Isolation and Characterization of the Escherichia coli msbB Gene, a ...

Vol. 174, No. 3

JOURNAL OF BACTERIOLOGY, Feb. 1992, p. 702-710

0021-9193/92/030702-09$02.00/0 Copyright C) 1992, American Society for Microbiology

Isolation and Characterization of the Escherichia coli msbB Gene, a Multicopy Suppressor of Null Mutations in the High-Temperature Requirement Gene htrB MARGARET KAROW* AND COSTA GEORGOPOULOS Department of Cellular, Viral and Molecular Biology, School of Medicine, University of Utah, Salt Lake City, Utah 84132 Received 23 September 1991/Accepted 22 November 1991

Previous work established that the htrB gene of Escherichia coli is required for growth in rich media at temperatures above 32.5°C but not at lower temperatures. In an effort to determine the functional role of the htrB gene product, we have isolated a multicopy suppressor of htrB, called msbB. The msbB gene has been mapped to 40.5 min on the E. coli genetic map, in a 12- to 15-kb gap of the genomic library made by Kohara et al. (Y. Kohara, K. Akiyama, and K. Isono, Cell 50:495-508, 1987). Mapping data show that the order of genes in the region is eda-edd-zwf-pykA-msbB. The msbB gene codes for a protein of 37,410 Da whose amino acid sequence is similar to that of HtrB and, like HtrB, the protein is very basic in nature. The similarity of the HtrB and MsbB proteins could indicate that they play functionally similar roles. Mutational analysis of msbB shows that the gene is not essential for E. coli growth; however, the htrB msbB double mutant exhibits a unique morphological phenotype at 30°C not seen with either of the single mutants. Analysis of both msbB and htrB mutants shows that these bacteria are resistant to four times more deoxycholate than wild-type bacteria but not to other hydrophobic substances. The addition of quaternary ammonium compounds rescues the temperaturesensitive phenotype of htrB bacteria, and this rescue is abolished by the simultaneous addition of Mg2' or Ca2+ . These results suggest that MsbB and HtrB play an important role in outer membrane structure and/or function.

hypothesis that HtrB is somehow involved in cell wall formation. To further our understanding of the htrB gene, we isolated several multicopy, extragenic suppressors of htrB insertion mutations. These genes, when cloned into multicopy vectors, suppress both the temperature-sensitive (Ts-) and the morphological phenotypes of htrB. We reasoned that the products of these genes may serve functions similar to those of HtrB, so their characterization may help clarify the function of HtrB in bacterial physiology. In this paper we present the characterization of one of these genes, which we have named msbB (multicopy suppressor of htrB).

The Escherichia coli htrB gene was identified during an insertional mutagenesis screening for new heat shock genes. Characterization of its pattern of transcription showed that htrB does not belong to either of the two known heat shock regulons (18). However, inactivation of the htrB gene causes E. coli to become uniquely temperature sensitive, growing in rich media at temperatures below 32.5°C but not at higher temperatures (17). Thus, htrB is a member of a new class of genes whose products are required for growth at high temperatures but that are not heat shock genes. We have been characterizing htrB and its mutant phenotypes to determine the role that it plays in bacterial physiology. Sequence analysis shows that it codes for a protein with an estimated molecular weight of 35,407 (18). The HtrB protein has a very basic nature, with an estimated isoelectric point of 10.31, and possesses the properties of an integral membrane protein. It does not share homology with any other proteins in the GenBank and SWISS-PROT data bases, thus offering us no suggestion as to its function. However, the morphology of htrB cells grown at high temperatures indicates that HtrB plays a role in cell wall formation. This change in morphology is density dependent. Cells in low-density cultures form bulges in the middle or at the ends, while cells in higher-density cultures form filaments. These changes are reminiscent of the mutant phenotypes of the pbpB gene, which is required for correct cell wall formation during septation (16). Mutations in pbpB can cause cells to form filaments (33), and double mutations in pbpB and another cell wall synthesis gene, pbpA, cause the bacteria to form bulges similar to those seen with htrB bacteria (1). The fact that the HtrB protein has the properties of an integral membrane protein is also consistent with the

MATERIALS AND METHODS Strains and media. The bacterial strains used in this study are shown in Table 1. Bacteria were grown in LB medium, unless otherwise indicated. Antibiotics were added, when needed, at the following final concentrations: ampicillin, 50 ,ug/ml; spectinomycin, 50 ,ug/ml; tetracycline, 10 ,ug/ml; chloramphenicol, 10 ,g/ml; and kanamycin, 50 ,ug/ml. Cell growth analysis. Growth and viability experiments and photography were performed as described previously (17). For the determination of the growth and viability of htrB mutant bacteria in the presence of tetradecyltrimethylammonium bromide, bacteria were grown at 30°C to an optical density at 595 nm (OD595) of 0.4 in LB medium and diluted to an OD595 of 0.05 in LB medium. Tetradecyltrimethylammonium bromide and/or MgCl2 was added to a final concentration of 10 ,ug/ml and 10 mM, respectively, and the cultures were shifted to 42°C at time zero. Plasmids. The pBS-233 plasmid is the original 2.6-kb PstI fragment of msbB cloned into Bluescript-KS (Stratagene). pBR-233 is the 2.6-kb PstI fragment cloned from pBS-233 by digestion with BamHI and SalI and then ligated with simi-

