hupA hupB Double Mutants Research Articles

HU decreases diffusible supercoiling density in bacterial chromosome

HU is an abundant protein in bacterial chromosome. It binds DNA and constrains negative supercoiling as well. In E.coli and Salmonella, it exists as a heterodimer composed of alpha and beta subunits. To study in vivo supercoiling that exists in hupA, hupB and hupAhupB double mutants, we employed the gamma delta resolution assay. Supercoil sensors were placed eight sites around the chromosome and resolution assay done to determine diffusible supercoil densities at these sites. Our studies showed that in addition to constraining supercoiling HU also enhances diffusible supercoiling either by its direct interaction with the chromosome or through gyrase activity. Supercoil density was measured to be − 0.035±0.002 in WT then reduced to −0.027±0.006, −0.022±0.007 and 0.013±0.004 in hupA, hupB and HupAhupB double mutants respectively. We observed significant differences between hupA and hupB subunits as well with hupA dimers constrains supercoiling better than hupB. We then measured transcription elongation rates in these mutants using beta galactosidase assay. HU double mutants have reduced transcription elongation rates 26 nuc/sec from WT 38–70 nuc/sec. Though hupA and hupB have similar reduced elongation rates (37 – 46 nuc/sec) alpha‐dimers had a slightly higher rate than beta‐dimers. Taken together, we were surprised that unlike eukaryotes where histone loss activates transcription, in bacteria this seems the opposite.This work was supported by National Science Foundation

Nucleoid-associated protein HU controls three regulons that coordinate virulence, response to stress and general physiology in Salmonella enterica serovar Typhimurium

The role of the HU nucleoid-associated proteins in gene regulation was examined in Salmonella enterica serovar Typhimurium. The dimeric HU protein consists of different combinations of its α and β subunits. Transcriptomic analysis was performed with cultures growing at 37 °C at 1, 4 and 6 h after inoculation with mutants that lack combinations of HU α and HU β. Distinct but overlapping patterns of gene expression were detected at each time point for each of the three mutants, revealing not one but three regulons of genes controlled by the HU proteins. Mutations in the hup genes altered the expression of regulatory and structural genes in both the SPI1 and SPI2 pathogenicity islands. The hupA hupB double mutant was defective in invasion of epithelial cell lines and in its ability to survive in macrophages. The double mutant also had defective swarming activity and a competitive fitness disadvantage compared with the wild-type. In contrast, inactivation of just the hupB gene resulted in increased fitness and correlated with the upregulation of members of the RpoS regulon in exponential-phase cultures. Our data show that HU coordinates the expression of genes involved in central metabolism and virulence and contributes to the success of S. enterica as a pathogen.

Escherichia coli HU protein suppresses DNA-gyrase-mediated illegitimate recombination and SOS induction.

The HU protein is an abundant DNA binding protein of bacteria and is a major constituent of the bacterial nucleoid. HU protein is known to be involved in several fundamental biological functions, including DNA supercoiling, DNA replication, site-specific DNA inversion, and transposition. It is generally thought that a functional relationship exists between HU protein and DNA gyrase. We found that an hupA hupB double mutant displays enhanced spontaneous illegitimate recombination during the formation of lambdabio transducing phage in Escherichia coli. Nucleotide sequence analysis of the resulting transducing phages showed that the E. coli bio and lambda recombination sites did not have any homologous sequence. This mutation also enhanced the spontaneous expression of SOS functions. Furthermore, either overproduced GyrA protein or a temperature-sensitive gyrB mutation suppressed the illegitimate recombination enhanced by the defect of HU protein. These results show that the defect of HU induces illegitimate recombination and SOS response, which are probably mediated by DNA gyrase, implying that HU protein plays roles in suppression of illegitimate recombination and SOS response through interaction with DNA gyrase.

The Escherichia coli histone-like protein HU affects DNA initiation, chromosome partitioning via MukB, and cell division via MinCDE.

Escherichia coli hupA hupB double mutants, lacking both subunits (HU1 and HU2) of the histone-like protein HU, accumulate secondary mutations. In some genetic backgrounds, these include mutations in the minCDE operon, inactivating this system of septation control and resulting in the formation of minicells. In the course of the characterization of hupA hupB mutants, we observed that the simultaneous absence of the HU2 subunit and the MukB protein, implicated in chromosome partitioning, is lethal for the bacteria; the integrity of either HU or MukB thus seems to be essential for bacterial growth. The HU protein has been shown to be involved in DNA replication in vitro; we show here that its inactivation in the hupA hupB double mutant disturbs the synchrony of replication initiation in vivo, as evaluated by flow cytometry. Our results suggest that global nucleoid structure, determined in part by the histone-like protein HU, plays a role in DNA replication initiation, in proper chromosome partitioning directed by the MukFEB proteins, and in correct septum placement directed by the MinCDE proteins.

