Biology Department, Concordia University, 1455 Maisonneuve Boulevard West, Montreal, Quebec, Canada H3G 1M8
Correspondence
Muriel B. Herrington
muriel{at}vax2.concordia.ca
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ABSTRACT |
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INTRODUCTION |
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Thus, E. coli switches between colonizing an animal host and water or soil. Colonization of a new habitat by bacterial cells is enhanced by their ability to attach to surfaces; such attachment is facilitated by fimbriae. E. coli can produce fimbriae called curli (Olsén et al., 1989). Salmonella enterica produces homologous fimbriae called thin aggregative fibres or SEF17 (Collinson et al., 1991
; Römling et al., 1998a
). We will use curli to refer to these fimbriae in both E. coli and S. enterica. Curli promote binding to a variety of host proteins, increase internalization of bacterial cells by eukaryotic cells in culture and trigger events leading to septic shock (Olsén et al., 1989
, 1998
; Sjöbring et al., 1994
; Ben Nasr et al., 1996
; Bian et al., 2000
; Gophna et al., 2001
; Uhlich et al., 2002
). Curli also promote clumping of bacterial cells in culture and binding to abiotic surfaces such as glass and polystyrene, making them important for biofilm formation (Vidal et al., 1998
; Römling et al., 1998b
; Austin et al., 1998
; Prigent-Combaret et al., 2000
).
Although E. coli, S. enterica serovars Typhimurium (Salty) and Enteritidis, and Shigella species have the genes required for curli synthesis, the expression of curli is not common to all strains. Many strains contain insertion sequence disruptions or deletions in the genes encoding curli components and assembly factors, or mutations elsewhere that block curli formation (Olsén et al., 1993b; Arnqvist et al., 1994
; La Ragione et al., 1999
; Uhlich et al., 2001
; Sakellaris et al., 2000
). Natural isolates of enterohaemorrhagic, enterotoxigenic and sepsis E. coli strains from humans, and isolates from avian and bovine infections, are frequently curliated, whereas those from enteroinvasive and enteropathogenic strains are not (Olsén et al., 1989
, 1993a
; Maurer, 1998
; Bian et al., 2000
; Uhlich et al., 2001
). E. coli K-12 strains generally are not curli-proficient but can produce curli as a result of mutations (Arnqvist et al., 1992
, 1994
; Vidal et al., 1998
). Expression of curli in E. coli and Salty normally occurs during stationary phase and requires growth at low temperature and low osmolarity, but some strains and S. enterica serovar Enteritidis can express curli at 37 °C (Olsén et al., 1989
; Collinson et al., 1991
; Römling et al., 1998b
; Uhlich et al., 2001
).
The csg (agf in S. enterica) genes are required for curli synthesis. They are organized in two adjacent divergently transcribed operons. The csgBA (agfBA) operon contains the gene (agfB) for a nucleator protein and the gene (agfA) for curlin, the major protein component of the curli fibre. The csgDEFG (agfDEFG) operon contains a putative regulatory gene (csgD), a gene (csgG) encoding a lipoprotein involved in secretion of curlin and CsgB, a gene (csgE) encoding a chaperone-like protein and a gene (csgF) encoding a second nucleator protein (Collinson et al., 1996, 1997
; Hammar et al., 1995
, 1996
; Loferer et al., 1997
; Römling et al., 1998a
; Chapman et al., 2002
).
Changes in the csgD (agfD) promoter region can influence curli formation. Some isolates of the enteroinvasive E. coli O157 : H7 show a phase shift between curliated and non-curliated cells that results from a single base pair change in the csgD promoter (Uhlich et al., 2002). A change in the agfD promoter in Salty enables the cells to make curli at 37 °C (Römling et al., 2000
).
The csgD-encoded protein is homologous to proteins of the FixJ/UhpA/LuxR family, suggesting that it is regulatory protein (Hammar et al., 1995). This role is supported by the observations that inactivation of csgD prevents the transcription of the csgBA operon (Hammar et al., 1995
) and of adrA, a putative regulatory gene required for cellulose synthesis (Römling et al., 2000
; Zogaj et al., 2001
). Cellulose is found in the matrix of E. coli and Salty biofilms. The FixJ/UhpA/LuxR family is characterized by a DNA-binding C-terminal domain and an N-terminal receiver domain. Depending on the regulator, the receiver domain is either phosphorylated or binds homoserine lactone or other small molecules (Kahn & Ditta, 1991
; Sitnikov et al., 1995
; Baikalov et al., 1996
; Ducros et al., 2001
). As yet there is no evidence for DNA binding by CsgD, so it is not known whether it controls the csgBA operon and the adrA gene directly or indirectly. Similarly, it is not known what ligand modulates its function. Expression of csgD or agfD increases in late exponential phase and is influenced by temperature, osmolarity and the availability of nutrients, oxygen and iron (Römling et al., 1998b
; Prigent-Combaret et al., 2001
; Gerstel & Römling, 2001
).
