~) INSTITUTPAS'rEtm/EIsEvJE~ Paris 1991
Res, Mlicrobiol. 1991, 142, 295-299
Alkaline Phosphatase Fusions in the Study of Cell Division Genes J . J . Barondess "1(2), M . C a r s o n (t), L . M . G u z m a n V e r d u z c o TM a n d J. Beckwith ()) ¢ODepartment o f Microbiology and Molecular Genetics and ~'~Program in Cell and Developmental Biology. Harvard Medical School, 200 Longwood, Ave., Boston, M A 02115
SUMMARY Alkaline phosphatase fusions have been used to analyse pla~mid- or phagecarried genes from the two-minute region of the EscherichM coll chromosome. Those studies have revealed the following: 1) Bacteriophage k carries two genes for cell envelope proteins, tom and bor, that are expressed in lysogens and probably contribute to the pathogenicity of its E. coil host. 2) The ftsQ and ftsl gone products are integral proteins of the cytoplasmic membrane with small cytoplasmic domains and large pariplasmi¢ domains. 3) The ftsO and ftsl gsne products are made in very small amounts, on the order of 25 molecules per cell. 4) The ftsQ gone product is essential for cell growth and is required throughout the formation of the cell septum. 5) An open reading frame just upstream from ttsl, thought to be invoired In cell divlsion, is expressed and probably codes for a cytoplasmic membrane protein. Key-words : Cell division, Alkaline phosphatase, Gene, Fusion; Serum resistance, Membrane proteins.
Major aspects of the process o f bacterial cell division are the regulation and orientation of the growth of the cell envelope. In Escberichie coli, the cell envelope is composed o f the cytoplasmic membrane, the periplasmic space, the outer membrane and the peptidoglycan layer. It seems quite likely that the c~oplasmic membrane plays an important role in cell division. Signals may be passed throu3h the membrane in either direction and some enzymes involved in the formation of the cell septum may be localized to this membrane. We are interested in analysing those proteins required for cell division that are found in the cytoplasmic membrane. From such studies,
we would hope to determine (1) •~,hether any of these proteins are localized to the cell septum itself, (2) the rote of these proteins, (3) whether any of these proteins interact with each other and (4) whether any ff)rm of transmembrane signalling takes place in the cell division process. Alkaline phospnatase fusions provide a tool for identifying and analyzing proteins localized to the cell envelope of E. cob ~Manoil et al., 1990). We have developed a transposon probe, T n p h o 4 , which allows the detection of genes located on plasmids or on bacterial chromosomes that code for pzotcins which contain export signals (Manoi! and Beckwith, 1985). These include
proteins of the two membranes and of the peripla~mic space. Since alkaline phosphatase is only enzymaticaliy active when it is t r a n s l o c a t e d across the cytopla.~mie membrane, fusions of this protein only exhibit enzymatic activity when the protein to which they are fused allows ihe export of alkaliae phosphatase. The alkaline phosphatase fusion approach also allows the analysis of the topology of cytoplasmic membrane proteins (Manoil and Beckwith, 1986). '~he n alkaline phosphatasc is fused to a periplasmic domain of a membrane protein, the h:,brid protein is enzymatica!ly active; when it is fused to a cytoplasmic domain, it has very
296
J.J. BARONDESS E T AL.
little activity. In Ihls way, the exlelual and interaal dmnalus of a Cylol~lasmic inenthrflne protein can be delcrmiued, thus allowing Ihe formulalion of topological models. We have used alkaline phosphatase fusions to analyse the cluster of genes involved in cell division and cell envelope biogenesis located at nlinnte two OU Ihe E. coil chromosome. In this paper, we summarize the results obtained so far, including some surprising findings on bacteriophage 3. happened upon during Ihese studies. We also show hnw Ihe fusion approach helps identify hitherto undetected genes in this region.
RESULTS TnphoA analysis o[ X 16-25 We began our studies with a 3. transducing phage isolated by Lmkenhaus el aL (1980) which carries the ftsQ, ftsA, and f:sZ genes (fig. 1). Insertions of the Iransposon TnphoA o n l o the phage genom¢ were obtained using the kanamyein-resistance marker for selections. Those insertions in which lysogens of the phage exhibited a blue color on alkaline phosphatase indicator (XP) plates were sludied further. Out of a collection of several dozen phages carrying active fusions~ a number were restriction-mapped and fusion joints were sequenced. Surprisingly, the highest proportion of fusions exhibiting alkaline phosphatase activity were locafized to genes in the X phage itself. This is surprising, because it has g~aerally been assumed that the only genes expressed during 3. lysogeny are cytoplasmically localized proteins mapping to the immunity onl--opcn rcadlns flame.
