- Email: [email protected]
[7] Identifying in Vivo expressed streptococcal genes in endocarditis
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Conclusions The genetic approach described in this article has the potential to expand our understanding of biofilm development profoundly. The methods outlined here are simple and can be applied readily to study many different types of bacteria that inhabit surface-associated communities. Subsequent microscopic and molecular analyses of the mutants uncovered by these screens are also straightforward. The results of these screens for mutants defective in biofilm formation in P. aeruginosa, P. fluorescens, E. coli, V. cholera, S. putrefaciens, and S. epiderrnidis have identified genes of both known and unknown function.2,4-6,8,12,4°,41 This suggests that by focusing on surface association, these screens have the potential to provide insight that will assist in assigning roles to genes of previously unknown function. These results also suggest that the study of biofilm formation represents the exploration of an aspect of microbial physiology not yet studied at the molecular genetic level. A molecular understanding of the control of biofilm development may eventually be applied to limit both the virulence and the distribution of pathogens in various environments, serve as a means to regulate microbial attachment to and degradation of solid materials, and improve our control of biofilm formation in industrial settings. 4o O. Vidal, R. Longin, C. Prigent-Combaret, C. Dorel, M. H o o r e m a n , and P. Lejune, J. BacterioL 180, 2442 (1998). 41 C. H e i l m a n n and C. Gotz, Zentbl. Bakteriol. 287, 69 (1998).
[7] I d e n t i f y i n g in V i v o E x p r e s s e d S t r e p t o c o c c a l G e n e s in Endocarditis By LIN TAO and MARK C. HERZBERG
Viridans streptococci normally reside in the oral cavity to form a special biofilm on the tooth called dental plaque. In the oral cavity, these streptococci are nonpathogenic except for the mutans group, which causes caries) In other anatomic sites, however, these streptococci may be pathogenic.2 For example, viridans streptococci are the most common organisms causing endocarditis. These bacteria can enter the circulation during dental procet S. H a m a d a and H. D. Slade, Microbiol. Rev. 44, 331 (1980). 2 R. Bayliss, C. Clarke, C. M. Oakley, W. Somerville, A. G. W. Whitfield, and S. E. J. Young, Br. Heart J. 50, 513 (1983).
METHODS IN ENZYMOLOGY,VOL. 310
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dures and cause transient bacteremia. If predisposing factors such as rheumatic or congenital heart diseases (valve defects) exist, the bacteria can colonize the heart by forming a different biofilm on the endocardium to cause endocarditis. Once the bacteria colonize the heart, virulence appears to depend in part on the expression of specific virulence genes. To date, the specific conditions that occur in vivo in the heart to modify streptococcal gene expression are largely undefined. While it will be of interest to know if virulence depends on streptococcal genes that are induced in the host, in vivo animal models 3-5 of infective endocarditis contain a robust set of environmental signals that may not be well simulated by in vitro bioassays.6'7 To learn about genes that are expressed specifically in vivo but not in vitro, new approaches are being developed. Mahan et al. s have reported the detection of host-induced Salmonella genes encoding potential virulence factors. This in vivo expression technology (IVET) uses an integration plasmid vector carrying two promoterless reporter genes, p u r A and lacZ, for both in vivo and in vitro selection. Using this and similar IVET systems, which target unique metabolic pathways or genetic markers of the seleted pathogens, many host-induced genes have been identified in several different bacterial species, including Salmonella typhimurium, s-l° Pseudomonas aeruginosa, ll Vibrio cholerae, 12 and Staphylococcus aureus. 13,14 Each IVET system for a specific bacterial genus or species must be designed to exploit certain fastidious, unique growth requirements or other traits that can be used for selection. Therefore, this article describes an IVET system applicable to studying experimental endocarditis and other biofilm problems, permitting selection of in vivo-induced (ivi) genes in the gram-positive Streptococcus gordonii. 3 H. A. Dewar, M. R. Jones, S. G. Griffin, A. Oxley, and J. Marriner, J. Comp. Pathol. 97, 567 (1987). 4 D. W. Durack and P. B. Beeson, Br. J. Exp. PathoL 53, 44 (1972). 5 M. C. Herzberg, G. D. MacFarlane, K. Gong, N. N. Armstrong, A. R. Witt, P. R. Erickson, and M. W. Meyer, Infect. Immun. 60, 4809 (1992). 6 M. C. Herzberg, K. Gong, G. D. MacFarlane, P. R. Erickson, A. H. Soberay, P. H. Krebsbach, G. Manjula, K. Schilling, and W. H. Bowen, Infect. Immun. 58, 515 (1990). 7 C. H. Ramirez-Ronda, J. Clin. Invest. 62, 805 (1978). 8 M. J. Mahan, J. M. Slauch, and J. J. Mekalanos, Science 259, 686 (1993). 9 E. M. Heithoff, C. P. Conner, P. C. Hanna, S. M. Julio, U, Hentschel, and M. J. Mahan, Proc. Natl. Acad. Sci. U.S.A. 94, 934 (1997). 10 R. H. Valdivia and S. Falkow, Science 277, 2007 (1997). al j. Wang, A. Mushegian, S. Lory, and S. Jin, Proc. Natl. Acad. Sci. U.S.A. 93, 10434 (1996). 12 A. Camilli and J. Mekalanos, Mol. Microbiol. 18, 671 (1995). 13 A. M. Lowe, D. T. Beattie, and R. L. Deresiewicz, Mol. Microbiol. 27, 967 (1998). 14M. A. Lane, K. W. Bayles, and R. E. Yasbin, Gene 100, 225 (1991).