In this study, we show that csgD is a multicopy suppressor of the glycine auxotrophy of a strain lacking dihydrofolate reductase (DHFR) activity. DHFR is required for the de novo synthesis of tetrahydrofolate (THF) and for the recycling of dihydrofolate produced during thymidylate synthesis. THF is converted to methylene THF during glycine sythesis or degradation. Methylene THF is required for thymidylate and pantothenate biosynthesis and is the precursor for methyl-THF and formyl-THF. Methyl-THF is required for methionine biosynthesis. Formyl-THF is used for purine (and, indirectly, histidine) synthesis and for the formylation of Met-tRNAfmet (reviewed by Matthews, 1996).
Strains with the DHFR gene (folA) deleted grow slowly on rich or supplemented minimal medium. The supplements required depend on the strain background, but most strains will grow with thymidine or a combination of folate end-products (FEP: thymine, glycine, histidine, methionine, adenine and pantothenic acid) (Ahrweiler & Frieden, 1988; Howell et al., 1988
; Hamm-Alvarez et al., 1990
; Krishnan & Berg, 1993
; Herrington & Chirwa, 1999
). When glycine or glycine and histidine are omitted from the mixture the cells grow extremely slowly, and when methionine or methionine and histidine are omitted the cells do not grow. Under these conditions the purine pathway is repressed and this could reduce turnover of formyl-THF (Herrington & Chirwa, 1999
). We show here that plasmids containing csgD sequences enabled faster growth in the absence of glycine. This results from an increased expression of glyA, the gene encoding serine hydroxymethyltransferase (SHMT, EC 2 . 1 . 2 . 1), suggesting that csgD also regulates glyA.
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METHODS |
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Genetic and molecular techniques.
P1 transductions (Miller, 1992) were performed with either P1CM or P1vir. P1CM lysates prepared from JC1089 were used to lysogenize other strains as needed. P1vir was obtained from C. G. Cupples, Concordia University, Montreal, Canada.
Normally, cells were rendered competent for transformation using CaCl2 (Sambrook, 1989). To avoid heat shock, the one-step PEG method (Chung et al., 1989
) was used to make strain GS162
glyAlacZ and its derivatives competent.
Strain construction.
P1vir-mediated transduction was used to transfer the purR6 : : Tn10 mutation from strain SØ5052 to strain MH829. We constructed strains MH910 and MH911 by, respectively, transducing csgA2 : : Tn105 and csgG1 : : Tn105 mutations into strain MH829 with P1vir lysates made from strains MHR204 and MHR210. Strains MH901 and MH902 were obtained by transferring these mutations to strain GS162glyAlacZ. P1CM lysates prepared on strain FB10186 were used to transduce the csgD : : Tn5<KAN-I-SceI> mutation to strains MH917 and GS162
glyAlacZ. Construction of the spectinomycin-resistant folA null mutant MH917 was similar to that of strain MH829 (Herrington & Chirwa, 1999
).
Isolation and identification of multicopy suppressors.
Strain PH43 is a double lysogen for Mu cts and the mini-Mu replicon MudII4042 which confers chloramphenicol resistance on the host strain (Groisman & Casadaban, 1987
). Lysates from strain
PH43 were used to prepare mini-Mu lysogens of MG1655. Mini-Mu lysates prepared on strain MG1655 were then used to transduce strain MH829/Mu cts at 31 °C. Chloramphenicol-resistant colonies were selected and then were replica-plated to screen for growth on various media.
Suppressing plasmids were restricted and the resulting fragments were ligated into pUC18. DH5 was transformed with the ligation mixture. Recombinant plasmids were tested for their effect on growth of strain MH829. Inserts in suppressor plasmids were sequenced by the York University Core Molecular Facility, Toronto, Canada.
Enzyme assays.
