JouRNAL OF BACTERIOLOGY , Dec. 1992 , p. 7697-7704-0021-9193 / 92/237697 -08 $ 02.00 / 0 Copyright © 1992 , American Society for Microbiology Vol .
174 , No. 23 Identification of Ancillary fim Genes Affecting fimA Expression in Salmonella typhimunum DANA L. SWENSON AND STEVEN CLEGG * Department of Microbiology , University of Iowa College of Medicine , Iowa City , Iowa 52242 Received 18 May 1992/Accepted 28 September 1992 Regulation of the gene , finL4 , encoding the major fimbrial subunit of S. typhimwuwm S6704 was examined by using a ?
Transformation of the Af?im4 - lacZ lysogen with various derivatives of the recombinant plasmid that encodes type 1 fimbrial expression , pISF101 , indicated that two regions of this plasmid alter P-galactosidase production .
One plasmid is a deletion resulting in the loss of a 28-kDa pobpeptide downstream of fim4 , while the other plasmid encodes a 24 .
and a 27-kDa polypeptide .
Northern ( RNA ) blot analyses indicated that the steady-state Jfn4 mRNA levels of these transformants were high .
In addition , phenotypic expression of type 1 fimbriae by agar-Wgrown cultures is observed only in those transformants bearing plasmids which show increased 13-galactosidase andfim4 mRNA levels .
Type 1 fimbriae are proteinaceous filaments produced by many members of the family Enterobacteriaceae .
The expression of type 1 fimbriae can be detected by the agglutination , in-vitro , of yeast cells or guinea pig erythrocytes , and this agglutination can be inhibited by the presence of mannose .
Therefore , type 1 fimbriae may mediate adherence to mannose-containing glycoproteins on eukaryotic cells , thereby facilitating colonization ( 30 ) .
In Escherichia coli the phenotypic expression of type 1 fimbriae is encoded by a cluster of genes .
A single gene , fimA , encodes the major fimbrial subunit , while fimF , fimG , and fimH encode minor fimbrial components ( 16 , 19 , 25 ) .
Of these genes , fimH has been shown to encode the adhesin which confers mannose sensitivity of the type 1 fimbrial adherence ( 18 ) .
Phenotypic expression of surface-associated fimbriae requires the production of ancillaryfim gene products most likely involved in assembly , transport , and regulation during the process of fimbrial biogenesis .
Many E. coli strains that produce type 1 fimbriae also demonstrate the ability to alternate between a fimbriate and a nonfimbriate phenotype , a phenomenon known as phase variation .
This conversion is mediated , in part , by the inversion of a 314-bp DNA sequence located immediately upstream of fimA ( 1 ) .
The orientation of this invertible fragment determines whether the fimA gene is transcribed ( 8 ) .
The products of the fimB and fimE genes and a cellular protein , integration host factor , facilitate a site-specific inversion of the region bearing the JimA promoter ( 9 , 15 ) .
However , recent evidence has indicated that the inversion mechanism is more complex than a simple on-and-off model , since FimB may mediate inversion in both directions while FimE acts primarily in one direction to turn off JimA transcription ( 23 ) .
As in E. coli , the Salmonella typhimurium fim genes have been cloned and the fimA gene has been sequenced ( 29 ) .
However , the identification of ancillary Jim genes affecting the expression of fimA has not been investigated , and therefore , it is unknown which genes are involved in the regulation of fimnA .
Although fimbrial phase variation is known to occur in S. typhimurium ( 26 , 27 ) , it is not known whether S. typhimurium possesses determinants analogous to the E. coil fimB and fimE genes .
For this report , the expression of fimA in S. typhimurium was examined by using recombinant plasmids and a XfimA-lacZ lysogen as a reporter system .
Deletion derivatives of the S. typhimuinum type 1 fimbrial gene cluster were used to identify regions of thefim gene cluster that alter , B-galactosi-dase production by the lysogen .
In addition , Northern ( RNA ) blots were used to confirm that the observed changes in 3-galactosidase activity correlated with the amounts of steady-state fimA mRNA present in S. typhimurium .
The expression of the S. typhimurium fimA gene appears to be affected byfim genes exhibiting no relatedness to the E. coi fimB and fimE genes and located at sites different from the position of fimB and fimE in the E. coli system .
MATERIALS AND METHODS Bacterial strains and media .
E. coli JM109 ( 35 ) was used for the isolation and characterization of the plasmid JimA-lacZ fusion molecules .
S. typhimurium LB5010 ( 2 ) was obtained from the Sabnonella Genetic Stock Center and was used as the host to prepare the Salmonella XJimA-lacZ lysogen .
