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Growth polarity and cell division in Streptomyces
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Growth polarity and cell division in Streptomyces Klas Fla¨rdh Streptomycetes are mycelial bacteria that resemble filamentous fungi in their apical growth, branching, and morphogenetic development. One inroad into the largely unknown mechanisms underlying this prokaryotic growth polarity is provided by Streptomyces DivIVA, a protein localized at hyphal tips and involved in tip extension. Another aspect is a proposed migration of nucleoids. During sporulation, the modes of growth and cell division are reorganised. This involves dynamic assembly of FtsZ into a multitude of cytokinetic rings. Controlled by developmental regulators and intriguingly coordinated with chromosome segregation, this leads to spores with a single chromosome each. Genome sequences have shed new light on these aspects and reinforced the role of Streptomyces in bacterial cell biology. Addresses Department of Cell and Molecular Biology, Uppsala University, Biomedical Centre Box 596, SE-751 24 Uppsala, Sweden e-mail: [email protected]
Current Opinion in Microbiology 2003, 6:564–571 This review comes from a themed issue on Growth and development Edited by Claudio Scazzocchio and Jeff Errington 1369-5274/$ – see front matter ß 2003 Elsevier Ltd. All rights reserved. DOI 10.1016/j.mib.2003.10.011
Introduction
mentation of proteomics [7,8], DNA microarray technology [9,10], and novel techniques for streamlined genetic engineering [11], the study of these complex processes has certainly become more tractable and rewarding. In this review, I revisit Streptomyces hyphal growth, cell division and related aspects of developmental biology from a cell biological perspective, summarise recent progress and highlight emerging issues.
Apical growth As demonstrated using pulse-labelling or lectin binding for example, the Streptomyces cell wall is polymerised at the hyphal apex [12,13]. Fluorescently labelled vancomycin was recently introduced as a probe to visualise sites of nascent peptidoglycan insertion into the cell wall sacculus in Gram-positive bacteria [14], and this further corroborated the pronounced tip extension in Streptomyces [14,15] (Figure 1a). Hence, although some turnover may occur in the lateral walls, hyphae are in principle tubes of inert murein that grow only at one extreme end. When disconnected from the apex by septation, a subapical cell appears unable to grow until it has created a new tip by lateral branching (Figure 1c). This pattern is in sharp contrast to rod-shaped bacteria such as Escherichia coli and Bacillus subtilis, which grow by intercalation of new murein along the lateral wall, while their polar caps are stable once they have been polymerised during cell division (see for example [14,16]).
Streptomycetes differ conspicuously from most other bacteria in their growth and developmental biology, and are superficially more reminiscent of filamentous fungi. Thus, they grow by tip extension to form a mycelium of branched hyphae. The mechanisms of apical growth and the inherent poleward transport processes have been extensively studied in fungi [1,2], but we have few clues about the corresponding mechanisms in a mycelial prokaryote. Furthermore, the modes of growth and cell division are dramatically modified during development of Streptomyces colonies, when specialized sporebearing hyphae emerge through the air–water interface to give a fluffy layer of aerial mycelium. This involves pronounced developmental regulation of morphogenetic and cell cycle-related processes [3].
Most models for Streptomyces tip extension assume that new material is incorporated at the apex in a flexible form, which becomes more rigid further back from the tip, and that stretching of the tip by turgor pressure is a major driving force for growth [12,17,18,19]. Staining with vancomycin gave patterns consistent with these models inasmuch as incorporation was highest in the tip itself, rather than at the leading edge of the cylindrical wall [14,15]. However, the molecular bases of tip extension remain unclear. Obviously, proteins involved in assembly of the cell envelope (e.g. penicillin-binding proteins) must be recruited to the apical growth zone, presumably forming part of larger apical complexes. It is also possible that assemblies of cytoplasmic or membrane proteins, in analogy with eukaryotic cytoskeletons, may provide a scaffold for the fragile tip.
With the revolution in our understanding of the bacterial cell that has been provoked by modern fluorescence microscopy [4], new opportunities are emerging to investigate growth polarity and differentiation in Streptomyces. In conjunction with the publication of two complete Streptomyces genome sequences [5,6], and the imple-
The first component of an apical protein complex has recently been identified. When tagged with enhanced green fluorescent protein (EGFP), the S. coelicolor DivIVA protein (denoted DivIVASC) formed distinct fluorescent foci at the hyphal tips, with an intensity indicative of the presence of large numbers of molecules [15] (Figure 1b).
Current Opinion in Microbiology 2003, 6:564–571
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Figure 1
(a)
t
(b)
Sp t t Sp t
(c)
Branching Tip extension
Cell division
Nucleoid migration
Apical growth in Streptomyces. (a) Staining of the sites of nascent peptidoglycan incorporation using fluorescently labelled vancomycin in vegetative hyphae of S. coelicolor. The fluorescence image is shown in inverted grey-scale. Hyphal tips are indicated by a ‘t’, crosswalls by arrowheads, and the spore from which the hyphae grew out by ‘Sp’. (b) Subcellular localisation of DivIVASC-EGFP (green colour) overlaid on a phase-contrast image of nascent mycelium growing out of a spore (Sp). Images in panels (a) and (b) have been published previously [15,62], and are reproduced with permission from Blackwell Publishing Ltd. Bars, 5 mm. (c) Simplified illustration of polarised growth in Streptomyces hyphae. The apical cell is extending its cell wall only at the tip (green). Once this cell has divided by forming a new hyphal cross wall, the subapical daughter cell becomes unable to grow, and eventually switches its polarity to generate a lateral branch with a new extending tip. A consequence of tip growth is that DNA, which replicates along most of the hyphal length, has to move towards the tip and into new branches - a process we propose to designate nucleoid migration. For clarity, only a few schematic nucleoids are drawn (red), and they are not meant to reflect the actual number of chromosomes per cell. Furthermore, individual nucleoids are typically not observed in vivo as separated bodies in growing hyphae.
Like the homologous DivIVA in B. subtilis [20], this protein is predicted to contain extensive coiled-coil structure and may oligomerise. DivIVASC was essential, and partial depletion gave hyphal morphologies strikingly similar to those observed in many fungal mutants defective in tip extension or nuclear migration [15]. Overexpression had dramatic effects on shape determination, leading to conspicuous swollen and pear-shaped cells. It was particularly compelling that induction of divIVASC overexpression in preformed hyphae rapidly gave rise to multiple new sites of wall growth, from which branch-like lateral outgrowths emerged. Thus, DivIVASC not only targets tips and affects morphogenesis, but also appears instrumental in establishment of new tips [15]. In B. subtilis, DivIVA sequesters the cell division inhibitors MinC and MinD at the cell poles, thereby allowing www.current-opinion.com
division only at central positions [21,22]. The Bacillus protein has no impact on vegetative growth in minC or minD mutants [23,24], although it has an additional role in chromosome localisation during sporulation [25]. DivIVASC works differently and did not detectably affect division or nucleoid localisation in S. coelicolor [15]. Intriguingly, all sequenced genomes of streptomycetes and related bacteria (actinomycetes, including pathogenic mycobacteria) to date lack minC, but still encode a DivIVA, which appears to be essential in both Mycobacterium tuberculosis (wag31) [26] and Brevibacterium lactofermentum (JA Gil, personal communication). The MinC-independent essential functions could relate to growth polarity also in rod-shaped cells, and Corynebacterium glutamicum (very closely related to B. lactofermentum) has indeed been demonstrated to grow apically at both poles [14]. Current Opinion in Microbiology 2003, 6:564–571
566 Growth and development
Given the prominent role of the actin-like MreB and Mbl proteins in directing cell wall assembly in B. subtilis [14], it is noteworthy that the sequenced genomes of rodshaped actinomycetes such as Mycobacterium and Corynebacterium lack such proteins, whereas the hyphal Streptomyces species encode two MreB/Mbl-like proteins each [5,6]. Although C. glutamicum and S. coelicolor both grow polarly, the former extends at the cell poles created by division, whereas the latter generates each tip de novo by branching or germ tube emergence (Figure 1), and does not require cell division for hyphal growth and branching [27]. This important distinction might relate to the differential conservation of MreB/Mbl. Alternatively, these proteins could have roles in morphological differentiation in Streptomyces.
