selection and endpoint distribution of bacterial ... - Europe PMC

Copyright 0 1983 by the Genetics Society of America

SELECTION AND ENDPOINT DISTRIBUTION OF BACTERIAL INVERSION MUTATIONS MOLLY B. SCHMID

AND

JOHN R. ROTH

Departtnetit o j Biology, Uiiiuersity of Utah, Salt Lake City, Utah 841 12 Manuscript received March 31, 1983 Revised copy accepted August 5 , 1983 ABSTRACT

This paper describes the isolation and characterization of spontaneous inversion mutants of Saltnotiella typhimurium. The mutants are selected by demanding that an unexpressed hisD gene acquire a new promoter. Chromosome rearrangements that juxtapose the hisD gene and a foreign promoter are obtained by this selection. Although a number of inversions are found, the frequency was lower than expected. The breakpoints of these inversions are not distributed randomly either in the his operon or on the chromosome. The his breakpoint lies in the hi&-hisD intercistronic region, a sequence known to occur at several places on the bacterial chromosome. In most of the inversions, the "non-his" breakpoint lies across the chromosome, so that the inverted region includes the origin or terminus of DNA replication. The significance of these results is discussed.

NVERSIONS are rare among bacterial mutations for unknown reasons. Iacteristics Strains carrying a bacterial inversion mutation have unique genetic char(ROTH and SCHMID SCHMIDand ROTH Using these

1981; 1983). characteristics, we believe that we can recognize bacterial inversions when they occur. We have selected spontaneous bacterial inversions with the hope that the map location of their endpoints might suggest a reason for the low frequency of inversion mutations. Two bits of data on previously characterized inversions suggest that only certain inversions will be viable. First, the I N 2 8 2 inversion, described in the previous paper, spans about 20% of the genetic map and has inversion breakpoints that bracket and are approximately equidistant from the and ROTH 1983). This makes the inverterminus of DNA replication (SCHMID sion very similar to the naturally occurring inversion that differentiates the E. coli and S. typhiinurium genetic maps (CASSE, PASCALand CHIPPAUX1973). Second, the results of KONRAD(1969, 1977) suggest that some inversions will not be viable. KONRADbuilt a strain carrying partial lac operons at two sites on the chromosome (at the normal lac region and at the @Watt site) in inverse orientation. T h e two partial lac regions cannot complement, so the strain is phenotypically Lac-. However, recombination should have yielded Lac+ mutants carrying an inversion between lac and 480att. Although an extensive search was made, these simple inversion mutants were never recovered. The

Genetics 105: 539-557 November, 1983.

540

M. B. SCHMID A N D J. R. ROTH

reason for this still remains a mystery. It suggests that a strain carrying an inversion between these two chromosome regions may not be viable. In selecting more inversion mutants, we have increased the number of inversion-carrying mutants that we can detect by using a genetic screen that is less demanding than that described in the previous paper. The screening method used here should detect inversion mutations that fuse the hisD gene to any distant nonessential transcript. These HisD' mutants are not required to show a new auxotrophy as was required previously (SCHMIDand ROTH 1983). MATERIALS A N D METHODS

Genetic techuigues: Basic genetic techniques are described in the previous paper (SCHMIDand ROTH 1983) and in the manual by DAVIS,BOTSTEINand ROTH (1980). Multiply marked bacterial strains used in this study are listed with their complete genotypes in Table 1. All of the inversion strains listed in Table 1 were independently isolated. Nomenclature conventions are described in the previous paper (SCHMID and ROTH 1983). A genetic map of the his operon is shown in Figure 1.

