Role of Gluconeogenesis and the Tricarboxylic Acid Cycle in the Virulence of \u3cem\u3eSalmonella enterica\u3c/em\u3e Serovar Typhimurium in BALB/c Mice

In Salmonella enterica serovar Typhimurium, the Cra protein (catabolite repressor/activator) regulates utilization of gluconeogenic carbon sources by activating transcription of genes in the gluconeogenic pathway, the glyoxylate bypass, the tricarboxylic acid (TCA) cycle, and electron transport and repressing genes encoding glycolytic enzymes. A serovar Typhimurium SR-11 Δcra mutant was recently reported to be avirulent in BALB/c mice via the peroral route, suggesting that gluconeogenesis may be required for virulence. In the present study, specific SR-11 genes in the gluconeogenic pathway were deleted (fbp, glpX, ppsA, and pckA), and the mutants were tested for virulence in BALB/c mice. The data show that SR-11 does not require gluconeogenesis to retain full virulence and suggest that as yet unidentified sugars are utilized by SR-11 for growth during infection of BALB/c mice. The data also suggest that the TCA cycle operates as a full cycle, i.e., a sucCD mutant, which prevents the conversion of succinyl coenzyme A to succinate, and an ΔsdhCDA mutant, which blocks the conversion of succinate to fumarate, were both attenuated, whereas both an SR-11 ΔaspA mutant and an SR-11 ΔfrdABC mutant, deficient in the ability to run the reductive branch of the TCA cycle, were fully virulent. Moreover, although it appears that SR-11 replenishes TCA cycle intermediates from substrates present in mouse tissues, fatty acid degradation and the glyoxylate bypass are not required, since an SR-11 ΔfadD mutant and an SR-11 ΔaceA mutant were both fully virulent

Wide Distribution of a Novel pmoA-Like Gene Copy among Type II Methanotrophs, and Its Expression in Methylocystis Strain SC2

Experiments were conducted to determine if a novel pmoA-like gene (pmoA2) recently discovered in the methane-oxidizing bacterium Methylocystis strain SC2 (P. F. Dunfield, M. Tchawa Yimga, S. D. Dedysh, U. Berger, W. Liesack, and J. Heyer, FEMS Microbiol. Ecol. 41:17-26, 2002) is present in other methane-oxidizing bacteria (MOB), and if it is expressed. A newly developed primer combination (pmoA206f-pmoA703b) allowed a differential detection of pmoA1 and pmoA2. By using this primer combination, we identified pmoA2 in a wide range of type II MOB of the Methylosinus-Methylocystis group. However, screening by PCR and by Southern hybridization using a newly developed pmoA2-specific oligonucleotide probe also showed that closely related type II MOB, exhibiting 16S rRNA gene sequence identities of higher than 97%, may or may not harbor pmoA2. No pmoA2 was detected in five type I MOB tested: Methylococcus capsulatus strain Bath, Methylocaldum strain E10A, Methylobacter luteus, Methylomicrobium album, and Methylomonas strain D1a. In comparative sequence analyses, all pmoA2-like sequences formed a coherent cluster clearly distinct from pmoA1 sequences of type I and type II MOB, and from amoA sequences of the Nitrosomonas-Nitrosospira group. Phylogenetic analysis using the paml model suggested that pmoA2 is subject to strong purifying selection and therefore has an important cellular function. We probed total RNA extracts of Methylocystis strain SC2 for gene expression of pmoA. A strong signal was observed for pmoA1 in Northern hybridization, while the results obtained for pmoA2 were ambiguous. However, reverse transcription-PCR confirmed that pmoA2 was expressed, albeit at lower level than pmoA1. This provided experimental evidence that the gene product of pmoA2 may be a functionally active enzyme

Role of Gluconeogenesis and the Tricarboxylic Acid Cycle in the Virulence of Salmonella enterica Serovar Typhimurium in BALB/c Mice

In Salmonella enterica serovar Typhimurium, the Cra protein (catabolite repressor/activator) regulates utilization of gluconeogenic carbon sources by activating transcription of genes in the gluconeogenic pathway, the glyoxylate bypass, the tricarboxylic acid (TCA) cycle, and electron transport and repressing genes encoding glycolytic enzymes. A serovar Typhimurium SR-11 Δcra mutant was recently reported to be avirulent in BALB/c mice via the peroral route, suggesting that gluconeogenesis may be required for virulence. In the present study, specific SR-11 genes in the gluconeogenic pathway were deleted (fbp, glpX, ppsA, and pckA), and the mutants were tested for virulence in BALB/c mice. The data show that SR-11 does not require gluconeogenesis to retain full virulence and suggest that as yet unidentified sugars are utilized by SR-11 for growth during infection of BALB/c mice. The data also suggest that the TCA cycle operates as a full cycle, i.e., a sucCD mutant, which prevents the conversion of succinyl coenzyme A to succinate, and an ΔsdhCDA mutant, which blocks the conversion of succinate to fumarate, were both attenuated, whereas both an SR-11 ΔaspA mutant and an SR-11 ΔfrdABC mutant, deficient in the ability to run the reductive branch of the TCA cycle, were fully virulent. Moreover, although it appears that SR-11 replenishes TCA cycle intermediates from substrates present in mouse tissues, fatty acid degradation and the glyoxylate bypass are not required, since an SR-11 ΔfadD mutant and an SR-11 ΔaceA mutant were both fully virulent