* Corresponding author. 702

VOL. 174, 1992

E. COLI msbB GENE TABLE 1. Strains

Strain

Genotype

Source or reference

W3110 MLK53 MLK1067 MLK986 JC7623

Our collection Karow et al. (17) This work This work Kushner et al. (21)

CAG18561 DH5a

Wild type W3110 htrBl::TnlO W3110 msbB::Q)cam MLK53 msbB::Qfcam AB1157 recB21 recC22 sbcBJ5 sbcC201 JC7623 (X RS45 msbB+) eda-3126::TnlOkan recAl

MC4100

recA+

MLK959

This work C. Gross Bethesda Research Laboratories M. Casadaban (4)

larly digested pBR322 (3) DNA. pGB-233 is the 2.6-kb PstI fragment cloned into the PstI site of pGB2 (5). The pRS415233 plasmid contains the PvuII-EcoRI fragment from pBS233 cloned into the EcoRI-SmaI sites of pRS415 (32). The pRS551-233 clone is the EcoRI fragment from pBS-233 that contains the msbB gene cloned into the EcoRI site of pRS551 (32). The omega cam cassette (flcam) insertion plasmid was made by partially digesting pBS-233 with XmnI. The linearized plasmid was isolated from a low-melting-point agarose (FMC) gel and ligated with the flcam DNA. The flcam DNA was isolated from a low-melting-point agarose gel after the plasmid containing it, pHP45fl-Cm (8), had been digested with BamHI and the ends had been made blunt with the Klenow fragment of E. coli PolI. msbB diploid strain. The msbB diploid strain was made by the method of Simons et al. (32). In brief, MC4100 bacteria carrying the pRS551-233 plasmid were infected with XRS45, and the resulting lysate was used to infect JC7623 bacteria. These bacteria were plated on L-kanamycin agar plates to select for A lysogens that had recombined a copy of the msbB gene onto themselves. The resulting diploid strain was used to make the msbB insertion mutation. DNA sequencing. All DNA sequencing was done with deletions of the pBS-233 plasmid constructed by the method of Hong (14). When necessary, several regions were sequenced from subclones of pBS-233 or by use of oligonucleotide primers. Sequencing reactions were carried out with Sequenase (version 2.1) as described by the manufacturer (United States Biochemical). Southern blot analysis. Genomic DNAs were isolated from msbB mutant bacteria and the isogenic wild-type parent by first treating 1.0 ml of an overnight culture of bacteria that had been centrifuged and resuspended in 200 Il of 25 mM Tris-HCl (pH 7.9)-10 mM EDTA-1% glucose with 2.5 mg of egg white lysozyme per ml for 10 min at room temperature. An equal volume of 50 mM Tris-HCl (pH 7.9)-40 mM EDTA-2% sodium dodecyl sulfate was added, and the suspension was mixed gently and placed at 55°C until the cells lysed, approximately 2 min. The lysed cells were extracted with an equal volume of phenol for 5 min, an equal volume of chloroform was added, and the cells were extracted for another 5 min. The suspension was centrifuged to create a biphase, and the aqueous layer was removed and reextracted as described above. The DNA was ethanol precipitated and treated with 10 p,g of RNase A per ml. After restriction enzyme digestion, approximately 1 ,ug of DNA was electrophoresed through a 0.9% agarose gel and transferred to a Hybond-N membrane (Amersham) in 20x SSPE (lx SSPE is 0.15 M NaCl, 10 mM NaPO4, and 1 mM EDTA

