CO CD 0) o o
co
COsz
o o o> o> COco
d) CO d a u g h te r c e ll fo rm a ti o ncellular expression consists of multicellular arrays of cells (Fig. 1.7) linked by prosthecae from the tips of which ovoid, peritrichously
flagellate swarmer cells are produced. Some strains of R m . vannielii are characterised by a "simplified" cell-cycle (fig. 1.7) in which the constitutive production of swarmer cells occurs in the absence of multicellular arrays. This can be "induced" in "complex" cultures by prolonged sub-culturing under conditions of high CO^ concentration and low light intensities (France, 1978; Dow & France, 1980), but once formed the condition is often stably maintained. The genetic and physiological bases for the simplified cycle are at present obscure. It is however, possible that the function of such a cell-cycle could be to ensure the dispersal of large numbers of swarmer cells upon the onset of potentially unfavourable conditions, as might occur in the centre of a dense multicellular array or microcolony, for example.
Under severe nutrient limitation, on poor carbon sources, or when strains are first isolated another cell-type can be formed; the exospore (Fig. 1.7). Exospores are angular, rather heat resistant structures produced from the prostheca tips and would appear to ensure the long term survival of the organism. They have a complex fine structure with a membrane system but much reduced photopigment content (Whlttenbury & Dow, 1977). Germination upon return of favourable conditions can occur in several ways, often sequentially from the apices by prostheca
outgrowth.
Rhodomlcrobium vannielii is therefore unique amongst photosynthetic bacteria in producing at least three types of differentiated cell (chain cell, swarmer cell and exospore) in two vegetative cell cycles (complex
growth - swarmer cell and exospore formation, or plug formation within the prosthecae - the physiological separation of cells within the
arrays). The morphological differences between the multicellular arrays and the swarmer cells has been exploited to enable the production of synchronized swarmer populations by a simple filtration procedure through glass wool (Dow, 1974).
It is the swarmer cell type that has received greatest attention with regard to its production, cell-cycle and the sequence of molecular events during differentiation which lead to the production of a mature reproductive unit.
1.5.3 The swarmer cell concept
Comparative biochemical and physiological studies of the cell cycles of Csulobacter. Hvphomlcroblum and Rhodomlcroblum have indicated that the daughter cells produced at division are not simply "immature" versions of the mother cell with a longer cell cycle (Dow & Whittenbury, 1980). They represent a specialized cell-type - the swarmer cell - the prime function of which is dispersal. Swarmer cells are usually highly motile, show low levels of endogenous mRNA and protein synthesis (Shapiro, 1976; Potts & Dow, 1979; Dow a l - , 1983; Porter, 1985) no DNA synthesis (Newton, 1972; Potts & Dow, 1979; Wall al. , 1980) and their nucleoid is in a highly condensed format with a high sedimentation value (Evinger & Agabian, 1979; Dow a£ al-> 1985). At least in R m . vannielii (Dow al-, 1983, 1985) rRNA synthesis is also suppressed in the swarmer cell. These features indicate a non-growing, metabollcally
presumably, tactic responses. From an ecological viewpoint it is the ability of the swarmer cell to remain in this condition until the environment becomes favourable for growth and reproduction - i.e. to initiate differentiation - that is its distinguishing characteristic (Dow & Whittenbury, 1980). Implicit in this view is a capacity for environmental sensing so that responses are possible over a relatively short time period. The life of a swarmer cell is thus transient and is not comparable to that of a spore, for example.
