genic and allelic interactions in the carotenogenic response of myxococcus xanthus … · 2002. 7....

Copyright 0 1989 by the Genetics Society of America

Genic and Allelic Interactions in the Carotenogenic Response of Myxococcus xanthus to Blue Light

Antonio Martinez-Laborda and Francisco J. Murillo

Departamento d e Genitica y Microbiologia, Facultad de Biologia, Universidad de Murcia, 30071 Murcia, Spain Manuscript received November 18, 1988 Accepted for publication March 16, 1989

ABSTRACT In the bacterium Myxococcus xanthus, the synthesis of carotenoids requires illumination with blue

light. This stimulates transcription of the carB locus, which is positively required for carotenogenesis. Mutations at the carR locus and the only mutation known at carA cause constitutive expression of carB and thus remove the light requirement for carotenoid accumulation (Car‘ phenotype). The carR locus is unlinked, and carA is linked, to carB. We have now identified a novel class of car mutation, closely linked to carR, that block accumulation of carotenoids and transcription of carB, and are epistatic over the Car‘ mutations at carR but not over the Car‘ mutation at carA. We also report here the cloning of a 16-kb DNA fragment that contains the entire carA gene in a shuttle vector for DNA transfer between Escherichia coli and M. xanthus. The study of allelic interactions at this gene strongly indicate that carA is a cis-acting element for the control of c a d expression. A regulatory model that

v

satisfies all the indicated data is presented.

W HEN illuminated with blue light, wild-type strains of the myxobacterium Myxococcus xan-

thus produce a complex mixture of carotenoids. This response results in a color change of the colonies or liquid cultures from yellow, due to a noncarotenoid pigment, to red (BURCHARD and DWORKIN 1966; BUR- CHARD and HENDRICKS 1969; REICHENBACH and KLEI- NIG 1984). The conspicuousness of the color change has greatly facilitated the isolation of mutants affected in the light response. On the other hand, genetic techniques have been developed for M. xanthus, mainly because of researchers’ interest in the social behavior of this organism. All this has made of M. xanthus a model system to understand the genetic and molecular basis of the response to blue light (ROSEN- BERG 1984; KAISER 1986; BALSALOBRE, RUIZ-VAZ- QUEZ and MURILLO 1987).

Three unlinked loci have been found to be involved in light induction of carotenogenesis in M. xanthus (see Figure 1). The locus called carR includes several independent mutations that render carotenoid production light independent. We will refer to this phenotype as constitutive or Car‘. Another mutation that also produces a Carc phenotype is located at gene carA, unlinked to carR. Gene carA itself is linked to a locus called carB; in this locus, several independent transposon insertions are known that block synthesis of colored carotenoids in otherwise wild-type strains and in all of the carA or carR constitutive mutants. Finally, a single transposon insertion is known in gene c a d , unlinked to the others, that also blocks carotenoid synthesis and is epistatic over Carc mutations. We will refer to the phenotype of carB and carC mutants as negative or Car- (MARTINEZ-LABORDA et

Genetics 122: 481-490 Uuly, 1989)

al. 1986; BALSALOBRE, RUIZ-VAZQUEZ and MURILLO 1987).

Using the “in vivo” promoter probe Tn5 lac (KROOS and KAISER 1984) we have recently shown that transcription of carB is strongly stimulated by light and becomes constitutive in the presence of Carc mutations either at carA or carR (BALSALOBRE, RUIZ-VAZQUEZ and MURILLO 1987).

All the available data for carotenogenesis in M. xanthus would satisfy a model in which carB and carC code for enzymes acting early in the pathway (before the first colored carotene is formed) and are activated at the level of transcription (at least for carB) through a light-driven mechanism in which the carA and carR loci are involved. It follows from the linkage data (Figure 1) that carR is likely to encode a trans-acting regulatory element. From the same data, and additional evidence suggesting that transcription of carB starts on the carA side, the possibility has been raised that carA might be a cis-acting regulatory element for expression of the carB locus (BALSALOBRE, RUIZ-VAZ- QUEZ and MURILLO 1987).

In an attempt to identify new genetic elements involved in the carotenogenic response of M. xanthus, we have searched for mutations that suppress the Car‘ phenotype of carR mutants. By a procedure that includes cloning of the carA region in a shuttle vector for DNA transfer between Escherichia coli and M. xanthus (GILL, CULL and FLY 1988) we have also constructed merodiploid strains that allow the study of carA allelic interactions. Reported here are the results of the genetic and phenotypic characterization of the indicated mutations and merodiploids. They suggest that close to carR there is a locus that codes

482 A. Martinez-Laborda and F. J. Murillo

QDK2836 I c a r B l ) , QMRLol (carB2J QDKL611 PMRl3 6

I c a r R 6 I I !

PMR,3L QMRL03 lcarC1) I

I I I I I

c a r A l I l l I I I I l

I l

FIGURE 1 .-Genetic map of mutations affecting carotenogenesis in M . xanthus. Three unlinked chromosomal regions are represented. Mutations at carA or carR cause Car‘ phenotype, whereas mutations at carB or carC cause Car- phenotype. Only mutations cited in this work are showzn. Underneath, allelic numbers of point mutations are indicated. For transposon mutations, both the notation of the insertion locus and the allelic number are given (Tn5 and Tn5 lac insertions are indicated by open and black circles, respectively). Insertions QDK46 1 1 and RMR134 affect no car genes. Insertion QMR136 produces some, but not all the effects of the carR mutations (see text). Also indicated are the cotransduction frequencies (in percentage) between some insertion sites and car mutations. The average frequencies for different carR mutations with QDK4611 and QMR136, and that between QMR134 and carA1, are taken from MARTINEZ-LABORDA et a l . (1986). Cotransduction between QMR40 1 and carAl is taken from data of BALSALOBRE, RUIZ-VAZQUEZ and MURILLO (1987) pooled with unpublished data from our laboratory.

for a positive regulatory element that interacts with carA to activate the expression of carB. The results also show that carA is, in fact, a cis-acting regulatory element.

