BackgroundThe structure and function of bacterial nucleoid are controlled by histone-like proteins of HU/IHF family, omnipresent in bacteria and also founding archaea and some eukaryotes.HU protein binds dsDNA without sequence specificity and avidly binds DNA structures with propensity to be inclined such as forks, three/four-way junctions, nicks, overhangs and DNA bulges. Sequence comparison of thousands of known histone-like proteins from diverse bacteria phyla reveals relation between HU/IHF sequence, DNA–binding properties and other protein features.Methodology and principal findingsPerformed alignment and clusterization of the protein sequences show that HU/IHF family proteins can be unambiguously divided into three groups, HU proteins, IHF_A and IHF_B proteins. HU proteins, IHF_A and IHF_B proteins are further partitioned into several clades for IHF and HU; such a subdivision is in good agreement with bacterial taxonomy. We also analyzed a hundred of 3D fold comparative models built for HU sequences from all revealed HU clades. It appears that HU fold remains similar in spite of the HU sequence variations. We studied DNA–binding properties of HU from N. gonorrhoeae, which sequence is similar to one of E.coli HU, and HU from M. gallisepticum and S. melliferum which sequences are distant from E.coli protein. We found that in respect to dsDNA binding, only S. melliferum HU essentially differs from E.coli HU. In respect to binding of distorted DNA structures, S. melliferum HU and E.coli HU have similar properties but essentially different from M. gallisepticum HU and N. gonorrhea HU. We found that in respect to dsDNA binding, only S. melliferum HU binds DNA in non-cooperative manner and both mycoplasma HU bend dsDNA stronger than E.coli and N. gonorrhoeae. In respect to binding to distorted DNA structures, each HU protein has its individual profile of affinities to various DNA-structures with the increased specificity to DNA junction.Conclusions and significanceHU/IHF family proteins sequence alignment and classification are updated. Comparative modeling demonstrates that HU protein 3D folding’s even more conservative than HU sequence. For the first time, DNA binding characteristics of HU from N. gonorrhoeae, M. gallisepticum and S. melliferum are studied. Here we provide detailed analysis of the similarity and variability of DNA-recognizing and bending of four HU proteins from closely and distantly related HU clades.