The control of swarmer cell formation and differentiation has been studied with respect to one environmental factor - light - in R m .
vannlelii. During growth in batch culture, the numbers of swarmer cells increase due to the light-limitation caused by self-shading which occurs towards the late-exponential phase of growth (Dow e£ a X . , 1983; Porter, 1985). Light-limitation inhibits swarmer cell differentiation by increasing the length of the variable I phase of the cell cycle (Porter, 1985). When kept under anaerobic dark conditions, swarmer cell
populations are prevented from initiating differentiation and remain motile and viable for several hours and several days respectively (Porter, 1985). However, when re-illuminated they synchronously (>90%; Whittenbury & Dow, 1977) initiate differentiation (Fig. 1.7). The major
landmark events are the loss of flagella (filament and hook) and thus motility, the synthesis of the prostheca at a polar location on the cell-surface and the production of a daughter cell from the distal end of the prostheca (Fig. 1.7). Further growth results in the formation of a new multicellular array but the process beyond the two-cell stage or "pair" is asynchronous (Whittenbury & Dow, 1977; Potts & Dow, 1979).
The generality of the swarroer cell concept was discussed by Dow & Whittenbury (1980). There are a number of species which appear to have a motile cell-stage during their cell-cycles and which multiply by polar growth, for example the genus Rhodopseudomonas as presently constituted
(Imhoff a l - . 1984); Nltrobacter and some other ammonia or nitrite oxidising bacteria; Bdellovibrio. which produces a free-living motile cell prior to lntraperlplasmic growth in other bacteria (Thomashow & Rittenburg, 1979); Sphaerotllus and Leptothrlx (Dow & Whittenbury, 1980), two genera of sheathed bacteria which produce motile dispersal phases and a number of genera of the non-photosynthetic prosethcate and budding bacteria (Morgan & Dow, 1985; Schmidt & Starr, 1985). In these microbes, the swarmer cell is clearly a dispersal vehicle. However, in view of the relationship between polar growth and differential cell type expression, discussed above, one may consider a very large group of bacteria to have cell-stages which possess at least some of the distinguishing features of swarmer cells if not overt motility. The general name "shut-down” or "growth precursor" cell has been proposed (Dow ££ a l .. 1983) for such stages. An important property is the phase of the cell-cycle that these cells maintain themselves in; the I phase. In the L. coll cell cycle this has been defined as the time from cell division to the initiation of chromosome replication and is when initiation components are synthesised and complexes assembled to allow replication to proceed at all chromosome origins within the cell
(Helmstetter a£ al. . 1979). In fact L. coll Itself may exhibit polar growth (Begg & Donachie, 1977) and a prolonged I phase at low dilution (growth) rates in chemostat culture (Skarstad at al- • 1983). Under these conditions it has been proposed (Dow at al- • 1983) that progeny cells may exhibit some of the properties of the more "classical” swarmer cells, thus indicating heterogeneity within such cultures.
Interestingly, Falkinham & Hoffman (1984) have recently drawn attention to the unique developmental characteristics of the "swarm" and "short" cells of Proteus Vt/tpar i s and Pj_ mi rabi 1 i s which differ from each other in outer membrane protein composition and enzyme complement. In their view, "swarming" in these species should be regarded as an example of differentiation.
Clearly, more evidence is needed in order to judge the validity of a generalized swarmer cell concept.
From the foregoing literature survey, the following points emerge which are of relevance to the aims of the present work.
(i) Most of the concepts of photosynthetic and respiratory energy conversion in the Rhodospirillaceae have been derived from work with two species of Rhodobacter which are assumed to be
representative of the family.
(ii) The notion that cultures of polarly growing photosynthetic bacteria may be heterogeneous with regard to cell-type has not been widely recognized.
(iii) Far more is known about the biogenesis of vesicular ICM systems than those of a lamellate nature.
(iv) Cell-cycle studies have only been performed with R b . sphaeroldes. largely using induction synchronization methods.
The present study was therefore aimed to redress the balance in favour of the "morphologically exciting" members of Rhodospirlllaceae.