E. coil HB101 ( 5 ) , E. coli ORN103 ( 21 ) , and S. typhimurinum LB5010 were used as host strains for the preparation of bacterial lysates .
All bacteria were grown in Luria-Bertani media containing the appropriate antibiotics .
The XJimA-lacZ lysogen was incubated at 30 °C ; all other incubations were performed at 37 °C .
Detection of surface-associated ype 1 flmbriae .
Bacteria were grown overnight on L agar supplemented with the appropriate antibiotics .
Harvested cells were resuspended in phosphate-buffered saline ( PBS ) to approximately 108 bacteria per ml , and 50 j.l of a 3 % ( vol/vol ) suspension of Candida albicans was incubated with equal volumes of bacterial suspension ( 17 ) .
Visible agglutination after 30 s indicated a positive reaction .
Inhibition of yeast cell agglutination was determined with a suspension of C. albicans in PBS containing 3 % ( wt/vol ) D-mannose .
Construction of the fimA4-acZ plasmid and the ?
The plasmid vector pMC1403 ( 3 ) , containing a promoterless lacZ gene , was used to construct the S. typhi-murium fimA-lacZ fusion .
Plasmid pISF101 encodes the phenotypic expression of S. typhimurium type 1 fimbriae , and the construction and characterization of this plasmid have been previously described ( 4 , 29 ) .
Initiation of transcription of the fimA gene was determined by the procedure of Weaver and Weissmann ( 34 ) and is located 195 nucleo-tides upstream of the fimA initiation codon within a 255-bp DNA fragment flanked by 10-bp inverted repeats ( 29 ) .
The nucleotide sequence of the fimA gene has been determined ( 29 ) , and this information was used to isolate a 500-bp RsaI DNA fragment carrying the fimA promoter and the first 33 codons of the fimA gene .
This DNA fragment was ligated into the unique SmaI site of pMC1403 , and E. coli transformants were selected on agar containing 100 , ug of ampicillin per ml and 50 , g of X-Gal ( 5-bromo-4-chloro-3-indolyl-p-D-galactopyranoside ) per ml .
Transformants possessing a recombinant molecule comprising an in-frame fusion between thefimA and lacZ genes were used to prepare plasmid DNA , and the fidelity of the fusion was determined by DNA sequencing through the fusion junction .
The fimA-lacZ fusion plasmid , pISF145 , was linearized with the restriction enzyme Sall .
Blunt ends were created by using the Klenow fragment of DNA polymerase , and EcoRI linkers were subsequently attached and ligated .
Following digestion of this plasmid with EcoRI , a 7-kbp DNA fragment was ligated into EcoRI-restricted Xgt2 ( 28 ) , and phage DNA was packaged by using a commercially available extract ( Stratagene , La Jolla , Calif. ) .
S. typhimurium LB5010 , transformed with pAMH70 to confer X sensitivity ( 14 ) , was used to construct the XfimA-lacZ lysogen .
S. typhimurium LB5010 was infected with recombinant lambda phage particles and plated on X-Gal-ampicillin agar at 37 °C .
Blue plaques were identified , and lysogens were obtained by plating on agar followed by incubation at 30 °C ( 31 ) .
E. coli JM109 or ORN103 XfimA-lacZ lysogens were also isolated by similar techniques except that the E. coli strains had not been transformed with pAMH70 .
The S. typhimurium AfimA-lacZ lysogen , designated ISF145 , or E. coli lysogens were transformed with pISF101 and various deletion derivatives of this plasmid .
Assays for P-galactosidase activity were performed in triplicate by the method of Miller ( 24 ) following growth in Luria-Bertani medium with the appropriate antibiotics .
RNA was isolated , by the method of Gosink et al. ( 13 ) , from the S. typhimurium LB5010 or the AfimA-lacZ lysogen containing deletion derivatives of pISF101 .
Twofold serial dilutions of 10 , ug of RNA were prepared , and following electrophoresis through a 1.2 % ( wt/vol ) agarose gel containing 0.66 M formaldehyde , the RNA was transferred to a nylon membrane according to the manufacturer 's instructions ( GeneScreen ; NEN Research Products , Boston , Mass. ) .
Two DNA fragments used as probes were isolated as follows .
The plasmid pMC1403 was restricted with EcoRV and EcoRI to yield a 1-kb DNA fragment containing only lacZ nucleotide sequences .
Also a S. typhimurium fimLA gene probe was isolated from pISF141 as previously described ( 12 ) .
This probe was composed solely of S. typhimurium fimA nucleotides .
DNA fragments were labeled by the random primer technique ( 31 ) .
All hybridizations and washes were performed under highstringency conditions as previously described ( 31 ) .
