The histone-like protein HU in Escherichia coli are encoded by the hupA and hupB genes. A hupA-hupB double deletion mutant has now been shown to express an immotile phenotype. The motility of hupA or hupB single mutants was similar to that of wild-type cells. SDS-polyacrylamide gel electrophoresis revealed that the amount of flagellin in the hupA-hupB double deletion mutant was markedly reduced compared with the wild-type strain, suggesting that the immotile phenotype of the double deletion mutant is caused by a loss of flagella.
Bacteria swim by the propeller-like action of their flagella, and this motility is important for the acquisition of nutrients and movement away from inadequate external conditions . The formation of flagella is controlled at the level of synthesis of the flagellin, a subunit of flagella , and genetic studies have revealed a cascade of genes responsible for regulation of the expression of the fliC gene, which encodes flagellin.
Genes that affect the expression of fliC include those important for DNA replication, such as the dnaA, dnaB, dnaC, and dnaG[3, 4], heat shock genes such as dnaK, dnaJ, and grpE, and those that contribute to phospholipid metabolism, such as pss and pgsA[6–8]. Mutations in these genes reduce the expression of fliC through inhibition of the expression of flhD, a master operon of the flagellin cascade [3, 5, 9–11].
A mutation in the gene that encodes the H-NS protein, one of the major histone-like proteins in Escherichia coli, also results in reduced expression of flhD and an immotile phenotype . The HU protein is another abundant histone-like protein and functions as a heterodimer of two highly homologous subunits, HUα and HUβ, encoded by the hupA and hupB genes, receptively . HU and H-NS show similar characteristics; both protein bind to DNA in a sequence-independent manner and contribute to maintenance of negative supercoiling of DNA [12, 14]. We considered the possibility that HU is also important for cell motility. We have therefore examined the effect of a hupA-hupB double deletion mutation on cell motility.
2Materials and methods
E. coli strain YK1340 (hupA16, hupB11)  was kindly provided by Dr. Y. Kano (Kyoto Pharmaceutical University). P1 phage-mediated transduction of hupA16 and hupB11 mutations to W3110 was performed as described previously [3, 16]. Mutants that contained hupA16, hupB11, or both of these mutations were designated SN0001, SN0002, and SN0003, respectively.
2.2Cell motility analysis
Formation of swarm rings was analyzed in semi-solid agar containing 1% tryptone, 0.5% NaCl, and 0.25% agar . After addition of full growth suspension (2 μl) to the plate, the cells were incubated at various temperatures.
2.3Determination of β-galactosidase activity in cells
β-Galactosidase activity in crude cell extracts was determined as described by Miller . Briefly, exponentially growing cells were chilled on ice when the optical density at 600 nm reached 1.0. After addition of a drop of toluene, the suspension was incubated at 37°C for 40 min. o-Nitrophenyl-β-d-galactose (0.8 mg) was added and the samples were then incubated at 28°C for 10 min.
2.4SDS-polyacrylamide gel electrophoresis of flagellin
Exponentially growing cells were harvested by centrifugation at 5000 rpm when the optical density at 600 nm reached 1.0. The cell pellets were suspended in 100 μl of 0.9% NaCl, and flagella were released from the cells by vigorous vortex mixing for 10 min . Cells were removed by centrifugation at 5000 rpm, and supernatants containing flagella were analyzed by SDS-polyacrylamide gel electrophoresis .
3.1Motility of E. coli lacking HU protein
We compared the motility phenotype of a hupA-hupB double deletion mutant (SN0003) with that of W3110, the isogenic parent strain, by measuring diameters of swarm rings in soft-agar plates. The swarm ring formed by the double mutant was markedly smaller than that by the wild-type strain (Fig. 1); the diameters of the rings were 8 and 31 mm, respectively, after incubation for 6 h at 37°C. Because the cell motility of E. coli is greatly affected by incubation temperature , we also performed the motility test at 28°C and 42°C. The mutant was immotile at both temperatures, whereas the wild-type strain was motile at 28°C but not at 42°C (data not shown). We also found that single mutants with a hupA16 or hupB11 mutation (SN0001 or SN0002, respectively) were motile, however, the diameters were a little smaller (28 or 24 mm, respectively) than that of the wild-type cells (Fig. 1).
3.2P1 phage-mediated transduction analysis of the motility phenotype of E. coli lacking HU protein
Next, we examined whether the immotile phenotype of SN0003 strain was co-transducible with the hupA16 and hupB11 mutations in P1 phage-mediated transduction experiments. P1 phages were grown in YK1340 cells (hupA16::KmR, hupB11::CmR), and transduced into SN0002 cells (hupB11::CmR), and the transductants were selected on kanamycin-containing plates. The motility phenotypes of the transductants were then examined. All 59 independent transductants showed an immotile phenotype. Furthermore, when the hupB11::CmR mutation in YK1340 was transduced into SN0001 cells (hupA16::KmR), all 15 independent transductants showed an immotile phenotype. These results indicate that the immotile phenotype of SN0003 is caused by double mutations in the hupA and hupB genes.
