Identification of potential drug targets in Salmonella enterica sv. Typhimurium using metabolic modelling and experimental validation

Salmonella enterica sv. Typhimurium is an established model organism for Gram-negative, intracellular pathogens. Owing to the rapid spread of resistance to antibiotics among this group of pathogens, new approaches to identify suitable target proteins are required. Based on the genome sequence of S. Typhimurium and associated databases, a genome-scale metabolic model was constructed. Output was based on an experimental determination of the biomass of Salmonella when growing in glucose minimal medium. Linear programming was used to simulate variations in the energy demand while growing in glucose minimal medium. By grouping reactions with similar flux responses, a subnetwork of 34 reactions responding to this variation was identified (the catabolic core). This network was used to identify sets of one and two reactions that when removed from the genome-scale model interfered with energy and biomass generation. Eleven such sets were found to be essential for the production of biomass precursors. Experimental investigation of seven of these showed that knockouts of the associated genes resulted in attenuated growth for four pairs of reactions, whilst three single reactions were shown to be essential for growth.

The Small RNA GcvB Regulates sstT mRNA Expression in Escherichia coli

ABSTRACT In Escherichia coli, the gcvB gene encodes a nontranslated RNA (referred to as GcvB) that regulates OppA and DppA, two periplasmic binding proteins for the oligopeptide and dipeptide transport systems. An additional regulatory target of GcvB, sstT, was found by microarray analysis of RNA isolated from a wild-type strain and a gcvB deletion strain grown to mid-log phase in Luria-Bertani broth. The SstT protein functions to transport l-serine and l-threonine by sodium transport into the cell. Reverse transcription-PCR and translational fusions confirmed that GcvB negatively regulates sstT mRNA levels in cells grown in Luria-Bertani broth. A series of transcriptional fusions identified a region of sstT mRNA upstream of the ribosome binding site needed for negative regulation by GcvB. Analysis of the GcvB RNA identified a sequence complementary to this region of the sstT mRNA. The region of GcvB complementary to sstT mRNA is the same region of GcvB identified to regulate the dppA and oppA mRNAs. Mutations predicted to disrupt base pairing between sstT mRNA and GcvB were made in gcvB, which resulted in the identification of a small region of GcvB necessary for negative regulation of sstT-lacZ. Additionally, the RNA chaperone protein Hfq was found to be necessary for GcvB to negatively regulate sstT-lacZ in Luria-Bertani broth and glucose minimal medium supplemented with glycine. The sstT mRNA is the first target found to be regulated by GcvB in glucose minimal medium supplemented with glycine.

YjjG, a dUMP Phosphatase, Is Critical for Thymine Utilization by Escherichia coli K-12

ABSTRACT Exogenous thymine must be converted to thymidine to enable a thyA (thymidylate synthase) mutant to grow. The deoxyribose in the thymidine comes from dUMP, which must first be dephosphorylated. The nucleotidase YjjG is critical for this step. A yjjG thyA mutant cannot use thymine for growth on a glucose minimal medium.

Mechanisms Causing Rapid and Parallel Losses of Ribose Catabolism in Evolving Populations of Escherichia coli B

ABSTRACT Twelve populations of Escherichia coli B all lostd-ribose catabolic function during 2,000 generations of evolution in glucose minimal medium. We sought to identify the population genetic processes and molecular genetic events that caused these rapid and parallel losses. Seven independent Rbs−mutants were isolated, and their competitive fitnesses were measured relative to that of their Rbs+ progenitor. These Rbs− mutants were all about 1 to 2% more fit than the progenitor. A fluctuation test revealed an unusually high rate, about 5 × 10−5 per cell generation, of mutation from Rbs+ to Rbs−, which contributed to rapid fixation. At the molecular level, the loss of ribose catabolic function involved the deletion of part or all of the ribose operon (rbs genes). The physical extent of the deletion varied between mutants, but each deletion was associated with an IS150 element located immediately upstream of therbs operon. The deletions apparently involved transposition into various locations within the rbs operon; recombination between the new IS150 copy and the one upstream of therbs operon then led to the deletion of the intervening sequence. To confirm that the beneficial fitness effect was caused by deletion of the rbs operon (and not some undetected mutation elsewhere), we used P1 transduction to restore the functionalrbs operon to two Rbs− mutants, and we constructed another Rbs− strain by gene replacement with a deletion not involving IS150. All three of these new constructs confirmed that Rbs− mutants have a competitive advantage relative to their Rbs+ counterparts in glucose minimal medium. The rapid and parallel evolutionary losses of ribose catabolic function thus involved both (i) an unusually high mutation rate, such that Rbs− mutants appeared repeatedly in all populations, and (ii) a selective advantage in glucose minimal medium that drove these mutants to fixation.