[7]
STREPTOCOCCALGENESIN ENDOCARDITIS
p15A,"'l Xbal
BamHI
amy
or'L
EcoRI
111
.Hindlll
[ cat To
EcoRI /
~ Hindlll
pAK36 (7.2 kb) FIG. 1. The streptococcal IVET vector pAK36.
Method Streptococcal I V E T Vector pAK36 In vivo-induced streptococcal genes can be identified using a specially designed IVET vector, pAK36 (Fig. 1). It has two promoterless reporter genes of gram-positive bacterial origin, amy and cat, from pRQ200 TM and pMH109,15 respectively. It also has a tetracycline (Tc) resistance marker and replication origin (ori) from the streptococcal integration vector pSF143.16 The plasmid can replicate in Escherichia coli with a medium copy number (the p15A ori), but it cannot replicate extrachromosomally in streptococci. The plasmid, however, can insert into the streptococcal chromosome by homologous, Campbell-like integration if it carries a streptococcal gene fragment. The cat reporter gene provides a positive in vivo selection for inducible promoter genes in the reporter gene-fused strains in an animal host to which chloramphenicol (Cm) is given. The amy gene confers amylase production, which is suitable for a negative selection in vitro and can be detected readily on agar plates containing 0.5% (w/v) starch by flooding with an iodine solution. Unlike constitutively expressed genes, the hostinduced genes only express in vivo, but not in vitro. Therefore, after selection in vivo, in vitro selection is also necessary to exclude from consideration constitutively expressed genes. Once a host-induced IVET-fusion clone is identified, the gene flanking the insertion site can be retrieved readily by self-ligating the Xbal-digested chromosomal D N A of the IVET-fusion clone and transforming E. coli and is characterized by subsequent sequencing or insertion inactivational analysis. 15M. C. Hudson and R. Curtiss III, Infect. Immun. 58, 464 (1990). 16L. Tao, D. J. LeBlanc,and J. J. Ferretti, Gene 120, 105 (1992).
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Construction of Reporter Gene-Fusion Strain Library in Streptococcus gordonii V288 Introduction. To identify streptococcal genes induced in vivo in endocarditis, a reporter gene-fusion library of more than 10,000 clones in one streptococcal strain is needed. In these clones, the IVET vector should be inserted randomly in the chromosome. Streptococcus gordonii strain V288 is recommended because it is highly competent for natural transformation but is low in background amylase activity. Procedure 1. Digest 10 tzg S. gordonii chromosomal DNA with 3 units of Sau3A1 overnight at 37°. 2. Digest 10 ~g pAK36 DNA with 3 units of BamHI overnight at 37°. 3. Purify the two restriction enzyme-digested DNA preparations with the GeneClean (Bio-101, Inc., Vista, CA) method to remove enzymes and buffering salts. 4. Ligate the two DNA preparations with 3 units of T4 ligase overnight at 14°. Test 1% of the ligation mixture on a 0.7% agarose gel to verify the ligation result and estimate the DNA concentration. The DNA in the remaining ligation mixture should be close to 20/zg. 5. Prepare competent S. gordonii V288 cells for natural transformation. [Inoculate a single colony from a blood agar plate into 2 ml Todd-Hewitt broth (THB) supplemented with 10% horse serum (THBS) in a glass test tube. Incubate the culture at 37° for 16 hr. Transfer the 2-ml culture into 80 ml of prewarmed THBS medium (in 10 test tubes). Incubate the 1 : 40 diluted culture at 37° for exactly 2 hr to achieve maximal competence.] 6. Add one-half of the ligation mixture (about 10/zg DNA) to the competent S. gordonii V288 cells and keep the other half at - 2 0 °. 7. Continue to incubate the culture at 37 ° for 1 hr. 8. Concentrate the culture by centrifugation and spread the cells on at least 100 Todd-Hewitt agar plates containing 10/zg/ml Tc. 9. Incubate the plates in candle jars or anaerobically for 24 hr at 37°. 10. Estimate the total number of transformant colonies. (If the procedures are followed correctly, a total of more than 50,000 transformant colonies should be obtained; proceed to step 11. If the colony count is less than 10,000, however, competence may have developed insufficiently. A low transformation rate may occur due to insufficient DNA, incorrect timing of addition of DNA, and impurity of the culture. Repeat steps 5 to 10 precisely using the remaining half of the ligation mixture.)
