Vol. 172, No. 10

JOURNAL OF BACTERIOLOGY, OCt. 1990, p. 5991-5998

0021-9193/90/105991-08$02.00/0 Copyright © 1990, American Society for Microbiology

TnlOOO-Mediated Insertion Mutagenesis of the Histidine Utilization (hut) Gene Cluster from Klebsiella aerogenes: Genetic Analysis of hut and Unusual Target Specificity of TnlOOO ANTHONY SCHWACHA, JILL A. COHEN,t KALLE B. GEHRING,t AND ROBERT A. BENDER* Department of Biology, The University of Michigan, Ann Arbor, Michigan 48109-1048 Received 24 August 1989/Accepted 27 June 1990

The histidine utilization (hut) genes from Klebsiella aerogenes were cloned in both orientations into the Hindlll site of plasmid pBR325, and the two resulting plasmids, pCB120 and pCB121, were subjected to mutagenesis with Tn1000. The insertion sites of TnlOOO into pCB121 were evenly distributed throughout the plasmid, but the insertion sites into pCB120 were not. There was a large excess of TnlOOO insertions in the "plus" or y8 orientation in a small, ca. 3.5-kilobase region of the plasmid. Genetic analysis of the TnlOOO insertions in pCB120 and pCB121 showed that the hutUH genes form an operon transcribed from hutU and that the hutC gene (encoding the hut-specific repressor) is independently transcribed from its own promoter. The hutlG cluster appears not to form an operon. Curiously, insertions in hutI gave two different phenotypes in complementation tests against hutG504, suggesting either that hutI contains two functionally distinct domains or that there may be another undefined locus within the hut cluster. The set of TnlOOO insertions allowed an assignment of the gene boundaries within the hut cluster, and minicell analysis of the polypeptides expressed from plasmids carrying insertions in the hut genes showed that the hutI, hutG, hutU, and hutH genes encode polypeptides of 43, 33, 57, and 54 kilodaltons, respectively.

our surprise, the insertion site specificity of Tn.1000 was decidedly nonrandom. Also surprisingly, the hutIGC cluster appears to contain at least two transcription units.

The histidine utilization (hut) gene cluster of Klebsiella aerogenes encodes the structural and regulatory proteins responsible for the inducible degradation of the amino acid L-histidine to glutamate, ammonia, and formamide (13). The hut genes are located between gal and bio on the K. aerogenes genetic map and are arranged in the order hut(M) IGC(P)UH (4, 7), where hut(M) and hut(P) are defined as the regions required for regulated transcription of hutI and hutU, respectively; hutl, hutG, hutU, and hutH encode the four enzymes of the pathway; and hutC encodes the hutspecific repressor which negatively regulates the entire pathway. In the closely related enteric bacterium Salmonella typhimurium, the hut cluster is arranged as two independent transcription units, one originating at hutIp [within the hut (M) region] and extending through hutIGC, and the other originating at hutUp [within the hut(P) region] and extending through hutUH (26, 27). Several lines of evidence led us to expect that the arrangement of transcription units in K. aerogenes would be the same as that in S. typhimurium: (i) the gene order is identical; (ii) hutU (but not hutI) expression is coordinate with hutH (7, 14); (iii) hutU and hutH do not require the hutIGC region for expression (4); and (iv) the hut DNA from K. aerogenes and S. typhimurium are so similar that under appropriate conditions, a continuous heteroduplex molecule can be formed across the entire length of the cluster (3). To identify the transcription units within the hut cluster and to provide a mobile priming site for later DNA sequence analysis of the hut genes, we isolated and characterized a set of transposon Tn1OOO (-y) insertions in the hut region. To