trans-Acting Factors Affecting Carbon Catabolite Repression of the hut Operon inBacillus subtilis

ABSTRACT In Bacillus subtilis, CcpA-dependent carbon catabolite repression (CCR) mediated at several cis-acting carbon repression elements (cre) requires the seryl-phosphorylated form of both the HPr (ptsH) and Crh (crh) proteins. During growth in minimal medium, theptsH1 mutation, which prevents seryl phosphorylation of HPr, partially relieves CCR of several genes regulated by CCR. Examination of the CCR of the histidine utilization (hut) enzymes in cells grown in minimal medium showed that neither theptsH1 nor the crh mutation individually had any affect on hut CCR but that hut CCR was abolished in a ptsH1 crh double mutant. In contrast, theptsH1 mutation completely relieved hut CCR in cells grown in Luria-Bertani medium. The ptsH1 crh double mutant exhibited several growth defects in glucose minimal medium, including reduced rates of growth and growth inhibition by high levels of glycerol or histidine. CCR is partially relieved in B. subtilis mutants which synthesize low levels of active glutamine synthetase (glnA). In addition, these glnAmutants grow more slowly than wild-type cells in glucose minimal medium. The defects in growth and CCR seen in these mutants are suppressed by mutational inactivation of TnrA, a global nitrogen regulatory protein. The inappropriate expression of TnrA-regulated genes in this class of glnA mutants may deplete intracellular pools of carbon metabolites and thereby result in the reduction of the growth rate and partial relief of CCR.

Construction of an Escherichia coli strain which excretes abnormally large amounts of adenosine 3′,5′-cyclic monophosphate

It has previously been shown that an Escherichia coli CRP− strain 5333 accumulates abnormally large amounts of adenosine 3′,5′-cyclic monophosphate (cAMP). Using P1 transduction, the CRP− character was transferred to E. coli Crookes strain which is deficient for cAMP phosphodiesterase (CPD−). The resulting strain HY22 (CRP−, CPD−) accumulates greater amounts of cAMP both intracellularly and extracellularly than does 5333. In glucose minimal medium, an HY22 cell accumulates 100 times more cAMP intracellularly and excretes cAMP 150 times faster than does a wild-type E. coli cell.

The intracellular reserve polysaccharide of Clostridium pasteurianum

An amylopectinlike polysaccharide (granulose) was the only glucan produced in significant quantities by six wild-type strains of Clostridium pasteurianum grown in glucose minimal medium. The intracellular polysaccharide granules laid down before sporulation contained only this amylopectin. No intracellular dextran was discovered in these wild-type strains, nor in a granulose-negative mutant strain of C. pasteurianum possessing an ADP glucose pyrophosphorylase (EC 2.7.7.27) but lacking a granulose synthase (i.e. ADPglucose-α-1,4-glucan glucosyl transferase, EC 2.4.1.21). Furthermore, methylation analysis demonstrated that (1 → 6) linked α-D-glucose units accounted for less than 2% of the entire glucose content of these organisms.

Effect of carbon source during growth on sensitivity of Pseudomonas fluorescens to actinomycin D

Sensitivity to actinomycin D (AD) varies in Pseudomonas fluorescens cells grown in glucose or succinate minimal salts medium. Growth is inhibited in succinate minimal medium by much lower concentrations of AD than in glucose minimal medium. Uptake of selected radioactive metabolites is inhibited by AD in cells incubated for 2 h in succinate medium containing AD but glucose-grown cells were not sensitive. EDTA treatment promotes increased sensitivity to AD in succinate-grown cells but does not alter sensitivity in glucose-grown cells. Succinate-grown cells bound 2–3 times as much 3H-AD as glucose-grown cells. Glucose-grown cells had much higher lipopolysaccharide levels in the envelope than succinate-grown cells. It is proposed that the lipopolysaccharide masks the binding sites and, therefore, is responsible for the difference in binding of AD by the glucose- and succinate-grown cells. The availability of the binding sites is also reflected in the sensitivity of the cells to the antibiotic.