Vol. 172, No. 5
JOURNAL OF BACTERIOLOGY, May 1990, p. 2594-2600
0021-9193/90/052594-07$02.00/0 Copyright C) 1990, American Society for Microbiology
Cloning and Expression of the Bacteroides fragilis TAL2480 Neuraminidase Gene, nanH, in Escherichia coli THOMAS A. RUSSO,1t JEFFREY S. THOMPSON,2 VERONICA G. GODOY,2 AND MICHAEL H. MALAMY2*
Department of Geographic Medicine and Infectious Diseases, New England Medical Center,1 and Department of Molecular Biology and Microbiology, Tufts University Health Sciences Campus,2 136 Harrison Avenue, Boston, Massachusetts 02111 Received 6 October 1989/Accepted 10 February 1990
We have cloned the Bacteroides fragilis TAL2480 neuraminidase (NANase) structural gene, nanH, in Escherichia coli. This was accomplished by using the cloning shuttle vector pJST61 and a partial Sau3A library of TAL2480 chromosomal inserts created in E. coli. The library was mobilized into the NANase-deficient B. fragilis TM4000 derivative TC2. NANase-producing colonies were enriched by taking advantage of the inability of TC2, but not the wild-type or NANase+ revertant, to grow in vitro in fluid aspirated from the rat granuloma pouch. Plasmids pJST61-TCN1 and pJST61-TCN3, containing inserts of 9.1 and 4.5 kilobases (kb), respectively, were found in the TC2 derivatives that grew in the rat pouch medium. In B. fragilis, NANase production from the two plasmids was inducible by free N-acetylneuraminic acid or sialic acid-containing substrates, just as in the parental TAL2480 strain. However, when these plasmids were transferred back to E. coli, NANase activity was barely detectable. A 3.5-kb portion of the insert in pJST61-TCN3 was subcloned in pJST61 to give plasmid pJST61-SC3C; NANase was produced from this plasmid both in E. coli and in B. fragilis. In E. coli, NANase expression was under the control of the vector promoter lambda PR and was therefore completely abolished by the presence of a lambda prophage. In B. fragilis, NANase production was inducible by free N-acetylneuraminic acid or sialic acid-containing substrates. By using deletion analysis and TnlOOO mutagenesis, the NANase structural gene and control region that functions in B. fragUis were localized to a 1.5- to 2.0-kb region of the insert. A partial nucleotide sequence of the NANase-deficient Tnl000 insertion mutants allowed us to identify the nanH gene and deduce the amino acid sequence of a portion of the NANase protein. We identified five regions showing great similarity to the Asp boxes, -Ser-X-Asp-X-Gly-X-Thr-Trp-, of other bacterial and viral NANase proteins.
Although Bacteroidesfragilis is a minor component of the normal intestinal flora of humans, it is the predominant obligately anaerobic organism isolated from intraabdominal infections (10). The high frequency of B. fragilis involvement at these sites reflects the actions of several virulence factors. The enzyme neuraminidase (NANase), the product of the nanH gene, is found in many pathogenic bacteria, including Vibrio cholerae (24), Salmonella typhimurium (18, 24), Corynebacterium diphtheriae (15), Streptococcus pneumoniae (3), and B. fragilis (9). It has been suggested that NANase plays a role in the pathogenicity of these bacteria as well as in infections caused by the influenza virus (1) and the protozoan Trypanosoma cruzi (6). The enzymatic activity of NANase, a glycohydrolase, cleaves sialic acid residues occurring at the termini of simple and complex oligosaccharides and on glycoproteins. Sialic acids are widely distributed on the surfaces of eucaryotic cells as terminal nonreducing residues and on important glycoproteins, such as immunoglobulin G and the Clq component of complement (5). Removal of sialic acid residues from the surfaces of cells and from these important glycoproteins may serve the nutritional requirements of the bacteria as well as disrupt many host functions (20). We have demonstrated that NANase activity in cultures of B. fragilis can be induced by free or bound sialic acid (D. Crook, T. A. Russo, F. P. Tally, and M. H. Malamy, unpublished data). To evaluate the possible role of NANase
in the pathogenesis of B. fragilis infections, it is necessary to use derivatives of B. fragilis that differ from the wild-type strain only by alterations in NANase production. Comparisons between otherwise isogenic strains could then be made in a variety of biological systems. As a first step in this process, we have cloned the B. fragilis TAL2480 NANase gene in E. coli by using a shuttle vector system recently
developed in this laboratory (22). This has allowed us to study the expression and regulation of NANase production in E. coli and B. fragilis and to identify the nanH gene and a portion of the sequence of the NANase protein. MATERIALS AND METHODS Bacterial strains and plasmids. B. fragilis and E. coli strains used in this study are summarized in Table 1. The construction and properties of the cloning vector pJST61 are presented in detail by Thompson and Malamy (22), and a partial map of the plasmid is given in Fig. 1. Growth media. (i) Enriched media. ML liquid and solid media for growth of E. coli and brain heart infusion broth (BHIS) and solid medium for growth of B. fragilis were prepared as previously described (12, 17). (ii) Minimal media. For growth of E. coli, medium M63 was supplemented with 0.002% Casamino Acids (Difco Laboratories, Detroit, Mich.) and 0.5% glucose or a combination of 0.5% N-acetylneuraminic acid (NANA; Sigma Chemical Co., St. Louis, Mo.) and 0.01% glucose as the carbon source. For growth of B. fragilis, AMM (23) with 0.5% glucose or 0.5% NANA was employed. AMM with 1% porcine gastric mucin (Sigma) and 5% laked sheep erythrocytes was also used. Rat pouch medium for growth of B.