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11. Harvest all colonies with a sterile cotton swab and resuspend the cells in 10 ml THB supplemented with 10 tzg/ml Tc. 12. Incubate the culture for 4 hr at 37° to adapt the cells to liquid medium. 13. Store the cells in 10 aliquots with 15% glycerol at -70 ° as the S. gordonii V288 IVET reporter gene-fusion library.
Comments. Normally, the ligation mixture can be amplified in E. coli to enrich the DNA to be transformed into streptococci. In the case of S. gordonii, however, amplification of the ligation mixture in E. coli can cause selective deletions to the cloned streptococcal DNA because some S. gordonii genes are toxic to E. coli and thus can create a bias in the overall representation within the library. To ensure a thorough representation of the reporter gene-fusion clone library, it is necessary to transform the ligation mixture directly into S. gordonii as described earlier. Because S. gordonii is highly competent for natural transformation, no amplification of ligated DNA in E. coli is needed. It is important, however, to start with a larger amount of highly purified DNA. Therefore, the CsC1 gradient ultraspeed centrifugation method is highly recommended to prepare the DNA before the restriction enzyme digestion procedure. Isolation of Streptococcus gordonii in Vivo-Induced Genes in Endocarditis Introduction. This section includes several major steps: an endocarditis animal model preparation, bacterial inoculation, in vivo selection, in vitro selection, and gene cloning and sequencing. Although different animals such as mice, rats, pigs, and dogs have been used to study endocarditis, we recommend the rabbit model because it has a larger heart than mice or rats, easily accessible ear veins for intravenous injections, and the anatomy is similar to humans. 17'a8 Procedure 1. Inoculate one sample (about 1 ml) frozen stock culture of S. gordonii V288 IVET reporter gene-fusion strain library into 10 ml THB supplemented with 10/zg/ml Tc and incubate for 16 hr at 37°. 2. Dilute the culture 1 : 4 in the same prewarmed broth and continue to incubate for 3 hr to reach the exponential phase.
17 T. G. Sarphie, A m . J. Anat. 1"/4, 145 (1985). 18 H. Masuda, Int. J. Biochem. 16, 99 (1984).
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3. Harvest the cells by centrifugation at 4000g for 10 min at room temperature and wash in sterile saline once. Resuspend in 2 ml sterile saline (about 109 cells/ml). 4. Inject the cell suspension intravenously through an ear vein into a New Zealand White rabbit that has been prepared for experimental endocarditis by placing an indwelling catheter as described previously. 5 5. Beginning 6 hr after the inoculation of the library, give the rabbit Cm intravenously two times a day for 3 days to a final serum level of 5/zg/ml. (The minimal inhibitory concentration of chloramphenicol for the wild-type S. gordonii V288 strain is 0.5/zg/ml.) 6. Euthanize the rabbit and dissect out the aortic valve vegetation. Immerse the valve specimen in sterile saline. Under aseptic conditions, disperse the specimen with a mortar and pestle to recover viable bacterial cells. 7. Spread the bacterial cells onto THB agar plates supplemented with 10/zg/ml Tc and incubate the plates in a candle jar at 37 ° for 24 hr. 8. Isolate single colonies and replica plate the colonies onto THB agar (master plate), THB agar supplemented with 5/zg/ml Cm, and THB supplemented with 0.5% (w/v) starch. Incubate the plates in candle jars for 24 hr at 37 °. 9. Detect amylase activity by flooding the starch plate with an iodine solution [0.2% (w/v) 12 and 0.2% (w/v) KI]. Identify bacterial colonies that display negative amylase activity and sensitivity to chloramphenicol. Select the same colonies from the master plate as the ivi clones. 10. Isolate chromosomal D N A from each of the selected clones. Digest the D N A with HindlII and hybridize the D N A with labeled pAK36 by the method of Southern. 19 Identify clones that display unique pAK36 insertion patterns to rule out redundant siblings. 11. Digest the chromosomal D N A of the ivi clones displaying unique pAK36 insertion patterns with the restriction enzyme XbaI or StyI. Remove the enzyme and buffering salt by the GeneClean method. Perform self-ligation with T4 ligase, and transform E. coli JM109 to TCR.2° If the transformation is not successful with JM109, use the E. coli recA-positive strain C600 instead. 12. Isolate plasmids from the Tc R transformant colonies with the Qiagen plasmid isolation kit (Qiagen, Inc., Chatsworth, CA) and analyze them by restriction enzyme digestion. 19 E. Southern, J. MoL BioL 98, 503 (1975). 20 C. T. Chung, S. L. Niemela, and R. H. Miller, Proc. Natl. Acad. Sci. U.S.A. 86, 2172 (1989).
