Vol. 135, No. 2
JOURNAL OF BACTERIOLOGY, Aug. 1978, p. 595-602 0021-9193/78/0135-0595$02.00/0 Copyright 0 1978 American Society for Microbiology
Printed in U.S.A.
Isolation and Characterization of Morphogenetic Mutants of Arthrobacter crystallopoietes E. C. ACHBERGERt AND P. E. KOLENBRANDERt* Department of Microbiology and Cell Biology, The Pennsylvania State University, University Park, Pennsylvania 16802
Received for publication 10 March 1978
Mutants of Arthrobacter crystallopoietes that exhibited altered ability to undergo the normal sphere-to-rod-to-sphere morphogenetic cycle were isolated. The procedure used to isolate these mutants involved velocity sedimentation in a sterile sucrose gradient to separate morphogenesis-deficient spherical cells from rod-shaped cells capable of normal morphogenesis. Three classes of mutants were obtained: (i) those that cannot form rods, (ii) those that cannot form long rods, and (iii) those that form long rods but exhibit more extensive rudimentary branching than the wild type. The isolation and characterization of these mutants are described, and the use of these mutants in the study of the morphogenetic cycle of arthrobacters is discussed.
Members of the genus Arthrobacter exhibit a unique sphere-to-rod-to-sphere morphogenetic life cycle. When spherical cells from the stationary phase of growth are inoculated into a rich organic medium, they elongate to form rods prior to the first cell division. Once they have formed rods, they continue to divide as rods throughout the exponential phase of growth. During stationary phase, several smaller spherical cells are formed from each rod by a process of reductive cell division or fragmentation. When inoculated into fresh media, these spheres are capable of again undergoing the morphogenetic cycle. The sphere-to-rod morphological transition can be nutritionally controlled in Arthrobacter crystallopoietes (3). Cells grown in a chemically defined minimal medium with glucose as a carbon source do not undergo the sphere-to-rod transition but grow solely as spheres throughout growth. Thus, cells of A. crystallopoietes are spherical shaped throughout growth in glucose minimal medium, whereas spheres are formed from rods only in the stationary phase of growth in all other media tested. When either exponentially growing spheres in glucose medium or stationary-phase spheres from other media are inoculated into minimal medium containing a rod-inducing carbon source, the spheres elongate to form rods and divide as rods throughout exponential growth. Some of the rod-inducing carbon sources that we have used are the sugar t Present address: Department of Microbiology and Immunology, University of Washington, Seattle, WA 98195. t Present address: Microbiology Section, Laboratory of Microbiology and Immunology, National Institute of Dental Research, Bethesda, MD 20014.
fructose, the amino acids L-asparaglne and Lphenylalanine, and the organic acids lactate, butyrate, and succinate. A. crystallopoietes is the only Arthrobacter species which exhibits nutritional control of this type. Thus, both spherical and rod-shaped cells grown under balanced growth conditions (exponential phase of growth) can be obtained and used to study certain physiological or chemical differences that may be related to cell shape. However, one potential disadvantage inherent in nutritional control is that the cells, although different in cell shape, were grown on different carbon sources. To eliminate this difficulty in a study of physiological events related to morphogenetic shape changes, it would be helpful to obtain cells of different shape but in the same physiological state when grown in identical media. This condition was obtained by isolating morphogenetically altered mutants which exhibited a different shape than the wild type during growth in media containing identical carbon sources. These mutants are the first reported morphogenetic mutants of A. crystallopoietes or any other Arthrobacter species. In this paper, we present the method of isolation and the characterization of these mutants.
