Bacterial chromosomes frequently contain arrays of contiguous genes that group according to related metabolic roles. We propose that clustering of genes for metabolically related functions confers thermodynamic advantage to the organism based upon our protein immobility model (PIM) of intracellular diffusion. This thermodynamic effect provides the selection force argument that is missing from previous models of gene clustering. The PIM posits that clustered genes produce local clusters of enzymes in bacteria owing to the co-linearity of transcription and translation, and to the relative immobility of large proteins released into the cytosol. We maintain that the resulting physical proximity of enzymes for related pathway steps minimizes the steady state level of reaction step intermediates and thus conserves the energy and material required for rapid growth and maintenance. Support for this idea comes from in silico experiments using the PIM applied to a model metabolic pathway A --> B --> C. The metabolites A, B, and C are small molecules that diffuse freely in a cytosol crowded with macromolecules, whereas the large enzyme molecules, E1 and E2, tend to remain in the vicinity of their point of release. Modeling E1 as a source of B from A, and E2 as a sink for B, numerical experiments suggest that the steady state concentration of B in the cytosol increases approximately in proportion to the square of the distance of the E1 and E2 separation. A further model prediction is that the steady state concentration of B is influenced by the geometric effects of the spatial location and orientation of E1 relative to E2. These results suggest that: (i) gene clustering reduces the energy and material costs of enzyme reactions linked by metabolic intermediates; (ii) gene clusters near ori, the origin of replication, utilize the geometric effect to conserve free energy by further reducing the steady state concentration of the intermediate; (iii) gene organization on a chromosome influences the organism's capacity to accelerate into steady state growth and is, in turn, influenced by the abundance and frequency of access to nutrients.

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A spontaneous mutant of a derivative of Escherichia coli strain K-12 resistant to 50 ug of ketomycin per ml was selected. The mutant displayed a two-to threefold derepression of the isoleucine-valine biosynthetic enzymes and a reduced growth rate in minimal medium. The lesion was found to lie in the gene (ilvE) specifying transaminase B and resulted in an isoleucine limitation. The presence of exogenous isoleucine during growth in minimal medium restored normal phenotypic properties. The reduced transaminase B activity is responsible for the resistance to ketomycin. An unusual derepression of the acetohydroxy acid synthetase in response to an isoleucine limitation was noted.

The Escherichia coli K-12 genetic map was divided into intervals of equal length to count the number of genes per interval. Plots of genes per interval at four sets of interval lengths revealed large-scale clustering of genes with the major clusters occurring at regularly spaced distances apart. Major gene cluster properties were analyzed at a scale of 100 intervals wherein each interval corresponded to a genetic map unit length of 1 min. In any major gene cluster, the highest gene concentration was observed at or near the midpoint interval, and the number of genes per interval was found to decline exponentially as a function of the linear distance from the midpoint or interval of peak gene concentration of that cluster. An autocorrelation analysis of gene content in first-neighbor intervals throughout the chromosome revealed an ordered first-neighbor relationship in comparison to 2,000 randomized interval versions of the chromosome. Attempts to simulate gene placement by a Gaussian model did not produce large-scale gene clustering in any way comparable to that observed on the chromosome. We propose that major gene clusters formed from smaller gene clusters, and the contemporary chromosome formed from fusion of homologous or heterologous major gene clusters.

Two genes (arg-3 and arp-6) marked by arginine auxotrophy and a gene (sac-2) marked by resistance to serum killing were found to be linked by cotransformation. Evidence derived from the linkage of these markers to spc (spectinomycin resistance) suggests that sac-2 may be genetically distinct from a sac gene previously described.