JOURNAL OF BACTERIOLOGY,0021-9193/01/$04.0010 DOI: 10.1128/JB.183.19.5554–5561.2001

Oct. 2001, p. 5554–5561 Vol. 183, No. 19

Copyright © 2001, American Society for Microbiology. All Rights Reserved.

Extragenic Suppressors of Growth Defects in msbB SalmonellaSEAN R. MURRAY,1 DAVID BERMUDES,2 KARIM SUWWAN DE FELIPE,3 AND K. BROOKS LOW4*

Departments of Biology,1 Molecular Biophysics and Biochemistry,3 and Therapeutic Radiology,4 Yale University,New Haven, Connecticut 06520, and Vion Pharmaceuticals,2 New Haven, Connecticut 06511

Received 9 April 2001/Accepted 7 May 2001

Lipid A, a potent endotoxin which can cause septic shock, anchors lipopolysaccharide (LPS) into the outerleaflet of the outer membrane of gram-negative bacteria. MsbB acylates (KDO)2-(lauroyl)-lipid IV-A withmyristate during lipid A biosynthesis. Reports of knockouts of the msbB gene describe effects on virulence butdescribe no evidence of growth defects in Escherichia coli K-12 or Salmonella. Our data confirm the general lackof growth defects in msbB E. coli K-12. In contrast, msbB Salmonella enterica serovar Typhimurium exhibitsmarked sensitivity to galactose-MacConkey and 6 mM EGTA media. At 37°C in Luria-Bertani (LB) broth,msbB Salmonella cells elongate, form bulges, and grow slowly. msbB Salmonella grow well on LB-no salt (LB-0)agar; however, under specific shaking conditions in LB-0 broth, many msbB Salmonella cells lyse duringexponential growth and a fraction of the cells form filaments. msbB Salmonella grow with a near-wild-typegrowth rate in MSB (LB-0 containing Mg21 and Ca21) broth (23 to 42°C). Extragenic compensatory mutations,which partially suppress the growth defects, spontaneously occur at high frequency, and mutants can beisolated on media selective for faster growing derivatives. One of the suppressor mutations maps at 19.8centisomes and is a recessive IS10 insertional mutation in somA, a gene of unknown function which corre-sponds to ybjX in E. coli. In addition, random Tn10 mutagenesis carried out in an unsuppressed msbB strainproduced a set of Tn10 inserts, not in msbB or somA, that correlate with different suppressor phenotypes. Thus,insertional mutations, in somA and other genes, can suppress the msbB phenotype.

Lipopolysaccharide (LPS) forms the outer leaflet of theouter membrane in gram-negative bacteria. Although theouter membrane is more permeable to small hydrophilic mol-ecules (because of the presence of porin channels) than theinner membrane, an intact LPS can protect gram-negative bac-teria from bile salts, hydrophobic antibiotics, and complement(12) and is associated with microbial virulence (27). In short,the LPS layer is a complex structure which is crucial for sur-vival, and its properties determine the permeability of theouter membrane to a wide variety of substances.

LPS consists of three major components: lipid A, core poly-saccharides, and O-linked polysaccharides. Lipid A is an en-dotoxin, and its fatty acids (lauric, myristic, and sometimespalmitic acid) anchor LPS into the outer membrane. Undernon-cold-shock conditions, the tightly regulated addition offatty acids to the lipid A precursor is catalyzed by the enzymesHtrB (lauric acid [3]), MsbB (myristic acid [4]), and PagP(palmitic acid [9]). htrB Escherichia coli and Salmonella arenonpermissive for growth on rich agar at or above 37°C (14,30). However, this growth defect can be suppressed in E. coliwith the msbB gene on a high-copy-number plasmid. msbB,also known as mlt (7), waaN (17), and lpxM (1), is one of twomulticopy suppressors of htrB E. coli isolated by Karow andGeorgopoulos (15).

MsbB can enzymatically add myristic (14:0; fast reaction) orlauric (12:0; slow reaction) acid to different positions on thelipid A precursor (demonstrated in vitro), whereas HtrB hasbeen shown to add lauric acid only to the lipid A precursor (4).

MsbB’s addition of lauric acid (slow reaction) to the lipid Aprecursor at the same position normally acylated by HtrB mayexplain msbB’s high-copy-number suppression of the htrB tem-perature-sensitive growth defect.

Several groups have studied msbB mutants in E. coli K-12and Salmonella choleraesuis (also known as enterica) serovarTyphimurium. Groups which have studied the growth of msbBknockouts have mentioned that there is no growth defect inmsbB E. coli K-12 (15, 28, 32). Khan et al. (17) concluded thatmsbB Salmonella serovar Typhimurium has a wild-type growthrate in BALB/c mice. In addition, Vaara and Numinen (32)noted that there is no defect in outer membrane permeabilitybarrier function in msbB E. coli K-12, and Somerville et al. (28)found no difference between minimum inhibitory concentra-tions for a spectrum of antibiotics in wild-type and msbB E. coliK-12. The only reported phenotypes attributed to msbB E. coliK-12 strains were increased deoxycholate resistance (15) and anonpyrogenic LPS (28). However, when Somerville et al. (29)knocked out the msbB gene in a clinical isolate of E. coli strainH16, they found that msbB H16 formed filaments at 37°C andhad a reduction in the level of the K1 capsule, an increase incomplement C3 deposition, and increases in opsonic and non-opsonic phagocytosis. Thus, filamentation in msbB E. coli H16was the first report of a growth defect in an msbB strain.

