Isolation of Salmonella typhimurium Strains That Utilize Exogenous

Vol. 169, No. 11

JOURNAL OF BACTERIOLOGY, Nov. 1987, p. 5060-5065 0021-9193/87/115060-06$02.00/0 Copyright © 1987, American Society for Microbiology

Isolation of Salmonella typhimurium Strains That Utilize Exogenous 3-Deoxy-D-manno-Octulosonate for Synthesis of Lipopolysaccharide ROBERT C. GOLDMAN* AND EDWARD M. DEVINE Anti-Infective Research Division, Pharmaceutical Discovery, Abbott Laboratories, Abbott Park, Illinois 60064 Received 16 April 1987/Accepted 31 July 1987

Spontaneous mutants of SalmoneUla typhimurium LT2 were selected for the ability to accumulate exogenous 3-deoxy-D-manno-octulosonate (KDO). Bacteria containing a gene (kdsA) which codes for a temperaturesensitive KDO-8-phosphate synthetase were plated at the restrictive temperature of 42°C on medium containing 5 mM KDO. Since bacteria containing the kdsA lesion are unable to grow at 42°C due to inhibition of lipopolysaccharide (LPS) synthesis and accumulation of lipid A precursor, this method allowed direct, positive selection of mutants capable of utilizing exogenous KDO for LPS synthesis. Spontaneous mutants, selected at a frequency of about 10-6, required exogenous KDO for growth at 42°C. The growth rate at 42°C was nearly normal in the presence of 20 mM KDO and was directly proportional to KDO concentrations below 20 mM. Exogenous KDO also suppressed accumulation of lipid A precursor. The apparent Km for KDO accumulation was 23 mM, and the maximum rate of transport was calculated to be 505 pmol of KDO per min per 108 cells. Bacteria incorporated exogenous [3H]KDO exclusively into LPS, with less than 10% dilution in specific activity due to residual endogenous KDO synthesis. The mutation giving rise to the ability to accumulate exogenous KDO was extremely useful in the direct screening for new mutations in the kdsA gene after localized mutagenesis. Five mutations in kdsA were isolated, four of which were new alleles as determined by on fine-structure analysis. The ability to introduce labeled (3H, 13C, and 14C) KDO in vivo should simplify and extend the analysis of this critical metabolic pathway in gram-negative bacteria. that membrane attack complex of complement can readily insert and cause cell death (R. C. Goldman and M. H. Miller, manuscript in preparation). Similar events occur when the pathway is inhibited by using a new class of antibacterial agent which specifically inhibits CMP-KDO synthetase (Goldman et al., Abstr. Annu. Meet. Am. Soc. Microbiol. 1987; Goldman et al., Nature [London], in press). The ability to specifically introduce a labeled probe into a metabolic pathway can render analysis of the pathway much less difficult. Radiolabel can be introduced into the KDO pathway via labeled arabinose 5-phosphate (26); however, some dissemination of radiolabel occurs, and since the first enzyme in the pathway (KDO-8-phosphate synthetase) must be functional, radiolabel is subject to dilution due to continuing endogenous synthesis of KDO. Highly specific, labeled probes of the KDO pathway have already been used in in vitro systems (4, 14), and the ability to use these same kinds of probes (3H, 13C, or 14C labeled) would be a distinct advantage in the study of the pathway in vivo. However, no strains of E. coli, Salmonella typhimurium, or any other gram-negative bacterium capable of utilizing exogenous KDO without extensive metabolism have been previously reported. We have selected spontaneous mutants of S. typhimurium LT2 which are capable of incorporating exogenous KDO in the absence of detectable metabolism to any pathway other than LPS. The selection, characterization, and potential uses of such mutants are described in this paper.