Cells were grown at 37 °C to an OD600 between 0·4 and 0·8 for all enzyme assays. Threonine dehydrogenase assays were measured in toluene-permeabilized cells (Ravnikar & Somerville, 1987). The SHMT- (Taylor & Weissbach, 1965
) and glycine-cleavage activities were assayed in crude extracts prepared by sonicating cells. Glycine cleavage was assayed by a modification of the SHMT assay. The reaction mix contained 50 mM potassium phosphate, pH 7·4, 5 mM DTT, 1·14 mM THF, 1 mM pyridoxal phosphate and 2-[14C]glycine (0·16 mCi mM-1). Reactions were incubated at 32 °C. Reactions were stopped and analysed as for SHMT. Protein was measured by the modified bicinchoninic acid procedure (Brown et al., 1989
).
-Galactosidase activity in reporter strains for glyA (Lorenz & Stauffer, 1995
) and gcvT (Jourdan & Stauffer, 1998
) expression was assayed as described by Miller (1992)
using the SDS/chloroform lysis method.
Curli formation.
Curli proficiency was assayed on Congo red plates (Hammar et al., 1995), by the ability of cells to bind to polystyrene (Vidal et al., 1998
) and by the binding of Congo red in solution (Gophna et al., 2001
). Congo red binding units were 1000 (
A500/OD600) where
A500 was the difference between the A500 value of the Congo red solution without cells and the A500 value measured after cells had been incubated in the solution and then removed. OD600 measurements for these assays were made on Milton Roy Spectronic 1001 Plus apparatus, using a 1 cm path length. Cells were diluted in 0·9 % saline.
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RESULTS |
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Mini-Mu plasmids can carry insertions of up to 22·3 kb (Groisman & Casadaban, 1987). To reduce the size of the inserts, HindIII fragments of suppressing plasmids were subcloned into the high-copy-number vector pUC18. The resultant recombinant plasmids were tested for their ability to correct the growth defect of strain MH829.
One recombinant plasmid, pSD6P, allowed growth on FEP-Gly but not on FEP-Met. It contained a 210 bp insert. The sequence of the insert was determined and a BLAST (Altschul et al., 1997) search of the E. coli genome sequence indicated that it matched part of the csgD gene (GenBank accession no. AE000205). When pSD6P was analysed for ORFs, two were observed. One corresponded to the bla gene of pUC18 (GenBank accession no. L08752). The other, which is in the opposite orientation to the sequence encoding the lacZ
fragment, initiated at the complement of nt 499 of pUC18, spanned the insert and terminated at complement of nt 143 of pUC18. The predicted chimaeric protein contains 70 aa corresponding to the central region of CsgD (Fig. 1
). The presence of a weak promoter located at the complement of nucleotide 746 of pUC18 suggests the chimaeric ORF could be transcribed.
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Plasmids containing intact csgD genes suppress
Colony formation on LB-Thy and FEP-Gly was monitored in strain MH829 transformed with different plasmids containing csgD or agfD sequences and with control plasmids. All transformants produced small colonies within 24 h on LB-Thy (Fig. 2a and Table 2
). Transformants with pSD6P (partial csgD), pCP994 (intact csgD coding region) or pCSGD (agfD from S. enterica serovar Typhimurium) formed colonies on FEP-Gly within 4 days. In contrast, the pUC18 control made no colonies (pUC18) and the pKK233-2 control made extremely small colonies after 5 days (Fig. 2b
and Table 2
). The efficiency of plating on LB-Thy and on FEP-Gly was similar. These results indicate that multiple copies of the intact csgD gene also suppress the glycine auxotrophy.
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Growth in the presence of purines and methionine normally represses SHMT (Mansouri et al., 1972; Miller & Newman, 1974
; Greene & Radovich, 1975
). We observed repression on FEP-Gly with strain GS162
glyAlacZ transformants, but not with the strain MH828 transformants. This suggests that the thyA mutation in strain MH828 affected the regulation of SHMT. Under the conditions used, strains with the thyA(ts) allele normally express 5 % of wild-type thymidylate synthase activity (Herrington & Chirwa, 1999
). The reduced demand for methylene THF for thymidylate synthesis could be perceived by the cell as a signal to provide more formyl-THF for protein synthesis; hence the upregulation of SHMT.
Expression of -galactosidase from a glyAlacZ fusion is also increased
Strain GS162glyAlacZ is lysogenized with the glyA reporter,
glyAlacZ. This phage contains the entire hmpglyA intergenic region and the sequence encoding the first 50 aa of SHMT fused in-frame with the lacZ gene (Lorenz & Stauffer, 1995
).
