Cell cycle regulation by the bacterial nucleoid

Highlights • Nucleoid occlusion prevents cell division over the bacterial chromosome.• Nucleoid occlusion factors identified in B. subtilis, E. coli and S. aureus.• Noc and SlmA are sequence specific DNA-binding proteins.• They both act as spatial and temporal regulators of cell division.• Using some basic general principles bacteria employ diverse regulatory mechanisms.


Introduction
How cell division is coordinated with the replication and segregation of chromosomes is a fundamental problem in biology. Bacteria are no exception. They employ sophisticated regulatory mechanisms to maximise the fitness of progeny by ensuring that they are suitably sized and inherit an intact copy of the genome. Bacteria typically contain a single circular chromosome that is replicated bidirectionally from a single origin of replication (oriC; 08). During replication the newly synthesised sister chromosomes rapidly segregate 'origin-first' in opposite directions, before the replication forks rendezvous and terminate in the terminus region (Ter; 1808). Once chromosome replication and segregation are complete the cell is ready to divide. In Bacteria this normally occurs by binary fission and in almost all species this is initiated by the assembly of the tubulin homologue FtsZ into a ring-like structure ('Z-ring') at the nascent division site ( Figure 1) [1]. The Z-ring then functions as a dynamic platform for assembly of the division machinery [2,3]. Its central role in division also allows FtsZ to serve as a regulatory hub for the majority of regulatory proteins identified to date [2,4]. Nevertheless, the precise ultrastructure of the Z-ring and whether or not it plays a direct role in force-generation during division remains controversial [5,6].
Although outwardly a simple process, the division site must be chosen carefully. Division at the pole would produce a non-viable anucleate 'mini-cell'. Conversely, division through the nucleoid would be catastrophic, generating at least one non-viable cell. In the best studied Gram-positive and Gram-negative rod-shaped model organisms, Bacillus subtilis and Escherichia coli, the division site is placed precisely at mid-cell [7][8][9]. Thus, division results in the production of two equally sized daughter cells. Such remarkable levels of precision are thought to result largely from the combined action of two negative regulatory systems. In the first, the Min system prevents division close to the cell poles, by inhibiting Z-ring assembly [10]. This is accomplished by the FtsZ inhibitor MinC, which is recruited into a membrane-associated complex by the ParA-like ATPase MinD [11]. In E. coli MinCD oscillates from pole-to-pole [12][13][14] whereas in B. subtilis it is recruited to both cell poles [15], but the net result is the same, with the active complex enriched at the cell poles (Figure 1a,b). The second regulatory system involves the long-standing observation that the nucleoid (bacterial chromosome) can itself act as a cell cycle 'checkpoint' and prevent division until the replicated sister chromosomes have segregated-a process termed nucleoid occlusion [16][17][18]. The foremost role of this process is in the 'anti-guillotine' checkpoint, which prevents catastrophic bisection of the genome by the division machinery. This is achieved by preventing assembly of the Z-ring over the nucleoid (Figure 1a,b). Consequently, nucleoid occlusion might not only act to protect DNA, but likely also acts positively to help identify the division site by directing the division machinery to the DNA-free zone that develops between the newly replicated chromosomes. Although widely recognised as a potentially critical regulatory system, it was only in the last decade that specific factors involved in this process were identified. Additionally, it is now known that bacterial chromosomes are subject to intricate largescale organisation, for example, structured macrodomains that occupy specific positions within the cell [19,20]. Moreover, translation also occurs in a spatially restricted manner [21]. Therefore, besides acting as a 'template' for specific regulatory proteins, the overall organisation and activity of the nucleoid may also play a more general role in regulating division. In this review we will describe recent progress in understanding the process of nucleoid occlusion as well as highlighting some of the diverse solutions employed by less-well studied bacteria.