* Corresponding author. t Present address: LCI, National Institute of Allergy and Infec-
tious Diseases, Bethesda, MD 20892. 2594
VOL. 172, 1990
B. FRAGILIS TAL2480 NEURAMINIDASE
2595
TABLE 1. Bacterial strains and plasmids Plasmid or bacterial strain
Plasmids pJST61 pJST61-TCN1 pJST61-TCN3 pJST61-SC3C pJST61-SC3C nanH: :TnO000 pJST61-SC3C::Tnl000 pTAR100 RK231 F' lac+
Construction or source
Relevant characteristics
Reeatcaatrsis(reference) Cloning shuttle vector, Ampr Clnr NANase+ in B. fragilis NANase+ in B. fragilis NANase+ in B. fragilis and E. coli NANase- TnlOO0 insertions NANase+ TnJOO0 insertions NANase- deletion of pJST61-SC3C Tra+, RP4 derivative, Kanr Tetr Amps Source of TnlOOO
Thompson and Malamy (22) This study This study Derived from pJST61-TCN3 Derived from pJST61-SC3C Derived from pJST61-SC3C Derived from pJST61-SC3C Guiney et al. (11) Laboratory strain
DW1030 KS303
recA rpsL recA rpsL (lambda) recA rpsE KS272, lpp-SSO8
Maniatis et al. (13) HB101 lysogenized with lambda wild type Robillard et al. (17) Strauch and Beckwith (21)
B. fragilis TAL2480
FoxjImpr, NANase+
Tufts Anaerobe Laboratory clinical isolate (7, 22) Strain 838rfm from M. Sebald, Paris, France Derived from TM4000 after NTG' mutagenesis Spontaneous NANase+ revertant from TC2
E. coli HB101
HB1O1(lambda)
TM4000 TC2 TClRb
Rifr, standard wild-type strain NANaseNANase+
a NTG, N-methyl-N'-nitro-N-nitrosoguanidine.
fragilis was obtained by aspirating the liquid contents from the pouch of the rat pouch granuloma model (8). The sterile aspirant was used immediately without further treatment or after storage at -70°C. When used, antibiotics were added to media at the following concentrations (,ug/ml): ampicillin, 200; gentamicin, 50; streptomycin, 200; clindamycin (CLN), 6; and ciprofloxacin, 1. Ciprofloxacin was obtained from Miles Pharmaceuticals, West Haven, Conn.; CLN was obtained from The Upjohn Co., Kalamazoo, Mich.; and all other antibiotics were purchased from Sigma Chemical Co. All cultures were incubated at 37°C. B. fragilis strains were grown in an anaerobic chamber (Coy Laboratory Products, Ann Arbor, Mich.) in an atmosphere of 85% N2, 10% C02, and 5% H2. Plasmid mobilization and transformation. Transfer of pJST61 and its derivatives from E. coli to B. fragilis was accomplished by conjugation, with RK231 as the mobilizing transfer factor (11). Filter matings were performed for 18 h under aerobic conditions. After suspension of the cells from the filter, the mixtures were plated to select for transconjugants containing pJST61 or its derivatives on BHIS plates with CLN, gentamicin, and streptomycin or BHIS with CLN and ciprofloxacin and grown under anaerobic conditions. Transformation of plasmid DNA into E. coli cells was performed by the protocol of Maniatis et al. (13). Plasmid isolation and analysis. Plasmids were purified from E. coli and B. fragilis strains by using the alkaline lysis technique described by Maniatis et al. (13) or by CsClethidium bromide equilibrium centrifugation by the method of Clewell and Helinski (4). All restriction enzymes were purchased from New England BioLabs, Inc. (Beverly, Mass.) and used as recommended by the supplier. Restriction digests were analyzed by electrophoresis on 0.9% horizontal agarose (SeaKem; FMC Corp., Marine Colloids Div., Rockland, Maine) gels after staining with ethidium bromide.