The 3-deoxy-D-manno-octulosonate (KDO) pathway is one of several metabolic pathways which are integrated in gram-negative bacteria to produce lipopolysaccharide (LPS). Four known enzymes are involved in the synthesis

and incorporation of KDO into LPS: (i) KDO-8-phosphate synthetase (22), (ii) KDO-8-phosphate phosphatase (23), (iii) CMP-KDO synthetase (24), and (iv) KDO-lipid A transferase (18; Fig. 1). Temperature-sensitive (TS) mutations in two of the genes coding for enzymes in the pathway have been isolated (9, 10, 16, 25, 28; M. J. Osborn, unpublished data). These mutations, kdsA and kdsB (coding for TS KDO-8phosphate synthetase and CMP-KDO synthetase, respectively), were mapped (30), and the corresponding genes were cloned from Escherichia coli (8, 32) and, in the case of kdsB, sequenced (7). Mutants carrying these mutations were prerequisites for determining the physiological effects of inhibition of the KDO pathway (17, 18, 20, 25-28), because specific inhibitors of the pathway were not available until recently (R. C. Goldman, C. C. Doran, J. 0. Capobianco, D. Halvorson, W. E. Kohlbrenner, P. A. Lartey, and A. G. Pernet, Abstr. Annu. Meet. Am. Soc. Microbiol. 1987, A113, p. 19; R. C. Goldman, W. E. Kahlbrenner, P. A. Lartay, and A. G. Pemet, Nature [London], in press). We now know that inhibition of the KDO pathway by using bacteria containing TS enzymes (20) or specific in vivo inhibitors (S. Kadam, manuscript in preparation) leads to accumulation of a lipid A precursor which is not efficiently translocated to the outer membrane. Precursor thus accumulates in the inner membrane, which may be the cause of growth stasis that occurs when the pathway is inhibited extensively (20). At the time of growth stasis, >90% of the total lipid A precursor accumulated is found in a unique fraction of lighter-density outer membrane (R. C. Goldman, manuscript in preparation) which is so perturbed in structure *

MATERIALS AND METHODS Bacterial strains and growth conditions. All bacterial strains used were derivatives of S. typhimurium LT2 and are listed in Table 1. Bacteria were grown in LB (3) broth (pH 7.4 with NaOH) containing 0.2% glucose and 0.5 mM N-acetyl-D-glucosamine when indicated or in PPBE broth (GIBCO Laboratories). Galactose was removed from PPBE

Corresponding author. 5060

INCORPORATION OF EXOGENOUS KDO INTO LIPOPOLYSACCHARIDE

VOL. 169, 1987

CHO HO

PEP

=aPo"I

coo

H

OH

ara-5-P

CH2

KDO-8-Phosphate Synthetase

HO

KD0-Phosphate Phosphatase

COCOOH

HO

OH

HO

CMP-KDO Synthetase

H~-OH

KDO

COOH HO

OH

HO~ _o.....

KDO-8-P

OH

O-CMP

CMP-KDO

COOH

Lipid A

B

HMA

FIG. 1. KDO pathway in gram-negative bacteria. PEP, Phosphoenolpyruvate; ara-5-P, arabinose 5-phosphate; BHMA, Phydroxymyristic acid.