MATERIALS AND METHODS Strains, plasmids, media, and enzymes. Table 1 lists the source and genotype of strains and plasmids used in this study. Rich medium was LB; minimal medium was W4 containing carbon sources at 0.4% (wt/vol), nitrogen sources at 0.2% (wt/vol), and supplements as described in the Results section. W4 salts are W salts (2) adjusted to pH 7.4. Restriction enzymes and T4 DNA ligase were obtained commercially and used as directed by the supplier. Tn1000 mutagenesis. Transposon mutagenesis was performed essentially as described by Guyer (8). In short, F152/KL253, a multiply auxotrophic Escherichia coli strain bearing F152 (an F' gal plasmid), was transformed to chloramphenicol and ampicillin resistance with either of the hut plasmids pCB120 or pCB121. The chloramphenicol and ampicillin resistances of the two resulting strains (EB1021, carrying pCB120, and EB1022, carrying pCB121) were mobilized by F152-mediated conjugation and thus transferred to E. coli RH202. Such mobilization is believed to occur by TnJOOO-mediated cointegrate formation (8). Resolution of the cointegrate results in a mobilized plasmid with a single Tn1000 insertion. The resulting transconjugants (with insertion mutations in pCB120 or pCB121) were purified, and their ability to grow with histidine or urocanate as the sole carbon source was tested. The location of each TnJOOO insertion was determined by restriction endonuclease analysis (15). Transfer of mutations from plasmid to chromosome. The TnJOOO insertion mutations were transferred from the plasmid to the chromosome in two steps essentially as described previously (21). In the first step, the plasmid was forced to integrate into the chromosome at hut via homologous recombination. This was done by taking advantage of the inability

Corresponding author. t Present address: University of Michigan Medical School, Ann Arbor, MI 48109. t Present address: Cell and Molecular Biology Division, Department of Biophysics, Lawrence Berkeley Laboratory, University of California, Berkeley, CA 94720. *

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TABLE 1. Strains and plasmids used in this studya E. coli

EB566 EB1021 EB1022 EB1098 EB1486 EB1536 EB1538 EB1591 EB1593 EB2326 EB2327 F152/KL253 KS302 MM383 RH202 K. aerogenes KC2151 KC2293 KC2328 KC2329 KC2457 KC2486 KC2488 KG2000 Plasmids pCB120 pCB121 pCB211 pCB214

Source or reference

Relevant characteristics

Strains

hutCS5Sb lac rpsL594 hsdR glk F152/KL253 with pCB120 F152/KL253 with pCB121 RH202 with pCB120.56 (Hut') MM383 with hut from EB566 KS302 with hutC+ As EB1486 but hutC+ As EB1486 but hutU72::TnlOOO As EB1486 but hutG84::Tn1000 As EB1486 but hutH64::Tn1000 As EB1486 but hutI58: :TnlO00 purD34 trp45 his-68 recAl thi-1 galK35 F'(lip-*gal) HfrH, A(gal-bio) lacZ53 rpsL151 thyA36 polA12 rha-5 deoC2 IN(rrnD-rrnE)J lacYI tonA21 thi-I supE44 hsdS

P1 MK9000 x EG47 (6) Transformation Transformation Transformation P1 EB566 x MM383 This study This study This study This study This study This study 10 D. Friedman 16 1

hut' hut'

R. A. Bender, unpublishedd This study (7) This study This study This study This study P1 KG2000 x KC2293 7

Apr Cmr Hut' Apr Cmr Hut' Apr hutC+U+ Apr hutC+ U+

This This This This

hutC515 recA3011 hutG504 KC2151 with pCB211 KC2151 with pCB214 KC2151 with pCB120 hutG504 recA3011

study studye study studye

a All E. coli strains were derived from strain K-12; all K. aerogenes strains were derived from strain W70; all plasmids were derived from plasmid pBR322 or pCB325. b The hut operons in this strain are derived from K. aerogenes. C A lysate of phage P1 grown on strain MK9000 was used to transduce strain EG47 to Hut+. d This strain will be characterized elsewhere. The recA allele was defined to be recA in that it makes strains carrying it very sensitive to UV light, it is tightly linked to srl by transduction, it reduces recombination frequencies at least 1,000-fold, and it is complemented by a cloned recA gene from E. coli. ePlasmids pCB120 and pCB121 are identical except for the orientation of the cloned fragment; the same is true for plasmids pCB211 and pCB214.