A 452-bp DNA fragment containing the promoter region of the S. typhimuiium fimnA4 gene was isolated following digestion of pISF141 with BstEII and RsaI .
This DNA fragment was end labeled with [ 32P ] dGTP by using the Klenow fragment of DNA polymerase ( 30 ) .
Cell sonicates were obtained from S. typhimunum and E. coli transformants as previously described ( 10 , 11 ) .
Gel retardation assays to detect DNA binding were performed according to previously published procedures ( 10 , 11 ) with the following modifications .
The addition of 1 mg of bovine serum albumin per ml to the reaction mixture was eliminated , and the volumes were adjusted with sterile H20 .
The DNA sequence of the fimA-lacZ fusion junction was determined by Maxam and Gilbert chemical cleavage ( 22 ) .
The DNA sequences of fimY and fimZ ( Fig. 1 ) were determined by dideoxy termination using standard techniques ( 32 ) .
Nucleotide sequence accession number .
The nucleotide sequences of fimY and fimZ have been deposited in the GenBank data library under accession number M90677 .
769 RESULTS Expression of 13-galactosidase by transformants of the S. typhimurium or E. coli AfimA-lacZ lysogens .
S. typhimurium ISF145 was transformed with pISF101 , which encodes the phenotypic expression of Salmonella type 1 fimbriae , and various deletion derivatives of this plasmid ( Fig. 1 ) .
Levels of 0-galactosidase activity of S. typhimurium ISF145 and its transformants are shown in Table 1 .
Transformation of S. typhimunum ISF145 by plasmid pISF121 or pISF161 resulted in significant increases in detectable 3-galactosidase activity compared with the activity of other transformants or the lysogen without a plasmid .
Plasmid pISF121 was constructed by linearization of pISF101 at a unique SmaI site and subsequent removal of approximately 500 bp by the exonuclease Bal 31 .
Using a minicell system we have previously demonstrated that a 28-kDa polypeptide immediately downstream of fimA is no longer produced by this plasmid ( 4 ) .
The,-galactosidase activity of S. typhimurium ISF145 bearing pISF121 shows a 40-fold increase in activity compared with that of the plasmidless strain or other transformants of the lysogen ( Table 1 ) .
A 25-fold increase in,-galactosidase activity is observed when the S. typhimurium AfimA-lacZ lysogen possesses the plasmid pISF161 .
This plasmid contains no fimA gene sequences and is predicted to encode a 24-and a 27-kDa protein ( Fig. 1 ) .
Both pISF161 and pISF162 are derivatives of pISF160 , yet no significant increase in 3-galactosidase activity was observed when pISF160 or pISF162 was used to transform the lysogen .
Plasmid pISF171 ( Fig. 1 ) was constructed by restriction of pISF161 with BglII and BamHI with subsequent recircularization of a 5-kbp DNA fragment .
This plasmid contains only the gene ( fimZ ) for the 24-kDa protein , and transformation of this construct into ISF145 resulted in no increase in 3-galac-tosidase levels over those observed with the lysogen alone .
Plasmid pISF172 ( Fig. 1 ) was constructed by digestion of pISF161 with the restriction enzymes BgIII and BamHI to isolate a 1.6-kbp DNA fragment .
This DNA fragment , containing only fimY , was ligated into the BamHI site of pACYC184 .
Transformation of the S. typhimurium fimA-lacZ lysogen resulted in significantly increased 0-galactosi-dase expression by the lysogen ( Table 1 ) .
Plasmid pISF173 ( Fig. 1 ) was constructed by linearization of pISF161 at a unique EcoRV site with the subsequent ligation of linkers containing a universal translation termination signal ( Pharmacia , Piscataway , N.J. ) .
Transformation of the S. typhimurium XfimA-lacZ lysogen with pISF173 , the plasmid possessing a translation termination signal in th gene encoding the 27-kDa polypeptide , showed a decrease in 3-galactosidase levels that are equivalent to those values observed in ISF145 alone .
Plasmid pISF174 ( Fig. 1 ) was constructed by linearization of pISF161 with the restriction enzyme PvuI , and subsequent removal of protruding nucleotides used T4 DNA polymerase .
Linkers possessing translation termination signals were inserted as described above .
Transformation of the S. typhimurium XflnA-lacZ lysogen with pISF174 , a plasmid possessing a translation termination signal in the gene encoding the 24-kDa polypeptide , resulted in increased 3-ga-lactosidase expression by S. typhimurium ISF145 ( Table 1 ) .
Transformation of S. typhimurium ISF145 with other derivatives of pISF101 resulted in no increase in 3-galactosi-dase activity ( data not shown ) .
