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Logo of nihpaAbout Author manuscriptsSubmit a manuscriptNIH Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
Mol Microbiol. Author manuscript; available in PMC May 1, 2012.
Published in final edited form as:
PMCID: PMC3097173
NIHMSID: NIHMS286553

Multiple orphan histidine kinases interact directly with Spo0A to control the initiation of endospore formation in Clostridium acetobutylicum

Abstract

The phosphorylated Spo0A transcription factor controls the initiation of endospore formation in Clostridium acetobutylicum, but genes encoding key phosphorelay components, Spo0F and Spo0B, are missing in the genome. We hypothesized that the five orphan histidine kinases of C. acetobutylicum interact directly with Spo0A to control its phosphorylation state. Sequential targeted gene disruption and gene expression profiling provided evidence for two pathways for Spo0A activation, one dependent on a histidine kinase encoded by cac0323, the other on both histidine kinases encoded by cac0903 and cac3319. Purified Cac0903 and Cac3319 kinases autophosphorylated and transferred phosphoryl groups to Spo0A in vitro, confirming their role in Spo0A activation in vivo. A cac0437 mutant hyper-sporulated, suggesting that Cac0437 is a modulator that prevents sporulation and maintains cellular Spo0A~P homeostasis during growth. Accordingly, Cac0437 has apparently lost the ability to autophosphorylate in vitro; instead it catalyses the ATP-dependent dephosphorylation of Spo0A~P releasing inorganic phosphate. Direct phosphorylation of Spo0A by histidine kinases and dephosphorylation by kinase-like proteins may be a common feature of the clostridia that may represent the ancestral state before the great oxygen event some 2.4 billion years ago, after which additional phosphorelay proteins were recruited in the evolutionary lineage that led to the bacilli.

Keywords: sporulation, clostridia, autophosphorylation, phosphotransfer, phosphorelay

Introduction

Bacillus subtilis forms endospores in response to nutrient (carbon, nitrogen or phosphorus) starvation (Schaeffer et al., 1965). The Spo0A transcription factor, acting in concert with a network of other regulatory proteins, controls the initiation of endospore formation in this organism (Perego, 1998; Hoch, 2002; Piggot and Hilbert, 2004), effectively ensuring that spore development is only initiated once alternative survival strategies have been fully explored (Hoch, 1993; Strauch and Hoch, 1993). Genes under direct spo0A control are characterised by the presence of one or more “0A boxes” (TGNCGAA) in their 5′ regulatory regions to which the activated form of the protein, Spo0A~P, binds (Strauch et al., 1990; Molle et al., 2003). Up-regulation occurs when the 0A box lies upstream (i.e. distal to the coding sequence) from the promoter; down-regulation occurs when the 0A box overlaps with or is located downstream (proximal to the coding sequence) from the promoter, sometimes within the coding sequence (Strauch et al., 1990; Fawcett et al., 2000; Molle et al., 2003).

In B. subtilis, Spo0A~P is generated via a phosphorelay, which integrates the various environmental, cell cycle and metabolic signals that control the onset of sporulation. In response to these signals, multiple sensor histidine kinases phosphorylate the Spo0F response regulator whose phosphoryl group is transferred to the sporulation transcription factor, Spo0A, via the Spo0B phospho-protein phosphotransferase (Burbulys et al., 1991; Hoch, 1993). Several additional proteins, including the Rap phosphatases that act on Spo0F~P (Perego et al., 1994), and the Spo0E, YnzD and YisI phosphatases that act on Spo0A~P (Perego, 2001), influence the flux of phosphate through the phosphorelay and control the phosphorylation status of B. subtilis Spo0A.

Much less is known about the initiation of endospore formation in Clostridium acetobutylicum. In laboratory batch culture volatile fatty acids (acetate and butyrate) are produced by the fermentation of glucose during exponential growth. They accumulate in the medium causing a drop in the culture pH. As the culture approaches stationary phase, solvent (butanol, acetone & ethanol) production is initiated (Jones and Woods, 1986). The acetate and butyrate produced previously are re-assimilated and as a result, the culture pH rises. At about this time, motility decreases, sporulation is initiated and granulose (a glycogen-like storage product) accumulates in swollen, phase-bright clostridial forms, within which endospores develop. C. acetobutylicum will only form endospores if nutrients are in excess (Jones and Woods, 1986), but the environmental trigger(s) that provokes endospore formation by this organism is not known.