broth as previously described (17). When indicated, bacteria were grown in MOPS (morpholinepropanesulfonic acid) defined medium (19) containing 0.2% (wt/vol) glucose. Selection of mutants capable of utilizing exogenous KDO. Strains RG113 and RG109 (both containing the same TS kdsASO lesion) were grown in LB broth at 30°C to saturation. Samples (0.1 ml) were then spread onto LB broth plates (1.5% agar) containing 1 to 5 mM KDO. Plates were incubated at 42°C for 24 h, at which time colonies were picked for further characterization. Individual colonies were plated onto LB broth plates with and without KDO and were incubated at 42°C. Colonies which grew at 420C only in the presence of KDO were selected for further study. Characterization of KDO transport. Bacteria were grown to an A600 (LB broth) or A420 (MOPS) of 1.0 and then concentrated 1Q-fold, by centrifugation, into the same medium and stored on ice. Transport experiments were initiated by adding cells to solutions of radiolabeled KDO. Typically, 1.9 ml of cells was added to 0.1 ml of KDO solution in a 50-ml Erlenmeyer flask to allow adequate aeration. Cells were agitated at 250 rpm at 42°C. Cell samples (0.1 ml in triplicate) were withdrawn at appropriate intervals, layered on top of 100 ,ul of silicone oil (13; Dow Coming 550 and 560, 5:7 [vol/vol]), and centrifuged for 3 min in a microcentrifuge at 13,000 x g. The microcentrifuge tubes were frozen immediately in a dry ice-ethanol bath for 10 min or longer. The tips of the tubes (containing the cell pellets) were cut off into scintillation vials and mixed vigorously with 1 ml of water. Instagel scintillation cocktail (Packard Instrument Co., Inc.; 10 ml) was added, and the samples were counted for radioactivity. The amount of cell-associated radioactivity at time zero was subtracted from that at each additional time point. These data were analyzed by least-squares fit with the Fortran program

5061

described by Cleland (2). The cell number was determined by plating for viable count in triplicate. The intracellular volume was determined in triplicate after incubation of cells with tritiated water for 15 min, followed by centrifugation through silicone oil and determination of radioactivity in the cell pellet. Tn1O insertions and selection of tetracycline-sensitive cells. The X TnJO vector 561 (b221 cI857: :TnJO Oam29 Pam8O) was obtained from N. Kleckner (Harvard University) and was used to make a pool of random TnJO inserts in strain TS736 as previously described (8). A TnlO insert cotransducible with kdsAS0 (30% cotransduction) was then constructed by standard procedures (3) with phage P22 int. Tetracyclinesensitive cells were selected on Bochner plates (3). Transductions. All transductions were carried out by using phage P22 int as described previously (3). Two-factor crosses were conducted by first selecting for tetracycline resistance and then screening for TS growth (at least 400 colonies per cross). Three-factor crosses were conducted with strain HD50 (kdsA50) and each of the new kdsA alleles. Localized mutagenesis near kdsA. Localized mutagenesis near kdsA, using hydroxylamine mutagenesis of transducing phage P22, was carried out as described previously (11). Enzyme assays. The assay of enzymes in the KDO pathway was essentially as described previously (22-24). Cells, grown in LB broth containing 50 mM KH2PO4, were suspended in 0.01 M Tris hydrochloride buffer (pH 7.2), and crude extracts were prepared after French press lysis (two passages at 16,000 lb/in2) and removal of unbroken cells and cell debris by centrifugation at 100,000 x g for 1 h. Supernatants were passed over Sephadex G-25 (Pharmacia PD10 columns) equilibrated with 0.01 M Tris acetate buffer (pH 7.2) containing 1 mM dithiothreitol. The void volume was assayed for protein and enzyme activities. Incorporation of [3H1KDO into LPS. A galE kdsA50 TnJO Kdol strain (RG134) was grown at 30°C in PPBE medium which was exhausted of galactose as described previously (17). Cells were shifted to 42°C at an A6,oo of 0.2, and after 30 min [3H]KDO (1,610 cpm/nmol) was added to a final concentration of 5 mM. After a defined period of growth, cells were harvested and extracted twice with ethanol, and LPS TABLE 1. Bacterial strains used Strain

Genotype and phenotype

Source

G30 TS736

galE his-6165 ilv-562 metA22 metE551 trpB2 ga1E496 xyl404 rpsLJ20flaA66 hsdL6 hsdSA29 malB F'112 kdsA50 uph(Con) galE metA trpB hisF ilvA pyrE xyl malA rpsL120 As RGlll; but kdsA50 zdj-3602::TrnlO As G30, but kdsB91 zbh-3601: :TnlO Wild type As HD5O, but zdj-3602::TnJO kdsA+ As G30, but zdj-3602::TnJO kdsA50 As RG112, but kdsA50 As G30, but zdj-3602::TnlO As RG116, but kdsA101 As RG116, but kdsA102 As RG116, but kdsA103 As RG116, but kdsA104 As RG116, but kdsA105 As RG113, but Kdol As RG134, but kdsA+ and lacks TnlO As RG109, but Kdo2 As RG139, but kdsA + and lacks TnlO