Nucleoid migration, segregation and replication In contrast to cell wall assembly, DNA replication takes place with similar rates along a large portion of the hyphae [12]. Thus, in analogy to nuclear migration in filamentous fungi [2], it has to be postulated that nucleoid migration occurs in Streptomyces, such that the chromosomes move in relation to the cell envelope to populate the extending tips and lateral branches (Figure 1c). Clarification of the driving forces and mechanisms for moving DNA along these prokaryotic cells would be of considerable interest.
[32,34]. The extreme ends of several Streptomyces chromosomes contain helicase-like genes, which might also contribute to terminal protein complexes [35]. Cytological evidence for co-localisation of the left and right telomeres has been presented [28], but it is not evident how chromosome linearity affects nucleoid segregation or migration. Strains with circularised chromosomes often show increased genetic instability, but generally display no or only small growth or sporulation disadvantage [31]. Although this suggests that internal regions like oriC, rather than the telomeres, are critical for partitioning during sporulation, further elucidation of these aspects are needed (see also Update).
Two modes of cell division in Streptomyces Streptomyces use at least two kinds of cell division: vegetative septation leading to crosswalls in the substrate mycelium and developmentally regulated sporulation septation. Both kinds involve the same basic division machinery, including FtsQ and FtsZ [27,36]. Additional Streptomyces homologues of known bacterial division proteins can be recognized in the genome sequences [5,6] (Table 1). The conservation of the tubulin homologue FtsZ, its polymerisation into the cytokinetic Z ring [13], and several membrane proteins apparently Table 1
In addition to poleward migration, newly replicated chromosomes segregate from each other [28]. This is poorly understood and difficult to visualise in vegetative hyphae, but during sporulation a single chromosome ends up in each of the spores. The S. coelicolor homologues of partitioning proteins ParA and ParB are involved at least in the developmental genome segregation [29]. Compared with other bacteria possessing chromosomal ParA– ParB systems, S. coelicolor has an unusually high concentration of binding sites for ParB (parS sites) around the oriC region, and ParB binds many if not all of these [30]. Evidence was also presented for a large ParB–parS nucleoprotein complex spanning sequences between the parS sites [30]. Not only proteins that bind near oriC, but also telomeric protein complexes could affect the behaviour of nucleoids. The linear Streptomyces chromosomes have covalently attached terminal proteins (Tpg) at the 50 ends, which are believed to prime the last Okazaki fragments, thereby providing a solution to the ‘endreplication problem’ of linear chromosomes [31]. The chromosomal tpg genes of S. rochei, S. lividans and S. coelicolor have now been identified [32,33]. Recently, the telomere-binding protein Tap, co-transcribed with tpg, was shown to interact with both Tpg and the telomeres [34]. Both tpg and tap are essential for maintenance of chromosome linearity, and mutants lacking any of these genes invariably had circularised chromosomes Current Opinion in Microbiology 2003, 6:564–571
Homologues of bacterial cell division genes present in the S. coelicolor and S. avermitilis genomes. E. coli
B. subtilis
S. coelicolor1 S. avermitilis1 References7
ftsA ftsB
ftsA
SCO3095 SCO2090 SCO57503 SCO2091 SCO2083 SCO20854 SCO2082 -5 SCO20776
ftsI ftsK ftsL ftsN ftsQ ftsW ftsZ zipA
minC minD minE
divIC ftsI spoIIIE ftsL divIB ftsW ftsZ zapA ezrA minC minD divIVA
SAV6104 SAV6116 SAV4542 SAV6115 SAV6123 SAV6121 SAV6124 SAV6129
McCormick, JR2 McCormick, JR2 McCormick, JR2 [36] [36] [27]
[15]
1
Gene designations show the homologues of known division genes that can be recognized in the two published Streptomyces genome sequences [5,6]. 2 JR McCormick, personal communication. 3 Additional members of this family are present in both genomes. 4 Three other genes related to the ftsW/spoVE/rodA family are present. 5 Some genes with similarity to minD are present, but lack motifs typical of the cell division-related minD. 6 Experimental data indicate no involvement in cell division in S. coelicolor. 7 Reference refers to S. coelicolor gene.
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involved in linking the Z ring to synthesis of septal peptidoglycan (FtsQ, FtsL, DivIC, FtsW and FtsI) [37], bear witness to a typical bacterial mechanism of cytokinesis. However, proteins implicated in the stability and/or bundling of FtsZ protofilaments are missing from Streptomyces. In addition to FtsA, these include ZipA in E. coli and ZapA and EzrA in B. subtilis [37,38]. Moreover, Streptomycetes lack a bona fide Min system and an overt nucleoid occlusion [39] — two negatively acting mechanisms that dictate the selection of division sites in many other bacteria [37]. Thus, new division proteins remain to be found that control assembly of Z rings in Streptomyces. Some of them are likely to be specific for a developmental stage.
