In contrast to organized hierarchical structure of eukaryotic chromosome, bacterial chromosomes are believed not to have such structures. The genomes of bacteria are condensed into a compact structure called the nucleoid. Among many architectural, histone-like proteins which associate with the chromosomal DNA is HU which is implicated in folding DNA into a compact structure by bending and wrapping DNA. Unlike the majority of other histone-like proteins, HU is highly conserved in eubacteria and unique in its ability to bind RNA. Furthermore, an HU mutation profoundly alters the cellular transcription profile and consequently has global effects on physiology and the lifestyle of E. coli. Here we provide a short overview of the mechanisms by which the nucleoid is organized into different topological domains. We propose that HU is a major player in creating domain-specific superhelicities and thus influences the transcription profile from the constituent promoters. This article is part of a Special Issue entitled: Chromatin in time and space.

SummaryReductions in growth rate caused by fusidic acidresistant EF-G mutants in Salmonella typhimurium correlate strongly with increased mean cell size. This is unusual because growth rate and cell size normally correlate positively. The global transcription regulator molecule ppGpp has a role in co-ordinating growth rate and division, and its basal level normally correlates inversely with cell size at division. We show that fusidic acid-resistant EF-G mutants have perturbed ppGpp basal levels during steady-state growth and perturbed induced levels during starvation. One mutation, fusA1, associated with the slowest growth rate and largest cell size, causes a reduction in the basal level of ppGpp to one-third of that found in the wild-type strain. Other fusA mutants with intermediate or wild-type growth rates and cell sizes have either normal or increased basal levels of ppGpp. There is an inverse relationship between the basal level of ppGpp in vivo and the degree to which translation dependent on mutant EF-G is inhibited by ppGpp in vitro. This enhanced interaction between mutant EF-G and ppGpp correlates with an increased K M for GTP. Our results suggest that mutant EF-G modulates the production of ppGpp by the RelA (PSI) pathway. In conclusion, fusidic acid-resistant EF-G mutations alter the level of ppGpp and break the normal relationship between growth rate and cell size at division. It would not be surprising if other phenotypes associated with these mutants, such as loss of virulence, were also related to perturbations in ppGpp levels effected through altered transcription patterns.

Some unidentified RNA molecules, together with the nucleoid protein HU, were suggested to be involved in the nucleoid structure of Escherichia coli. HU is a conserved protein known for its role in binding to DNA and maintaining negative supercoils in the latter. HU also binds to a few RNAs, but the full spectrum of its binding targets in the cell is not known. To understand any interaction of HU with RNA in the nucleoid structure, we immunoprecipitated potential HU-RNA complexes from cells and examined bound RNAs by hybridization to whole-genome tiling arrays. We identified associations between HU and 10 new intragenic and intergenic noncoding RNAs (ncRNAs), 2 of which are homologous to the annotated bacterial interspersed mosaic elements (BIMEs) and boxC DNA repeat elements. We confirmed direct binding of HU to BIME RNA in vitro. We also studied the nucleoid shape of HU and two of the ncRNA mutants (nc1 and nc5) by transmission electron microscopy and showed that both HU and the two ncRNAs play a role in nucleoid morphology. We propose that at least two of the ncRNA species complex with HU and help the formation or maintenance of the architecture of the E. coli chromosome. We also observed binding of HU with rRNA and tRNA segments, a few small RNAs, and a distinct small set of mRNAs, although the significance, if any, of these associations is not known. HU, a well-conserved and abundant nucleoid protein in Escherichia coli, is composed of two 9-kDa homologous subunits, ␣ and ␤ (9,18,40,41,48,49). HU showed nonspecific interactions with DNA with micromolar affinity (8, 52) and specific interactions with nicked, cruciform, and kinked DNA with nanomolar affinities (5,29,30,45,52,55,60). HU also showed interactions with tRNAs and dsrA and rpoS mRNA (4, 5).It was suggested that RNA molecules, together with HU and other nucleoid proteins, are involved in maintaining E. coli chromosome structure, although the identities of the RNAs have not been established (44). These and subsequent studies showed that 100-to 300-nucleotide (nt)-long RNA chains are part of the nucleoid (43,44). Treatment of the nucleoid with RNase caused a decondensation with a dramatic decrease in the sedimentation constant of the isolated chromosome from 1600S to approximately 400S to 500S (44, 65). In this work, we followed the idea that some HU-RNA complexes are of importance in nucleoid structure with the aim of identifying the RNAs that bind to HU and then understanding the role of such complexes in the nucleoid.We took the ribonomic approach, also referred to as RNA immunoprecipitation (IP) followed by microarray (Chip) profiling (RIP-Chip) assay, which involves immunoprecipitation of ribonucleoprotein (RNP) complexes with antibodies against specific proteins, extraction of RNA, and hybridization to microarrays (58). The assay helped with global identification of putative endogenous RNA targets of specific proteins in several systems (11,12,24,25,32,33,50,51,53,57,66).We report associations of HU with several RNAs and their identification: a s...