Specific nucleoid occlusion factors
About 10 years ago the first nucleoid occlusion proteins were identified. Noc in B. subtilis [22 ], and in parallel work, SlmA (synthetic-lethal with min) in E. coli [23 ].
The absence of these proteins allows cell division to occur over the nucleoid under conditions in which DNA replication or cell division are perturbed [22 ,23 ]. Both proteins inhibit division when overproduced and, as might be expected, are synthetic-lethal with defects in the Min system and other genes involved in division site selection; a phenotype that facilitated their initial identification [22 ,23 ]. Contrary to expectations, however, the loss of two regulatory systems does not lead to unfettered division. Instead, it causes a severe division block, apparently because FtsZ assembles indiscriminately throughout the cell, such that it is unable to form a productive structure at any one particular site [22 ,23 ].
To function properly nucleoid occlusion factors must act in a controlled manner. An obvious mechanism would be to link their activity to DNA binding. In B. subtilis chromatin affinity precipitation followed by microarray analysis   Importantly, the introduction of a multi-copy plasmid carrying a single NBS led to a severe division defect, which was dependent on both the ability of Noc to bind DNA and on the presence of the NBS on the plasmid [24 ]. These findings indicated that Noc activity is coupled to specific DNA binding and are consistent with the idea that the relatively mild division defect caused by Noc overproduction is due to the spatial constraints imposed by the nucleoid. Likewise, in E. coli SlmA binds specifically to around 24-52 palindromic SlmA binding sites (SBSs) (Figure 2a)

Noc associates with both DNA and the cell membrane
Understanding the mechanism by which Noc acts has proved more challenging. In B. subtilis Noc is an abundant protein (4500 molecules per cell) and forms large nucleoprotein complexes at the NBSs [24 ]. It localises to the nucleoid and forms dynamic foci at the overlying cell periphery (Figure 2b) [24 ]. An early hypothesis was that these foci represented sites of interaction between Noc and its target. Nevertheless, all attempts to identify a direct protein target have so far been unsuccessful [24 ,30]. Recently, however, evidence has been discovered that Noc associates directly with the cell membrane and that complex assembly at NBSs controls this activity [Adams, Wu and Errington, unpublished]. Even so, in the absence of a defined target, the question remains-how does Noc inhibit division?
The developmental lifestyle of B. subtilis poses additional challenges (Figure 1b). During sporulation the chromosomes adopt an elongated configuration with the oriC regions tethered to the poles and the Ter regions at midcell. An asymmetric septum then forms close to one of the poles, trapping roughly one-third of the chromosome. The remaining DNA is then 'pumped' into the prespore compartment by the DNA-translocase SpoIIIE [31]. Thus, Noc activity must be relieved at the cell poles and Z-ring assembly prevented at mid-cell. Noc activity may be attenuated by the down-regulation of noc expression [32] coupled with an underrepresentation of NBSs in the regions close to the trapping event (Figure 1b) [24 ]. Alternatively, the altered chromosome organisation might itself inactivate Noc. Interestingly, the TetR-like protein RefZ (regulator of FtsZ) was recently shown to promote the redistribution of Z-rings from mid-cell to the cell poles ( Figure 1b) [33]. It appears to act on FtsZ directly via a DNA-dependent mechanism, suggesting that other DNA-binding proteins may use the specific conformation of the nucleoid during sporulation to help specify the division site [33].

Critical role for Noc in S. aureus
Since the chromosome occupies the majority of the cytoplasm in coccoid bacteria such as Staphylococcus aureus, nucleoid occlusion might be expected to play a more central role, especially given that in many cocci there is no Min system [34]. Indeed, Veiga et al. [35 ] recently showed that the deletion of noc results in a significant increase in cell size and strikingly, around 15% of cells contained division septa assembled over the nucleoid. Importantly, the detection of a similar frequency of DNA breaks confirmed that the DNA was bisected by the division machinery and not just trapped by it, thus indicating that even during normal growth conditions Noc plays a critical role [35 ]. Given its clinical importance it will be interesting to test whether the noc mutant is attenuated for virulence. Similar to B. subtilis, microscopy suggests that Noc is absent from the terminus region of the S. aureus chromosome [35 ], consistent with the prediction of a highly asymmetric distribution of NBSs [30]. Interestingly, S. aureus divides in three consecutive perpendicular planes. Veiga et al. [35 ] propose a model in which segregation of the chromosomes parallel to the incipient division septum provides only one possible plane free from nucleoid occlusion, which in combination with another geometric cue, perhaps a division 'scar' [36], then restricts the selection of the next division plane.

