@ INSTITUTPas'IEI;~/EI Sl v'I:R

Res. Microbiot. 1992, 143, 743 753

Paris 1992

Characterization of high molecular weights of complexes and polymers of cytoplasmic proteins in Escherichia coil R. Imamura (o, H. Niki o), M. Kitaoka (z~, K, Yamanaka tt~, T. Ogura ~L~ and S. Hiraga '='-.r

-.,l~--plO0-A

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p77

66

p52 49_.

:/ i:~ :::;

30 i £2 -~. • 2

Fig. 2. Proteins in fractions eluted through a DEAE-Sephacel column. Fraction I (fig. 1B) was loaded onto a Dl~AE-Sephacelcolumn. Fractions eluted with 0.1 M and 0.5 M NaCIwere analysed in SDS-PAGE(fig. 1A). Proteins in the gel were stained with Coomassie brilliant blue. Lane 1: the sample eluted with 0.1 M NaCI. Lane 2: the sample eluted with 0.5 M NaC1.

cycles of polymerization and depo!ymerization according to Tomioka (!~1). The resulting sample was compared with the sample containing p100-1~, p77 and p52 in SDS-PAGE. The 3 major proteins in the former showed the same mobilities as pl00-B, p77 and p52 in the latter, respectively (data not shown). The results support the above speculation.

Electron microscopic complexes

observation

of HMW

The sample prepared by repeated cycles of polymerization and depolymerization was kept in the presence of 100 mM KC1 and observed under an electron microscope. Numerous long fila-

ments of 6 t~m in diameter and sheets were observcd as described by Tomioka (1991) (data not s~.owr,). However, we could not observe such long filaments and sheets in the DEAE sample containing pl00-B, p77 and p52 even in the presence of 100 mM KCI. In fraction I and in the DEAE sample eluted with 0.5 M NaC1, small vesicles, dense rods (approximately 50 nm in length), and unusual vesicles having a dense rod or patch on the surface were observed (,fig. 4C, D, E and F). Similar unusual vesicles with a dense patch have been described by tshidate et aL (1986). We found a large number of spiral filamentous particles (50-300 nm in length) in the pl00-A sample eluted with O.i M NaC{ from the

R. IMAMURA E T AL.

748

A kDa

M

]

200 116

2

3

•

. . ~ . -

4 .

, ~

66

pl00-A, B p77 p52

42

30 ~

....

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17 --..1~

B kD,a

1

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5

200 --..!1-116 ~

pl00-B

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Fi X. 3. lmmunoblo~'~i~.: e f pr2teins~ A) Samples were electmphoresed in SDS-PAGE [llJ %), and then slained with Coomassie brilliant blue. B) Proteins in a gel were analysed by the immunoluminescence method using the antiserum a~ainst the 100K protein described by Tomioka (1991). Lane M : molecular markers, Lane 1 : the sample eluted with 0.1 M NaCI from the DEAE-Sephacel column. Lane 2; the sample eluted with 0.5 M NaCI from the column. Lane 3: |he cell lysate of tbe JE2217 s!rain. Lane 4 : the ceil lysatc of the CY377 strain. Lane 5 : the cell lysate of the SHH31 $tlail~.

POLYMERS OF E. COLI C Y T O P L A S ~ i C PROTEINS

DEAE column (fig. 4A a r d B). The spiral filaments were morphologically very similar to spirosomes, which had beer. f ~ n d in various species of Gram-positive and -negad-~e bacteria including F... coil (Kawata et al.. 1976; Kawata e t a L , 1979; Kawata et aL, 1975; Kessel etal., 1981; Matayoshi and Oda, 1985; Ueda and Takagi, 1972; Oeki e t a l . , 1982).