MATERIALS AND METHODS

Strains and culture conditions: The pigmentation of wild-type strains of M. xanthus is usually not a stable char- acter and both “tan” and “yellow” colonies may arise from a single colony (BURCHARD and DWORKIN 1966). Our standard strain, DK1050, was isolated as a spontaneous “stable yellow” strain and thus is specially useful for studies of carotenogenesis (MARTINEZ-LABORDA et al. 1986). Other strains used in this work are listed in Table 1, together with their phenotype, genotype and origin. Car+ strains form yellow colonies when grown in the dark and red colonies when grown in the light. Car‘ strains form red colonies both in the dark and in the light and Car- strains form yellow colonies in both conditions. Insertion of the transposon Tn5 or Tn5 lac causes resistance to kanamycin (KmR) and that of Tn5-132, resistance to oxytetracycline (TcR). The corresponding insertion sites are identified in Table 1 by letters and numbers, preceded by Q [see AVERY and KAISER (1983) for details on genetic notation]. The Tn5 lac insertion in MR40 1 causes light-inducible production of P-galactosidase (LacZ’ in Table 1). LacZ- strains produce very low, and LacZ‘ strains high, levels of P-galactosidase in the dark and in the light (see RESULTS and BALSALOBRE, RUIZ-VAZ- QUEZ and MURILLO 1987).

Media, culture conditions, and illumination with visible light were as previously described (MARTINEZ-LABORDA et al. 1986; BALSALOBRE, RUIZ-VAZQUEZ and MURILLO 1987). “Zn vivo” genetic manipulations: Transduction of M.

xanthus strains was carried out with the generalized transducing phage Mx4-LA27 (AVERY and KAISER 1983). Selec- tion for KmR was performed as previously described (MAR- TINEZ-LABORDA e t al. 1986) and the Car phenotype of the transductant colonies was assigned by picking them in du- plicate plates, one to be incubated in the dark and the other in the light. Data for each cross shown in the Tables were pooled from two or more independent transduction experiments.

“Zn situ” replacement of Tn5 by Tn5- 132 was performed by mixing Pl::Tn5-132 phages with cells of the strain carrying the Tn5 insertion to be replaced, and selecting for the TcR KmS phenotype, as described in MARTINEZ-LABORDA et al. (1 986).

To transfer plasmids from E. coli to M. xanthus, specialized transducing particles of coliphage P1 were used, following the method described by SHIMKETS, GILL and KAISER (1983). Transductants were selected for the KmR determinant present in the plasmids, using the procedure detailed in STEPHENS and KAISER (1987).

Cloning of the carA gene: We followed the “in situ” cloning strategy diagrammed in Figure 3 and previously described by GILL, CULL and FLY (1988). M. xanthus strain MR148 contains a Tn5-132 insertion (QDK2836) in the carB locus, linked to carA (Figure 1). The 13-kb plasmid vector pREG429 (kindly supplied by RON GILL) carries the incompatibility region of phage P1 and a DNA fragment from Tn5 that includes the KmR gene and a large portion of the 1S50L element. The plasmid was transferred from E. coli to MR148, where it does not replicate but can integrate by homologous recombination into one of the two IS50 elements of ODK2836::Tn5-132 to generate KmR transductants. Five independent KmR TcR transductants were sepa- rately grown and their DNA was extracted and digested with the restriction enzyme BamHI (Boehringer Mann- heim). As can be seen in Figure 3, the single BamHI site in the plasmid vector is located in such a manner that the indicated treatment should produce BamHI fragments containing the vector DNA linked to M. xanthus DNA that originally lay to one or the other side of the Tn5-132 insertion QDK2836. The BamHI treated samples were then ligated with T4 DNA ligase (Boehringer Mannheim) at low DNA concentration (1-5 pg/ml) to favor intramolecular with respect to intermolecular reactions. The ligation mix- tures were used to transform E. coli MC1061 (CASADABAN and COHEN 1980) selecting for KmR, and plasmids present in the transformants were isolated and characterized by restriction mapping on agarose gel electrophoresis. Several E. coli colonies, transformed with either of two of the DNA samples, appeared to carry the same chimeric plasmid containing the vector and an additional piece of DNA 16 kb long. When this plasmid was transferred back to M . xanthus, it could rescue the Car‘ phenotype of a carA mutant (see

Control Genes for Light Response 483

RESULTS). For all these manipulations, we followed the methods described by MANIATIS, FRITSCH and SAMBROOK (1982). M . xanthus DNA was extracted according to YEE and INOUYE

&Galactosidase assay: For colony screening of P-galactosidase production, we used plates containing 40 pg/ml of 5-bromo-4-chloro-3-indolyl P-D-galactoside (X-Gal) and, after incubation, colonies were examined for the blue color (indigo) formed by the enzymatic cleavage of X-Gal. Quan- titative analysis of P-galactosidase was carried out, using o- nitrophenyl Pa-galactoside, as previously described (BAL- SALOBRE, RUIZ-VAZQUEZ and MURILLO 1987). Specific activity is expressed as nmol of o-nitrophenol produced per min per mg of protein.

(1981).

