Molec. gen. Genet. 152, 105-108 (1977) © by Springer-Verlag 1977
Chromosomal Location of Gene Governing the Trehalose Utilization in Escherichia coli K12 L. Becerra de Lares*, J. Ratouchniak, and F. Casse** Laboratoire de Chimie Bact6rienne, C.N.R.S., F-13274 Marseille Cedex 2, France
Summary. Several mutants unable to utilize trehalose were isolated from Escherichia coli. Their genetic analysis led to determine the following gene order on the chromosomal map: pur B-dad R-tre-hem A-trp. Furthermore, the tre gene belongs to the inversion of the trp chromosomal region between E. coli and S. typhimurium.
Introduction Attempting to estimate the length of the chromosomal inverted segment between Escherichia coli and Salmonella typhimurium (Casse et al., 1973), we had noted that the gene governing the utilization of trehalose has been mapped only in the last organism (St Pierre, 1968). In order to verify whether this tre gene is located at the homologous site on the chromosome of E. coli, the isolation and genetic analysis of mutants from E. coli K12 unable to ferment this dissacharide were undertaken.
Material. and Methods a) Strains. Table 1 gives the genotype and mating type of the strains used. b) Isolation of Mutants. An exponentially growing culture of bacteria was treated with N-methyl-N'-nitro-N-nitrosoguanidine (NTG) as described by Adelberg et al. (1965) and the relative number of mutants in the culture was increased by a penicillin treatment according to Gorini and Kaufmann (1960). e) Conjugation Experiments. Exponential cultures of the Hfr and F strains were mixed in the proportion of 1 donor for 20 recipient * Present address: Departamento de Biologia y Quimica, Instituto Pedagogico Nacional, Caracas, Venezuela ** To whom reprints will be requested
cells and left for 90 min at 37 ° C; then the mixture is plated on selective medium.
d) Transduction Experiments. The procedure of Lennox (1955) was employed for transduction experiments. e) Media. Minimal medium of Davis and Mingioli (1950) was used for the selection of auxotrophic characters. The E.M.B. medium of Lederberg (1950) was employed for fermentation tests. The hem + recombinants were selected on 6 aminolevulinic acid free rich medium.
Results and Discussion After N T G mutagenesis of E. eoli K12 strain CB356 and subsequent penicillin treatment, several mutant colonies were found, colourless on trehalose supplemented EMB medium, i.e., unable to ferment this sugar. In fact all the mutants obtained still slightly grow on minimal medium with trehalose as sole carbon source. The tre- strains develop a light black colour on EMB if this medium is devoid of phosphate buffer. However, the buffer effect of the usual EMB medium is sufficient to mask the acidification due to the residual fermentating activity of our tre mutants, and this medium was employed all along this work to determine the tre character of the recombinants. A first genetic analysis of one of our tre mutants (CB959) was undertaken by conjugation. Results of several mating experiments with different Hfr strains as donors are summarized in Table 2. The tre + allele being incorporated in recombinants as an early marker by Hfr B9 and Hfr KL99, the tre mutation is located between the origins of transfer of these two donors, i.e. between 22 and 30 min on the chromosomal map of E. coli K12. As shown of Figure 1, these preliminary results exclude for the E. coli lre gene a location corresponding to that found in S. typhimurium i.e. between tlp and his operons.
106
L. Becerra de Lares et al. : Chromosomal Location of Gene Governing the Trehalose Utilization in E. coli K12
Table 1. List of the strains used Number
Sex type
Genotype
Origin
CB29
F
pur B52 , trp E3, trp A2, dad Ra, tna
JK268 Kuhn
CB104
F-
hem As, met B1, trp A43 , lac Ys, mal As, str134
SHSP18 Sasarman
CB107
F
met B1, trp A43 , lac Y1, real As, strx34, trel
104 x P1962 hem +
CBll2
F-
met B, str134, lac 11, real A, hem As, chl C, ana
104xP1 ana- chl C- trp +
CBll9
F-
met B, lac I1, mal A, stria4, hem As, chl C, ana, tre
107xP1 112trp +
CB322
HffH
thi
Hayes
CB356
F-
this, thrs, leu 6, his, arg B CEH, pur E, pro, lac Y1, real A1, xyl7, arab3, mtl2, gal6, ton A2, str
PA601 Wollman
CB392
Hfr G1
his145
Wollman
CB459
F-
lac2~ pur B, trp C, pyr F, his, tyr A, mal, thi, str
X149 Jacob
CB915
Hfr B9
met B, lam, phx, rel,
KA70 Wijsman
CB923
Hfr KL99
thi, rel 1, lac42
CGSC4242 Bachmann
CB959
F-
thr, leu, thi, his, arg, pro, pur E, lac Y, gal B, mtl, ara, xyl, trel
CB356NTG penillin
CB962
F
thr, leu, thi, his, arg, pro, pur E, lac Y, gal B, mtl, ara, xyl, trel, chl C
CB959/C103
Table 2. Results of mating experiments with strain CB959 as F Donor strain
Percent of unselected recombinants
Selected recombinants
Number
Polarity
CB322
H
recipient
thr + leu +
pro +
lac +
gal +
his +
tre +
gal + str-r thr + leu + str-r his + str-r
55
98
61
-
16
18
-
30
40
38
23
32
69
2
2
3
5
-
84
CB915
B9
gal + str-r
86
94
-
-
89
CB392
G1
gal + str-r his + str-r
-
-
96
-
93
-
-
82
80
-
76 99
his + str-r
.
