We applied whole-genome resequencing of Escherichia coli to monitor the acquisition and fixation of mutations that conveyed a selective growth advantage during adaptation to a glycerol-based growth medium. We identified 13 different de novo mutations in five different E. coli strains and monitored their fixation over a 44-d period of adaptation. We obtained proof that the observed spontaneous mutations were responsible for improved fitness by creating single, double and triple site-directed mutants that had growth rates matching those of the evolved strains. The success of this new genome-scale approach indicates that real-time evolution studies will now be practical in a wide variety of contexts.Comparative genomics has been almost entirely focused on genomic changes over long periods of time, on the order of millions of years. A new microarray-based method of whole-genome resequencing called comparative genome sequencing (CGS) 1 now makes it cost efficient to monitor bacterial evolution comprehensively over short time periods as well. This capability is important because many microbial phenomena, such as the emergence of new pathogens and the acquisition of antibiotic resistance factors, can occur over relatively short time scales. Experimental evolution of bacteria and viruses 2,3 is a facile approach to study these topics. It has been used to test predictions of evolutionary theory 4 and to study parallel changes in populations evolved for 20,000 generations 5 , acquisition of antibiotic resistance 1 and in vitro symbiosis 6 . Our laboratory has used experimental evolution as a tool for metabolic engineering 7 and to study the recovery of strains with gene knockouts in central metabolic genes 8 . Nevertheless, much remains unknown about genome plasticity over short evolutionary timescales.It is a common mistake to think of bacteria as static; that is, to assume that a culture grown overnight is the same as it was the day before. It has been estimated that nearly 10% of the individual bacteria in a Salmonella enterica population carry large-scale genome rearrangements 9 , and in a suboptimal environment, selection can alter a population very rapidly. The 10-20 generations that occur in the process of growing a bacterial culture are sufficient to create a heterogeneous population, depending on the magnitude of the selective advantage of adaptive mutations. This problem is avoided by using strains of bacteria that are adapted to common laboratory media, but there are interesting cases where a seemingly straightforward growth medium poses a great challenge to a bacterium.An example is E. coli K-12 grown in minimal medium supplemented with glycerol as the carbon and energy source. Despite a complete pathway for glycerol catabolism, large variations in the growth rates of various strains have been noted 10 . Growth of the sequenced strain MG1655 has been observed to differ from computational predictions based on flux balance analysis 11 . Upon extended logarithmic growth in glycerol minimal medium, the growth rate...

Background: Infections with Salmonella cause significant morbidity and mortality worldwide. Replication of Salmonella typhimurium inside its host cell is a model system for studying the pathogenesis of intracellular bacterial infections. Genome-scale modeling of bacterial metabolic networks provides a powerful tool to identify and analyze pathways required for successful intracellular replication during host-pathogen interaction.

BackgroundMetabolic reconstructions (MRs) are common denominators in systems biology and represent biochemical, genetic, and genomic (BiGG) knowledge-bases for target organisms by capturing currently available information in a consistent, structured manner. Salmonella enterica subspecies I serovar Typhimurium is a human pathogen, causes various diseases and its increasing antibiotic resistance poses a public health problem.ResultsHere, we describe a community-driven effort, in which more than 20 experts in S. Typhimurium biology and systems biology collaborated to reconcile and expand the S. Typhimurium BiGG knowledge-base. The consensus MR was obtained starting from two independently developed MRs for S. Typhimurium. Key results of this reconstruction jamboree include i) development and implementation of a community-based workflow for MR annotation and reconciliation; ii) incorporation of thermodynamic information; and iii) use of the consensus MR to identify potential multi-target drug therapy approaches.ConclusionTaken together, with the growing number of parallel MRs a structured, community-driven approach will be necessary to maximize quality while increasing adoption of MRs in experimental design and interpretation.

The intermediary metabolism of Haemophilus influenzae strain Rd KW20 was studied by a combination of protein expression analysis using a recently developed direct proteomics approach, mutational analysis, and mathematical modeling. Special emphasis was placed on carbon utilization, sugar fermentation, TCA cycle, and electron transport of H. influenzae cells grown microaerobically and anaerobically in a rich medium. The data indicate that several H. influenzae metabolic proteins similar to Escherichia coli proteins, known to be regulated by low concentrations of oxygen, were well expressed in both growth conditions in H. influenzae. An in silico model of the H. influenzae metabolic network was used to study the effects of selective deletion of certain enzymatic steps. This allowed us to define proteins predicted to be essential or non-essential for cell growth and to address numerous unresolved questions about intermediary metabolism of H. influenzae. Comparison of data from in vivo protein expression with the protein list associated with a genome-scale metabolic model showed significant coverage of the known metabolic proteome. This study demonstrates the significance of an integrated approach to the characterization of H. influenzae metabolism.