MATERIALS AND METHODS Bacteria and bacteriophage. A. crystallopoietes (ATCC 15481), A. globifornis (ATCC 8010), A. pyridinolis, and A. viridescens were obtained from laboratory culture stocks. Bacteriophage AC-1, AG-1, and AV-3 were isolated from local soil using, as the host species, A. crystallopoietes, A. globiformis, and A. viridescens, respectively. Growth media and culture conditions. Cultures 595
596
ACHBERGER AND KOLENBRANDER
were grown in either a complex broth medium (Trypticase soy broth [BBL]) or a defined mineral salts phosphate (MSP) medium consisting of (grams per liter of 0.05 M potassium phosphate buffer, pH 7.2): (NH4)2SO4, 1.0; MgSO4 7H20, 0.1; carbon source, 5.0; and a trace salts solution, 10 ml, containing 2 g of CaCl2 2H20, 1.0 g of MnSO4 H20, and 0.5 g of FeSO4 7H20 dissolved in 1 liter of 0.1 N HCl. The carbon source, MSP medium, and trace salts were sterilized separately. Cultures were grown at 30°C with shaking aeration in Erlenmeyer flasks fitted with a tubular side arm. The culture volume never exceeded 10% of the flask capacity. Cell growth was monitored with a Klett-Summerson colorimeter with a red filter (660 nm). Mutant isolation. Spherical cells of A. crystallopoietes harvested during exponential growth in glucose-MSP medium were mutagenized with ethyl methane sulfonate (15). Cells from 10 ml of the culture were suspended in 10 ml of MSP buffer (MSP with no carbon source) containing 0.15 ml of ethyl methane sulfonate. The cells were incubated for 90 min with shaking at 30°C, washed in MSP buffer to remove the ethyl methane sulfonate, and inoculated into fresh glucose-MSP medium. After allowing two doublings of cell mass, the culture was diluted with 2 volumes of succinate-MSP medium to induce the formation of rods in cells capable of morphogenesis. Cell growth was permitted until the turbidity increased from 10 to 70 Klett units. Cells were washed free of the growth medium and suspended in 0.1 volume of MSP buffer. A sample (0.5 ml) of the cell suspension was layered onto either a sterile 15 to 40% linear sucrose gradient or one made by repeatedly (three times) freezing and thawing 28% (wt/vol) sucrose (2). The sucrose gradients were centrifuged at 2,500 x g for 5 min to separate spheres from rods. Above a broad layer of cells near the bottom of the gradient that contained rods there was a narrow, welldefined band that contained spheres. The spherical cells in this top layer were collected, washed free of sucrose, and plated onto succinate-MSP agar or succinate-MSP agar containing 0.8% (vol/vol) nutrient broth. After incubation at 30°C for several days, the resulting colonies were transferred to complex broth medium by using thin, sterile slivers cut from tygon tubing and incubated until the cultures reached midexponential growth. By using phase-contrast microscopy, the cultures were examined for cells with abnormal morphology. Those cultures containing cell populations with abnormal morphology were tested for purity by repeated single-colony transfers onto complex media solidified with agar. Sensitivity to bacteriophage. The wild type and each of the morphogenetically altered mutants, as well as A. viridescens and A. globiformis, were tested for sensitivity to three different bacteriophage, using a routine spot test assay and the soft-agar overlay technique (1). A 0.01-ml sample of each of a series of 10fold dilutions, 100 to 10-9, of a given bacteriophage stock suspension, 1012 plaque-forming units/ml, was spotted onto a 0.75% (wt/vol) agar overlay containing complex medium and one of the bacterial indicators. Sensitivity to bacteriophage was determined by the presence of cell lysis in the spotted zones.
J. BACTERIOL.
Cesium chloride buoyant density gradient centrifugation. Each of the mutants and A. pyridinolis were labeled by addition of 10 jiCi of carrier-free H:,''P04 per ml to cells growing in a low-phosphate medium adopted from Singer and Smith (13). The low-phosphate medium was prepared in two parts. Part A was filter sterilized as a 1X-concentrated solution and contained, per liter of distilled water: Ntris(hydroxymethyl)-methyl-2-aminoethane sulfonic acid, 274 g; NH4Cl, 10.7 g; peptone (Difco), 4 g; Na2SO4, 0.71 g; KH2PO4, 272 mg; and KCl, 14.9 mg; adjusted to a final pH of 7.5. Part B (40x concentrated) contained, per liter of distilled water: MgCl2 6H20, 16.0 g; CaCl2, 0.44 g; and FeCl;, 6H20, 32 mg. Each of the three components of part B was autoclaved separately, and then aseptically combined, to make the 40x-concentrated solution. The two parts were then combined in the proper proportions. Filter-sterilized D-fructose was added to give a final concentration of 0.5Y (wt/vol), and the final culture volume was adjusted with sterile distilled water. ;32'P-labeled cells from 10 ml of culture were washed with MSP buffer and lysed by using a slightly modified lysozyme-ethylenediaminetetraacetic acid-sodium dodecyl sulfate procedure (4). After 20 min of incubation at 37°C, the cell lysate was centrifuged at 10,000 x g for 10 min, and the resulting supernatant fluid (cell extract) was dialyzed overnight against three changes (2-liter volumes) of TEN buffer composed of 0.05 M tris(hydroxymethyl)aminomethane, 0.005 M ethylenediaminetetraacetic acid, and 0.05 M NaCl, adjusted to a final pH of 8.0. The volume of the extract after dialysis was adjusted to 4.93 ml with TEN buffer in a cellulose nitrate ultracentrifuge tube. The following was then added: 0.33 ml of 0.7 M phosphate buffer, pH 7.3; 7.1 g of CsCl; and 0.1 ml (5.0 x 105 cpm) of 'H-labeled A. crystallopoietes chromosomal DNA. The gradients were centrifuged at 42,000 rpm for 40 to 48 h at 15°C in a type 65 fixed-angle rotor. After centrifugation, the fractions were collected and processed as described in detail previously (8). These procedures included hydrolysis of RNA with KOH, precipitation of DNA with trichloroacetic acid, collection of precipitate on glassfiber filters, drying of filters, and determination of radioactivity by liquid scintillation counting.