703

[pH 7.4]). The blot was probed with the 1,931-bp PvuII fragment that contained the msbB gene and that was labeled with [a-32P]dATP by use of a random primer kit, Prime-it, from Stratagene. Transfer, hybridization, and washing of the blot were performed as described previously (30). Drug and detergent sensitivities. Bacteria grown in LB medium to the late log phase were spotted and then streaked on L agar plates with the appropriate concentrations of drugs or detergents. All experiments were repeated a minimum of three times, with qualitatively identical results. Polymerase chain reaction. Polymerase chain reactions were carried out by the method of Innis and Gelfand (15). The zwf primer has the sequence 5'-TGCGTTGAATCATA CACGGG-3', and the pykA primer has the sequence 5'GGATGTCGTATAAGATTAGG-3'. Computer analysis. Nucleic acid and protein sequences were analyzed with the Intelligenetics, Inc., PC/gene program package. GenBank searches were done with the FASTA method of Pearson and Lipman (26). Nucleotide sequence accession number. The DNA sequence reported in this paper is held in the GenBank data bank under accession number M77039. RESULTS Isolation of multicopy suppressors of htrB. During the original cloning of the htrB gene, two clones that complemented the HtrB Ts- phenotype but did not contain the htrB gene were isolated from a low-copy-number cosmid library. Restriction maps of these two clones showed that they carried very similar fragments of DNA and most likely encoded the same suppressing gene. We were able to subclone a rescuing 4.5-kb PstI fragment from these cosmid clones onto the low-copy-number pGB2 vector but were unable to clone this fragment onto higher-copy-number vectors, such as Bluescript-KS. During the attempt to clone this fragment onto Bluescript-KS, we accidentally isolated a 2.6-kb PstI fragment of E. coli DNA that also complemented the HtrB Ts- phenotype. The former gene has been named msbA (multicopy suppressor of htrB), and the latter gene has been named msbB. This paper focuses on the msbB gene; cloning and characterization of the msbA gene are in progress. Suppression of the HtrB phenotypes by msbB. To determine whether the msbB gene could complement htrB when cloned onto vectors with copy numbers lower than that of Bluescript-KS (500 to 700 copies per cell), we cloned the 2.6-kb PstI fragment onto pGB2 (5 copies per cell) and pBR322 (30 to 50 copies per cell). When the 2.6-kb fragment was cloned onto pGB2 (pGB-233), it complemented htrB at 37°C but not at 42°C. However, when the fragment was cloned onto pBR322 (pBR-233), complementation was seen at all temperatures. Hence, a 5-fold increase in the msbB gene copy number is insufficient at 42°C for full rescue of the HtrB Tsphenotype, but a 30- to 50-fold increase is sufficient. The extent of complementation of the HtrB Ts- phenotype by msbB is shown in Fig. 1A. htrB bacteria carrying pBR-233 grew as well as wild-type cells at 42°C. The pBR-233 clone complemented not only the Ts- phenotype of htrB but also all of the morphological alterations seen at nonpermissive temperatures. Figure 1B shows the morphology of htrB bacteria transformed with pBR322 or pBR-233 and grown at 42°C for 2.75 h. htrB bacteria carrying pBR322 formed the bulges that are characteristic of htrB mutants, while those carrying pBR-233 looked like the isogenic wildtype cells.

704

KAROW AND GEORGOPOULOS

J. BACTERIOL.

A

B htrB +pBR322

WT +pBR322

+F

htrB +pBR-233

WT +pBR322 htrB +pBR322 FIG. 1. Suppression of the Ts- and morphological phenotypes of htrB null mutations by the msbB gene. (A) Growth at 42°C on L agar of htrB mutant bacteria carrying pBR322 or pBR-233 and isogenic wild-type (WT) bacteria carrying pBR322. (B) Morphology of htrB mutant bacteria carrying pBR322 or pBR-233 and isogenic wild-type (WT) bacteria carrying pBR322.

Sequence analysis of the msbB clone. We sequenced the original 2.6-kb fragment as described in Materials and Methods. The resulting sequence is shown in Fig. 2. There are three open reading frames (ORFs) encoded on this fragment; only one is complete. This ORF starts at bp 999, located 170 bp downstream of the EcoRI site, and ends at bp 1968, located 268 bp upstream of the PvuII site, as shown in Fig. 2 and 3. Seven base pairs upstream of the initiation codon for this ORF is a putative Shine-Dalgarno sequence, GGAA. Further upstream are DNA sequences with homology to the - 10 and -35 consensus promoter sequences for (x70-containing RNA polymerase (Fig. 2) (12). Just upstream of the -35 sequence is a palindromic sequence followed by 7 thymine residues (Fig. 2), a typical rho-independent terminator structure (27). Downstream of the ORF is yet another palindromic sequence (Fig. 2) that is homologous to the family of repetitive extragenic palindromic sequences (34). These sequences have been proposed to be DNA gyrase binding sites and points of recA-independent recombination (31, 38). To determine whether the ORF from bp 999 to 1968 is indeed the msbB gene, we subcloned the 1.4-kb EcoRI-PvuII fragment onto pRS415 (pRS415-233) (32). This vector is a pBR322-based vector constructed to prevent readthrough transcription from other promoters. The 1.4-kb fragment complements the HtrB Ts- phenotype, showing both that this ORF is the msbB gene and that the gene is fully contained within this fragment. This ORF codes for a protein with an estimated molecular weight of 37,410. Consistent with this result, in vivo protein labeling with the T7 promoter-polymerase system of Tabor and Richardson (35) showed that only one protein, with an approximate molecular weight of 37,000, is produced from the 2.6-kb fragment (data not shown). An 879-bp ORF upstream of, and in the same orientation as, the msbB gene did not produce a protein in the abovedescribed labeling experiments. We have named this ORF orfU, for upstream ORF. The protein was not labeled because the 2.6-kb fragment does not contain the amino terminus of the ORF. The portion of the ORF that we have sequenced codes for a polypeptide of 292 amino acids that is similar to the Staphylococcus simulans and Staphylococcus