Noc/SlmA independent nucleoid occlusion
In B. subtilis or E. coli cells lacking both a functional Min and nucleoid occlusion systems FtsZ assembly still exhibits a clear bias towards the inter-nucleoid spaces [22 ,23 ,37 ], indicating that there are probably further mechanisms governing division site selection in these organisms. Intriguingly, in E. coli cells with unusual shapes the nucleoid appears to be the primary factor influencing division site selection [38]. Likewise, experiments with blocked or non-replicating nucleoids in B. subtilis [39] and E. coli [40] have shown that the nucleoid prevents Z-ring assembly in its vicinity, independently of both Noc/SlmA and the SOS-response. One possibility is that there are additional, but as yet uncharacterised, nucleoid occlusion factors present. Indeed, some possible candidates exist, though any involvement in division site selection is yet to be determined [41,42].
Another possibility is that the activity or organisation of the nucleoid may itself play a role in restricting Z-ring assembly [43]. One classical proposal is that the presence of large membrane-associated complexes in the vicinity of the nucleoid resulting from transertion; the coupled transcription, translation and insertion of membrane proteins, generates a short range inhibitor of cell division [16][17][18]44]. The recent demonstration that loci encoding membrane proteins are repositioned towards the membrane upon induction lends support to this idea [45]. Alternatively, specific loci or chromosomal domains may play an active role. MatP, a protein that organises the Ter macrodomain (MD) of E. coli [46] is recruited to the Z-ring via a direct interaction with ZapB [47 ]. This could simply serve as a convenient way to retain the Ter MD at mid-cell so that it can be processed by FtsK [48][49][50]. However, recent work by Bailey et al. [51], suggests that this association can also act positively by providing a 'landmark' for Z-ring assembly between the replicated chromosomes. Although normally this process appears to be relatively weak, it plays a more prominent role in cells lacking MinC and SlmA [51]. Interestingly, since the Ter appears to leave mid-cell just prior to constriction, might this process also communicate the completion of chromosome segregation? [47 ].
A third distinct possibility is that DNA-translocases present in the division machinery such as FtsK/SpoIIIE proteins might clear the DNA from beneath the closing septum [52,53]. In Streptococcus pneumoniae this process may play a more prominent role, especially given the small volume of these cells and the observation that the Z-ring appears to assemble on top of unsegregated nucleoids [34,54,55]. However, while DNA translocation provides a valuable 'fail-safe' mechanism, another level of redundancy would seem desirable, particularly since results in other organisms demonstrate that DNA translocases are not always 100% effective [22 ,23 ,35 ].

Positive regulation of division site selection
In contrast to the well-studied systems that negatively regulate division site selection, it has been proposed that additional mechanisms might exist that act positively, for example, by contributing an essential division component or by counteracting an inhibitor [37 ,56]. Rodrigues and Harry [37 ] recently showed that in a B. subtilis min noc background the frequency of Z-ring assembly was both dramatically reduced and subject to a significant delay. Nevertheless, in a small population of cells there was a substantial preference for Z-ring assembly at mid-cell, independently of either the Min system or the nucleoid [37 ], which the authors propose might result from unknown factors that actively identify the division site [37 ]. Currently, however, only two examples of positively acting systems have been reported in bacteria.
In Streptomyces spp., which are multi-nucleate filamentous bacteria that produce long chains of spores, FtsZ initially assembles extended 'spiral' structures on top of the nucleoids during sporulation, before forming a 'ladder' of Z-rings between the segregated nucleoids [57].
Remarkably, SsgA directs the localisation of a membrane-associated protein, SsgB, into these inter-nucleoid spaces where it recruits FtsZ, and possibly enhances Z-ring assembly [58 ]. But how does SsgA direct its partner to the correct site? Willemse et al. [58 ] highlight that in a filamentous bacterium where the cell poles are distant the nucleoid presents an ideal candidate to control this critical task. A comparable mechanism is used by Schizosaccharomyces pombe, whereby the nucleus restricts the localisation of Mid1p, which acts positively to position the division site [59,60]. More recently, the ParA-like protein PomZ (Positioning at midcell of FtsZ) was found to positively regulate Z-ring positioning in Myxococcus xanthus, although it probably acts indirectly since a direct interaction with FtsZ could not be established [61]. Interestingly, PomZ first localises over the nucleoid, before moving to mid-cell ahead of FtsZ, raising the possibility that this might be triggered by the completion of chromosome replication/segregation. Furthermore, abnormal cell divisions in DpomZ cells never occurred over the nucleoids, indicating that M. xanthus probably contains a nucleoid occlusion system [61].
A role for ParABS systems?
Although Caulobacter crescentus lacks obvious homologues of MinCD or nucleoid occlusion factors, an alternative mechanism has been identified that combines aspects of both systems. MipZ (mid-cell positioning of FtsZ), a divergent ParA/MinD family ATPase, is essential for the correct placement of the division site [62 ]. It forms ATP-dependent dimers that interact with and perturb FtsZ polymers by stimulating their GTPase activity [62 ]. Strikingly, dimer formation is stimulated by ParB, which is localised at the cell poles with the origin [63]. Consequently, MipZ dimers emanate outwards from the cell poles on DNA such that their lowest concentration is towards mid-cell (Figure 1c) [62 ,63]. Nevertheless, since cell division apparently initiates with the chromosome still present at mid-cell, other factors are likely to be involved [64]. Interestingly, ParA-like proteins may also play a role in division site selection in Corynebacterineae (see for [65] indepth review). In Corynebacterium glutamicum PldP, an orphan ParA-like division protein, localises over the nucleoid and at the nascent division site [66]. Since null mutants exhibit a division defect, PldP might help to link chromosome segregation and cell division in C. glutamicum [66,67]. Similarly, a ParA-like protein also seems to have a division specific role in Mycobacterium smegmatis, but whether it acts directly remains unclear [68].