RESULTS Morphogenetically altered mutants. The mutagenesis treatment of spherical cells of A. crystallopoietes with ethyl methane sulfonate resulted in about 90% killing as determined by viable cell counts obtained by plating samples of the spherical cell population before and after mutagenesis. The initial purpose of the mutant isolation procedure described in Materials and Methods was to enrich for and isolate morphogenetically altered (designated Mph) mutants which were unable to change from spherical shape to rod shape in routinely used rod-inducing media. For example, in complex broth medium, the wild type grows as long pleomorphic rods during exponential phase. Thus, mutants
VOL. 135, 1978
MORPHOGENETIC MUTANTS OF A. CRYSTALLOPOIETES
unable to form rods in this medium would be easily recognized by phase-contrast microscopy. Accordingly, samples of 2,400 colonies from succinate-MSP agar plates were inoculated into culture tubes containing complex broth medium, and after reaching exponential growth, the morphology of the growing cells was examined microscopically. In addition to the desired spherical mutant, other morphological types were observed. The mutants isolated can be grouped into three morphological classes: (i) those unable to form rods, (ii) those unable to form long rods, and (iii) those that form long pleomorphic rods but exhibit more extensive rudimentary branching than the wild type. The morphology of some of the mutants representative of each class is presented in Fig. 1 and 2. In Fig. 1, cell populations of the mutants and the wild type at four stages (inoculum spheres and midexponential, early stationary, and late stationary phases) during growth in complex broth are shown. Photographs of mutant and wild-type cells observed in midexponential growth phase in glucose- and succinate-MSP media are depicted in Fig. 2. A complete description of the mutants and comparison with the wild type are given below. The following mutants, representative of the three classes, are described: Mph-3 and Mph-4 (class 1), Mph-5 and Mph-6 (class 2), and Mph-8 (class 3). The two mutants, Mph-3 and Mph-4, that are unable to undergo sphere-to-rod-to-sphere morphogenesis grow exclusively as spheres on all media tested. The morphology of these two mutants has been examined by using cells grown in the rod-inducing carbon sources lactate, butyrate, succinate, malate, fructose, asparagine, and phenylalanine. The cell shapes of these two mutants (Fig. 1, rows 1 and 2) compared to the wild type (Fig. 1, row 6) at various times of growth in complex broth medium are depicted. The wild type exhibits a normal morphogenetic cycle including elongation of inoculum spheres to typical long pleomorphic rods arranged in characteristic V-formations during the exponential phase of growth (Fig. 1, row 6 column B), followed by shorter rods in early stationary phase (Fig. 1, row 6, column C), and, finally, spherical cells in late stationary phase (Fig. 1, row 6, column D). In comparison, Mph-3 and Mph-4 remain spherical at all stages of growth (Fig. 1, rows 1 and 2). Although both strains grow as spheres, they are morphologically distinguishable. Mph-3 grows as well-isolated spheres, whereas Mph-4 grows as clumps of spheres with the presence of large swollen cells with diameters ranging from 2.0 to 2.8 t,m. These two mutants generally have cell diameters of 1.4 to 1.7
597
,um depending on the growth medium and doubling time. The incubation temperature (20 to 37°C) has no effect on the inability of these two mutants to form rods. The morphology of Mph3 growing on glucose minimal medium is identical to that of the wild type growing as spheres on the medium (Fig. 2, rows 1 and 5, column B). However, in minimal medium containing succinate, a rod-inducing carbon source for the wild type, the morphological difference between the mutant strain and the wild type is clearly apparent (Fig. 2, rows 1 and 5, column A). In addition to the spherical mutants, several other mutants were isolated that were capable of morphogenesis but formed only short rods when grown in a rod-inducing medium. The most carefully characterized of these mutants is designated Mph-5. During exponential growth in complex broth medium, it forms rods of less than one-half the length of the wild type (Fig. 1, rows 3 and 6, column B). However, the sphereto-rod-to-sphere morphogenetic cycle is evident (Fig. 1, row 3, columns A, B, C, and D). When growing in a chemically defined medium