JOURNAL OF BACTERIOLOGY, Feb. 1991, p. 1561-1564 0021-9193/91/041561-04$02.00/0 Copyright © 1991, American Society for Microbiology

Vol. 173, No. 4

NOTES A Novel Approach to Insertional Mutagenesis of Haemophilus influenzae CHRISTINE SHARETZSKY,"2 THOMAS D. EDLIND,2 JOHN J. LiPUMA,1'2 AND TERRENCE L.

STULL'.2*

Departments of Pediatrics' and MicrobiologylImmunology,2 The Medical College of Pennsylvania, Philadelphia, Pennsylvania 19129 Received 2 August 1990/Accepted 12 December 1990

Insertional mutagenesis of the Haemophilus influenzae chromosome was accomplished by a novel method employing a 2.2-kbp element, TSTE. This element, consisting of the neo gene of TnS flanked by Haemophilusspecific uptake sequences, was ligated to circularized chromosomal fragments before transformation into the homologous strain. Eight mutants defective in the production of haemocin were detected. This strategy provides an efficient mechanism for the insertional mutagenesis of H. influenzae.

Generation of mutants by insertion of a transposable antibiotic marker is a useful approach in the characterization of bacterial genes in a wide range of bacterial species (9, 13, 17, 25, 33, 35). Transposons have been introduced into Haemophilus influenzae DNA that was cloned into Esche-

richia coli; these chimeric fragments were subsequently reintroduced into H. influenzae by natural transformation (20, 31). Recently the transposon Tn916 (25), a 16.4-kbp Streptococcus transposon, was used to interrupt the chromosomes of Haemophilus parainfluenzae and H. influenzae (14). Although this approach is useful, the large size of Tn9O6 limits its utility as a marker for cloning the flanking DNA, which is important for further characterization of the mutagenized gene. Furthermore, the use of transposons is limited, since certain transposons are inherently unstable (4, 8) and nonrandom insertions due to hot spots are common (15, 19, 26, 32, 36). Insertional mutagenesis without transposons has been applied to Streptococcus pneumoniae and other species that are naturally transformed. This method employs transformation of bacteria with genomic DNA fragments ligated to an antibiotic resistance determinant (16, 31). In this study, we report the use of a novel mutagenesis element that is ligated at high frequency into random positions of the H. influenzae chromosome. The bacterial strains and plasmids used are listed in Table 1. Plasmid pCS was constructed by interruption of a random

7.8-kbp EcoRI chromosomal fragment of H. influenzae Ela,

previously cloned into pJl-8, by inserting the TSTE element in place of an internal 1.2-kbp BglII fragment. H. influenzae was grown in brain heart infusion medium (Difco Laboratories, Detroit, Mich.) supplemented with 10 jig each of hemin and P-NAD per ml (sBHI). For the selection of antibioticresistant H. influenzae, carbenicillin (Cb; 3 fxg/ml) and ribostamycin sulfate (Rb; 15 pg/ml) were added. E. coli was cultured in Luria-Bertani broth containing 15 ,g of Cb or 40 ,ug of Rb per ml when appropriate. Competence was developed in H. influenzae by using MIT medium (28), and transformations were performed with *

