Growth of Escherichia coli MG1655 on LB medium: monitoring utilization of sugars, alcohols, and organic acids with transcriptional microarrays.

Microorganisms respond to environmental changes by reprogramming their metabolism primarily through altered patterns of gene expression. DNA microarrays provide a tool for exploiting microorganisms as living sensors of their environment. The potential of DNA microarrays to reflect availability of nutrient components during fermentations on complex media was examined by monitoring global gene expression throughout batch cultivation of Escherichia coli MG1655 on Luria-Bertani (LB) medium. Gene expression profiles group into pathways that clearly demonstrate the metabolic changes occurring in the course of fermentation. Functional analysis of the gene expression related to metabolism of sugars, alcohols, and organic acids revealed that E. coli growing on LB medium switches from a sequential mode of substrate utilization to the simultaneous one in the course of the growth. Maltose and maltodextrins are the first of these substrates to support growth. Utilization of these nutrients associated with the highest growth rate of the culture was followed by simultaneous induction of enzymes involved in assimilation of a large group of other carbon sources including D-mannose, melibiose, D-galactose, L-fucose, L-rhamnose, D-mannitol, amino sugars, trehalose, L-arabinose, glycerol, and lactate. Availability of these nutrients to the cells was monitored by induction of corresponding transport and/or catabolic systems specific for each of the compounds.

Growth of Escherichia coli MG1655 on LB medium: determining metabolic strategy with transcriptional microarrays.

Expression profiles of genes related to stress responses, substrate assimilation, acetate metabolism, and biosynthesis were obtained by monitoring growth of Escherichia coli MG1655 in Luria-Bertani (LB) medium with transcriptional microarrays. Superimposing gene expression profiles on a plot of specific growth rate demonstrates that the cells pass through four distinct physiological states during fermentation before entering stationary phase. Each of these states can be characterized by specific patterns of substrate utilization and cellular biosynthesis corresponding to the nutrient status of the medium. These data allow the growth phases of the classical microbial growth curve to be redefined in terms of the physiological states and environmental changes commonly occurring during bacterial growth in batch culture on LB medium.

Growth of Escherichia coli MG1655 on LB medium: monitoring utilization of amino acids, peptides, and nucleotides with transcriptional microarrays.

Analysis of gene expression data related to assimilation and biosynthesis of nitrogen-containing compounds amino acids, peptides, and nucleotides was used to monitor availability of these nutrients to Escherichia coli MG1655 growing on Luria-Bertani medium. The data indicate that free amino acids and nucleotides only transiently support the nitrogen requirement for growth and are no longer available by 3.5 h of fermentation. The resulting shortage of available nitrogen sources induces the Ntr response, which involves induction of the glnALG, glnK-amtB, dppABCDF, and oppABCDF operons as well as the genes coding for outer membrane proteins, porins OmpA and OmpC, and proteases OmpP and OmpT. The increased uptake of peptides facilitated by the products of dppABCDF, oppABCDF, ompA, ompC, ompP, and ompT alleviates nitrogen limitation of the growth.

From genetic footprinting to antimicrobial drug targets: examples in cofactor biosynthetic pathways.

Novel drug targets are required in order to design new defenses against antibiotic-resistant pathogens. Comparative genomics provides new opportunities for finding optimal targets among previously unexplored cellular functions, based on an understanding of related biological processes in bacterial pathogens and their hosts. We describe an integrated approach to identification and prioritization of broad-spectrum drug targets. Our strategy is based on genetic footprinting in Escherichia coli followed by metabolic context analysis of essential gene orthologs in various species. Genes required for viability of E. coli in rich medium were identified on a whole-genome scale using the genetic footprinting technique. Potential target pathways were deduced from these data and compared with a panel of representative bacterial pathogens by using metabolic reconstructions from genomic data. Conserved and indispensable functions revealed by this analysis potentially represent broad-spectrum antibacterial targets. Further target prioritization involves comparison of the corresponding pathways and individual functions between pathogens and the human host. The most promising targets are validated by direct knockouts in model pathogens. The efficacy of this approach is illustrated using examples from metabolism of adenylate cofactors NAD(P), coenzyme A, and flavin adenine dinucleotide. Several drug targets within these pathways, including three distantly related adenylyltransferases (orthologs of the E. coli genes nadD, coaD, and ribF), are discussed in detail.

Effect of growth conditions on the synthesis of terminal oxidases in Methylobacillus flagellatus KT.

Membranes of the obligate methylotroph Methylobacillus flagellatus KT contained hemes B, O, and C and cytochromes b, o, and c both in batch and in continuous cultures. Neither heme A nor heme D was detected in the membranes. The cytochromes o and bb were the main components reversibly binding carbon monoxide (CO) in the terminal part of the respiratory chain. The alpha-region and especially the alpha-peaks at 568 and 573 nm and the alpha-troughs at 586 and 592 on the CO-difference spectra were diagnostic for the cytochromes o and bb, respectively. The cytochrome o content increased up to 1.8 times upon increasing the dilution rate of the culture from 0.15 to 0.55 h(-1) under methanol limitation. By contrast, the level of the CO-binding cytochrome bb was not affected by methanol concentration but its content increased up to 1.9 times when the level of oxygen decreased from 95 to 21 microM under the constant dilution rate (mu = 0.55 h(-1)). The maximum ratio between the cytochromes o and bb reached 2 during continuous cultivation under methanol-limited conditions (mu = 0.55 h(-1)), whereas the minimum ratio between them was about 0.7 during batch cultivation at stationary phase of growth. The synthesis of the CO-binding cytochrome bb but not of the cytochrome o in M. flagellatus KT was assumed to depend on the ambient redox potential of the medium. The cytochrome o synthesis was supposed to depend on the transmembrane gradient of protons (Delta(mu)H+).