John Innes Centre, Norwich Research Park, Colney, Norwich NR4 7UH, UK1
Author for correspondence: Keith F. Chater. Tel: +44 1603 452571. Fax: +44 1603 456844. e-mail: chater{at}bbsrc.ac.uk
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ABSTRACT |
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Keywords: cell division, whi genes, aerial mycelium, checkpoints
Abbreviations: 7-AAD, 7-aminoactinomycin D; DAPI, 4',6-diamidino-2-phenylindole; Fluo-WGA, fluorescein-conjugated wheat germ agglutinin
a Present address: Dept of Cell and Molecular Biology, Uppsala University, Biomedical Center, Box 596, S-75124 Uppsala, Sweden.
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INTRODUCTION |
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The situation for streptomycetes is further complicated by their reproductive strategy, with which this paper is concerned. From the older parts of the substrate mycelium, new branches grow into the air to form an aerial mycelium, which eventually generates long chains of unigenomic spores. In the extensively studied organism Streptomyces coelicolor A3(2), some aspects of aerial hyphal wall structure change after an initial growth period, because a typical aerial hypha has a straight stalk surmounted by a somewhat coiled apical region (Wildermuth, 1970 ; Wildermuth & Hopwood, 1970
). The onset of sporulation in such aerial hyphae requires a switch to a different mode of septation and chromosome partitioning from that in vegetative hyphae. The key cell division protein FtsZ assembles into a large number of rings (sometimes as many as 50), forming a regular ladder (Schwedock et al., 1997
). The rings are the precursors of sporulation septa, which delimit prespore compartments into which single nucleoids are partitioned. Sporulation septa differ morphologically from vegetative septa, having a structure that leads to full separation of adjacent cells and not simply to hyphal crosswalls (Wildermuth & Hopwood, 1970
). Maturation of the spores involves induction of late sporulation genes, rounding up and thickening of the cell wall and synthesis of a grey polyketide spore pigment (for reviews, see Chater, 1989
, 1993
, 1998
; Chater & Losick, 1997
).
Several so-called whi (white) mutants have been isolated which form sporulation-defective aerial hyphae lacking the dark grey spore pigment (Hopwood et al., 1970 ; Chater, 1972
). Previous work indicated that mutations in whiG, whiA, whiB or whiH block development at early stages and prevent sporulation septation (Chater, 1972
; Hopwood et al., 1970
; McVittie, 1974
), and more recently Schwedock et al. (1997
) used immunofluorescence microscopy to show that whiG, whiB and whiH mutants fail to assemble ladders of FtsZ rings in aerial hyphae. Of these early whi genes, whiG encodes an alternative sigma factor (Chater et al., 1989
); whiA encodes a protein of unknown function with orthologues in several other Gram-positive bacteria (N. J. Ryding, J. A. Aínsa, N. Hartley, C. J. Bruton & K. F. Chater, unpublished); whiB belongs to a group of genes unique to actinomycetes, which code for small highly charged and cysteine-rich proteins of unknown function (Davis & Chater, 1992
; J. Soliveri, J. Gomez, W. R. Bishai & K. F. Chater, unpublished); and whiH encodes a member of the GntR family of transcription factors (Ryding et al., 1998
). It would be of considerable interest to understand how these genes, directly or indirectly, interact with the cell division machinery to control sporulation septation.