Corresponding author. 1561

saturating quantities of DNA (.0.5 ,ug/ml). Transformation of E. coli was performed by the method of Hanahan (10). DNA fragments were separated in agarose gels and transferred to nitrocellulose by the method of Southern (27). Hybridization probes were prepared by nick translation (Boehringer Mannheim) using [ox-32P]dCTP as instructed by the manufacturer, and hybridization bands were detected by autoradiography with Kodak X-Omat AR film. Haemocin production by H. influenzae was detected by using an in vitro bioassay as described previously (18). Ribostamycin selection for aminoglycoside phosphotransferase activity. In initial experiments, we observed that kanamycin was lethal to a significant fraction of TnS-containing H. influenzae. Aminoglycoside 3'-phosphotransferase encoded by TnS is capable of modifying a variety of other aminoglycoside substrates (7), and the aminoglycoside most useful for the selection of this marker varies with the species (24). Aminoglycoside selectivity in H. influenzae was investigated by plating approximately 108 CFU of H. influenzae Ela and approximately 103 CFU of H. influenzae 1613 OMP Pl::TnS tac I (1613 TnS), either separately or after mixing, onto media containing 5 or 15 ,ug of butirosin sulfate, gentamicin sulfate, hygromycin B, kanamycin sulfate, kasugamycin, neomycin sulfate, Rb, or sisomicin sulfate (Sigma, St. Louis, Mo.) per ml. Selection on 15 p.g of Rb per ml most accurately detected the H. influenzae with the TnS neo gene. Similarly, selection of E. coli transformants was more accurate by using 40 ,ug of Rb per ml than 50 jig of kanamycin per ml. Construction of the TSTE element. The TSTE element contains the neo gene from TnS, encoding aminoglycoside resistance, flanked by synthetic oligonucleotides containing the uptake sequences specific for transformation into H. influenzae (Fig. 1). To construct the TSTE element, TnS was first digested with HindIll, removing 80% of the insertion sequence inverted repeats to prevent transposition and reduce the size of the insert; the remaining Hindlll fragment was cloned into pBR322. Similarly, an internal fragment was deleted by complete digestion with BamHI and partial digestion with BglII, followed by self-ligation of the plasmid and transformation into E. coli HB101. Two largely complementary oligonucleotides were synthesized with the following

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J. BACTERIOL.

NOTES

TABLE 1. Bacterial strains and plasmids used Strain or plasmid

E. coli HB101 JM109 K-12 H. influenzae Ela Rd Rd Hmc+ 1613 TOS Plasmids pBR322

Genotype and phenotypea

p

F- recAl3 lacYI Smr recAl laclq lacZAM15 F+

20 34 2

Serotype b, Smr Nonserotypable, HmcNonserotypable, Hmc+ OMP PI::Tn5 tac I Neor

29 1 18 22

pMB1 replicon; lacZ' Apr pUC19 pUC19::TSTE Neor 3.8-kbp E. coli-H. influenzae pJl-8

pCS

CHROMOSOMAL DNA

Reference

8HI 8aalmt1 0I 00

BnI lmH

4:.-p r

us

us

TSTE MUTAGENIZING ELEMENT

s

3, 30 34 This study

12

shuttle vector

TSTE interruption of H. influenzae This study chromosomal insert

Smr, Streptomycin resistance at 50 pLg/ml in E. coli and H. irjfluenzae; OMP PI::TnS tac I, Tn5 tac I is contained within the gene encoding protein I; Neor, neomycin resistance; Apr, ampicillin resistance at 40 pLg/ml in E. coli. a

I,fdigeson

p POMM

Pp

p

I

P

p

I fdigesfion

P s P s s CHRMSML FRAGMENTS

PS 8

2°lgaon TSTE

TSTE

TSTE

SIp

-p

S

Syp

T_1f| Selction wkh Rb or TSTE Inwtons

sequences: 5'-GATCCAAGTGCGGTATTT-3' and 3'-GT TCACGCCATAAATCGA-5'. The DNA hybrid formed by these oligonucleotides includes (i) a BamHI restriction site, (ii) HindIll sticky ends, and (iii) the Haemophilus 9-bp transformation uptake sequence and an A+T-rich 3' end (5, 6). The oligonucleotides were phosphorylated with T4 kinase and ligated to the deleted Tn5 HindIII fragment; following this ligation, the HindIlI site is lost. The resulting BamHI fragment was ligated to BamHI-digested pUC19 and transformed into E. coli JM109. Plasmid pUC19::TSTE was isolated from one Cb- and Rb-resistant transformant, and digestion with BamHI yielded the predicted 2.2-k-bp insert. The nucleotide sequences of the ends constructed with the synthetic oligonucleotides were identical to one another and to the sequence defined above. The TSTE element lacks internal restriction sites for various enzymes, including Ball, EcoRI, Hindlll, KpnI, and PvuI (11). These restriction enzymes can be used for cloning the insertion element along with flanking DNA. . The strategy for insertion Inseronal mutgeness s of the TSTE element into the chromosome of H. influenzae is illustrated in Fig. 1. Restriction fragments resulting from primary digestion of chromosomal DNA from H. influenzae Ela were ligated at a concentration (1 ,Lg/300 ,lI) that promotes intramolecular ligation. The resultant circular molecules were subjected to secondary digestion with enzymes that produce GATC sticky ends, such as BamHI, BclI, BgllI, MboI, XhoII, and Sau3A. This provides complementary ends for ligation to BamHI-digested TSTE. The TSTE element was ligated to this DNA at a concentration that promotes intermolecular ligation (1 p4/20 ,l). Approximately 10 competent H. influenzae were transformed with 0.5 to 1 ,ug of the recombinant molecules. Organisms containing TSTE insertions were detected by growth on sBHI agar containing 15 pg of Rb per ml. Chromosomi al_of a TSTE-interrupted gment. To determine whether a TSTE-interrupted DNA fragment would faithfully recombine into the H. influenzae chromosome, strain Ela was transformed with the TSTEinterrupted EcoRI chromosomal fragment of pCS. Chromosomal DNA from 40 Rb-resistant, Cb-sensitive transformants was digested with EcoRI, and the fragments were