The reported apparent lack of growth defects, in msbB E.coli K-12 or Salmonella, was quite surprising because the otherreported lipid A mutations were either lethal or exhibitedconditional (temperature-sensitive) phenotypes, such as thatseen with htrB (14, 30). The published data seem to suggestthat the myristic acid moiety, added to lipid A by the MsbBenzyme, does not play a significant role in outer membranebarrier function, since msbB E. coli K-12 mutants were re-ported to have no growth phenotype other than increased

* Corresponding author. Mailing address: Radiobiology Laborato-ries, Yale University School of Medicine, 333 Cedar St., New Haven,CT 06520. Phone: (203) 785-2976. Fax: (203) 785-6309. E-mail: brooks.low@yale.edu.

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deoxycholate resistance. As we report below, newly purifiedmsbB Salmonella cultures actually exhibit significant specificgrowth abnormalities in vitro, whereas msbB E. coli K-12 (andE. coli B) does not.

In the course of tests of Salmonella as an anticancer agent(26), an msbB knockout mutational block in lipid A biosynthe-sis was introduced in order to decrease the stimulation of theseptic shock response and thereby increase the safety for use ofSalmonella in humans (21). Spontaneous faster growing deriv-atives of msbB recombinants were described and used in theconstruction of some of these strains, which in spite of theirgood growth characteristics are still as nonpyrogenic as theparental unsuppressed msbB strains (2, 21). We find that theuse of Luria-Bertani (LB) broth and no salt (LB-0) or MSB(LB-0 supplemented with Mg21 and Ca21) agar and MSBbroth support good growth of unsuppressed msbB strains.Since there is little enrichment for derivatives in these media,the primary de novo phenotype of msbB can be studied. Wefind that msbB mutations, especially in Salmonella, do in factconfer marked changes in physiology. As we report here, ex-tragenic suppressor mutations occur at a high frequency andconfer a more normal growth phenotype in the msbB back-ground.

MATERIALS AND METHODS

Bacterial strains, phage, and media. The bacterial strains used in this study arelisted in Table 1. The Salmonella msbB insertion-deletion for tetracycline resis-tance was constructed as described by Low et al. (21), and the E. coli msbBinsertion-deletion was provided by Costa Georgopoulos and is described byKarow and Georgopoulos (15). P22 mutant HT105/1int201 (obtained from theSalmonella Genetic Stock Center, Calgary, Canada) was used for Salmonellatransductions, and P1vir (gift of J. Tomizawa) was used for E. coli transductions.Salmonella serovar Typhimurium and E. coli strains were grown on LB-0 or MSBagar or in MSB broth. MSB medium consists of LB medium (22) with no NaCland supplemented with 2 mM MgSO4 and 2 mM CaCl2. LB-0 is LB medium withno NaCl. MSB broth and agar were used for the growth of strains under non-selective conditions. LB-0 agar was used when using selective antibiotics intransductions and transformations; Mg21 and Ca21 were found to increasephage contamination in transductions (5) and to decrease the effectiveness ofcertain antibiotics, such as ampicillin and tetracycline. Plates were solidified with

1.5% agar. LB-0 agar or MSB broth were supplemented as needed with ampi-cillin or carbenicillin (20 or 50 mg/ml), tetracycline (3, 5, or 20 mg/ml), chloram-phenicol (15 mg/ml in broth; 25 mg/ml in agar), ethylene glycol-bis(b-aminoethylether)-N,N,N9,N9-tetraacetic acid (EGTA, free acid) (Sigma, St. Louis, Mo.) (6mM or 6.5 mM), or deoxycholate (80,000 mg/ml). A 350 mM stock of EGTA, pH8.0 (adjusted with NaOH), was dissolved and then autoclaved. Antibiotics andsodium deoxycholate were added to LB-0 agar after cooling to 45°C. MacConkeyagar base (Difco) was used to prepare galactose-MacConkey agar.

Plasmids. A multiple cloning site containing NotI and SfiI sites on each side ofa BamHI site was cloned into the EcoRI and HindIII sites of the high-copy-number vector pSP72 (Promega) and the low-copy-number vector pHSG576 (31)(;8 copies per cell, as reported in reference 19) to facilitate shuttling insertsbetween the two vectors. These new vectors are named pSM1 and pSM2, re-spectively. pSM2 containing the cloned wild-type msbB gene is designatedpSM21, and pSM2 with the cloned wild-type somA gene is designated pSM22(Table 2).