Determination of N.terminal sequences of these protein.,

amino

acid

We determined the amino acid sequences of the N-terminal r,ortions o f p 100-B and p52 and searched for homology in the Swiss-Prot protein sequence data base. As shown in figure 5, thc N-terminal sequence of pl00-B is identical with the N-terminal portion of pyruvate dehydrogenase, i.e, the polypeptide (99.6 kDa) encoded by the aceE gone (Stephens et aL, 1983a) located at., rain on the E. coil chromosome. The N-terminal sequence of p52 is identical with that o f lipoamide dehydrogenase, i.e. the polypeptide (50.6 kDa) encoded by the Ipd gone (Stvpnens et al., 1983c) located at 3 min. We did not succeed in determining the N-terminal sequence o f p77. The N-terminal sequence of pl00-A is identical to that of alcohol dehydrogenase, i.e. the polypeptide (95.9 kDa) encoded by the adhE gene (Goodlove el al., 1989), which is located at 27 min. Alcohol dehydrogenase thus forms spirosomes. As shown in figure 3B (lanes 3 to 5), a 100-kDa protein which cross-reacted with the anti-i~%)K ar~lisetunt ,,vas detecteo in cell lysates o f strain JE2217 and the a a d h E mutant (strain SHH31), but not detected in the cell lysate of the AaceEF mutant (strain CY377), supporting the above conclusion. As can be seen in figure 3A, protein bands at 100 and 77 kDa were strongly stained with Coomassie brilliant blue in cell lysates o f strain JE2217 and the AadhE mutant, but these bands were very faint in the AaceEF mutant. This suggests that these b: nds correspond to pyruvate dehydrogenase (the aceE gone product) and dihydrolipoamide acyltransferase (the aceF gone product}, respectively.

749

Property o|" the aceE or adhE deletion mutant

We observed :~rowing cells of the AaceEF or AadhE mutant by the fluorescence and phasecorgrast combineti method after the DAPI staining. These mutants showed normal morphology in L medium and in minimal salt medium (medium E) containing 0.5 % glucose and 0.2 % Casamino acids. Anucleate cells (chromosomeless cells) :ver,~ not observed in the cultures (data not shown}. These results indicated that pyruvale dehydrogenase and alcohol dehydrogenase are not essential at least for celt morphology, cell division, and chromosome partition. DISCUSSION

Individual chromosomes are fixed ia position within the celt both before and during replicalion, but they are rapidly moved from midceli to cell quarter positions immediately after replication has been completed (Begg and Donachie, 1991 ; Donachie and Begg, 1989; Hiraga et al., 1990; for a review, see Hiraga, 1996). This suggests that E. coil cells have an apparatus for chromosome positioning, which acts as the spindle apparatus of chromosome segregation in eukaryotic ceils. We reported previously that the MukB protein of 177 kDa is involved in correct partitioning of the E. coil chromosome (Niki etaL, 1991). The predicted secoadary structure of MukB suggests that the protein is the first candidate in eubacteria for a force-generating enzyme which moves daughter chromosomes from midcell to cell quarter positions, and the N-terminal dnmain ;s expected to act as a " m o t o r " domain (Niki etaL, 1991 ; for reviews, see Hiragaetal.. 1991 ; Hiraga, 1992). The amino acid sequence of the N-terminal domain of MukB is significantly homologous to dynamin (DI00) of rat brain, which is a microtubule-associated mechanochcmical enzyme (Niki et al., !991; Ober etal., 1990}. Our recent results (unpublished data) on the purified MukB protein indicate that it forms a homodimer with a rodand-hinge structure having a pair of C-terminal globular domains at one end and a pair of Nterminal globular domains at the opposite end :

R. I M , ¢ M U R A E T AL .

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Fig. 4, Electron micrographs of HMW complexes fraetiona~,e~ From the cell extract. A and B : spirosome-like particles observed in the pl00-A sample eluted with 0.1 M NaCI through a DEAE-Sephacel column; C, D. E, and F: fraction ! shown in figure lB. These figures are in same scale. Black arrow heads show dense rods. Black arrows shaw unusual vesicles wi~h a dense rod or patch. White arrows show spirosome-like particles. Solid bars represent 1130 nm.

/

POL YMERS OF E. COLI CYTOPLASMIC PROTEINS

751

plOO-B AceE

MSERFPNDVDF!ETRDWLQAIESVIREEGVERAQYL

....