RESULTS

Car- mutations epistatic over Car' mutations at the carR locus: M. xanthus strains DK406, DK2834, DK46 13, and DK6000 all carry mutations at the carR locus that result in constitutive synthesis of carotenoids. They thus produce colonies which are red when grown either in the dark or in the light. Cultures of these strains were used to look for spontaneous mutants showing in the dark a yellow, carotenoid-less, phenotype. Around 40,000 colonies were screened in each case and mutants were found at an average frequency of loT4. From each experiment, a single colony was selected and purified. The four mutants were given the names MR132, MR135, MR166, and MR170 (Table 1) . When grown in the light, none of these mutants produced colored carotenoids. The Car- phenotype indicates that the mutants are not carR revertants but each has acquired a second car mutation that has rendered them defective in a carotenoid biosynthetic gene or in a regulatory element acting at a later step of that controlled by carR.

T o locate the new Car- mutations, several transduction experiments were carried out taking advan- tage of the kanamycin resistance (KmR) marker pro- vided by insertions of Tn5 at particular sites in the M. xanthus chromosome (Figure 1) . In fact, two of the new mutants, MRl66 and MR170, already contained insertion QDK46 1 1 ::Tn5, which is loosely linked (about 9% cotransduction frequency) to the carR locus. When KmR was transduced from MR170 into wild-type strain DK1050, both wild type (Car+) and Car- colonies were found among the transductants (Table 2, cross 17). Their relative frequencies indicate a loose linkage between QDK4611 and the locus affected by the novel car mutation. In the same cross, a single transductant, out of 165, showed a Carc phenotype. This confirms that the original carR mutation is still present in MR 170 and suggests that the epistatic Car- mutation is itself closely linked to carR. A similar experiment performed with MR166 (results not shown) indicates that the novel Car- mutation in this strain is not linked to RDK4611 (see below).

Car+ T n 5 carrying strains MR134, DK4611, and MR136 (Figure 1 and Table 1 ) were used to transduce

KmR into the four novel Car- mutants, and the transductants were screened for their Car phenotype (Table 2). Notice that MR166 and MR170 had to undergo Tc-replacement (MR168 and MR17 1, respectively) before they could be used for KmR transduction. We should also note that MR136 represents a special case of Car+ strain. In the dark, MR136 colonies are of a similar, although darker, color to that of wild-type colonies. This dark-yellow phenotype itself is caused by the Tn5 insertion present in MRl36 (MARTINEZ-LABORDA et al. 1986) and is probably due to the accumulation of small amounts of {-carotene and neurosporene. These are colored precursors in the carotenogenic pathway (KLEINIG 1975) absent in dark-grown cultures of the wild type. In the light, MR136 colonies are conspicuously red and they contain the same carotenoids found in light-grown wild-type cultures (our manuscript in preparation). In a previous paper, we considered insertion QMR136::Tn5 as a carR mutation, since it is closely linked to that locus and causes constitutive expression of carB (BALSALOBRE, RUIZ-VAZQUEZ and MURILLO 1987). We do not know why the color phenotype of MR 136 is different from the phenotype produced by other mutations that cause constitutive expression of carB. But since the presence of Tn5 a t QMR 136 does not conceal the light-inducible phenotype, it provides a good marker for linkage analysis of Car- mutations. So, for the sake of clarity, we have included MR136 as a Car+ strain in Table 1 .

When MR132, MR135, and MR171 were used as recipients in crosses with MR134 (Table 2, crosses 1- 3) all the KmR transductants showed the same Car- phenotype, thus indicating lack of linkage between the involved markers. When the same strains were crossed with DK4611 or MR136, both negative and carotenoid producing transductants arose (Table 2, crosses 5-7 and 9-1 1). The corresponding cotransduction frequencies indicate that the Car- mutations present in these three strains are loosely linked to QDK46 1 1 and closely linked to QMR 136, and therefore to the carR locus.

When MR136 was the donor, both Car+ and Carc phenotypes were found among the carotenoid producing transductants from MRl32 and MR17 1 (Table 2, crosses 9 and 1 1 ) . The Car+ colonies should have gained, by cotransduction with QMR136, the wild-type alleles of the two mutated loci. Carc transductants should have gained the wild-type allele for the epistatic Car- locus but still retain the carR mutation (negative transductants should have retained at least the Car- mutation). In these two crosses, the proportions of Car' transductants were similar to those of Car- transductants. This argues against carR being located between the Car- mutations and QMRl36, as a double recombination event between QMR 136 and the Car- locus would be required in this


