Vol. 138, No. 2

JOURNAL OF BACTERIOLOGY, May 1979, p. 305-313 0021-9193/79/05-0305/09$02.00/0

Genetic Studies of an Escherichia coli K-12 TemperatureSensitive Mutant Defective in Membrane Protein Synthesis TAKASHI SATO,' MISAO OHKI,2 TAKASHI YURA,' * AND KOREAKI ITO' Institute for Virus Research, Kyoto University, Kyoto,' and National Cancer Center Research Institute,

Tokyo,2 Japan

Received for publication 17 February 1979

The mutant divE42(Ts) of Escherichia coli K-12, defective in the synthesis of membrane proteins and in the transcription of the lac operon at high temperature, has been further characterized. It was found that a mutation (divE42) located at about min 22 on the E. coli chromosome map is responsible for the Lacphenotype and temperature-sensitive growth. The mutation could be cotransduced with serC, pyrD, or pyrC by phage P1 at a frequency of 4, 16, or 0.5%, respectively, the gene order being serC-pyrD-ompA-sulA-divE-pyrC. Examination of temperature-independent revertants and Pyr+ transductants revealed that all the mutant phenotypes examined (deficiencies in the increase of activities of some membrane enzymes, expression of the lac operon, and synthesis of several other proteins) are due to a single mutation (divF,42) which is recessive to the wild-type (divE+) allele. Protein synthesis in the mutant was also analyzed by dodecyl sulfate-polyacrylamide gel electrophoresis. Synthesis of a number of proteins, including membrane proteins, was found to decrease significantly, whereas that of an elongation factor, EF-Tu, increased upon transfer of a logphase culture to high temperature (4200). These effects of temperature shift-up on protein synthesis were evident within 5 min under the conditions used.

Some proteins, including several membrane proteins, are synthesized specifically at a particular stage in the cell cycle of Escherichia coli (7, 8, 14, 24, 27). A temperature-sensitive mutant (divE42) of Escherichia coli K-12 was previously shown to be deficient in the synthesis of a number of membrane proteins, including several "stage-specific" proteins, and mRNA transcribed from the lac operon at the restrictive temperature (25, 26; I. Yamato, Y. Anraku, and M. Ohki, J. Biol. Chem., in press). The proteins affected by this mutation include cytochrome bi, the L-a-glycerophosphate uptake system, L-aglycerophosphate dehydrogenase, succinate dehydrogenase, and D-lactate dehydrogenase. The amounts of these proteins in the wild-type cell may be doubled at a specific stage before the onset of cell division (24; M. Ohki, I. Yamato, and M. Futai, submitted for publication). When a mutant culture grown at 300C was transferred to 420C, the growth seemed to continue until most of the cells reached a certain stage of the cell cycle (25). This mutant strain therefore appears to carry a lesion in some process related to

cell division, affecting the stage-specific synthesis of some of the membrane proteins. The present paper reports on the results of further genetic and physiological characteriza-

tion of this mutant strain. The chromosomal location of the mutation divE42 (division, EFTu) (previously called tsC42) and analysis of temperature-independent revertants and of transductants provided evidence that all the mutant phenotypes observed are due to a single mutation located at about min 22 on the E. coli chromosome (1). Dodecyl sulfate-polyacrylamide gel electrophoresis of proteins synthesized at the restrictive temperature revealed a rapid and striking decrease in the rates of synthesis of a number of proteins that can be separated on the gel.

MATERIALS AND METHODS Bacterial strains. The bacterial strains used were all derivatives of E. coli K-12 and are listed in Table 1. Strains KL181, KL208, and KL259(KLF43) were supplied by B. J. Bachmann, and phage TuII* was from U. Henning. Strain CSH59 was obtained from the Cold Spring Harbor Laboratory (21). Media. Polypeptone-glucose medium, L broth, and synthetic medium E were described previously (23). Medium A (32) supplemented with 1% Casamino Acids was used for growing cells for assay of membraneassociated enzymes. These media were supplemented with 25 jg of thymine or uracil per ml for strains carrying thy orpyrC or pyrD, respectively. In the case of serC strains, either nutrient broth containing 2 ,tg 305

306

SATO ET AL.