Despite an early suggestion to the contrary (Chater, 1972 ), it has become increasingly clear that the phenotypes of early whi mutants do not directly correspond to intermediate stages of normal development of wild-type spore chains. Rather, it seems that certain growth and/or morphological processes continue in the mutants after the point at which normal development is blocked, leading to mutation-specific terminal phenotypes. With the exception of the work by Schwedock et al. (1997
), previous descriptions of these phenotypes have utilized only phase-contrast microscopy or transmission electron microscopy and have mostly dealt with uncharacterized presumptive point mutations which may retain residual activity to various degrees (Chater, 1972
, 1975
; Hopwood et al., 1970
; McVittie, 1974
; Plaskitt & Chater, 1995
; Ryding et al., 1998
). The availability of defined gene disruptions and the recent development of a technique for transformation with denatured chromosomal DNA (Oh & Chater, 1997
) have made it straightforward to construct a series of defined null whiG, whiA, whiB and whiH mutants in an isogenic background. We have analysed hyphal morphology, septation patterns and distribution of nuclear material in fully developed aerial mycelium of these mutants using scanning electron and fluorescence microscopy. Based on these analyses, the whiG, whiA and whiB genes can be associated with blocks at specific early stages of morphological development, which may represent developmental decision points in the sporulation process. Furthermore, it is shown that whiH affects a different stage of sporulation than previously thought, and is not absolutely required for some of the later sporulation processes.
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METHODS |
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Microscopical methods.
For scanning electron microscopy, colonies were mounted on the surface of an aluminium stub with O. C. T. compound (Agar Scientific) as the mounting medium. The stub was then plunged into liquid nitrogen slush at approximately -210 °C to cryopreserve the material. The sample was then transferred onto the cryostage of a CT1500HF cryo-transfer system (Oxford Instruments) attached to a Philips XL30 FEG scanning electron microscope (Philips Electron Optics). Sublimation of surface frost was performed at -95 °C for 3 min before sputter-coating the sample with platinum for 2 min at 10 mA, at colder than -110 °C. After sputter-coating, the sample was moved onto the cryo-stage in the main chamber of the microscope, held at approximately -140 °C. The sample was viewed at 3 kV and photographs were taken using Ilford FP4 120 roll film.
Cultures for light and fluorescence microscopy were set up by inserting a sterile coverslip at a 45 ° angle into MM agar and inoculating in the acute angle along the glass surface (Chater, 1972 ). Coverslips were removed after 24 d incubation at 30 °C and cells on the coverslip surface were either stained with 4',6-diamidino-2-phenylindole (DAPI) for nucleoids (Kwak & Kendrick, 1996
), or stained for the cell wall, with fluorescein-conjugated wheat germ agglutinin (Fluo-WGA) after fixation, treatment with lysozyme and blocking with BSA (Schwedock et al., 1997
). To stain DNA in the Fluo-WGA-treated material, 7-aminoactinomycin D (7-AAD) was included at a final concentration of 10 µg ml-1 (both fluorescent reagents were obtained from Molecular Probes). The samples were then washed five times with PBS (Sambrook et al., 1989
), and mounted for microscopy in PBS containing 50% glycerol. Samples were studied and photographed using a Zeiss Axiophot microscope equipped for phase-contrast and epifluorescence microscopy. No filters were used for observation of DAPI staining, Zeiss filter set 09 with an added filter cutting off at 560 nm was used for Fluo-WGA, and filter set 15 was used for 7-AAD. Photographs were taken with Kodak Technical pan film; exposure was at 25 ASA with normal development for phase-contrast illumination, and at 100 ASA with development in HC-110, dilution D, 6 min, for fluorescence illumination. Negative images were scanned, processed using Adobe Photoshop software and assigned monochromatic red, green or blue colours to resemble the samples as observed in the microscope.