As is the case in the bacilli, Spo0A controls the initiation of endospore formation in the clostridia. Genes directly controlled by Spo0A have the same characteristic 0A boxes in their promoter regions (Brown et al., 1994; Wilkinson et al., 1995; Ravagnani et al., 2000; Harris et al., 2002; Huang and Sarker, 2006). However, the available genome sequence information provides no evidence that clostridia have a phosphorelay similar to that found throughout the bacilli (Stephenson and Hoch, 2002; Stragier, 2002; Dürre and Hollergschwandner, 2004). Although all clostridia contain spo0A, most species lack spo0B and none contain an obvious counterpart of spo0F (Stephenson and Hoch, 2002). This raises the intriguing question of how clostridia phosphorylate Spo0A. Several mechanisms have been suggested. The possible presence of a novel phosphorelay has been considered (Stephenson and Lewis, 2005), as has a potential role for metabolites such as butyryl phosphate or acetyl phosphate (Lukat et al., 1992; Paredes et al., 2005; Zhao et al., 2005). Another proposal, which has some experimental support, is that one or more of the orphan kinases found in these organisms might phosphorylate Spo0A directly (Dürre and Hollergschwandner, 2004; Stephenson and Lewis, 2005). Firstly, there is evidence of functional interaction between one of the orphan kinases of Clostridium botulinum, CBO1120, and C. botulinum Spo0A when both genes are co-expressed in B. subtilis (Wörner et al., 2006). Secondly, phosphorylation of Clostridium difficile Spo0A by one of its cognate orphan kinases (CD1579) has been demonstrated in vitro, although a role for this particular kinase in spore development was not established (Underwood et al., 2009). Thirdly, there is evidence for direct interaction between sensory histidine kinases and Spo0A in B. subtilis (Perego et al., 1989; Kobayashi et al., 1995; Jiang et al., 2000).

The kinases that feed phosphate into the B. subtilis phosphorelay are all orphans, lacking a cognate response regulator (Fabret et al., 1999). C. acetobutylicum encodes 35 histidine kinases, of which five are orphans (Nolling et al., 2001). Gene expression profiles have been published, covering the shift from exponential growth to stationary phase in laboratory culture, but analysis of these data has not provided any clear evidence that one or more of these orphan kinases is involved, either directly or indirectly, in Spo0A phosphorylation (Paredes et al., 2005; Jones et al., 2008). Here we use a combination of in vivo and in vitro approaches to show that the orphan kinases of C. acetobutylicum positively and negatively control the initiation of endospore formation by interacting with Spo0A directly to catalyse its phosphorylation and dephosphorylation.

Results

Phenotypic consequences of histidine kinase deletions

Five histidine kinase mutants were characterised using the wild type and a spo0A mutant (Heap et al., 2007) as reference strains. The sporulation frequency of strains harbouring mutations in either cac0323, or cac0903, or cac3319 was reduced to 4%, 5% and 1% of the wild type frequency, respectively (Fig. 1), indicating that the products of these three genes promote spore formation. Surprisingly, the sporulation frequency of the cac0437 mutant was enhanced more than 30-fold over wild type levels, with conversion of almost the entire bacterial population into endospores, suggesting that the protein encoded by cac0437 functions to inhibit spore formation rather than promote it. The cac2730 mutant sporulated at about the same frequency as the wild type (Fig. 1). The phenotypes of all five mutants were confirmed by microscopy and by tests for granulose production (Supplementary Fig. S1).

Figure 1
Sporulation phenotypes of mutant strains. Single mutants (grey bars) and double mutants (black bars) were generated by inactivation of the indicated genes. To elucidate the function of the Cac0437 kinase (white bars), a multi-copy plasmid carrying wild ...

Several double mutants were constructed to further explore the functions of the five orphan kinases. Disruption of cac0323 together with either cac0903 or cac3319 completely abolished sporulation, whereas a strain lacking both cac0903 and cac3319 formed spores at a frequency that was only slightly reduced compared with that observed in the single mutants (Fig. 1). These results suggested that two separate pathways control the initiation of sporulation in liquid medium, one dependent on cac0323 and the other dependent on both cac0903 and cac3319.

The sporulation frequencies of mutants lacking either cac0903 or cac3319 were restored to greater than wild type levels by the introduction of the cac0437 mutation, confirming an antagonistic role for the cac0437 product in the initiation of sporulation (Fig. 1). This conclusion was further reinforced by the introduction of multiple copies of cac0437 into the wild type strain as a result of which, the spore frequency was reduced to about 0.02% of the wild type level (Fig. 1). Finally, the cac0323 and cac3319 phenotypes were effectively complemented when wild type copies of either cac0323 or cac3319 were re-introduced on an autonomous plasmid into a variety of single, double and triple mutants (Supplementary Fig. S2).

Instability of the cac0437 mutant

The hypersporulation phenotype associated with inactivation of cac0437 (Fig. 1) was readily lost upon subculture or storage of the strain and in some cases surviving colonies had completely lost the ability to form spores and granulose (data not shown). This instability was evident in three different cac0437 mutants, each with an intron insertion at a different position within the coding sequence. The data reported here were obtained with a strain harbouring an insertion in the region encoding the HisKA domain (see Supplementary Fig. S3). Because of its intrinsic instability, this mutant had to be re-isolated afresh for each experiment. The underlying basis of the instability was not studied extensively, but in three independent stable derivatives the spo0A gene had been affected. Two of these isolates harboured different frameshift mutations near the 5′ end of the coding sequence and in the third, a 1276 bp segment encompassing the entire spo0A gene had been deleted as a result of recombination between two imperfect 18/19 bp repeats – ATATATAG(t)GAGAAGGATA – located upstream and downstream of the coding sequence. The cac0437 mutant embarked upon massive sporulation inappropriately early (Fig. 1 & Supplementary Fig. S1) and there was therefore strong selective pressure for overgrowth by spontaneous variants harbouring compensatory mutations that permitted the organism to continue growing when the parental strain was sporulating.