Growth analysis. Phenotypes of strains were confirmed by replica plating.Master plates were made on either MSB or LB-0 agar. Replica plating was

TABLE 1. Bacterial strains

Strain Parental strain Genotype or phenotype Derivation or source

S. enterica serovarTyphimurium

ATCC 14028 14028 Wild type ATCCYS8211 14028 msbB1::Vtet Low et al. (21)YS1 14028 msbB1::Vtet P22 z YS8211 3 140283Tet5

r, where YS8211 5 donor and14028 5 recipient in P22 transduction

YS1456 14028 DmsbB2 DpurI3252 somA1 Spontaneous EGTAr derivative of msbB1::Vtet DpurI3252derivative of 14028; replacement of msbB1::Vtet by DmsbB2by homologous recombination (6)

YS871 14028 DmsbB2 DpurI3252 somA1 zbj10:Tn10 P22 z 14028 Tn10 pool 3 YS14563Tet20r (EGTAs)

YS872 14028 DmsbB2 DpurI3252 somA1 zbj10:Tn10 P22 z YS871 3 YS14563Tet20r (EGTAr)

YS873 14028 msbB1::Vtet somA1 zbj10:Tn10 P22 z YS871 3 YS13Tet20r (EGTAr)

YS1170 14028 msbB1::Vtet; unknown suppressor(s) From YS8211, spontaneous selection on EGTA platesTT16812 LT2 recD541::Tn10dCm Salmonella Genetic Stock Center, Calgary, CanadaYS2 LT2 msbB1::Vtet recD541::Tn10dCm P22 z YS8211 3 TT168123Tet5

r

SL1344 SL1344 his Strr; mouse virulent Sunshine et al. (30)YS3 SL1344 msbB1::Vtet his Strr; mouse virulent P22 z YS8211 3 SL13443Tet5

r

E. coli K-12MLK1067 W3110 F2 l2 rph-1 IN(rrnD-rrnE) msbB1::Vcam Karow and Georgopoulos (15)MG1655 MG1655 F2 l2 rph-1 Guyer et al. (10)KL423 MG1655 F2 l2 rph-1 msbB1::Vcam P1vir z MLK1067 3 MG16553Camr

TABLE 2. Plasmids

Plasmid Relevant characteristic(s) Source

pNK2883 Ampr, inducible mini-Tn10 withisopropyl-b-D-thiogalactopyranosidepromoter

Kleckner et al.(18)

pSP72 High copy number, Ampr PromegapHSG576 Low copy number, Cmr, pSC101

derivativeTakeshita et al.

(31)pSM1 pSP72 with a modified multiple

cloning site replacing the pSP72sites between the EcoRI andHindIII sites with the followingrestriction sites: NotI, SfiI, BamHI,NotI, and SfiI

This study

pSM2 pHSG576 with a modified multiplecloning site replacing the pHSG576sites between the EcoRI andHindIII sites with the followingrestriction sites: NotI, SfiI, BamHI,NotI, and SfiI

This study

pSM3 pSP72 with msbB1 This studypSM21 pSM2 with msbB1 This studypSM22 pSM2 with somA1 This study

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performed using the double velvet technique (20). To test for LB agar sensitivity,triple velvet replica plating, which uses an unincubated double velvet plate toreplica plate onto various media, was used. Plates were incubated for 10 h at 28,30, 37, or 42°C or for 1 to 2 days at 23°C. To generate growth curves, 10-ml MSBbroth tubes were inoculated from patches from new clones, with verified phe-notypes, and grown on a slant without movement overnight at 37°C. Tubes(2.5-cm diameter) with 10 ml of broth were then inoculated with cells to achievean optical density at 600 nm (OD600) of 0.05. Cells were held on ice until allinoculations were completed. Then the cultures were placed in a 37°C or roomtemperature (21 to 23°C) water bath on a 30° angle with 100 rpm of translationalmovement. OD600 was measured every 30 min for 420 min.

Restoring msbB1 genotype. In order to confirm that the observed MsbBphenotypes result simply from knocking out MsbB function, a fragment contain-ing wild-type msbB (21) was digested with EcoRV and cloned into pSP72 to testfor complementation in YS1 on EGTA and galactose-MacConkey plates. pSP72carrying wild-type msbB was named pSM3. After observing complementation inYS1 and confirming the insert by sequencing, wild-type msbB was again EcoRVdigested and blunt-end ligated into HindIII-digested pSM2, thus producing plas-mid pSM21.

Microscopic observation. Strains ATCC 14028, YS1 (msbB1::Vtet), andYS1456 (msbB2 purI3252 somA1) were grown, as described above for growthcurves, to an OD600 of 0.40. Then the cells were stained with nigrosin (11) andobserved with an Olympus AX70 microscope.