[7]
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STREPTOCOCCAL GENES IN ENDOCARDITIS
13. Sequence the cloned genes with both forward.and reverse primers designed according to the sequence of amy and ori genes, which flank the cloned streptococcal gene fragment in the plasmid. To sequence DNA fragments upstream to the amy gene, an oligonucleotide, 5'-AGC GCA A A T A A C AGC GTC AGC AA-3', complementary to the 5' end of the amy coding region of Bacillus licheniformis (GenBank accession A17930), can be used as the reverse primer. Another oligonucleotide, 5'-CAA G A G ATT ACG CGC A G A CC-3', derived from the plasmid ori sequence of pACYC184 (X06403), can be used as the forward primer. Nucleotide sequences can be determined either by the dideoxynucleotide chain-termina-
TcR
~~.~CC'amvIcatJ •
Sau3A1 -digested S. gordonii DNA
.
4r
,~ S. gordonfi chromosome ~
4r
MCS
pAK36
To R
Transforming S. gordonfi To R
o@
x' { ~mvl cat
X÷
~lncubate for 6 hr> Challenging with I.V. chloramphenicol In¢ sal Tc R
Cloning the target ~r gene fragment [-> x',
Screening foramy-in vitro
FIG. 2. Isolation of S. gordonii ivi genes induced in endocarditis.
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[7]
tion method 21 or by a DNA sequencing service using an automated DNA sequencer. 14. Analyze the DNA sequence by translation in all six reading frames to search the nonredundant sequence database (National Center for Biotechnology Information, National Institute of Health) by the BLASTX program. 22 Comments. Certain streptococcal genes may be lethal to E. coli and thus cannot be cloned in E. coli without deletions or rearrangement. Because these genes may not be cloned with the E. coli recA- strain JM109, an alternative recA +E. coli strain C600 can be used. Deletions of S. gordonii genes in E. coli C600, however, may occur in about one-half of the plasmids, in which a portion of the amy gene may also be lost. 23 Therefore, these plasmids can be sequenced only by the ori gene-derived forward primer. In this case, the gene sequenced with the forward primer may not be proximal upstream to the ivi gene fused to the amy-cat cassette and induced during endocarditis. This can be determined from a comparison of the size of the entire inserted DNA fragment with the size and orientation of the sequenced gene. The ivi gene, if deleted, may also be lethal to E. coli and not be cloned readily in that host. The gene sequence can be obtained by chromosomal walking with the inverse polymerase chain reaction method 24 once the sequence of its adjacent gene is available. We have used this method successfully to identify 13 S. gordonii V288 ivi genes induced in endocarditis with the rabbit model (see Fig. 2). 23 The availability of the new IVET system for S. gordonii has expanded the repertoire of genetic tools for the identification of in vivo expressed microbial genes. The plasmid pAK36 may also be applicable to the identification of in vivo-induced genes from other streptococci and closely related grampositive bacterial species in endocarditis or other types of infections.
Acknowledgments This study was supported by NIH Grants DEI1336, DE05501, DE08590, and DE00270 and by a Grant-in-Aid (KS-96-GB-56) from the American Heart Association, Kansas Affiliate, Inc.
21 F. Sanger, S. Nicklen, and A. R. Coulson, Proc. Natl. Acad. Sci. U.S.A. 74, 5463 (1977). 22 S. F. Altsehul, W. Gish, W. Miller, E. W. Myers, and D. J. Lipman, J. Mol. BioL 215, 403 (1990). 23 A. O. Kiliq, M. C. Herzberg, M. W. Meyer, X. Zhao, and L. Tao, Plasmid, in press (1999). 24 H. Ochman, A. S. Gerber, and D. L. Hartl, Genetics 120, 621 (1988).