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RESULTS |
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A whiH null mutant produces perceptible amounts of the whiE-encoded grey spore pigment
The production of aerial hyphal fragments with spore-like characteristics in the whiH::hyg strain J2210 indicated that whiH may not be required for all late sporulation processes. This was further supported by the previous observations that whiH mutants develop a slightly grey aerial mycelium while whiG, whiA and whiB colonies remain pure white (Ryding et al., 1998 ) and by low, but detectable, transcription of the major operon of whiE spore pigment genes and of sigF, encoding a late sporulation
factor, in a whiH point mutant (Kelemen et al., 1996
, 1998
). However, the possibility could not be excluded that previously studied whiH mutants retained residual whiH activity because they contained either missense mutations, a frameshift in the C-terminal half, or uncharacterized mutations (Chater, 1972
; Ryding et al., 1998
). Also, the whiH::hyg insertion mutation, which gives rise to a very low level of grey pigmentation, allows expression of the N-terminal half of WhiH, including a putative DNA-binding motif (Ryding et al., 1998
). A knockout mutation of whiH was therefore constructed by deleting the promoter and the first half of the gene and replacing it with the ermE gene. When introduced into M145 to produce strain J2408, this
whiH::ermE allele gave aerial hyphae which were morphologically and cytologically very similar to those of other whiH mutants (see Fig. 4cd
and below). The aerial mycelium of J2408 colonies accumulated readily perceptible grey pigment, to a level intermediate between J2210 (whiH::hyg) and M145 (Fig. 6
). The extent of grey pigmentation was medium-dependent (e.g. J2408 appears white on soya flour mannitol agar), which may explain why whiH mutants had appeared no less white than any other white colony mutants in the original screen (Hopwood et al., 1970
; see also Ryding et al., 1998
).
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Condensation and aberrant partitioning of nucleoids in aerial hyphae of whiH mutants
Spore formation involves accurate segregation of one nucleoid to each prespore compartment (although occasional abnormal events occur, as is evident from Fig. 3ac). We used DAPI staining of methanol-fixed aerial hyphae to examine how nucleoid partitioning was affected by the whi mutations. As reported for several Streptomyces species (Kwak & Kendrick, 1996
; Miguélez et al., 1998
; Schwedock et al., 1997
), separation of nucleoids is not seen in the wild-type strains until septa have started forming. Thereafter, nucleoids stain more intensely and become smaller (Fig. 7ab
). No such condensation was seen in J2400 (whiG), J2401 (whiA) and J2402 (whiB), nuclear material remaining more or less continuous throughout the straight or coiled aerial hyphae (Fig. 7ce
). However, away from apical parts, in particular in J2400, and in older parts of hyphae, the staining was more patchy and unevenly distributed (data not shown). Results obtained with 7-AAD staining of paraformaldehyde/glutaraldehyde-fixed mutant aerial hyphae agreed with the results from DAPI staining (data not shown). In contrast, the whiH mutant J2210 displayed a distinctly different pattern of nucleoid staining. In the phase-dark and often loosely coiled fragments that formed in many aerial branches, nucleoids were partitioned into bodies of variable sizes and distributed unevenly along the length of the fragments, leaving large DNA-free spaces between them (Fig. 7f
). There were no nucleoids close to the poles of the phase-dark fragments, and the total length of DNA-containing sections was about equal to the total length of DNA-free sections. Otherwise, no obvious pattern of distribution could be discerned. The nucleoids fluoresced intensely and appeared to be condensed, contrasting sharply with the non-condensed DNA in the hyphal stems leading up to the phase-dark fragments. Often, the stems appeared to be lysed in ageing material, while the phase-dark fragments persisted. Further analysis revealed this pattern of DNA staining in all of 13 independent whiH mutants obtained after UV or NTG (nitrosoguanidine) mutagenesis (listed in Methods section; representative examples shown in Fig. 7 gj
).