region of the phage, Restriction mapping and sequencing of these fusions localized them to two sites on the prophage (fig. 1). Some of the fusions mapped to the lore gone, a previously discovered gone which codes for an outer membrane protein in minicell infection experiments, but not k n o w n to be expressed during lysogeny (Reeve and Shaw, 1979). The remaining fusions mapping in X were to an open reading frame we have named her (for blue open rending frame). No previous work had indicated a product or expression of this open reading frame. The finding of active alkaline phosphatase fusions to these two proteins suggested that both proteins were located in the cell envelope. However~ as we have pointed out elsewhere, it is important to determine the specific activity of the fusions (enzymatic activity related to alkaline phosphatase cross-reacting material) to be sure that the blue colour seen represents efficient export (San Millan etaL, 1989), Both classes of fusions exhibited specific activities close to that of wildtype alkaline phosphatase. These activities can also be used 1o estimate the level of expression of
the two genes, The results suggest that Bor is made in 2,000 molecules per cell and Lore at 100 molecules per cell. We believe that both Bor and Lore are proteins that may contribute to the pathogenic properties of E. colt (Barondess and Beckwith, 1990). Others had shown that the D N A sequence of the her ORF was 93% identical to the gone iss, located on colicin V plasmids (Chuba e t a L , 1989), The iss gone product is known to contribute to bacterial virulence by conferring serum resistance on hOStS that carry the gene. We have found that X lysogens expressing bor are 20-fold more resistant to serum complement killing in vitro than non-lysogens or lysogens in which bor is not expressed. We have no direct evidence for pathogenic properties of Lore, but it shares extensive sequence homology with proteins of two other pathogenic bacteria that are involved in entry or survival within mammalian cells. Th--se results extend previous findings of Mekalanos and coworkers on the utility of T n p h o A for detecting genes involved in bac:eria! pathogenesis (Manoil et ,'1., 1990). They
3. 16-25
/am d~ ~ t~A f~ZenvA~ N
in~t
bet
~nS
Fig. l. Map of X 16-25. The structure of this phage is described in Lutkenhaus et aL (1980l.
ALKALINE PHOSPHATASE FUSIONS FOR CELL DIVISION GENES also suggest that lysogenie phages, in general, may carry hitherto undetected genes contributing to the virulence of their host bacteria, Previous examples of lysogenic phages contributing to pathogenicity include phage ~ o f Corynebacterium diphtheriae which codes for diphteria toxin, It is notable that there are still significant portions of the h genome, functions for which have not been established. The possibility should be considered that there are still other ). genes involved in the pathogenicity of its host, E. coil. T n p h o A analysis of the f l s l region We have isolated a series o f T n p h o A insertions in a piasmid which ca.ries a 7.5-kb fragment including the f t s l gene. Seven fusions in the f l s l gene all had alkaline phosphatase fused to points beyond the hydrophohic stretch f o u n d early in the sequence (fig. 2). Our results are consistent with earlier finding5 (Bowler and Spratt, 1989) that Ftsl is a cytoplasmic membrane protein with a small cytoplasmic domain and a large peripl~snaic domain. One advantage of working with these fusions is that we can use antibody to alkaline p h o s p h a t a s e to e x a m i n e the behaviour of hybrid proteins and d ~ u c ¢ J~forrn.at.~n about the native protein. We have asked whether the early hydrophobic sequence m i g h t represent a cleaved s i g n a l sequence by examining the behaviour of a hybrid Ftsl-UhoA protein in a mutant defective in secretion, seeAfs. In this secAts mutant background, e,~,.ry other protein with a cleavable signal sequence tested has been blocked in the processing of the signal sequence at high temperature. (The small phage fl coat protein is the sole exception). We find that these Ftsl-PhoA hybrid proteins are not altered in mobility on SDS