STREPTOCOCCAL GENES IN ENDOCARDITIS
109
Conclusions The genetic approach described in this article has the potential to expand our understanding of biofilm development profoundly. The methods outlined here are simple and can be applied readily to study many different types of bacteria that inhabit surface-associated communities. Subsequent microscopic and molecular analyses of the mutants uncovered by these screens are also straightforward. The results of these screens for mutants defective in biofilm formation in P. aeruginosa, P. fluorescens, E. coli, V. cholera, S. putrefaciens, and S. epiderrnidis have identified genes of both known and unknown function.2,4-6,8,12,4°,41 This suggests that by focusing on surface association, these screens have the potential to provide insight that will assist in assigning roles to genes of previously unknown function. These results also suggest that the study of biofilm formation represents the exploration of an aspect of microbial physiology not yet studied at the molecular genetic level. A molecular understanding of the control of biofilm development may eventually be applied to limit both the virulence and the distribution of pathogens in various environments, serve as a means to regulate microbial attachment to and degradation of solid materials, and improve our control of biofilm formation in industrial settings. 4o O. Vidal, R. Longin, C. Prigent-Combaret, C. Dorel, M. H o o r e m a n , and P. Lejune, J. BacterioL 180, 2442 (1998). 41 C. H e i l m a n n and C. Gotz, Zentbl. Bakteriol. 287, 69 (1998).
[7] I d e n t i f y i n g in V i v o E x p r e s s e d S t r e p t o c o c c a l G e n e s in Endocarditis By LIN TAO and MARK C. HERZBERG
Viridans streptococci normally reside in the oral cavity to form a special biofilm on the tooth called dental plaque. In the oral cavity, these streptococci are nonpathogenic except for the mutans group, which causes caries) In other anatomic sites, however, these streptococci may be pathogenic.2 For example, viridans streptococci are the most common organisms causing endocarditis. These bacteria can enter the circulation during dental procet S. H a m a d a and H. D. Slade, Microbiol. Rev. 44, 331 (1980). 2 R. Bayliss, C. Clarke, C. M. Oakley, W. Somerville, A. G. W. Whitfield, and S. E. J. Young, Br. Heart J. 50, 513 (1983).
METHODS IN ENZYMOLOGY,VOL. 310
Copyright © 1999by AcademicPress All rights of reproduction in any form reserved. 0076-6879/99$30.00
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[71
dures and cause transient bacteremia. If predisposing factors such as rheumatic or congenital heart diseases (valve defects) exist, the bacteria can colonize the heart by forming a different biofilm on the endocardium to cause endocarditis. Once the bacteria colonize the heart, virulence appears to depend in part on the expression of specific virulence genes. To date, the specific conditions that occur in vivo in the heart to modify streptococcal gene expression are largely undefined. While it will be of interest to know if virulence depends on streptococcal genes that are induced in the host, in vivo animal models 3-5 of infective endocarditis contain a robust set of environmental signals that may not be well simulated by in vitro bioassays.6'7 To learn about genes that are expressed specifically in vivo but not in vitro, new approaches are being developed. Mahan et al. s have reported the detection of host-induced Salmonella genes encoding potential virulence factors. This in vivo expression technology (IVET) uses an integration plasmid vector carrying two promoterless reporter genes, p u r A and lacZ, for both in vivo and in vitro selection. Using this and similar IVET systems, which target unique metabolic pathways or genetic markers of the seleted pathogens, many host-induced genes have been identified in several different bacterial species, including Salmonella typhimurium, s-l° Pseudomonas aeruginosa, ll Vibrio cholerae, 12 and Staphylococcus aureus. 13,14 Each IVET system for a specific bacterial genus or species must be designed to exploit certain fastidious, unique growth requirements or other traits that can be used for selection. Therefore, this article describes an IVET system applicable to studying experimental endocarditis and other biofilm problems, permitting selection of in vivo-induced (ivi) genes in the gram-positive Streptococcus gordonii. 3 H. A. Dewar, M. R. Jones, S. G. Griffin, A. Oxley, and J. Marriner, J. Comp. Pathol. 97, 567 (1987). 4 D. W. Durack and P. B. Beeson, Br. J. Exp. PathoL 53, 44 (1972). 5 M. C. Herzberg, G. D. MacFarlane, K. Gong, N. N. Armstrong, A. R. Witt, P. R. Erickson, and M. W. Meyer, Infect. Immun. 60, 4809 (1992). 6 M. C. Herzberg, K. Gong, G. D. MacFarlane, P. R. Erickson, A. H. Soberay, P. H. Krebsbach, G. Manjula, K. Schilling, and W. H. Bowen, Infect. Immun. 58, 515 (1990). 7 C. H. Ramirez-Ronda, J. Clin. Invest. 62, 805 (1978). 8 M. J. Mahan, J. M. Slauch, and J. J. Mekalanos, Science 259, 686 (1993). 9 E. M. Heithoff, C. P. Conner, P. C. Hanna, S. M. Julio, U, Hentschel, and M. J. Mahan, Proc. Natl. Acad. Sci. U.S.A. 94, 934 (1997). 10 R. H. Valdivia and S. Falkow, Science 277, 2007 (1997). al j. Wang, A. Mushegian, S. Lory, and S. Jin, Proc. Natl. Acad. Sci. U.S.A. 93, 10434 (1996). 12 A. Camilli and J. Mekalanos, Mol. Microbiol. 18, 671 (1995). 13 A. M. Lowe, D. T. Beattie, and R. L. Deresiewicz, Mol. Microbiol. 27, 967 (1998). 14M. A. Lane, K. W. Bayles, and R. E. Yasbin, Gene 100, 225 (1991).