Developmental control of cell division Recent progress related to aerial mycelium formation is summarised in an excellent review by Chater and Horinouchi [40] and papers by Elliot et al. and Claessen et al. [9,41]. Once aerial hyphae have formed, their differentiation into spores involves the synchronous formation of several tens of sporulation septa, normally one between each of the chromosomes. This is preceded by a ladder of uniformly spaced Z rings in sporogenic cells [13], which can be observed in vivo using a translational fusion of FtsZ to EGFP [39] (Figure 2). The massive assembly of Z
rings requires a cell-specific upregulation of ftsZ transcription (from one of three promoters) in both S. coelicolor and S. griseus [42,43]. Six early developmental regulators are needed to stimulate ftsZ transcription, providing a plausible explanation for the failure of mutants lacking any of these regulators to produce sporulation septa [42]. There are also differences in the way Z rings assemble during development, as demonstrated by an ftsZ missense allele that prevented sporulation [39]. Growth and aerial mycelium formation appeared normal, but the ftsZ17(Spo) mutant was largely defective in sporulation septation and synthesis of the grey spore pigment [39] (i.e. it displayed a classical white mutant phenotype [3]). Bacterial FtsZ rings are remarkably dynamic in vivo [44] and B. subtilis sporulation involves reshaping a central Z ring by moving spiral-shaped intermediates into two bipolarly localized rings [45]. Close examination of Figure 2 reveals several examples of short spirals rather than rings, and our recent data demonstrate that a phase of dynamic remodelling of spiral-like FtsZ polymers precedes the regularly spaced Z rings in sporogenic aerial hyphae (N Grantcharova and K Fla¨ rdh, unpublished data). The ftsZ17(Spo) mutant behaved normally up to this stage of remodelling, but was then unable to assemble correct Z rings [39]. The distinct phenotypic manifestations of this defect suggest
Figure 2
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(b)
Current Opinion in Microbiology
Visualization of Z ring assembly during an early stage of aerial hyphal sporulation in S. coelicolor using an FtsZ-EGFP fusion protein. In the phase-contrast image (a) no signs of constrictions are visible, but the fluorescence image (b) reveals three important aspects of the developmental regulation of cell division: (i) A cell-type specific upregulation of FtsZ-EGFP expression is seen as an increased level of fluorescence throughout the apical part of the hypha, from the tip down to a presumed basal septum just above the emerging lateral branch. (ii) Multiple Z rings are being assembled at regular intervals. (iii) As a part of this process, several examples of short apparently spiral-shaped intermediates are visible (arrowheads in enlarged inset in panel b). The details of the FtsZ-EGFP fusion are described elsewhere [40]. Bar ¼ 10 mm. www.current-opinion.com
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that Streptomyces sporulation offers a useful system for analysing the in vivo dynamics of FtsZ. New genes have recently been found to affect septation in aerial hyphae and give rise to white phenotypes when disrupted: an iclR-like regulatory gene in S. coelicolor (samR) [46]; ssgA and ssgB, encoding members of an actinomycete-specific protein family with unknown mode of action [47,48]; and ssgR (ssfR in S. griseus), lying upstream of ssgA and encoding another IclR-like regulator Figure 3
[49,50]. It remains to be determined if these, or any of the previously described early whi genes [3], interact directly with cell division genes or proteins. SsgA and SsgB are of particular interest as overexpression of the former triggers sporulation-like septation in vegetative hyphae [48], and both are developmentally regulated. Transcription of ssgB coincides with aerial mycelium formation and depends on sH [51], a s factor that is itself both transcriptionally and post-transcriptionally controlled during development [52,53], but paradoxically not required for normal sporulation [54]. In S. griseus, ssgA was directly controlled by AdpA, a transcriptional activator that mediates many of the responses to the gbutyrolactone A-factor [50]. In addition to the stimulation in sporogenic hyphae, it has been suggested that septation is specifically repressed in vegetative hyphae, and that the relief of this by genetic modifications gives rise to ectopic sporulation in the substrate mycelium [55,56].
Interplay between chromosomes and Z ring assembly? Can the regular placement of sporulation septation be explained without postulating the existence of fixed landmarks along the sporogenic hypha? In a simple model, chromosomes and the division machinery interplay to affect the positions of each other. Consistent with this, DNA condensation in mutants that fail to make sporulation septa revealed irregularly separated nucleoids [39,42,57], and a mutant defective in chromosome partitioning showed examples of abnormal spacing between septa [29] (see also Update). Although the initial ingrowth of septa normally occurs over non-separated nuclear material [58] (Figure 3), I suggest that a chromosomal locus, such as oriC, is regularly positioned in coordination with the dynamic FtsZ polymers at an early stage of sporulation. In this view, a mutual occlusion between Z rings and this locus could position both chromosomes and division planes. One of the ftsK/spoIIIE-like genes in Streptomyces could then work as a DNA translocator, equivalent to SpoIIIE during B. subtilis sporulation [59], and move the remainder of the chromosomes out of the closing septa and into the correct compartments.
Conclusions
Transmission electron micrograph showing the initial closure of sporulation septa over non-segregated nuclear material in an aerial hypha of a normally sporulating strain of S. coelicolor. The image was kindly provided by Mark Buttner (John Innes Centre, Norwich, UK). Current Opinion in Microbiology 2003, 6:564–571
The molecular cell biology of Streptomyces highlights intriguing processes such as the basis for cell polarisation, establishment and maintenance of growth polarity, nucleoid migration, and the dynamic regulation of cell division and its interplay with genome segregation during sporulation. A molecular understanding of such phenomena will not only reveal peculiarities of Streptomyces and actinomycetes, but also shed light on conserved fundamental functions of the bacterial cell. Concerning the biotechnological importance of streptomycetes and their relatives, the mycelial growth habit www.current-opinion.com
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strongly affects their behaviour and productivity in large-scale fermentations. A mechanistic and physiological understanding of tip growth, hyphal branching and mycelium fragmentation (e.g. by cell division) should significantly improve their value as producers of antibiotics and other natural products, as underlined by two recent reports [60,61].
Update In a study of genetic instability of Streptomyces, Leblond and co-workers have characterised strains of S. ambofaciens in which the end of one arm of the chromosome was lost and the remainder fused end-to-end to produce a duplicated linear chromosome of around 13 Mb, containing two replication origins (Wenner et al., unpublished results). These strains had obvious defects in segregation of DNA into spores, showed strong heterogeneity in spore sizes and were genetically unstable. This was suggested to be a result of the duplication of the parAB-oriC region involved in partitioning of chromosomes, hence giving rise to dicentric chromosomes (Wenner et al., unpublished results).
Acknowledgements The author would like to thank Rolf Bernander, Keith Chater, Santanu Dasgupta and Joe McCormick for critical reading of the manuscript. Colleagues who have communicated results and manuscripts are gratefully acknowledged. However, space constraints regretfully made it impossible to include all of the interesting recent developments in this field.
References and recommended reading Papers of particular interest, published within the annual period of review, have been highlighted as: of special interest of outstanding interest 1.
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Ikeda H, Ishikawa J, Hanamoto A, Shinose M, Kikuchi H, Shiba T, Sakaki Y, Hattori M, Omura S: Complete genome sequence and comparative analysis of the industrial microorganism Streptomyces avermitilis. Nat Biotechnol 2003, 21:526-531. This genome sequence of an industrially important streptomycete confirmed that many novel features described in Bentley et al. (2002) [6] are widespread in this group of organisms, and underlined the enormous richness of their secondary metabolic capacity. 6.
Bentley SD, Chater KF, Cerdeno-Tarraga AM, Challis GL, Thomson NR, James KD, Harris DE, Quail MA, Kieser H, Harper D et al.: Complete genome sequence of the model actinomycete Streptomyces coelicolor A3(2). Nature 2002, 417:141-147. This milestone paper reveals many unusual features of the linear genome of the major genetic model organism of Streptomyces. 7.
Novotna J, Vohradsky J, Berndt P, Gramajo H, Langen H, Li X-M, Minas W, Orsaria L, Roeder D, Thompson CJ: Proteomic studies of diauxic lag in the differentiating prokaryote Streptomyces coelicolor reveal a regulatory network of stress-induced proteins and central metabolic enzymes. Mol Microbiol 2003, 48:1289-1303.
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A proteomic and physiological analysis of diauxic growth in liquid medium. This paper also introduces a web-server for Streptomyces proteomics. 8.
Hesketh AR, Chandra G, Shaw AD, Rowland JJ, B. KD, Bibb MJ, Chater KF: Primary and secondary metabolism, and posttranslational protein modifications, as portrayed by proteomic analysis of Streptomyces coelicolor. Mol Microbiol 2002, 46:917-932. A large-scale proteomic study revealing extensive post-translational modifications and forming the basis for an on-line proteome map. 9.