gels when secretion is defective, s u g g e s t i n g that the early hydrophobic stretch is a m e m b r a n e - s p a n n i n g segment rather than a cleavable signal sequence. On a multicopy pie,staid, the a l ka l i ne p h o s p h a t a s e units produced by a n f t s l - p h o A fusion indicate that it is making about 500 copies of the prot e i n. A s s u m i n g a copy n u m b e r between 20 and 50, we can estimate that the amount of Fts[ in single copy may be from 10 to 25 molecules. These calculations should be considered with some caution umill measurements are made on single-copy versions of the fusions. We have also found alkaline phosphatase fusions to other open reading frames on t~e JIM plasmid (fig. 2). One of these is located downstream from f t s l in a region which has been sequenced, but in which the function of not all the ORF is known (lkcda et aL. 1990). At this time, the most interesting fusions to us are three located in the region of an open-reading frame just upstream from ftsL This is a region which inelucies a gene complementing a new c;ass of ceil division mutants (lshino
297
et al., 1989). We have sequenced two of these fusions and find that they have alkaline phosphatase fl~sed to the only open reading frame of significant length found in this fragment preceding flsL The location of the active fusion joints and the sequence of this ORF suggest that is codes for a cytoplasmic membrane protein with p e r i p l a s m i c and cytoplasmic domains of approximately equal size. The nature of this protein raises the possibility that it is involved in transmembrane signalling. Since it is the only O R F in the fragment that complements the fts36 mutation of Ishino et al. (1989), it seems likely that it corresponds to this gene involved in cell division, Fusions of aikaiine phosphntase to F b Q We have analysed the structure of the FtsQ protein and other aspects o f it5 function using al ka l i n e p h o s p h a t a s e fusions (Carson er al., 1991. in press). These fusions were obtained on a plasmid that carried the 2.4-kb HindlIl-Bglll fragment that inelude~ ftsQ, a portiGa of StsA and a small amoun: of material preceding
Aclive alkaline pho~phalasegene fusiul,s i,~ the remton o t Jlsl
ftsl
I
tllndnl
fff
FIT f ff
÷*"
.....
rff ....
I
EcoRt
Fig. 2. Position of active alkaline phosphat~e fusions to genes on an frI plasmid. Fusions were obtained and sequenced as described previo~iy (Carson et aL, in press).
298
.ft.sQ We have obtained a set of
J.J. BARONDESS ET AL.
active lusions in which the alkaline i~hosphatase is always fLtsed at a positinn ill the amino acid sequence past an early hydrophohic strelch of amino acids. As is the case for the F t s l - P h o A fusion prnteios, when we block secretion, s~e do not see alteration in tile mobility of FIsQPhoA fusion proteins in SDSPAGE. Thus, the hydrophobie stretch located early in the FtsQ protein (Yi el at., 1985) is also likely to he a m e m b r a n e spanning stretch rather than a cleavable signal sequence. Also consistent with a membrane location, we find that an FtsQ-PhoA fusion protein fractionates with the pellet in an osmotic shock fractionation (Manoil and Beckwith, 1986), under conditions which release other periplasmic proteins. Tltese results suggest that the FtsQ protein has a structure similar to that of Ftsl. That is, the protein is located in the cytoplasmic membrane, has a small cytoplasmic d~,,,tain and a large periplasmic domain. On this basis, the p er ip lasmic domain of FtsQ could be an enzyme, as is the case with Ftsl, or a peripla~mic structural or assembly component of the cell division apparatus.
wilh lhe hypothesis (Donachie et aL, 1984) that its product is required throughout the retinalion of tile cell septum (Donachie el aL, 1984). Strains with insertion mutation in the ftsQ gene complemented by a plasmid with the ftsQ gene under control of the ara promoter, form long non-septate filaments when the inducer is removed, indicating a defect early in the septation process. On the other hand, multiseptate filaments accumulate either when the ftsQ temperature-sensitive mutant is grown at semi-permissive temperatures or when the FtsQ protein is overexpressed a b o u t 200-fold from the ara promoter in a wild-type background. The chromosomal inserts of the TnphoA fusion also allow us to measure the level of expression of ftsQ and its regulation. The ftsQ.phoA fusions make approximately 3 units of alkaline phosphatase when located in the normal ftsQ chromosomal site. T h e specific activity of the hybrid proteins is again comparable to that of wild-type alkaline phosphatase. This activity corresponds to about 25 molecules of the hybrid protein, which suggests that is the level of ftsQ expression.