Individual DNA fragments used in subcloning and hybridization experiments were purified from 0.7% low-meltingpoint agarose (SeaPlaque; FMC Corp.) gels (13). Construction of DNA libraries from B. fragiis TAL2480. Chromosomal DNA from B. fragilis TAL2480 was purified by using CsCl-ethidium bromide centrifugation. A partial Sau3A digest of this DNA was fractionated on a 10 to 40% (wt/vol) sucrose gradient to yield two DNA pools containing fragments from 1 to 15 kilobases (kb) and >15 kb, respectively. DNAs from these pools were ligated to the unique BglII site within the endRI gene of pJST61 (Fig. la) (22). Ligation mixes were used to transform E. coli HB101 (RK231) and KS303(RK231) for Ampr colonies. Because of the lethality of the endRI gene product, endonuclease EcoRI, in E. coli lambda nonlysogens, only transformants containing a pJST61 plasmid with a B. fragilis chromosomal insert survive. The colonies from plates containing many Ampr transformants were resuspended in ML broth to create the TAL2480 chromosomal library. Assays for NANase activity. Qualitative plate assays were performed by a modification of the method of Myers et al. (16) and used filter paper (3MM; Whatman, Inc., Clifton, N.J.) moistened with a solution of 0.17 M sodium acetate buffer (pH 5.4) containing 2'(4-methylumbelliferyl)N-acetylneuraminic acid (Sigma) (1 ,iM for B. fragilis and 2 ,uM for E. coli). Colonies to be tested were overlaid with the filter paper, which was then removed, and incubated at 37°C (1 min for B. fragilis and 10 min for E. coli). The reaction was stopped by soaking the filter paper in a solution containing 0.085 M glycine and 0.2 M sodium carbonate (pH 9.4). Colonies containing significant amounts of NANase activity were detected by their fluorescence when examined by long-wave-length UV light. Quantitative NANase assays were performed by a minor modification of the method of Warner and O'Brien (25): 0.08 ml of 0.17 M sodium acetate buffer (pH 5.4), 0.01 ml of 1 mM 2'(4-methylumbelliferyl)Nacetylneuraminic acid, and 0.01 ml of cellular extract were mixed on ice and then incubated at 37°C for 10 min. The
2596
RUSSO ET AL.
pBR322 Ori
bla
a pJST61 11.4 kb
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endRI Pr -m_
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pJST61 -TCN3 15.9 kb C
~ ~ ~ IL B. fragilis pBFTM2006
ermF
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J. BACTERIOL.
B2
-
FIG. 1. (a) Restriction and functional map of the cloning vector pJST61 as described previously (22). The bla gene leads to Amp resistance in E. coli, while the ermF gene leads to CLN resistance in B. fragilis. The RK2 oriT sequence permits efficient trans-mobilization of the plasmid from E. coli to E. coli or B. fragilis recipients. The endRI gene under the control of the lambda PR promoter results in efficient expression of endonuclease EcoRI product in E. coli cells lacking lambda repressor. (b) Plasmid pJST61-TCN3 contains a 4.5-kb insert of chromosomal DNA from B. fragilis TAL2480. (c) Plasmid pJST61-SC3C was derived by subcloning a Sau3A restriction fragment from the BglII-BamHI fragment of pJST61-TCN3, as indicated by the arrows, into the BglII site of pJST61. (d) Plasmid pTAR100 was derived from pJST61-SC3C by deleting the BstEII fragment starting within pBFTM2006 up to the BstEII site at 1.75 kb within the insert. All restriction maps are drawn to scale. Restriction enzyme abbreviations: A, AvaI; B2, BglII; BE2, BstEII; BYI, BstYI; C, ClaI; E, EcoRI; H, HindIll; HI, BamHI; H2, HaeII; N/X, NheI-XbaI; P, PstI; Sau3A, Sau3AI.
reaction mixture was placed on ice and stopped with 1.9 ml of a solution containing 0.085 M glycine and 0.2 M sodium carbonate (pH 9.4). Fluorescence was measured on a fluorometer. Suitable enzyme dilutions were used so that fluorescence was measured in the linear region of the enzyme process curve. NANase-specific activity is expressed as fluorescence units per milliliter per milligram of protein. The same assay conditions were used for B. fragilis or E. coli extracts.
Transposon TnlOOO mutagenesis. Transposon TnO000 mutagenesis of pJST61 derivatives with B. fragilis chromosomal inserts was performed as previously described (22). Transconjugants were screened for NANase activity by using the qualitative plate test; NANase-deficient candidates were confirmed by using the quantitative assay. The location of the Tn1000 inserts was determined by restriction analysis. Construction of a deletion derivative of pJST61-SC3C. pTAR100 (Fig. ld) carries a 4.55-kb deletion extending from the BstEII site in the pBFTM2006 portion of pJST61 to the first BstEII site in the insert, thereby deleting the 1.75 kb of TAL2480 DNA most distal to PR' It was constructed by partial digestion of the parental plasmid with BstEII, followed by transformation of the cleaved DNA without ligation into HB101. Several Ampr but NANase-deficient candidates were isolated. The extent of the deletion in pTAR100 was established by restriction analysis. DNA sequencing. Sequencing of double-stranded CsCl-