An Additional Role of Transcriptional Activation of ori-λ in the Regulation of λ-Plasmid Replication in Escherichia coli

Initiation of replication of plasmids derived from coliphage λ in vivo is dependent on transcription at or near the replication origin, oriλ. However, this transcriptional activation is dispensable for λ plasmid DNA replication reconstituted in vitro from purified λ and Escherichia coli proteins. It was proposed previously that histone-like protein HU interferes with the assembly or function of the pre-primosomal complex, and transcription at or near oriλ abolishes HU-mediated inhibition of λ DNA replication. We found that during λ plasmid replication driven by the previously assembled replication complex (in amino acid-starved relA mutants), when the inhibition by HU protein should not be observed, the synthesis of λ DNA was still dependent on transcriptional activation. Moreover, in hupAhupB double mutants the transcription is necessary for the initiation of λ plasmid DNA replication perpetuated by the replication complex inherited by one of two daughter copies after a replication round. We conclude that transcriptional activation of oriλ has an additional role in the initiation of λ plasmid DNA replication beside the abolition of HU-mediated inhibition.

Subunit-specific phenotypes of Salmonella typhimurium HU mutants

Salmonella hupA and hupB mutants were studied to determine the reasons for the high degree of conservation in HU structure in bacteria. We found one HU-1-specific effect; the F'128 plasmid was 25-fold less stable in hupB compared with hupA or wild-type cells. F' plasmids were 120-fold more unstable in hupA hupB double mutants compared with wild-type cells, and the double mutant also had a significant alteration in plasmid DNA structure. pBR322 DNA isolated from hupA hupB strains was deficient in supercoiling by 10 to 15% compared with wild-type cells, and the topoisomer distribution was significantly more heterogeneous than in wild-type or single-mutant strains. Other systems altered by HU inactivation included flagellar phase variation and phage Mu transposition. However, Mu transposition rates were only about fourfold lower in Salmonella HU double mutants. One reason that Salmonella HU double mutants may be less defective for Mu transposition than E. coli is the synthesis in double mutants of a new, small, basic heat-stable protein, which might partially compensate for the loss of HU. The results indicate that although either HU-1 or HU-2 subunit alone may accommodate the cellular need for general chromosomal organization, the selective pressure to conserve HU-1 and HU-2 structure during evolution could involve specialized roles of the individual subunits.

HU-1 mutants of Escherichia coli deficient in DNA binding

We constructed four mutants of the Escherichia coli hupB gene, encoding HU-1 protein, by synthetic oligodeoxyribonucleicotide-directed, site-specific mutagenesis on M13mp18 vectors. The HupBR45 protein contained alterations of Arg 58 → Gly and Arg 61 → Gly, and the HupBF3, HupBK2 and HupBK2 and HupBA1 proteins contained Phe 47 → Thr, Lys 37 → Gln and Ala 30 → Asp alterations, respectively. HupBF3 and HupBR45 were unable to maintain normal cell growth in a hupA-hupB-himA triple mutant at 42°C, mini-F or RSF1010 proliferation, or Mu phage development in a hupA-hupB double mutant, whereas HupBA1 and HupBK2 supported these cellular activities. DNA-affinity column chromatography showed that the HupBF3 and HupBR45 had reduced affinities to DNA. These observations indicate that two highly conserved Arg residues in the arm structure of the C-terminal half of the HU-1 molecule and a Phe residue in the short β-sheet connecting the two halves of the molecule are important for the DNA-binding ability and biological functions of this protein.

Participation of hup gene product in replicative transposition of Mu phage in Escherichia coli

The closely related Escherichia coli genes hupA and hupB each encode a bacterial histone-like protein HU. We report here that mutator phage Mucts62 was unable to replicate in a hupA hupB double mutant, although it could replicate in hupA or hupB single mutant as efficiently as in the wild-type strain. Mu cts62 was able to lysogenize the double mutant at 30°C; cell killing occurred when the lysogen was incubated at 42°C, but did not result in phage production. High-frequency non-replicative integration of Mu into host genomic DNA soon after infection could not be detected in the hupAB double mutant. These results provide the evidence that HU protein is essential for replicative transposition of Mu phage in E. coli, and also participates in high-frequency conservative integration.

Participation of the hup gene product in site-specific DNA inversion in Escherichia coli

The closely related Escherichia coli genes hupA and hupB each encode a bacterial histone-like protein HU. We report here that DNA inversion mediated by hin, gin, pin and rci but not by cin is blocked in a hupA hupB double mutant, although inversions in these systems occur in the hupA or hupB single mutant as efficiently as in the wild-type strain. These findings show that HU protein participates in site-specific DNA inversion in E. coli and that only one subunit, either HU-1 or HU-2, is sufficient for this inversion.

Participation of hup gene product in ori2-dependent replication of fertility plasmid F.

The fertility plasmid F'gal was not stably maintained in a hupA-hupB double mutant of Escherichia coli. Moreover, mini-F plasmids pFZY1, pFTC1 and pFTC2 were unable to transform the double mutant, though these plasmids efficiently transformed cells harboring a hupA or hupB single mutation. The composite plasmid pFHS1, which consists of the f5 DNA fragment of F plasmid and the whole DNA of a pSC101 derivative that carries a temperature-sensitive mutation for DNA replication, was not stably maintained in the hup double mutant at 42 degrees C. These findings strongly suggest that HU protein is required for ori2-dependent replication of the F plasmid.