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whiG, whiA and whiB are epistatic to whiH in morphological studies of double mutants
Since whiH was not absolutely required for parts of the wild-type sporulation processes, while whiG, whiA and whiB mutants were completely blocked at early stages of sporulation, whiG, whiA and whiB mutations would be expected to be epistatic to whiH mutations. However, early phase-contrast microscopic studies of double mutants had indicated that the epistasis pattern was G>H>A,B (Chater, 1975 ). Those results were based on highly non-isogenic strains arising from genetic crosses. Exploiting our ability to construct strains in an isogenic background, and using the easily scorable DAPI staining pattern, the epistasis was re-examined. The whiG::hyg, whiA::hyg and whiB::hyg mutations were transferred to strain C119, which contains a point mutation (whiH119) causing an amino acid substitution near the supposed WhiH DNA-binding domain, and has a phenotype like that of the whiH null mutant (Ryding et al., 1998
). Derivatives of the double mutants containing functional alleles of the relevant whi genes on low-copy-number plasmids were also studied as controls expected to show the relevant single mutant phenotypes. In agreement with previous epistasis data, whiG was epistatic to whiH: the aerial hyphae of strain J2404 (whiG whiH) were indistinguishable in morphology and DNA staining pattern from those of J2400 (whiG) (Fig. 8a
). The phenotype of the double mutant was not affected by introduction of whiH+ on pIJ6201 (not shown), but the complementation of whiG by introducing pIJ6301 gave a characteristic whiH phenotype (Fig. 8b
). J2405 (whiA whiH) was similar to the whiA single mutant, although the coils were slightly less tight (Fig. 8c
). The whiB whiH strain J2406 had a clearly reduced tightness of the coils in comparison to the whiB single mutants (Fig. 8e
). However, the coils of the whiA whiH and whiB whiH mutants were very long, appeared non-septated and had continuously dispersed nuclear material, in this respect closely resembling the whiA and whiB single mutants. Complementation of whiA::hyg in J2405 by pIJ6204, and of whiB::hyg in J2406 by pIJ2157, restored the whiH phenotypes completely (Fig. 8d
, f
), while introduction into either strain of the whiH+ plasmid pIJ6201 only increased the tightness of the coils (data not shown). In summary, whiG, whiA and whiB mutations were epistatic to whiH119 on the criteria that they completely abolished the formation of phase-dark hyphal fragments, the occasional sporulation-like septation, and the separation and condensation of nucleoids that occurred in the whiH119 single mutant. In addition, they abolished the pale grey pigmentation of C119 and gave pure white aerial mycelium (data not shown).
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DISCUSSION |
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The whiG gene product is an RNA polymerase factor,
WhiG (Chater et al., 1989
; Tan et al., 1998
), of a subclass whose homologues in other bacteria (e.g.
FliA in Salmonella typhimurium) are subject to regulation by anti-
factors responsive to morphological checkpoints (Hughes et al., 1993
). It has previously been suggested that the morphologically coupled release of
WhiG from an anti-
factor may be critical in initiating sporulation (Losick & Shapiro, 1993
; Kelemen et al., 1996
). As one result,
WhiG RNA polymerase would transcribe a gene or genes influencing cell wall structure. We suggest that the transition from straight to curled growth of a hypha occurs very soon after
WhiG RNA polymerase has become active, and provides a visible record of the time at which the checkpoint was successfully negotiated. Another whiG-dependent gene encodes a possible glycine-betaine-binding protein, perhaps contributing to turgor for continued growth of the apical compartment after it has been cut off from direct contact with the substrate mycelium (Tan et al., 1998
). Indeed, this septation event might itself provide the checkpoint, as suggested by Losick & Shapiro (1993
); a consequent transient limitation of nutrients or of turgor might be more direct sources of the
WhiG-activating signal. Further
WhiG targets include the sporulation regulatory gene whiH (Ryding et al., 1998
).
A putative dependence of sporulation septation on growth cessation may provide the basis of a whiA- and whiB-mediated developmental checkpoint
The whiG-dependent switch in cell wall structure apparently works in whiA and whiB mutants since these develop tightly coiled aerial hyphae. The epistasis of whiG to whiA and whiB is consistent with this conclusion (Chater, 1975 ). Mutants in whiA or whiB lack sporulation septation or nucleoid segregation and condensation, and develop abnormally long and coiled aerial hyphae. This identifies a putative second developmental decision point required for formation of sporulation septa and for appropriate limitation of aerial growth (Fig. 9
). On the other hand, aerial hyphae of whiH mutants are not abnormally long, so they appear to retain proper control of the decision to stop aerial growth.
The fact that whiH mutants make reduced numbers of sporulation septa, yet cease growth properly, indicates that a full commitment to sporulation septation is not a prerequisite for orderly growth cessation. This leads us to suggest tentatively that, instead, growth cessation is a prerequisite for sporulation septation, and that whiA and whiB affect elongation more directly than septation. Nevertheless, it remains possible that a whiA/whiB-dependent initiation of sporulation septation (even if just a few septa are formed as in whiH mutants) could lead to cessation of hyphal elongation.