containing a rod-inducing carbon source, the shape of the rods of Mph-5 is similar to that of the wild type, but cell measurements of these rods show that both length and width are shorter than those of the wild type by 10 to 20% (Fig. 2, rows 3 and 5, column A). As was found for class 1 mutants, cells of Mph-5 grown in glucose-MSP medium are indistinguishable from wild-type cells grown in the same medium (Fig. 2, rows 3 and 5, column B). The mutant Mph-6 was also originally isolated because of its inability to form long rods in complex media (Fig. 1, row 4). It was later identified as an auxotroph. Addition of low levels of L-methionine relieved the auxotrophic requirement in defined minimal media, but addition of L-methionine to complex medium did not restore the long-rod morphology of Mph-6. The morphologies of other amino acid auxotrophs isolated by different selective techniques have been examined on complex medium, and some, but not all, were unable to form long rods. Prototrophic revertants of Mph-6 regained long-rod morphology. Another class 2 mutant, Mph-7, exhibits extremely slow growth properties in all media tested. Although its doubling time is about three times as long as that of the wild type in glucose, succinate, fructose, and complex media, its shape is similar to that of the wild type in each medium except complex medium, where it is a short rod. It is interesting to note that this mutant is a short rod at doubling times much slower than Mph-3 or wild type in complex media, where the latter two are spherical and long pleomorphic
F .S t
w --
-
-
i-
--::
0.
i.
. >5W,
|
.
|B
~~
0
*C
JOURNAL OF BACTERIOLOGY, Aug. 1978, p. 595-602 0021-9193/78/0135-0595$02.00/0 Copyright 0 1978 American Society for Microbiology
Printed in U.S.A.
Isolation and Characterization of Morphogenetic Mutants of Arthrobacter crystallopoietes E. C. ACHBERGERt AND P. E. KOLENBRANDERt* Department of Microbiology and Cell Biology, The Pennsylvania State University, University Park, Pennsylvania 16802
Received for publication 10 March 1978
Mutants of Arthrobacter crystallopoietes that exhibited altered ability to undergo the normal sphere-to-rod-to-sphere morphogenetic cycle were isolated. The procedure used to isolate these mutants involved velocity sedimentation in a sterile sucrose gradient to separate morphogenesis-deficient spherical cells from rod-shaped cells capable of normal morphogenesis. Three classes of mutants were obtained: (i) those that cannot form rods, (ii) those that cannot form long rods, and (iii) those that form long rods but exhibit more extensive rudimentary branching than the wild type. The isolation and characterization of these mutants are described, and the use of these mutants in the study of the morphogenetic cycle of arthrobacters is discussed.
Members of the genus Arthrobacter exhibit a unique sphere-to-rod-to-sphere morphogenetic life cycle. When spherical cells from the stationary phase of growth are inoculated into a rich organic medium, they elongate to form rods prior to the first cell division. Once they have formed rods, they continue to divide as rods throughout the exponential phase of growth. During stationary phase, several smaller spherical cells are formed from each rod by a process of reductive cell division or fragmentation. When inoculated into fresh media, these spheres are capable of again undergoing the morphogenetic cycle. The sphere-to-rod morphological transition can be nutritionally controlled in Arthrobacter crystallopoietes (3). Cells grown in a chemically defined minimal medium with glucose as a carbon source do not undergo the sphere-to-rod transition but grow solely as spheres throughout growth. Thus, cells of A. crystallopoietes are spherical shaped throughout growth in glucose minimal medium, whereas spheres are formed from rods only in the stationary phase of growth in all other media tested. When either exponentially growing spheres in glucose medium or stationary-phase spheres from other media are inoculated into minimal medium containing a rod-inducing carbon source, the spheres elongate to form rods and divide as rods throughout exponential growth. Some of the rod-inducing carbon sources that we have used are the sugar t Present address: Department of Microbiology and Immunology, University of Washington, Seattle, WA 98195. t Present address: Microbiology Section, Laboratory of Microbiology and Immunology, National Institute of Dental Research, Bethesda, MD 20014.