M. Osborn B. Wanner

HD50

RG109 RG110 RG111 RG112 RG113

RG115 RG116 RG117 RG118 RG119

RG120 RG121 RG134 RG136 RG139 RG141

M. Osborn

This study This study This study This study This study This study This study This study This study This study This study This study This study This study This study This study

5062

GOLDMAN AND DEVINE

was extracted twice by the phenol-chloroform-petroleum ether (PCP) method (5). Chloroform and petroleum ether were evaporated from the combined PCP extracts, and LPS was precipitated with cold ethanol (80% final concentration). After 1 h on ice, LPS was collected by centrifugation (10,000 x g for 10 min at 4°C). The dried pellet was assayed for 3H and for KDO content (13) after hydrolysis in 1 M HCl at 100°C for 30 min. Purification of lipid A precursor for qualitative analysis. Cells were grown at the permissive temperature (30°C) in LB medium to an A6wo of 0.2 to 0.3 and then shifted to the restrictive temperature (42°C). After 30 min, 35 ,uCi of [3H]glucosamine hydrochloride (30 Ci/mmol) was added to 100 ml of culture. When growth stasis occurred, cultures were chilled, harvested, and washed twice with LB medium. Combined pellets from 400 ml of cells were either extracted immediately or stored at -20°C. Cell pellets were first delipidated by three extractions (15 ml each) with ethanol (30 min for each extraction with vortexing every 10 min). The pellet of delipidated cells was extracted twice with 10 ml of PCP and finally with 90% hot phenol. Extracts (ethanol, PCP, and the aqueous phase from the 90% phenol extraction) were assayed for radioactivity. Chloroform and petroleum ether were evaporated from PCP extracts at 35°C under a stream of N2. Chloroform (0.5 ml) was added, and the sample was mixed and applied to a 2-ml (bed volume) column of DEAE-cellulose (Bio-Rad polypropylene column, 4 by 8 cm) which was prepared in methanol and equilibrated with 15 ml of methanol. Columns were eluted in steps (twice with 8 ml each) of ethanol containing 0.0, 0.05, 0.1, 0.15, 0.35, 0.45, and 0.8 M ammonium acetate. Portions of each step were counted for radio-

activity. Pooled fractions eluting from DEAE columns between 0.15 and 0.45 M ammonium acetate were analyzed for fatty acids after methylation with methanolic HCl (100°C for 17 h) and for glucosamine and phosphate as described previously

(1).

Radiolabeling and quantitation of lipid A precursor. Cells were grown in LB broth or MOPS medium containing 0.2% (wt/vol) glucose and 0.5 mM N-acetyl-D-glucosamine. NAcetyl-D-[3H]glucosamine was added (at the time of temperature shift) to 4 pXCi/ml in order to radiolabel lipid A precursor. Lipid A precursor was extracted from cells as described previously (21) and analyzed by chromatography on Silica Gel H (Merck & Co., Inc.) (21). The relative amount of each lipid A precursor species was monitored by summing the amount of tritium in each peak and normalizing to cell protein. Chemicals. The syntheses of KDO and [8-3H]KDO are described elsewhere (1). The synthesis of 2-deoxy-KDO will be described elsewhere (P. Lartey et al., manuscript in preparation). N-Acetyl-D-[1-3H]glucosamine (TRK.376) was from Amersham Corp. RESULTS AND DISCUSSION Selection of strains that can utilize exogenous KDO. S. typhimurium LT2 does not accumulate exogenous KDO, and thus previously described kdsA mutants could not be rescued by exogenous KDO (26). We successfully isolated spontaneous mutants that could utilize sufficient exogenous KDO to support LPS synthesis at a nearly normal rate. This was accomplished by plating kdsASO mutants (strain RG109 or RG113) at 42°C on LB broth agar containing 1 to 5 mM KDO. Colonies arose at a frequency of about 10-6 and were