0de! I Sa_"fmws

spof

FIG. 1. TSTE mutagenesis strategy. TSTE is a 2.2-kbp BamHI fiagment consisting of the neo gene of TOS flanked by Haemophilusspecific uptake sequences. Chromosomal DNA was purified from the H. influenzae strain to be mutagenized. Restriction fragments (primary digestion) of this chromosomal DNA were ligated at a concentration favoring intramolecular ligation. These molecules were linearized (secondary digestion) and ligated to TSTE. The homologous strain was transformed with the ligation mixture, and transformants were selected on Rb. Symbols: US, Haemophilusspecific uptake sequences; B, BamHI; Bg, BglH; 1°, primary; P, primary digestion site; 2°, secondary; S, secondary digestion site; FJ, TSTE DNA; m, gene of interest.

analyzed by Southern blot hybridization. All of the transformants, but not wild-type H. influenzae, contained a single EcoRI fragment of 8.4 kbp which hybridized to 32P-labeled TSTE element. As predicted, a hybridization band of approximately 7.2 kbp was evident in the wild-type strain after hybridization with the 32P-labeled chromosomal DNA fragment from pCS (data not shown). This altered size represents the difference resulting from the deletion of a section of the chromosomal insert and the insertion of the TSTE element during the construction of pCS. Characterizatio of TSTE chromosomal insertons. The number of insertion sites per chromosome and the average fragment size that promotes efficient transformation were determined in experiments using the mutagenesis strategy outlined in Fig. 1. EcoRI and Bgll were used at the primary and secondary digestion steps, respectively. Southern blot analysis of EcoRI-digested chromosomal fagments from 17 Rb-resistant transformants revealed a single band that hybridized to radiolabeled TSTE, indicating the presence of one insertion per transformant. The bands ranged in size from 7.4 to 25 kbp. Random chromosomal insertion of the TSTE element. To improve the randomness of insertions, an alternative to

VOL. 173, 1991

A B

NOTES CD

E

F GH

J

K

L

MN

p

13 11 -

6.6-

4.4_ 2.3_

FIG. 2. Insertions of TSTE into Sau3A sites of the Ela chromoShown is an autoradiograph of a Southern blot of BglIIdigested chromosomal DNA from 15 RbF transfonnants (lanes B through R) probed with 32P-labeled pUC::TSTE. The transformants were detected after transforming strain Ela with chromosomal DNA constructed as shown in Fig. 1, using Sau3A partial digestion at the primary and secondary steps of digestion. Lane A contains chromosomal DNA from parent strain Ela; lane P contains BamHI-digested pUC::TSTE. Lanes A to 0 each contain approximately 4 ,g and some.