Some of these plasmids contain fimA sequences as well as sequences shared with pISF121 or pISF161 .
In addition , transformants of S. typhi-murium ISF145 possessing ancillaryfim genes from the type 1 fimbrial system of Klebsiella pneumoniae ( 4 ) demonstrated no increase in 3-galactosidase activity compared with that of the lysogen alone .
E. coli ORN103 , a genotypically nonfimbriate strain ( 21 ) , was also used to construct a XfimA-lacZ lysogen .
However , the fusion molecule was unstable in this strain of E. coli , and following growth on L agar or in L broth , nonlysogenic variants arose at a high frequency .
Following overnight growth , 60 to 70 % of the bacteria had lost phage DNA .
This instability was not observed by using E. coli JM109 ( 35 ) to construct the lysogen .
As for the Salmonella lysogen , pISF121 transformants of the XfimA-lacZ E. coli lysogen demonstrated a greater expression of 3-galactosidase activity compared with that in other transformants .
A 50-fold increase in enzyme activity was observed when the E. coli possessed pISF121 compared with the activity of transformants possessing other derivatives of pISF101 .
However , pISF161 , a plasmid imparting increased fimA expression in S. typhimurium LB5010 , did not increase 3-galactosidase activity in E. coli .
The addition of a second plasmid , pISF102 ( Fig. 1 ) , to the E. coli lysogen possessing pISF161 did result in a marked increase in enzyme activity .
This double transformant exhibited 140 times the activity of the E. coli strain transformed with pISF161 or pISF102 alone fimbriae by the XfimA-lacZ lysogen of S. typhimurinum ISF145 , B-Galactosidase Yeast cell activity ' agglutinationb None 1.0 pISF101 1.4 pISF121 40 pISF160 5.0 pISF161 25 pISF162 1.0 pISF171 1.0 pISF172 70 + pISF173 1.0 pISF174 51 + a Values of enzyme activity are expressed relative to the activity of the XfsnL4-lacZ lysogen with no plasmid , this value being arbitrarily designated 1.0 .
b Agglutination of a 3 % ( vol/vol ) suspension of C. albicans by agar-grown bacterial suspensions .
+ , yeast cells were agglutinated within 30 s at the ambient temperature but were not agglutinated in the presence of D-mannose ; yeast cells were not agglutinated at all .
174 , 1992 7699 ANCILLARY SALMONELLA fim GENES 8 0 C2 2 -- a-i IA C0L ) .
lU plSF101 D W Y ¬ H F z -- 20 24 m 36 ¬ 30 21 28 82 27 24 - > + < > < ¬ plSF1 02 pISF1 05 pISF1 08 pISF1 21 pISF148 pISF149 pISFI 60 pISF1 61 pISF1 62 pISFI 71 pISF1 72 pISFI 73 pISFI 74 x 1 kb FIG. 1 .
Genetic organization of S. typhimuriumfim genes .
The sizes of the polypeptides encoded by the genes are shown below the solid boxes ; fimA is the gene encoding the major fimbrial subunit , whereas fimZ andfimY are those described in the text .
The arrows indicate the direction of transcription derived by S1-nuclease-mapping for fimA and predicted from the nucleotide sequence for fimY and fimZ .
The derivatives of pISF101 are indicated below the map , with the solid lines representing the DNA retained by each derivative .
For pISF173 and pISF174 the crosses indicate the location of the inserted translation termination linkers .
Representative Northern blots of total RNA from S. typhimurium LB5010 .
Equal amounts of RNA ( 10 p.g in lanes 1 through 1.25 , g in lanes 4 ) from S. typhimunum transformed with pISF160 ( A ) and pISF161 ( B ) were loaded onto the gels .
The DNA probe is composed solely of nucleotides from fimA ( see Materials and Methods ) .
The numbers on the left indicate the size ( in kilobases ) of RNA standards run through the gel , and the length of the fimA transcript is approximately 800 bases .
Analysis of the fimA mRNA produced by transformants .
RNA was isolated from S. typhimurium LB5010 transformed with derivatives of pISF101 .
Specific fimA mRNA was detected in these transformants by using a DNA probe comprising nucleotides derived solely from the fimA gene ( 12 ) .
As shown in Fig. 2 , the steady-state mRNA level of fimA was increased in pISF161 transformants compared with that in S. typhimurium LB5010 possessing pISF160 .
Since pISF121 , a plasmid resulting in increased P-galac-tosidase production by the XfimA-lacZ lysogen , possesses an intact copy of fimA , an analysis of only chromosomal fimA expression , by observation of fimA mRNA , was not possible .
Consequently , for pISF121 transformants , RNA was isolated from the XfimA-lacZ lysogen and a DNA probe derived from the lacZ gene was used to detect fimzA-lacZ mRNA .