Elliot MA, Karoonuthaisiri N, Huang J, Bibb MJ, Cohen SN, Kao CM, Buttner MJ: The chaplins: a family of hydrophobic cell-surface proteins involved in aerial mycelium formation in Streptomyces coelicolor. Genes Dev 2003, 17:1727-1740. The first reported DNA microarray analysis of Streptomyces development on solid medium. This report together with Claessen D et al. (2003) [41] led to the identification of a new family of cell-surface proteins involved in aerial mycelium formation. 10. Huang J, Lih CJ, Pan KH, Cohen SN: Global analysis of growth phase responsive gene expression and regulation of antibiotic biosynthetic pathways in Streptomyces coelicolor using DNA microarrays. Genes Dev 2001, 15:3183-3192. 11. Gust B, Challis GL, Fowler K, Kieser T, Chater KF: PCR-targeted Streptomyces gene replacement identifies a protein domain needed for biosynthesis of the sesquiterpene soil odor geosmin. Proc Natl Acad Sci USA 2003, 100:1541-1546. An elegant adaptation of a l-Red-based PCR-targeting system for making a purpose-built system for efficient gene disruption and other modifications of Streptomyces genomes. 12. Prosser JI, Tough AJ: Growth mechanics and growth kinetics of filamentous microorganisms. Crit Rev Biotechnol 1991, 10:253-274. 13. Schwedock J, McCormick JR, Angert EA, Nodwell JR, Losick R: Assembly of the cell division protein FtsZ into ladder-like structures in the aerial hyphae of Streptomyces coelicolor. Mol Microbiol 1997, 25:847-858. 14. Daniel RA, Errington J: Control of cell morphogenesis in bacteria: two distinct ways to make a rod-shaped cell. Cell 2003, 113:767-776. A very important paper that uses vancomycin-based staining to visualise the architecture of murein assembly, and to demonstrate the crucial role of the actin-like Mbl protein in this process in B. subtilis. Furthermore, it demonstrates polar growth of the cell wall sacculus in both Streptomyces and Corynebacterium. 15. Fla¨ rdh K: Essential role of DivIVA in polar growth and morphogenesis in Streptomyces coelicolor A3(2). Mol Microbiol 2003, 49:1523-1536. The first report of a protein specifically localised at hyphal tips in Streptomyces. The S. coelicolor homologue of DivIVA is shown to be essential and to have strong impact on tip extension, branching patterns and cell shape determination. 16. de Pedro MA, Young KD, Ho¨ ltje J-V, Schwarz H: Branching of Escherichia coli cells arises from multiple sites of inert peptidoglycan. J Bacteriol 2003, 185:1147-1152. An analysis of branching in mutant strains of E. coli, indicating that branches arise from sites where murein metabolism is severely reduced. This is the inverse of the situation in Streptomyces, where branches appear to be formed by creation of new growth zones in the lateral wall. 17. Goriely A, Tabor M: Biomechanical models of hyphal growth in actinomycetes. J Theor Biol 2003, 222:211-218. A recent model of hyphal growth, based on large deformation membrane theory. 18. Migue´ lez EM, Martin C, Manzanal MB, Hardison C: Growth and morphogenesis in Streptomyces. FEMS Microbiol Lett 1992, 79:351-360. 19. Koch AL: Apical growth of streptomycetes and fungi. In Bacterial Growth and Form. New York: Chapman & Hall; 1995:311-325. 20. Muchova´ K, Kutejova´ E, Scott DJ, Brannigan JA, Lewis RJ, Wilkinson AJ, Bara´ k I: Oligomerization of the Bacillus subtilis division protein DivIVA. Microbiol 2002, 148:807-813. 21. Marston AL, Thomaides HB, Edwards DH, Sharpe ME, Errington J: Polar localization of the MinD protein of Bacillus subtilis and its Current Opinion in Microbiology 2003, 6:564–571
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role in selection of the mid-cell division site. Genes Dev 1998, 12:3419-3430. 22. Marston AL, Errington J: Selection of the mid-cell division site in Bacillus subtilis through MinD-dependent polar localization and activation of MinC. Mol Microbiol 1999, 33:84-96. 23. Edwards DH, Errington J: The Bacillus subtilis DivIVA protein targets to the division septum and controls the site specificity of cell division. Mol Microbiol 1997, 24:905-915. 24. Cha J-H, Stewart GC: The divIVA minicell locus of Bacillus subtilis. J Bacteriol 1997, 179:1671-1683. 25. Thomaides HB, Freeman M, El Karoui M, Errington J: Division site selection protein DivIVA of Bacillus subtilis has a second distinct function in chromosome segregation during sporulation. Genes Dev 2001, 15:1662-1673. 26. Sassetti CM, Boyd DH, Rubin EJ: Genes required for mycobacterial growth defined by high density mutagenesis. Mol Microbiol 2003, 48:77-84. 27. McCormick JR, Su EP, Driks A, Losick R: Growth and viability of Streptomyces coelicolor mutant for the cell division gene ftsZ. Mol Microbiol 1994, 14:243-254. 28. Yang MC, Losick R: Cytological evidence for association of the ends of the linear chromosome in Streptomyces coelicolor. J Bacteriol 2001, 183:5180-5186. 29. Kim HJ, Calcutt MJ, Schmidt FJ, Chater KF: Partitioning of the linear chromosome during sporulation of Streptomyces coelicolor A3(2) involves an oriC-linked parAB locus. J Bacteriol 2000, 182:1313-1320. 30. Jakimowicz D, Chater K, Zakrzewska-Czerwinska J: The ParB protein of Streptomyces coelicolor A3(2) recognizes a cluster of parS sequences within the origin-proximal region of the linear chromosome. Mol Microbiol 2002, 45:1365-1377. This paper shows by different methods that S. coelicolor ParB binds both in vitro and in vivo to most of the parS sites surrounding the oriC region. It is suggested that it forms a large partitioning complex that may facilitate the separation of chromosomes after replication. 31. Volff J-N, Altenbuchner J: A new beginning with new ends: linearisation of circular chromosomes during bacterial evolution. FEMS Microbiol Lett 2000, 186:143-150. 32. Bao K, Cohen SN: Terminal proteins essential for the replication of linear plasmids and chromosomes in Streptomyces. Genes Dev 2001, 15:1518-1527. 33. Yang C-C, Huang C-H, Li C-Y, Tsay Y-G, Lee S-C, Chen CW: The terminal proteins of linear Streptomyces chromosomes and plasmids: a novel class of replication priming proteins. Mol Microbiol 2002, 43:297-305. This paper reports the purification of the terminal protein of S. coelicolor, demonstrates that it is covalently attached to the 50 -end of the chromosome, and identifies the corresponding gene tpgC, which is similar to previously reported tpg genes [32]. 34. Bao K, Cohen SN: Recruitment of terminal protein to the ends of Streptomyces linear plasmids and chromosomes by a novel telomere-binding protein essential for linear DNA replication. Genes Dev 2003, 17:774-785. An impressive paper that identifies a telomere-associated protein, provides evidence that it binds both telomeric DNA and the terminal protein Tpg to form a telomeric protein complex, and shows that it is required for chromosome linearity. In addition, it includes the use of microarray-based chromosome mapping. 35. Yeats C, Bentley S, Bateman A: New knowledge from old: in silico discovery of novel protein domains in Streptomyces coelicolor. BMC Microbiol 2003, 3:3. A bioinformatic study of the S. coelicolor genome sequence that identifies several new protein domains and protein families. 36. McCormick JR, Losick R: Cell division gene ftsQ is required for efficient sporulation but not growth and viability in Streptomyces coelicolor A3(2). J Bacteriol 1996, 178:5295-5301. 37. Errington J, Daniel RA, Scheffers D-J: Cytokinesis in bacteria. Microbiol Mol Biol Rev 2003, 67:52-65. Current Opinion in Microbiology 2003, 6:564–571
