548 SHORT COMMUNICATI( )N>

Weise von n iedermolekularen Su l fhydry lve rb indungen oder Mikrosomenpro te inen fiber SH-Gruppen gebunden werden, wobei wasserl6sliche Stoffwechselprodukte ent- s tehen k6nnen. Die N a t u r der die Bindung eingehenden Oes t ronabk6mml inge ist noch unbekann t . Obwohl verschiedene Beobach tungen darauf h indeuten , dass sic im Verlauf der mikrosomalen 2 -Hydroxy l i e rung des Oestrons gebi ldet werdenT, 8 ist cs nach vorl iegenden Befunden und Ergebnissen von anderer Seite 9 wenig wahrschein- lich, dass es sich um o-Chinone handel t . Die exper imente l len Da ten sprechen eher ffir rad ika l i sche Zwischenprodukte .

Biochemisches Ins t i tu t des Deutschen Krebsforschungszentrums, Heidelberg, und Max-Planck-[ns t i tu t f i i r Biochemie, Miinehen (Deutschland)

ERICH HECKER GERNOT WALTER

FRIEDRICH MARKS

i H. BREUER, Vitamins Hormones, 20 (1962) 285. 2 G. RUMNEY, Federation Proc., 15 (1956) 343. 3 E. HECKER UND S. M. A. D. ZAYED, Z. Physiol. Chem., 325 (1961) 209. 4 E. HECKER UND F. MARKS, Z. Physiol. Chem., 34 ° (1965) 229. 5 C. LAZIER UND P. H. JELLINCK, Can. J. 13iochem. Physiol., 43 (1965) 281. 6 P. H. JELLINCK UND I. LUClEER, J. Endocrinol., 32 (1965) 9I. 7 F. MARKS, Dissertation, UniversitSt Mi~nchen, 1964. S.3If.f. 8 E. HECKER END F. MARKS, Biochem. Z., im Druck. 9 P. H. JELLINCK, Biochim. Biophys. Acta, 78 (I963) 778.

Eingegangen am 5. August , 1965

Biochim. Biophys. Acta, I 1 ~ (I965) 546-548

SC 23 Z72

Biosynthesis of thymidine diphosphate L-rhamnose in Escherichia coli K-12

In Ente robac te r iaceae , the m u t a t i o n which t ransforms smooth wild t ype in to rough s t ra ins results in a loss b y cell wall l ipopolysacchar ide of i ts "S-specific s ide- cha ins" . These s ide-chains are known to conta in f requent ly , in Escherichia coli as well as in Salmonel la , sugars such as mannose, rhamnose , fucose, and 3 ,6-dideoxyhexoses; the loss of these s ide-chains leaves a " r o u g h - t y p e " or "RI I core" l ipopolysacchar ide , conta in ing only glucose, galactose, a ldoheptose , 2-keto-3-deoxyoctonic acid, and glucosamine as monosacchar ide uni ts 1,~.

E. coli K - I 2 and all i ts subs t ra ins are rough organisms; t h e y show auto- agg lu t ina t ion in saline, and in none of t hem have any specific O-ant igens been de- t e c t ed 3. Al though s t ra in K - I 2 p re sumab ly der ives from a smooth organism, this parent s t ra in is not known.

The b iosynthes is of L-rhamnose involves two steps 4. F i r s t TDP-glucose is con- ve r ted into TDP-4-ke to-6-deoxyglucose ; then the l a t t e r is t r ans fo rmed into TDP- rhamnose. OKAZAKI et al.* r epor t ed t h a t a subs t ra in of I{-12, s t ra in Y-Io , had a metabol ic block in the second of these steps. I t is t e m p t i n g to specula te t ha t this defect might be the cause of the roughness of K - I 2 strains. I f rhamnose is an integral componen t of the S-specific s ide-chains of cell wall l ipopolysacchar ide in the hypo- the t ica l smooth pa ren t s t ra in of K- I2 , the inab i l i t y to synthesize TDP- rhamnose

Biochim. Biophys. Acta, i R l (i905) 548--551

SHORT COMMUNICATIONS 549

should lead to the almost complete loss of the side-chains, leaving the rough-type lipopolysaccharide*.

Several substrains of K-I2, however, were found to contain a small, but sig- nificant amount of L-rhamnose in their cell wall lipopolysaccharideS,L During the course of other experiments we found also that extracts of a few substrains of K- I2 could readily synthesize TDP-rhamnose. These results suggested that the block in strain Y- io might be due to a secondary mutation which occurred after the true "rough" mutation.

W 4 > W 7 o o 2 > H f r C - - + K I o ~ E I 5

(58) (58-161 )

W7ooo

(the original K-I2)

W 5 > WI2 > W7oo4-+ - - > - - - - - - ' - ~ C 6 o o - - - + W l I 7 7

(679) (679-680) (Y-Io)

Scheme I. The "genealogy" of K-I2 substrains. The older nomencla ture is shown in parenthesis. The reader is referred to the paper by J. LEDERBERG $ for the properties of most substrains.