CB923
KL99
thr 0/90
lac
gal
10
pur B hem A trp 20
30
his 40
H
G1
)
B9
)
(
KL 99
1. Chromosomal map of E. coli: Some markers are indicated and the inverted segment as compared with S. typhimurium is thickly drawn on the upper line; origins and direction of transfer of the used Hfr are indicated below Fig.
.
.
.
.
54
To precise the location of this tre mutation, it was necessary to analyze comparatively several markers of this region. For this purpose, a derivative of strain CB959 was selected with a mutation in the chl C gene which is located in the tip region. This specific nitrate reductase mutant was obtained among mutants selected by chlorate resistance in anaerobic conditions (Piechaud et al., 1967). It was differenciated from pleiotropic chl mutants by its ability to produce Hz from formate in anaerobiosis and its genetic localization near 26 min. (Puig et al., 1969). Transductions were then performed with this tre chl C strain (CB962) as donor and strain CB104 carrying trp A and hem A mutations as recipient. The linkage of tre- and chl C - alleles with both markers was analyzed. Results reported in Table 3a and 3b strongly suggest the trp-chl- C-hem A-tre sequence of genes.
107
L. Becerra de Lares et al. : C h r o m o s o m a l Location of Gene Governing the Trehalose Utilization in E. coli K I 2 Table 3. Results of transduction experiments Donor strain
Recipient strain
Selected recombinants
Percent of unselected marker trp A
ana
chl C
hem A
ire
a) CB962 CB962
CB104 CB104
hem A +
7.7
-
trp A +
-
-
33.8 51
10
18.75 1.1
b) CBI12
CB107
trp A +
-
64
53
16
2.8
Another transduction experiment was performed with a trp- tre- derivative as recipient and a hem A - chl C- ana- strain (Casse et al., 1976) as donor. The linkage of the tre ÷ allele with all these known markers was observed among the trp + selected transductants and the results (Table 3 b and 4b), confirmed the gene order: trp-ana-chl C-hem A-tre reported on Figure 2. Cotransduction with pur B was then measured using strain CB459 as recipient and strain CB962 as donor. Among the pur B ÷ selected recombinants, 10.7 per cent had acquired the tre- allele, while none became chl C-. As pur B and hem A markers are not cotransducible by phage P1, the studied tre mutation which is cotranduced with both characters must lie between these two genes on the chromosome of E. coli. This pur B-hem A region includes gene dad R (Kuhn and Somerville, 1971). Therefore mapping of the tre mutation had to be done comparatively with dad R. Strain CB29 was used as recipient and the donor employed was a ire hem A chl C ana recombinant (CB 119). Among the 558 selected pur B ÷ recombinants 100 had received the tre mutation while 184 were unable to utilize D-tryptophan (dad R ÷) and none acquired the hem A chl C or ana mutations. Most of the tre- recombinants had coinherited the dad R ÷ character from the donor, while only 3 recombinants were found to have the tre- dad R - phenotype, resulting from a double crossing over in the pur B tre segment. To determine whether the other seven independant mutations affecting the trehalose utilization are located in the same chromosomal region, P1 mediated transductions were performed with each tre mutant as donor and the linkage with trp, hem A and pur B was measured. The results reported in Table 5 show that all the selected tre strains do bear mutations lying between pur B and hem A genes. In each case, the frequency of cotransduction of tre with pur B never exceeds 15 per cent. As dad R is 33 per cent cotransducible with pur B, all those results clearly indicate the pur B-dad R-tre-hem A gene order on the chromosomal map of E. coli, as noted on Figure 2. The 8 tre mutations can be considered as lying between dad R and hem A. Therefore, it can be
trp
ana
chl C
hem A
tre
dad R
pur B
7.7 33.8 18.75 64
)
53 10-16 1.1-2.8
Chromosomal Location of Gene Governing the Trehalose Utilization in Escherichia coli K12 L. Becerra de Lares*, J. Ratouchniak, and F. Casse** Laboratoire de Chimie Bact6rienne, C.N.R.S., F-13274 Marseille Cedex 2, France
Summary. Several mutants unable to utilize trehalose were isolated from Escherichia coli. Their genetic analysis led to determine the following gene order on the chromosomal map: pur B-dad R-tre-hem A-trp. Furthermore, the tre gene belongs to the inversion of the trp chromosomal region between E. coli and S. typhimurium.