of the pBR325-derived plasmids pCB120 and pCB121 to replicate in polA(Ts) strains at nonpermissive temperatures. In the second step, the plasmid was forced to excise, again by homologous recombination in the region. This was done by taking advantage of the lethal character of integrated pBR325 replicons in polA(Ts) strains at permissive temperatures. Excisants either restored the original hut' operon of the chromosome or left behind the Tn1000 mutant form from the plasmid. The presence of the mutant form of hut was confirmed physically (by Southern blot analysis), phenotypically (by failure to grow on histidine as sole carbon source), or by both methods. The hutC' allele was transferred from pCB120 to the chromosome of strain EB1486 by a similar procedure, using the deletion mutation found on plasmid pCB103 (4) as an intermediate. Wild-type E. coli strains lack hut DNA; the hut operon was transferred to the initial E. coli strain as described previously (29). Assays. Histidase was assayed as described by Boylan and Bender (4) and formiminoglutamate hydrolase was assayed by the method of Lund and Magasanik (12). Whole cells were assayed for protein by the method of Lowry et al. (11) with bovine serum albumin as a standard. Genetic methods. The procedures for conjugation, P1mediated transduction, and transformation of competent cells with DNA have been described elsewhere (4, 28).

RESULTS

TnlOOO mutagenesis. Plasmids pCB120 and pCB121 were

constructed by cloning the 7.88-kilobase-pair (kb) fragment carrying the hut gene cluster from plasmid pCB101 (5) into

the HindlIl site of pBR325. The hut genes in these two plasmids lie in opposite orientations with respect to pBR325. Both plasmids were subjected to transposon TnJOOO mutagenesis as described in Materials and Methods. Experiments with either strain EB1021 (carrying pCB120) or EB1022 (carrying pCB121) as the donor yielded transconjugants (i.e., mutants) at frequencies between 0.003 and 0.15% per donor. Virtually all of the transconjugants tested showed a single Tn1000 insertion in the plasmid, since a single 5.7-kb insertion was detected in 144 of 145 plasmids tested by restriction enzyme digestion. Moreover, each of the insertions appeared to be at a unique site (see below). The mutant transconjugants arising from this mutagenesis could be divided into two distinguishable phenotypes: Hut+, which could use histidine as the sole carbon source, and Hut-, which could not. The Hut- class could be further subdivided into two classes: HutH-, which could use urocanic acid (the first intermediate in the pathway) as the sole carbon source, and HutIGU-, which could use neither histidine nor urocanate. Table 2 summarizes the results of 10 independent experiments (5 each with pCB120 and pCB121)

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UNUSUAL SPECIFICITY FOR yB INSERTION IN hut

TABLE 2. Distribution of phenotypic classes obtained from TnlOOO mutagenesisa Phenotype

% of transconjugants of mutagenized strain: EB1021(pCB120)

Total Hut' Total HutHutHHutIGU-

22.0 78.0 18.8 59.2

± ± ± ±

EB1022(pCB121)

4.6 4.6 1.6 4.4

65.4 34.6 7.0 27.6

± ± ± ±

7.0 7.0 3.3 7.2

a Plasmids pCB120 and pCB121 were mutagenized with TnlO00 as described in Materials and Methods, and the phenotypes of the resulting transconjugants were determined. The data shown represent the average of five different experiments, with a total of 337 EB1021(pCB120) transconjugants and 323 EB1022(pCB121) transconjugants. The three phenotypes were distinguished as follows: Hut', able to use both histidine and urocanate as the sole carbon source; HutH-, able to use urocanate but not histidine as the sole carbon source; HutIGU-, able to use neither histidine nor urocanate as the sole carbon source.

carried out over a 4-year period, with 50 to 100 transconjugants scored per experiment. There was a marked, reproducible difference in the distribution of phenotypes when different plasmids were mutagenized. Mutagenesis of pCB120 invariably resulted in more than twice as many Hut- mutations (78%) as did mutagenesis of pCB121 (35% Hut- mutations). The only difference in these two sets of experiments is the orientation of the cloned hut region relative to the vector sequences in the plasmid. To confirm this difference in the distribution of phenotypes, we analyzed the site of insertion of the Tn1000 element in a randomly selected set of mutants of both pCB120 and pCB121. Distribution of insertion sites in pCB120 and pCB121. The map of the TnJOOO element available to us (8) lacked information about restriction sites for PstI, BglIl, and AvaI, three enzymes that are particularly useful for mapping the hut cluster. We therefore extended the TnJOOO map to include these sites (Fig. 1) and confirmed the locations of the cleavage sites for BamHI, EcoRI, HindIII, Sall, and XhoI. These latter sites agreed with the known map for Tn1000 (8), confirming that the element on F'152(lip-gal) is typical of the elements previously characterized. A total of 76 randomly chosen transconjugants from mutagenesis of pCB120 and 68 from mutagenesis of pCB121 were purified on selective media, their phenotypes were determined, and the location of their insertions was determined by restriction endonuclease analysis of plasmid DNA. The sites of insertion and the orientations of these 144 insertions are shown in Fig. 2. The distributions of the sites of insertion (Fig. 3) are noticeably different between the two plasmids, consistent with the difference in the distribution of phenotypes shown in Table 2. The region believed to encode the urocanase gene (kb 3.6 to 5.2 on the hut restriction map