T)zphoA insertions also provide us with null mutations in the gone of iutel¢gt. Vqe have recomhioed certain of the frsQp/teA fusions onto the chromosome in a strain that carries a secono copy of ftsQ on a ;',transducing phage. At the same time, we have constructen plasmids in which the frsQ gene is under :he control of the tightlyregulated araB,A,D promoter. When the chromosomal TnphoA insertion is ir.troduced into a strain that carries ftsQ under ara control, the strains are dependent for tiaeir survival on the ar--~binose,-induced expression of ftsQ. Studies on the phenotype cf ce~;~ with different levels of ftsQ expression are consistent
We have initiated studies on cell division by obtaining fusions of alkaline phosphatase to protcios coded for in the minute 2 region of the E. colt chromosome. These studies have revealed a n,~mber of features of the genes in this region and an unexpected finding concerning bacteriophage k. First, with respect to ~., we have discovered two genes coding for cell envelope proteins which are expressed in the lysogenie state. These results counter the prevailing notion that it is only the immunity geoe, CI, and
DISCUSSION
the closely linked rex gent that are expressed in lysogens. There are many other O R F in X, functions for which have not been determined. It many well be that a n u m b e r of these are also expressed from the prophage. We have presented evidence that one of these genes, her, confers increased serum-resistance on E. colt in vitro. Serum-resistance is a known factor involved in bacterial virulence in animal hosts. While wc have not directly identified a function for the lore gene, the finding of sequence similarity between it and genes of other bacteria involved in pathogenesis suggests a similar role for il. Thus, 3. carries at least two genes which m a y enhance the pathogenicity of its host, E. colt, and thus the survival of both hosL and phage. Other temperate phages carry regions of their genome similar to the regions of ;', in which Iota or her map, raising the possibility that a previously unrecognized feature o f t e m p e r a t e phages, in general, may be their contribution to the pathogenicity of their bacterial hosts. The still large regions of phages such as ~. carrying a number of O R F may correspond, in part, to clusters of genes having this function. Our studies on alkaline phosphatase fusions to the ~wo cell division genes, ftsl and flsQ, give us information on the structure and function of the nroteins. Both geoc products appear to be cytoplasmic m e m b r a n e proteins with small cytoplasmic domains and large periplasmic domains. The amount of fusion proteins made indicates that both p r o t e i n s are m a d e in s m a l l amounts, approximately 10-25 molecules per cell. These levels are appropriate to proteins which have a rate-limiting catalytic step or are limited to particular locations in the ccU such as the septurn. In addition, we have used the ftsQ-phoA fusion as a null mutation when recombined into the chromosome. Such strains
ALKALINE PHOSPI-IATASE FUSIONS FOR CELL DIVISION G~NES have allowed us to show the essentiality of f t s Q and In study its function. Controlling the amount o f f t s Q expression using the era promoter allows us to either make or not make FtsQ in the null background. Studies on cell morphology show that FtsQ is likely to be required throughout ihe formation of the cell sopturn. Such a property is consistent with the idea t h a t the protein is an enzyme involved in the synthesis of this special structure. Obviously, other functions for this protein are also consistent with our findings. TnphoA fusions have also allowed us to identify an expressed ORF that precedes the f t M gene. This ORF, in all likelihood, is responsible for the corn. plemcntation of the fts36 mutation, a cell division mutation described by Ishino et aL (1989), From the properties of fusions and from the sequence, we expect that this protein, like FtsQ and Ftsl, is a transrnembrahe protein. We are currently testing the possibility that there are interactions between these different membrane proteins and developing approaches to assay function of ~he cytoplasmic domains and the membranespPnu~ng s.cgnle,tts.
Acknowledgements This work was supporled by an American Cancer Society gram and a
grant from Merck and Co., Inc. to J.g.
References Barondess, J.J. & llgckwith, J. (199P~ A bacterial virulence determinant encoded by lysogenie coliphage X Natnre (Lond.), ]46, 871-874. Bowler, L.D. & Spratt, B,G. (19891, Membrane topology of penicillin-binding protein 5 of ~scherichia coil MoL MicrobioL, 3, 1277-1286. Carson~ M., llarondess, J, & fleckwith, J. (19911, The FtsQ protein: a cytoplasmic membrane protein required throughout septum formation in Escherichia coll. Genes Develop. J, Bact., in press. Chuha, P., L~on, M.. Baner~ee. A. & Palchaudhuri, S. (1989), Cloning and DNA sequence of plasrnid determinant iss, coding for increased serum survival ann surt~lee exclusion, which has homology with lambda DNA. MoL gun. Genetics, 216. 287-292. Donachie, W.D,, llegg, K.J. & Sullivan, N.F. (1984), Mot phogenes of Escherichia L~li, m ~ Microbial Development , (R. Losick, and L. Shapiro), (pp. 27-621. Cold Spring Harbor Laboratory, New York. lkeda, M., Wachi, M., Ishino, F, & Matsuhashi, M. (1990), Nucleotide sequence involving murD and aa open reading frame ORF- Y spacing m u f f and fts IV in Escherichia coil Nucl. Acids Reg., 18, 1058, [shino. F., Jung, H.K., lkeda, M.,