spo0A expression is cac0903-dependent

Although the strains harbouring cac0323, cac0903 or cac3319 mutations had similar sporulation frequencies, measured after 5 days (Fig. 1), early spo gene expression measured by quantitative real time PCR (qRT-PCR) during the first few hours of sporulation was affected much more drastically in the cac0903 mutant than in the cac0323 or cac3319 mutants (Fig. 2). This probably accounts for the observed delay in granulose production and sporulation of the cac0903 mutant (Supplementary Fig. S1). Expression of spo0A was severely depressed compared with wild type in the cac0903 mutant, whereas it was almost unaffected in the cac0323, cac3319 and cac2730 mutants (Fig. 2A and data not shown), indicating that spo0A expression is dependent on cac0903, but not on cac0323, cac3319 or cac2730. In B. subtilis, the expression of spoIIAA and sigG is either directly or indirectly dependent on Spo0A (Grossman, 1995; Hilbert and Piggot, 2004) and this may also be the case in C. acetobutylicum, since the cac0903 mutant only expressed spoIIAA and sigG to the same basal level as seen in the spo0A control over the time period of the experiment (Fig. 2B, C). The cac0323 and cac3319 mutants did express spoIIAA and sigG albeit to reduced levels as compared with the wild type (Fig. 2B, C), whereas the cac2730 mutant showed reduced expression of spoIIAA but normal expression of spoIIIG as compared with the wild type (data not shown). Massive precocious expression of spo0A, spoIIAA and sigG was observed in the cac0437 mutant (Fig. 2), consonant with its hyper-sporulation phenotype and with previous work showing that spo0A hyper-expression in C. acetobutylicum was associated with enhanced and accelerated sporulation (Harris et al., 2002; Alsaker et al., 2004; Alsaker and Papoutsakis, 2005). Based on the expression profiles of spoIIAA, sigG and cac0903 (see below) in the wild type strain (Fig. 2D) we deduced that sporulation was initiated at about 4h in these experiments.

Figure 2
Expression of early spo genes in the kinase mutants. The expression of spo0A (A), spoIIAA (B) and sigG (C) was monitored by qRT-PCR during the transition from exponential to stationary phase in three replicate laboratory batch cultures for each strain: ...

Expression of cac3319, cac0323 and cac0437 is cac0903-dependent

To gain further insight into the roles of the various orphan kinases, the expression profiles of their encoding genes were monitored during the transition from exponential to stationary phase. Data for the three informative kinases, encoded by cac0903, cac0437 and cac3319, are given in Fig. 3. Two distinct phases of cac0903 expression occurred in the wild type. There was an initial plateau during exponential growth followed by a period of elevated expression during early stationary phase. In most strains, the early phase of cac0903 expression (pre-4h in Fig. 3A) was similar to wild type. However, it was substantially increased in the cac0437 mutant, indicating that Cac0437 normally limits the extent of cac0903 expression and hence also the expression of spo0A and all the other Spo0A-dependent genes. This is consistent with the elevated spo0A expression and the precocious onset of sporulation seen in the cac0437 mutant (Fig. 1). The late phase of enhanced cac0903 expression (post-4 h in Fig. 3A) was abolished in strains harbouring mutations in spo0A or cac0323 or cac3319 and was therefore dependent on these three genes.

Figure 3
Expression of genes encoding orphan histidine kinases in the mutant strains. The expression of cac0903 (A), cac0437 (B), and cac3319 (C) was monitored by qRT-PCR during the transition from exponential to stationary phase in three replicate laboratory ...

The expression of cac0437 peaked in the wild type as the bacteria entered stationary phase and was essentially unaffected by mutations in cac0323, cac3319 and cac2730 and therefore independent of these kinases (Fig. 3B). However, cac0437 expression remained at a basal level in the cac0903 and spo0A mutants, suggesting that its induction is spo0A-dependent, and therefore also dependent on cac0903.

The results obtained for cac3319 (Fig. 3C) were broadly similar to those obtained for cac0437. The elevated expression that occurred in the wild type as the cells entered stationary phase was completely abolished in the cac0903 and the spo0A mutants, consistent with spo0A-dependence, and more or less normal in the cac0323 and cac2730 mutants. However, expression was elevated throughout the time course in the cac0437 mutant.

The expression of the cac0323 kinase increased over the first 4 h in all strains and then subsided, whereas that of the cac2730 kinase remained relatively constant throughout, never falling below 60 % of its maximal value (Fig. 3D and data not shown). Finally, in Fig. 3D the expression levels of all five kinases (as a percent of the maximum) are compared with those of spo0A in the wild type strain, from which it may be deduced that cac0323, cac0437, cac3319 and cac2730 show maximal expression before that of spo0A, whereas cac0903 and spo0A expression levels peak at the same time.

The basic picture that emerges from these studies of the initiation of sporulation is firstly the dominance of histidine kinase Cac0903 activity and secondly, the need for the product of the cac0437 gene to modulate the expression of the spo0A gene and hence also, the Spo0A-controlled spoIIA gene. At this time and in these conditions the other histidine kinases have only minor effects.