purified DNA was performed by the dideoxynucleotidechain termination method of Sanger et al. (19) with Sequenase (U.S. Biochemical Corp., Cleveland, Ohio). Oligonucleotide primers complementary to unique sequences near the ends of Tn1000 were as previously described (22). RESULTS Enrichment protocol to isolate clones expressing NANase activity in B. fragiis. We have determined that the fluid aspirated from a sterile rat granuloma pouch can serve as a complete growth medium for B. fragilis TM4000. A plateau level of greater than 2 x 109 CFU/ml is reached from a low inoculum (2 x 106 CFU/ml) in 20 h at 37°C in the anaerobic chamber (Fig. 2). Under these growth conditions, NANase activity is fully induced. In contrast, the NANase-deficient strain TC2, which contains multiple mutations including the inability to synthesize NANase and a requirement for cysteine (unpublished observations), grows poorly in the rat pouch fluid with or without cysteine (0.5 mg/ml), reaching levels of only 2 x 107 CFU/ml even after prolonged incubation. We determined that the lack of NANase was an important factor in the inability of TC2 to grow in this medium, since strain TClRb, a spontaneous revertant of TC2 with restored NANase activity, grows as well as TM4000 in the rat pouch fluid if the fluid is supplemented with cysteine. We used the inability of TC2 to grow to high
VOL. 172, 1990
B. FRAGILIS TAL2480 NEURAMINIDASE
9.1 or 4.5 kb (data not shown); a representative of the larger insert was chosen and designated pJST61-TCN1, and a plasmid with the smaller insert'was designated pJST61TCN3. Restriction analysis showed that the 4.5-kb insert was contained within the 9.1-kb insert; therefore, further studies used pJST61-TCN3 exclusively. To verify that pJST61-TCN1 and pJST61-TCN3 carried the NANase gene, these plasmids were introduced into E. coli HB1O1(RK231) by transformation and again mobilized to TC2. All of the resultant Clnr B. fragilis transconjugants acquired NANase activity. When the same procedure was followed by using KS303(RK231) cells carrying the B. fragilis TAL2480 chromosomal library of small (1- to 15-kb) Sau3A fragments, no cells capable of growth in the rat pouch fluid were found. Subcloning and mapping the B. fragiis NANase structural gene. Plasmid pJST61-TCN3 containing the 4.5-kb chromosomal insert was used for detailed restriction mapping and subcloning. A partial restriction map of the 4.5-kb insert is presented in Fig. lb. A 3.8-kb BglII-BamHI fragment from the insert in pJST61-TCN3 was purified by gel electrophoresis and digested to completion with Sau3A. The digestion products were purified'from agarose gels and ligated into the unique BglII site of pJST61. The DNA was used to transform E. coli KS303 to Ampr. Screening of transformants for NANase activity in E. coli led to the isolation of pJST61SC3C, which contains the leftmost 3.5-kb of the 4.5-kb insert (Fig. lc). Plasmid pJST61-SC3C was used as the target for Tn1000 mutagenesis' to further localize the NANase gene. The positions of TnJ000 insertions are given in Fig. 3. 'Insertions within a 2.2-kb portion of the 3.5-kb fragment led to loss of NANase activity in E. coli. The leftmost 0.8 kb of the' insert is not necessary for NANase expression in' E. coli, since strains carrying Tn1000 insertions in this region still produced NANase. Plasmid pTAR100 (Fig. ld) which lacks the lefthand portion of the chromosomal insert up to the BstEII site at 1.75 kb did not produce NANase in either E. coli or B.
101 C 109 U-
U9
TC2
10 7
106 20
0
40
60
80
Time (hours) FIG. 2. Growth of wild-type B. fragilis TM4000 and mutants TC2 and TClRb in rat pouch fluid. Cultures were grown in BHIS broth to mid-log stage, harvested by centrifugation, washed with MPBS, and then used to inoculate 5 ml of fresh rat pouch aspirant in 50-ml flasks. For TC2 and TClRb, cysteine (0.5 mg/ml) was added before incubation. Cells were grown in the anaerobic chamber at 37°C. Samples were taken and plated on BHIS solid medium for viable counts.
density in rat pouch fluid supplemented with cysteine as the selective condition to isolate plasmids containing an expressed NANase gene. A B. fragilis TAL2480 chromosomal library with an insert size of >15 kb in pJST61 (see Materials and Methods) was mobilized from E. coli KS303(RK231) to B. fragilis TC2, selecting for Clnr. Approximately 103 transconjugant colonies were resuspended in BHIS broth, and 1.7 x 103 CFU of this resuspension was inoculated into 1 ml of rat pouch fluid and incubated anaerobically. After 23 h of incubation, the cell titer had increased 3 orders of magnitude to 1.2 x 106 CFU/ml. Individual colonies isolated from the initial inoculum and the 23-h cultures were tested for NANase activity: 0 of 30 colonies from the initial time point scored positive for NANase, while 28 of 30 colonies from the 23-h sample were NANase positive. Restriction analysis of 15 NANase-positive candidates revealed that all contained inserts of either
pJST61
B. fragilis
pBR322 oriV pRK2 oriT
bla
2597
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C
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I
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pJST61 -SC3C 14.9 kb
p BF B2
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L2.2kbg FIG. 3. Locations of TnlO00 insertions in pJST61-SC3C. Each independent TnlO00 insertion was mapped by restriction enzyme analysis. Arrowheads point to the positions of the insertions within the cloned B. fragilis insert, except for 6, which inserted into the endRI gene. Arrowheads above the line represent insertions that do not prevent NANase expression in E. coli or B. fragilis, while arrowheads below the line represent insertions that result in loss of NANase expression in E. coli and B. fragilis with the exception of 6, which is NANase- in E. coli but NANase+ in B. fragilis. Insertions marked with an asterisk were used for DNA sequencing. Restriction enzyme abbreviations are as defined in the legend to Fig. 1.
J. BACTERIOL.
RUSSO ET AL.