-Oyruva~ dehydrogenase ~9,614 Dzl p52

STEI ~ T W ~ m ~ A G P A G X S A

Lpd

aTE IKTQVVVLGAC? AG YSA/~FRCA DL6LETVI VZ ....

Llpoamlde dehydrogermse [5C.557 Da] pl00-A AdhE

Av'gttv~v~ AVTNVAE LNA[,VERVKKAQ~Y ;%SFT~EQVD K I CR ....

Alcohol d e h y d ~ e n a a e

(05,995 Da)

I:i~, $, The N-terminal amino acid sequences of pl00-A, pl00-B and p52. Proteins ha~ing homology with those are also shown (see text). X represents an unidentified amino acid residue,

it tends to bend at a middle hinge site of the rod section, and moreover possesses DNA-binding and ATP/GTP-binding activities. These results are consistent witlt the above idea of the MukB function. This gives impetus to the search for cytoplasmic filaments o f protein polymers which play a role, cooperatively with MukB, in correct chromosome partitioning. T o search for unknown E. coil proteins which form cytoplasmic polymers relating '~o chromosome positioning and/or other importam cellular mechanisms, we have analysed proteins in the HMW complexes which, in this work, were fractionated by gel filtration. We show here evidence that pl00-B and p52 are identical to pyruvate dehydrogenase and lipoaraide dehydrogenase, respectively, and that pl00-B is identical with Tomioka's 100K protein. Tomioka (per3onal communication) recently determined the N-terminal amiao acid sequence of his 100K protein and co~firrned our conclusion. It is known that the pyruvate dehydrogenase and 2-oxogiutarate dehydrogenase multienzyme complexes catalyse homologous reactions. Each complex comprises 3 components: a specific del,ydrogenase, a dihydro-

tipoamide acyltransferase and a hpoamide dehydrogenase (for a review, see Nimmo, 1987). AhhGugh we did not succeed in determining the N-terminal sequence of p77, our results in the A6t¢ruEFir|titalil. (Fig. 3A) ~uggc3t tha: p77, ,:'h]:h is co-purified with pl00-B and p52, is identical with dihydrolipoamide acyltransferase, i.e. the polypeptide (65.9 kDa) encoded by the aceFgene (Stephens etal., 1983b) located at 3 min. Protein p77 migrates in a broad band or a few hands ~r, aDS-PAGE. Discrepancy between the molecular weight and mobility can be explained by the anomalous property of dihydrolipoamlde aeyltransferase in electrophoresis. We could observe neither long filaments (6 nm in diameter} nor sheets in the DEAE s~anple containing pl00-B, p77 and p52 even in the presence of 100 mM KCI, being inconsistent with tile conclusion described by Tomioka (1991). In the ',,3EAE sampl," and in fraction I containing pl00-B, p77 and p52, numerous dense rods of about 50 nm in leng',h were observed. It is not clear yet whether the dense rods relate to HMW complexes of these 3 proteins. We also show evidence that pl00-A is identical with alcohol dehydrogenase and forms fine