staphylolyticus lysostaphin proteins (13, 28). Lysostaphin is a cell wall-degrading enzyme that is specific for Staphylococcus cell walls. The OrfU protein may also be involved in cell wall degradation or formation. On the other DNA strand and 3' to msbB is another ORF. FASTA searches (26) of the GenBank data bank showed that this region, from bp 2054 to the 3' end of the clone, contains the E. coli pykA gene (accession number M63703), encoding pyruvate kinase type II. The restriction map in Fig. 3 summarizes the ORFs and their positions relative to the msbB gene. MsbB protein. A comparison of the MsbB protein sequence with the HtrB protein sequence shows that the two are similar. The alignment of the two protein sequences is shown in Fig. 4A. The two proteins are 27.5% identical, with a total similarity of 42.2%. The hydropathy plots of both proteins are also strikingly similar (Fig. 4B). The similarity of MsbB and HtrB is also reflected in their overall charge. Both proteins are very basic; HtrB has an estimated isoelectric point of 10.31, and MsbB has an estimated isoelectric point of 10.07. Isolation of an msbB insertion mutation. The similarity of MsbB and HtrB could indicate that the two proteins are functionally similar. This similarity could be reflected by the phenotype of an msbB mutation. An msbB insertion mutation was made by first creating an insertion in the cloned copy of the gene and then recombining this mutation into the endogenous chromosomal copy. The DNA that was inserted is an omega fragment containing the chloramphenicol acetyltransferase gene (8). The omega cassette has both transcriptional and translational termination signals flanking the drug resistance gene (8). We inserted this cassette into the amino-terminal end of the msbB ORF, at the XmnI site located 27 bp downstream of the translational start. Using the procedure described by Oden et al. (25), we transformed this cloned insertion mutation into MLK959, a recBC sbcBC strain made diploid for the msbB gene (as described in Materials and Methods). Transformants were repeatedly selected for Camr and screened for Amps. Camr AmpS colonies arise when the insertion mutation has recombined into one of the two copies of the msbB gene, the endogenous

E. COLI msbB GENE

VOL. 174, 1992

orfU

CTGCAGCGCCTCACCTGGGAAGTGTCTCGTCGTGAAACCCGAACCTATGACCGTACTGCCGCTAACGGTTTTAAAATGACCAGCGAAATGCAGCAAGGAGAG L Q R L T W B V S R R B T R T Y D R T A A N G F K N T S K N Q Q G E TGGGTTAACAATCTGCTGAAAGGTACCGTCGGGGGAAGCTTTGTTGCCAGCGCCAGAAACGCCGGTTTAACCAGCGCCGAAGTGAGCGCAGTGATTAAAGCC W V N N L L I G T V G G S F V A S A R N A G L T S A B V S A V I K A ATGCAGTGGCAAATGGATTTCCGCAAIsCTGA1AAAAGCGATGAATTTGCGGTGTTAATGTCTCGAGAAATGCTTGATGGTAAACGTGAGCAAUGCCAGCTG N Q W

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122

TCCGATCGTCGCGAATTCCTGGCGCAGGCCAAAGAGATTGTGCCGCAGCTACGGTTGATTAATTAACATCCATTCGCAGCCGGT&CGCAGTCAGW L A Q A K B I V P Q L R F D TTTTTTTATTTGGTGCGGGGCAAGTTGCGCCGCTACACTATCACCAGATTGATTTTTGCCTTATCCGA S

.aB

612

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GGACGTTCAACCGGGCCGvCATCTGCACTATGAAGTATGGATAAACCAGCAGGCCGTAAACCCGCTGACGGCAAAACTGCCGCGTACCGAAGGGCTGACCGGC G

5 10

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GGTCGCAGCTACACCACGCGTTATATGCACTTGCGCAAGATTCTGGTGAAACCGGGACAGAAGGTGAAACGTGGCGACCGTATCGCGCTTTCCGGTAATACC G

40 8

D

TTCGCCATGCCGCAGGGTACGCCAGTGCTTTCAGTGGGTGACGGTGAAGTGGTGGTTGCCAAACGCAGTGGCGCAGCAGGTTATTATGTGGCTATTCGTCAT F

306

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TTGCGQTTCCCGACGGCGAAACAGTTCCGTATCTCGTCTAATTTTAACCCGCGTCGTACTAATCCGGTGACCGGTCGCGTTGCACCACACAGAGGTGTTGAT L