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[Fig. 3]) seems to be a hot spot for insertion into pCB120, particularly in the plus or yb orientation, with a gradual reduction of insertion frequency in the hutH region and a very sharp reduction in the hutIGC region. In fact, only 2 Hut- insertions of the 76 tested (3%) lay in the 3.6 kb of cloned hut DNA located between the left end of hut and the hutUH cluster (33% of the available target). When an additional 60 Hut- insertions in pCB120 were tested, 5 more insertions in the region from kb 0.0 to 2.5 were found, showing that insertion into this region in pCB120 is rare but not lethal. The distribution of insertion sites in pCB121 was more random than that in pCB120, and insertions were found throughout the hut region, including the interval from kb 0.0 to 2.5 (Fig. 3B). When all the pCB120 and pCB121 insertion sites were combined, the collection of Tn1000 insertions covered the entire hut region, with insertions at intervals of less than 100 to 200 base pairs, except for one small region (from kb 1.5 to 2.05) defining the hutI-hutG boundary (see below) and one large region (from kb 2.55 to 3.55) known to contain the hutC (repressor) gene (4). Extent of the hut genes. (i) hutUH. The hut cluster as a whole must lie between kb 0.4 and approximately 6.8 on the physical map of this region. An insertion at position 0.4 is still Hut', but all insertions mapping to the right of this insertion (including another that also lies at about kb 0.4) are Hut- (Fig. 2). The right end of the cluster is similarly defined by the fact that all insertions to the right of position 6.8 are Hut' while all insertions located to the left of this insertion (including one that also lies at about kb 6.8) are Hut- (Fig. 2). The promoter of the hutU gene was assumed to lie at position 3.5 (18). An insertion located very near this site (isolated in plasmid pCB120.135) was the leftmost insertion identifiable as being in the hutUH operon, perhaps defining the promoter. To locate this insertion more precisely, a PstI junction fragment including the fy end of TnJOOO was cloned and the DNA sequence of this junction was determined and compared with the published sequence of the hut(P) region (17). The insertion in pCB120.135 lies 13 bp to the left of the PvuII site at kb 3.59, within the fourth codon of the urocanase gene. The Hut- phenotype of this insertion confirms that the region previously identified at hutUp is indeed required for hutU transcription. The data summarized in Fig. 2 show that all hutUH insertions that lie to the right of position 5.3 cause the HutH phenotype (i.e., are defective in the hutH gene) and that all hutUH insertions to the left of position 5.0 cause the HutIGU phenotype (and must therefore affect hutU). Therefore the boundary between hutU and hutH must lie at about position 5.2. (ii) hutIGC cluster. Since insertion into hutI and hutG have identical phenotypes (HutIGU-), these two classes were

N cq N

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I

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(A

a. cx, a. ~~~~~~(1 c,

cn

CIE,

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,oin

8

(5.7) (OQ) FIG. 1. Restriction map of Tn1000 from F152. Sites without asterisks were determined by Guyer (8) and confirmed by us for this isolate. Sites with asterisks were determined in this work.

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pCB120 -01

i . pBR325

CB121

la.