As described previously (SCHMID and ROTH 1983), HisD+ mutants of strain his-203 were selected on minimal medium (E) plates supplemented with amino acids, nucleosides and histidinol. In all cases, auxotrophic h i 0 mutants could have been recovered but were not found. T h e screening procedure identified histidinol-utilizing mutants (HisD+) in which a chromosome rearrangement led to the HisD+ phenotype. To be detected by our screening procedure, the chromosome rearrangement must prevent repair of the his operon by PZZ-mediated generalized transduction. HisDC mutants were picked from the selection plates and purified once on the same medium by streaking for single colonies. A single colony from each streak was tested for the ability to be transduced to His'. Massive testing was accomplished by cross-streaking each mutant against P22 H T int- transducing phage that was grown on a strain carrying the his constitutive mutation, h i s 0 2 2 4 2 . T h e rationale of this procedure is described in RESULTS. Most HisD+ mutants readily give His+ transductants that have a rough colony morphology. Any mutants showing very reduced transducibility to His+ or yielding His+ transductants with a smooth colony morphology were purified on nonselective medium and retested in full plate transduction tests. Potential inversion mutants were frozen in 8% dimethyl sulfoxide (DMSO) at -70". Strciius used to itlop the his h e r s i o n breakpoint: To construct the strains used to map the his inversion breakpoint, the HisD+ inversion mutants were transduced to TetR with phage grown on strain TTI 889 (his02242, rer-2::TnlO). All transductants that inherit the zee-2::TnlO insertion must also inherit the h i s 0 1 2 4 2 mutation, because both markers lie in the sequences deleted by the h i - 2 0 3 deletion. TetRHisDC His- strains were saved. These strains were purified, and a P22 phage lysate was grown on the strains for use in the mapping crosses shown in Table 6. Strciiris used to mcrp the "mi-his" inversion bredpoint: To construct strains for use in mapping the non-his inversion breakpoint, the inversion strains were transduced to TetR with phage growth on strains TT3003 and TT3010. These two strains carry the hisoCD646 deletion and a hisC::TnlU insertion in either the "A" (TT3003) or "B" (TT3010) orientation. HisD+ transductants were saved. Genetic analysis shows that these transductants retain the inversion causing the HisD+ phenotype and acquire the hisC::TnlO insertion mutation. Phqsicril techniques: The procedures for restriction fragment hybridization and nick-translation have been described (SCHMIDand ROTH 1983). T h e cloned his region (his0GD) present on M13Ho168 was used as the hybridization probe (BARNES1978, 1979). RESULTS

The genetic selection: A functional hisD protein enables a strain to utilize histidinol as a source of histidine. Deletion mutation his-203 removes the his

BACTERIAL INVERSION MUTATIONS

541

TABLE 1 Bacterial strains Strain TT1889 TT3003 TT30 10 TR5886 TR5891 TR5892 TR5895 TR5898 TR5901 TR6346 TR6403 TR6405 TR6412

Genotype his01242 zee-2::TnlO his-646 hisC8691: :Tn 10 his-646 hisC8667: :Tn 10 lNV285 [hisD+, ilvA] his-203 INv290 [hisD+, ilvA] his-203 IN11291 [hisD+, unknown] his-203 IiW294 [hisD+, metC] his-203 INV297 [hisD+, ilvA] his-203 IW300 [hisD+, rJb] his-203 IhV383 [hisD+, iZvA] hk-203 INV385 [hisD+,unknown] his-203 INV387 [hid)+,ihA] his-203 INV393 [hisD+, unknown] his-203

TT3707-TT3872 TT600 1 TT6003 TT6006 TT6008

and ROTH 1983) (see SCHMID INV285, zee-2: :Tn 10 INV290, zee-2::TnlO INV294, zee-2::TnlO INV297, zee-2::TnlO

Multiply marked bacterial strains are listed with their complete genotypes. All strains are derived from S. typhitnztriuvt strain LT2.

zee-2::Tn/O 4 G ' D C B H A F I E his-203

MI3hol68 FIGURE1.-A genetic map of the his operon of S . tyfihitnurium. The extent of the his-203 deletion and the position of the ztc2::TnlO insertion are shown. The hk-01242 mutation is to the right of zee-2::TnIO but within the region deleted by the his-203 mutation. Transcription normally proceeds from left to right. The his-203 deletion removes the promoter, his control region and the first part of the hisC gene. The extent of the his region cloned on the M13Ho168 phage is shown.