102 204

L

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S R A E R B A I V D R N F A T A P Q A N A N N A E L A I R G P R AAATTCAGCCGCGCGTTGACTGGCAACGGCTGGAGATCATCGAGGTGCGGCGTAATAACGAGAAAGTTATCTTTCTGGTGCCGCACGGTTGGGCCGTCG R E N R R N N R K V I F L V P H G W A V K I Q P R V D W Q G L F I I ATATTCCTGCCATGCTGATGGCCTCGCAAGGGCAGACTGGCAGCGATGTTCCATAATCAGGGCAACCCGGTTTTTGATTATGTCTGGAACACGGTGCGTC I N A A N F H N Q G N P V F D Y V W N T V R D I P A N L N A S Q G Q GTCGCTTTGGCGGTCGTCTGCATGCGAGAAATGACGGTATTAAACCATTCATCCAGTCGGTACGTCAGGGGTACTGGGGATATTATTTACCCGATCAGGATC R R F G G R L H A R N D G I K P F I Q S V R Q G Y W G Y Y L P D Q D ATGGCCCAGAGCACAGCGAATTTGT GGATTTCTTTGCCAC CTATAAAGCGACGTTGCCCGCGATTGGTCGTTTGATGAAAGTGTGCCGTGCGCGCGTTGTAC R H S B F V D F F A T Y K A T L P A I G R L N K V C R A R V V H G P B

705

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GGTACTCATCACGTCGCCCTGGGTGACAATCACCAGGTCACCAGA7XTCAAGTAACCTTTATCGCGCAGCAGATTAACCGCTTCGCTGGCAGCTGCTACGCC

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AACGGTTTCTGACGG&TACTGCCCAGCGGCAGTTTCTGC

142 8

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2142 2244 234 6 244 8 2 550 2589

FIG. 2. Sequence of the 2.6-kb DNA fragment containing the msbB gene. The deduced protein sequences for MsbB and OrfU are shown below the DNA sequence. Between the orfU and msbB genes is a sequence that is similar to a rho-independent terminator (indicated by heavy lines and arrows below the DNA sequence). The -10 and -35 sequences that are similar to the consensus sequence for a70_type promoters are marked and underlined below the DNA sequence. Below the DNA sequence, a sequence with homology to the family of repetitive extragenic palindromic sequences is underlined with a thin line, and the inverted repeats are delineated with arrows.

chromosomal copy or the second copy present at the X attachment site, with the concomitant loss of the plasmid. Of 20 Camr colonies isolated following the transformation, 4 eventually became Amps but remained Camr. To determine which copy the mutation recombined into, we took advantage of the fact that the A lysogen carrying the second copy of msbB is marked with Kanr. If the fcam insertion was in the diploid copy, Camr should cotransduce with Kanr. In all four cases, the insertion mutation must have recombined

with the endogenous chromosomal copy because none of the four strains contained closely linked Kanr and Camr markers. The recipient for these P1 transduction experiments was a haploid wild-type strain. The ability to transduce the msbB insertion mutation into this strain indicates that msbB is not an essential gene. To ensure that the insertion mutation truly inactivated the msbB gene, we performed a Southern blot analysis of the haploid wild-type strain with and without the msbB inser-

orJU

msbB P Pv

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msbB

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m | 500 bp

i

p 1 kb

FIG. 3. Restriction maps of the 2.6-kb DNA fragment containing the msbB gene and the region of the E. coli chromosome near msbB. The upper map shows the locations of the pykA, msbB, and orfU genes on the 2.6-kb msbB DNA fragment and the direction of transcription of each gene. PstI (P), PvuII (Pv), and EcoRI (E) restriction sites are marked below the line. The lower map indicates the locations and directions of transcription of other nearby genes. PstI (P) restriction sites are marked below the line.

J. BACTERIOL.

KAROW AND GEORGOPOULOS

706

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138

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229

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LDRKD-LKGMIKALKKGEVVWYAPDHDYGPRSSVFVPLFAVEQAAT

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275

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200

300

FIG. 4. Similarity of the MsbB and HtrB proteins. (A) Alignment of the MsbB and HtrB protein sequences by use of the structuregenetic matrix described by Feng et al. (9), with an open gap and a unit gap cost of 10. Vertical lines indicate identical amino-acids; colons indicate similar amino acids. (B) Hydropathy plots of the MsbB and HtrB proteins. Plots were produced by the method of Kyte and Doolittle (22), with an interval of 13 amino acids.

tion. The results (Fig. 5) indicated that the msbB gene had indeed been insertionally inactivated. The two genomic DNAs were digested with EcoRV, PvuII, or PstI and probed with the 1,931-bp PvuII fragment containing the msbB gene. flcam has both an EcoRV site and a PvuII site. Thus, when the genomic insertion DNA was cut with either of these enzymes, two DNA fragments hybridized with the probe instead of the one fragment seen with the wild-type DNA. Because there is no PstI site in Qicam, the PstI digest of the insertion DNA yielded a fragment that was 3.85 kb larger (the size of flcam) than the corresponding wild-type DNA fragment. The results from all three digests are consistent with the replacement of the endogenous wild-type copy of msbB with the insertion copy. We refer to this mutation as msbB: :Qcam. Genomic mapping of the msbB gene. When we originally