Mutation frequency determination. A frozen stock of YS1 was streaked onMSB medium and incubated overnight at 37°C to isolate individual clones. Tenmilliliters of MSB broth, in 2.5-cm diameter tubes, were inoculated with inde-pendent YS1 colonies. They were grown in tubes at a 30° angle in a 37°C waterbath with 100 rpm of translational movement until an OD600 of 0.10 wasachieved. The tubes were then placed on ice and diluted in ice-cold MSB broth.Dilutions (2 3 1026) were plated onto MSB agar to calculate the number of CFUper milliliter. Dilutions (1022, 1023, and 1024) were plated on 6.5 mM EGTA,galactose-MacConkey, and LB plates and incubated overnight at 37°C. Thisconcentration of EGTA (6.5 mM) was used to minimize the chances of gettinggrowth of nonmutated survivors (based on a series of CFU tests, data notshown). Approximately 20 clones arising on each type of plate were used to makea master plate on MSB medium to determine what percentage of the clones weremutants.

Preparation of electroporation-competent cells. The technique of O’Callaghanand Charbit (25) was used for preparation of electroporation-competent cellswith the following modifications. Overnight cultures in MSB broth were pre-pared as described above. The next morning, 2 ml of the overnight culture wasused to inoculate 100 ml of MSB broth, which was grown in a 37°C water bathwith 100 rpm of translational movement until the cells reached an OD600 of 0.6.Cells were rinsed with ice-cold 1% glycerol instead of distilled water, because 1%glycerol was found to increase the survival of msbB Salmonella and to helpmaintain the unsuppressed phenotype (data not shown).

Transduction and transformation. Salmonella P22 transductions were per-formed by the method of Davis et al. (5), and E. coli P1 transductions wereperformed by the method of Miller (22), except that LB-0 plates supplementedwith the appropriate antibiotic were used. EGTA was not added to the antibioticplates for transductions. A Bio-Rad Gene Pulser was used for electroporationwith the following settings: 2.5 kV, 1,000 V, and 25 mF for transformation of YS1,and 2.5 kV, 400 V, and 25 mF for YS1456 and 14028.

Tn10 mutagenesis. A transposon pool of ATCC 14048 was made usingpNK2883 by following the technique of Kleckner et al. (18), except that MSBbroth and LB-0-Tet20 agar (contains 20 mg of tetracycline per ml) were usedinstead of LB broth and LB-Tet20 agar (contains 20 mg of tetracycline per ml).Over 65,000 tetracycline resistant (Tetr) clones of ATCC 14028 were pooled, anda P22 lysate was made. The pool was screened for auxotrophy for differentbiosynthetic pathways by replica plating onto minimal media and media contain-ing various pools of amino acids and bases (5).

Linkage of the YS1456 suppressor mutation to a Tn10. A P22 lysate was madefrom the ATCC 14028 Tn10 library and transduced into strain YS1456. Tetr

transductants were screened for EGTA sensitivity by replica plating. Upon iso-lation of a Tn10 which showed linkage to a suppressor gene, the formula of Wu(34) was used to estimate the distance between the Tn10 and the suppressorgene. Additional P22 lysates were made from both suppressed and nonsup-pressed recombinants; thus, the Tn10 was linked to the wild-type allele of theYS1456 suppressor gene (strain YS871) and to the YS1456 suppressor mutation(strain YS872).

Cloning of somA. YS871 (with a Tn10 approximately 3.0 kb away from thewild-type allele of the YS1456 suppressor gene) genomic DNA was cloned intopSM1, and YS872 (with a Tn10 approximately 3.0 kb away from the YS1456

suppressor mutation) genomic DNA was cloned into pSP72 to test for comple-mentation. Genomic DNA was partially digested with Sau3AI, size selected, andcloned into the BamHI site of pSM1 or pSP72. The libraries were transformedinto maximum efficiency DH5a (Gibco-BRL) and plated onto LB-0-Amp20-Tet3

agar in order to select for inserts with Tn10s. Tn10 results in 20 mg/ml Tetr

(resistance to 20 mg of tetracycline per ml) when incorporated into the chromo-some but 2 to 5 mg/ml Tetr when present on a high-copy-number plasmid (18).PCR was used to test transformants arising on LB-0-Amp20-Tet3 agar to confirmthe presence of the Tn10 before sequencing and transforming the inserts intoeither YS1456 (YS871 library) or YS1 (YS872 library). The YS871 library wastested for complementation in strain YS1456 to screen for an EGTA-sensitivephenotype, and the YS872 library was transformed into YS1 to screen for thesuppressor phenotype. The location of the suppressor gene was mapped bysequencing and aligning inserts which did or did not complement the phenotype.Inserts with the suppressor or wild-type allele of the YS1456 suppressor genewere sequenced at Yale’s Keck Facility. The putative suppressor gene, somA, wasthen cloned into a low-copy-number vector, pSM2, via PCR and retested forcomplementation. Genomic DNA clones were sequenced on both strands andsubmitted to GenBank under S. enterica serovar Typhimurium somA accessionnumber AF360548.

