Multiple defects in Escherichia coli mutants lacking HU protein

Abstract

The HU protein isolated from Escherichia coli, composed of two partially homologous subunits, ot and 0, shares some of the properties of eucaryotic histones and is a major constituent of the bacterial nucleoid. We report here the construction of double mutants totally lacking both subunits of HU protein. These mutants exhibited poor growth and a pertubation of cell division, resulting in the formation of anucleate cells. In the absence of HU, phage Mu was unable to grow, to lysogenize, or to carry out transposition.The Escherichia coli HU protein is the most abundant DNA-binding protein present in the bacterial cell (38). This small, basic, dimeric protein interacts with double-stranded DNA, single-stranded DNA, and RNA. As with mixtures of the four core eucaryotic histones, HU can introduce negative supercoils into a relaxed closed circular DNA in vitro if topoisomerase I is present, and the resulting condensed structures resemble nucleosomes (5, 40). In addition, just as the histones in eucaryotes are conserved, the HU protein is highly conserved among procaryotic organisms and also in mitochondria and chloroplasts (10). It was shown earlier that HU is associated with the E. coli nucleoid (37, 47) (although some cytological observations were interpreted as locating HU on the edge rather than inside of the nucleoid [11]), but whether it has a role in maintaining the structure of the chromosome as a whole is not known.Biochemical studies have suggested a role for HU in the site-specific recognition of nucleotide sequences by other proteins (for a review, see reference 10). Among these interactions are the formation of protein-DNA complexes involved in the initiation of replication of the bacterial chromosome at oriC, in the hin-mediated gene inversion, and in the transposition of transposon TnJO and bacteriophage Mu. More precisely, replication from oriC (9, 13) and the transposition of TnJO (29,34) are moderately stimulated by HU protein, whereas the flagellar phase variation in Salmonella typhimurium (21) MATERIALS AND METHODSStrains and bacteria. The bacterial strains, plasmids, and phages used in this study are listed in Table 1. Bacteria were grown in LB (10 g of Bactotryptone [Difco Laboratories], 5 g of yeast extract, and 5 g of NaCl per liter) and counted on L agar (LA) (28). Minimal medium was M9 supplemented with thiamine (10 ,ug/ml), amino acids (100 pg/ml) or casamino acids (0.4%, wt/vol), and 0.4% (wt/vol) sugar (glycerol, maltose, lactose, or glucose). Kanamycin (25 p.g/ml), chloramphenicol (12.5 pLg/ml), streptomycin (200 pig/ml), and spectinomycin (100 ,ug/ml) were included when appropriate.Methods for Mu assays. The general methods used to manipulate the phage have been described previously (6,12 (ii) Mu transposition assay. The frequency of transposition onto the pUZ8 plasmid was measured by a mating-out assay. The Mu cts62pApl lysogens (or the bacteria infected with the bacteriophage X mini-Mu Apr) carrying pUZ8 were mated with the MFG625B recipient on a filter. After incubation for 2 h at 42...

Micromechanical Analysis of the Binding of DNA-Bending Proteins HMGB1, NHP6A, and HU Reveals Their Ability To Form Highly Stable DNA−Protein Complexes

“…Consistent with an important role of HU in chromosome compaction, hupAB mutants lacking both subunits of HU display decondensed nucleoids and reduced superhelical densities (4,(15)(16)(17). Moreover, in many of the specialized roles that HU performs in vivo, one or a small number of HU dimers are clearly functioning by inducing DNA bends.…”

“…HU's DNA-bending activity is central to its role in a number of different reactions involving assembly of higher-order nucleoprotein complexes in bacteria; notably, HMGB proteins can substitute for HU in some of these reactions (6,8,9). HU mutant phenotypes have implicated HU as being a determinant in bacterial chromosome condensation (4,(15)(16)(17).…”

The nonspecific DNA-binding and -bending proteins HMG1 and HMG2 promote the assembly of complex nucleoprotein structures.

“…In addition to its function in Hin-mediated DNA inversion, HU has been shown to play important roles in other specific reactions such as phage Mu transposition (Craigie et al 1985), Tnl 0 transposition (Morisato and Kleckner 1987), and chromosomal DNA replication (Dixon and Kornberg 1984). A general role in mediating chromosome compaction in the bacterial nucleoid has also been implicated, although mutants deficient in the synthesis of both subunits remain viable (Rouvihre-Yaniv et al 1979;Wada et al 1988;Huisman et al 1989). …”

Organization of the Bacterial Chromosome

“…By contrast, double mutants are severely enfeebled under some growth conditions (especially at temperatures at the extremes of the permissible range). Double mutants contain less ordered nucleoids and form both filamentous cells and anucleate cells (160,164,361). The value of the double mutants is that the roles of the HU proteins can be assessed in vivo rather than solely in model systems.…”