1 T_ -

-

I Q Q

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FIG. 2. Physical location of TnlO00 insertions in the hut-containing plasmids pCB12O and pCB121. The restriction map of the hut region (5) is shown at the bottom. Above the restriction map, the deduced extents of the five hut genes are indicated (see text). The positions of the TnlO00 insertions relative to the restriction map are indicated in the two upper lines of the figures, and their resulting genotypes are encoded in the symbols as follows: A\, hutH (can grow with urocanate but not histidine as the sole carbon source); 0, hutU (cannot grow with either histidine or urocanate as the sole carbon source); V, hutG (able to complement hutIS19 but not hutGSO4 for growth on urocanate); O, hutl (able to complement hutGS04 but not hutIS19 for growth on urocanate). Symbols above the line represent insertions in the plus (yb) orientation, and those below the line represent insertions in the minus (b^y) orientation. Multiple symbols on a single stem represent insertions at near but not identical sites. Solid symbols (0) represent Hut+ insertion mutants.

resolved by complementation against known hutI and hutG mutations. All plasmids with mutations mapping between kb 0.2 and 3.5 were transferred into strains KG2005 (hutIS19) (7) and KC2293 (hutG504) (7), and the growth of the resulting transformants was tested on urocanate minimal medium. Mutations that lay between kb 0.4 and 1.5 were able to complement hutGS04 but not hutIS19, showing that these insertions lie in hutI. The ability of insertion mutations in hutI to complement mutations in hutG was unexpected since the data from the related hut cluster from S. typhimurium strongly suggested that hutI and hutG formed a single operon transcribed in the order hutIG (27). Insertions mapping between kb 2.05 and 2.55 complemented hutI519 but not hutG504 and were therefore assigned to the hutG gene. No insertions were isolated in the region between kb 1.5 and 2.05, leaving the boundary between hutI and hutG somewhat imprecise. Considering the size of the polypeptides encoded by hutI and hutG (see below), the boundary is likely to lie near kb 1.7. Insertions in the hutC gene were not expected to give a Hut- phenotype, but this could not be directly confirmed with this set of insertions as no insertions in the hutC region were found. Still, since a mutation at kb 2.55 is clearly in hutG and one at kb 3.5 is clearly in hutU, the maximum possible extent of hutC would be from kb 2.55 to 3.5. (iii) hutC gene. Since no insertions in hutC were recovered from the mapped sets of insertions described above, either

the repressor region is a transpositional cold spot or insertions into the repressor gene are deleterious to cell growth. If the repressor protein were essential for cells carrying pCB120 and pCB121, the presence of hutC+ in trans on the chromosome might allow isolation of a hutC::TnlO00 mutation on the plasmid. To test this possibility, we mutagenized pCB121 by F-mediated conjugal transfer from strain EB1022 into EB1536, an E. coli strain that has a copy of the K. aerogenes wild-type hut genes integrated into the chromosome. Seventy-two transconjugants were screened by restriction analysis and agarose gel electrophoresis for insertions located in the smallest Sall fragment (including the region from kb 2.0 to 3.35). Seven such insertions were found, and two were subsequently mapped to positions 2.75 and 2.9. E. coli strains which carried a chromosomal hutC+ allele as well as either of the two hutC: :Tn1000 plasmids had no obvious growth defect. However, E. coli strains lacking a chromosomal hutC+ allele and carrying either of the two hutC::Tn1000 plasmids formed small, mucoid colonies that would not easily repurify on selective media. This observation explains why no hutC::Tnl000 mutants were initially obtained. An insertion at position 7.37. We were able to resolve a remaining ambiguity in the published restriction map of the hut region (5). In this restriction map, the BamHI fragments between kb 7.28 and 7.74 could not be ordered and the BamHI site at kb 7.44 was arbitrarily placed. The insertion in

VOL . 172 1990

UNUSUAL SPECIFICITY FOR

,

+ Wlui _ _-nOK4 M§\OTUI GLC U

H

Cm

INSERTION IN hut

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TABLE 3. Regulation of histidase formation in hut::Tnl000 mutant strainsa

Ap

r-

20

yb

Sp act' of:

z w

Strain

0

EB1538 EB1486 EB2326 EB1591 EB1593 EB2327

1

10

z

5

-

hut genotype

hut' hutCSJS

hutH64::Tnl000 hutU72::Tnl000

hutG84::Tnl000 hutIS8::Tnl000

FIGlu hydrolase

Histidase

-U

+U

-U

+U

Tn1000-mediated insertion mutagenesis of the histidine utilization (hut) gene cluster from Klebsiella aerogenes: genetic analysis of hut and unusual target specificity of Tn1000.

The histidine utilization (hut) genes from Klebsiella aerogenes were cloned in both orientations into the HindIII site of plasmid pBR325, and the two ...
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