38. Gueiros-Filho FJ, Losick R: A widely conserved bacterial cell division protein that promotes assembly of the tubulin-like protein FtsZ. Genes Dev 2002, 16:2544-2556. 39. Grantcharova N, Ubhayasekera W, Mowbray SL, McCormick JR, Fla¨ rdh K: A missense mutation in ftsZ differentially affects vegetative and developmentally controlled cell division in Streptomyces coelicolor A3(2). Mol Microbiol 2003, 47:645-656. This report describes a genetic strategy for isolation of non-sporulating ftsZ mutants. One mutant allele is characterised which strongly affects the assembly of FtsZ rings during sporulation, but has only a limited effect on vegetative septation. 40. Chater KF, Horinouchi S: Signalling early developmental events in two highly diverged Streptomyces species. Mol Microbiol 2003, 48:9-15. As summarised in this review, significant progress has recently been made in understanding the control of aerial mycelium formation and its coordination with secondary metabolism. 41. Claessen D, Rink R, de Jong W, Siebring J, de Vreugd P, Boersma FGH, Dijkhuizen L, Wo¨ sten HAB: A novel class of secreted hydrophobic proteins is involved in aerial mycelium formation in Streptomyces coelicolor by forming amyloid-like fibrils. Genes Dev 2003, 17:1714-1726. Together with Elliot et al. (2003) [9] this work identified a new family of cell-surface proteins involved in aerial mycelium formation. 42. Fla¨ rdh K, Leibovitz E, Buttner MJ, Chater KF: Generation of a nonsporulating strain of Streptomyces coelicolor A3(2) by the manipulation of a developmentally controlled ftsZ promoter. Mol Microbiol 2000, 38:737-749. 43. Kwak J, Dharmatilake AJ, Jiang H, Kendrick KE: Differential regulation of ftsZ transcription during septation of Streptomyces griseus. J Bacteriol 2001, 183:5092-5101. 44. Stricker J, Maddox P, Salmon ED, Erickson HP: Rapid assembly dynamics of the Escherichia coli FtsZ-ring demonstrated by fluorescence recovery after photobleaching. Proc Natl Acad Sci USA 2002, 99:3171-3175. It is demonstrated that bacterial FtsZ rings, in similarity to microtubules, are extremely dynamic structures, and may remodel themselves with a half-time of 30 s. The remodelling was slower in a mutant protein with defective GTPase activity. 45. Ben-Yehuda S, Losick R: Asymmetric cell division in B. subtilis involves a spiral-like intermediate of the cytokinetic protein FtsZ. Cell 2002, 109:257-266. This paper beautifully shows that asymmetric cell division during B. subtilis sporulation involves dynamic remodelling of spiral-shaped FtsZ polymers that eventually stabilise as two bipolar Z rings, one of which is chosen for division. The SpoIIE protein and a developmentally controlled upregulation of ftsZ expression were required and together apparently sufficient to trigger this remodelling. 46. Tan H, Tian Y, Yang H, Liu G, Nie L: A novel Streptomyces gene, samR, with different effects on differentiation of Streptomyces ansochromogenes and Streptomyces coelicolor. Arch Microbiol 2002, 177:274-278. 47. Keijser BJF, Noens EEE, Kraal B, Koerten HK, van Wezel GP: The Streptomyces coelicolor ssgB gene is required for early stages of sporulation. FEMS Microbiol Lett 2003, 225:59-67. An additional member of the actinomycete-specific family of SsgA-like proteins [48,49] is shown to be required for the developmentally induced cell division in sporogenic aerial hyphae. 48. van Wezel GP, van der Meulen J, Kawamoto S, Luiten RG, Koerten HK, Kraal B: ssgA is essential for sporulation of Streptomyces coelicolor A3(2) and affects hyphal development by stimulating septum formation. J Bacteriol 2000, 182:5653-5662. 49. Jiang H, Kendrick KE: Characterization of ssfR and ssgA, two genes involved in sporulation of S. griseus. J Bacteriol 2000, 182:5521-5529. 50. Yamazaki H, Ohnishi Y, Horinouchi S: Transcriptional switch on of ssgA by A-factor, which is essential for spore septum formation in Streptomyces griseus. J Bacteriol 2003, 185:1273-1283. The paper reports on the developmental control of ssgA transcription in S. griseus. It shows direct regulation by AdpA, a transcriptional activator mediating the response to the quorum-sensing signal A-factor, as well as a dependence on the ECF s factor AdsA/BldN. www.current-opinion.com
Growth polarity and cell division in Streptomyces Fla¨ rdh 571
51. Kormanec J, Sevcikova B: The stress-response sigma factor rH controls the expression of ssgB, a homologue of the sporulation-specific cell division gene ssgA, in Streptomyces coelicolor A3(2). Mol Genet Genomics 2002, 267:536-543. This work shows that ssgB is developmentally regulated during sporulation on solid medium. This was not dependent on a group of known developmental regulators often referred to as the early whi genes, but rather on the stress-related sigma factor sH.
58. Migue´ lez EM, Rueda B, Hardisson C, Manzanal MB: Nucleoid partitioning and the later stages of sporulation septum synthesis are closely associated events in the sporulating hyphae of Streptomyces carpinensis. FEMS Microbiol Lett 1998, 159:59-62.
52. Kelemen GH, Viollier PH, Tenor JL, Marri L, Buttner MJ, Thompson CJ: A connection between stress and development in the multicellular prokaryote Streptomyces coelicolor A3(2). Mol Microbiol 2001, 40:804-814.
60. Jonsbu E, McIntyre M, Nielsen J: The influence of carbon sources and morphology on nystatin production by Streptomyces noursei. J Biotechnol 2002, 95:133-144.
53. Viollier PH, Weihofen A, Folcher M, Thompson CJ: Posttranscriptional regulation of the Streptomyces coelicolor stress responsive sigma factor, SigH, involves translational control, proteolytic processing, and an anti-sigma factor homolog. J Mol Biol 2003, 325:637-649.
59. Errington J, Bath J, Wu LJ: DNA transport in bacteria. Nature Reviews Molecular Cell Biology 2001, 2:538-545.
61. Wardell JN, Stocks SM, Thomas CR, Bushell ME: Decreasing the hyphal branching rate of Saccharopolyspora erythraea NRRL 2338 leads to increased resistance to breakage and increased antibiotic production. Biotechnol Bioeng 2002, 78:141-146. 62. Figge RM, Gober JW: Cell shape, division and development: the 2002 American Society for Microbiology (ASM) conference on prokaryotic development. Mol Microbiol 2003, 47:1475-1483.
54. Viollier PH, Kelemen GH, Dale GE, Nguyen KT, Buttner MJ, Thompson CJ: Specialized osmotic stress response systems involve multiple SigB-like sigma factors in Streptomyces coelicolor. Mol Microbiol 2003, 47:699-714.
Now in press
55. Ohnishi Y, Seo J-W, Horinouchi S: Deprogrammed sporulation in Streptomyces. FEMS Microbiol Lett 2002, 216:1-7.
The work referred to in the text as (JA Gil, personal communication) and (Wenner et al. unpublished results) is now in press [63,64]:
56. Seo J-W, Ohnishi Y, Hirata A, Horinouchi S: ATP-binding cassette transport system involved in regulation of morphological differentiation in response to glucose in Streptomyces griseus. J Bacteriol 2002, 184:91-103.
63. Ramos A, Honrubia MP, Valbuena N, Vaquera J, Mateos LM, Gil JA: Involvement of DivIVA in the morphology of the rod-shaped actinomycete Brevibacterium lactofermentum. Microbiol 2003, 149:in press.
57. Fla¨ rdh K, Findlay KC, Chater KF: Association of early sporulation genes with suggested developmental decision points in Streptomyces coelicolor A3(2). Microbiol 1999, 145:2229-2243.