2598
TABLE 2. Neuraminidase activity in B. fragilis derivativesa Strain
Growth medium (AMM plus indicated C source [0.5%])
NANase activity (units/mg)
Glucose NANA Glucose NANA Glucose NANA Glucose NANA
3.6 x 102 4.5 X 104
JOURNAL OF BACTERIOLOGY, May 1990, p. 2594-2600
0021-9193/90/052594-07$02.00/0 Copyright C) 1990, American Society for Microbiology
Cloning and Expression of the Bacteroides fragilis TAL2480 Neuraminidase Gene, nanH, in Escherichia coli THOMAS A. RUSSO,1t JEFFREY S. THOMPSON,2 VERONICA G. GODOY,2 AND MICHAEL H. MALAMY2*
Department of Geographic Medicine and Infectious Diseases, New England Medical Center,1 and Department of Molecular Biology and Microbiology, Tufts University Health Sciences Campus,2 136 Harrison Avenue, Boston, Massachusetts 02111 Received 6 October 1989/Accepted 10 February 1990
We have cloned the Bacteroides fragilis TAL2480 neuraminidase (NANase) structural gene, nanH, in Escherichia coli. This was accomplished by using the cloning shuttle vector pJST61 and a partial Sau3A library of TAL2480 chromosomal inserts created in E. coli. The library was mobilized into the NANase-deficient B. fragilis TM4000 derivative TC2. NANase-producing colonies were enriched by taking advantage of the inability of TC2, but not the wild-type or NANase+ revertant, to grow in vitro in fluid aspirated from the rat granuloma pouch. Plasmids pJST61-TCN1 and pJST61-TCN3, containing inserts of 9.1 and 4.5 kilobases (kb), respectively, were found in the TC2 derivatives that grew in the rat pouch medium. In B. fragilis, NANase production from the two plasmids was inducible by free N-acetylneuraminic acid or sialic acid-containing substrates, just as in the parental TAL2480 strain. However, when these plasmids were transferred back to E. coli, NANase activity was barely detectable. A 3.5-kb portion of the insert in pJST61-TCN3 was subcloned in pJST61 to give plasmid pJST61-SC3C; NANase was produced from this plasmid both in E. coli and in B. fragilis. In E. coli, NANase expression was under the control of the vector promoter lambda PR and was therefore completely abolished by the presence of a lambda prophage. In B. fragilis, NANase production was inducible by free N-acetylneuraminic acid or sialic acid-containing substrates. By using deletion analysis and TnlOOO mutagenesis, the NANase structural gene and control region that functions in B. fragUis were localized to a 1.5- to 2.0-kb region of the insert. A partial nucleotide sequence of the NANase-deficient Tnl000 insertion mutants allowed us to identify the nanH gene and deduce the amino acid sequence of a portion of the NANase protein. We identified five regions showing great similarity to the Asp boxes, -Ser-X-Asp-X-Gly-X-Thr-Trp-, of other bacterial and viral NANase proteins.
Although Bacteroidesfragilis is a minor component of the normal intestinal flora of humans, it is the predominant obligately anaerobic organism isolated from intraabdominal infections (10). The high frequency of B. fragilis involvement at these sites reflects the actions of several virulence factors. The enzyme neuraminidase (NANase), the product of the nanH gene, is found in many pathogenic bacteria, including Vibrio cholerae (24), Salmonella typhimurium (18, 24), Corynebacterium diphtheriae (15), Streptococcus pneumoniae (3), and B. fragilis (9). It has been suggested that NANase plays a role in the pathogenicity of these bacteria as well as in infections caused by the influenza virus (1) and the protozoan Trypanosoma cruzi (6). The enzymatic activity of NANase, a glycohydrolase, cleaves sialic acid residues occurring at the termini of simple and complex oligosaccharides and on glycoproteins. Sialic acids are widely distributed on the surfaces of eucaryotic cells as terminal nonreducing residues and on important glycoproteins, such as immunoglobulin G and the Clq component of complement (5). Removal of sialic acid residues from the surfaces of cells and from these important glycoproteins may serve the nutritional requirements of the bacteria as well as disrupt many host functions (20). We have demonstrated that NANase activity in cultures of B. fragilis can be induced by free or bound sialic acid (D. Crook, T. A. Russo, F. P. Tally, and M. H. Malamy, unpublished data). To evaluate the possible role of NANase
in the pathogenesis of B. fragilis infections, it is necessary to use derivatives of B. fragilis that differ from the wild-type strain only by alterations in NANase production. Comparisons between otherwise isogenic strains could then be made in a variety of biological systems. As a first step in this process, we have cloned the B. fragilis TAL2480 NANase gene in E. coli by using a shuttle vector system recently
developed in this laboratory (22). This has allowed us to study the expression and regulation of NANase production in E. coli and B. fragilis and to identify the nanH gene and a portion of the sequence of the NANase protein. MATERIALS AND METHODS Bacterial strains and plasmids. B. fragilis and E. coli strains used in this study are summarized in Table 1. The construction and properties of the cloning vector pJST61 are presented in detail by Thompson and Malamy (22), and a partial map of the plasmid is given in Fig. 1. Growth media. (i) Enriched media. ML liquid and solid media for growth of E. coli and brain heart infusion broth (BHIS) and solid medium for growth of B. fragilis were prepared as previously described (12, 17). (ii) Minimal media. For growth of E. coli, medium M63 was supplemented with 0.002% Casamino Acids (Difco Laboratories, Detroit, Mich.) and 0.5% glucose or a combination of 0.5% N-acetylneuraminic acid (NANA; Sigma Chemical Co., St. Louis, Mo.) and 0.01% glucose as the carbon source. For growth of B. fragilis, AMM (23) with 0.5% glucose or 0.5% NANA was employed. AMM with 1% porcine gastric mucin (Sigma) and 5% laked sheep erythrocytes was also used. Rat pouch medium for growth of B.