In this way it was possible to demonstrate that the increased P-galactosidase activity of this transformant was due to increased fimA-lacZ gene expression , since lacZ mRNA could be detected only in this transformant .
All plasmids not resulting in increased P-galactosidase expression by the lysogen also did not increasefimA mRNA expression when thefimA DNA probe was used .
Even those transformants bearing fimA as part of the plasmid molecule produced undetectable amounts of fimA mRNA , indicating thatfimA gene expression is not constitutive in this strain of S. typhimurium .
Surface expression of type 1 fimbriae .
The phenotypic expression of type 1 fimbriae by S. typhimurium ISF145 and various transformants was determined by yeast cell suspension agglutination and reactivity with specific antifimbrial serum .
S. typhimurium LB5010 produces surface-associated fimbriae only following serial cultivation in liquid medium ; therefore , agar-grown cultures of this strain did not agglutinate yeast cells .
However , transformants of S. typhimurinum bearing ISF101 , pISF121 , pISF161 , pISF172 , or pISF174 invariably resulted in expression of functional type 1 fimbriae regardless of the method of cultivation ( Table 1 ) .
All remaining transformants were phenotypically nonfimbriate following growth on solid media .
Cell lysates were prepared from typhimurium transformants of S. LB5010 , E. coli HB101 , or E. coli ORN103 .
These lysates were used to detect the presence of proteins binding to the S. typhimurium fimA promoter .
All lysates derived from Salmonella or E. coli ORN103 transformants resulted in electrophoretic retardation of a DNA fragment possessing the fimA promoter ( Fig. 3 ) .
However , with E. coli HB101 lysates , only those preparations derived from E. coli transformed with pISF101 or pISF121 resulted in any change in mobility of the DNA fragment possessing the fimA promoter ( Fig. 3 ) .
All other lysates , including that of the E. coli HB101 transformant possessing pISF161 , had no effect upon the electrophoretic mobility .
Sequence analysis of pISF161 .
The DNA sequence of pISF161 is shown in Fig. 4 .
Only two large open reading frames were identified : one may encode a 27-kDa polypeptide , and the second may encode a 24-kDa polypeptide .
Comparison of the probable amino-acid sequences of the two proteins with those found in the GenBank data base ( 6 ) showed no sequence similarity to any E. coli type 1 fimbrial gene products .
Figure 1 illustrates the location of the genes encoding the 27-and 24-kDa polypeptides in relation to the fimbrial subunit gene ( fimA ) .
Representative Northern blots of total RNA from S. typhimurium LB5010 .
Equal amounts of RNA ( 10 p.g in lanes 1 through 1.25 , g in lanes 4 ) from S. typhimunum transformed with pISF160 ( A ) and pISF161 ( B ) were loaded onto the gels .
The DNA probe is composed solely of nucleotides from fimA ( see Materials and Methods ) .
The numbers on the left indicate the size ( in kilobases ) of RNA standards run through the gel , and the length of the fimA transcript is approximately 800 bases .
A B C 1 2 3 4 1 2 3 in : 6 FIG. 3 .
Gel mobility-shift assays using labeled DNA containing the fimA promoter region and bacterial lysates from E. coli HB101 transformed with pISF101 ( A ) and pISF121 ( B ) .
Serial twofold dilutions of bacterial lysate were used for each transformant , with the highest concentration of protein in lanes 1 .
Nucleotide sequences offimYandfimZ and predicted amino-acids of the gene products .
The termination codons of each gene are indicated by asterisks .
DISCUSSION The phenotypic production of type 1 fimbriae requires the expression of severalfim gene products most likely involved in the transport and assembly of Fim polypeptides .
With the exception of E. coli , the regulation of fimbrial biogenesis in members of the Enterobacteriaceae has not been investigated .
In this communication we show thatfimA expression in S. typhimurium appears to be regulated by fim genes located at sites different from those found in E. coli .
In E. coli , the regulation of fimA expression has been shown to be mediated by at least two genes , fimB and fimE which are located immediately upstream offimA ( 9 , 15 ) .
The products of these genes regulate transcription of fimA by mediating a phase inversion of the fimA promoter .
FimB mediates inversion from an on to an off position as well as off to on , while FimE preferentially orients the promoter to the off position ( 23 ) .
The inversion of the DNA fragment possessing the fimA promoter also appears to be dependent upon at least one host protein , integration host factor ( 7 ) .
No Salmonella strain that is genotypically nonfimbriate is currently available , as we have recently demonstrated that the nonfimbriate S. typhimunium FIRN and non-FIRN strains do possess fim sequences ( 33 ) .