TABLE 1

Myxococcus xanthus strains used in this work

Strain" Phenotype car genotype Tn5-wt Tn5-132 Tn5 lac Source or derivationb

Basic strains: DK 1050

DK406 DK2834 DK4613 DK6000

MR132 M R 1 3 5 M R 1 6 6 M R 1 7 0

DK46 1 1 MR134 M R I 36

Other strains: MR7 MR146 MR148 MR168

MR171

MR172

M R 1 7 7

M R 1 7 8

M R I 9 8

M R I 9 9

MR401 MR407 MR409 MR410 MR454

M R 4 5 5

MR456

MR457

MR458

MR459

Car+

Car': Car" Car" KmR CarC KmR

Car- Car- Car- KmR Car- KmR

Car+ KmR Car+ KmR Car+ KmR

Ca rc CarC KmR Car- TcR Car- TcR

Car- TcR

Car- KmR

Car- KmR TcR

Car' TcR

Car- KmR

Car- KmR TcR

Car- L a d ' KmR Car- LacZ" KmR Car- LacZC KmR Car- LacZ" KmR Car- LacZ- KmR

Car- LacZC KmR

Car- LacZ- KmR TcR

Car- LacZC KmR TcR

Car- LacZ- KmR

Car- LacZ" KmR

carR4 carR6 carR5 QDK4611 carR4 QK46 1 1

carR4, car-I1 carRb?, car-l2 carR5, carB4 QDK46 1 1 carR4, car-13 ClDK4611

QDK46 1 1 QMR134 QMR136

carAI carAI QMR134 carBl carR5. carB4

carR4, car-13

carR6?, car-l2 QDK46 1 1

carR5, carB4 QMR134

QDK2836 QDK46 1 1

QDK46 1 1

RDK4611

QDK46 1 1

carR4, car-l I QMR136

carR4. car- l3 QMR136

carB2 carR4, carB2 carR6, carB2 carAl, carB2 carR4, carB2, car-I1

carR4, carB2, carAl, car-I1

carR4, carB2, car-13

carR4, carB2, carAl, car-l3

carRh?, carB2, car-l2

carRG?, carB2, carAI, car-l2

RDK46 1 1

RMR401 RMR401 QMR401 QMR401 RMR401

ClMR401

QDK46 1 1 QMR40 1

RDK46 1 1 ClMR40 1

ClMR401

QMR401

MERM

MERM MERM MERM MERM

DK406 (spontaneous) DK2834 (spontaneous)

DK6000 (spontaneous)

MERM MERM MERM

DK46 1 3 (SpOntdllKOuS)

MERM MERM MERM PI::Tn5-132 X M R 1 6 6 + +

TcR (Km')

TcR (Km')

-+ KmR (Car-)

KmR (Car-)

+ TcR (Km')

KmR (Car-)

KmR (Car-)

Pl::Tn5-132 X M R 1 7 0 ++

Mx4(DK4611) X M R 1 3 5 +

Mx4(MR134) X M R 1 6 8 -++

PI::Tn5-132 X DK4611 +

Mx4(MR136) X M R 1 3 2 + +

Mx4(MR136) X MR171 ++

BRM BRM BRM BRM Mx4(MR401) X M R 1 3 2 ++

Mx4(MR410) X M R 1 3 2 + +

Mx4(MR401) X MR171 ++

Mx4(MR410) X MR171 ++

Mx4(MR401) X M R 1 3 5 -+ -+

Mx4(MR410) X M R 1 3 5 ++

KmR

KmR (LacZ")

KmR

KmR(LacZ")

KmR

KmR(LacZ")

Basic strains include the wild type, the four carR strains used to look for mutations suppressing their Car" phenotype, the four Car- strains carrying these mutations, and the three T n 5 carrying strains used to map the Car- loci. It is not certain that M R 1 3 5 carries the mutation carR6 (see the text). Other strains are listed in numeric order.

References in this column are: MERM, MARTINEZ-LABORDA et al. (1986); BRM, BALSALOBRE, RUIZ-VAZQUEZ and MURILLO (1987). For strains obtained in this work, brief descriptions of the way in which they were derived are given. For example, the notation Mx4(DK4611) X M R 1 3 5 + KmR (Car-) indicates that phages grown in DK4611 were used to transduce the KmR determinant into M R I 35 and among the KmR transductants, the Car- phenotype was screened for.

case to generate the Car' transductants. Given the two alternatives, three-factor crosses were carried out close linkage between RMRl36 and both the carR in which the insertion sites Q M R l 3 6 and RDK4611, and the Car- loci (Figure 1; Table 2) that event should and the Car- mutation present in MR 132 or MR17 1, be extremely rare. Thus, the Car- mutations should were involved (Table 3). In these two crosses, the be located either between carR and RMR136 or on frequency of recombination between RDK4611 and the other side of OMR136. To decide between these RMR136 was found to be higher (an average of 69%)


TABLE 2

Linkage of the novel Car- mutations to Tn5 insertion sites RMR134, RDK4611, and RMR136

KmR transductants

(donor X recipient) Car+ CarC Car- frequency (W)* Cross" Cotransduction

1. MR134 X MR132 2. MR134 X MR135 3. MR134 X MR171 4. MR134 X MR168

5. DK4611 X MR132 6. DK4611 X MR135 7. DK4611 X MR171 8. DK4611 X MR168

9. MR136 X MR132 10. MR136 X MR135 11. MR136 X MR171 12. MR136 X MR168

13. MR177 X DK1050 14. MR172 X DK1050 15. MR198 X DK1050 16. MR199 X DK1050 17. MR170 X DK1050

0 0 0 0

15 8

17 0

307 37 1 357

0

14 93 24 27

147

0 224 0 1300 0 128

251 35

0 198 0 138 0 180 0 778

47 38 0 0

46 66 0 440

0 46 0 7 - 185 - 108

1 17

< l C O . 1 <l 88

7 5 9

<0.2

90 >99

86 <O , .5

77 7

89 80 10

may have been removed as a consequence of the spontaneous mutational event that gave rise to the Car- phenotype of MR135. The fact that the Car- phenotype of MR135 showed no segregation with the KmR marker of MR136 (Table 2, cross 10) supports the hypothesis of a deletion in MR135 covering QMR136 itself and extending into the carR locus.

The results of similar transduction experiments with MR168 (Table 2, crosses 4, 8, and 12) clearly showed that the Car- mutation of this strain is closely linked to QMR134. Thus it has probably affected the previously identified carB locus (Figure 1).