* Corresponding author. t Present address: LCI, National Institute of Allergy and Infec-
tious Diseases, Bethesda, MD 20892. 2594
VOL. 172, 1990
B. FRAGILIS TAL2480 NEURAMINIDASE
2595
TABLE 1. Bacterial strains and plasmids Plasmid or bacterial strain
Plasmids pJST61 pJST61-TCN1 pJST61-TCN3 pJST61-SC3C pJST61-SC3C nanH: :TnO000 pJST61-SC3C::Tnl000 pTAR100 RK231 F' lac+
Construction or source
Relevant characteristics
Reeatcaatrsis(reference) Cloning shuttle vector, Ampr Clnr NANase+ in B. fragilis NANase+ in B. fragilis NANase+ in B. fragilis and E. coli NANase- TnlOO0 insertions NANase+ TnJOO0 insertions NANase- deletion of pJST61-SC3C Tra+, RP4 derivative, Kanr Tetr Amps Source of TnlOOO
Thompson and Malamy (22) This study This study Derived from pJST61-TCN3 Derived from pJST61-SC3C Derived from pJST61-SC3C Derived from pJST61-SC3C Guiney et al. (11) Laboratory strain
DW1030 KS303
recA rpsL recA rpsL (lambda) recA rpsE KS272, lpp-SSO8
Maniatis et al. (13) HB101 lysogenized with lambda wild type Robillard et al. (17) Strauch and Beckwith (21)
B. fragilis TAL2480
FoxjImpr, NANase+
Tufts Anaerobe Laboratory clinical isolate (7, 22) Strain 838rfm from M. Sebald, Paris, France Derived from TM4000 after NTG' mutagenesis Spontaneous NANase+ revertant from TC2
E. coli HB101
HB1O1(lambda)
TM4000 TC2 TClRb
Rifr, standard wild-type strain NANaseNANase+
a NTG, N-methyl-N'-nitro-N-nitrosoguanidine.
fragilis was obtained by aspirating the liquid contents from the pouch of the rat pouch granuloma model (8). The sterile aspirant was used immediately without further treatment or after storage at -70°C. When used, antibiotics were added to media at the following concentrations (,ug/ml): ampicillin, 200; gentamicin, 50; streptomycin, 200; clindamycin (CLN), 6; and ciprofloxacin, 1. Ciprofloxacin was obtained from Miles Pharmaceuticals, West Haven, Conn.; CLN was obtained from The Upjohn Co., Kalamazoo, Mich.; and all other antibiotics were purchased from Sigma Chemical Co. All cultures were incubated at 37°C. B. fragilis strains were grown in an anaerobic chamber (Coy Laboratory Products, Ann Arbor, Mich.) in an atmosphere of 85% N2, 10% C02, and 5% H2. Plasmid mobilization and transformation. Transfer of pJST61 and its derivatives from E. coli to B. fragilis was accomplished by conjugation, with RK231 as the mobilizing transfer factor (11). Filter matings were performed for 18 h under aerobic conditions. After suspension of the cells from the filter, the mixtures were plated to select for transconjugants containing pJST61 or its derivatives on BHIS plates with CLN, gentamicin, and streptomycin or BHIS with CLN and ciprofloxacin and grown under anaerobic conditions. Transformation of plasmid DNA into E. coli cells was performed by the protocol of Maniatis et al. (13). Plasmid isolation and analysis. Plasmids were purified from E. coli and B. fragilis strains by using the alkaline lysis technique described by Maniatis et al. (13) or by CsClethidium bromide equilibrium centrifugation by the method of Clewell and Helinski (4). All restriction enzymes were purchased from New England BioLabs, Inc. (Beverly, Mass.) and used as recommended by the supplier. Restriction digests were analyzed by electrophoresis on 0.9% horizontal agarose (SeaKem; FMC Corp., Marine Colloids Div., Rockland, Maine) gels after staining with ethidium bromide.