Introduction Attempting to estimate the length of the chromosomal inverted segment between Escherichia coli and Salmonella typhimurium (Casse et al., 1973), we had noted that the gene governing the utilization of trehalose has been mapped only in the last organism (St Pierre, 1968). In order to verify whether this tre gene is located at the homologous site on the chromosome of E. coli, the isolation and genetic analysis of mutants from E. coli K12 unable to ferment this dissacharide were undertaken.
Material. and Methods a) Strains. Table 1 gives the genotype and mating type of the strains used. b) Isolation of Mutants. An exponentially growing culture of bacteria was treated with N-methyl-N'-nitro-N-nitrosoguanidine (NTG) as described by Adelberg et al. (1965) and the relative number of mutants in the culture was increased by a penicillin treatment according to Gorini and Kaufmann (1960). e) Conjugation Experiments. Exponential cultures of the Hfr and F strains were mixed in the proportion of 1 donor for 20 recipient * Present address: Departamento de Biologia y Quimica, Instituto Pedagogico Nacional, Caracas, Venezuela ** To whom reprints will be requested
cells and left for 90 min at 37 ° C; then the mixture is plated on selective medium.
d) Transduction Experiments. The procedure of Lennox (1955) was employed for transduction experiments. e) Media. Minimal medium of Davis and Mingioli (1950) was used for the selection of auxotrophic characters. The E.M.B. medium of Lederberg (1950) was employed for fermentation tests. The hem + recombinants were selected on 6 aminolevulinic acid free rich medium.
Results and Discussion After N T G mutagenesis of E. eoli K12 strain CB356 and subsequent penicillin treatment, several mutant colonies were found, colourless on trehalose supplemented EMB medium, i.e., unable to ferment this sugar. In fact all the mutants obtained still slightly grow on minimal medium with trehalose as sole carbon source. The tre- strains develop a light black colour on EMB if this medium is devoid of phosphate buffer. However, the buffer effect of the usual EMB medium is sufficient to mask the acidification due to the residual fermentating activity of our tre mutants, and this medium was employed all along this work to determine the tre character of the recombinants. A first genetic analysis of one of our tre mutants (CB959) was undertaken by conjugation. Results of several mating experiments with different Hfr strains as donors are summarized in Table 2. The tre + allele being incorporated in recombinants as an early marker by Hfr B9 and Hfr KL99, the tre mutation is located between the origins of transfer of these two donors, i.e. between 22 and 30 min on the chromosomal map of E. coli K12. As shown of Figure 1, these preliminary results exclude for the E. coli lre gene a location corresponding to that found in S. typhimurium i.e. between tlp and his operons.
106
L. Becerra de Lares et al. : Chromosomal Location of Gene Governing the Trehalose Utilization in E. coli K12
Table 1. List of the strains used Number
Sex type
Genotype
Origin
CB29
F
pur B52 , trp E3, trp A2, dad Ra, tna
JK268 Kuhn
CB104
F-
hem As, met B1, trp A43 , lac Ys, mal As, str134
SHSP18 Sasarman
CB107
F
met B1, trp A43 , lac Y1, real As, strx34, trel
104 x P1962 hem +
CBll2
F-
met B, str134, lac 11, real A, hem As, chl C, ana
104xP1 ana- chl C- trp +
CBll9
F-
met B, lac I1, mal A, stria4, hem As, chl C, ana, tre
107xP1 112trp +
CB322
HffH
thi
Hayes
CB356
F-
this, thrs, leu 6, his, arg B CEH, pur E, pro, lac Y1, real A1, xyl7, arab3, mtl2, gal6, ton A2, str
PA601 Wollman
CB392
Hfr G1
his145
Wollman
CB459
F-
lac2~ pur B, trp C, pyr F, his, tyr A, mal, thi, str
X149 Jacob
CB915
Hfr B9
met B, lam, phx, rel,
KA70 Wijsman
CB923
Hfr KL99
thi, rel 1, lac42
CGSC4242 Bachmann
CB959
F-
thr, leu, thi, his, arg, pro, pur E, lac Y, gal B, mtl, ara, xyl, trel
CB356NTG penillin
CB962
F
thr, leu, thi, his, arg, pro, pur E, lac Y, gal B, mtl, ara, xyl, trel, chl C
CB959/C103
Table 2. Results of mating experiments with strain CB959 as F Donor strain
Percent of unselected recombinants
Selected recombinants
Number
Polarity
CB322
H
recipient
thr + leu +
pro +
lac +
gal +
his +
tre +
gal + str-r thr + leu + str-r his + str-r
55
98
61
-
16
18
-
30
40
38
23
32
69
2
2
3
5
-
84
CB915
B9
gal + str-r
86
94
-
-
89
CB392
G1
gal + str-r his + str-r
-
-
96
-
93
-
-
82
80
-
76 99
his + str-r
.