299
Doi, M., Wachi, M. & Matsuhashi, M. {19891, New mutatiolls fts-36, its-M, and ftsW chlstercd in the tnra region of the Escherichia coil chromosome induce Ihermnsensitive cell growlh and division, J. tlact., 171, 5523-5350. Lutkenhaus, J,P., Wolf-Watz, H, & Donachie, W.D. (19801, Organization of genes in lhe fl~A-envA region of the Eseherichia coli genetic map and identification of a new fts locus fftsZ). ~L Bart., 142, hl5-620, Manod, C, & lleckwith, J. (19851, TnphoA: a I1ansposon probe for protein export signals, Proe, nat, Aead. Sci. (Wash.), 82, g129-glgg. Mav~oil, C. & Beekwirh, J. (1986l, A genetic approach to analyzing membrane protein topology. Science, 233, 1405-1408, Manoil, C,, Mekalanos, J.J. & Beckwith, J. (1990l, Alkaline phosphatase fusions; sensors of subcellular location. J. Bart., 172, 515-51g. Reeve, .I.N. & Shaw, J.E. (1979), Lambda encodes an outer membrane protein: The Iota gone, MoL gun. Generics, 172, 243-248. San Millan, J.L,, lloyd, D., Dalbey, R., Wickner, W. and lleckwilh, J. ~.19891.Use of phoA fusions to study the topology of the Escherichia c~li inner membrane protein leader peptidase. Z BtzcterioL 171, 5516-5541. Yi, Q.M. Rockenbach, S., Ward Jr. , J.E. & Lutkenhaus, J. (19851, StruCture and expression of the cell division genes ftsQ. ftsA and ftaZ. 3. Mol. Biol. 184., 399412.
Res, Mlicrobiol. 1991, 142, 295-299
Alkaline Phosphatase Fusions in the Study of Cell Division Genes J . J . Barondess "1(2), M . C a r s o n (t), L . M . G u z m a n V e r d u z c o TM a n d J. Beckwith ()) ¢ODepartment o f Microbiology and Molecular Genetics and ~'~Program in Cell and Developmental Biology. Harvard Medical School, 200 Longwood, Ave., Boston, M A 02115
SUMMARY Alkaline phosphatase fusions have been used to analyse pla~mid- or phagecarried genes from the two-minute region of the EscherichM coll chromosome. Those studies have revealed the following: 1) Bacteriophage k carries two genes for cell envelope proteins, tom and bor, that are expressed in lysogens and probably contribute to the pathogenicity of its E. coil host. 2) The ftsQ and ftsl gone products are integral proteins of the cytoplasmic membrane with small cytoplasmic domains and large pariplasmi¢ domains. 3) The ftsO and ftsl gsne products are made in very small amounts, on the order of 25 molecules per cell. 4) The ftsQ gone product is essential for cell growth and is required throughout the formation of the cell septum. 5) An open reading frame just upstream from ttsl, thought to be invoired In cell divlsion, is expressed and probably codes for a cytoplasmic membrane protein. Key-words : Cell division, Alkaline phosphatase, Gene, Fusion; Serum resistance, Membrane proteins.
Major aspects of the process o f bacterial cell division are the regulation and orientation of the growth of the cell envelope. In Escberichie coli, the cell envelope is composed o f the cytoplasmic membrane, the periplasmic space, the outer membrane and the peptidoglycan layer. It seems quite likely that the c~oplasmic membrane plays an important role in cell division. Signals may be passed throu3h the membrane in either direction and some enzymes involved in the formation of the cell septum may be localized to this membrane. We are interested in analysing those proteins required for cell division that are found in the cytoplasmic membrane. From such studies,
we would hope to determine (1) •~,hether any of these proteins are localized to the cell septum itself, (2) the rote of these proteins, (3) whether any of these proteins interact with each other and (4) whether any ff)rm of transmembrane signalling takes place in the cell division process. Alkaline phospnatase fusions provide a tool for identifying and analyzing proteins localized to the cell envelope of E. cob ~Manoil et al., 1990). We have developed a transposon probe, T n p h o 4 , which allows the detection of genes located on plasmids or on bacterial chromosomes that code for pzotcins which contain export signals (Manoi! and Beckwith, 1985). These include
proteins of the two membranes and of the peripla~mic space. Since alkaline phosphatase is only enzymaticaliy active when it is t r a n s l o c a t e d across the cytopla.~mie membrane, fusions of this protein only exhibit enzymatic activity when the protein to which they are fused allows ihe export of alkaliae phosphatase. The alkaline phosphatase fusion approach also allows the analysis of the topology of cytoplasmic membrane proteins (Manoil and Beckwith, 1986). '~he n alkaline phosphatasc is fused to a periplasmic domain of a membrane protein, the h:,brid protein is enzymatica!ly active; when it is fused to a cytoplasmic domain, it has very
296
J.J. BARONDESS E T AL.