[7]
STREPTOCOCCALGENESIN ENDOCARDITIS
p15A,"'l Xbal
BamHI
amy
or'L
EcoRI
111
.Hindlll
[ cat To
EcoRI /
~ Hindlll
pAK36 (7.2 kb) FIG. 1. The streptococcal IVET vector pAK36.
Method Streptococcal I V E T Vector pAK36 In vivo-induced streptococcal genes can be identified using a specially designed IVET vector, pAK36 (Fig. 1). It has two promoterless reporter genes of gram-positive bacterial origin, amy and cat, from pRQ200 TM and pMH109,15 respectively. It also has a tetracycline (Tc) resistance marker and replication origin (ori) from the streptococcal integration vector pSF143.16 The plasmid can replicate in Escherichia coli with a medium copy number (the p15A ori), but it cannot replicate extrachromosomally in streptococci. The plasmid, however, can insert into the streptococcal chromosome by homologous, Campbell-like integration if it carries a streptococcal gene fragment. The cat reporter gene provides a positive in vivo selection for inducible promoter genes in the reporter gene-fused strains in an animal host to which chloramphenicol (Cm) is given. The amy gene confers amylase production, which is suitable for a negative selection in vitro and can be detected readily on agar plates containing 0.5% (w/v) starch by flooding with an iodine solution. Unlike constitutively expressed genes, the hostinduced genes only express in vivo, but not in vitro. Therefore, after selection in vivo, in vitro selection is also necessary to exclude from consideration constitutively expressed genes. Once a host-induced IVET-fusion clone is identified, the gene flanking the insertion site can be retrieved readily by self-ligating the Xbal-digested chromosomal D N A of the IVET-fusion clone and transforming E. coli and is characterized by subsequent sequencing or insertion inactivational analysis. 15M. C. Hudson and R. Curtiss III, Infect. Immun. 58, 464 (1990). 16L. Tao, D. J. LeBlanc,and J. J. Ferretti, Gene 120, 105 (1992).
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Construction of Reporter Gene-Fusion Strain Library in Streptococcus gordonii V288 Introduction. To identify streptococcal genes induced in vivo in endocarditis, a reporter gene-fusion library of more than 10,000 clones in one streptococcal strain is needed. In these clones, the IVET vector should be inserted randomly in the chromosome. Streptococcus gordonii strain V288 is recommended because it is highly competent for natural transformation but is low in background amylase activity. Procedure 1. Digest 10 tzg S. gordonii chromosomal DNA with 3 units of Sau3A1 overnight at 37°. 2. Digest 10 ~g pAK36 DNA with 3 units of BamHI overnight at 37°. 3. Purify the two restriction enzyme-digested DNA preparations with the GeneClean (Bio-101, Inc., Vista, CA) method to remove enzymes and buffering salts. 4. Ligate the two DNA preparations with 3 units of T4 ligase overnight at 14°. Test 1% of the ligation mixture on a 0.7% agarose gel to verify the ligation result and estimate the DNA concentration. The DNA in the remaining ligation mixture should be close to 20/zg. 5. Prepare competent S. gordonii V288 cells for natural transformation. [Inoculate a single colony from a blood agar plate into 2 ml Todd-Hewitt broth (THB) supplemented with 10% horse serum (THBS) in a glass test tube. Incubate the culture at 37° for 16 hr. Transfer the 2-ml culture into 80 ml of prewarmed THBS medium (in 10 test tubes). Incubate the 1 : 40 diluted culture at 37° for exactly 2 hr to achieve maximal competence.] 6. Add one-half of the ligation mixture (about 10/zg DNA) to the competent S. gordonii V288 cells and keep the other half at - 2 0 °. 7. Continue to incubate the culture at 37 ° for 1 hr. 8. Concentrate the culture by centrifugation and spread the cells on at least 100 Todd-Hewitt agar plates containing 10/zg/ml Tc. 9. Incubate the plates in candle jars or anaerobically for 24 hr at 37°. 10. Estimate the total number of transformant colonies. (If the procedures are followed correctly, a total of more than 50,000 transformant colonies should be obtained; proceed to step 11. If the colony count is less than 10,000, however, competence may have developed insufficiently. A low transformation rate may occur due to insufficient DNA, incorrect timing of addition of DNA, and impurity of the culture. Repeat steps 5 to 10 precisely using the remaining half of the ligation mixture.)