-Galactosidase activity (Table 4
) in strain GS162
glyAlacZ transformed with pKK233-2 and pCP994 paralleled SHMT activity (Table 3
). In all media tested, the presence of pCP994 elicited 1·5- to 3-fold more
-galactosidase activity than the control plasmid pKK233-2. A similar but smaller increase (1·31·4-fold) in
-galactosidase activity was observed with pSD6P transformants compared to pUC18 transformants (data not shown).
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Increased SHMT activity was sufficient for growth of strain MH829 on FEP-Gly
Several lines of evidence indicate that increasing the SHMT activity above a threshold level is sufficient to enable strain MH829 to make enough glycine and methylene THF to grow on FEP-Gly. SHMT activity could be increased by transforming cells with the glyA-containing plasmid, pGS29 (Stauffer et al., 1981), by inactivating the PurR repressor by mutation of purR (Steiert et al., 1990
), or by growth in FEP-His-Gly (Table 4
). Strain MH829 grows on FEP-His-Gly (Herrington & Chirwa, 1999
) and grew on FEP-Gly when transformed with pGS29 or made purR- (Table 2
), indicating that the increase in SHMT activity was sufficient to compensate for the lack of glycine in the medium.
Are there alternative sources of glycine in cells transformed with a csgD-containing plasmid?
Glycine can also be produced from threonine via threonine dehydrogenase (Fraser & Newman, 1975; Ravnikar & Somerville, 1987
) or can be spared by reducing glycine cleavage (Stauffer, 1996
). We assayed threonine dehydrogenase activity and glycine-cleavage activity in transformants of MH829 and
-galactosidase activity in transformants of GS162
glyTlacZ. These activities were very low, and there were no reproducible differences between control and csgD transformants when transformants were grown in FEP-Gly (data not shown). Therefore, glycine was not provided by either of these routes.
Are curli produced in transformants?
Curli are not expressed by most laboratory strains of E. coli, but expression of csgD from a plasmid induces their formation (Prigent-Combaret et al., 1999). We tested for curli formation by spotting cells on Congo red plates (Hammar et al., 1995
), by measuring Congo red binding (Gophna et al., 2001
) and by monitoring binding to polystyrene (Prigent-Combaret et al., 1999
). On Congo red plates, spots of strains MH829 (
folA : : kan3) and MH828 (folA+) were white, indicating that they do not produce curli (data not shown). Both strains transformed with the control plasmids pUC18 and pCP994 pKK233-2 were white, whereas pSD6P and pCP994 transformants were red (Fig. 2c
and data not shown). Strain MH828 transformed with control plasmids pUC18 and pKK233-2 bound 49±6·5 and 47±0·1 Congo red units, respectively, whereas pSD6P and pCP994 transformants bound 67±7·0 and 96±32 units, respectively. The csgD plasmids also promoted binding to plastic whereas the control plasmids did not (data not shown). These results indicate that our strains made curli when the csgD plasmids were present.
Does strain MH829 have to produce curli to grow on FEP-Gly?
To determine whether the ability to make curli was necessary for growth on FEP-Gly or increasing glyA expression we constructed csgA- and csgG- derivatives of strains MH829 and GS162glyAlacZ. Both genes are essential for curli biogenesis. Transformants of strains MH910(
folA csgA) and MH911(
folA csgG) were tested for growth on FEP-Gly (Table 2
). The csg mutations did not affect growth.
-Galactosidase activity was higher in pCP994 transformants of the glyAlacZ fusion strains MH901 and MH902 than in control transformants (Table 4
). We conclude that formation of curli fibres was not necessary for the CsgD-mediated suppression of glycine auxotrophy or elevation of glyA expression.
Is chromosomally encoded CsgD involved?
To determine if the chromosomal csgD gene was required for suppression by the plasmids pSD6P and pCP994, we constructed csgD- derivatives of strains MH917 and GS162glyAlacZ, and transformed the resultant strains with pSD6P, pCP94 and control plasmids. The pCP994 transformants of strain MH937 (
folA : : aadA csgD-) grew as well as transformants of MH829 (Table 2
) and were red on Congo red plates (data not shown). In contrast, pSD6P transformants grew poorly and did not stain as strongly on the Congo red plates. The activity of
-galactosidase in pCP994 transformants of the csgD- reporter strain MH938 was higher than in the controls (Table 5
) and the magnitude of the increase was similar to that observed in the csgD+ strain GS162
glyAlacZ (Table 4
). In contrast, pSD6P enhanced the activity slightly in two of the three growth conditions tested (Table 5
). Taken together, these results suggest that the chimaeric protein coded by pSD6P is not sufficient for optimum suppression, curli formation and glyA expression, and that it influences the expression or activity of the chromosomally encoded CsgD protein.