J. BACTERIOL.

0.5A

0.1

0.05

30

90

150

210

TIME AFTER SHIFT TO 42C

(MINUTES) 2. FIG. Restoration of cell growth by exogenous KDO. Strain RG139 was grown in LB medium at 30°C to an A600 of 0.2 to 0.3. Portions of this culture were diluted (to an A600 of 0.05) into fresh, prewarmed (42°C) medium containing 0, 1, 5, or 20 mM KDO. Samples with an A600 of >0.9 were diluted 1:10 before the A6(w was read. Growth stasis for kdsA strains lacking the Kdo phenotype was identical to the control curve for strain RG139, even in the presence of 20 mM KDO.

screened for retention of the kdsA allele by being plated at 420C in the absence of KDO. Two independent isolates, RG134 and RG139 (derived from RG113 and RG109, respectively), were selected for further study. Strains RG134 and RG139 both contained stable genetic alterations which allowed utilization of exogenous KDO. The kdsA lesions were removed by transduction with P22 phage grown on strain RG111, with selection for temperature-resistant growth and screening for sensitivity to tetracycline, yielding strains RG136 and RG141. These strains were passed several times on LB plates, and the kdsA and kdsB alleles from strains RG113 and RG110, respectively, were introduced via their linkage to TnlO. All transductants inheriting kdsA grew at 42°C in the presence of 5 mM KDO, while those inheriting kdsB did not. The growth rate at 42°C was directly correlated with the amount of exogenous KDO added to cultures of strains containing the kdsAS0 lesion and the Kdol or Kdo2 alterations, which allowed utilization of KDO (Fig. 2). Nearly normal growth rates (,u = 1.2) were attained at 20 mM exogenous KDO in either LB medium (Fig. 2) or MOPS defined medium (,u = 0.75; data not shown). In contrast, growth stasis occurred even when up to 20 mM KDO was added to strains containing kdsA but lacking the Kdo alteration (data not shown). Direct measurement of KDO accumulation. Strains containing Kdo mutations accumulated exogenous KDO in a concentration-dependent manner which was saturable at high KDO concentrations (Fig. 3). Strain RG139 (kdsASO Kdo2) was grown at 30°C and shifted to 42°C for 20 min to inactivate the TS KDO-8-phosphate synthetase and deplete intracellular KDO pools. These cells accumulated exogenous KDO at a maximum rate of 505 pmol/min per 10-8 cells,

5063


VOL. 169, 1987

3 9 A

50

-

I

I I I

I I I'

I',

C)

I

40-

'.9

400

600

800

I

I

2 0

I

I

30-

1000

PL

1/[KDO], (M)-'

FIG. 3. Characterization of KDO accumulation. Strain RG139 in LB medium at 30°C, shifted to 42°C for 20 min, and concentrated by centrifugation to an A6w of 10. Accumulation of KDO was monitored after addition of KDO (15, 10, 5, 2.5, or 1 mM) containing [3H]KDO (at specific activities of 321, 514, 944, 1,654, and 4,417 cpm/nmol of KDO, respectively). Samples were incubated at 42°C with vigorous shaking, and portions (0.1 ml) were taken in triplicate at 8, 16, and 24 min and pelleted immediately through silicone oil. Radioactivity in the cell pellets was determined, and these data were used to determine rates of KDO accumulation versus extracellular KDO concentration. Cell numbers were determined by viable counts in triplicate. Km and Vmax were calculated by using these data. Strain RG109, the parent of RG139, accumulated KDO at less than 5% of the rate of strain RG139 (limit of detection) under identical conditions.