promoter, his control region and most of the hisG gene and leaves the hisD gene intact but unexpressed. Mutants of deletion strain his-203 that could use histidinol as a source of histidine were selected. A new promoter must express the hisD gene in these mutant strains. This new promoter can arise in a number of ways. It can arise by a base substitution in the sequence preceding the hisD gene (ST. PIERRE1968; AMES, HARTMAN and JACOB 1963), by deletion that fuses the hisD gene to a new promoter (AMES,HARTMAN and JACOB 1963), or by duplication in which the hisD gene is fused to a new promoter at the

542

M. B. SCHMID AND J. R. ROTH

duplication join point (ANDERSON and ROTH 1978). These three types of mutations account for about 90% of all of the histidinol-utilizing mutants that arise from this selection. In principle, inversion mutations should satisfy this selection by causing the hisD gene to be fused to a new promoter at an inversion join point. Such an inversion would have one inversion breakpoint within the sequences immediately preceding the hisD gene; the second inversion breakpoint would be near a properly oriented promoter whose transcript is not essential. Presumably, this foreign promoter could be located anywhere on the chromosome. Genetir screen for inversion mutants: The HisD+ mutants isolated by the selection will express the hisD gene and, presumably, the rest of the his genes downstream of hisD. However, the hisG gene, partially removed by the his-203 deletion, will still be defective, so the HkD+ mutants will remain phenotypically His-. The genetic screen used to find inversion mutants in these experiments detected mutants in which the hisG gene is separated from the rest of the his genes. This was accomplished by searching for HisD+ mutants in which the his operon can no longer be repaired by PPP-mediated generalized transduction. In most mutants from this selection (point mutations, deletions and duplications) the his-203 deletion can be repaired, yielding His+ transductants. A large inversion will behave differently, since the his-203 deletion (and thus the hisG gene) will be separated from the rest of the his genes. Repair of the his203 deletion by transduction will give two different results depending on the position of the inversion breakpoint in the his operon. If the inversion breakpoint lies within the hisG coding sequence, repair of the his-203 deletion will not result in expression of a functional hisG protein. Such an inversion strain will yield no His+ transductants (except by rare two-fragment transduction events). If the inversion breakpoint lies within the 100-base pair hisG-hisD intercistronic region (BARNES1978; W. BARNES,unpublished results), no hiscoding sequences will be disrupted. This type of inversion mutant can be transduced to His+ because repair of the his-203 deletion (at one end of the inverted segment) will lead to expression of an intact hZsG gene. Presumably, the promoter that initiates transcription of the hisD gene will transcribe the other his genes as a polycistronic message. This has been shown for the INV282 inversion (M. SCHMID,unpublished data). Complementation between these two parts of the his operon (at opposite ends of the inverted segment) should lead to a His+ phenotype. To detect either type of inversion mutation, the HisD+ mutants were tested for transducibility to His+ with a P22 lysate grown on a strain carrying the ha01242 mutation. The his01242 mutation causes a high level of his transcription, normally leading to a high level of hisH and hisF expression. When these two genes are expressed at a high level, a strain has a rough colony morphology (MURRAYand HARTMAN 1972). However, the hisH and hisF genes will not be linked to the his01242 mutation in an inversion mutant from this selection. Mutants with an inversion breakpoint in the hzsG-hisD intercistronic region may yield His+ transductants because of complementation. However, these transductants can be identified by their characteristic smooth colony morphology,