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DISCUSSION |
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The glycine auxotrophy of strain MH829 can also be alleviated by mutations resulting in curli proficiency. Curli-proficient mutants of strain MH829 grew on FEP-Gly, and some mutants selected for growth on FEP-Gly were curli-proficient (T. MacRae, C. Zambrana, N. T. Chirwa and M. B. Herrington, unpublished results). We have not yet tested whether glyA is upregulated in these mutants, but it is reasonable to expect that some could express higher levels of SHMT.
We propose that upregulation of glyA is an integral response to signals eliciting curli formation. Cells make curli in stationary phase, suggesting that the demand for amino acids is high at a time when their availability could be limited. Glycine makes up 11·5 % of the amino acid residues in total E. coli protein (Neidhardt & Umbarger, 1996). Curlin, the major protein of curli, contains 1·7 times as much glycine, and cells make a lot of curli, suggesting the demand for glycine is particularly high. Thus, increasing the cell's ability to make glycine could facilitate its production of curli.
Expression of csgD is highly regulated. A variety of environmental factors including nutrient limitation, low osmotic strength, low temperature, microaerophilic conditions and iron limitation increase expression of csgD or its Salty homologue, agfD (Römling et al., 1998b; Prigent-Combaret et al., 2001
; Gerstel & Römling, 2001
). At least four regulatory proteins (RpoS, OmpR, Cpx and Mlr) control CsgD expression (Arnqvist et al., 1994
; Brown et al., 2001
; Prigent-Combaret et al., 2001
) (Fig. 4
). AgfD is detectable by Western blotting in late exponential and stationary phases, but transcript measurements and reporter constructs suggest low levels of AgfD and CsgD are made even during exponential phase (Prigent-Combaret et al., 2001
; Gerstel & Römling, 2001
).
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In the cell, CsgD presumably can have ligand bound to it (CsgD[L]) or not (CsgD[L]). We propose that CsgD[ ] activates transcription of target genes whereas CsgD[L] is inactive (Fig. 4). Normally, growing cells contain very little CsgD (Prigent-Combaret, 2001
) so CsgD[L] predominates. In curli-proficient cells, activation of csgDEFG transcription by OmpR, MlrA or other activators results in increased amounts of CsgD so that the active form predominates. CsgD[L] then activates transcription of csgBA, adrA and possibly other genes. In curli non-proficient cells, CsgD[L] predominates because either ligand levels are high or csgD expression remains very low.
When curli non-proficient cells are transformed with pCP994, the amount of CsgD made is higher than the amount of ligand so CsgD[L] will activate curli synthesis and glyA expression. In cells transformed with pSD6P, the chimaeric protein will bind the ligand and there will be a mixture of chimaeric protein and chromosomally encoded CsgD in the activator form. The net activation will be weaker than in cells transformed with pCP994 because the chimaeric protein is a poor activator. This is demonstrated by eliminating CsgD. In the csgD null mutant, the response to pSD6P was weaker than in the csgD+ strain.
How does CsgD regulate glyA expression? CsgD[L] could activate glyA directly. Alternatively, it could alter known regulation either by binding one of the ligands or by altering expression of one of the regulatory proteins. MetR and its co-activator, homocysteine, activates transcription of glyA and PurR and its co-repressor, guanine or inosine, represses transcription. Other, as yet unidentified, factors also influence glyA transcription ((Stauffer, 1996). A search of the Conserved Domain Database (Marchler-Bauer, 2002
) using the N-terminal region of CsgD returned no hits, suggesting that the receiver domain does not resemble any proteins known to bind homocysteine, guanine, inosine or a related molecule. This suggests that CsgD does not titrate out a known ligand needed for glyA expression. CsgD[L] could enhance expression of glyA by activating metR or repressing purR (Fig. 4
).
Our results are the first to demonstrate that CsgD alters the expression of a gene not directly involved in multicellular behaviour. Our observation that a chimaeric protein containing only 70 aa of CsgD is sufficient to stimulate curli formation is of interest because it defines a functionally important region of the protein.
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ACKNOWLEDGEMENTS |
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Received 28 June 2002;
revised 25 October 2002;
accepted 30 October 2002.