Spo0A phosphorylation in vitro

The five orphan kinases show 38 % sequence similarity in the α-1 helix regions of their HisKA domains (Supplementary Fig. S4), suggesting that they may all interact with the same target protein. A signature sequence, mainly corresponding to residues on the exposed surface of the α-1 helix of Spo0B that makes contact with Spo0F in the co-crystal between these two B. subtilis proteins (Zapf et al., 2000; Hoch and Varughese, 2001), is also present in the Cac3319 kinase of C. acetobutylicum, suggesting that Spo0A is the likely target for this protein and potentially also for the other four orphan kinases found in this organism.

To test this prediction and to understand how the orphan kinases of C. acetobutylicum control the initiation of endospore formation, recombinant histidine-tagged versions of four of the proteins were produced and tested for their ability to autophosphorylate using ATP and to transfer their phosphoryl group to Spo0A. Cac0437 and Cac3319 were made as full length proteins. Truncated versions of the Cac0323 and Cac0903 proteins were made that lack their N-terminal transmembrane helices (see Experimental Procedures and Supplementary Fig. S3). Since the cac2730 mutant lacked a clear sporulation phenotype, Cac2730 was not investigated further.

The Cac0903 and Cac3319 kinases showed both autophosphorylation and phosphotransfer activities (Fig. 4A). The Cac3319 reactions were essentially complete after 15 min, whereas for Cac0903, neither autophosphorylation nor phosphotransfer to Spo0A was complete after 30 min (Fig. 4A, B). Autophosphorylation activity was not detected with either the Cac0323 or the Cac0437 kinases (data not shown and Fig. 5 legend). We suspect that the Cac0323 protein was simply inactive, possibly as a result of the truncation required to express it. The function of Cac0437 is addressed below.

Figure 4
Cac0903 and Cac3319 autophosphorylation and phosphotransfer to Spo0A. (A) Cac0903 and Cac3319 were incubated with [γ-32P]-ATP in the absence (autophosphorylation) or presence (phosphate transfer) of C. acetobutylicum Spo0A (CaSpo0A) for the times ...
Figure 5
Cac0437 does not affect the autophosphorylation or the phosphotransfer activities of Cac3319. (A) Kinetics of phosphotransfer from Cac3319 to C. acetobutylicum Spo0A (CaSpo0A) in the absence or presence of Cac0437. Proteins were added (Spo0A, 8 μM; ...

The results of a series of control reactions involving three native B. subtilis proteins, KinA, Spo0F and Spo0A, in which the specificity of the kinase/Spo0A interaction was explored, are shown in Fig. 4B. Efficient autophosphorylation of B. subtilis KinA in the presence of ATP, and transfer of its phosphoryl group to its natural acceptor, Spo0F, occurred as expected (Fig. 4A, B) (Burbulys et al., 1991). Moreover, B. subtilis KinA was unable to transfer its phosphoryl group directly to B. subtilis Spo0A or to C. acetobutylicum Spo0A. C. acetobutylicum Spo0A was unable to autophosphorylate and the two clostridial kinases, Cac0903 and Cac3319, were unable to transfer phosphoryl groups to B. subtilis Spo0F. Interestingly, the C. acetobutylicum and B. subtilis Spo0A proteins appear to be very effective phosphoacceptors for both Cac0903 and Cac3319 (Fig. 4B). Consistent with this finding, these two Spo0A proteins share 53.7 % sequence identity and 73 % sequence similarity (Brown et al., 1994) and they bind to the same 0A box sequences in the promoter regions of genes under their direct control (Wilkinson et al., 1995; Ravagnani et al., 2000).

The kinetics of phosphotransfer from Cac0903~P and Cac3319~P to Spo0A are shown in Fig. 4C. The reaction was essentially complete within 15 s for Cac3319 and after 1 min for the truncated Cac0903 protein, indicating that Spo0A is probably the natural phosphoacceptor for both kinases.

Mode of action of the Cac0437 kinase

The lack of Cac0437 autophosphorylation (and phosphotransfer to Spo0A) in vitro was consistent with, but did not serve to explain, its inferred inhibitory effect on spore formation in vivo (Fig. 1). To test whether Cac0437 interferes with Spo0A~P production by adversely affecting kinase autophosphorylation or phosphotransfer, these activities were monitored in the presence and in the absence of Cac0437, using Cac3319~P as phosphate donor. The rate of Spo0A~P accumulation in the presence of Cac0437 was reduced to about 40% of that observed in the control (Fig. 5A, B). Cac3319 autophosphorylation and phosphotransfer were then monitored with and without Cac0437 over shorter time periods and these data indicated that Cac0437 did not inhibit either reaction (Fig. 5C). We therefore inferred that the inhibition of Spo0A~P accumulation seen in Fig. 5A & B must involve the removal of phosphoryl groups from Spo0A~P to regenerate Spo0A.