543

even though they have inherited the his01242 mutation. Mutants with an inversion breakpoint within the hisG gene will yield no (or very few) His+ transductants. HisD' mutants that arose because of most other mutational events will give only rough His' transductants, because the recipient his-203 deletion removes the site of the his01242 mutation. Thus, all transductants that have repaired the his-203 deletion must inherit the his01242 mutation. All HisD' mutants that gave no His+ transductants and those that gave any His' transductants with smooth colony morphology were saved and analyzed further. The inversions that can be detected by nontransducibility by this screening method must be larger than the size of a P22-transduced fragment. A P22-transduced fragment carries about 48 kb of bacterial DNA (EBEL-TSIPIS, BOTSTEINand Fox 1972; SUSSKIND and BOTSTEIN1978). Frequency of inversion mutation: From these experiments, 17 independent hisD+ mutants survived the initial screening procedure out of 2000 histidinol-utilizing mutants in 268 independent groups. Some of the mutants carry chromosome rearrangements that cause the hisD+ phenotype, whereas others are nonrearrangement mutants that survived the initial screen for a variety of reasons. According to the following data, the mutations in ten of these 17 strains have the characteristics of chromosomal inversion mutations. The detailed data for the strains carrying inversion mutations will be presented. T h e noninversion strains are described only briefly here. A detailed description of the noninversion mutants appears elsewhere (SCHMID1981). Linkage disruption in the his operon: The data in Table 2 show that all the inversion strains found yield His+ transductants that have a smooth colony morphology even though they have inherited the his01242 mutation. The presence of the his01242 mutation was confirmed in these transductants (data not shown). The occurrence of these His' transductants suggests that the inversions all have a breakpoint between the hisG and hisD genes that does not disrupt hi&-coding sequences. One of the initial 17 isolates (strain TR5901) was found to be resistant to bacteriophage P22 infection. This explains its ability to survive the screening procedure since no transductants can arise without phage infection. However, this mutant is still interesting, since physical analysis demonstrates that the his operon is disrupted in this strain. This mutation may have arisen by fusion of the hisD gene to the $I transcript near the end of the his operon. T h e rJb and his mRNAs are transcribed in opposite directions (LEVINTHAL and NIKAIDO 1969). All other potential inversion mutants plate P22 phage as well as wild type (SCHMID1981). Linkage disruption in the his operon also is apparent when the mutants are used as donors to transduce the parent strain, his-203, to histidinol utilization. The data in Table 3 show that these mutants donate their HisD' phenotype to the parent strain, his-203, very poorly. One strain, TR6403, donates its HisD' phenotype 100-fold better than the other mutants. This strain is believed to carry a short inversion that is about the same size as a single P22transduced fragment. Occasionally, this HisD+ inversion is carried in toto by a single transduced fragment and is inherited by the recipient strain. We believe

544


TABLE 2 Trriiisdurtioii of inversion mutonts to His+ No. o f His+ transductants

Recipient strain

his-203

TR5886 (IAVZ85) TR5891 (IAV290) TR5892 (INV.291) TR5895 (1AV.294) TR5898 (IiW.297) TR5901 (Ii\V300) TR6346 (LW"83) TR6403 (IiW385) TR6405 (1XV387) TR64 12 (IiW393)

Transductant colony morphology

Rough Smooth Smooth (Uncertain) Smooth Smooth

5000 50 50 75 100 30 0 100 500 30 100

Smooth 95% rough, 5% smooth Smooth Smooth

T h e parent strain, hs-203, and HisD' inversion mutants were crossed with a phage lysate grown on a strain carrying his01242. His+ transductants were selected. T h e rationale for using the his01242 mutation is explained In the text. T h e number of His+ transductants and the colony morphology of these transductants were scored. In all cases, a constant amount of donor phage was used. Approximately 10' cells were infected at a multiplicity of 10. TABLE 3 IJIVPTTIOH inutciritP os troizsductzonol donors

Donor

TR5886 (1>W.285) TR5891 (IW.290) TR5892 (1XV291) TR5895 ( I ~ W 2 9 4 ) TR6346 (1i\'V383) TR6403 (1~VV385) TR6405 (1NV387) TR64 12 (1AV393) LT2

of

HiqDD+

Multiplicity of infection

No. o f transductants

4.1 2.5 2.3 6.0 1.9 2.2 2.0 4.6 3.9

19 22 1 26 2 500 4 21 10,000

No. of transductants/pfu

2.7 x 5.3 x 2.6 X 2.6 X 7x 1.4 x 1.1 x 2.7 x 1.6 x

10lo-* lo-' lo-' 10-9 lo-* 10-5

Phage lysates made on the HisD+ inversion mutants listed in the first column were used t o transduce strain /lis-203 to HisD+ (histidinol utilization). T h e multiplicity of infection is shown and was between 2 and 6 in all cases. T h e frequency of HisD+ transductants per plaque forming unit (pfu) at the indicated multiplicity of infection is shown. T h e number of transductants per plaque forming unit rises with increasing multiplicity of infection in all cases except TR6403 (INv385) (data not shown).