Concluding remarks
The identification of Noc and SlmA provided a molecular basis for nucleoid occlusion and considerable progress has been made in the last decade in understanding the mechanisms by which these factors act. While the primary role of nucleoid occlusion is almost certainly to prevent catastrophic guillotining of the genetic material, in B. subtilis and E. coli, Noc and SlmA also play integral roles in helping to specify the spatial and temporal organisation of division. Noc may also play a role in restricting division to one plane in S. aureus. Nonetheless, it is clear that in some bacteria other factors may act to protect the DNA at later stages of division, for example, DNA translocases. One important area of future research will be how nucleoid occlusion varies with growth rate, particularly since during slow growth, in E. coli at least, the chromosomes appear to segregate as cell division initiates [69]. Another open question is whether nucleoid occlusion is active in bacteria with fundamentally different modes of growth and division, for example, symbiotic bacteria that grow in width [70] or in bacteria that divide independently of FtsZ [71,72]. Similarly, how does nucleoid occlusion differ in bacteria such as V. cholerae that have more than one chromosome? As the range of organisms studied and the development of novel genetic and cell-biology tools continues to rapidly expand, we anticipate new insights into how diverse organisms tackle this fundamental problem.

27.
Cho H, Bernhardt TG: Identification of the SlmA active site responsible for blocking bacterial cytokinetic ring assembly over the chromosome. PLoS Genet 2013, 9:e1003304. This paper identified the FtsZ interaction site on SlmA and showed that its proximity to the DNA-binding domain provides a simple mechanism for DNA-dependent activation. The work also showed that even though SlmA acts as a dimer, a single FtsZ-binding site is necessary and sufficient for SlmA function.

28.
Tonthat NK, Milam SL, Chinnam N, Whitfill T, Margolin W, Schumacher MA: SlmA forms a higher-order structure on DNA that inhibits cytokinetic Z-ring formation over the nucleoid. Proc Natl Acad Sci U S A 2013, 110:10586-10591. This paper reports co-crystal structures of SlmA and SBS DNA and makes the unexpected finding that by distorting DNA, SlmA is able to bind to SBSs as a dimer of dimers.

29.
Du S, Lutkenhaus J: SlmA antagonism of FtsZ assembly employs a two-pronged mechanism like MinCD. PLoS Genet 2014, 10:e1004460. This paper resolves the uncertainty regarding how SlmA modulates Z-ring assembly by demonstrating that SlmA inhibits FtsZ polymerisation under all conditions previously tested and by isolating FtsZ mutants that can still interact with SlmA but are no longer sensitive to its inhibitory effect. The work also shows that FtsZ interacts with SlmA via its conserved Cterminal tail.