fructose, the amino acids L-asparaglne and Lphenylalanine, and the organic acids lactate, butyrate, and succinate. A. crystallopoietes is the only Arthrobacter species which exhibits nutritional control of this type. Thus, both spherical and rod-shaped cells grown under balanced growth conditions (exponential phase of growth) can be obtained and used to study certain physiological or chemical differences that may be related to cell shape. However, one potential disadvantage inherent in nutritional control is that the cells, although different in cell shape, were grown on different carbon sources. To eliminate this difficulty in a study of physiological events related to morphogenetic shape changes, it would be helpful to obtain cells of different shape but in the same physiological state when grown in identical media. This condition was obtained by isolating morphogenetically altered mutants which exhibited a different shape than the wild type during growth in media containing identical carbon sources. These mutants are the first reported morphogenetic mutants of A. crystallopoietes or any other Arthrobacter species. In this paper, we present the method of isolation and the characterization of these mutants.
MATERIALS AND METHODS Bacteria and bacteriophage. A. crystallopoietes (ATCC 15481), A. globifornis (ATCC 8010), A. pyridinolis, and A. viridescens were obtained from laboratory culture stocks. Bacteriophage AC-1, AG-1, and AV-3 were isolated from local soil using, as the host species, A. crystallopoietes, A. globiformis, and A. viridescens, respectively. Growth media and culture conditions. Cultures 595
596
ACHBERGER AND KOLENBRANDER
were grown in either a complex broth medium (Trypticase soy broth [BBL]) or a defined mineral salts phosphate (MSP) medium consisting of (grams per liter of 0.05 M potassium phosphate buffer, pH 7.2): (NH4)2SO4, 1.0; MgSO4 7H20, 0.1; carbon source, 5.0; and a trace salts solution, 10 ml, containing 2 g of CaCl2 2H20, 1.0 g of MnSO4 H20, and 0.5 g of FeSO4 7H20 dissolved in 1 liter of 0.1 N HCl. The carbon source, MSP medium, and trace salts were sterilized separately. Cultures were grown at 30°C with shaking aeration in Erlenmeyer flasks fitted with a tubular side arm. The culture volume never exceeded 10% of the flask capacity. Cell growth was monitored with a Klett-Summerson colorimeter with a red filter (660 nm). Mutant isolation. Spherical cells of A. crystallopoietes harvested during exponential growth in glucose-MSP medium were mutagenized with ethyl methane sulfonate (15). Cells from 10 ml of the culture were suspended in 10 ml of MSP buffer (MSP with no carbon source) containing 0.15 ml of ethyl methane sulfonate. The cells were incubated for 90 min with shaking at 30°C, washed in MSP buffer to remove the ethyl methane sulfonate, and inoculated into fresh glucose-MSP medium. After allowing two doublings of cell mass, the culture was diluted with 2 volumes of succinate-MSP medium to induce the formation of rods in cells capable of morphogenesis. Cell growth was permitted until the turbidity increased from 10 to 70 Klett units. Cells were washed free of the growth medium and suspended in 0.1 volume of MSP buffer. A sample (0.5 ml) of the cell suspension was layered onto either a sterile 15 to 40% linear sucrose gradient or one made by repeatedly (three times) freezing and thawing 28% (wt/vol) sucrose (2). The sucrose gradients were centrifuged at 2,500 x g for 5 min to separate spheres from rods. Above a broad layer of cells near the bottom of the gradient that contained rods there was a narrow, welldefined band that contained spheres. The spherical cells in this top layer were collected, washed free of sucrose, and plated onto succinate-MSP agar or succinate-MSP agar containing 0.8% (vol/vol) nutrient broth. After incubation at 30°C for several days, the resulting colonies were transferred to complex broth medium by using thin, sterile slivers cut from tygon tubing and incubated until the cultures reached midexponential growth. By using phase-contrast microscopy, the cultures were examined for cells with abnormal morphology. Those cultures containing cell populations with abnormal morphology were tested for purity by repeated single-colony transfers onto complex media solidified with agar. Sensitivity to bacteriophage. The wild type and each of the morphogenetically altered mutants, as well as A. viridescens and A. globiformis, were tested for sensitivity to three different bacteriophage, using a routine spot test assay and the soft-agar overlay technique (1). A 0.01-ml sample of each of a series of 10fold dilutions, 100 to 10-9, of a given bacteriophage stock suspension, 1012 plaque-forming units/ml, was spotted onto a 0.75% (wt/vol) agar overlay containing complex medium and one of the bacterial indicators. Sensitivity to bacteriophage was determined by the presence of cell lysis in the spotted zones.