lane P contains approximately 30 of HindIIl

of DNA. Sizes (kilobase pairs) fragments of lambda DNA are shown on the left. ng

EcoRI and BglII digestion in the mutagenesis strategy was developed by using partial Sau3A digestion (one cleavage per 15 kbp) at each step. The internal BglII site of TSTE was used to assess the randomness of insertion. BglII-digested chromosomal fragments from Rb-resistant transformants were subjected to Southern blot analysis. Southern blots of the DNAs from 40 random transformants were probed with 32P-labeled TSTE; two bands were detected for each transformant, with no evidence of tandem TSTE sequences (Fig. 2). The site of TSTE insertion in the transformants was highly variable. In experiments using a twofold-higher concentration of TSTE (6:1 TSTE/chromosomal fragment ratio) during ligation, three bands were detected in some transformants; one band corresponded to the 2.2-kbp TSTE element, indicating tandem repeats of the TSTE element (data not shown). Efficiency of TSTE insertions. The efficiency of natural transformation varies among strains of H. influenzae (23). Strain Rd can be transformed 1 to 2 orders of magnitude more efficiently than the type b strain Ela. The transformation frequency of H. influenzae Ela using Sau3A as described above was 1.5 x 10-6 transformants per cell. In experiments with ligation of TSTE after the primary digestion, i.e., omitting circularization, the transformation frequency was 3.9 x 1O-7 transformants per cell. After circularization, secondary digestion, and ligation with the TSTE element, introduction of the DNA constructs into Rd Hmc+ (see below) occurred at a frequency of 4.4 x 1O' transformants per cell, or approximately 2 orders of magnitude greater than the frequency obtained with strain Ela. Thus, to improve the frequency of TSTE insertions, derivatives of strain Rd were used for detecting mutants. Isolation of haemocin-deficient mutants. To determine the utility of this insertional mutagenesis strategy for generating mutants of H. influenzae, the genes encoding production of the H. influenzae bacteriocin haemocin were investigated. H. influenzae Rd Hmc+ is a haemocin-producing transformant of strain Rd (18). By using homologous DNA, strain Rd Hmc+ was subjected to TSTE mutagenesis. Eight Hmcmutants were isolated by screening a library of 3,250 Rb-

1563

resistant transformants. After introduction of chromosomal DNA from one of these Rd::TSTE Hmc- mutants into Rd Hmc+, 25 Rb-resistant transformants were screened for haemocin production. None of these transformants produced detectable haemocin, demonstrating that the TSTE element mediated the mutations. Chromosomal DNA from five of the secondary transformants was further analyzed by Southern blot hybridization; when probed with radiolabeled TSTE, all transformants demonstrated the same banding pattern as the initial transformant from which they were derived. Insertions obtained by using the TSTE element with this mutagenesis method were stable, easily detectable, distributed randomly, inserted only once per genome, and obtained in high frequency. The effectiveness of this novel method has been demonstrated by the detection of hmc mutants of H. influenzae. We anticipate that this approach will be useful in other bacterial species; we have recently used TSTE to construct a lac mutant of wild-type E. coli K-12 (data not

shown).

We thank Sol Goodgal for insightful discussions and critique of the manuscript. Support for this project was provided by grant 22140 from the National Institute of Allergy and Infectious Diseases. REFERENCES 1. Alexander, H. E., and G. Leidy. 1953. Induction of streptomycin resistance in sensitive Haemophilus influenzae by extracts containing deoxyribonucleic acid from resistant Haemophilus influenzae. J. Exp. Med. 97:17-31. 2. Bachmann, B. J. 1972. Pedigrees of some mutant strains of Escherichia coli K-12. Bacteriol. Rev. 36:525-557. 3. Bolivar, F., R. L. Rodriguez, P. J. Greene, M. C. Betlach, H. L. Heynecker, H. W. Boyer, J. H. Crosa, and S. Falkow. 1977. Construction and characterization of new cloning vehicles, a multi purpose cloning system. Gene 2:95. 4. Bukhari, A. I. 1976. Bacteriophage Mu as a transposition element. Annu. Rev. Genet. 10:389-412. 5. Danner, D. B., R. A. Deich, K. L. Sisco, and H. 0. Smith. 1980. An eleven-base-pair sequence determines the specificity of DNA uptake in Haemophilus transformation. Gene 11:311-318. 6. Danner, D. B., H. 0. Smith, and S. A. Narang. 1982. Construction of DNA recognition sites active in Haemophilus transfor-

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A novel approach to insertional mutagenesis of Haemophilus influenzae.

Insertional mutagenesis of the Haemophilus influenzae chromosome was accomplished by a novel method employing a 2.2-kbp element, TSTE. This element, c...
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