Several reciprocal transduction experiments (Table 2, crosses 13-1 7) confirm the above conclusions on genetic linkage and indicate that in all cases the Car- phenotype is due to a single mutation. Alleles for the Car- mutations in MRl32, MR135 and MR170 have been named car-11, car-12, and car-13, respectively, whereas that in MR166 has been named carB4.

Car- mutations linked to carR suppress expression of the distant gene carB Using the transposon Tn5 lac, that carries a lac2 transcrimional Drobe for

, ~ " tank. Reciprocal crosses (If-17): Recipient is the standard Car' inducible promoter and becomes constitutively ex- Kms strain. Donors in crosses 13, 14, 15, and 16 are Car- KmR transductants from direct crosses 4, 6, 9, and 11, respectively.

pressed in the presence of carA or carR mutations Strain MRI 70 (cross 17) carries the same Car- mutation of MR17 1 (BALSALOBRE, Rurz-VAzQuEz and MURrLLo 987; and Tn5 (KmR) at QDK46 1 1. Table 4).

1-12 and percentage of Car- transductants in crosses 13-17.

> ,

Given as percentage of Car+ plus CarC transductants in crosses The original strain having Tn5 lac inserted at the carB locus is called MR401. We used this strain to

than the corresponding frequencies between QDK4611 and either of the Car- mutations (55% and 58%). Therefore, the Car- mutations present in MRl32 and MR17 1 are both located between QDK4611 and QMRl36. Altogether, the data argue for the order QDK46 1 1 -carR-Car-locus-QMR 136.

None of the transductants from crosses of MR135 with DK4611 or MR136 showed a Carc phenotype (Table 2, crosses 6 and 10). This could be explained if the corresponding carR and Car- mutations were more closely linked in MR135 than in MR132 or MR17 1. Alternatively, the original carR mutation

transduce the carB expression probe into strains MR132, MR135, and MR171, selecting for the KmR determinant present in Tn5 lac. With rare exceptions due to transposition to a new locus, the site and orientation of Tn5 lac in the transductants should be the same as in the donor strain. For each experiment, one hundred randomly chosen KmR transductants were tested for expression of @-galactosidase in X-Gal plates incubated in the dark or in the light. All transductants showed little expression of the lac2 gene at both conditions, as was evident from the low intensity of the blue color formed by enzymatic cleavage of X-

TABLE 3

Three factor crosses involving Car- mutations in M. santhus strains MR132 and MR170

KmR transductants Recombination unitsb

TcS TcR

(donor X recipient) Total Car- Car* CarC Car- Car* CarC RMR136 RDK4611 RDK4611

MRl98 X MR178 207 69 0 0 113 25 0 12 55 67 MR1.36 X MR171 266 1 76 1 36 127 25 14 58 71

Cross" Car- Car- RMR136

Transductant colonies were selected for the KmR determinant (QMRI 36::Tn5) of donor strains, and then screened for their Tc and Car phenotype. Donor strain MR198 also carries the Car- mutation of MR132. Both recipients carry insertion QDK4611::Tn5-132 (TcR). Recipient strain MR171 also carries the Car- mutation from MR170 (Table 1).

'Given as percentage of colonies showing nonparental phenotype for the corresponding pair of genetic markers indicated below. Both Car+ and CarC transductants are pooled in a single class for calculations involving the Car- locus.


TABLE 4

@-Galactosidase activity of various strains of M. santhus carrying a lacZ expression probe in gene carB

Relative level of @-galactosidaseb

Strain Relevant genotype" Dark Light

MR401 1 6.4 MR410 carAI 6.5 6.4 MR407 carR4 6.8 7.1 MR409 carR6 16.1 16.1

MR454 carR4, car-l l 0.6 0.5 MR456 carR4, car-13 2.3 2.3 MR458 carR6, car-12 1 .0 1.3

MR455 carAI, carR4, car-l1 6.2 6.9 MR457 carA1, carR4, car-l3 15.5 15.7 MR459 carAl, carR6, car-I2 8.2 7.6

All the listed strains also carry the lacZ transcriptional probe QMR401::Tn5 lac in gene carE. It is not certain if MR458 and MR459 contain the carR6 mutation (see text). ' Extracts of exponentially growing dark cultures that were in-

cubated in the dark or in the light for 6 hr more. Data are the ratio of the specific activity found in each case to that found for MR401 in the dark (this varies very little around 10 nmol . min" . mg").

Gal. The blue color of MR401 colonies, used as control, was also low in the dark but very intense in the light. These observations were corroborated by quantitative analysis in liquid cultures, whose results are shown in Table 4 (compare strains MR454, MR456, and MR458 with strain MR401). Time course of p- galactosidase production in some control strains and in one of the Car- transductants (MR454) are shown in Figure 2 (MR456 and MR458 behaved as MR454 in this experiment). Thus, as happens for carotenoid production, mutations car-l 1, car-12, and car- l3 have a negative effect on carB expression that is epistatic over the constitutive effect of carR mutations.

Mutation carA2 is epistatic over the Car- mutations linked to carR: A single mutation has been identified at gene carA (carAl) and it causes constitu- tiveness for both production of colored carotenoids and expression of the carB locus (MARTINEZ-LABORDA et al. 1986; BALSALOBRE, RUIZ-VAZQUEZ and MURILLO 1987). Gene carA is linked to carB and to QMR134, a Tn5 insertion that affects no car genes (Figure 1). So the carAI allele can be easily transferred to other strains by cotransduction with KmR determinants at QMRl34 or carB.