J. BACTERIOL.
Cesium chloride buoyant density gradient centrifugation. Each of the mutants and A. pyridinolis were labeled by addition of 10 jiCi of carrier-free H:,''P04 per ml to cells growing in a low-phosphate medium adopted from Singer and Smith (13). The low-phosphate medium was prepared in two parts. Part A was filter sterilized as a 1X-concentrated solution and contained, per liter of distilled water: Ntris(hydroxymethyl)-methyl-2-aminoethane sulfonic acid, 274 g; NH4Cl, 10.7 g; peptone (Difco), 4 g; Na2SO4, 0.71 g; KH2PO4, 272 mg; and KCl, 14.9 mg; adjusted to a final pH of 7.5. Part B (40x concentrated) contained, per liter of distilled water: MgCl2 6H20, 16.0 g; CaCl2, 0.44 g; and FeCl;, 6H20, 32 mg. Each of the three components of part B was autoclaved separately, and then aseptically combined, to make the 40x-concentrated solution. The two parts were then combined in the proper proportions. Filter-sterilized D-fructose was added to give a final concentration of 0.5Y (wt/vol), and the final culture volume was adjusted with sterile distilled water. ;32'P-labeled cells from 10 ml of culture were washed with MSP buffer and lysed by using a slightly modified lysozyme-ethylenediaminetetraacetic acid-sodium dodecyl sulfate procedure (4). After 20 min of incubation at 37°C, the cell lysate was centrifuged at 10,000 x g for 10 min, and the resulting supernatant fluid (cell extract) was dialyzed overnight against three changes (2-liter volumes) of TEN buffer composed of 0.05 M tris(hydroxymethyl)aminomethane, 0.005 M ethylenediaminetetraacetic acid, and 0.05 M NaCl, adjusted to a final pH of 8.0. The volume of the extract after dialysis was adjusted to 4.93 ml with TEN buffer in a cellulose nitrate ultracentrifuge tube. The following was then added: 0.33 ml of 0.7 M phosphate buffer, pH 7.3; 7.1 g of CsCl; and 0.1 ml (5.0 x 105 cpm) of 'H-labeled A. crystallopoietes chromosomal DNA. The gradients were centrifuged at 42,000 rpm for 40 to 48 h at 15°C in a type 65 fixed-angle rotor. After centrifugation, the fractions were collected and processed as described in detail previously (8). These procedures included hydrolysis of RNA with KOH, precipitation of DNA with trichloroacetic acid, collection of precipitate on glassfiber filters, drying of filters, and determination of radioactivity by liquid scintillation counting.