J. BACTERIOL.

TABLE 1. Bacterial strains Reference or derivation

Sex

Known genetic markers'

AT713

F-

cysC39 argA21 lysA10 maLAI xyl-4 rpsL9 met

M. Abe

N42 KY2304

FFt

divE42, other markers as in AT713

KY2305

Hfr

metE rpoB endAllf10 divE42; (O-dsd-

KY2318

F-

KY2323 KY2329

F' F-

thr metE thy endAl100 rpoB tsx serC13 pyrD34 KL181/F' trp+ pyrD+ (KYF2323) pyrD+ divE42, other markers as in

25, 26 Spontaneous trp revertant of KL259 (KLF43) Derivative of KH326 (10) x KL983 (19) Derivative of KH326 (10)

KY2343 KY2503

FF-

thy+ recAl; other markers as in KY2329 serC+ divE4Z; other markers as in

KY2506

F-

serC+; other markers as in KY2329

KY2510

F-

KY2514

F-

pyrD34 thi argE mtl xyl rpsL non-9 ompA pyrD+; other markers as in KY2510

KY2515

F-

pyrD+ divE42, other markers

KY2530

F-

KY2510 sulA Ion pyrD his rpsL

KY2547c

F-

divE42 Ion pyrD rpsL

KL181

F-

KL208 KL259 (KLF43)

Hfr F'

thi-1 pyrD34 his-68 trp-45 recAl mtl-2 xyl- 7 malAl galK35 rpsL18 (O-rac-trp---) thi-l tyrA2 pyrD34 his-38 thyA33 trp-45 mtl-2 xyl-7 malAI galK35 recAl (F'

Strain

trp+; other markers as in KL259 (KLF43) his.-)b

KY2318

KY2329

as

in

KL181 x KL208 KY2318, transduced with P1 grown on KY2305 KY2329 x KL16-99 (18) KY2329, transduced with P1 grown on KY2304 KY2329, transduced with P1 grown on KY2304 Derivative of P400 (4)

KY2510, transduced with P1 grown on KY2305 KY2510, transduced with P1 grown on KY2305 Spontaneous methylmethane sulfonate-resistant derivative of SG13009 (donated by S. Gottesman) Derivative of SG13009 obtained by two-step crosses CGSC4275d

CGSC4314 (19) CGSC4291

thy+ tyr+) For gene symbols, see Bachmann et al. (1). These indicate the direction of transfer of markers. 'This strain was resistant to 0.04% methylmethane sulfonate, although the Ion mutation was retained. d CGSC numbers are those of E. Coli Genetic Stock Center, Yale University, New Haven, Conn. b

of pyridoxine-HCl per ml or a synthetic medium con- 10% trichloroacetic acid in an ice bath and, after taining L-serine (300 ug/ml) and pyridoxine-HCl (2 standing for at least 1 h, collected by centrifugation at ,ug/ml) was used. For other amino acid auxotrophs, 25 3,000 rpm for 15 min. The precipitates were washed ,ug of each amino acid per ml was added to a synthetic with acetone and dissolved in sample buffer for gel medium. electrophoresis. Preparation of subeellular fractions. ProceConjugation and transduction. Procedures for conjugation were described previously (28), and trans- dures for fractionation of proteins into soluble, inner duction with phage Plvir was carried out according to membrane and outer membrane fractions were deIkeda and Tomizawa (11). scribed previously (29). Cells were converted to spherPreparation of pulse-labeled whole proteins. oplasts by lysozyme-EDTA and disrupted by sonicaCells were grown at 30°C in minimal medium supple- tion. Soluble fraction was separated from whole memmented with the required nutrients. When the turbid- branes by centrifugation through sucrose bilayers (15 ity reached about 40 Klett units (no. 54 filter), cultures and 70%). Further separations of whole membranes into inner and outer membrane fractions were carried were divided into two portions; one was transferred to 42°C, and the other was kept at 30°C. A 1-,uCi amount out by aubsequent centrifugation through 53 and 70% of L-[4,5-3H]leucine per ml (58 Ci/mmol; Radiochem- sucrose bilayers. Fractions obtained were treated with ical Centre, Amersham, England) was added to the trichloroacetic acid and analyzed by gel electrophoresample. After 3 min of incubation at 30°C, L-leucine sis. Polyacrylamide gel electrophoresis and fluo(200 tg/ml) and L-isoleucine (50 ug/ml) were added, and incubation was continued for an additional 2 min. rography. Dodecyl sulfate-polyacrylamide gel elecThe labeled cells were mixed with an equal volume of trophoresis was carried out using 12% polyacrylamide