Individual DNA fragments used in subcloning and hybridization experiments were purified from 0.7% low-meltingpoint agarose (SeaPlaque; FMC Corp.) gels (13). Construction of DNA libraries from B. fragiis TAL2480. Chromosomal DNA from B. fragilis TAL2480 was purified by using CsCl-ethidium bromide centrifugation. A partial Sau3A digest of this DNA was fractionated on a 10 to 40% (wt/vol) sucrose gradient to yield two DNA pools containing fragments from 1 to 15 kilobases (kb) and >15 kb, respectively. DNAs from these pools were ligated to the unique BglII site within the endRI gene of pJST61 (Fig. la) (22). Ligation mixes were used to transform E. coli HB101 (RK231) and KS303(RK231) for Ampr colonies. Because of the lethality of the endRI gene product, endonuclease EcoRI, in E. coli lambda nonlysogens, only transformants containing a pJST61 plasmid with a B. fragilis chromosomal insert survive. The colonies from plates containing many Ampr transformants were resuspended in ML broth to create the TAL2480 chromosomal library. Assays for NANase activity. Qualitative plate assays were performed by a modification of the method of Myers et al. (16) and used filter paper (3MM; Whatman, Inc., Clifton, N.J.) moistened with a solution of 0.17 M sodium acetate buffer (pH 5.4) containing 2'(4-methylumbelliferyl)N-acetylneuraminic acid (Sigma) (1 ,iM for B. fragilis and 2 ,uM for E. coli). Colonies to be tested were overlaid with the filter paper, which was then removed, and incubated at 37°C (1 min for B. fragilis and 10 min for E. coli). The reaction was stopped by soaking the filter paper in a solution containing 0.085 M glycine and 0.2 M sodium carbonate (pH 9.4). Colonies containing significant amounts of NANase activity were detected by their fluorescence when examined by long-wave-length UV light. Quantitative NANase assays were performed by a minor modification of the method of Warner and O'Brien (25): 0.08 ml of 0.17 M sodium acetate buffer (pH 5.4), 0.01 ml of 1 mM 2'(4-methylumbelliferyl)Nacetylneuraminic acid, and 0.01 ml of cellular extract were mixed on ice and then incubated at 37°C for 10 min. The
2596
RUSSO ET AL.
pBR322 Ori
bla
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J. BACTERIOL.
B2
-
FIG. 1. (a) Restriction and functional map of the cloning vector pJST61 as described previously (22). The bla gene leads to Amp resistance in E. coli, while the ermF gene leads to CLN resistance in B. fragilis. The RK2 oriT sequence permits efficient trans-mobilization of the plasmid from E. coli to E. coli or B. fragilis recipients. The endRI gene under the control of the lambda PR promoter results in efficient expression of endonuclease EcoRI product in E. coli cells lacking lambda repressor. (b) Plasmid pJST61-TCN3 contains a 4.5-kb insert of chromosomal DNA from B. fragilis TAL2480. (c) Plasmid pJST61-SC3C was derived by subcloning a Sau3A restriction fragment from the BglII-BamHI fragment of pJST61-TCN3, as indicated by the arrows, into the BglII site of pJST61. (d) Plasmid pTAR100 was derived from pJST61-SC3C by deleting the BstEII fragment starting within pBFTM2006 up to the BstEII site at 1.75 kb within the insert. All restriction maps are drawn to scale. Restriction enzyme abbreviations: A, AvaI; B2, BglII; BE2, BstEII; BYI, BstYI; C, ClaI; E, EcoRI; H, HindIll; HI, BamHI; H2, HaeII; N/X, NheI-XbaI; P, PstI; Sau3A, Sau3AI.
reaction mixture was placed on ice and stopped with 1.9 ml of a solution containing 0.085 M glycine and 0.2 M sodium carbonate (pH 9.4). Fluorescence was measured on a fluorometer. Suitable enzyme dilutions were used so that fluorescence was measured in the linear region of the enzyme process curve. NANase-specific activity is expressed as fluorescence units per milliliter per milligram of protein. The same assay conditions were used for B. fragilis or E. coli extracts.
Transposon TnlOOO mutagenesis. Transposon TnO000 mutagenesis of pJST61 derivatives with B. fragilis chromosomal inserts was performed as previously described (22). Transconjugants were screened for NANase activity by using the qualitative plate test; NANase-deficient candidates were confirmed by using the quantitative assay. The location of the Tn1000 inserts was determined by restriction analysis. Construction of a deletion derivative of pJST61-SC3C. pTAR100 (Fig. ld) carries a 4.55-kb deletion extending from the BstEII site in the pBFTM2006 portion of pJST61 to the first BstEII site in the insert, thereby deleting the 1.75 kb of TAL2480 DNA most distal to PR' It was constructed by partial digestion of the parental plasmid with BstEII, followed by transformation of the cleaved DNA without ligation into HB101. Several Ampr but NANase-deficient candidates were isolated. The extent of the deletion in pTAR100 was established by restriction analysis. DNA sequencing. Sequencing of double-stranded CsCl-
