contrast to crr mutants that lack IIIGlc. The IIIGlc of the iex strain functions normally in glucose and methyl alpha-glucoside transport, and the specific activity in in vitro phosphorylation is approximately 60% of that of the parent. The IIIGlc activity of the iex strain is, however, heat labile, in contrast to the parental IIIGlc, suggesting that the mutant contains an altered IIIGlc. This is supported by the observation that IIIGlc from the iex strain cannot bind to the lactose carrier. Thus it cannot inhibit the carrier, and this explains why the uptake of non-phosphotransferase system compounds in an iex strain is resistant to phosphotransferase system sugars. The introduction of a plasmid containing a wild-type crr+ allele into the iex strain restores the iex phenotype to that of the iex+ parent. The IIIGlc produced from the plasmid in the iex strain is heat stable and binds normally to the lactose carrier. These results lead to the conclusion that the iex mutation is most likely allelic with crr and results in an altered, temperature-sensitive IIIGlc that is still able to function D-glucose and methyl alpha-glucoside uptake and phosphorylation and in the activation of adenylate cyclase, but is unable to bind to and inhibit the lactose carrier.     

Glucose transport in crabtree-positive and crabtree-negative yeasts

The kinetic parameters of glucose transport in four Crabtree-positive and four Crabtree-negative yeasts were determined. The organisms were grown in aerobic glucose-limited chemostats at a dilution rate of 0.1 h-1. The results show a clear correlation between the presence of high-affinity glucose transport systems and the absence of aerobic fermentation upon addition of excess glucose to steady-state cultures. The presence of these H+-symport systems could be established by determination of intracellular accumulation of 6-deoxy-[3H]glucose and alkalinization of buffered cell suspensions upon addition of glucose. In contrast, the yeasts that did show aerobic alcoholic fermentation during these glucose pulse experiments had low-affinity facilitated-diffusion carriers only. In the yeasts examined the capacity of the glucose transport carriers was higher than the actual glucose consumption rates during the glucose pulse experiments. The relationship between the rate of sugar consumption and the rate of alcoholic fermentation was studied in detail with Saccharomyces cerevisiae. When S. cerevisiae was pulsed with low amounts of glucose or mannose, in order to obtain submaximal sugar consumption rates, fermentation was already occurring at sugar consumption rates just above those which were maintained in the glucose-limited steady-state culture. The results are interpreted in relation with the Crabtree effect. In Crabtree-positive yeasts, an increase in the external glucose concentration may lead to unrestricted glucose uptake by facilitated diffusion and hence, to aerobic fermentation. In contrast, Crabtree-negative yeasts may restrict the entry of glucose by their regulated H+-symport systems and thus prevent the occurrence of overflow metabolism.