RESULTS Morphogenetically altered mutants. The mutagenesis treatment of spherical cells of A. crystallopoietes with ethyl methane sulfonate resulted in about 90% killing as determined by viable cell counts obtained by plating samples of the spherical cell population before and after mutagenesis. The initial purpose of the mutant isolation procedure described in Materials and Methods was to enrich for and isolate morphogenetically altered (designated Mph) mutants which were unable to change from spherical shape to rod shape in routinely used rod-inducing media. For example, in complex broth medium, the wild type grows as long pleomorphic rods during exponential phase. Thus, mutants
VOL. 135, 1978
MORPHOGENETIC MUTANTS OF A. CRYSTALLOPOIETES
unable to form rods in this medium would be easily recognized by phase-contrast microscopy. Accordingly, samples of 2,400 colonies from succinate-MSP agar plates were inoculated into culture tubes containing complex broth medium, and after reaching exponential growth, the morphology of the growing cells was examined microscopically. In addition to the desired spherical mutant, other morphological types were observed. The mutants isolated can be grouped into three morphological classes: (i) those unable to form rods, (ii) those unable to form long rods, and (iii) those that form long pleomorphic rods but exhibit more extensive rudimentary branching than the wild type. The morphology of some of the mutants representative of each class is presented in Fig. 1 and 2. In Fig. 1, cell populations of the mutants and the wild type at four stages (inoculum spheres and midexponential, early stationary, and late stationary phases) during growth in complex broth are shown. Photographs of mutant and wild-type cells observed in midexponential growth phase in glucose- and succinate-MSP media are depicted in Fig. 2. A complete description of the mutants and comparison with the wild type are given below. The following mutants, representative of the three classes, are described: Mph-3 and Mph-4 (class 1), Mph-5 and Mph-6 (class 2), and Mph-8 (class 3). The two mutants, Mph-3 and Mph-4, that are unable to undergo sphere-to-rod-to-sphere morphogenesis grow exclusively as spheres on all media tested. The morphology of these two mutants has been examined by using cells grown in the rod-inducing carbon sources lactate, butyrate, succinate, malate, fructose, asparagine, and phenylalanine. The cell shapes of these two mutants (Fig. 1, rows 1 and 2) compared to the wild type (Fig. 1, row 6) at various times of growth in complex broth medium are depicted. The wild type exhibits a normal morphogenetic cycle including elongation of inoculum spheres to typical long pleomorphic rods arranged in characteristic V-formations during the exponential phase of growth (Fig. 1, row 6 column B), followed by shorter rods in early stationary phase (Fig. 1, row 6, column C), and, finally, spherical cells in late stationary phase (Fig. 1, row 6, column D). In comparison, Mph-3 and Mph-4 remain spherical at all stages of growth (Fig. 1, rows 1 and 2). Although both strains grow as spheres, they are morphologically distinguishable. Mph-3 grows as well-isolated spheres, whereas Mph-4 grows as clumps of spheres with the presence of large swollen cells with diameters ranging from 2.0 to 2.8 t,m. These two mutants generally have cell diameters of 1.4 to 1.7
597
,um depending on the growth medium and doubling time. The incubation temperature (20 to 37°C) has no effect on the inability of these two mutants to form rods. The morphology of Mph3 growing on glucose minimal medium is identical to that of the wild type growing as spheres on the medium (Fig. 2, rows 1 and 5, column B). However, in minimal medium containing succinate, a rod-inducing carbon source for the wild type, the morphological difference between the mutant strain and the wild type is clearly apparent (Fig. 2, rows 1 and 5, column A). In addition to the spherical mutants, several other mutants were isolated that were capable of morphogenesis but formed only short rods when grown in a rod-inducing medium. The most carefully characterized of these mutants is designated Mph-5. During exponential growth in complex broth medium, it forms rods of less than one-half the length of the wild type (Fig. 1, rows 3 and 6, column B). However, the sphereto-rod-to-sphere morphogenetic cycle is evident (Fig. 1, row 3, columns A, B, C, and D). When growing in a chemically defined medium containing a rod-inducing carbon source, the shape of the rods of Mph-5 is similar to that of the wild type, but cell measurements of these rods show that both length and width are shorter than those of the wild type by 10 to 20% (Fig. 2, rows 3 and 5, column A). As was found for class 1 mutants, cells of Mph-5 grown in glucose-MSP medium are indistinguishable from wild-type cells grown in the same medium (Fig. 2, rows 3 and 5, column B). The mutant Mph-6 was also originally isolated because of its inability to form long rods in complex media (Fig. 1, row 4). It was later identified as an auxotroph. Addition of low levels of L-methionine relieved the auxotrophic requirement in defined minimal media, but addition of L-methionine to complex medium did not restore the long-rod morphology of Mph-6. The morphologies of other amino acid auxotrophs isolated by different selective techniques have been examined on complex medium, and some, but not all, were unable to form long rods. Prototrophic revertants of Mph-6 regained long-rod morphology. Another class 2 mutant, Mph-7, exhibits extremely slow growth properties in all media tested. Although its doubling time is about three times as long as that of the wild type in glucose, succinate, fructose, and complex media, its shape is similar to that of the wild type in each medium except complex medium, where it is a short rod. It is interesting to note that this mutant is a short rod at doubling times much slower than Mph-3 or wild type in complex media, where the latter two are spherical and long pleomorphic
F .S t
w --
-
-
i-
--::
0.
i.
. >5W,
|
.
|B
~~
0
*C