Consequently , to investigate the regulation of S. typhimurium fimA expression , a XfimA-lacZ lysogen was constructed in S. typhimu-rium LB5010 , a genotypically defined strain which does produce type 1 fimbriae .
In this system the fimA-lacZ fusion is a reporter gene offimA expression in an appropriate host .
Thus , all gene products examined in this study that regulate fimA expression , as determined by quantitation of 3-galac-tosidase activity , exert such an effect by acting in trans .
Relatively low levels of,3-galactosidase activity are detected in the lysogen following growth on agar or for short periods of time in broth .
This is consistent with the lack of fimbrial expression by this strain when grown under these conditions .
The XfimA-lacZ DNA does integrate into the chromosome of S. typhimurium LB5010 , since analysis of pAMH70 from the lysogen indicates no increase in the size of the plasmid DNA .
In addition , the Salmonella lysogen does retain the ability to produce type 1 fimbriae ( see below ) , and therefore , it is unlikely that integration at the site of the parental fimA has occurred .
The results obtained with the Salmonella AfimA-lacZ lysogen demonstrate that two regions play a role in fimA expression .
One region is a deletion of nucleotides , encoding a 28-kDa polypeptide , downstream of fimA ( Fig. 1 ) .
The absence of this gene product increases 3-galactosidase activity almost 40-fold .
Therefore , the absence of this-28-kDa polypeptide allows expression offimA to be increased .
Such a Fim polypeptide could be a negative regulator of fimA expression .
A similar result was observed with the E. coli lysogen .
A second region of the fim gene cluster that alters n-ga-lactosidase activity in the Salmonella lysogen possesses only two ancillary fim genes whose gene products are 24-and 27-kDa polypeptides , and these determinants are located at the opposite end of the gene cluster from fimA ( Fig. 1 ) .
A plasmid possessing these two genes increases P-galactosi-dase activity approximately 25-fold compared with the activity of either the lysogen alone or the lysogen transformed with other plasmids ( Table 1 ) .
A possible explanation for the increase in 3-galactosidase activity is that one of the gene products encoded by pISF161 acts directly upon fimA to increase expression .
Analysis of the probable amino-acid sequence of this region indicated that the 24-kDa Fim polypeptide exhibits an amino-acid sequence similar to that found in many transcriptional activators ( 6 ) .
However , a mutation in this gene does not significantly alter 3-galactosi-dase activity , thereby suggesting that this gene product may exert its effect upon a fimbrial gene other than fimA .
Interestingly , a mutation in the 27-kDa polypeptide decreases,-galactosidase activity to the level of the lysogen alone .
This gene product may exert its effect directly upon fimA or may indirectly result in increasedfimA expression , although , as discussed below , a direct effect upon fimA can not be established .
Since the 27-kDa polypeptide appears to affect fimA expression in S. typhimurium , we decided to determine whether the plasmid ( pISF161 ) encoding this protein could , in the absence of other fim genes and Salmonella host factors , directly increase fimA expression .
To answer this question we attempted to construct a fimA-lacZ lysogen in the genotypically nonfimbriate strain E. coli ORN103 .
However , these lysogens were found to be unstable , and under the conditions of growth used in these studies , nonlysogenic variants arose at a relatively high frequency .
Therefore , it was not possible to construct and maintain a stable lysogen in a nonfimbriate strain .
The reasons for this instability are unclear but may reflect the absence of fim genes in E. coli ORN103 .
The fimbriate strain E. coli JM109 was a suitable host for the XfimA-lacZ fusion .
Also , the host E. coli fim genes in this strain did not appear to affect enzyme expression by the XfimA-lacZ fusion , since strongly fimbriate cultures did not produce detectable levels of 0-galactosidase .
Interestingly , E. coli transformants possessing pISF161 also did not produce high levels of 3-galactosidase , unlike S. typhinunm transformants .
However , the addition of a second plasmid did increase expression of the fimA-lacZ fusion by a factor greater than 100 .
Consequently , the effect of the 27-kDa polypeptide uponfimA expression is not likely to be due to the direct action of this gene product upon fimA .
In addition , the simple presence of pISF161 , as a multicopy plasmid , in the Salmonella lysogen is not likely to explain the increase in,-galactosidase production , since a similar effect is not observed in E. coli To confirm that the increase offimA expression , observed in both pISF121 and pISF161 transformants , was at the level of transcription , steady-state levels offimA mRNA or fusion product mRNA were determined .
As noted above , S. typhi-murium LB5010 possesses functional type 1 fimbrial gene sequences .
Therefore , in transformants possessing no fimA sequences on the transforming plasmid , it was possible to use afimA gene probe to detect transcription of the wild-type fimA gene .
