JOURNAL OF BACTERIOLOGY, Jan. 1976, p. 282-289 Copyright © 1976 American Society for Microbiolog-Y
Vol. 125, No. 1 Printed in U.S.A.
Isolation and Description of a Menaquinone Mutant from Bacillus licheniformis STEVEN R. GOODMAN, BARRY L. MARRS, ROMAN J. NARCONIS, AND ROBERT E. OLSON* Department of Biochemistry, St. Louis University School of Medicine, St. Louis, Missouri 63104 Received for publication 6 October 1975
A menaquinone mutant (SG1) of Bacillus licheniformis has been isolated by selecting for colonies that are resistant to low levels of kanamycin (1.5 ,g/ml) but sensitive to the same concentration of kanamycin in the presence of shikimate (25 jg/ml). The wild type (IU1) contained 0.38 + 0.02 nmol of menaquinone-7 (MK-7) per mg (dry weight) of cells when grown ± shikimate, whereas SG1 had
404
95
30 .
5
201
H.I
-. -a "
.1L
40
120
80
6433
ter
bL *
225
i,2!
200
160
240
xgr,
260
320
srs
360
i
5rt9
~~~ ~ ~~443 5.1
400
440
400
440
480
520
560
520
560
600
640
600
640
m/r
AUTHENTIC MENAQUNONE-7 Dired Probe, KX0C, 70v >- 90
,F,80
Z
70
W 60. ir50
>
40
5
30
I
,,,
60
ia I L 120
. 4t7 L& 160
223 239 -
200
373
307
16
240
280
320
360
443
646
573
Sit
440
m/e
FIG. 2. Mass spectra of bacterial and authentic menaquinone. Mass spectra of menaquinone isolated by TLC of a cold acetone extract of B. licheniformis were run at 100 C, with an electron impact of 70 eV, using the direct probe.
MENAQUINONE MUTANT FROM B. LICHENIFORMIS
VOL. 125, 1976
285
TABLE 1. Growth of shikimate auxotroph on TBABG platea Growth on TBABG plates (CFU/Klett unit) Mutant
+Kanamycin
No addition
+Shikimate
Wild type
510,000 (±+40,000)
530,000 (±+20,000)
7 (±1)
11 (±i 1)
Wild type + shikimate
500,000 (±40,000)
510,000 (-20,000)
14 (±1)
8 (±2)
Men-
920,000 (±30,000)
970,000 (±40,000)
900,000 (±30,000)
750 (+50)
Men- + shikimate
500,000 (±20,000)
490,000 (±40,000)
498 (±14)
456 (±15)
+Kanamycin
+shikimate
a Wild type (IU1) and Men- (SG1) were grown + shikimate (25 Ag/ml) to 100 Klett units in TMG media at 37 C. The cells were then plated on TBABG plates, with shikimate (25 ug/ml) and kanamycin (1.5 yg/ml) added in conjunction and separately, and the plates were allowed to incubate for 15 h at 37 C before colonies were counted. Values presented are the mean of triplicate plates. Numbers in parentheses represent the range of three determinations. CFU, Colony-forming units.
TMG + shikimate (25 jg/ml), to give an initial cell density of 1 Klett unit. In the experiment described in Table 1, the cells were allowed to grow at 37 C to 100 Klett units and then were plated. The data in Table 1 show that: (i) IU1 is unaffected by having shikimate present in either the liquid or solid media; (ii) IU1 is sensitive to kanamycin (1.5 ug/ml); (iii) SG1 is resistant to kanamycin (1.5 Ag/ml); (iv) SG1 is sensitive to kanamycin (1.5 jg/ml) in the presence of shikimate (25 jug/ml), but is not killed by shikimate (25 ,g/ml) alone; (v) SG1, when grown with shikimate (25 ug/ml) in the liquid medium, behaves in a manner similar to MUl; (vi) there is -1.8 times as many colony-forming units per Klett unit for SG1 grown without shikimate as compared to IU1 i shikimate or SG1 + shikimate. Results (i) through (v) suggest that SG1 is a menaquinone auxotroph blocked prior to shikimate in the synthetic pathway, whereas (vi) suggests that a morphological alteration also exists in SG1. MK-7 content of IUI and SG1. The concentration of MK-7 in IU1 and SG1 grown + shikimate is presented in Table 2. IU1 grown with or without shikimate had -0.38 nmol of vitamin K2 per mg (dry weight) of cells. However, for SG1 grown without shikimate, there was no detectable menaquinone in an acetone extract of cells derived from a 1-liter culture harvested 1 h into stationary phase. There was no spot comigrating with MK-7 on TLC viewed with a UV lamp, and, when the region where MK-7 should run was scraped from the plate and eluted, no vitamin K2 spectra were observed on scanning the UV range (200 to 320 nm) with a Beckman DB spectrometer. The
TABLE 2. MK-7 in IUI and SG1 grown Mutant Mutant
Wild type ...................... Wild type + shikimate (25 'Ug/ml) ...................... Men . ....................... Men- + shikimate (25 Ag/ ml) .......................
i
shikimatea
~~MK-7 (nmol/mg
[dry wt] of cells) 0.38
+
0.02 (5)
0.37 ± 0.02 (3)
Vol. 125, No. 1 Printed in U.S.A.