VOL. 138, 1979

E. COLI TEMPERATURE-SENSITIVE MEMBRANE MUTANT

307

slab gel (140 by 180 mm) in the Laemmli discontinuous examined. It was found that divE42 is located buffer system (15) unless otherwise indicated. Electro- distal to suA from pyrD (Table 2, experiment phoresis was performed at 20 or 30 mA, and gels were 4). Finally, the results of a transduction experistained and destained as described by Sato et al. (29). ment using CSH59 (pyrC; 21) as recipient and Standard proteins used to estimate molecular weights KY2515 (divE42) as donor suggest that the frewere described previously (29); their molecular weights are indicated on Fig. 2 and 4 (left). Fluorog- quency of cotransduction of divE42 with pyrC raphy was performed according to Bonner and Laskey may be about 0.5% (data not shown). Figure 1 (2) as modified by Laskey and Miles (16) by exposing summarizes the mapping data presented above. Dominance test. To determine if divE42 is Fuji X-ray films to the dried gels for appropriate periods. recessive to the wild-type allele (divE+), we first Enzyme assays. Activities of succinate dehydro- isolated an F' plasmid carrying the pyrD region genase, L-a-glycerophosphate dehydrogenase, and the of the E. coli chromosome following the method L-a-glycerophosphate uptake system as well as total of Low (18). Cells of KL181 (F- pyrD trp his proteins were assayed as described previously (25). recA rpsL) and KL208 (Hfr) were mated, and Transport of,-galactoside into cells was measured Pyr+ Trp+ Strr (streptomycin-resistant) recomaccording to Ohki and Sato (26). binants were selected (see Fig. 1). They were purified on nutrient agar not supplemented with RESULTS and screened for the clones that were Mapping ofthe divE42 mutation. To locate uracil to UV light (Rec-) and capable of sensitive the divE42 mutation on the E. coli chromosome, transfering serC+ and trp+ into another F-

Hfr strain carrying the divE42 mutation was constructed and used for mating with an Fstrain carrying serC (or pyrC tip). Examination of Ser+ (or Pyr+ Trp+) recombinants for unselected markers suggested that divE42 is located in the serC-pyrC region (data not shown). Determination of 8-galactosidase induction at 420C with 50 Pyr+ recombinants showed a strict correlation between inability to induce ,B-galactosidase at 420C and the temperature sensitivity of growth. Further mapping of divE42 was carried out by a series of P1-mediated transduction experiments. First, cells of KY2329 (serC divE42) were infected with phage P1 grown on KY2304 (pyrD), and Ser+ transductants obtained were examined for distribution of unselected markers. The gene order serC-pyrD-divE was suggested from this experiment, and the frequency of cotransduction for divE42 and serC was about 4% (Table 2, experiment 1). We then tried to determine the location of divE42 relative to ompA (tolG), which is known to be closely linked to pyrD (5). Cells of KY2510 (pyrD ompA) were infected with phage P1 grown on KY2305 (divE42), and Pyr+ transductants were examined for unselected markers. The results indicate the gene orderpyrD-ompAdivE, the cotransduction frequency forpyrD and divE42 being about 22% (Table 2, experiment 2). The cross between KY2547 (pyrD divE42) and KY2514 (ompA) gave essentially the same results (Table 2, experiment 3), though the cotransduction frequency for divE42 and pyrD was somewhat lower (9%), possibly due to the difference in the strains used. To map the location of divE42 relative to sulA, cells of KY2530 (pyrD sulA Ion) were infected with P1 grown on KY2515 (divE42), and Pyr+ transductants were an

TABLE 2. Transductional mapping of the divE42 mutation' No. of

Expt

tion

1

Ser+

2

Pyr+

3

Pyr+

ompA

divE42

-

+ +

+ +

-

-

-

ompA

divE42 -

+

_ +

4

Pyr+

Fre-

Unselected markers transduc- quency (%) tants pyrD divE42 + 157 91 9 5 _ _ + 5 3 + + 1 1

+ +

36 25 15 2

46 32 19 3

67 22 9 0

68 22 9

Genetic studies of an Escherichia coli K-12 temperature-sensitive mutant defective in membrane protein synthesis.

Vol. 138, No. 2 JOURNAL OF BACTERIOLOGY, May 1979, p. 305-313 0021-9193/79/05-0305/09$02.00/0 Genetic Studies of an Escherichia coli K-12 Temperatur...
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