CB923
KL99
thr 0/90
lac
gal
10
pur B hem A trp 20
30
his 40
H
G1
)
B9
)
(
KL 99
1. Chromosomal map of E. coli: Some markers are indicated and the inverted segment as compared with S. typhimurium is thickly drawn on the upper line; origins and direction of transfer of the used Hfr are indicated below Fig.
.
.
.
.
54
To precise the location of this tre mutation, it was necessary to analyze comparatively several markers of this region. For this purpose, a derivative of strain CB959 was selected with a mutation in the chl C gene which is located in the tip region. This specific nitrate reductase mutant was obtained among mutants selected by chlorate resistance in anaerobic conditions (Piechaud et al., 1967). It was differenciated from pleiotropic chl mutants by its ability to produce Hz from formate in anaerobiosis and its genetic localization near 26 min. (Puig et al., 1969). Transductions were then performed with this tre chl C strain (CB962) as donor and strain CB104 carrying trp A and hem A mutations as recipient. The linkage of tre- and chl C - alleles with both markers was analyzed. Results reported in Table 3a and 3b strongly suggest the trp-chl- C-hem A-tre sequence of genes.
107
L. Becerra de Lares et al. : C h r o m o s o m a l Location of Gene Governing the Trehalose Utilization in E. coli K I 2 Table 3. Results of transduction experiments Donor strain
Recipient strain
Selected recombinants
Percent of unselected marker trp A
ana
chl C
hem A
ire
a) CB962 CB962
CB104 CB104
hem A +
7.7
-
trp A +
-
-
33.8 51
10
18.75 1.1
b) CBI12
CB107
trp A +
-
64
53
16
2.8
Another transduction experiment was performed with a trp- tre- derivative as recipient and a hem A - chl C- ana- strain (Casse et al., 1976) as donor. The linkage of the tre ÷ allele with all these known markers was observed among the trp + selected transductants and the results (Table 3 b and 4b), confirmed the gene order: trp-ana-chl C-hem A-tre reported on Figure 2. Cotransduction with pur B was then measured using strain CB459 as recipient and strain CB962 as donor. Among the pur B ÷ selected recombinants, 10.7 per cent had acquired the tre- allele, while none became chl C-. As pur B and hem A markers are not cotransducible by phage P1, the studied tre mutation which is cotranduced with both characters must lie between these two genes on the chromosome of E. coli. This pur B-hem A region includes gene dad R (Kuhn and Somerville, 1971). Therefore mapping of the tre mutation had to be done comparatively with dad R. Strain CB29 was used as recipient and the donor employed was a ire hem A chl C ana recombinant (CB 119). Among the 558 selected pur B ÷ recombinants 100 had received the tre mutation while 184 were unable to utilize D-tryptophan (dad R ÷) and none acquired the hem A chl C or ana mutations. Most of the tre- recombinants had coinherited the dad R ÷ character from the donor, while only 3 recombinants were found to have the tre- dad R - phenotype, resulting from a double crossing over in the pur B tre segment. To determine whether the other seven independant mutations affecting the trehalose utilization are located in the same chromosomal region, P1 mediated transductions were performed with each tre mutant as donor and the linkage with trp, hem A and pur B was measured. The results reported in Table 5 show that all the selected tre strains do bear mutations lying between pur B and hem A genes. In each case, the frequency of cotransduction of tre with pur B never exceeds 15 per cent. As dad R is 33 per cent cotransducible with pur B, all those results clearly indicate the pur B-dad R-tre-hem A gene order on the chromosomal map of E. coli, as noted on Figure 2. The 8 tre mutations can be considered as lying between dad R and hem A. Therefore, it can be
trp
ana
chl C
hem A
tre
dad R
pur B
7.7 33.8 18.75 64
)
53 10-16 1.1-2.8
