DNA-binding proteins are central regulators of chromosome organization; however, in genome-reduced bacteria their diversity is largely diminished. Whether the chromosomes of such bacteria adopt defined three-dimensional structures remains unexplored. Here we combine Hi-C and super-resolution microscopy to determine the structure of the Mycoplasma pneumoniae chromosome at a 10 kb resolution. We find a defined structure, with a global symmetry between two arms that connect opposite poles, one bearing the chromosomal Ori and the other the midpoint. Analysis of local structures at a 3 kb resolution indicates that the chromosome is organized into domains ranging from 15 to 33 kb. We provide evidence that genes within the same domain tend to be co-regulated, suggesting that chromosome organization influences transcriptional regulation, and that supercoiling regulates local organization. This study extends the current understanding of bacterial genome organization and demonstrates that a defined chromosomal structure is a universal feature of living systems.

The research leading to these results was funded by the European Union Seventh Framework Programme (FP7/2007-2013 to L.S.), through the European Research Council, under grant agreement 232913 to L.S. and 609989 to M.A.M-R., the European Union's Horizon 2020 research and innovation program under Grant Agreement No. 634942 to L.S, the Fundación Botín to L.S., the Spanish Ministry of Economy and Competitiveness (BIO2007-61762 to L.S. and BFU2013-47736-P to M.A.M.-R., the National Plan of R+D+i, the ISCIII-Subdirección General de Evaluación y Fomento de la Investigación- (PI10/01702 to L.S.), the Human Frontiers Science Program (RGP0044 to M.A.M.-R.), the ERASynBio/MINECO Grant PCIN-2015-125 to L.S, and the European Regional Development Fund (ERDF) to L.S. We acknowledge support from the Spanish Ministry of Economy and Competitiveness, 'Centro de Excelencia Severo Ochoa 2013-2017' (SEV-2012-0208). We acknowledge the support of the CERCA Programme / Generalitat de Catalunya.