64. Wenner T, Roth V, Fischer G, Fourrier C, Aigle B, Decaris B, Leblond P: End-to end fusion of linear deleted chromosomes initiates a cycle of genome instability in Streptomyces ambofaciens. Mol Microbiol 2003, 50:in press.
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Current Opinion in Microbiology 2003, 6:564–571
Growth polarity and cell division in Streptomyces Klas Fla¨rdh Streptomycetes are mycelial bacteria that resemble filamentous fungi in their apical growth, branching, and morphogenetic development. One inroad into the largely unknown mechanisms underlying this prokaryotic growth polarity is provided by Streptomyces DivIVA, a protein localized at hyphal tips and involved in tip extension. Another aspect is a proposed migration of nucleoids. During sporulation, the modes of growth and cell division are reorganised. This involves dynamic assembly of FtsZ into a multitude of cytokinetic rings. Controlled by developmental regulators and intriguingly coordinated with chromosome segregation, this leads to spores with a single chromosome each. Genome sequences have shed new light on these aspects and reinforced the role of Streptomyces in bacterial cell biology. Addresses Department of Cell and Molecular Biology, Uppsala University, Biomedical Centre Box 596, SE-751 24 Uppsala, Sweden e-mail: [email protected]
Current Opinion in Microbiology 2003, 6:564–571 This review comes from a themed issue on Growth and development Edited by Claudio Scazzocchio and Jeff Errington 1369-5274/$ – see front matter ß 2003 Elsevier Ltd. All rights reserved. DOI 10.1016/j.mib.2003.10.011
Introduction
mentation of proteomics [7,8], DNA microarray technology [9,10], and novel techniques for streamlined genetic engineering [11], the study of these complex processes has certainly become more tractable and rewarding. In this review, I revisit Streptomyces hyphal growth, cell division and related aspects of developmental biology from a cell biological perspective, summarise recent progress and highlight emerging issues.
Apical growth As demonstrated using pulse-labelling or lectin binding for example, the Streptomyces cell wall is polymerised at the hyphal apex [12,13]. Fluorescently labelled vancomycin was recently introduced as a probe to visualise sites of nascent peptidoglycan insertion into the cell wall sacculus in Gram-positive bacteria [14], and this further corroborated the pronounced tip extension in Streptomyces [14,15] (Figure 1a). Hence, although some turnover may occur in the lateral walls, hyphae are in principle tubes of inert murein that grow only at one extreme end. When disconnected from the apex by septation, a subapical cell appears unable to grow until it has created a new tip by lateral branching (Figure 1c). This pattern is in sharp contrast to rod-shaped bacteria such as Escherichia coli and Bacillus subtilis, which grow by intercalation of new murein along the lateral wall, while their polar caps are stable once they have been polymerised during cell division (see for example [14,16]).
Streptomycetes differ conspicuously from most other bacteria in their growth and developmental biology, and are superficially more reminiscent of filamentous fungi. Thus, they grow by tip extension to form a mycelium of branched hyphae. The mechanisms of apical growth and the inherent poleward transport processes have been extensively studied in fungi [1,2], but we have few clues about the corresponding mechanisms in a mycelial prokaryote. Furthermore, the modes of growth and cell division are dramatically modified during development of Streptomyces colonies, when specialized sporebearing hyphae emerge through the air–water interface to give a fluffy layer of aerial mycelium. This involves pronounced developmental regulation of morphogenetic and cell cycle-related processes [3].
Most models for Streptomyces tip extension assume that new material is incorporated at the apex in a flexible form, which becomes more rigid further back from the tip, and that stretching of the tip by turgor pressure is a major driving force for growth [12,17,18,19]. Staining with vancomycin gave patterns consistent with these models inasmuch as incorporation was highest in the tip itself, rather than at the leading edge of the cylindrical wall [14,15]. However, the molecular bases of tip extension remain unclear. Obviously, proteins involved in assembly of the cell envelope (e.g. penicillin-binding proteins) must be recruited to the apical growth zone, presumably forming part of larger apical complexes. It is also possible that assemblies of cytoplasmic or membrane proteins, in analogy with eukaryotic cytoskeletons, may provide a scaffold for the fragile tip.
With the revolution in our understanding of the bacterial cell that has been provoked by modern fluorescence microscopy [4], new opportunities are emerging to investigate growth polarity and differentiation in Streptomyces. In conjunction with the publication of two complete Streptomyces genome sequences [5,6], and the imple-
The first component of an apical protein complex has recently been identified. When tagged with enhanced green fluorescent protein (EGFP), the S. coelicolor DivIVA protein (denoted DivIVASC) formed distinct fluorescent foci at the hyphal tips, with an intensity indicative of the presence of large numbers of molecules [15] (Figure 1b).
Current Opinion in Microbiology 2003, 6:564–571
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Growth polarity and cell division in Streptomyces Fla¨ rdh 565
Figure 1
(a)
t
(b)
Sp t t Sp t
(c)
Branching Tip extension
Cell division
Nucleoid migration
Apical growth in Streptomyces. (a) Staining of the sites of nascent peptidoglycan incorporation using fluorescently labelled vancomycin in vegetative hyphae of S. coelicolor. The fluorescence image is shown in inverted grey-scale. Hyphal tips are indicated by a ‘t’, crosswalls by arrowheads, and the spore from which the hyphae grew out by ‘Sp’. (b) Subcellular localisation of DivIVASC-EGFP (green colour) overlaid on a phase-contrast image of nascent mycelium growing out of a spore (Sp). Images in panels (a) and (b) have been published previously [15,62], and are reproduced with permission from Blackwell Publishing Ltd. Bars, 5 mm. (c) Simplified illustration of polarised growth in Streptomyces hyphae. The apical cell is extending its cell wall only at the tip (green). Once this cell has divided by forming a new hyphal cross wall, the subapical daughter cell becomes unable to grow, and eventually switches its polarity to generate a lateral branch with a new extending tip. A consequence of tip growth is that DNA, which replicates along most of the hyphal length, has to move towards the tip and into new branches - a process we propose to designate nucleoid migration. For clarity, only a few schematic nucleoids are drawn (red), and they are not meant to reflect the actual number of chromosomes per cell. Furthermore, individual nucleoids are typically not observed in vivo as separated bodies in growing hyphae.
Like the homologous DivIVA in B. subtilis [20], this protein is predicted to contain extensive coiled-coil structure and may oligomerise. DivIVASC was essential, and partial depletion gave hyphal morphologies strikingly similar to those observed in many fungal mutants defective in tip extension or nuclear migration [15]. Overexpression had dramatic effects on shape determination, leading to conspicuous swollen and pear-shaped cells. It was particularly compelling that induction of divIVASC overexpression in preformed hyphae rapidly gave rise to multiple new sites of wall growth, from which branch-like lateral outgrowths emerged. Thus, DivIVASC not only targets tips and affects morphogenesis, but also appears instrumental in establishment of new tips [15]. In B. subtilis, DivIVA sequesters the cell division inhibitors MinC and MinD at the cell poles, thereby allowing www.current-opinion.com
division only at central positions [21,22]. The Bacillus protein has no impact on vegetative growth in minC or minD mutants [23,24], although it has an additional role in chromosome localisation during sporulation [25]. DivIVASC works differently and did not detectably affect division or nucleoid localisation in S. coelicolor [15]. Intriguingly, all sequenced genomes of streptomycetes and related bacteria (actinomycetes, including pathogenic mycobacteria) to date lack minC, but still encode a DivIVA, which appears to be essential in both Mycobacterium tuberculosis (wag31) [26] and Brevibacterium lactofermentum (JA Gil, personal communication). The MinC-independent essential functions could relate to growth polarity also in rod-shaped cells, and Corynebacterium glutamicum (very closely related to B. lactofermentum) has indeed been demonstrated to grow apically at both poles [14]. Current Opinion in Microbiology 2003, 6:564–571
566 Growth and development
Given the prominent role of the actin-like MreB and Mbl proteins in directing cell wall assembly in B. subtilis [14], it is noteworthy that the sequenced genomes of rodshaped actinomycetes such as Mycobacterium and Corynebacterium lack such proteins, whereas the hyphal Streptomyces species encode two MreB/Mbl-like proteins each [5,6]. Although C. glutamicum and S. coelicolor both grow polarly, the former extends at the cell poles created by division, whereas the latter generates each tip de novo by branching or germ tube emergence (Figure 1), and does not require cell division for hyphal growth and branching [27]. This important distinction might relate to the differential conservation of MreB/Mbl. Alternatively, these proteins could have roles in morphological differentiation in Streptomyces.