To test this notion, we compared the abilities of the various kinases to accept phosphoryl groups from Spo0A~P in the presence and absence of ADP and ATP (Fig. 6A). This is, incidentally, a specificity test, which showed that all three kinases were able to accept phosphoryl groups from Spo0A~P. Addition of ADP to the reaction with Cac3319 resulted in a normal back reaction that drained the phosphate from the kinase with the concomitant production of ATP (Fig. 6B). To a lesser extent, Cac0903 was also capable of generating ATP. However, Cac0437 appeared devoid of this back reaction. This suggested that Cac0437 may dephosphorylate Spo0A~P directly and not through the intervention of ADP. To test this possibility we incubated Spo0A~P and Cac0437 expecting to observe loss of label in Spo0A~P and the production of inorganic phosphate (Fig. 6C, D). However, label was not removed from Spo0A~P, even after incubation for two hours (lane 2, Fig. 6C). The addition of ATP mobilized the transfer of label to the kinase and the disappearance of label from Spo0A~P within five minutes (Fig. 6C). Moreover, the label went directly to inorganic phosphate with no evidence of new labeled nucleotide (Fig. 6D). Thus Cac0437 neither autophosphorylates with ATP nor forms ATP from kinase~P but possesses an ATP stimulated phosphatase activity of Spo0A~P. Whether kinase~P is an intermediate in this reaction cannot be determined from the present data.

Figure 6
Cac0437 removes phosphoryl groups from Spo0A~P. (A) Dephosphorylation of C. acetobutylicum Spo0A~P (CaSpo0A~P). All proteins were added at a concentration of 2 μM. ADP or ATP was added at 0.5 mM and reactions were allowed to proceed for 60 min. ...

Discussion

We have shown that C. acetobutylicum Spo0A is phosphorylated by direct interaction with orphan histidine kinases. In vivo experiments indicated that Cac0323, Cac0903 and Cac3319 fulfil this role (Fig. 1) and this was confirmed for the latter two proteins by kinase autophosphorylation and phosphotransfer to Spo0A in vitro (Figs. 4 & 5). Under the in vitro conditions employed here, the Cac3319 kinase appeared to phosphorylate Spo0A more efficiently than the Cac0903 kinase. However, it is unlikely that this reflects their relative activities in vivo, since Cac0903 was a truncated protein lacking its N-terminal trans-membrane helices. Moreover, neither protein was assayed in the presence of its (unknown) natural ligand.

To maintain control over the phosphorylation status of Spo0A, which is required to prevent the occurrence of endospore formation under inappropriate conditions, the compensatory process of Spo0A~P dephosphorylation must also occur. In B. subtilis, with its phosphorelay (Burbulys et al., 1991), a variety of protein aspartyl phosphatases undertake this function, including Spo0E, YnzD and YisI that act directly on Spo0A~P (Perego et al., 1994; Perego, 1998; 2001). In C. acetobutylicum this role is assumed by the Cac0437 protein. In vivo experiments showed that Cac0437 exerts an inhibitory effect on spore formation: cac0437 inactivation caused extensive and accelerated sporulation whereas the presence of multiple copies of cac0437 in the wild type inhibited sporulation (Fig. 1). Although annotated as a histidine kinase, we and others (Wurfl, 2008) have been unable to demonstrate Cac0437 autophosphorylation activity in vitro. Surprisingly, Cac0437 dephosphorylated Spo0A~P in vitro in an ATP-dependent fashion (Fig. 6). The reaction mechanism remains to be established.

The complex RedCDEF signalling system of Myxococcus xanthus also contains a histidine kinase-like protein, RedE, which is apparently unable to autophosphorylate but it can accept a phosphoryl group from another protein, the RedD response regulator. The phosphorylated form of RedE functions to remove the phosphoryl group from RedF~P, which is involved in controlling M. xanthus development (Jagadeesan et al., 2009).

According to the qRT-PCR data in Fig. 3, the cac0437, cac0903 and cac3319 genes are actively transcribed (and presumably the cognate proteins are therefore present) during exponential growth. Their competing activities, with Cac0903 and Cac3319 transferring phosphoryl groups to Spo0A and Cac0437 removing them, provide a simple mechanism for controlling the phosphorylation status of Spo0A and maintaining Spo0A~P homeostasis during bacterial growth.

The expression of spo0A was strongly cac0903-dependent (Fig. 2A), suggesting that Spo0A~P induces spo0A expression, as is the case in B. subtilis (Strauch et al., 1992). The presence of a partial 0A box (aGGCGAA) in what appears to be a promoter-proximal position (only 16 bp upstream from the start codon) invites speculation that cac0903 expression is also down-regulated by Spo0A~P. This would have the effect of preventing spo0A hyper-expression and the untimely initiation of sporulation during exponential growth.

The expression of cac0437 was strongly spo0A-dependent (Fig. 3B) and therefore, indirectly dependent on the Cac0903 kinase. The cac0437 gene has two (probably promoter-distal) 0A boxes located in tandem, 156 (TGACGAA) and 166 (TGTCGtA) bp upstream from the start codon, consistent with its observed spo0A-dependence and suggestive of up-regulation mediated by Spo0A~P. If the Spo0A~P concentration rises, so too will the concentration of Cac0437 and this will promote the dephosphorylation of Spo0A~P to maintain homeostasis. As above, the net effect would be to prevent the inappropriate accumulation of Spo0A~P during exponential growth.