Strain MR146 (Table 1) simultaneously carries the carAI mutation and a Tn5 inserted at QMR134. MR146 was used as donor to transduce KmR into strains MR132, MR135, and MR171. The KmR transductants were screened for their Car phenotype and the results are shown in Table 5 (crosses 1-3). In all three cases, the transductants fell into two classes, some of them showing a Car- phenotype and the others a Carc phenotype. The proportion of the latter class (around 50%) corresponds to the known cotransduction frequency between QMRl34 and carAI (Fig-

TABLE 5

Epistatic effect of carAl over the Car- mutations linked to CarR

Carotenoid production KmR transductants

Cross Cotransduction (donor X recipient) Constitutive Negative frequency (%)"

1. MR146 X MR132 112 94 54 2. MR146 X MR135 110 100 52 3. MR146 X MR171 89 95 48

@-Galactosidase production

Constitutive Negative

4. MR410 X MR132 37 19 66 5. MR410 X MR135 257 114 69 6. MR410 X MR171 27 15 64

a Given as percentage of transductant colonies that, according to their constitutive phenotype, should have incorporated both the KmR determinant and the carAI mutation from the donor strains.

ure 1). Therefore, one should conclude that the constitutive effect of carAI on carotenogenesis is epistatic over the negative effect of mutations car-11, car-12, and car-13.

Strain MR410 (Table 1) simultaneously carries the carAI allele and the Tn5 lac expression probe at carB. Thus, 0-galactosidase is expressed in this strain at a high level and in a light-independent manner (Table 4; Figure 2). When MR410 was used to transduce KmR into strains MR132, MR135, and MR171, all the transductants showed the Car- phenotype expected from the disruption of gene carB. When these transductants were tested for 0-galactosidase expression in X-Gal plates, about 32% of them showed low activity in the dark and in the light, whereas the rest produced high level of the enzyme in both conditions (Table 5, crosses 4-6). These latter transductants should have gained the carAI allele along with the Tn5 lac insertion probe. Their proportions in the indicated crosses are in agreement with the known cotransduction frequency between carA I and carB::Tn5 lac (Figure 1). Quantitative analysis of p- galactosidase produced by a constitutive transductant from each of these crosses confirm that carAI is also epistatic over the Car- mutations as to their effect on carB expression (Table 4, strains MR455, MR457, and MR459; see also Figure 2 for a time course experiment with MR455).

Cis-dominance of the carA2 allele: As indicated in the Introduction, it has been speculated that carA may be a cis-acting element, such as a promoter, operator, or both, for light control of the expression of gene carB. T o further test this hypothesis, we have studied the interaction of mutant and wild-type alleles of carA in merodiploid strains.

In M . xanthus, merodiploids can be obtained after cloning the region of interest in a plasmid that repli- cates in E. coli and that carries a selectable marker and the Inc region of phage P1 (GILL, CULL and FLY


80

60

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A

B

A -A- A-A-A-

A A A t

I - +- +- 0-0-6-

0 2 L 6 8 10

Time (hr) FIGURE 2.-Time course of 0-galactosidase activity in several M .

xanthus strains. Zero time indicates the moment at which dark, early stationary cultures were diluted fivefold in fresh medium and incubated in the dark. At the time indicated by the arrows, the cultures were divided in two, one for dark (black symbols) and the other for light (open symbols) incubation. Panel A shows the results obtained with strains MR401 (W, 0) and MR410 (0, 0). Those obtained with MR454 (e, 0) and MR455 (A, A) are shown in panel B.

1988). This allows DNA transfer from E. coli to M . xanthus by P1 mediated specialized transduction. Once inside the M. xanthus cells, the chimeric plasmid cannot replicate and the selected transductants arise by integrative homologous recombination that gen- erates a tandem duplication of the cloned DNA (see for example, SHIMKETS, GILL and KAISER 1983; BLACKHART and ZUSMAN 1985; STEPHENS and KAISER 1987).

To clone the carA region, we used vector pREG429 and followed the "in situ" cloning strategy schemati- cally represented in Figure 3. Through this procedure, we cloned, from wild-type DK1050, a DNA fragment with one endpoint at the Tn5 insertion site ODK2836, which interrupts gene carB, and the other end point at the proximal BamHI site, 16 kb apart on

aDK2836

carA c a r 8

U 8amH1 digestion and intramolecular llgatton

Pllnc # .""

I \ 8 KmR

carA

FIGURE 3.-Cloning of the carA region. Shown on top is the arrangement in MR148 of gene carA (open box) and QDK2836::Tn5-132 (TcR) that interrupts the carB locus (striped box). The limits of carA and carB have been arbitrarily drawn. Plasmid pREG429 (13 kb) was introduced into strain MR148 by P1-mediated KmR transduction. Transductants should arise by integrative recombination of the plasmid into one of the IS50 elements (thick lines) of Tn5-132 (only integration at the IS50 closer to carA is represented). DNA was extracted from several KmR TcR transductants, digested with BarnH1, ligated and used to transform E . coli for KmR (see MATERIALS AND METHODS). Plasmid pMARlOO (29 kb) was present in colonies of E. coli transformed with two independent DNA samples. Since this plasmid could rescue a carA1 strain (see RESULTS) the extra piece of DNA (16 kb) should have come from the carA side of QDK2836. Shown in the bottom is a restriction map of the cloned DNA. Numbers below the map are the size (in kb) of the XhoI fragments. B, X and S stand for restriction enzymes BarnHI, XhoI and SalI , respectively.

the carA side of carB (Figure 3). Since the cotransduction frequency between ODK2836 and carA1 predicts an approximate distance of 7 kb between these two sites (MARTINEZ-LABORDA et al. 1986; WU 1966), the cloned DNA fragment is likely to include the complete carA locus. The chimeric plasmid was named pMAR 100.

