Interruption of the phosphoglucose isomerase gene results in glucose auxotrophy in Mycobacterium smegmatis

Abstract

Two glycerol utilization mutants of Mycobacterium smegmatis that were unable to utilize most carbon sources except glucose were isolated. Supplementation of these media with small amounts of glucose restored growth in the mutants; these strains are therefore glucose auxotrophs. The mutant phenotype is complemented by the gene encoding phosphoglucose isomerase (pgi), and direct measurement of enzyme activities in the mutants suggests that this gene product is absent in the auxotrophic strains. Mapping of the mutant allele by Southern analysis demonstrates the presence of a 1-kb deletion extending into the coding sequence of pgi. The possible roles of phosphoglucose isomerase in mycobacterial cell wall synthesis and metabolic regulation are discussed.The resurgence of diseases caused by mycobacteria has spurred a new interest in these acid-fast organisms. Although current research is largely directed towards virulence mechanisms in pathogenic strains of mycobacteria, basic biochemical information about these organisms is essential for understanding their interactions with their hosts and for developing new therapeutics. Knowledge of intermediary metabolism of the mycobacteria has been fragmentary, in large part due to the low growth rates, tendency to clump in culture, and lack of genetics systems of members of this genus. Mycobacteria exhibit unusual and complex patterns of carbohydrate utilization. In Mycobacterium tuberculosis, Mycobacterium phlei, and Mycobacterium smegmatis, glycerol utilization is blocked until all other carbon sources, including glutamate and asparagine, have been consumed (3,23,38). Nevertheless, glycerol is considered the preferred carbon source under laboratory conditions. Growth of most species on this substrate results in greater mass per unit volume of growth medium (32).Glycerol and its metabolic derivatives are at the interface of catabolic (glycolytic and oxidative pathways) and anabolic (lipid biosynthesis and gluconeogenesis) processes and therefore play important growth regulatory roles in cells grown on a variety of carbon sources. In addition, glycerol is an important precursor of key cell wall constituents. The presence of glycolipids such as the acyl glycerols and acylglucoses varies widely depending on the carbon source in the medium (4). A number of the enzymes of glycolysis, the hexose monophosphate shunt, and the Entner-Doudoroff pathways have been studied (reviewed in reference 26). However, carbon metabolism in mycobacteria has not been characterized at the genetic level and few genes encoding glycolytic enzymes have been cloned.We have initiated a genetic approach to the study of carbohydrate metabolism in M. smegmatis by isolating and characterizing mutants unable to utilize certain substrates as sole carbon sources. In this study, we describe two strains obtained in a screen for glycerol utilization mutants. Unexpectedly, these mutants are unable to use any carbohydrate except glucose as a sole carbon source. Genetic complementation of the growth defect on glycerol ...

Molecular Analysis of the Gene Encoding F 420 -Dependent Glucose-6-Phosphate Dehydrogenase from Mycobacterium smegmatis

“…In E. coli, GTG rarely functions as an initiation codon, and when it does it is less efficient than ATG (23). However, Mycobacterium species use GTG as the initiation codon for many genes (9,25,35,59). The GϩC content of fgd was 65%, which is in the range of the GϩC content of Mycobacterium chromosomes (62 to 70%).…”

Differential expression of iron-, carbon-, and oxygen-responsive mycobacterial genes in the lungs of chronically infected mice and tuberculosis patients

“…The observations that ICL is essential for MTB persistence in vivo (15), and that normalized levels of icl1 mRNA increase during infection, suggest that glucose might be unavailable in vivo. Glucose 6-phosphate is an essential intermediate in a number of metabolic pathways; if an external source of glucose is unavailable, glucose 6-phosphate is synthesized de novo by gluconeogenesis (34). Carbon derived from ␤-oxidation of fatty acids is diverted into gluconeogenesis through phosphoenolpyruvate carboxykinase (PckA), which converts oxaloacetate to phosphoenolpyruvate.…”

Glucose 6-Phosphate Accumulation in Mycobacteria

“…Glucose 6-phosphate, derived from glucose through the glycolytic enzymes hexokinase or glucokinase and through gluconeogenesis, generates reducing power via the pentose phosphate pathway. In mycobacteria, G6P is also an important precursor of cell wall components L-rhamnose and the galactan of arabinogalac-tan (20). Thus, because G6P is a central intermediate with many possible fates, we sought to understand the role of Fgd in G6P metabolism.…”