Isolation and Description of a Menaquinone Mutant from Bacillus licheniformis STEVEN R. GOODMAN, BARRY L. MARRS, ROMAN J. NARCONIS, AND ROBERT E. OLSON* Department of Biochemistry, St. Louis University School of Medicine, St. Louis, Missouri 63104 Received for publication 6 October 1975
A menaquinone mutant (SG1) of Bacillus licheniformis has been isolated by selecting for colonies that are resistant to low levels of kanamycin (1.5 ,g/ml) but sensitive to the same concentration of kanamycin in the presence of shikimate (25 jg/ml). The wild type (IU1) contained 0.38 + 0.02 nmol of menaquinone-7 (MK-7) per mg (dry weight) of cells when grown ± shikimate, whereas SG1 had
404
95
30 .
5
201
H.I
-. -a "
.1L
40
120
80
6433
ter
bL *
225
i,2!
200
160
240
xgr,
260
320
srs
360
i
5rt9
~~~ ~ ~~443 5.1
400
440
400
440
480
520
560
520
560
600
640
600
640
m/r
AUTHENTIC MENAQUNONE-7 Dired Probe, KX0C, 70v >- 90
,F,80
Z
70
W 60. ir50
>
40
5
30
I
,,,
60
ia I L 120
. 4t7 L& 160
223 239 -
200
373
307
16
240
280
320
360
443
646
573
Sit
440
m/e
FIG. 2. Mass spectra of bacterial and authentic menaquinone. Mass spectra of menaquinone isolated by TLC of a cold acetone extract of B. licheniformis were run at 100 C, with an electron impact of 70 eV, using the direct probe.
MENAQUINONE MUTANT FROM B. LICHENIFORMIS
VOL. 125, 1976
285
TABLE 1. Growth of shikimate auxotroph on TBABG platea Growth on TBABG plates (CFU/Klett unit) Mutant
+Kanamycin
No addition
+Shikimate
Wild type
510,000 (±+40,000)
530,000 (±+20,000)
7 (±1)
11 (±i 1)
Wild type + shikimate
500,000 (±40,000)
510,000 (-20,000)
14 (±1)
8 (±2)
Men-
920,000 (±30,000)
970,000 (±40,000)
900,000 (±30,000)
750 (+50)
Men- + shikimate
500,000 (±20,000)
490,000 (±40,000)
498 (±14)
456 (±15)
+Kanamycin
+shikimate
a Wild type (IU1) and Men- (SG1) were grown + shikimate (25 Ag/ml) to 100 Klett units in TMG media at 37 C. The cells were then plated on TBABG plates, with shikimate (25 ug/ml) and kanamycin (1.5 yg/ml) added in conjunction and separately, and the plates were allowed to incubate for 15 h at 37 C before colonies were counted. Values presented are the mean of triplicate plates. Numbers in parentheses represent the range of three determinations. CFU, Colony-forming units.
TMG + shikimate (25 jg/ml), to give an initial cell density of 1 Klett unit. In the experiment described in Table 1, the cells were allowed to grow at 37 C to 100 Klett units and then were plated. The data in Table 1 show that: (i) IU1 is unaffected by having shikimate present in either the liquid or solid media; (ii) IU1 is sensitive to kanamycin (1.5 ug/ml); (iii) SG1 is resistant to kanamycin (1.5 Ag/ml); (iv) SG1 is sensitive to kanamycin (1.5 jg/ml) in the presence of shikimate (25 jug/ml), but is not killed by shikimate (25 ,g/ml) alone; (v) SG1, when grown with shikimate (25 ug/ml) in the liquid medium, behaves in a manner similar to MUl; (vi) there is -1.8 times as many colony-forming units per Klett unit for SG1 grown without shikimate as compared to IU1 i shikimate or SG1 + shikimate. Results (i) through (v) suggest that SG1 is a menaquinone auxotroph blocked prior to shikimate in the synthetic pathway, whereas (vi) suggests that a morphological alteration also exists in SG1. MK-7 content of IUI and SG1. The concentration of MK-7 in IU1 and SG1 grown + shikimate is presented in Table 2. IU1 grown with or without shikimate had -0.38 nmol of vitamin K2 per mg (dry weight) of cells. However, for SG1 grown without shikimate, there was no detectable menaquinone in an acetone extract of cells derived from a 1-liter culture harvested 1 h into stationary phase. There was no spot comigrating with MK-7 on TLC viewed with a UV lamp, and, when the region where MK-7 should run was scraped from the plate and eluted, no vitamin K2 spectra were observed on scanning the UV range (200 to 320 nm) with a Beckman DB spectrometer. The
TABLE 2. MK-7 in IUI and SG1 grown Mutant Mutant
Wild type ...................... Wild type + shikimate (25 'Ug/ml) ...................... Men . ....................... Men- + shikimate (25 Ag/ ml) .......................
i
shikimatea
~~MK-7 (nmol/mg
[dry wt] of cells) 0.38
+
0.02 (5)
0.37 ± 0.02 (3)