little activity. In Ihls way, the exlelual and interaal dmnalus of a Cylol~lasmic inenthrflne protein can be delcrmiued, thus allowing Ihe formulalion of topological models. We have used alkaline phosphatase fusions to analyse the cluster of genes involved in cell division and cell envelope biogenesis located at nlinnte two OU Ihe E. coil chromosome. In this paper, we summarize the results obtained so far, including some surprising findings on bacteriophage 3. happened upon during Ihese studies. We also show hnw Ihe fusion approach helps identify hitherto undetected genes in this region.
RESULTS TnphoA analysis o[ X 16-25 We began our studies with a 3. transducing phage isolated by Lmkenhaus el aL (1980) which carries the ftsQ, ftsA, and f:sZ genes (fig. 1). Insertions of the Iransposon TnphoA o n l o the phage genom¢ were obtained using the kanamyein-resistance marker for selections. Those insertions in which lysogens of the phage exhibited a blue color on alkaline phosphatase indicator (XP) plates were sludied further. Out of a collection of several dozen phages carrying active fusions~ a number were restriction-mapped and fusion joints were sequenced. Surprisingly, the highest proportion of fusions exhibiting alkaline phosphatase activity were locafized to genes in the X phage itself. This is surprising, because it has g~aerally been assumed that the only genes expressed during 3. lysogeny are cytoplasmically localized proteins mapping to the immunity onl--opcn rcadlns flame.
region of the phage, Restriction mapping and sequencing of these fusions localized them to two sites on the prophage (fig. 1). Some of the fusions mapped to the lore gone, a previously discovered gone which codes for an outer membrane protein in minicell infection experiments, but not k n o w n to be expressed during lysogeny (Reeve and Shaw, 1979). The remaining fusions mapping in X were to an open reading frame we have named her (for blue open rending frame). No previous work had indicated a product or expression of this open reading frame. The finding of active alkaline phosphatase fusions to these two proteins suggested that both proteins were located in the cell envelope. However~ as we have pointed out elsewhere, it is important to determine the specific activity of the fusions (enzymatic activity related to alkaline phosphatase cross-reacting material) to be sure that the blue colour seen represents efficient export (San Millan etaL, 1989), Both classes of fusions exhibited specific activities close to that of wildtype alkaline phosphatase. These activities can also be used 1o estimate the level of expression of
the two genes, The results suggest that Bor is made in 2,000 molecules per cell and Lore at 100 molecules per cell. We believe that both Bor and Lore are proteins that may contribute to the pathogenic properties of E. colt (Barondess and Beckwith, 1990). Others had shown that the D N A sequence of the her ORF was 93% identical to the gone iss, located on colicin V plasmids (Chuba e t a L , 1989), The iss gone product is known to contribute to bacterial virulence by conferring serum resistance on hOStS that carry the gene. We have found that X lysogens expressing bor are 20-fold more resistant to serum complement killing in vitro than non-lysogens or lysogens in which bor is not expressed. We have no direct evidence for pathogenic properties of Lore, but it shares extensive sequence homology with proteins of two other pathogenic bacteria that are involved in entry or survival within mammalian cells. Th--se results extend previous findings of Mekalanos and coworkers on the utility of T n p h o A for detecting genes involved in bac:eria! pathogenesis (Manoil et ,'1., 1990). They
3. 16-25
/am d~ ~ t~A f~ZenvA~ N
in~t
bet
~nS
Fig. l. Map of X 16-25. The structure of this phage is described in Lutkenhaus et aL (1980l.