1 kb FIG. 5. Southern blot analysis of the msbB::flcam insertion mutation. Genomic DNAs from the msbB::flcam insertion mutant (-) and the isogenic wild-type parent (+) were digested with EcoRV (Ev), PvuII (Pv), or PstI (P) and probed with the 1,931-bp PvuIIlabeled fragment containing the msbB gene. The resulting autoradiogram is shown above a schematic drawing of the plasmid DNA (I) recombining with the chromosomal DNA (II), forming the insertion mutation (III). The 2.6-kb msbB DNA fragment is indicated by a solid bar, plasmid sequences are indicated by a broken line, chromosomal sequences are indicated by a thin line, the flcam cassette is indicated by an open bar, and the msbB ORF and direction of transcription are marked above the drawings with an arrow. Molecular weights of DNA size standards are indicated to the left of the autoradiogram.

isolated the msbB gene, we attempted to map its position on the E. coli genomic map by probing the overlapping clones of the genomic library made by Kohara et al. (20). The 2.6-kb fragment did not hybridize to any of the clones, implying that msbB lies in one of the gaps in the library. A comparison of the msbB restriction map with those of the cosmid libraries of Birkenbihl and Vielmetter (2) and Knott et al. (19) that cover these gaps showed that the best match is to the Knott et al. map of the DNA bridging the gap at 40.5 min. Linkage analysis of a Kanr insertion in the eda gene, which lies within this gap, confirmed this conclusion. The msbB::QIcam mutation is 75% linked to the eda gene by P1 transduction. Genetic and molecular analyses of this region placed two other genes, edd and zwf, in this region (6, 10). The three genes are very closely linked, and their order is eda-edd-zwf, clockwise on the genetic map. The zwf gene has recently been sequenced (29). A comparison of the restriction maps led us to conclude that the 5' end of the zwf sequence would be near the 5' end of the pykA sequence. To determine

E. COLI msbB GENE

VOL. 174, 1992

whether this conclusion is true and also how large the distance is between the two sequences, we made oligonucleotide primers that are homologous to the 5' ends of the two sequences and performed a polymerase chain reaction. There was a distance of approximately 480 bp between the two sequences (data not shown). Thus, the deduced order of the six genes is eda-edd-zwf-pykA-msbB-orfU, as shown in Fig. 3, and all of the genes, except for pykA, are transcribed in a counterclockwise direction. Analysis of the msbB::flcam insertion mutation. An initial characterization of the msbB mutation showed that the msbB gene is not essential for bacterial growth at any temperature between 20 and 42°C, nor is it required for growth in minimal medium with a variety of carbon sources. The lack of a phenotype for the msbB: :fcam mutation could indicate that the msbB gene product plays a role redundant with that of HtrB. Thus, in the presence of a wild-type copy of htrB, the msbB mutation will have no effect. If this is the case, we could expect that an msbB htrB double mutant would be lethal, or there could be the appearance of a new phenotype that is not seen with either of the single mutants. Although the htrB msbB double mutant is not lethal, the cells do show an altered morphology at 30°C, a temperature at which both of the single mutants are completely wild type. The doublemutant cells show a range of mutant morphologies including some filamentous cells and fat, misshapen cells, some of which appear to lyse (Fig. 6A). In addition, there are also cells whose morphology is similar to that of wild-type cells. This mixture of wild-type and mutant cells may account for the viability of the double mutant, as well as the slightly slower growth rate and the slight decrease in viability (Fig. 6B and C). The heterogeneity in cell morphologies exhibited by the htrB msbB double mutant could be due to the continuous accumulation of extragenic suppressors, allowing for better growth. However, even if this were the case, the majority of the htrB msbB double-mutant cells must be viable, since the cotransduction frequency of the htrB mutation and a nearby linked Kanr marker is roughly the same when either wild-type or msbB::fQcam bacteria are used as the recipients (data not shown). At 42°C, htrB is epistatic to msbB; both the morphology and the killing of the double mutant essentially mimic those of the single htrB mutant (data not shown). Deoxycholate resistance of the msbB and htrB mutants. In the hope of identifying a phenotype for the msbB mutation, we tested bacterial growth under a variety of conditions. In general, the msbB mutation had no effect on cell growth. However, we did find that at 30°C it conferred resistance to deoxycholate, that is, msbB bacteria were able to grow in the presence of four times more deoxycholate than the wild-type isogenic parent can tolerate (Table 2). We found that htrB bacteria were also able to grow at this high concentration of deoxycholate. To determine whether this resistance is a general property of the htrB and msbB insertion mutations, we checked for resistance to a variety of hydrophobic drugs and detergents. The results of such experiments are shown in Table 2. The msbB bacteria showed a slight sensitivity to all of the substances tested, except for deoxycholate. Although this was true at both 30 and 42°C, the sensitivities of both wild-type and msbB bacteria were less severe at 42°C. This was especially true for the sensitivity of wild-type bacteria to deoxycholate. At 30°C, wild-type bacterial growth was inhibited by the addition of deoxycholate to 2%, while at 42°C, it was inhibited by 8% deoxycholate. In general, the sensitivities exhibited by htrB bacteria were similar to those exhibited by msbB bacteria, except that htrB