PCR. PCR was performed using whole bacteria. Clones were tested for thepresence of Tn10 using primers specific for the regulatory region. The Tn10primers 59-GGATCCTTAAGACCCACTTTCACATTTAAGT-39 and 59-GGTTCCATGGTTCACTTTTCTCTATCAC-39 yield a 721-bp product. somA wasamplified with primers containing NotI restriction sites: 59-GGGGGCGGCCGCCGGATTTGGCGATTGAAGTC-39 and 59-GGGGGCGGCCGCGATAAGTTGGCAGCGGGG-39. These primers generate a 1,329-bp product when am-plifying the wild-type allele and a 2,298-bp product from the YS1456 suppressorallele. The Tn10 primers were kindly provided by Caroline Clairmont, VionPharmaceuticals. All primers were made by the Yale University Keck Facility.PCRs were performed using Ready-To-Go PCR beads (Amersham PharmaciaBiotech Inc.).

DNA sequencing. DNA sequencing was performed at the Yale UniversityKeck Facility using fluorescent dye terminated thermocycle sequencing. To se-quence DNA flanking Tn10s on cloned inserts, the following primers were used:TnL1, 59-CCCACCTAAATGGAACGGCGTT-39, and TnR2, 59-GGCACCTTTGGTCACCAACGCTT-39. These primers were provided by Stanley Lin ofVion Pharmaceuticals. SP6 and T7 primers were used to sequence from the endsof inserts in pSM1, which is a derivative of pSP72. For sequencing inserts in thelow-copy-number vector, pSM2 (derivative of pHSG576), the following primers,obtained from Joann Sweasy, were used: M13, 59-GCGGATAACAATTTCATATAGG-39, and U17, 59-GTAAAACGACGGCCAGT-39.

RESULTS AND DISCUSSION

Growth phenotypes of msbB strains. During the engineeringof msbB Salmonella a variety of colony sizes were observed onLB plates. As shown in Fig. 1, when plating dilutions onto LBagar from an msbB broth culture with an OD600 of 0.1 (usinggrowth conditions as described in the “Mutation frequencydetermination” section of Materials and Methods), a few col-onies arise after 15 h, and by 27 h a variety of colony sizes areapparent. Subsequent experiments revealed that the smallercolonies (unsuppressed msbB clones) were sensitive to 6 mMEGTA and galactose-MacConkey media, whereas the largercolonies (suppressed msbB clones) grew well on these media.

Sensitivity of YS1 (msbB1) to 6 mM EGTA (Fig. 2B), ga-lactose-MacConkey (Fig. 2C and D), and LB medium (Fig. 2F)is shown in a replica plate series. Growth of YS1 is inhibited orretarded on these media after 10 h of incubation at 28, 30, 37,or 42°C. Similarly, strains YS2 (LT2 msbB1) and YS3 (SL1344msbB1) are also sensitive to 6 mM EGTA, galactose-MacCon-key, and LB medium (data not shown). Thus, these growthdefects, which are observed in three different strain back-grounds, are not strain-specific phenomena. No growth inhibi-tion is observed on 6 mM EGTA or LB plates at 23°C, butsensitivity to galactose-MacConkey is maintained at 23°C (datanot shown). As shown in Fig. 2A, YS1 grows well on LB-0 agar

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at 30°C (and also at 23, 28, 37, and 42°C; data not shown). Thephenotype of msbB Salmonella strains is in sharp contrast tothe reported phenotype of mutants of htrB, which encodesanother LPS biosynthetic enzyme, since htrB Salmonella has atemperature-sensitive growth defect on rich agar at or above37°C (30).

In contrast to the above phenotypes of simple msbB mu-tants, spontaneously derived suppressor strains YS1456 andYS1170 (both msbB with suppressor mutations) have interme-diate resistance phenotypes, showing that suppressor muta-tions can partially compensate for the msbB growth defect.Strain YS1456 grows stronger on 6 mM EGTA than YS1170(Fig. 2B), and YS1170 grows stronger on galactose-MacCon-key than YS1456 (Fig. 2C and D).

In order to test if the phenotypes observed result solely fromthe disruption of the msbB gene, we cloned msbB1 into pSM2(generating the vector pSM21) and tested for complementation.Wild-type msbB on a low-copy-number vector complements the 6mM EGTA and galactose-MacConkey growth defects (data not

shown), demonstrating that the phenotypes observed are due todisruption of the msbB gene and not a polarity effect.