ALKALINE PHOSPHATASE FUSIONS FOR CELL DIVISION GENES also suggest that lysogenie phages, in general, may carry hitherto undetected genes contributing to the virulence of their host bacteria, Previous examples of lysogenic phages contributing to pathogenicity include phage ~ o f Corynebacterium diphtheriae which codes for diphteria toxin, It is notable that there are still significant portions of the h genome, functions for which have not been established. The possibility should be considered that there are still other ). genes involved in the pathogenicity of its host, E. coil. T n p h o A analysis of the f l s l region We have isolated a series o f T n p h o A insertions in a piasmid which ca.ries a 7.5-kb fragment including the f t s l gene. Seven fusions in the f l s l gene all had alkaline phosphatase fused to points beyond the hydrophohic stretch f o u n d early in the sequence (fig. 2). Our results are consistent with earlier finding5 (Bowler and Spratt, 1989) that Ftsl is a cytoplasmic membrane protein with a small cytoplasmic domain and a large peripl~snaic domain. One advantage of working with these fusions is that we can use antibody to alkaline p h o s p h a t a s e to e x a m i n e the behaviour of hybrid proteins and d ~ u c ¢ J~forrn.at.~n about the native protein. We have asked whether the early hydrophobic sequence m i g h t represent a cleaved s i g n a l sequence by examining the behaviour of a hybrid Ftsl-UhoA protein in a mutant defective in secretion, seeAfs. In this secAts mutant background, e,~,.ry other protein with a cleavable signal sequence tested has been blocked in the processing of the signal sequence at high temperature. (The small phage fl coat protein is the sole exception). We find that these Ftsl-PhoA hybrid proteins are not altered in mobility on SDS
gels when secretion is defective, s u g g e s t i n g that the early hydrophobic stretch is a m e m b r a n e - s p a n n i n g segment rather than a cleavable signal sequence. On a multicopy pie,staid, the a l ka l i ne p h o s p h a t a s e units produced by a n f t s l - p h o A fusion indicate that it is making about 500 copies of the prot e i n. A s s u m i n g a copy n u m b e r between 20 and 50, we can estimate that the amount of Fts[ in single copy may be from 10 to 25 molecules. These calculations should be considered with some caution umill measurements are made on single-copy versions of the fusions. We have also found alkaline phosphatase fusions to other open reading frames on t~e JIM plasmid (fig. 2). One of these is located downstream from f t s l in a region which has been sequenced, but in which the function of not all the ORF is known (lkcda et aL. 1990). At this time, the most interesting fusions to us are three located in the region of an open-reading frame just upstream from ftsL This is a region which inelucies a gene complementing a new c;ass of ceil division mutants (lshino
297
et al., 1989). We have sequenced two of these fusions and find that they have alkaline phosphatase fl~sed to the only open reading frame of significant length found in this fragment preceding flsL The location of the active fusion joints and the sequence of this ORF suggest that is codes for a cytoplasmic membrane protein with p e r i p l a s m i c and cytoplasmic domains of approximately equal size. The nature of this protein raises the possibility that it is involved in transmembrane signalling. Since it is the only O R F in the fragment that complements the fts36 mutation of Ishino et al. (1989), it seems likely that it corresponds to this gene involved in cell division, Fusions of aikaiine phosphntase to F b Q We have analysed the structure of the FtsQ protein and other aspects o f it5 function using al ka l i n e p h o s p h a t a s e fusions (Carson er al., 1991. in press). These fusions were obtained on a plasmid that carried the 2.4-kb HindlIl-Bglll fragment that inelude~ ftsQ, a portiGa of StsA and a small amoun: of material preceding
Aclive alkaline pho~phalasegene fusiul,s i,~ the remton o t Jlsl
ftsl
I
tllndnl
fff
FIT f ff
÷*"
.....
rff ....
I
EcoRt
Fig. 2. Position of active alkaline phosphat~e fusions to genes on an frI plasmid. Fusions were obtained and sequenced as described previo~iy (Carson et aL, in press).
298
.ft.sQ We have obtained a set of
J.J. BARONDESS ET AL.
active lusions in which the alkaline i~hosphatase is always fLtsed at a positinn ill the amino acid sequence past an early hydrophohic strelch of amino acids. As is the case for the F t s l - P h o A fusion prnteios, when we block secretion, s~e do not see alteration in tile mobility of FIsQPhoA fusion proteins in SDSPAGE. Thus, the hydrophobie stretch located early in the FtsQ protein (Yi el at., 1985) is also likely to he a m e m b r a n e spanning stretch rather than a cleavable signal sequence. Also consistent with a membrane location, we find that an FtsQ-PhoA fusion protein fractionates with the pellet in an osmotic shock fractionation (Manoil and Beckwith, 1986), under conditions which release other periplasmic proteins. Tltese results suggest that the FtsQ protein has a structure similar to that of Ftsl. That is, the protein is located in the cytoplasmic membrane, has a small cytoplasmic d~,,,tain and a large periplasmic domain. On this basis, the p er ip lasmic domain of FtsQ could be an enzyme, as is the case with Ftsl, or a peripla~mic structural or assembly component of the cell division apparatus.