was grown

with an apparent Km of 23 mM as determined by kinetic analysis (Fig. 3), and transport did not require prior exposure to KDO. Similar values were obtained with strain RG134 (data not shown). Although not examined in detail, the intracellular level of free KDO reached 100 to 200 p.M when RG139 was shifted to 42°C in the presence of 20 mM KDO (data not shown). KDO accumulation was not inhibited by an equal molar amount of 2-deoxy-KDO, an analog of KDO which is locked in the ,-pyranose form. Since 2deoxy-KDO is locked in this stereochemical configuration, one of the other three forms of KDO in solution (4) is more likely to be the transported species. In contrast, an equal molar amount of sialic acid, a structurally related sugar, caused greater than 90% inhibition of KDO accumulation. The ability of sialic acid to inhibit KDO accumulation indicated that it would also prevent restoration of cell growth by KDO at 42°C. When RG139 was shifted to 42°C in the presence of 20 mM KDO, the addition of as little as 0.3 mM sialic acid detectably reduced the growth rate, and addition of 2 mM or greater resulted in growth stasis. These data suggest that KDO accumulation is related to sialic acid transport, which is known to occur in E. coli and S. typhimurium (31). Sialic acid (2 mM) would serve as a sole carbon and energy source for RG111, RG109, and RG139, whereas KDO would not. Since nanA and nanT (sialic acid aldolase and transport, respectively) map at 69 min in E. coli, we checked for genetic linkage of the Kdo phenotype to the analogous chromosomal region in S. typhimurium. Phage grown on S. typhimurium containing TnJO insertion 3163 (this insert maps at 69 min [15]) was used to transduce a tetracycline-sensitive variant of RG139 to tetracycline resistance; 32% of the 125 transductants were unable to grow at 42°C in the presence of 5 mM KDO. However, further biochemical and genetic analyses are required to determine

25

FRACTION NUMBER FIG. 4. Analysis of lipid A precursor accumulation. Strain RG139 was grown in LB medium (10 ml containing 0.2% glucose and 0.5 mM N-acetylglucosamine) at 30°C to an A600 of 0.2 to 0.3. The culture was then split, with 5 ml shifted to 42°C without KDO (0) and 5 ml shifted to 420C with 20 mM KDO (0). N-Acetyl[3H] glucosamine was added at the time of shift to label lipid A precursor, which was extracted from the cells and analyzed by chromatography on Silica Gel H. The major precursor species are labeled 1 through 4. Peak 4 comigrates with authentic species IVA (see text). PL, Phospholipid.

the precise relationship between KDO and sialic acid transport. Suppression of accumulation of lipid A precursor by exogenous KDO. The addition of exogenous KDO at the time of shift to 42°C suppressed accumulation of lipid A precursor in Kdo kdsASO strains (Fig. 4 and Table 2) but not in strains lacking the Kdo alteration (data not shown). Accumulation of lipid A precursor at 30°C was caused by below-normal activity of the altered KDO-8-phosphate synthetase enzyme (about 5% of the normal level), even when cells were grown at 300C. As expected, the level of lipid A precursor increased significantly when cells were incubated at 42°C (Table 2); the mass of lipid A precursor accumulated at 42°C was 3 to 5 ,ug/100 ,ug of cell protein. Accumulation of precursor was suppressed when exogenous KDO (20 mM) was present at the time of temperature shift, and the cells then grew at a nearly normal rate (Fig. 2). The level of precursor in cells at 30°C was also suppressed by exogenous KDO (Table 2), and TABLE 2. Quantitation of lipid A precursor species Amt (kcpm/100 ,ug of protein) of the following Incubation conditionsa

30°C, no KDO 30°C, with KDO 42°C, no KDO 420C, with KDO

lipid A precursor speciesb:

1

2

3

4

8.6 1.9 118.8 1.5

25.8 6.4 247.9 4.8

212.9 116.4 442.4 21.3

52.5 17.7 394.8 7.1

a KDO was present at 20 mM when indicated. b The amount of each precursor species was quantitated by separation of radiolabeled precursor on Silica Gel H and summing of the radioactivity under each peak. Species 4 is identical to IVA. Species 1, 2, and 3 should correspond to IA. IIIA, and IIA, respectively, based on order of migrations on Silica Gel H (see text). Values represent the average of three experiments.