In contrast to Salmonella, KL423 (E. coli msbB) does notshow sensitivity to 6 mM EGTA (Fig. 2B) but does have aparticular temperature-dependent phenotype for galactose-MacConkey sensitivity: its growth is inhibited on galactose-MacConkey medium at 28 (Fig. 2D) but not at 30 (Fig. 2C), 23,37, or 42°C (data not shown). As reported by Karow andGeorgopoulos (15), we find that KL423 has increased resis-tance to deoxycholate at 30°C; this is also true for Salmonellasuppressor strain YS1456. No increased resistance to deoxy-cholate was observed in unsuppressed msbB Salmonella strainYS1 (data not shown). Furthermore, KL423 (E. coli msbB) wasfound to have no apparent growth defect in LB, LB-0, or MSBbroth at 37°C (data not shown), confirming the results reportedby Karow and Georgopoulos (15). One possible interpretationof the species difference is that wild-type E. coli (at least strainsK-12 and B [strain WA837; data not shown]) carry alleles thatcompensate for msbB in a way similar to that of the Salmonella

FIG. 1. Dilution (1024) of a YS1 culture (OD600 of 0.10) plated onto LB agar after a 15-h (A) or 27-h (B) incubation at 37°C. Arrows pointto colonies with spontaneous suppressor mutations arising after 15 h.

FIG. 2. Replica plate series. (A) LB-0 medium, 30°C; (B) 6 mM EGTA medium, 30°C; (C) galactose-MacConkey (Gal. Mac.) medium, 30°C;(D) galactose-MacConkey medium, 28°C; (E) LB-0 medium, triple velvet (t.v.), 30°C; (F) LB medium, triple velvet, 30°C. The master plate wasmade on MSB agar. w.t., wild type.

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suppressor mutations, thereby preventing the specific growthdefects which are seen in simple msbB Salmonella mutants.

A number of specific growth defects of msbB Salmonellastrains were also observed in liquid growth media. LB-0 me-dium compensates for the LB slow growth defect in agar (Fig.2A and E) but not always in broth. At 37°C, the growth of YS1is inhibited in LB broth (Fig. 3A) and the strain lyses in LB-0broth under our specific conditions (see Materials and Meth-ods and Fig. 3B). If the starter culture is from a plate, the lysisphenomenon is not observed (data not shown). We also testedother unrelated Salmonella serovar Typhimurium strains YS2(LT2 msbB) and YS3 (SL1344 msbB) and found that they alsolyse in LB-0 broth and have poor growth in LB broth (data notshown). The somA1 suppressor mutation in strain YS1456 (seebelow) compensates for the msbB lysis phenomenon in LB-0broth (Fig. 3B) and substantially compensates for the LB sen-sitivity (Fig. 3A). In order to test for the possibility that the purImutation in YS1456 played a role in these growth properties, apurI1 YS1456 strain (YS873) was constructed and was foundto have growth curves identical to those observed withpurI3252 YS1456 (data not shown). YS1 log phase lysis doesnot occur at 23°C, but sensitivity to LB broth is maintained at23°C (data not shown).

In an attempt to compensate for the specific in vitro msbBgrowth defects in LB and LB-0 broth (Fig. 3A and B), LB-0medium was supplemented with 2 mM MgSO4 and 2 mMCaCl2 (called MSB medium). In MSB broth (Fig. 3C), YS1(msbB) has a near-wild-type growth rate when grown in ashaker with 100 rpm of translational movement. However,even in MSB broth YS1 cultures accumulate some debris whengrown with rapid rotational movement at 37°C (data notshown). Thus, the extent of shear forces and oxygenation ap-pears to be important for maintaining the de novo msbB phe-notype.

The phenotype of msbB cells is presumably a result of theabsence of the myristoyl group on lipid A. Lateral interactionsbetween neighboring lipid A molecules can presumably beweakened by having fewer acyl groups anchoring lipid A intothe outer membrane or by a shortage in divalent cations. An

excess of Mg21 or Ca21 might stabilize the LPS moieties inmsbB strains, which have one less fatty acid than the wild type,since it is possible that these divalent cations could increase thenumber divalent-cation cross-linked phosphate groups thatdecorate lipid A and the LPS core. This inverse relationshipbetween levels of cations and acyl groups is consistent with thereported increase of lipid A palmitate levels (by PagP) inwild-type Salmonella under low Mg21 conditions (9).

Previous work has shown that Mg21 and Ca21 have differenteffects on the stability of the outer membrane in Salmonellaand E. coli (24). We have found that either CaCl2 or MgCl2 cancompensate for msbB growth defects. However, much higherconcentrations of MgCl2 are needed to yield compensationsimilar to that with CaCl2-containing media (data not shown).Since either Mg21 or Ca21 can cross-link the phosphategroups of lipid A, it is possible that Ca21 may be able tocompensate by an additional, Ca21-specific, pathway. For ex-ample, Kanipes et al. (13) recently reported that Ca21-, but notMg21-, containing media resulted in increased phosphoeth-anolamine substitution on LPS’s inner core.

Morphological abnormalities in msbB Salmonella. To fur-ther investigate the phenotype of msbB cells in LB and LB-0broth, we observed the morphology of cells grown in thesemedia and also MSB broth at 37°C with 100 rpm of transla-tional movement at an OD600 of 0.40. Unusual morphologywas observed in YS1 cultures in all three broths tested andsuggests that a loss-of-function mutation in msbB can lead toproblems in cell division.