752

R. IMAMURA ET AL.

spiral particles. Kawata (personal communicalion) recently determined the Noterminal sequences of spirosins prepared from E. coil and Yersinia enterocolitica. We recently found that the N-terminal sequence of the E. coil spirosin determined by Kawata and his collaborators is identical to that of alcohol dehydrogenase. It has been described that pyruvate-formate-lyasedeactivase and acetyl-CoA reductase activities o f E. coil reside on a polymeric protein particle encoded by the adhE gene and that the protein polymers show spirosome-like structures (Kessler el al., 1991). Both the enzyme activity of alcohol dehydrogenase and the amount of spirosome are higher under anaerobic conditions than under aerobic conditions (Clark and Cronan, 1980; Matayoshi e¢ al., 1989). Our results in the AaceEFand AadhE mutants suggest that neither pyruvate dehydrogenase nor alcohol dehydrogenase is essential for cell morphology, cell division and chromosome pm'tition. We have prepared a rabbit antiserum, A - I , cross-reacting with a 51-kDa protein o r E . coil The 51-kDa protein was distributed broadly from slowly sedimenting fractions through fast sedimenting fractions near the bottom of the tube in a sucrose gradient centrifugation in the presence of i00 mM KCI (B. Ezaki, our unpublished data). This suggests that the 51-kDa prorein f o r m s v a r i o u s m o l e c u l a r weight homopolymers or complexes with other proteins. As can be seen in figure 1B, a 51-kDa protein, named p51, existed in the fraction II, but not in the fraction I. Protein p51 strongly crossreacted with this antiserum (our unpublished dma). When 100 mM KCI was added to the fraction I1, filaments and sheets were effectively formed, though proteins which form the filaments and sheets are not yet elucidated. Okada et al. (M. Wachi, personal communication) recently observed, by electron micrography, bundles of cytoplasmic filaments in uttrasections of ceils harbour'~ng a high copy number plasmid carrying the orfF gene. The orfF gene is located in the order of mreB-mreCmreD-orfE~orfF on the E. coil chromosome. The apv=rent motecuiar weight of the orfF gene product is 51,000. We therefore tested whether the orfFgene product is identical with p5 i. The

O r f f~ protein migrated more slowly than p51 in SDS-PAGE. The OrfF protein did not react with the antiserum A-I (our unpublished data). The OrfF protein is thus not identical with p51. Although we identified only the major proteins existing in the fractions of HMW complexes in this work, many other proteins in these fractions (see fig. I B) remain for further study. Acknowledgements We are grateful to Dr. ShigeoT0mioka for the antiserum against the 10OK protein, to Drs. Shizuko Iyobe, A. Nashimura, M.R. Leonardo and M. Wa::hifor the bacterial strains, and to Dr. Sumio Tanase for delermination of the amino acid sequence. We thank Chiyomelchinose, Akiko Matsusaka and Tomokc Tsunenari for assistance. This work was supported by a Grant-in-Aid ['orScienlific Research on Priority Areas and a Grant-in-Aid for Scientific Research B from the Ministry of Education, Science and Culture of Japan.

Caractdrisation des hauls poids mol~enlaires des complexes et polym~res des prot~ines cytoplasmiques de Escherichia ¢oli En rue de l'isolement de polym~res filamenteux de prot6ine, cytoplasmiques de Escherichla coil, des hauts poids mol6culaires (> 670 kDa) de complexes prot~iniques d'extraits cellulaires ont 6t6 d~finis par fractionnement (filtration sur gel ou ehromztographie sur colonnes 6changeuses d'ions). Des prot~ines de 100, 77 et 52 kDa ont el6 co-purifi~es. Les prot~ines de i00 et 52 kDa ont ~te identifi6es eomme etant respectivement la pyruvate-d~shydrog~nase et Is lipoamide-d~shydrog~nase, gr.~ce fi la d6termination des s6quences N-terminates. La proteine de 77 kDa correspond ~ ia dihydrolipoamidetransf6rase. La prot~ine de 100 kDa s'av~re identique A ia 100K d~erite pat" Tomioka, en rapport avec la formation de structures filarnenteuses et membranaires en presence de KCI ~ (00 raM. Mais fi l'encontre des conclusions de Tomioka, nous n'avons pus observ6 de telLes structures duns nos 6chantillons contenant ces enzymes. Une autre prot6ine de 100 kDa formant de longues particutes qui ont l'aspect de spirosomes a ~t6 purifi~e: scion les s6quences N:erminates, cUe correspond /~ t'alcool-d~shydrog6nase.

Mots-clds: Cytoplasme, Prot6ine, Spirosome, Filament; Escheriehia coil, Pyruvate-d~shydrogdnase, tipoamide-d(~shydrogenase, Dihydrolipoamide-acyltrans f~rase, Alcool-d~shydrogtnase.

P O L Y M E R S O F E. COLI C Y T O P L A S M I C P R O T E I N S

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753

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