2.1 Organisms and Media
Table 2.1 lists the members of the Rhodospirillaceae used in this study. Bacteria were grown routinely in pyruvate-malate mineral salts (PM) medium (Whittenbury & Dow, 1977) which contained;
gl ^ distilled water
n h4c i 0.5
MgS04.7H20 0 .4
NaCl 0.4
CaCl2.2H2 0 0.05
sodium hydrogen malate 1.5
sodium pyruvate 1.5
For all species except R m . vannielll. which has no growth factor requirements, the medium was supplemented with 0.1* (w/v) yeast extract. The pH of the medium was adjusted to 6.8-6.9 with potassium hydroxide
(KOH) pellets prior to autoclaving at 121°C for 15 min. After cooling, sterile phosphate buffer was added aseptically to a final concentration of 5 m M .
0.1 M phosphate buffer stock solution contained; sodium dlhydrogen phosphate NaHjPO^.2 ^ 0 , 7.8 g l a n d dlsodlum hydrogen phosphate Na2HP04 , 7.1 g l* 1 pH 6.8.
For solid media, Dlfco Bacto-agar was added (1.5*w/v) before autoclaving and plates or agar deeps poured after cooling to 60°C.
T A B L E 2.1. Sources of strains used in this study
Spec ies Strain number Reference
Ks. rubrum NCIB 8255 Kc. tenuis ATCC 25093 Kc. gelatinosus NCIB 8290 Kb. sphaeroides NCIB 8253 Kb. sphaeroides
"cordata"/81-1 ATCC 33575 Gest et al. Kp. acidophila ATCC 25092
Kp. palustris NCIB 8288 Kp. viridis ATCC 19567
Km. vannielii Km5 Dow (1974),
2.2 Maintenance of Cultures
Bacteria were maintained as stab cultures in PM agar deeps contained in universals. After inoculation from exponential phase liquid cultures, they were incubated at 30°C for 3 d under an incident light intensity of
-2 -1
35 ii E m s then kept at room temperature. Culture purity was checked by phase contrast microscopy and streaking to obtain isolated colonies on PM agar plates. Plates were incubated under anaerobic conditions in the light using the anaerobic bag technique described by Westmacott & Primrose (1975). Plates were placed on a tray within transparent nylon bags (Portex Ltd., Hythe, Kent) which were heat sealed and flushed with C>2 free Ng for 15 minutes. A beaker containing saturated pyrogallol solution (15 ml) and 15% (w/v) potassium carbonate / 10% (w/v) sodium hydroxide solution (15 ml) was also Included within the bag to remove traces of oxygen. Incubation was at 30°C at a light intensity of 35 n E
2.3 Photoheterotrophic Growth Conditions
Small scale cultures were grown in 100 ml PM contained in 250 ml B19 Qulckflt conical flasks. After inoculation (1% v/v) the flasks were capped with rubber Suba Seals (William Freeman & C o . , Barnsley, W. Yorkshire) and flushed with 0 ^ free Nj for 15 min through inserted syringe needles. Flasks were Incubated in a shaking water bath at 30 C
-2 -1
under a light intensity of 25 p E m s
Larger scale photosynthetic cultures were grown in 5, 10 or 20 1 flat- bottomed vessels (244/1350, Baird and Tatlock)with Quickflt tops which
could be sealed with Suba-Seals. After Inoculation (0.2-0.4% v/v) the vessels were flushed with 0 ^ free Nj for 30 rain and incubated at an
incident light intensity of 3 5 u E m ^s ^ in a warm room at 3 0 ° C . All cultures were stirred continuously by magnetic stirrers. Growth was followed by the optical density of cultures at 540 or 650 nra in a Pye- Unicam SP500 spectrophotometer.
For growth experiments at different light intensities, PM contained in flat pyrex Roux tissue culture bottles (1 1) was inoculated (0.2% v/v) and incubated with stirring at 30°C in a warm room. Incident light
-2 -1
intensities of 85, 35, 17 and 6.5 u, E m s were obtained by varying the distance between the tungsten light source ( 1 0 0 W bulbs) and the culture vessel. At the highest light intensity, a heat-filter consisting of a water-filled Roux bottle was placed in front of the lamp. Water-filled culture vessels were initially set up to check that the temperature within them remained at 30°C.