that this is the reason for the higher frequency of HisD+ donation. The few HisD+ transductants that arise in all other cases are probably due to twofragment transduction events. T h e ability to donate the HisD+ phenotype to his-203 is dependent on the multiplicity of infection of the transduction (except for TR6403) (data not shown), as expected of recombination events requiring two transduced fragments (see preceding paper by SCHMIDand ROTH 1983). Physirctl nnctljsis of inu~rsioizs:As described in the accompanying paper, restriction fragment hybridization can be used to demonstrate that the his operon has been disrupted. The his promoter, control region and the first two genes


545

of the Ais operon, hisC and hisD, have been cloned onto the single-stranded phage M13 (BARNES1978, 1979). Rearrangements of the his operon can be differentiated from other mutants that survived the screening procedure using the double-stranded replicative form of this phage (M 13Ho168) to probe DNA from the histidinol-utilizing mutants. DNA from the mutant strains was digested with restriction enzyme EcoRI, subjected to electrophoresis, blotted and hybridized with '*P-labeled DNA from the M 13Ho168 phage. The restriction fragment hybridizations are shown in Figure 2. The EcoRI restriction enzyme does not cleave within the cloned his region. The parent strain, his-203 shows only one band hybridizing to the cloned his region. A number of the mutants show two restriction fragments that hybridize with the M 13Ho168 DNA. These strains include TR5886 ( I W Z S S ) , TR5891 (IiW290). TR5895 (IiWZ94), TR5898 (IiW297), TR5901 (INv300), TR6346 (LW383).TR6403 (I,W385), TR6405 (IiW387) and TR64 12 (IlW393), which are shown in Figure 2, as well as TR5892 (I'W291) (data not shown). (The single "band" present in DNA from strain TR5895 is actually a doublet; two bands can clearly be resolved by longer electrophoresis time or by digestion of the DNA with a different restriction enzyme.) These HisD+ mutants must

1234567891011

FIGURE2.-Chromosomal DNA was digested with restriciton enzyme EmRI, electrophoresed on a I % agarose gel, then blotted onto a nitrocellulose filter. "P-labeled M13Ho168 DNA was hybridized with the chromosomal DNA. The source of DNA in each lane is as follows: lane 1 , his203; lane 2. TR5835 (LW282); lane 3, TR5886 (IW285); lane 4, TR5891 (I~W290);lane 5, TR5895 (ISV294);lane 6, TR5898 (I'W297); lane 7, TR5901 (IW300); lane 8, TR6346 (hW383); lane 9. TR6403 ( I A ~ 3 8 5 )lane ; IO, TR6405 (ISV387); lane 1 I . TR6412 (hW393).