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11. Harvest all colonies with a sterile cotton swab and resuspend the cells in 10 ml THB supplemented with 10 tzg/ml Tc. 12. Incubate the culture for 4 hr at 37° to adapt the cells to liquid medium. 13. Store the cells in 10 aliquots with 15% glycerol at -70 ° as the S. gordonii V288 IVET reporter gene-fusion library.
Comments. Normally, the ligation mixture can be amplified in E. coli to enrich the DNA to be transformed into streptococci. In the case of S. gordonii, however, amplification of the ligation mixture in E. coli can cause selective deletions to the cloned streptococcal DNA because some S. gordonii genes are toxic to E. coli and thus can create a bias in the overall representation within the library. To ensure a thorough representation of the reporter gene-fusion clone library, it is necessary to transform the ligation mixture directly into S. gordonii as described earlier. Because S. gordonii is highly competent for natural transformation, no amplification of ligated DNA in E. coli is needed. It is important, however, to start with a larger amount of highly purified DNA. Therefore, the CsC1 gradient ultraspeed centrifugation method is highly recommended to prepare the DNA before the restriction enzyme digestion procedure. Isolation of Streptococcus gordonii in Vivo-Induced Genes in Endocarditis Introduction. This section includes several major steps: an endocarditis animal model preparation, bacterial inoculation, in vivo selection, in vitro selection, and gene cloning and sequencing. Although different animals such as mice, rats, pigs, and dogs have been used to study endocarditis, we recommend the rabbit model because it has a larger heart than mice or rats, easily accessible ear veins for intravenous injections, and the anatomy is similar to humans. 17'a8 Procedure 1. Inoculate one sample (about 1 ml) frozen stock culture of S. gordonii V288 IVET reporter gene-fusion strain library into 10 ml THB supplemented with 10/zg/ml Tc and incubate for 16 hr at 37°. 2. Dilute the culture 1 : 4 in the same prewarmed broth and continue to incubate for 3 hr to reach the exponential phase.
17 T. G. Sarphie, A m . J. Anat. 1"/4, 145 (1985). 18 H. Masuda, Int. J. Biochem. 16, 99 (1984).
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MOLECULAR BIOLOGY FOR BIOFILM BACTERIA
[7]
3. Harvest the cells by centrifugation at 4000g for 10 min at room temperature and wash in sterile saline once. Resuspend in 2 ml sterile saline (about 109 cells/ml). 4. Inject the cell suspension intravenously through an ear vein into a New Zealand White rabbit that has been prepared for experimental endocarditis by placing an indwelling catheter as described previously. 5 5. Beginning 6 hr after the inoculation of the library, give the rabbit Cm intravenously two times a day for 3 days to a final serum level of 5/zg/ml. (The minimal inhibitory concentration of chloramphenicol for the wild-type S. gordonii V288 strain is 0.5/zg/ml.) 6. Euthanize the rabbit and dissect out the aortic valve vegetation. Immerse the valve specimen in sterile saline. Under aseptic conditions, disperse the specimen with a mortar and pestle to recover viable bacterial cells. 7. Spread the bacterial cells onto THB agar plates supplemented with 10/zg/ml Tc and incubate the plates in a candle jar at 37 ° for 24 hr. 8. Isolate single colonies and replica plate the colonies onto THB agar (master plate), THB agar supplemented with 5/zg/ml Cm, and THB supplemented with 0.5% (w/v) starch. Incubate the plates in candle jars for 24 hr at 37 °. 9. Detect amylase activity by flooding the starch plate with an iodine solution [0.2% (w/v) 12 and 0.2% (w/v) KI]. Identify bacterial colonies that display negative amylase activity and sensitivity to chloramphenicol. Select the same colonies from the master plate as the ivi clones. 10. Isolate chromosomal D N A from each of the selected clones. Digest the D N A with HindlII and hybridize the D N A with labeled pAK36 by the method of Southern. 19 Identify clones that display unique pAK36 insertion patterns to rule out redundant siblings. 11. Digest the chromosomal D N A of the ivi clones displaying unique pAK36 insertion patterns with the restriction enzyme XbaI or StyI. Remove the enzyme and buffering salt by the GeneClean method. Perform self-ligation with T4 ligase, and transform E. coli JM109 to TCR.2° If the transformation is not successful with JM109, use the E. coli recA-positive strain C600 instead. 12. Isolate plasmids from the Tc R transformant colonies with the Qiagen plasmid isolation kit (Qiagen, Inc., Chatsworth, CA) and analyze them by restriction enzyme digestion. 19 E. Southern, J. MoL BioL 98, 503 (1975). 20 C. T. Chung, S. L. Niemela, and R. H. Miller, Proc. Natl. Acad. Sci. U.S.A. 86, 2172 (1989).