At 37°C, ;3 to 30% of various clones of YS1 cells in MSBbroth form filaments (Fig. 4B). Filaments were defined as cellswhich were at least three times the length of neighboring cells.In addition, many filaments have cross-sections that are largerthan normal. The YS1456 suppressor mutation suppresses fila-mentation, since only ;1 to 3% of YS1456 cells form filamentsin MSB broth compared to ;3 to 30% in YS1 and ;0% inwild-type ATCC 14028 (Fig. 4A). The length of wild-type cellsranged from ;2 to 4 mM, and the average width was ;1 mM.In contrast, the length of YS1 cells ranged from ;1 to 36 mM,and the width ranged from ;1 to 2 mM in MSB broth.

FIG. 3. Growth curves of 14028, YS1, and YS1456 at 37°C in LB broth (A), LB-0 broth (B), or MSB broth (C).

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Filamentation of YS1 may contribute to small (approximate-ly twofold) decreases in CFU per OD unit observed in MSBbroth at 37°C. No filaments were observed when YS1 wasgrown under identical conditions at 23°C (data not shown). Asimilar finding was observed in the H16 clinical isolate of the E.coli msbB mutant, where filamentation occurs at 37°C but notat 30°C (29). In contrast, Karow and Georgopoulos (15) didnot report any filamentation in their K-12 W3110 msbB mu-tants, and we observed a low percentage (1 to 2%) of filamentformation in our K-12 MG1655 msbB mutant strain in MSBbroth (data not shown).

In LB broth at 37°C, all YS1 cells appear to be elongatedand curved and formed bulges (Fig. 4D). In contrast to YS1,;22% of YS1456 cells form filaments, with some swelling inLB broth (data not shown). 14028 cells have normal morphol-ogy in LB broth (Fig. 4C). In LB broth, the length of wild-typecells ranged from 2 to 4 mM, and the average width was ;1mM. In contrast, the length of YS1 cells ranged from ;6 to 10mM, and the average width was ;2 mM. In LB broth at 23°C,YS1 forms filaments similar to those observed in MSB broth at37°C but not elongated bulging cells (data not shown). Beforelog phase lysis in LB-0 broth at 37°C, YS1 forms both filamentsand curvy, elongated, bulging cells (data not shown).

Mutation frequency determination. Having noticed a seem-ingly high frequency of faster-growing derivatives of msbBstrains upon streaking on various media, we then measured thefrequency of mutants exhibiting faster growth. Our results (Ta-ble 3) show that using freshly isolated clones, the averagenumber of 6.5 mM EGTA-resistant mutants from stabilizedclones grown in MSB broth is ;4 3 1024, and the averagenumber of galactose-MacConkey-resistant mutants is ;1 31024. However, the observed frequency of LB-resistant mu-tants grown similarly in MSB broth but plated on LB agar,which allows slow growth, is ;1.0 3 1022, which is approxi-mately 25- to 100-fold higher than the frequency of the directlyselected EGTA- or galactose-MacConkey-resistant mutants.After streaking the EGTA-, galactose-MacConkey-, or LB-resistant colonies on MSB agar, we found that ;88% of theEGTA-resistant colonies, 100% of the galactose-MacConkey-resistant colonies, and ;73% of the LB-resistant coloniesmaintained their phenotype after streaking, showing that themajority of resistant colonies are relatively stable mutants.Thus, the data suggest that our mutation frequency estimatesreflect the number of mutants in the cultures, except for theselection on LB, which allows slow growth of unmutated clones(as shown in Fig. 1). Since YS1 cells can undergo a significant

FIG. 4. Morphology of 14028 and YS1 in MSB or LB broth at 37°C. Magnification, 34,400. (A) 14028, MSB broth; (B) YS1, MSB broth; (C)14028, LB broth; (D) YS1, LB broth, Bar, 10 mM.

TABLE 3. Measurements of mutation frequency in strain YS1 (msbB; LB, EGTA, and galactose-MacConkey sensitive) to LB, EGTA,or galactose-MacConkey resistance

Selective mediumFrequency of mutants for YS1 clone: Avg mutation

frequencyAvg no. of mutants

per 10,000 cellsA B C

EGTA (6.5 mM) 3.8 3 1024 2.8 3 1024 4.3 3 1024 3.6 3 1024 4Galactose-MacConkey 2.0 3 1024 1.2 3 1024 4.5 3 1025 1.2 3 1024 1LB 1.0 3 1022 1.7 3 1022 2.9 3 1023 9.9 3 1023 99

