Proc. Natl. Acad. Sci. USA Vol. 94, pp. 6712–6717, June 1997 Biochemistry Alterations of the outer membrane composition in Escherichia coli lacking the histone-like protein HU (bacterial chromosomal proteinyhypersensitivity to antibioticsyOmpF porinymicF RNA) ERIC PAINBENI*, MARTINE CAROFF†, AND JOSETTE ROUVIERE-YANIV*‡ *Unite´Propre de Recherche 7090, Centre National de la Recherche Scientifique, Laboratoire de Physiologie Bacterienne, Institut de Biologie Physico-Chimique, 13 rue Pierre et Marie Curie, 75005 Paris, France; and †Unite´de Recherche Associe´e1116, Centre National de la Recherche Scientifique, Batiment 432 Universite´de Paris XI, 91405 Orsay, France Communicated by Jonathan Beckwith, Harvard Medical School, Boston, MA, April 11, 1997 (received for review March 1, 1997) ABSTRACT Escherichia coli cells lacking the histone-like containing chloramphenicol, the hupAB mutants exhibited protein HU form filaments and have an abnormal number of extreme sensitivity to chloramphenicol. This sensitivity was anucleate cells. Furthermore, their phenotype resembles that not observed with the single hupA mutant even though both of rfa mutants, the well-characterized deep-rough phenotype, contained the same chloramphenicol-resistance cassette. To as they show an enhanced permeability that renders them check if this sensitivity was due to a low expression of hypersensitive to chloramphenicol, novobiocin, and deter- chloramphenicol acetyltransferase carried by the resistance gents. We show that, unlike rfa mutants, hupAB mutants do cassette, we measured the level of chloramphenicol acetyl- not have a truncated lipopolysaccharide but do have an transferase activity in sonicated extracts and found no differ- abnormal abundance of OmpF porin in their outer membrane. ence between the hupA and hupAB mutants (12). Therefore, While the complete absence of HU does not abolish the we concluded that the permeability to chloramphenicol was osmoregulation of OmpF protein synthesis, the steady-state much increased in the hupAB mutant compared with hupA level of micF RNA, the negative regulator of OmpF, decreases strains. In the course of further work using these double in bacteria lacking HU, increasing the basal level of this mutants, several striking facts highlighted the fragility of the membrane protein. These findings demonstrate a novel link double hupAB mutants. For these reasons, we investigated a between a bacterial chromosomal protein and the outer possible change in the cell envelope composition of the hup membrane composition. mutants. The envelope of Gram-negative bacteria consists of three The Escherichia coli HU protein is one of the most abundant layers: the outer membrane (OM), the peptidoglycan, and the DNA-binding proteins associated with the bacterial chromo- inner cytoplasmic membrane. The inner membrane contains some (1, 2). This small, basic, dimeric protein, composed of most of the transport systems and machinery for protein two closely related subunits, shares with histones the ability to export. The peptidoglycan is a large heteropolymer that con- introduce negative supercoiling into relaxed DNA molecules in fers the rigidity of the cell envelope, hence the shape of the cell, the presence of topoisomerase I in vitro (3). This property and protects it from osmotic lysis. In Gram-negative bacteria, makes HU one of the best candidates for constraining DNA it consists of a network of amino sugars and amino acids. The supercoils in the bacterial chromosome. In addition, it has OM, which plays an important role in the physiology of these recently been shown that a relationship exists between the level bacteria, is a strong permeability barrier for all nutrients of HU protein in vivo and the activity of DNA topoisomerase (andyor antibiotics) on their way from the medium to the I. Thus, in addition to restraining negative supercoils, HU may, periplasm and prevents leakage of periplasmic proteins. In in vivo, communicate with topoisomerase I to regulate the enterobacteria, the OM is an asymmetric bilayer located global level of supercoiling in E. coli (4). Furthermore, bio- outside the peptidoglycan. It is composed of glycerophospho- chemical and genetic studies have shown that HU participates lipids, lipopolysaccharides (LPS), and proteins that are re- in several specific processes, including oriC-dependent DNA sponsible for selective permeability to nutrients and antibiot- replication, hin-mediated gene inversion, transposition of bac- ics. This function is fulfilled by a few major proteins called teriophage Mu, and the transposon 10 and DNA repair (5–9), porins. probably as a component of active multiprotein complexes. We show here that the increased sensitivity to antibiotics The hupB gene encoding the HUb subunit and the hupA and detergents of the hupAB mutants is related to an overex- gene encoding HUa subunit were isolated and, respectively, pression of OmpF, one OM porin. This high level of OmpF is mapped at 10.5 and 90.7 min on the E. coli chromosome (10, due