Cac0437 down-regulates the expression of both spo0A and cac0903, since cac0437 inactivation enhanced the expression of both genes (Figs. 2A, ,3A).3A). The data presented here indicate that by their concerted action, the Cac0903 (Spo0A phosphorylation) and Cac0437 (Spo0A~P dephosphorylation) proteins control spo0A expression and Spo0A activity, effectively preventing an excessive accumulation of Spo0A~P that would provoke the activation of sporulation under growth conditions: this is precisely what happens in a cac0437 mutant.

We propose that Cac0903 plays the dominant role in the induction of spore formation at or soon after 4 h under the experimental conditions employed here, since its expression level increases rapidly at this time (Fig. 3A, D) coincident with the up-regulation of spoIIAA and sigG. Regardless of the capacity of the other histidine kinases to phosphorylate Spo0A, the expression of the Spo0A~P-dependent spoIIA gene in a cac0903 mutant does not occur even 9 hours into the experiment (Fig. 2B). This seems inconsistent with the results of Fig. 1, which showed decreased sporulation in a cac0903 mutant but not to a greater extent than that observed in a cac0323 or cac3319 mutant. The results in Fig. 1 were obtained after incubation for 5 days and they suggest that prolonged incubation changes the environment sufficiently to allow kinases other than Cac0903 to function. It is noteworthy in this context that on an agar-solidified medium the Cac3319 kinase is required for sporulation and granulose formation, whereas the Cac0323 kinase is not (Supplementary Fig. 1S).

In B. subtilis the various kinases that feed phosphoryl groups into the phosphorelay act independently (Hoch, 1993; 2002). However, in C. acetobutylicum the data indicate the presence of two distinct pathways for Spo0A activation under the experimental conditions we have employed, one dependent on both Cac0903 and Cac3319 and the other on Cac0323 alone. Either pathway on its own can support a low level of sporulation but both must be functional for high level sporulation (Fig. 1) and high level post-exponential phase expression of cac0903 (Fig. 3A).

We have demonstrated that in C. acetobutylicum, and probably also therefore in other clostridia, the initiation of endospore formation is controlled by direct interaction between orphan kinases and Spo0A, circumventing the need for the additional phosphorelay proteins, Spo0F and Spo0B which, are absent from most clostridia (Stephenson and Hoch, 2002; Stragier, 2002; Dürre and Hollergschwandner, 2004). The direct phosphorylation of Spo0A by sensory histidine kinases may represent the ancestral state: the Spo0F and Spo0B proteins were probably recruited subsequently, in the lineage that gave rise to the bacilli, providing additional controls over the level of Spo0A phosphorylation in response to the metabolic changes that occurred as oxygen accumulated in the Earth’s atmosphere, some 2.4 billion years ago (Stephenson and Lewis, 2005).

Experimental procedures

Organisms and Media

Clostridium acetobutylicum ATCC 824 was routinely grown on clostridial basal medium (CBM) (O’Brien and Morris, 1971) or 2×YTG (tryptone, 16 g/l; yeast extract, 10 g/l, NaCl, 5 g/l; glucose, 10 g/l) adjusted to pH5.2 (broth) or pH5.8 (agar-solidified medium) in an anaerobic cabinet (Don Whitley Scientific) at 37 °C under an atmosphere of 80% N2, 10% CO2 and 10% H2. Thiamphenicol (15 μg/ml) and erythomycin (10 μg/ml for CBM; 40 μg/ml for 2×YTG) were added as required. Escherichia coli strains TOP10 (Invitrogen) and Rosetta 2 (DE3) (Novagen) were grown in Luria-Bertani medium (LB) supplemented as appropriate with 100 μg ampicillin/ml, 10 μg chloramphenicol/ml and 25 μg tetracycline/ml. All strains were kept as cell suspensions in 10% glycerol at −80 °C.

DNA isolation

Plasmid DNA from E. coli was prepared using the QIAprep Spin Miniprep Kit (Qiagen). Genomic DNA was isolated from C. acetobutylicum strains using the DNeasy Blood & Tissue Kit (Qiagen).

Bacterial transformation

E. coli strains were transformed by standard procedures. C. acetobutylicum was transformed by electroporation as previously described (Mermelstein et al., 1992) with slight modifications. Briefly, C. acetobutylicum was grown in 2×YTG to an OD600 of 0.9. Cells were harvested by centrifugation, washed and resuspended in ice-cold ETB buffer. The cell suspension (570 μl) was mixed with approximately 1 μg plasmid DNA, placed in a Gene Pulser cuvette (0.4 cm electrode gap; Biorad) and incubated on ice for 2 min. The pulse was delivered with a Gene Pulser (Biorad) at the following settings: 2.5 kV, 25 F, ∞ Ω. After electroporation, cells were immediately transferred into pre-warmed 2×YTG and left to recover for 4 h before plating on antibiotic-supplemented 2×YTG. All plasmids used for clostridial transformation were methylated with pAN-2 (Heap et al., 2007), a derivative of the methylation plasmid pAN-1 (Mermelstein and Papoutsakis, 1993).