5064

GOLDMAN AND DEVINE

the magnitude of suppression of lipid A precursor was directly related to the concentration of exogenous KDO at 30 or 42°C (data not shown). Although the accumulation of all four major species of lipid A precursor was suppressed by exogenous KDO, the magnitude of suppression of one of these four species (species 3) was significantly less than that of the other three (Fig. 4 and Table 2). Precursor species 4 (Fig. 4 and Table 2) was identified as IVA (21) by comigration on Silica Gel H (IVA was kindly provided by K. Takayama). Species IVA is 0-(2-amino-2-deoxy-,-D-glucopyranosyl)(1--6)-2-amino-2-deoxy-a-D-glucose, acylated at positions 2, 3, 2', and 3' with P-hydroxymyristoyl moieties and containing phosphate groups at positions 1 and 4' (21). The structures of the other three major species, which migrate more slowly on Silica Gel H, are known (21); however, standards of these species were not available. Based on order of mobility, species 1, 2, 3, and 4 (Fig. 4 and Table 2) should correlate with species IA, IIIA, IIA, and IVA, respectively, described by Raetz et al. (21). Species IIIA, IIA, and IA are related to IVA as follows: IIIA contains phosphoethanolamine, IIA contains aminopentose, and IA contains both phosphoethanolamine and aminopentose (21). The order of appearance of species 1 through 4 is 4 before 2 and 3 before 1 (R. C. Goldman, C. C. Doran, and J. 0. Capobianco, manuscript in preparation) after inhibition of CMP-KDO synthetase by an antibacterial agent which we have designed (Goldman et al., Abstr. Annu. Meet. Am. Soc. Microbiol. 1987). These data suggest that species 4 (IVA) is the normal acceptor of KDO in vivo and that species 1, 2, and 3 (IA, IIIA, and IIA) represent products of aberrant side reactions which occur due to accumulation of IVA. The differential suppressive effect of exogenous KDO on the levels of the four major precursor species (Table 2), therefore, is likely due to a differential flow of IVA towards LPS (normal pathway) and species 1 through 3 (aberrant pathway due to accumulation of IVA) under conditions of partial restoration by KDO. Incorporation of exogenous KDO into LPS. The results described above predict that exogenous [3H]KDO would be incorporated directly into LPS. Strain RG134 (Kdol kdsA50 galE) was grown in PPBE medium which was previously exhausted of galactose to suppress production of 0 antigen. This was necessary because the abequose present in 0 antigen will interfere with the accurate quantitation of KDO present in LPS. When cells were shifted to 42°C in the presence of 5 mM KDO (1,610 cpm of [3H]KDO per nmol), labeled KDO was incorporated into LPS at nearly the same specific activity (1,500 cpm/nmol), and greater than 90% of the [3H]KDO incorporated into these cells was recovered in LPS. Although S. typhimurium and E. coli may contain an inducible aldolase capable of degrading KDO to D-arabinose and pyruvate (6), less than 10% of the incorporated KDO is metabolized via this route under the conditions employed in this study. Isolation and screening of TS mutants with mutations near kdsA. The lack of a method for directly selecting, or screening, for kdsA mutants has required the development and use of less efficient, indirect methods (9, 16, 26, 27). The results described above showed that the kdsA50 mutation could be rescued by exogenous KDO when the bacteria contained a Kdo alteration which allows accumulation of exogenous KDO. In contrast, it is unlikely that mutations in genes coding for other enzymatic steps in the KDO pathway would be rescued in the same Kdo genetic background. We have coupled the use of localized mutagenesis and screening for rescue of growth by exogenous KDO in order to isolate kdsA