Nucleoid migration, segregation and replication In contrast to cell wall assembly, DNA replication takes place with similar rates along a large portion of the hyphae [12]. Thus, in analogy to nuclear migration in filamentous fungi [2], it has to be postulated that nucleoid migration occurs in Streptomyces, such that the chromosomes move in relation to the cell envelope to populate the extending tips and lateral branches (Figure 1c). Clarification of the driving forces and mechanisms for moving DNA along these prokaryotic cells would be of considerable interest.
[32,34]. The extreme ends of several Streptomyces chromosomes contain helicase-like genes, which might also contribute to terminal protein complexes [35]. Cytological evidence for co-localisation of the left and right telomeres has been presented [28], but it is not evident how chromosome linearity affects nucleoid segregation or migration. Strains with circularised chromosomes often show increased genetic instability, but generally display no or only small growth or sporulation disadvantage [31]. Although this suggests that internal regions like oriC, rather than the telomeres, are critical for partitioning during sporulation, further elucidation of these aspects are needed (see also Update).
Two modes of cell division in Streptomyces Streptomyces use at least two kinds of cell division: vegetative septation leading to crosswalls in the substrate mycelium and developmentally regulated sporulation septation. Both kinds involve the same basic division machinery, including FtsQ and FtsZ [27,36]. Additional Streptomyces homologues of known bacterial division proteins can be recognized in the genome sequences [5,6] (Table 1). The conservation of the tubulin homologue FtsZ, its polymerisation into the cytokinetic Z ring [13], and several membrane proteins apparently Table 1
In addition to poleward migration, newly replicated chromosomes segregate from each other [28]. This is poorly understood and difficult to visualise in vegetative hyphae, but during sporulation a single chromosome ends up in each of the spores. The S. coelicolor homologues of partitioning proteins ParA and ParB are involved at least in the developmental genome segregation [29]. Compared with other bacteria possessing chromosomal ParA– ParB systems, S. coelicolor has an unusually high concentration of binding sites for ParB (parS sites) around the oriC region, and ParB binds many if not all of these [30]. Evidence was also presented for a large ParB–parS nucleoprotein complex spanning sequences between the parS sites [30]. Not only proteins that bind near oriC, but also telomeric protein complexes could affect the behaviour of nucleoids. The linear Streptomyces chromosomes have covalently attached terminal proteins (Tpg) at the 50 ends, which are believed to prime the last Okazaki fragments, thereby providing a solution to the ‘endreplication problem’ of linear chromosomes [31]. The chromosomal tpg genes of S. rochei, S. lividans and S. coelicolor have now been identified [32,33]. Recently, the telomere-binding protein Tap, co-transcribed with tpg, was shown to interact with both Tpg and the telomeres [34]. Both tpg and tap are essential for maintenance of chromosome linearity, and mutants lacking any of these genes invariably had circularised chromosomes Current Opinion in Microbiology 2003, 6:564–571
Homologues of bacterial cell division genes present in the S. coelicolor and S. avermitilis genomes. E. coli
B. subtilis
S. coelicolor1 S. avermitilis1 References7
ftsA ftsB
ftsA
SCO3095 SCO2090 SCO57503 SCO2091 SCO2083 SCO20854 SCO2082 -5 SCO20776
ftsI ftsK ftsL ftsN ftsQ ftsW ftsZ zipA
minC minD minE
divIC ftsI spoIIIE ftsL divIB ftsW ftsZ zapA ezrA minC minD divIVA
SAV6104 SAV6116 SAV4542 SAV6115 SAV6123 SAV6121 SAV6124 SAV6129
McCormick, JR2 McCormick, JR2 McCormick, JR2 [36] [36] [27]
[15]
1
Gene designations show the homologues of known division genes that can be recognized in the two published Streptomyces genome sequences [5,6]. 2 JR McCormick, personal communication. 3 Additional members of this family are present in both genomes. 4 Three other genes related to the ftsW/spoVE/rodA family are present. 5 Some genes with similarity to minD are present, but lack motifs typical of the cell division-related minD. 6 Experimental data indicate no involvement in cell division in S. coelicolor. 7 Reference refers to S. coelicolor gene.
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involved in linking the Z ring to synthesis of septal peptidoglycan (FtsQ, FtsL, DivIC, FtsW and FtsI) [37], bear witness to a typical bacterial mechanism of cytokinesis. However, proteins implicated in the stability and/or bundling of FtsZ protofilaments are missing from Streptomyces. In addition to FtsA, these include ZipA in E. coli and ZapA and EzrA in B. subtilis [37,38]. Moreover, Streptomycetes lack a bona fide Min system and an overt nucleoid occlusion [39] — two negatively acting mechanisms that dictate the selection of division sites in many other bacteria [37]. Thus, new division proteins remain to be found that control assembly of Z rings in Streptomyces. Some of them are likely to be specific for a developmental stage.
