Compared to cytokinesis at the mid-cell of a unicellular bacterium, the conversion of a long syncytial hyphal cell into a chain of unigenomic prespore compartments places particular demands on how cell division is orchestrated during sporogenesis in Streptomyces
. Indeed, our knowledge of how multiple, regular-spaced septa are formed and how this is coordinated with chromosome replication and segregation is limited. Developmental upregulation of ftsZ
transcription occurs in sporogenic hyphae, dependent on several whi
genes that encode early-stage sporulation regulators (10
). The regular placement of multiple FtsZ rings is preceded by a protracted phase during which spiral-shaped FtsZ filaments are formed along the length of an aerial hyphal cell (12
). Remodelling of these filaments is believed to generate an array of regular-spaced Z rings, formation of which immediately precedes cytokinesis. The identity of factors that guide regular placement of Z rings is unclear. Some irregularly placed septa are observed in mutants defective in chromosome partitioning (22
), suggesting some influence by this process.
In addition to the orchestration of Z-ring placement in sporogenic cells, the early events of cytokinesis must be coordinated with growth arrest of an aerial hypha and limited to apical syncytial cells. Genetic and cytological evidence indicates that CrgA has a critical function in coordinating this aspect of development, particularly during growth on glucose-containing medium. A crgA mutant produces a greater abundance of hyphae in which Z-ring formation and subsequent regular septation occurs, and the length of the zones of these hyphae in which cytokinesis occurs is significantly greater than in the wild type. The latter observation may be a consequence of delayed growth arrest of aerial hyphae in the mutant so that apical sporogenic cells are longer. Expression of CrgA in the wild type is primarily regulated at the level of transcription, to peak immediately before the appearance of aerial hyphae. The translated integral membrane protein then localizes to discrete foci away from the emerging hyphal tips. Evidence that CrgA inhibits Z-ring formation comes from ectopic overexpression, leading to CrgA foci distributed throughout the length of the aerial hyphae. In this case developmental upregulation of ftsZ in sporogenic cells was evident, but the protein was not assembled into regularly placed rings. Evidently, in these cells CrgA affects either correct remodeling of spirals or the stability of FtsZ polymers; the failure to form rings underlies the inability for these cells to undergo sporulation septation.
CrgA expression not only influences the dynamics of Z-ring formation, but also has a dramatic effect on the timing of FtsZ expression and its turnover. After 24 h growth, in the absence of CrgA, the organism undergoes precocious development and concomitant transient upregulation of FtsZ expression. The level of FtsZ protein in the mutant subsequently declines rapidly; this level of turnover is not seen for another 48 h in the wild type. Protein turnover after transcriptional upregulation of ftsZ
has a precedent in another differentiating bacterium, Caulobacter crescentus
). In this organism, transcription of ftsZ
is upregulated to allow the initiation of cell division in stalked cells. Immediately afterwards, FtsZ undergoes rapid proteolysis, especially in progeny motile swarmer cells that are consequently unable to initiate cytokinesis until after an obligatory gap period. Developmentally programmed proteolysis of FtsZ in spore-forming cells of S. coelicolor
is likely to be important in establishing dormancy, as the spores mature prior to their dispersal.
In contrast to precocious upregulation of FtsZ expression in the absence of CrgA, overexpression of the latter reduces the levels of FtsZ protein in sporogenic hyphae. This is not a consequence of a failure to upregulate transcription. Rather, the observed reduction in levels of FtsZ is presumably a consequence of higher turnover of the protein. This turnover may result from the failure to remodel FtsZ spiral filaments into productive Z rings, stabilized by association with other cell division proteins (the divisome).