Intron re-targeting and RAM removal

The ClosTron (Heap et al., 2007; Heap et al., 2010) was employed for gene inactivation in C. acetobutylicum (http://www.clostron.com/). It is based on the procedure initially devised by Lambowitz and colleagues (Karberg et al., 2001) for insertional inactivation of genes using the mobile group II intron from the ltrB gene of Lactococcus lactis, which can easily be re-programmed for insertion into any target site. The introduction of an antibiotic resistance gene that is, itself, interrupted by a group I intron, couples the acquisition of antibiotic resistance to integration/gene inactivation events (Zhong et al., 2003). This retrotransposition-activated marker (RAM) allows highly efficient positive selection for mutants.

The coding sequences of the five C. acetobutylicum ATCC 824 genes, cac0323, cac0437, cac0903, cac2730, & cac3319, encoding orphan kinases were submitted to the Sigma TargeTron Design Site (http://www.sigma-genosys.com/targetron/) to identify suitable target sites within them. The PCR for intron re-targeting was performed according to the TargeTron Gene Knockout System manual (Sigma-Aldrich) and using the FailSafe PCR Enzyme Mix (Epicentre). The primers used are listed in Supplementary Table S1. The 350 bp PCR products were purified with the QIAquick Gel Extraction Kit (Qiagen) cloned into pGEM-T Easy (Promega) and the integrity of the inserts verified by sequencing. The 350 bp fragments were then sub-cloned into pMTL007C-E2 using the unique HindIII and BsrGI restriction sites in both vector and insert (Heap et al., 2007; Heap et al., 2010) to generate the required re-targeted ClosTron derivatives. For the generation of double and triple mutants, the ermB RAM, which is flanked by FLP recognition target (FRT) sites, was removed following transformation with a FLP recombinase expression vector (Heap et al. 2010). Loss of the RAM was confirmed by PCR and testing for erythromycin-sensitivity and loss of the FLP recombinase-encoding plasmid was confirmed by testing for thiamphenicol-sensitivity, thus permitting the sequential disruption of several genes in one strain.

Generation and confirmation of knockout strains

The locations of the selected intron insertion sites are given in Supplementary Fig. S3. Insertion was in the antisense orientation in cac2730 and in the sense orientation for the remaining genes. Derivatives of pMTL007C-E2 (Heap et al., 2010) harbouring the re-targeted introns were methylated (see above) and electro-transformed into the wild type strain (C. acetobutylicum ATCC 824). Gene-specific PCR primers (Supplementary Table S2) flanking the intron insertion site were employed to distinguish between wild-type and intron-interrupted genes (Supplementary Fig. S5A). GoTaq DNA polymerase (Promega) was used for DNA amplification by thermal cycling: 5 min at 94 °C followed by 30 cycles of 30 s at 94 °C, 30 s at 52 °C & 4 min at 72 °C and finally, 7 min at 72 °C. Southern hybridization with an intron-specific probe revealed that all transformants harboured the desired mutation and that there was only one intron copy per strain (Supplementary Fig. S5B). DNA fragments were transferred to a positively charged nylon membrane (Boehringer) and hybridized using as probe a digoxigenin-labelled 2 kb fragment of the group II intron amplified from pMTL007 with primers ErmRAM-F and CAC3319-771s-IBS (see Supplementary Tables S1 and S2). The labelling and detection kit (Roche) was employed according to the manufacturer’s instructions.

Details of the plasmids employed for complementation experiments are given in the online Supplementary Material (Appendix 1).

Sporulation assay and granulose stain

C. acetobutylicum strains were grown in modified CBM broth containing 5% glucose and 0.5% CaCO3. After 5 days, heat-resistant cfu were determined by incubating samples for 10 min in a tightly lidded 80 °C water bath, serially diluted and plating on CBM agar. The detection limit was 10 spores per ml.

Granulose production coincides with the initiation of spore formation and may be employed to monitor this process. For the granulose assay, colonies grown on CBM agar for 1–2 d were exposed to I2 vapour, which stains granulose-containing colonies brown (Robson et al., 1974).

Gene expression profiling

RNA was extracted from strains growing in batch culture in CBM supplemented with 5% glucose and 0.5% CaCO3 (3 independent experiments for each strain). Samples (0.5 ml) were removed at 80-min intervals starting when the culture reached mid-exponential phase (ca. OD600 ~ 0.4 – see Supplementary Fig. S6A) and added to 1 ml RNAlater (Ambion). RNA from each time series, covering the period when sporulation was initiated, was subsequently extracted and purified using an RNeasy mini kit (Qiagen) and any residual contaminating DNA was removed by an additional treatment with TurboDNase (Ambion). RNA quality and quantity were monitored using an Agilent BioAnalyser 1200 and a Nanodrop ND1000 Spectrophotometer (Thermo Scientific). An iScript onestep RT-PCR kit (BioRad) was employed for qRT-PCR. Each reaction contained 100 ng RNA and 20 μMol each of the paired gene-specific primers (Supplementary Table S4). Product accumulation was monitored in a BioRad iCycler using SYBR green and the quality of each PCR product was verified by melt curve analysis. The PCR cycle at which the amplification threshold was attained was converted to copy number using standard curves prepared with C. acetobutylicum genomic DNA (1 pg C. acetobutylicum DNA corresponds to ca. 225 genome equivalents). Since mRNA specific for each gene was not used to generate the standard curves, between-gene comparisons have been avoided.