J. BACTERIOL.

mutations in a more direct manner than previously available (9, 16, 26, 27). When phage P22, grown on strain RG116, was mutagenized with hydroxylamine to 5% survival, approximately 1% of the tetracycline-resistant transductants (transduced into strain G30) were TS for growth. The TS mutations were first tested for linkage to TnJO by transduction back into G30, and the transduction frequency was recorded. Twenty percent of the TS growth mutations were rescued by exogenous KDO when transduced into a Kdo background (strain RG136). Five such mutant strains (RG117 to RG121) were selected for further study. All five mutations were in the kdsA gene based on the following results: (i) LPS synthesis ceased after a shift to 42°C (measured by inhibition of [3H]galactose incorporation in a galE background), (ii) four major species of lipid A precursor accumulated after a shift to 42°C, (iii) all mutant strains showed reduced (>90%) KDO-8-phosphate synthetase activity in crude extracts prepared from cells grown at 30°C (normal activity in extracts of wild-type strain RG111 = 17.5 nmol of KDO-8-phosphate produced per min per mg of protein), (iv) all mutailt strains had normal activities of CMP-KDO-synthetase activity (7 to 9 nmol of CMP-KDO produced per min per mg of protein) and KDO-8-phosphate phosphatase activity (18 to 25 nmol of KDO produced per min per mg of protein), and (v) all five mutations were closely linked to TnlO (cotransduction frequencies of 0.1 to 0.3; kdsASO frequency = 0.3 [see below]). The four major lipid A precursor species accumulated were analogous to those described by Raetz et al. (21), having the basic structure

0-(2-amino-2-deoxy-p-D-glucopyranosyl)-(1-*6)-2-

amino-2-deoxy-a-D-glucose, acylated at positions 2, 3, 2', and 3' with P-hydroxymyristic acid, and bearing phosphate at positions 1 and 4'. This structure was confirmed by compositional analysis (glucosamine/phosphate/3-hydroxymyristic acid ratio of 1:1:2) and negative-ion fast atom bombardment mass spectroscopy ([M - H]- at mlz 1404). Four of the five new mutations isolated in the kdsA gene

represented genetic alterations at a different site than in kdsASO and than in one another. Mutations in strains HD50, RG117, RG120, and RG121 represented different alleles of kdsA, since all could recombine with each other to yield temperature-resistant growth. The mutations in strains RG118 and RG119 could recombine with alleles in HD50, RG117, RG120, and RG121, but apparently not with each other. Three-factor crosses, using strains HD50 and RG117 through RG121, showed that lesions in RG117 through RG121 all mapped to the TnJO-distal side of the allele in HD50, with the most likely order being HD50, RG118 and RG119, RG121, RG120, and RG117. Data describing fine-structure analysis of previously described kdsA mutations have not been reported, and the present data reveal that several sites of potential genetic alteration in structure are available for structure-function analysis. Although several other mutations occurring in the chromosotne region near kdsA caused TS growth, none were specifically defective in LPS synthesis. Since several mutations were introduced in the kdsA gene, these data suggest that a second gene coding for another enzyme in the KDO pathway is not clustered with kdsA, as might have been expected based on gene clustering in bacteria (29). In conclusion, S. typhimurium strains were selected which are capable of accumulating exogenous KDO at a sufficient rate to support near-normal growth and LPS synthesis in the absence of endogenous KDO production. The use of such strains for screening, along with the technique of localized

VOL. 169, 1987


mutagenesis, simplified the isolation of mutations at different loci in the kdsA gene. Such strains will also incorporate exogenous [3H]KDO solely into LPS, a technique that should prove advantageous for studying KDO metabolism in vivo by using labeled substrates (3H, 13C, and '4C), as has previously been done in vitro (4, 14). LITERATURE CITED 1. Capobianco, J. O., R. P. Darveau, R. C. Goldman, P. A. Lartey, and A. G. Pernet. 1987. Inhibition of exogenous 3-deoxy-D-

2. 3. 4.

5.

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