wilh lhe hypothesis (Donachie et aL, 1984) that its product is required throughout the retinalion of tile cell septum (Donachie el aL, 1984). Strains with insertion mutation in the ftsQ gene complemented by a plasmid with the ftsQ gene under control of the ara promoter, form long non-septate filaments when the inducer is removed, indicating a defect early in the septation process. On the other hand, multiseptate filaments accumulate either when the ftsQ temperature-sensitive mutant is grown at semi-permissive temperatures or when the FtsQ protein is overexpressed a b o u t 200-fold from the ara promoter in a wild-type background. The chromosomal inserts of the TnphoA fusion also allow us to measure the level of expression of ftsQ and its regulation. The ftsQ.phoA fusions make approximately 3 units of alkaline phosphatase when located in the normal ftsQ chromosomal site. T h e specific activity of the hybrid proteins is again comparable to that of wild-type alkaline phosphatase. This activity corresponds to about 25 molecules of the hybrid protein, which suggests that is the level of ftsQ expression.
T)zphoA insertions also provide us with null mutations in the gone of iutel¢gt. Vqe have recomhioed certain of the frsQp/teA fusions onto the chromosome in a strain that carries a secono copy of ftsQ on a ;',transducing phage. At the same time, we have constructen plasmids in which the frsQ gene is under :he control of the tightlyregulated araB,A,D promoter. When the chromosomal TnphoA insertion is ir.troduced into a strain that carries ftsQ under ara control, the strains are dependent for tiaeir survival on the ar--~binose,-induced expression of ftsQ. Studies on the phenotype cf ce~;~ with different levels of ftsQ expression are consistent
We have initiated studies on cell division by obtaining fusions of alkaline phosphatase to protcios coded for in the minute 2 region of the E. colt chromosome. These studies have revealed a n,~mber of features of the genes in this region and an unexpected finding concerning bacteriophage k. First, with respect to ~., we have discovered two genes coding for cell envelope proteins which are expressed in the lysogenie state. These results counter the prevailing notion that it is only the immunity geoe, CI, and
DISCUSSION
the closely linked rex gent that are expressed in lysogens. There are many other O R F in X, functions for which have not been determined. It many well be that a n u m b e r of these are also expressed from the prophage. We have presented evidence that one of these genes, her, confers increased serum-resistance on E. colt in vitro. Serum-resistance is a known factor involved in bacterial virulence in animal hosts. While wc have not directly identified a function for the lore gene, the finding of sequence similarity between it and genes of other bacteria involved in pathogenesis suggests a similar role for il. Thus, 3. carries at least two genes which m a y enhance the pathogenicity of its host, E. colt, and thus the survival of both hosL and phage. Other temperate phages carry regions of their genome similar to the regions of ;', in which Iota or her map, raising the possibility that a previously unrecognized feature o f t e m p e r a t e phages, in general, may be their contribution to the pathogenicity of their bacterial hosts. The still large regions of phages such as ~. carrying a number of O R F may correspond, in part, to clusters of genes having this function. Our studies on alkaline phosphatase fusions to the ~wo cell division genes, ftsl and flsQ, give us information on the structure and function of the nroteins. Both geoc products appear to be cytoplasmic m e m b r a n e proteins with small cytoplasmic domains and large periplasmic domains. The amount of fusion proteins made indicates that both p r o t e i n s are m a d e in s m a l l amounts, approximately 10-25 molecules per cell. These levels are appropriate to proteins which have a rate-limiting catalytic step or are limited to particular locations in the ccU such as the septurn. In addition, we have used the ftsQ-phoA fusion as a null mutation when recombined into the chromosome. Such strains
ALKALINE PHOSPI-IATASE FUSIONS FOR CELL DIVISION G~NES have allowed us to show the essentiality of f t s Q and In study its function. Controlling the amount o f f t s Q expression using the era promoter allows us to either make or not make FtsQ in the null background. Studies on cell morphology show that FtsQ is likely to be required throughout ihe formation of the cell sopturn. Such a property is consistent with the idea t h a t the protein is an enzyme involved in the synthesis of this special structure. Obviously, other functions for this protein are also consistent with our findings. TnphoA fusions have also allowed us to identify an expressed ORF that precedes the f t M gene. This ORF, in all likelihood, is responsible for the corn. plemcntation of the fts36 mutation, a cell division mutation described by Ishino et aL (1989), From the properties of fusions and from the sequence, we expect that this protein, like FtsQ and Ftsl, is a transrnembrahe protein. We are currently testing the possibility that there are interactions between these different membrane proteins and developing approaches to assay function of ~he cytoplasmic domains and the membranespPnu~ng s.cgnle,tts.
Acknowledgements This work was supporled by an American Cancer Society gram and a
grant from Merck and Co., Inc. to J.g.
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