3.3The amount of flagellin in E. coli lacking HU protein
To investigate whether the immotile phenotype of SN0003 is due to a lack of flagella, we analyzed flagella fractions of SN0001, SN0002, SN0003, and the wild-type strain by SDS-polyacrylamide gel electrophoresis. The amount of flagellin in the SN0003 cells was much less than that in the wild type strain (Fig. 2). The amount of this protein in each single mutant (SN0001 and SN0002) was a little less than that in the wild type strain (Fig. 2). Analysis of total cell protein from these strains by SDS-polyacrylamide gel electrophoresis showed that flagellin is an abundant protein in SN0001, SN0002, and the wild-type strain but not in SN0003 (data not shown). These results suggest that the immotile phenotype of SN0003 is due to a lack of flagellin.
3.4Promoter activity of the fliC gene in E. coli lacking HU protein
The synthesis of flagellin is regulated at the level of transcription . Thus, we examined the promoter activity of fliC in SN0003. Either hupA16, hupB11, or both mutations were introduced by P1 phage-mediated transduction into strain YK3421, in which the open reading frame of the lacZ gene is fused to the promoter of fliC. The resulting transductants were named SN1001 (YK3421, hupA16), SN1002 (YK3421, hupB11), and SN1003 (YK3421, hupA16, hupB11). The promoter activity of fliC were determined by measuring the activity of β-galactosidase in cell extracts. The β-galactosidase activity in an SN1003 extract was 53% of that in a wild-type extract (Table 1). The enzyme activities in SN1001 and SN1002 extracts were a little less than that in the wild-type strain (Table 1), corresponding to results in Fig. 2.
Table 1. Promoter activity of the fliC gene
β-Galactosidase activity (units)
SN1001 (hupA16, fliC-lacZ), SN1002 (hupB11, fliC-lacZ), SN1003 (hupA16, hupB11, fliC-lacZ) and the isogenic parent strain (YK3421) were grown at 37°C, after which the activity of β-galactosidase in crude extracts was measured . Data are means of duplicates.
YK3421 hupA hupB
We have shown that the hupA-hupB double deletion mutant of E. coli is immotile. P1 phage-mediated transduction experiments indicated that the mutations in both hupA and hupB are responsible for the immotile phenotype. SDS-polyacrylamide gel electrophoresis analysis indicated that the immotile phenotype of the double mutant is caused by a marked decrease in the amount of flagellin. Together with the previous observation that a mutant lacking the H-NS protein is involved in the synthesis of flagellin , our data suggest that histone-like proteins in general contribute to synthesis of flagellin.
The motility of hupA or hupB single mutants was similar to that of wild-type cells. The immotile phenotype thus requires mutations in both genes as do other aspects of phenotype of the double mutant, such as effects on cell growth, DNA replication, transposition, and site-specific recombination . The HUα or HUβ homodimers thus appear able to substitute functionally for the HUα-HUβ heterodimer in many cellular process, including the expression of flagella.
Our observation that the promoter activity of fliC was reduced in the hupA-hupB double deletion mutant suggests that the decreased abundance of flagellin in the mutant is, at least in part, due to inhibition of the fliC promoter. However, the amount of flagellin drastically decreased in SN0003 (Fig. 2), whereas the promoter activity of fliC decreased to only 53% of that of the wild-type strain (Table 1). Therefore, the hupA-hupB double deletion mutation must cause the decrease in the amount of flagellin not only through inhibition of promoter activity of the fliC gene but also through other mechanism. Since H-NS protein inhibit the translation reaction in vitro , we consider that HU protein inhibit the translation of mRNA of the fliC gene in cells.
DNA in the hupA-hupB double deletion mutant is more relaxed than in wild-type cells , and alterations in the extent of DNA supercoiling greatly affect the promoter activities of various genes [25, 26]. The expression of fliC appears to be affected by changes in DNA supercoiling, because it is inhibited by various stress or mutations that affect this parameter in cells [3, 5, 11, 23, 27, 28]. We, therefore, propose that the reduced expression of fliC in the hupA-hupB double deletion mutant results from the alteration in DNA supercoiling.
We thank Drs. F. Imamoto (Life Technologies Oriental, Tokyo, Japan), K. Kutsukake (Hiroshima University, Hiroshima, Japan) and Y. Kano (Kyoto Pharmaceutical University, Kyoto, Japan) for providing bacterial strains and helpful discussion. This work was supported in part by Grants-in-Aid for Scientific Research from the Ministry of Education, Science, and Culture of Japan.