How does CrgA function? A precedent for a membrane-associated inhibitor of Z-ring formation is EzrA of Bacillus subtilis
). This protein can interact directly with FtsZ, preventing its polymerization. However, our evidence suggests a different mode of action for CrgA. Firstly, FtsZ polymer spirals, but not productive Z rings, are formed in hyphae in which CrgA is overexpressed. Secondly, EzrA is distributed throughout the plasma membrane of dividing cells, but it also concentrates at the cytokinetic ring in an FtsZ-dependent manner (14
). This localization pattern is interpreted as implying that EzrA prevents Z-ring assembly anywhere along the inner surface of the membrane. A Z ring is formed at the mid-cell due to the activity of an as-yet-unidentified temporally regulated positive factor that can overcome the inhibitory activity of EzrA. The latter can then interact with FtsZ polymers but not promote disassembly. In contrast, CrgA is localized to foci in hyphal cells in which Z rings that precede sporulation septa are absent. Lastly, EzrA and CrgA have quite different topologies. The former has a single 26-residue membrane-spanning domain. Although deletion of this anchor interferes with the protein's function in vivo, the remaining 236-residue cytoplasmic domain is sufficient to inhibit FtsZ assembly in vitro (14
). In contrast, the two transmembrane domains of CrgA comprise half of the protein, and the only significant cytoplasmic portion of the protein is a 30-residue nonconserved N-terminal domain. Overexpression of this cytoplasmic domain, after deletion of both transmembrane domains, has no effect on sporulation (results not shown). Identity between full-length S. coelicolor
CrgA and nonstreptomycete orthologs in other actinobacteria is between 28 and 40%. This contrasts with the >65% identity shared between FtsZ proteins from the same species. The conserved residues of CrgA are all immediately at the boundaries of or within the transmembrane domains. Several of these residues are likely to promote intimate interhelical interactions between TM1 and TM2, supporting a hairpin-like topology of the protein. The internal half of TM1 is rich in large branched chain residues at appropriate depths to enable their interaction with glycine residues (including the conserved Gly 76 and widely found Gly 71) located in the internal half of TM2. Conversely, at the middle of the membrane, there is a conserved glycine residue (Gly 42) on TM1 that is predicted to interact with either the conserved Phe 70 or Val 68, both at the appropriate membrane depth, on TM2. Towards the outer halves of the two transmembrane domains there is decidedly less scope for close helix packing. Indeed, the successive conserved bulky Trp 64 and polar Asn 65 residues of TM2, opposite a bulky and hydrophobic portion of TM1, suggest a possible role for this region in specific interactions with other proteins. Importantly, the topology predictions and absence of any conservation of the cytoplasmic domain both suggest that, in contrast to EzrA, CrgA itself does not directly interact with cytoplasmic FtsZ. Instead, interactions, possibly with other membrane-associated protein components of the divisome, may be critical in preventing remodelling of FtsZ spirals. In E. coli
, both ZipA and FtsA are implicated in tethering Z rings to the membrane via their respective transmembrane domains (27
). The fully sequenced actinomycetes lack orthologs of these proteins. FtsZ from M. tuberculosis
can interact with FtsW (Rv2154c), an integral membrane protein, through sequences in these proteins that appear to be unique to this genus (5
). The identity of the anchor in Streptomyces
is unclear: there are four ftsW/rodA
-like genes in S. coelicolor
, but the corresponding proteins do not possess a similar C-terminal tail to that of the M. tuberculosis
FtsW implicated in FtsZ interaction.
is present as a single copy in all sequenced actinomycete genomes, including intracellular pathogens with severely reduced genomes such as T. whipplei
and M. leprae
, but not other prokaryotes. All the sequenced actinomycete genomes also lack orthologs of minC
. We propose that CrgA has an important role as an inhibitor of Z-ring formation in actinomycetes generally, coordinating growth with cytokinesis. A conservation of function is supported by the conserved location of the gene, close to oriC
and bordering a conserved morphogenic cluster. This cluster includes genes encoding RodA (SCO3846 in S. coelicolor
; Rv0017c in M. tuberculosis
) and a penicillin binding protein, both of which that have been implicated in peptidoglycan synthesis during bacterial growth (7
), and a signaling kinase containing PASTA (penicillin-binding protein and serine-threonine kinase associated) domains involved in the control of cell shape in M. tuberculosis
). Further insight into CrgA function is of particular importance in understanding cell division in pathogenic actinomycetes such as M. tuberculosis
, against which there is an urgent need to develop new chemotherapeutics.