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115
STREPTOCOCCAL GENES IN ENDOCARDITIS
13. Sequence the cloned genes with both forward.and reverse primers designed according to the sequence of amy and ori genes, which flank the cloned streptococcal gene fragment in the plasmid. To sequence DNA fragments upstream to the amy gene, an oligonucleotide, 5'-AGC GCA A A T A A C AGC GTC AGC AA-3', complementary to the 5' end of the amy coding region of Bacillus licheniformis (GenBank accession A17930), can be used as the reverse primer. Another oligonucleotide, 5'-CAA G A G ATT ACG CGC A G A CC-3', derived from the plasmid ori sequence of pACYC184 (X06403), can be used as the forward primer. Nucleotide sequences can be determined either by the dideoxynucleotide chain-termina-
TcR
~~.~CC'amvIcatJ •
Sau3A1 -digested S. gordonii DNA
.
4r
,~ S. gordonfi chromosome ~
4r
MCS
pAK36
To R
Transforming S. gordonfi To R
o@
x' { ~mvl cat
X÷
~lncubate for 6 hr> Challenging with I.V. chloramphenicol In¢ sal Tc R
Cloning the target ~r gene fragment [-> x',
Screening foramy-in vitro
FIG. 2. Isolation of S. gordonii ivi genes induced in endocarditis.
116
MOLECULAR BIOLOGY FOR BIOFILM BACTERIA
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tion method 21 or by a DNA sequencing service using an automated DNA sequencer. 14. Analyze the DNA sequence by translation in all six reading frames to search the nonredundant sequence database (National Center for Biotechnology Information, National Institute of Health) by the BLASTX program. 22 Comments. Certain streptococcal genes may be lethal to E. coli and thus cannot be cloned in E. coli without deletions or rearrangement. Because these genes may not be cloned with the E. coli recA- strain JM109, an alternative recA +E. coli strain C600 can be used. Deletions of S. gordonii genes in E. coli C600, however, may occur in about one-half of the plasmids, in which a portion of the amy gene may also be lost. 23 Therefore, these plasmids can be sequenced only by the ori gene-derived forward primer. In this case, the gene sequenced with the forward primer may not be proximal upstream to the ivi gene fused to the amy-cat cassette and induced during endocarditis. This can be determined from a comparison of the size of the entire inserted DNA fragment with the size and orientation of the sequenced gene. The ivi gene, if deleted, may also be lethal to E. coli and not be cloned readily in that host. The gene sequence can be obtained by chromosomal walking with the inverse polymerase chain reaction method 24 once the sequence of its adjacent gene is available. We have used this method successfully to identify 13 S. gordonii V288 ivi genes induced in endocarditis with the rabbit model (see Fig. 2). 23 The availability of the new IVET system for S. gordonii has expanded the repertoire of genetic tools for the identification of in vivo expressed microbial genes. The plasmid pAK36 may also be applicable to the identification of in vivo-induced genes from other streptococci and closely related grampositive bacterial species in endocarditis or other types of infections.
Acknowledgments This study was supported by NIH Grants DEI1336, DE05501, DE08590, and DE00270 and by a Grant-in-Aid (KS-96-GB-56) from the American Heart Association, Kansas Affiliate, Inc.
21 F. Sanger, S. Nicklen, and A. R. Coulson, Proc. Natl. Acad. Sci. U.S.A. 74, 5463 (1977). 22 S. F. Altsehul, W. Gish, W. Miller, E. W. Myers, and D. J. Lipman, J. Mol. BioL 215, 403 (1990). 23 A. O. Kiliq, M. C. Herzberg, M. W. Meyer, X. Zhao, and L. Tao, Plasmid, in press (1999). 24 H. Ochman, A. S. Gerber, and D. L. Hartl, Genetics 120, 621 (1988).