Merodiploids containing tandem duplications of the cloned 16-kb fragment were generated, both in wild- type and carAI background, by selecting for the KmR determinant carried by pMAR100 in P1-mediated


A

""++"" Car' c e r A c a r 6

KmR

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X

c a r A I c a r 6 p MR7

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0

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FIGURE 4.-Tandem duplications of the carA region. The tandem duplications were generated by P1 mediated KmR transduction of plasmid p M A R 1 0 0 into the wild-type strain DK1050 (panel A) or the carA1 strain M R 7 (panel B). In panel B, the two types of transductants will arise when integration of p M A R l O 0 in the chro- mosonle of MR7 takes place by a recombination event to tli'e right (type 1) or to the left (type 11) of carAl . The indicated Cay phenotypes are those expected if the constitutive activation of carB produced by mutation carAI were cis-dominant. Symbols as in Figure 3.

transductions of strains DK1050 and MR7. Figure 4 shows the expected outcome of these crosses. It can be seen in this figure that the structure of the tandem duplication should be the same in all the transductants derived from DK1050 (Figure 4A), whereas two types of transductants are expected from MR7 (Figure 4B). These differ in the relative arrangement of the wild- type and mutant allele of carA with respect to the functional copy of the carB locus (Figure 4B).

Random samples of KmR colonies from the two indicated transductions were tested for production of colored carotenoids in the dark and in the light. Using DK1050 as the recipient, a total of 193 merodiploid transductants were screened and they all showed a Car+ phenotype. Apparently, chromosomal disruption by the vector DNA did not affect carotenogenesis, so we conclude that the cloned DNA fragment contains all the cis-acting elements that are required for the correct expression of the carB region (Figure 4A).

Two classes of transductants were found when pMAR100 was transduced into strain MR7. The first class includes the merodiploids that showed a Car+

phenotype and was represented by 114 out of 272 transductants (42%). The rest of the transductants (58%) showed a Car' phenotype. These two phenotypic classes might correspond to the two expected alternatives depicted in Figure 4B. However, one should consider the possibility of gene conversion, which has been observed in similar experiments with M . xanthus (STEPHENS and KAISER 1987).

To reveal the presence of both alleles, carA and carAI, in the merodiploids derived from MR7, one may rely on the spontaneous loss of the duplication by intramolecular recombination. This will produce the loss of the KmR determinant and also of one of the two copies of the carA gene, depending on the particular site in the duplicated region where the intramolecular exchange has occurred. Four independent Car+ or Car' merodiploids from MR7 were grown individually, in the absence of kanamycin, for about 25 generations. Then, the cultures were plated in the dark, also without selection, to visualize single colonies. Three of the Car+ cultures produced red colonies in the dark (Car') at a frequency that varies between 0.7% and 6.4%. When tested, most of these colonies were KmS, but a few were KmR and thus should have originated by gene conversion. These red colonies provide evidence for the presence of the mutant allele carAI in at least some of the original merodiploids that showed a Car+ phenotype. Simi- larly, two of the Car' merodiploids tested produced some colonies that in the dark showed a yellow, carotenoid-less phenotype (0.4% in one case and 3.7% in the other). Here we also found both KmR and KmS colonies. All these yellow colonies produced colored carotenoids upon illumination and thus were considered as Car+. These results strongly indicate that at least some of the Carc merodiploids from MR7 did in fact contain a wild-type allele of carA. Therefore, their phenotype cannot be explained by conversion of that allele to the mutated condition during the P1 mediated transduction of pMARlOO into strain MR7.

Altogether, the results presented above show that carA is a cis-acting genetic element and they could not be explained by carAI being recessive or trans-dominant over the corresponding wild-type allele.

All things considered, namely the Car phenotype and the organization of the carA-carB region in strains DK1050 and MR7, and the merodiploids derived from them (Figure 4), one may conclude that the Car+ and Carc merodiploids from MR7 correspond, respectively, to the type I and type I1 of transductants depicted in Figure 4B. In type I merodiploids, the mutant carAI allele will affect the expression of the adjacent, non-functional carB allele, but will not affect the wild-type copy of that gene. This one, under the cis-control of wild-type carA will be expressed only after illumination. In type I1 merodiploids, the carAI allele will cause constitutive expression of the adjacent


wild-type copy of carB and thus should produce the same Carc phenotype as MR7. In this model, assuming a random distribution of the integrative recombination events along both sides of carAI (Figure 4B) we could use the relative proportions of Car+ and Carc merodiploids to predict a distance of 6.7 kb between RDK2836 and the carA2 site. That prediction is in good agreement with the distance of 7 kb between those two sites that was deduced from transductional analysis, as mentioned above.

DISCUSSION

We have previously identified several genetic loci involved in the carotenogenic response of M. xanthus to illumination with blue light (MARTINEZ-LABORDA et a l . 1986; BALSALOBRE, RUIZ-VAZQUEZ and MURILLO 1987; Figure 1) . One of these loci, named carR, is defined by several closely linked mutations that cause constitutive synthesis of carotenoids. Here we have reported the isolation and characterization of four independent strains, derived from carR mutants and selected for loss of the Carc phenotype. We have shown that in the four cases single mutational events occurred that have not reverted the carR impairment but instead have blocked production of carotenoids. The four strains then carry Car- mutations that are epistatic over mutations at the carR locus.