In order to investigate this possibility, various substrains of K-I2 were system- atically examined; their derivation is shown in Scheme I. Table I shows the ability of extracts of these strains to catalyze the overall conversion : TDP-glucose -+ TDP- rhamnose. The first step of the conversion was also assayed in several cases, to as- certain tha t the defect was not in this step.

T A B L E I

ENZYMATIC SYNTHESIS OF T D P - R H A M N O S E BY VARIOUS SUBSTRAINS OF K - I 2

The cells were grown and extracts were prepared as previously described s. Activities were assayed according to OKAZAKI et al. 4.

Strain F-status Specific activity (mt~moles/mg protein per h)

TDP-glucose ~ TDP-glucose --+ TDP-rhamnose TDP-4-keto-6-deoxyglucose

W7ooo F + 36 820 W7oo2 F+ 39 630 K i o Hfr 29 n.d. E I 5 Hfr 31 n.d. ~r 5 F - 36 690 W I 2 F - < 2 74 ° W7oo 4 F - < 2 74 ° W I I 7 7 F - < 2 n.d.

n.d. s tands for not determined.

The results suggest that the defect in TDP-rhamnose biosynthesis was intro- duced accidentally when strain WI2 (requiring both threonine and leucine for growth) was isolated from W5 (requiring only threonine). I t is seen, however, that all the strains which could not synthesize TDP-rhamnose were F- , whereas most of the strains which could were either F + or Hfr. In order to see whether the ability to synthesize TDP-rhamnose is related to the presence of F-factor, the episomal factor

* In fact, a m u t a n t of Salmonella typhimurium was recently found which synthesizes rough- type lipopolysaccharide because of defective synthesis of TDP-rhamnose 6.

Biochim. Biophys. Acta, x i i (1965) 548-551

550

T A B L E II

Q U A N T I T A T I V E A N A L Y S I S ()F' ( I~:LI. VVALL I_IPOPOI.YSAC( 'H: \RIDI '2

S u g a r s were d e t e r m i n e d as d e s c r i b e d l n ' c v i o u s l y li.

(e: l wall lipopolysacckaride % q/: lipopolysaccharides Molar rail . D'om s/rain

Hep/osc He.roses Rhamm~se Heptose

SHORT ( ( ) M M U N I( ' .VII I )N >

He,roses [~h~lt}lll(ISt'

\~YTO00 I 3" 5 I 5 ' 2 0 "37 2 . 0 0 2 ( I 7 ~1,(I 7

W 7 o o 2 I : 7 14.7 o. 74 2.oo 2.7 ' , ,. t 5 W 7 o o 4 13.2 17. 4 - 0.0 5 2.oo 3.o7 • o.(ll \V 1 2 I 5..~; i I),O O.O 5 2 .O0 2 "4~ ().0 I

was introduced from \\'7oo2 into WI2 by mixed culture; extracts of two resulting F + derivatives of WI2 still failed to convert TDP-glucose into TDP-rhamnose. In a complementary experiment, the F-factor was eliminated from W7oo2 by acridine treatmentg; extracts of two F - derivatives of W7oo2 could synthesize TDP-rhamnose at the normal rate. Thus, the presence of the F-factor is not related to the host 's ability to synthesize L-rhamnose.

Cell wall lipopolysaccharide preparations were isolated from some of the strains by the hot phenol procedure of \ V E s T P H A L , Lt)DERITZ A N D B I S T E R 10, purified by repeated ultracentrifugations, and analyzed by colorimetric reactions. Tile results given in Table I I show clearly that the strains which can make TDP-rhanmose, in- corporate small but significant amounts of rhamnose into lipopolysaccharides, whereas those which cannot have no detectable rhamnose in their lipopolysaecharides. Acid hydrolysates of lipopolysaccharides from these strains were examined by thin-layer and paper chromatography. No rhamnose could be detected in lipopolysaecharides from W7oo 4 and \VI2, whereas it was clearly observed in lipopolysaecharides from \V7ooo and W7oo2. In other respects, all the cell wall lipopolysaecharide preparations examined showed a sugar composition typical of rough-type lipopolysaccharides; heptose, glucose, galactose, glucosamine, 2-keto-3-deoxyoctonic acid, and small amounts of an unidentified, fast-running component were detected chromatographi- cally.

In conclusion, the defect in rhamnose synthesis was only found in the K-I2 suhstrains which derived from strain \VI2*. All K-I2 substrains, however, including those able to make rhamnose are rough strains and synthesize essentially rough-type lipopolysaccharides, presumably owing to yet uncharacterized defect(s) in the later stages of synthesis of the S-specific side-chain.