to a decreased accumulation of micF RNA, a negative 11). To better understand the role of HU in vivo we attempted regulator of OmpF translation. to construct mutants lacking this protein (12). The hupB and the hupA genes were, respectively, disrupted by a kanamycin and a chloramphenicol-resistance cassette. Surprisingly, E. coli MATERIALS AND METHODS cells survived, albeit poorly, in the absence of HU. These Bacterial Strains, Plasmids, and Growth Conditions. The mutants, as well as those constructed by Wada et al. (13), bacteria used in this study were E. coli K12, C600 (F- thr leu exhibited perturbations of growth and cell division and defects tonA rpsL supE lacY) and its hup derivatives, which were in transposition of phage Mu. Hence, the evidence seemed to described by Huisman et al. (12). Unless otherwise noted, cells show that HU was not essential for growth. In the course of this were grown in LB medium (10 g of tryptoney5 g of yeast work, we observed an astonishing property of these double extracty5 g of NaCl per liter) at 37°C. ‘‘LB-0’’ corresponds to mutants. When plated on Luria–Bertani (LB) medium agar the LB medium without NaCl. The ompC and ompF mutants were, respectively, MH225 (MC4100 malQ7 F(ompC::lacZ) The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked ‘‘advertisement’’ in Abbreviations: OM, outer membrane; OMP, outer membrane protein; accordance with 18 U.S.C. §1734 solely to indicate this fact. LPS, lipopolysaccharide; IHF, integration host factor. © 1997 by The National Academy of Sciences 0027-8424y97y946712-6$2.00y0 ‡To whom reprint requests should be addressed. 6712 Downloaded by guest on September 28, 2021 Biochemistry: Painbeni et al. Proc. Natl. Acad. Sci. USA 94 (1997) 6713 10–25 and MH 513 (MC4100 araD1 F(ompF::lacZ) 16–13. RESULTS The plasmid pompF is a kanamycin-resistant tetracycline- HU Mutants Resemble the Deep-Rough Phenotype. During sensitive derivative of pLG361 (14). The BamHI–BamHI the construction of the hup mutants we found that the double fragment from pUC4K (carrying the kanamycin cassette) was hupAB mutant lacking the two subunits of HU was abnormally inserted into the BamHI site of pLG361. sensitive to chloramphenicol, even though it contained the Osmolarity-Dependent Tetracycline Sensitivity. Exponen- resistance gene. In fact, a difference of nearly two logs in tial cultures of strains C600 and C600 hupAB were diluted viability on LByagar plates, containing chloramphenicol (12.5 100-fold in ‘‘liquid’’ LB agar medium without (0 mM) or with mgyml) and NaCl (5 gyliter), was found between the hupAB (300 mM) NaCl and quickly poured into Petri dishes. What- and hupA mutants, although both had their hupA gene inter- man 3MM 5-mm disks were soaked into tetracycline solution rupted with a chloramphenicol-resistance cassette. This sen- (0.1 mgyml in 50% ethanol) and then deposited on top of the sitivity was not due to a defect in the synthesis of the acetylase, solidified agar. Plates were incubated overnight at 37°C. The which confers resistance to this antibiotic (12). A further halo diameter represents the relative sensitivity to the antibi- observation was that hupAB mutants were not only hypersen- otic. sitive to chloramphenicol, even when the cassette was perfectly Temperature-Dependent Tetracycline Sensitivity. Five mi- expressed, but were generally oversensitive to all the antibi- croliters of strains C600, C600 hupAB, and C600 transformed otics tested, even when present at a very low concentration. by pompF from overnight cultures in LB medium were spotted This suggested that the double mutant may have increased on LB plates with or without tetracycline (1 mgyml) and permeability. It is possible that defects in the cell envelope incubated overnight at 30°C, 37°C, and 42°C. could have rendered these mutants more fragile or more Phenotype Tests. Exponential cultures of C600 (wt), hupB, sensitive to drugs. It was intriguing that this hypersensitivity hupA, and hupAB were diluted (1:100) in LB liquid medium was almost eliminated when the NaCl concentration of the LB with an increasing concentration of cholic acid (3a,7a,12a- agar plates was increased from 5 gyliter to 10 gyliter, suggesting trihydroxy-5b-cholan-24-oic acid; Sigma) (0–15%) or SDS that salt could somehow compensate for this fragility in the (0–3%). Viability was assayed by measuring the absorbance at hupAB mutant. Fig. 1 illustrates the hypersensitivity of hupAB 600 nm after overnight incubation. cells to a low concentration of tetracycline (1mgyml) compared Extraction of the OM Proteins (OMP). C600 and C600 with wild-type cells in low salt medium. In the presence of 300 hupAB were grown overnight in LB liquid medium at 37°C. mM NaCl, the observed difference due to the absence of HU Then an aliquot corresponding to 50 OD600 was centrifuged. is virtually undetectable. Similarly, the hupAB mutant, con- The pellets were washed twice in 10 ml of TH buffer (10 mM structed in a C600 background, grew very poorly when plated EDTAyHepesy10 mM NaOH, pH 7.4).