Properly controlled aerial hyphal cell wall structure, as reflected in the extent of coiling of aerial hyphae, as well as other sporulation events such as sporulation septation, nucleoid condensation, spore pigment synthesis and transcription of sigF and whiE, all require whiA and whiB (Kelemen et al., 1996 , 1998
). Although these dependences may conceivably be fairly directly exerted at the level of transcription of the relevant genes, it is quite possible that some or all of the events are more directly affected by the morphological checkpoint in which whiA and whiB are implicated. The sequences of the whiA and whiB genes are known but have not yet provided any unambiguous clues about their molecular function (Davis & Chater, 1992
; N. J. Ryding, J. A. Aínsa, N. Hartley, C. J. Bruton & K. F. Chater, unpublished).
Some later sporulation processes are initiated in whiH mutants
Inactivation of whiH does not fully block all aspects of spore chain development. First, all whiH alleles examined to date, including the whiH::ermE null allele, allow synthesis of detectable amounts of a grey spore pigment. We show here that this pigmentation is directed by the whiE gene cluster which is responsible for the grey polyketide pigment of wild-type spores. Consistent with this, very low levels of transcription of the late sporulation loci sigF and whiE were detectable in a whiH point mutant (Kelemen et al., 1996
, 1998
), whereas no transcription of these loci was detected in whiG, whiA and whiB strains. Second, a few irregularly spaced apparent sporulation septa divide whiH mutant aerial hyphae into shorter fragments, and only a few widely spaced FtsZ rings were reported in whiH::hyg aerial hyphae by Schwedock et al. (1997
). Some of these rings may have corresponded to the occasional putative sporulation septa formed in whiH strains. Third, the fragments formed from whiH aerial hyphae become phase-dark and their cell wall becomes resistant to staining with Fluo-WGA in a manner reminiscent of developing spore chains. Fourth, nucleoids in these fragments condense and are partitioned (albeit aberrantly).
We interpret these observations and the epistasis of whiG, whiA and whiB to all these aspects of whiH to mean that the first two developmental decision points do not require whiH (Fig. 9). The whiH mutants initiate later sporulation processes, but execute them incorrectly or only to a limited extent. In the simplest model, whiH would act after whiG, whiA and whiB and be dependent on these genes. Indeed, a direct dependence of whiH transcription on
WhiG has been demonstrated (Ryding et al., 1998
). The situation appears, however, to be more complex than this, as whiH mutations clearly reduce the tight coiling that develops in whiA and whiB aerial mycelium, indicating that the wild-type whiH gene promotes this hypercoiling, and is therefore likely to be active during aerial hyphal growth, at least in whiA and whiB mutants. However, this situation in the mutants may not reflect a normal activity: whiH single mutants show a degree of coiling similar to that of developing wild-type aerial hyphae.
WhiH resembles a family of DNA-binding regulatory proteins responsive to carboxylate-containing intermediates of carbon metabolism (Ryding et al., 1998 ). It has therefore been suggested that WhiH may sense some such metabolic intermediate, whose concentration changes during aerial growth (particularly at growth cessation), giving rise to two forms of WhiH: the form whose proportion increases later can be designated WhiH*. It is possible that later events are regulated by or indirectly require WhiH*. These would include increased expression of genes required for sporulation septation and for orchestration of the partitioning of DNA after the final one or two rounds of replication. Possibly, the level of WhiH* may be important in transmitting information about whiA/whiB-dependent cessation of tip growth to genes whose up-regulation mediates subsequent events. On this view, whiH would provide a point of information exchange to coordinate the whiG-dependent regulatory cascade (of which whiH is a part) with the apparently whiG-independent cascade represented by whiB (Soliveri et al., 1992
) and, perhaps, whiA (J. A. Aínsa & K. F. Chater, unpublished).
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ACKNOWLEDGEMENTS |
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Received 9 March 1999;
accepted 11 May 1999.