Cell wall lipopolysaccharides from the TDP-rhamnose-synthesizing strains con- tain small amounts of rhamnose. Although the classical rough or RII mutants (of Salmonella) synthesize the Rn core lipopolysaecharide 1~ which does not contain rhamnose, it should be noted that lipopolysaccharide from some of the more or less "rough" mutants were recently found to contain small amounts of additional sugars. Two different classes have been described among such mutants : (I) the class D

* A f t e r t h i s w o r k w a s c o m p l e t e d , wc n o t e d a n a b s t r a c t b y HIROTA, SUG1MOTO AND ()KAZAK111 ,

in w h i c h i t is r e p o r t e d t h a t " g e n e s d e t e r m i n i n g T D P - r h a m n o s e b i o s y n t h e s i s a r e l o c a t e d close t~ t h e his loci in s t r a i n K - 1 2 " . I t is p o s s i b l e t h a t t h e s e a u t h o r s a l so m a d e o b s e r v a t i o n s s i m i l a r to ours .

Bi'ochim. B iopkys . . 4 c/a, I [ I (1905) 548--551

SHORT COMMUNICATIONS 551

mutants, which synthesize lipopolysaccharides with few but long S-specific side-chains attached to the RII core n; (2) the class C mutants, whose lipopolysaccharides contain many but uniformly short side-chains n. In addition, evidence suggests the presence of a third class of mutants whose RII core carries few and short side-chains; a Salmo- nella typhimurium mutant unable to synthesize GDP-mannose was reported to contain a small amount of rhamnose in its lipopolysaccharide in and may belong to this class. It is possible that K-I2 belongs to a class similar to one of these, most likely to the last one; but more study will be necessary to establish the structure of the lipo- polysaccharides of K-I2 substrains.

This work was supported by grants from U.S. Public Health Service. We are indebted to Drs. E. M. LEDERBERG and E. C. C. LIN for the gift of the strains. The excellent technical assistance of Mr. K. NAKANE is gratefully acknowledged.

Biochemical Research Laboratory, Massachusetts General Hospital and Harvard Medical School, Boston, Mass. (U.S.A.)

H . NIKAIDO

K. NIKAIDO A. M. C. RAPIN

I F. KAUFFMANN, L. KR/JGER, O. LUDERITZ AND O. WESTPHAL, Zentr. Bakteriol. Parasitenk., Abt. I Orig., 182 (I96I) 57.

2 E. C. HEATH AND M. A. GHALAMBOR, Biochem. Biophys. Res. Commun., IO (1963) 34 o. 3 F. ORSKOV AND I. Q~RSKOV, Acta Pathol. Microbiol. Scand., 55 (1962) 99. 4 R. OKAZAKI, T. OKAZAKI, J. L. STROMINGER AND A. M. MICHELSON, J. Biol. Chem., 237 (I962)

3o14. 5 H. NIKAIDO, K. NIKAIDO, T. V. SUBBAIAH AND B. A. D. STOCKER, Nature, 2Ol (1964) i3oi . 6 T. A. SUNDARARAJAN, A. M. C. RAPIN AND H. M. KALCKAR, Proc. Natl. Acad. Sci. U.S., 48

(1962) 2187. 7 A. M. C. RAPIN AND H. MAYER, Federation Proc., 24 (1965) 479- 8 J. LEDERBERG, Genetics, 32 (I947) 505 . 9 Y. HIROTA, Proc. Natl. Acad. Sci. U.S., 46 (196o) 57.

lO O. WESTPHAL, O. L/3DERITZ AND F. BISTER, Z. Naturforsch., 7b (1952) 148. i i Y. NAIDE, H. NIKAIDO, P. H. MAKELA, R. G. WILKINSON AND B. A. D. STOCKER, Proc. Natl.

Acad. Sci. U.S., 53 (1965) 147. 12 I. BECKMANN, O. L/2DERITZ AND O. WESTPHAL, Biochem. Z., 339 (1964) 4 ol- 13 M. J. OSBORN, S. M. ROSEN, L. ROTHFIELD, L. D. ZELEZNICK AND B. L. HORECKER, Science,

145 (1964) 783 • 14 Y. HIROTA, K. SUGIMOTO AND R. OKAZAKI, Japan. J. Genetics, 39 (1964) 344.

Received August 9th, 1965 Biochim. Biophys. Acta, z z z (1965)548-551

sc 23176

A modified Sakaguchi spray

For several years the details of a modified Sakaguchi spray as developed by the present author has been quoted in the literature as forthcoming 1-4. This note describes the procedure.

Stock reagents: (I) o.o125 % a-naphthol in absolute ethanol. This solution is stable for a week when kept cold in a brown bottle. (2) 1. 5 N NaOC1. A commercial household bleaching agent such as "Chlorox" was found to be a good and ready source of hypochlorite for this work. Several bottles of "Chlorox" bought at random showed

Biochim. Biophys. Acta, i I i (i965) 551-552