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Time (min) FIG. 6. Morphology and growth characteristics of the msbB htrB double mutant at 30°C. (A) Morphology of msbB htrB mutant bacteria grown at 30°C from an OD595 of 0.05 to an OD595 of 0.4. (B and C) Growth curves (B) and viability curves (C) of isogenic wild-type bacteria (O), htrB mutant bacteria (*), msbB mutant bacteria (U), and htrB msbB mutant bacteria (O).

bacteria tended to be slightly more sensitive to cationic detergents such as tetradecyltrimethylammonium bromide and benzalkonium chloride. The htrB msbB double mutant was, for the most part, slightly more sensitive to all of the hydrophobic substances than were the two single mutants (data not shown). Although the double mutant grew on 10% deoxycholate, it did so poorly. This result probably reflects the slightly poorer growth of the double mutant in general. Rescue of htrB bacteria by cationic detergents. The most striking result of this analysis is the finding that two cationic detergents, benzalkonium chloride and tetradecyltrimethylammonium bromide, rescue the Ts- phenotype of htrB bacteria, allowing them to grow at 42°C. Figures 7A and B

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J. BACTERIOL.

TABLE 2. Resistance of msbB and htrB mutants to drugs and detergents MIC (>Lg/ml) for the indicated strain at:

420C

300C

Drug or detergent

Deoxycholic acid Tetradecyltrimethylammonium bromide Benzalkonium chloride Caprylic acid Rifampin Crystal violet

W3110

htrB

msbB

W3110

htrB

msbB

25,000 80

>100,000 40

>100,000 70

75,000 80

_a 40

>100,000 80

70 12,000 3 6

30 10,000 2 5

40 12,000 2 4

70 12,000 2 10

30

50 12,000 2 8

-

a _, nonpermissive temperature for htrB mutant bacteria.

show both growth and viability curves of wild-type and htrB bacteria grown at 42°C with and without the addition of 10 ,ug of tetradecyltrimethylammonium bromide per ml. Both the growth and the viability of the htrB mutant were restored after the addition of the detergent. It was not possible to determine whether the quatemary ammonium compounds fully rescue the morphological phenotypes of htrB. Both mutant and wild-type cells go through a period of extensive filamentation. However, after this period, both cell types resolve the filaments into wild-type cells. The nature of the

A

period of filamentation is not understood, nor do we know how it might relate to rescue by these compounds. These cationic detergents are quatemary ammonium compounds which carry a strong positive charge. The positive charge appears to be important, because other molecules, such as procaine, spermidine, and polylysine, which are tertiary ammonium compounds, also rescue (data not shown). Since these molecules are not as highly charged, the concentration required for rescue is much higher and the rescue is not as complete as that seen with the quaternary

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Time (hrs) Time (hrs) FIG. 7. Effects of the cationic detergent tetradecyltrimethylammonium bromide on the growth and viability of htrB mutant bacteria at 42°C. (A and B) Growth curves (A) and viability curves (B) of htrB mutant bacteria in the presence (0) or the absence (U) of tetradecyltrimethylammonium bromide, compared with those of isogenic wild-type bacteria grown in the presence (0) or the absence (U) of the detergent. (C and D) Growth curves (C) and viability curves (D) of htrB bacteria grown in the absence of the detergent (U), in the presence of the detergent (0), in the presence of the detergent and MgCl2 (A), and in the presence of MgCI2 without the detergent (*).