For those transformants possessing fimA as part of a plasmid , a DNA probe comprising only lacZ nucleotides was used to detectfimA-lacZ mRNA in the lysogen .
Thus , in both cases only the transcription of the chromosomally borne allele under control of afimA promoter was detected .
No detectable fimA mRNA was observed in the untransformed S. typhimurium LB5010 following overnight growth in broth .
This is consistent with the lack of yeast cell agglutinating activity and low expression of , B-galactosidase activity .
S. typhimurium transformed with pISF121 or pISF161 demonstrated an increase in steady-state fimA mRNA production .
Transformants possessing pISF160 or pISF162 showed no increase infimA mRNA expression , and thus fimA transcription in these bacteria was very similar to that of the plasmidless strain .
Consequently , fimA promoter activity in pISF161 and pISF121 transformants correlated with measured , B-galactosidase activity and surface fimbrial expression .
However , we can not rule out the possibility that pISF121 and pISF161 increase the stability of fimA mRNA rather than increase fimA transcription .
The results indicate altered fimA expression due to two discrete regions of the gene cluster .
To determine whether this activation of fimA was achieved by binding of Fim polypeptides to the promoter of fimA , gel-shift-mobility assays were performed .
However , it was not possible to demonstrate that increased fimA expression of pISF121 or pISF161 transformants was due to the production of DNA-binding proteins by the fim sequences of these plasmids .
In fact , the results with S. typhimurium or E. coli ORN103 transformants suggest that a nonfimbrial polypeptide ma bind to the fimA promoter , and similar results have been observed in the E. coli system ( 7 , 9 ) .
Further assays must be performed , using S. typhimurium mutants with known deficiencies in DNA-binding proteins , to determine the nature of any polypeptide interaction with the fimA promoter .
In E. coli the host protein integration host factor has been shown to be required for efficient fimbrial phase variation and therefore is required for fimA regulation .
It is unknown whether integration host factor is involved in the regulation of fimA in S. typhimurium .
The , B-galactosidase expression by S. typhimurium possessing pISF161 or pISF160 is interesting , since the former plasmid is a derivative of the latter ( Fig. 1 ) .
However , pISF160 transformants demonstrated no increase in 1-galac-tosidase expression compared with that of the lysogen alone and also did not produce detectable amounts offimA mRNA .
In addition , pISF160 transformants were consistently Fim-when grown under conditions that resulted in phenotypic expression of type 1 fimbriae by pISF161 transformants .
Consequently , the presence of additional fim nucleotide sequences on pISF160 results in the abrogation of fimA activation observed in pISF161 transformants .
These results demonstrate that the control of expression of fimA in S. typhimurium is due , in part , to the interaction of a number of fimbrial gene products .
A similar complex mechanism of regulation of the E. coli fimA gene is indicated ( 23 ) .
However , in E. coli two genes , fimB and fimE , immediately upstream offimA are needed for mediating the inversion of a DNA fragment containing the fimA promoter .
Genes downstream offimA have not been implicated in the control of the subunit gene .
Neither of the fimbrial polypeptides encoded by pISF161 demonstrates any significant amino-acid se-E .
coli FimB FimE , and have quence agreement to or we not been able to demonstrate that mutations within the S. typhimurium fim gene cluster immediately upstream offimA result in altered fimA expression .
Therefore , it appears that the control of fimA expression in S. typhimurium has evolved differently from that in E. coli .
However , the results of,3-galactosidase expression by the S. typhimurium and E. coli lysogens do indicate that the control of the S. typhimu-rium fimA gene is a complex process requiring more than one ancillaryfim gene and that , as in E. coli , specific Salmonella host factors also be involved .
mentioned As above , may although the E. coli XfimA-lacZ lysogen possesses its own complement of fim genes , these did not appear to affect the production of enzyme by the fusion molecule .
All transformants of the E. coli lysogen were strongly fimbriate even when grown on agar , indicating that the host fimA gene was expressed , but only two transformants produced detectable levels of,-galactosidase .
The location of a fim gene that affects fimA expression at the opposite end of the gene cluster to the site of fimA is intriguing .
This gene lies immediately downstream of a determinant encoding a 33-kDa polypeptide that is identical in size to the Salmonella fimbrial adhesin ( 20 ) .
Therefore , it is possible that this determinant is involved in integrating adhesin and fimbrial subunit assembly in S. typhimurium .
Experiments are currently under way to characterize the S. typhimurium fimbrial adhesin gene and to determine whether its expression is coupled to that of fimA .
Bullas , L. R , and J.-I .
Salmonella typhimurium LT2 strains which are r-m + for all three chromosomally located systems of DNA restriction and modification .