purified DNA was performed by the dideoxynucleotidechain termination method of Sanger et al. (19) with Sequenase (U.S. Biochemical Corp., Cleveland, Ohio). Oligonucleotide primers complementary to unique sequences near the ends of Tn1000 were as previously described (22). RESULTS Enrichment protocol to isolate clones expressing NANase activity in B. fragiis. We have determined that the fluid aspirated from a sterile rat granuloma pouch can serve as a complete growth medium for B. fragilis TM4000. A plateau level of greater than 2 x 109 CFU/ml is reached from a low inoculum (2 x 106 CFU/ml) in 20 h at 37°C in the anaerobic chamber (Fig. 2). Under these growth conditions, NANase activity is fully induced. In contrast, the NANase-deficient strain TC2, which contains multiple mutations including the inability to synthesize NANase and a requirement for cysteine (unpublished observations), grows poorly in the rat pouch fluid with or without cysteine (0.5 mg/ml), reaching levels of only 2 x 107 CFU/ml even after prolonged incubation. We determined that the lack of NANase was an important factor in the inability of TC2 to grow in this medium, since strain TClRb, a spontaneous revertant of TC2 with restored NANase activity, grows as well as TM4000 in the rat pouch fluid if the fluid is supplemented with cysteine. We used the inability of TC2 to grow to high
VOL. 172, 1990
B. FRAGILIS TAL2480 NEURAMINIDASE
9.1 or 4.5 kb (data not shown); a representative of the larger insert was chosen and designated pJST61-TCN1, and a plasmid with the smaller insert'was designated pJST61TCN3. Restriction analysis showed that the 4.5-kb insert was contained within the 9.1-kb insert; therefore, further studies used pJST61-TCN3 exclusively. To verify that pJST61-TCN1 and pJST61-TCN3 carried the NANase gene, these plasmids were introduced into E. coli HB1O1(RK231) by transformation and again mobilized to TC2. All of the resultant Clnr B. fragilis transconjugants acquired NANase activity. When the same procedure was followed by using KS303(RK231) cells carrying the B. fragilis TAL2480 chromosomal library of small (1- to 15-kb) Sau3A fragments, no cells capable of growth in the rat pouch fluid were found. Subcloning and mapping the B. fragiis NANase structural gene. Plasmid pJST61-TCN3 containing the 4.5-kb chromosomal insert was used for detailed restriction mapping and subcloning. A partial restriction map of the 4.5-kb insert is presented in Fig. lb. A 3.8-kb BglII-BamHI fragment from the insert in pJST61-TCN3 was purified by gel electrophoresis and digested to completion with Sau3A. The digestion products were purified'from agarose gels and ligated into the unique BglII site of pJST61. The DNA was used to transform E. coli KS303 to Ampr. Screening of transformants for NANase activity in E. coli led to the isolation of pJST61SC3C, which contains the leftmost 3.5-kb of the 4.5-kb insert (Fig. lc). Plasmid pJST61-SC3C was used as the target for Tn1000 mutagenesis' to further localize the NANase gene. The positions of TnJ000 insertions are given in Fig. 3. 'Insertions within a 2.2-kb portion of the 3.5-kb fragment led to loss of NANase activity in E. coli. The leftmost 0.8 kb of the' insert is not necessary for NANase expression in' E. coli, since strains carrying Tn1000 insertions in this region still produced NANase. Plasmid pTAR100 (Fig. ld) which lacks the lefthand portion of the chromosomal insert up to the BstEII site at 1.75 kb did not produce NANase in either E. coli or B.
101 C 109 U-
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TC2
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106 20
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80
Time (hours) FIG. 2. Growth of wild-type B. fragilis TM4000 and mutants TC2 and TClRb in rat pouch fluid. Cultures were grown in BHIS broth to mid-log stage, harvested by centrifugation, washed with MPBS, and then used to inoculate 5 ml of fresh rat pouch aspirant in 50-ml flasks. For TC2 and TClRb, cysteine (0.5 mg/ml) was added before incubation. Cells were grown in the anaerobic chamber at 37°C. Samples were taken and plated on BHIS solid medium for viable counts.
density in rat pouch fluid supplemented with cysteine as the selective condition to isolate plasmids containing an expressed NANase gene. A B. fragilis TAL2480 chromosomal library with an insert size of >15 kb in pJST61 (see Materials and Methods) was mobilized from E. coli KS303(RK231) to B. fragilis TC2, selecting for Clnr. Approximately 103 transconjugant colonies were resuspended in BHIS broth, and 1.7 x 103 CFU of this resuspension was inoculated into 1 ml of rat pouch fluid and incubated anaerobically. After 23 h of incubation, the cell titer had increased 3 orders of magnitude to 1.2 x 106 CFU/ml. Individual colonies isolated from the initial inoculum and the 23-h cultures were tested for NANase activity: 0 of 30 colonies from the initial time point scored positive for NANase, while 28 of 30 colonies from the 23-h sample were NANase positive. Restriction analysis of 15 NANase-positive candidates revealed that all contained inserts of either
pJST61
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L2.2kbg FIG. 3. Locations of TnlO00 insertions in pJST61-SC3C. Each independent TnlO00 insertion was mapped by restriction enzyme analysis. Arrowheads point to the positions of the insertions within the cloned B. fragilis insert, except for 6, which inserted into the endRI gene. Arrowheads above the line represent insertions that do not prevent NANase expression in E. coli or B. fragilis, while arrowheads below the line represent insertions that result in loss of NANase expression in E. coli and B. fragilis with the exception of 6, which is NANase- in E. coli but NANase+ in B. fragilis. Insertions marked with an asterisk were used for DNA sequencing. Restriction enzyme abbreviations are as defined in the legend to Fig. 1.
J. BACTERIOL.
RUSSO ET AL.
2598
TABLE 2. Neuraminidase activity in B. fragilis derivativesa Strain
Growth medium (AMM plus indicated C source [0.5%])
NANase activity (units/mg)
Glucose NANA Glucose NANA Glucose NANA Glucose NANA
3.6 x 102 4.5 X 104