VOL. 174, 1992

ammonium compounds. One possible mechanism of htrB rescue is that these molecules disrupt the lipopolysaccharide (LPS) layer of the outer membrane or reorganize it in such a way as to alleviate the lethal effects of the htrB mutation. This effect may be mediated through an interaction with the negatively charged groups of LPS. These groups are normally associated with Mg2" and Ca2" ions, and such interactions have been shown to be important for the integrity of the LPS layer (24). We reasoned that the addition of Mg2" or Ca2" may interfere with the effects of the detergents. This turns out to be the case. The addition of either Mg2+ or Ca2+ reverses the effects of tetradecyltrimethylammonium bromide. This reversal is shown in Fig. 7C and D for MgCl2. Like the untreated cells, 95% of the detergent-plus-Mg2+treated cells die in the first 3 h, although the kinetics of killing may be slightly altered. The addition of Mg2+ alone also affects the lysis and killing kinetics of the htrB bacteria. This result could reflect a change in the LPS layer as well. DISCUSSION The msbB gene, when cloned onto plasmid vectors such as pBR322 and Bluescript, can complement the Ts- phenotype of htrB bacteria and the morphological alterations that accompany the lethality of the htrB mutation. We have mapped msbB to 40.5 min on the E. coli chromosome, in one of the gaps in the library of Kohara et al. (20). Through genetic and molecular approaches, the order of the genes in this region has been determined to be eda-edd-zwf-pykAmsbB-orfU. The eda and edd genes encode the two enzymes of the Entner-Doudoroff pathway for the metabolism of gluconate, the zwf gene encodes glucose-6-phosphate dehydrogenase, one of the enzymes in the pentose phosphate pathway, and the pykA gene encodes pyruvate kinase type II, one of two isoenzymes in the glycolytic pathway (11). The significance of this clustering of carbohydrate metabolism genes is unknown. The linkage of msbB to this cluster could indicate that its product also participates in these pathways. It is unlikely that msbB and, by inference, htrB are directly involved in carbohydrate metabolism, because the genes in these pathways have been either mapped to other chromosomal regions or cloned. However, it is possible that the msbB and htrB gene products indirectly affect or are affected by carbohydrate metabolism, especially if they play roles in the structure or function of the LPS layer, which is largely formed of sugars. The similarities between the MsbB and HtrB protein sequences and structures led us to propose that these proteins play similar and possibly redundant roles in E. coli physiology. The appearance of a new phenotype associated with the msbB htrB double mutant supports such a proposal. Although both of the single mutants have completely wildtype growth properties and morphologies at 30°C, the double mutant shows morphological alterations and has a slightly slower growth rate. Redundancy could also explain why htrB bacteria are completely wild type at temperatures below 33°C, since MsbB would substitute for the missing HtrB function. At higher temperatures or at faster growth rates, the increased requirement for HtrB can only be met by increasing the amount of MsbB. Although we found that in the presence of a wild-type copy of the htrB gene the msbB gene is not essential, we did discover that msbB and htrB mutant cells are much more resistant to deoxycholate than wild-type cells. This resistance is specific for deoxycholate; both htrB and msbB bacteria are slightly more sensitive to other hydrophobic

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substances. During these studies, we also found that the mdoH insertion mutation mdoA200:TnJO (23), which results in the lack of membrane-derived oligosaccharides, is more sensitive to deoxycholate but not to other hydrophobic substances. These results imply that the permeability barrier to deoxycholate may differ from that to the other hydrophobic substances examined and that HtrB, MsbB, and MdoH affect this barrier. Interestingly, the mdoA locus is located very near htrB on the E. coli map. For gram-negative bacteria, the barrier to most hydrophobic molecules is thought to be the LPS layer. Most mutations affecting LPS synthesis and structure cause the bacteria to become highly sensitive to hydrophobic substances, such as deoxycholate (24). Although rare, there are mutations that cause increased resistance to hydrophobic molecules (7, 36). The best-studied example is the pmrA mutant of Salmonella typhimurium (36). This mutant has an increased resistance to polymyxin, a polycationic antibiotic, and some cationic substances, such as polylysine. The pmrA mutation affects the lipid A portion of the LPS molecule by increasing the number of ester-linked phosphate groups that have been substituted with 4-amino-4-deoxy-L-arabinose from 10 to 15% to 60 to 70% (37). This substitution alters the charge of the LPS, and it has been proposed that this alteration affects the ability of basic molecules to interact with LPS (36). While the LPS of E. coli is not thought to have this substitution, it is possible that htrB and msbB exert a similar type of effect on LPS, leading to increased deoxycholate resistance. The ability of cationic detergents to rescue htrB bacteria supports the notion that HtrB affects the LPS layer. Cationic detergents are highly charged molecules that are very effective antiseptics because of their ability to disrupt bacterial membranes. With the addition of sublethal concentrations of these compounds, there may be a reorganization of the outer membrane, countering the effects of the htrB mutation. Consistent with the idea that LPS is the target for these compounds is the fact that either Mg2" or Ca2" ions can reverse this rescue. Both Mg2" and Ca2" are associated with the LPS layer, countering the electrostatic repulsion between the negatively charged LPS molecules. This interaction appears to be important for the organization and function of the outer membrane (24). Through the continued study of the htrB and msbB mutations and the identification and characterization of other suppressors of htrB, we hope to determine what these outer membrane alterations are and why the absence of HtrB function leads to cell lysis at temperatures above 32.5°C. ACKNOWLEDGMENTS We thank Ken Rudd for help in mapping the msbB gene. This work was supported by NIH grants A121029 (to C.G.) and GM07464 (to M.K.).

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