Transcription generates local topological and mechanical constraints on the DNA fiber, leading to the formation of supercoiled domains in bacterial chromosomes. However, the global impact of transcription on chromosome organization remains unclear due to the limited resolution of chromosomal contact maps. This study combines sub-kb Hi-C contact maps with chromosome engineering to visualize individual transcriptional units (TUs). It shows that TUs form discrete three-dimensional transcription-induced domains (TIDs) that impose mechanical and topological constraints on neighboring sequences, influencing their localization and dynamics. These TIDs are primary building blocks of bacterial chromosome folding. Bacterial genomes are organized into the nucleoid, a membrane-less compartment where DNA, RNA, and proteins interact to shape the chromosome. Transcription modulates DNA supercoiling, creating twin domains spanning 25 kb. Topoisomerases maintain supercoiling homeostasis. Hi-C contact maps reveal higher-order organization in bacterial chromosomes, with directionality index (DI) analysis identifying ~30 chromosome self-interacting domains (CIDs). Highly expressed genes (HEGs) correlate with CID boundaries, though not systematically. Transcription level correlates with contact frequencies between adjacent DNA segments. Inhibition of transcription by rifampicin abrogates domains and decondenses nucleoids, suggesting a direct role for transcription in chromosome folding. High-resolution Hi-C maps of E. coli cells reveal strong heterogeneity in short-range contact signals, with bundled domains correlated with transcriptional activity. These domains range in size from 1 to 20 kb and are distributed across the genome. A plaid-like pattern is observed, corresponding to enrichment in contacts between transcribed and non-transcribed regions. These observations suggest that neighboring transcribed regions tend to contact each other, possibly due to relocation to the nucleoid periphery or transcription-dependent clustering. TIDs explain CID detection in low-resolution maps. TIDs are separated by non-transcribed regions but can interact if genomic distance is small. A single TU is sufficient to imprint a Hi-C domain. T7 promoter induction creates a bundled domain, with Hi-C signals shaped like an arrowhead. The presence of rifampicin highlights a single transcriptional unit, showing that endogenous RNA polymerase blocks T7-induced transcription. TIDs impose mechanical constraints on adjacent regions. Live imaging shows that T7 transcription affects gene localization, bringing flanking regions closer. T7 expression increases colocalization of distant regions, suggesting T7-mediated folding influences chromosome organization. TIDs are structurally dependent on transcription level and genomic context, with TIDs forming the center of twin-supercoiled domains. These findings suggest that transcription shapes bacterial chromosomes by imposing local constraints, influencing chromosome organization and dynamics. The study highlights the role of transcription in bacterial chromosome folding and the mechanical constraints it imposes on neighboring regions.Transcription generates local topological and mechanical constraints on the DNA fiber, leading to the formation of supercoiled domains in bacterial chromosomes. However, the global impact of transcription on chromosome organization remains unclear due to the limited resolution of chromosomal contact maps. This study combines sub-kb Hi-C contact maps with chromosome engineering to visualize individual transcriptional units (TUs). It shows that TUs form discrete three-dimensional transcription-induced domains (TIDs) that impose mechanical and topological constraints on neighboring sequences, influencing their localization and dynamics. These TIDs are primary building blocks of bacterial chromosome folding. Bacterial genomes are organized into the nucleoid, a membrane-less compartment where DNA, RNA, and proteins interact to shape the chromosome. Transcription modulates DNA supercoiling, creating twin domains spanning 25 kb. Topoisomerases maintain supercoiling homeostasis. Hi-C contact maps reveal higher-order organization in bacterial chromosomes, with directionality index (DI) analysis identifying ~30 chromosome self-interacting domains (CIDs). Highly expressed genes (HEGs) correlate with CID boundaries, though not systematically. Transcription level correlates with contact frequencies between adjacent DNA segments. Inhibition of transcription by rifampicin abrogates domains and decondenses nucleoids, suggesting a direct role for transcription in chromosome folding. High-resolution Hi-C maps of E. coli cells reveal strong heterogeneity in short-range contact signals, with bundled domains correlated with transcriptional activity. These domains range in size from 1 to 20 kb and are distributed across the genome. A plaid-like pattern is observed, corresponding to enrichment in contacts between transcribed and non-transcribed regions. These observations suggest that neighboring transcribed regions tend to contact each other, possibly due to relocation to the nucleoid periphery or transcription-dependent clustering. TIDs explain CID detection in low-resolution maps. TIDs are separated by non-transcribed regions but can interact if genomic distance is small. A single TU is sufficient to imprint a Hi-C domain. T7 promoter induction creates a bundled domain, with Hi-C signals shaped like an arrowhead. The presence of rifampicin highlights a single transcriptional unit, showing that endogenous RNA polymerase blocks T7-induced transcription. TIDs impose mechanical constraints on adjacent regions. Live imaging shows that T7 transcription affects gene localization, bringing flanking regions closer. T7 expression increases colocalization of distant regions, suggesting T7-mediated folding influences chromosome organization. TIDs are structurally dependent on transcription level and genomic context, with TIDs forming the center of twin-supercoiled domains. These findings suggest that transcription shapes bacterial chromosomes by imposing local constraints, influencing chromosome organization and dynamics. The study highlights the role of transcription in bacterial chromosome folding and the mechanical constraints it imposes on neighboring regions.