Chromosomes are compacted hundreds of times to fit in the cell, packaged into dynamic folds whose structures are largely unknown. Here, we examine patterns in gene locations to infer large-scale features of bacterial chromosomes. Specifically, we analyzed >100 genomes and identified thousands of gene pairs that display two types of evolutionary correlations: a tendency to co-occur and a tendency to be located close together in many genomes. We then analyzed the detailed distribution of these pairs in Escherichia coli and found that genes in a pair tend to be separated by integral multiples of 117 kb along the genome and to be positioned in a 117-kb grid of genomic locations. In addition, the most pair-dense locations coincide with regions of intense transcriptional activity and the positions of top transcribed and conserved genes. These patterns suggest that the E. coli chromosome may be organized into a 117-kb helix-like topology that localizes a subset of the most essential and highly transcribed genes along a specific face of this structure. Our approach indicates an evolutionarily maintained preference in the spacing of genes along the chromosome and offers a general comparative genomics framework for studying chromosome structure, broadly applicable to other organisms.