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In vitro gene fusions that join an enzymatically active 13-galactosidase segment to amino-terminal fragments of exogenous proteins : Esch-erichia coli plasmid vectors for the detection and cloning of translation initiation signals .
Clegg , S. , S. Hull , R. Hull , and J. Pruckler .
Construction and comparison of recombinant plasmids encoding type 1 fimbriae of members of the family Enterobacteniaceae .
Davis , R. W. , D. Botstein , and J. Roth .
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Cold Spring Harbor Laboratory , Cold Spring Harbor , N.Y. 6 .
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Dorman , C. J. , and C. F. Higgins .
Fimbrial phase variation in Escherichia coli : dependence on integration host factor and homologies with other site-specific recombinases .
Phase variation of type 1 fimbriae in Escherichia coli is under transcriptional control .
Eisenstein , B. I. , D. S. Sweet , V. Vaughn , and D. I. Friedman .
Integration host factor is required for the DNA inversion that controls phase variation in Escherichia coli .
Fried , M. , and D. Crothers .
Equilibrium and kinetics of lac repressor-operator interactions by polyacrylamide gel electrophoresis .
Garner , M. , and A. Revzin .
A gel electrophoresis method for quantifying the binding of proteins to specific DNA regions : application to components of the E. coli lactose operon regulatory system .
Gerlach , G. F. , S. Clegg , N. J. Ness , D. L. Swenson , B. L. Allen , and W. A. Nichols .
Expression of type 1 fimbriae and mannose-sensitive hemagglutination by recombinant plasmids .
Gosink , M. M. , N. M. Franklin , and G. P. Roberts .
The product of the Klebsiella pneumoniae nifX gene is a negative regulator of the nitrogen fixation ( nif ) regulon .
Harkki , A. , H. Karkku , and E. T. Palva .
Use of vehicles to isolate ompC-lacZ gene fusions in Salmonella typhimunum LT2 .
Two regulatory fim genes , fimB and fimE , control to phase variation of type 1 fimbriae in Escherichia coli .
Klemm , P. , and G. Christiansen .
Threefim genes required for the regulation of length and mediation of adhesion of Eschenchia coli type 1 fimbriae .
Yeast cell agglutination by purified enterobacterial pili .
Krogfelt , K. A. , H. Bergmans , and P. Klemm .
Direct evidence that the FimH protein is the mannose-specific adhesin of Escherichia coli type 1 fimbriae .
Krogfelt , K. A. , and P. Klemm .
Investigation of minor components of Escherichia coli type 1 fimbriae : protein chemical and immunological aspects .
Lockman , H. A. , and R. Curtiss m. 1992 .
Isolation and characterization of conditional adherent and non-type 1 fimbriated Salmonella typhimurium mutants .
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Identification and characterization of genes determining receptor binding and pilus length of Escherichia coli type 1 pili .
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A new method for sequencing DNA .
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Roles offimB andfimE in site-specific DNA inversion associated wit phase variation of type 1 fimbriae in Escherichia coli .
Experiments in molecular genetics .
Cold Spring Harbor Laboratory , Cold Spring Harbor , N.Y. 25 .
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Selective outgrowth of fimbriate bacteria in static liquid medium .
Old , D. C. , and J. P. Duguld .
Transduction of fimbriation demonstrating common ancestry in FIRN strains of Salmonella typhimurium .
Panasenko , S. M. , J. R. Cameron , R. W. Davis , and I. R. Lehman .
Five hundred-fold overproduction of DNA ligase after induction of a hybrid lambda lysogen constructed in-vitro .
Purcell , B. K. , J. Pruckler , and S. Clegg .
Nucleotide sequences of the gene encoding type 1 fimbrial subunits of KIebsiella pneumoniae and Salmonella typhimurium .
Rauvala , H. , and J. Finne .
Structural similarity of the terminal carbohydrate sequence of glycoproteins and glycolip-ids .
Sambrook , J. , E. F. Fritsch , and T. Maniatis .
Molecular cloning : a laboratory manual .
Cold Spring Harbor Laboratory , Cold Spring Harbor , N.Y. 32 .
Sanger , F. , S. Nicilen , and A. R Coulson .
DNA sequencing with chain-terminating inhibitors .
Swenson , D. L. , S. Clegg , and D. C. Old .
The frequency of fim genes among Salmonella serovars .
Weaver , R. F. , and C. Weissmann .
Mapping of RNA by a modification of the Berk-Sharp procedure .
Yanisch-Perron , C. , J. Vleira , and J. Messing .
Improved M13 phage cloning vectors and host strains : nucleotide sequences of the M13mpl8 and pUC19 vectors .