The Car- mutation in one of the indicated strains, MRl66, maps at a locus previously identified and named carB (Figure 1). The function of carB is not known yet. It might code for a positive regulatory element or for an enzyme acting early in the pathway, before the first colored carotenoid is formed. Since transcription of carB, which requires illumination with blue light, is already under the control of two regulatory elements, carA and carR (BALSALOBRE, RUIZ- VAZQUEZ and MURILLO 1987), the idea of an enzyme- coding function for carB is the more attractive, but this remains to be proved.

The epistatic Car- mutations present in the other three strains are all closely linked to the carR locus. Those carried by MR132 or MR170 (car-I1 and car- l?) are located between carR and the closely linked Tn5 insertion site RMR136 (Tables 2 and 3). The Car-mutation carried by MRl35 (car-12) shows 100% cotransduction both with RMR136 and the carR mutation originally present in the parental strain of MR135. This would be easily explained if car - l2 were a deletion of the carR-RMR136 region, although other explanations are possible. The Car- phenotype caused by mutations car-11, car-12 or car-13 is very probably due to their effect on the distant gene carB. As said before, this gene, required for carotenogenesis and normally transcribed only upon illumination, is constitutively expressed in carR mutants. Our results show that any of the three Car- mutations linked to

carR suppresses expression of carB (Table 4 and Fig- ure 2).

Given the lack of linkage between carR and carB, we assume that the Car- locus linked to carR codes for a trans-acting regulatory element that mediates the effect of blue-light on carB expression. As a simple model, we suggest that this element is a positive regulator (activator) of carB that in the wild type is produced (or becomes functional) only in the presence of light. However, other explanations might be dis- cussed, particularly as mutations at the closely linked carR locus have quite the opposite effect (they make constitutive both the synthesis of carotenoids and the expression of carB). A simple alternative hypothesis would be that carB is under the control of a negative regulator (repressor) that in the wild type is present (or active) only in the dark. In this model, the carR mutations would destroy the repressor function, whereas the Car- mutations would affect the repressor in such a way that it is now present (or active) both in the dark and in the light. We consider this very unlikely, as rather sophisticated intra- or intergenic interactions would be required in this case to explain the epistasis of the Car- over the Carc mutations.

The apparent dual function of the whole region that includes the carR and the Car- loci, and the epistatic interaction between their corresponding mutations, can be explained in terms of either one or two gene products. Considering data from another author (see below) only the second hypothesis will be dis- cussed. In this hypothesis, carR would be a gene distinct from that affected by the Car- mutations. The product of carR, which should be present or active in the wild type only in the dark, will normally inhibit the expression of the neighboring gene coding for the carB activator, or else block its action. That an effect on gene expression is operating is supported by data of D. A. HODGSON (1987 and personal communica- tion) who has found two open reading frames in a cloned DNA fragment that contains the carR locus. Using a plasmid based lac2 promoter probe, he identified a light-inducible promoter closed to one of the open reading frames. This might correspond to the promoter of the activator gene. The same author has created specific deletions in the carR region that, like the mutations described here, produce a Car- phenotype.

We have also shown in this paper that gene carA, linked to carB and involved in stimulation by light of carB expression as well (BALSALOBRE, RUIZ-VAZQUEZ and MURILLO 1987), acts in a regulatory cascade after the activator gene. The single mutation known in carA, which causes constitutive expression of carB and a Car' phenotype, is epistatic over the Car- mutations linked to carR (Tables 4 and 5; Figure 2). Cloning of a DNA fragment that extends several kilobases along both sides of carA, has allowed us to construct mero-


diploids heterozygous for that gene (Figures 3 and 4). The Car phenotype of the merodiploids (see RESULTS) strongly suggests that carA is a cis-acting element for the control of carB. A simple hypothesis is that carA is the site of interaction for the trans-activator of carB and that mutation carAI has made that interaction unnecessary. Recent data from our group (our manuscript in preparation) discard the other simple alternative that carAI is producing an antitermination effect on a near transcriptional unit.

Gene carA is several kilobases apart from carB. T o explain the cis-action of carA, one may consider either an enhancer-like element, reported both in prokar- yotes and eukaryotes (PTASHNE 1986) or an operon that contains several genes. By manipulation of the plasmid pMAR100, we have recently managed to introduce a ZacZ transcriptional probe at different sites between carA and carB and have studied the “in vivo” production of 0-galactosidase and carotenoids in a wild-type, carA, and carR genetic background. The results (F. J. Murillo, in preparation) indicate that between carA and carB there are several genes coding for carotenogenic enzymes and coordinately regu- lated by light. A similar cluster of carotenogenic genes has been described in Rhodobacter capsulatus, although in this case most of the genes are organized as single transcriptional units (GUILIANO et al. 1988).

As shown by the kind of data included in this and our previous works, M. xanthus is a useful system to understand the genetic and molecular basis of a blue- light response (SENGER 1987; HOOBER and PHINNEY 1988). Cloning of cis- and trans-acting regulatory regions will allow structural analysis of the involved elements. It also provides a variety of tools to probe the regulatory mechanisms that operate in this system at the gene and also at the protein level (photorecep- tion and signal transduction). Very little information is presently available on the nature of carotenogenic enzymes (BRAMLEY and MACKENZIE 1988) and the chances are that M. xanthus could also contribute to the progress in this field.

We thank M. CARRETERO for technical assistance and M. ELIAS for revising the manuscript. We also thank our colleagues in Murcia and D. A. HODGSON for useful discussions, and the latter author for sharing unpublished results. This work was supported by a grant (PB86-0416) from the Spanish Direccibn General de Investigacih Cientifica y Tkcnica.

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Communicating editor: N. KLECKNER

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