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number of generations of growth on LB plates, there are manymore cell divisions in which mutations could occur in the earlyhours of incubation, thus giving an artificially high calculatedfrequency of mutation from simply counting the number ofcolonies after 15 h. This interpretation is strengthened by find-ing that the vast majority of the suppressed derivatives ob-tained on LB are resistant to 6 mM EGTA and/or galactose-MacConkey medium, and the frequency of these mutations,when selected directly, was shown to be much lower (1024

versus 1022).High mutation frequencies in YS1 cultures for resistance to

6.5 mM EGTA (4 3 1024) and galactose-MacConkey (1 31024) agar suggest that there may be many genetic targets forsuppression of the msbB Salmonella phenotype. The EGTAand galactose-MacConkey mutation frequencies are similar tothat observed for temperature-sensitive compensatory muta-tion in htrB E. coli, which was ;1.0 3 1024 (16). It is possiblethat many of the YS1 suppressor mutations are loss-of-func-tion mutations, since there are many more targets for loss-of-function than gain-of-function mutations. Furthermore, thehigh frequency of spontaneous suppressor mutations may ex-plain why other groups (8, 17, 33) did not mention the pheno-types discussed in this report. It is possible that these authorsused cultures that had been inadvertently overgrown withfaster growing derivatives, which rapidly arise on LB plates anddo not exhibit strong specific growth defects in vitro.

Mapping of one of the mutations which suppresses msbB.We used a Tn10 pool to obtain linkage to the suppressormutation, denoted somA1 for suppressor of msbB, in YS1456.The presence of 0.7% auxotrophy involving a variety of bio-synthetic pathways in our 14028 transposon pool (data notshown) suggested that Tn10s had integrated into the 14028chromosome rather randomly. In order to link a Tn10 markerto the YS1456 suppressor mutation, we transduced DNA frag-ments, from this transposon library in 14028, into YS1456. Twoout of 300 transductants were EGTA sensitive from a trans-duction bringing the wild-type P22 Tn10 library into strainYS1456. The EGTA-sensitive phenotype results from replac-ing the YS1456 suppressor gene with wild-type DNA linked toparticular Tn10s from the library. Cotransduction frequenciesof 79 and 82% were obtained, suggesting that the two Tn10s lie;3.2 and ;3.0 kb, respectively, away from the YS1456 sup-pressor gene. The Tn10 lying ;3.0 kb away was selected for usein gene linkage experiments. Transductants with the Tn10linked to wild-type or mutant alleles of somA were used tomake genomic libraries.

Cloning and sequencing of somA. When plasmids with Tn10linked to the YS1456 allele of somA were electroporated intoYS1, no suppressor phenotypes were observed (data notshown). (The electroporation conditions used select for thetransformation of unsuppressed msbB cells, therefore makingspontaneously suppressed mutants nearly undetectable in ourscreens.) However, when plasmid clones carrying the Tn10linked to wild-type alleles of the YS1456 suppressor gene wereelectroporated into strain YS1456, EGTA sensitivity was ob-served, thus showing that YS1456 has a recessive suppressormutation. Both mutant and wild-type versions of the 19.8-centisome (Cs) region were sequenced, and the sequence datarevealed that YS1456 has an IS10 insertion sequence at nucle-otide 922 in an open reading frame homologous to ybjX of E.

coli, which we call somA (suppressor of msbB). The IS10 in-sertion may have occurred at some point in the ancestry ofYS1456 in which a purI::Tn10 mutation was present. The Sal-monella somA gene, which maps close to 19.8 Cs, has ;59%nucleotide homology to E. coli ybjX and at the level of proteinhas 56% identity. somA was then amplified by PCR and clonedinto a low-copy-number vector to demonstrate complementa-tion (restoration of EGTA and galactose-MacConkey sensitiv-ity [data not shown]) in strain YS1456.

somA, a gene encoding a protein of unknown function, is thefirst example of an identified spontaneous suppressor of msbB.No growth defect was found for the somA1 mutation in anmsbB1 background (data not shown), and no obvious trans-membrane domains or signal sequences were apparent frombasic sequence analyses. In a search of GenBank sequences,Salmonella somA was found to have ;46% nucleotide homol-ogy to serovar Typhi virK, and at the level of protein there is34% identity. Serovar Typhi virK maps at approximately 61 Cs,in the iroN upstream region, on the Salmonella chromosome(GenBank accession no. AF029845). As part of the Shigellavirulence plasmid, virK was found to be a virulence factorwhich mediates intracellular spreading by posttranscriptionallyregulating the virG gene product (23). A possible relationshipbetween somA and virK is, at this point, unclear.

We have begun further analysis along these lines, and Tn10mutagenesis in YS1 has produced Tn10-induced suppressormutations which yield phenotypes and genetic targets distinctfrom that of the somA suppressor mutation, indicating thatsomA is only one example of an extragenic insertional suppres-sor mutation (data not shown). Experiments to infer the func-tion of somA are in progress.

ACKNOWLEDGMENTS

This research was supported by a grant from Vion Pharmaceuticals.S.R.M. was supported by a National Institutes of Health PredoctoralTraining Grant in Genetics (5 T32 GM07499) and a Yale UniversityFellowship.

We thank Martina Ittensohn and Jeremy Pike of Vion Pharmaceu-ticals for technical support and Michel Slotman, Timothy Gorton, andJoann Sweasy of Yale University for their helpful ideas and sugges-tions.

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