Genetic Evidence for a Link Between Glycolysis and DNA Replication

BACKGROUND: A challenging goal in biology is to understand how the principal cellular functions are integrated so that cells achieve viability and optimal fitness in a wide range of nutritional conditions. METHODOLOGY/PRINCIPAL FINDINGS: We report here a tight link between glycolysis and DNA synthesis. The link, discovered during an analysis of suppressors of thermosensitive replication mutants in bacterium Bacillus subtilis, is very strong as some metabolic alterations fully restore viability to replication mutants in which a lethal arrest of DNA synthesis otherwise occurs at a high, restrictive, temperature. Full restoration of viability by such alterations was limited to cells with mutations in three elongation factors (the lagging strand DnaE polymerase, the primase and the helicase) out of a large set of thermosensitive mutants affected in most of the replication proteins. Restoration of viability resulted, at least in part, from maintenance of replication protein activity at high temperature. Physiological studies suggested that this restoration depended on the activity of the three-carbon part of the glycolysis/gluconeogenesis pathway and occurred in both glycolytic and gluconeogenic regimens. Restoration took place abruptly over a narrow range of expression of genes in the three-carbon part of glycolysis. However, the absolute value of this range varied greatly with the allele in question. Finally, restoration of cell viability did not appear to be the result of a decrease in growth rate or an induction of major stress responses. CONCLUSIONS/SIGNIFICANCE: Our findings provide the first evidence for a genetic system that connects DNA chain elongation to glycolysis. Its role may be to modulate some aspect of DNA synthesis in response to the energy provided by the environment and the underlying mechanism is discussed. It is proposed that related systems are ubiquitous

PCR primers for detection and characterisation of IncP-9 plasmids.

Abstract IncP-9 plasmids are best known as the vehicles for spreading biodegradation functions among Pseudomonas species but can also carry resistance determinants. New PCR primer systems targeting different replicon-specific regions were designed to allow detection of IncP-9 plasmids. Their specificity was checked against a range of IncP-9 plasmids as well as representatives of incompatibility groups IncFI, IncFII, IncN, IncQ, IncP-1K, IncP-1L, IncP-2, IncP-7, IncP-13, IncW, IncU, IncX and IncZ. Products obtained for plasmids assigned to IncP-9 group by traditional incompatibility testing varied in size and restriction pattern suggesting diversity in the 'core' sequence among related replicons. Specific primer pairs were applied to community DNA extracted from a range of environments including those subject to strong selective pressure, caused by antibiotics, metals and organic pollutants. Abundant products were observed in manure and sewage, independently of the presence of antibiotics and metals, but could also be detected in coastal water and streptomycin-treated soil. Community DNA from faeces of piglets treated and non-treated with Zn gave particularly strong PCR product with IncP-9 rep primers. Therefore, an attempt was made to isolate bacteria carrying the IncP-9-like plasmids, but this was not successful. The results of application of these newly designed primer pairs to plasmid isolates as well as community DNA indicate that the IncP-9-related plasmids are a diverse family prevalent in various environments and widely distributed geographically