546


have sequences present on the M13Hul68 clone (hisO, hisG and hisD) in two different chromosome locations. In general, one of the two bands shows less hybridization with the cloned his region. When the sequence deleted by the his-203 deletion and the fact that the inversion breakpoint must lie promoter proximal to the hisD gene are taken into account, the band corresponding to the upstream portion of the his operon can have no more than 750 base pairs of homology with the cloned region. The downstream part of the operon should have more than 1200 base pairs of homology with the cloned region. Thus, we believe that the weaker hybridizing fragment corresponds to the upstream portion of the his operon, and the strongly hybridizing fragment corresponds to the downstream HisD+ restriction fragment. All of the mutants showing two bands when cut with EcoRI also show two bands when cut with NindIII, an enzyme that also has no cleavage site within the cloned region (data not shown). iMupping the non-his breakpoint: The data shown so far demonstrate that a chromosome rearrangement is associated with the his operon in the inversion mutants. The mapping scheme described in the accompanying paper was used to determine the chromosomal location of the promoter expressing the hzsD gene and to provide evidence that the his operon is inverted relative to wild type. T h e mapping procedure is diagrammed in Figure 3 and described in detail in the preceding paper (SCHMIDand ROTH 1983). A hisC::TnlO-containing derivative of the rearrangment strain is used as a donor of the hisD+ phenotype. In the donor, the hisC::TnlO and the foreign promoter are linked. Between these two regions lies the hisD+ gene. When a TnlO insertion is placed at an appropriate point in the recipient chromosome, this HisD+ donor fragment can be inherited. Recombination can occur between the foreign promoter region of the donor and recipient and between the two TnlO insertions, thereby introducing the hisD+ gene. A large deletion will be generated by these exchanges if the recipient TnlO lies counterclockwise of the foreign promoter on the standard genetic map. The deletion will extend between the site of the recipient TnlO insertion and the foreign promoter. If the deletion removes any essential genes, the resulting strain will be inviable. If the TnlO insertion is clockwise of the foreign promoter, a duplication will be generated between the foreign promoter and the TnlO insertion. Strains carrying duplications between many arbitrarily chosen sites are known to be viable (SCHMID 1981). A set of recipient strains is used, each of which carries a single TnlO insertion at a known location. T h e recipient strains also carry a nontransducible deletion of the his operon that prevents inheritance of the HisD+ phenotype by simple repair of the recipient his region. For inversions extending clockwise from the his operon, the HisD+ inversion join point is located between the most his-proximal T n 20 insertion that gives hisD+ transductants and the most distant TnlO that gives no hisD+ transductants. Two donors were used for each rearrangement tested. These two donors differ in the orientation of their hisC::TnlO insertion. One of the two donors will provide properly oriented TnlO homology with any recipient TnlO inser-

547


inversion donor fragment

(TniO)

recipient chromosome

m:Recipient TnlO to the m t of

his-9707

Jvl-

C d : Recipient

F--

to the @j

esz TnlO

Product: lorge lethal deletion

L

of

TnK)

7

TnlO

-P

TnlO

P

Product: large

duplication

No HisD* transductonts recovered HisD* transductants recovered FIGURE3.-Mapping method used for locating inversion endpoints. Inheritance of a functional donor hisD gene requires properly oriented TnlO elements in donor and recipient. The recipient TnlO insertion must lie clockwise of the foreign promoter expressing hisD if a duplication is to form. If the recipient T n l O lies counterclockwise from the promoter, a deletion will form; in most cases these deletions will be lethal. The pattern of recipient TnlO strains that show hkD+ transductants permits approximate mapping of the foreign promoter and the inversion breakpoint. tion. Thus, the presence or absence of hisD+ transductants should depend solely on the relative positions of the foreign promoter and the TnlO insertion. Table 4 shows the results of the mapping crosses. T h e formation of HisD+ transductants depends on the presence of the TnlO insertion in the recipient; none of the donors yielded HisD+ transductants with the recipient that lacks a TnlO insertion (TT3707). The formation of HisD+ transductants also depends on the presence of a TnlO insertion in the donor strain (data not shown), as well as a rearranged his operon. Strains T T l l Z 7 and' T T 1 1 5 1 carry hisC::TnlO insertions in an otherwise wild-type his operon (the strains are HisD+). These two donors yield no HisD+ transductants with any of the recipient strains. For each rearrangement, a block of recipient strains gave no HisD+ transductants. As the recipient TnlO insertion was moved clockwise on the genetic map, a block of strains was found that gave HisD+ transductants with one of the two donors. The foreign promoter is believed to lie in the chromosome region between these two sets of T n 10 insertions. T h e inversion breakpoint in five of these strains [TR5886 (ZNV285), TR5891 (ZNV29O), TR5898 (ZNV297),TR6346 (ZNV383)and TR6405 (INV387)l mapped between 83 and 88 min on the genetic map. The inversion breakpoint of I N 2 9 4 (TR5895) maps between 66 and 68 min. From these mapping data, the breakpoint of ZNV385 (TR6403) appears to be very close to the his operon. Genetic

548

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