Developmental control of cell division Recent progress related to aerial mycelium formation is summarised in an excellent review by Chater and Horinouchi [40] and papers by Elliot et al. and Claessen et al. [9,41]. Once aerial hyphae have formed, their differentiation into spores involves the synchronous formation of several tens of sporulation septa, normally one between each of the chromosomes. This is preceded by a ladder of uniformly spaced Z rings in sporogenic cells [13], which can be observed in vivo using a translational fusion of FtsZ to EGFP [39] (Figure 2). The massive assembly of Z
rings requires a cell-specific upregulation of ftsZ transcription (from one of three promoters) in both S. coelicolor and S. griseus [42,43]. Six early developmental regulators are needed to stimulate ftsZ transcription, providing a plausible explanation for the failure of mutants lacking any of these regulators to produce sporulation septa [42]. There are also differences in the way Z rings assemble during development, as demonstrated by an ftsZ missense allele that prevented sporulation [39]. Growth and aerial mycelium formation appeared normal, but the ftsZ17(Spo) mutant was largely defective in sporulation septation and synthesis of the grey spore pigment [39] (i.e. it displayed a classical white mutant phenotype [3]). Bacterial FtsZ rings are remarkably dynamic in vivo [44] and B. subtilis sporulation involves reshaping a central Z ring by moving spiral-shaped intermediates into two bipolarly localized rings [45]. Close examination of Figure 2 reveals several examples of short spirals rather than rings, and our recent data demonstrate that a phase of dynamic remodelling of spiral-like FtsZ polymers precedes the regularly spaced Z rings in sporogenic aerial hyphae (N Grantcharova and K Fla¨ rdh, unpublished data). The ftsZ17(Spo) mutant behaved normally up to this stage of remodelling, but was then unable to assemble correct Z rings [39]. The distinct phenotypic manifestations of this defect suggest
Figure 2
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(b)
Current Opinion in Microbiology
Visualization of Z ring assembly during an early stage of aerial hyphal sporulation in S. coelicolor using an FtsZ-EGFP fusion protein. In the phase-contrast image (a) no signs of constrictions are visible, but the fluorescence image (b) reveals three important aspects of the developmental regulation of cell division: (i) A cell-type specific upregulation of FtsZ-EGFP expression is seen as an increased level of fluorescence throughout the apical part of the hypha, from the tip down to a presumed basal septum just above the emerging lateral branch. (ii) Multiple Z rings are being assembled at regular intervals. (iii) As a part of this process, several examples of short apparently spiral-shaped intermediates are visible (arrowheads in enlarged inset in panel b). The details of the FtsZ-EGFP fusion are described elsewhere [40]. Bar ¼ 10 mm. www.current-opinion.com
Current Opinion in Microbiology 2003, 6:564–571
568 Growth and development
that Streptomyces sporulation offers a useful system for analysing the in vivo dynamics of FtsZ. New genes have recently been found to affect septation in aerial hyphae and give rise to white phenotypes when disrupted: an iclR-like regulatory gene in S. coelicolor (samR) [46]; ssgA and ssgB, encoding members of an actinomycete-specific protein family with unknown mode of action [47,48]; and ssgR (ssfR in S. griseus), lying upstream of ssgA and encoding another IclR-like regulator Figure 3
[49,50]. It remains to be determined if these, or any of the previously described early whi genes [3], interact directly with cell division genes or proteins. SsgA and SsgB are of particular interest as overexpression of the former triggers sporulation-like septation in vegetative hyphae [48], and both are developmentally regulated. Transcription of ssgB coincides with aerial mycelium formation and depends on sH [51], a s factor that is itself both transcriptionally and post-transcriptionally controlled during development [52,53], but paradoxically not required for normal sporulation [54]. In S. griseus, ssgA was directly controlled by AdpA, a transcriptional activator that mediates many of the responses to the gbutyrolactone A-factor [50]. In addition to the stimulation in sporogenic hyphae, it has been suggested that septation is specifically repressed in vegetative hyphae, and that the relief of this by genetic modifications gives rise to ectopic sporulation in the substrate mycelium [55,56].
Interplay between chromosomes and Z ring assembly? Can the regular placement of sporulation septation be explained without postulating the existence of fixed landmarks along the sporogenic hypha? In a simple model, chromosomes and the division machinery interplay to affect the positions of each other. Consistent with this, DNA condensation in mutants that fail to make sporulation septa revealed irregularly separated nucleoids [39,42,57], and a mutant defective in chromosome partitioning showed examples of abnormal spacing between septa [29] (see also Update). Although the initial ingrowth of septa normally occurs over non-separated nuclear material [58] (Figure 3), I suggest that a chromosomal locus, such as oriC, is regularly positioned in coordination with the dynamic FtsZ polymers at an early stage of sporulation. In this view, a mutual occlusion between Z rings and this locus could position both chromosomes and division planes. One of the ftsK/spoIIIE-like genes in Streptomyces could then work as a DNA translocator, equivalent to SpoIIIE during B. subtilis sporulation [59], and move the remainder of the chromosomes out of the closing septa and into the correct compartments.
Conclusions
Transmission electron micrograph showing the initial closure of sporulation septa over non-segregated nuclear material in an aerial hypha of a normally sporulating strain of S. coelicolor. The image was kindly provided by Mark Buttner (John Innes Centre, Norwich, UK). Current Opinion in Microbiology 2003, 6:564–571
The molecular cell biology of Streptomyces highlights intriguing processes such as the basis for cell polarisation, establishment and maintenance of growth polarity, nucleoid migration, and the dynamic regulation of cell division and its interplay with genome segregation during sporulation. A molecular understanding of such phenomena will not only reveal peculiarities of Streptomyces and actinomycetes, but also shed light on conserved fundamental functions of the bacterial cell. Concerning the biotechnological importance of streptomycetes and their relatives, the mycelial growth habit www.current-opinion.com
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strongly affects their behaviour and productivity in large-scale fermentations. A mechanistic and physiological understanding of tip growth, hyphal branching and mycelium fragmentation (e.g. by cell division) should significantly improve their value as producers of antibiotics and other natural products, as underlined by two recent reports [60,61].
Update In a study of genetic instability of Streptomyces, Leblond and co-workers have characterised strains of S. ambofaciens in which the end of one arm of the chromosome was lost and the remainder fused end-to-end to produce a duplicated linear chromosome of around 13 Mb, containing two replication origins (Wenner et al., unpublished results). These strains had obvious defects in segregation of DNA into spores, showed strong heterogeneity in spore sizes and were genetically unstable. This was suggested to be a result of the duplication of the parAB-oriC region involved in partitioning of chromosomes, hence giving rise to dicentric chromosomes (Wenner et al., unpublished results).
Acknowledgements The author would like to thank Rolf Bernander, Keith Chater, Santanu Dasgupta and Joe McCormick for critical reading of the manuscript. Colleagues who have communicated results and manuscripts are gratefully acknowledged. However, space constraints regretfully made it impossible to include all of the interesting recent developments in this field.
References and recommended reading Papers of particular interest, published within the annual period of review, have been highlighted as: of special interest of outstanding interest 1.
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A proteomic and physiological analysis of diauxic growth in liquid medium. This paper also introduces a web-server for Streptomyces proteomics. 8.
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Growth polarity and cell division in Streptomyces Fla¨ rdh 571
51. Kormanec J, Sevcikova B: The stress-response sigma factor rH controls the expression of ssgB, a homologue of the sporulation-specific cell division gene ssgA, in Streptomyces coelicolor A3(2). Mol Genet Genomics 2002, 267:536-543. This work shows that ssgB is developmentally regulated during sporulation on solid medium. This was not dependent on a group of known developmental regulators often referred to as the early whi genes, but rather on the stress-related sigma factor sH.
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Now in press
55. Ohnishi Y, Seo J-W, Horinouchi S: Deprogrammed sporulation in Streptomyces. FEMS Microbiol Lett 2002, 216:1-7.
The work referred to in the text as (JA Gil, personal communication) and (Wenner et al. unpublished results) is now in press [63,64]:
56. Seo J-W, Ohnishi Y, Hirata A, Horinouchi S: ATP-binding cassette transport system involved in regulation of morphological differentiation in response to glucose in Streptomyces griseus. J Bacteriol 2002, 184:91-103.
63. Ramos A, Honrubia MP, Valbuena N, Vaquera J, Mateos LM, Gil JA: Involvement of DivIVA in the morphology of the rod-shaped actinomycete Brevibacterium lactofermentum. Microbiol 2003, 149:in press.
57. Fla¨ rdh K, Findlay KC, Chater KF: Association of early sporulation genes with suggested developmental decision points in Streptomyces coelicolor A3(2). Microbiol 1999, 145:2229-2243.
64. Wenner T, Roth V, Fischer G, Fourrier C, Aigle B, Decaris B, Leblond P: End-to end fusion of linear deleted chromosomes initiates a cycle of genome instability in Streptomyces ambofaciens. Mol Microbiol 2003, 50:in press.
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Current Opinion in Microbiology 2003, 6:564–571