The sigA gene is frequently employed for data normalisation and in previous C. acetobutylicum microarray experiments the thl signal was employed for this purpose (Paredes et al., 2005). Under the growth conditions employed here, both the thl and the sigA signals showed significant and systematic variation with time, so copy numbers were simply expressed per ng RNA. The data shown are average values for three replicate experiments; examples of the between experiment variation are shown in Supplementary Fig. S6B.

Recombinant protein production

Full length cac0437, cac2071 (spo0A), & cac3319 and truncated versions of cac0323 (residues 232–654) and cac0903 (residues 244–683), lacking the sequences encoding their N-terminal transmembrane segments, were amplified from genomic DNA using gene-specific primers (Supplementary Table S3), trimmed with NdeI + BamHI and cloned into similarly digested pET19b (Novagen). Following sequence verification, plasmids were transformed into E. coli Rosetta 2 (DE3) (Novagen). Details of the plasmid and strain constructions are given in the online Supplementary Material (Appendix 1). For expression of these amino-terminally, His10-tagged proteins, strains were grown in Overnight Express Instant TB medium (Novagen) without IPTG induction at 18 – 22 °C for 18 – 24 h. Cell pellets obtained by centrifugation were frozen at −20 °C. Proteins were isolated and purified using divalent metal ion chelation chromatography. Briefly, cell pellets were resuspended in 5 vol cold binding buffer (BB; 20 mM Tris/HCl pH8.0, 500 mM NaCl, 60 mM imidazole) supplemented with a protease inhibitor cocktail (Roche) and disrupted using a French press. The soluble fraction was cleared by centrifugation at 27,000 g for 30 min and His10-tagged proteins bound by incubation with 0.5 ml of Ni-NTA agarose (Qiagen), pre-equilibrated with BB for 1 h at 4 °C with gentle agitation. The gel was packed into a disposable column and after washing with 200 ml BB, proteins were eluted using a concentration gradient from 0.1 M to 1 M imidazole in 20 mM Tris/HCl pH8.0, 500 mM NaCl. Proteins were dialysed against 3 changes of 20 mM Tris/HCl, 500 mM NaCl, 1 mM EDTA, 1 mM dithiothreitol adjusted to pH8.0 (Cac0437, Cac0903 & Cac2071) or to pH7.2 (Cac0323 & Cac3319) to remove imidazole. Proteins were concentrated by ultrafiltration using a 30 kDal membrane (Centriprep, Amicon) and stored at 4 °C. Protein concentrations were determined using the Bradford reagent. Cac0437, Cac2071 and Cac3319 were obtained at greater than 90% purity; Cac0903 & Cac0323 were obtained at ca. 80% & 50% purity, respectively.

Protein autophosphorylation and the Spo0A phosphotransfer reaction

Assays were carried out as previously described (Grimshaw et al., 1998). Briefly, the standard fixed time point assay mixture reaction (20-μl total volume for each time point) contained 20 mM MgCl2, 0.1 mM EDTA, 5% (v/v) glycerol, 100 μCi [γ-32P]-ATP (6000Ci/mmol; PerkinElmer) diluted to a final concentration of 1 mM with unlabelled ATP and 50 mM EPPS buffer pH8.5 (Cac0437, Cac0903 and B. subtilis KinA) or pH7.5 (Cac0323 and Cac3319). Unless otherwise stated, kinases (alone or in combination) and Spo0A were employed at final concentrations of 2 μM and 4 μM, respectively. After incubation at 25 °C for the designated period of time, a 15-μL sample was withdrawn and the reaction stopped with 5 μL of 4-fold concentrated Laemmli sample buffer. Samples (10 μL) of the reaction products were loaded and separated by electrophoresis (15% SDS PAGE) and dried gels were subjected to autoradiography using a Storm Model 840 PhosphorImager. ImageQuant software was employed for data quantification and analysis. Native B. subtilis KinA, Spo0A and Spo0F proteins purified as detailed elsewhere were employed for control reactions (Grimshaw et al., 1998).

Spo0A~P dephosphorylation assay and thin-layer chromatography

Spo0A (10 μM) was phospho-labelled using the Cac3319 kinase (1 μM) as described above, except that the reaction volume was increased to 200 μl and the reaction was allowed to proceed for 1 h at room temperature. Spo0A~P was separated from Cac3319 and excess ATP by gel-filtration (Sephadex G100 column equilibrated with 20 mM Tris/HCl, 500 mM NaCl) and then concentrated by ultrafiltration using a Centricon YM-10 centrifugal filter device according to the manufacturer’s instructions. The protein concentration was measured using the Bradford reagent. Dephosphorylation assays were performed in a reaction buffer containing 20 mM MgCl2, 0.1 mM EDTA, 5% (v/v) glycerol, and 50 mM EPPS buffer pH8.5 (Cac0437, Cac0903) or pH7.5 (Cac0323 and Cac3319) containing radioactively labelled Spo0A~P at 2 μM (nominal final concentration) in the presence or absence of ADP, GDP or ATP (0.5 mM) and in the presence or absence of the C. acetobutylicum kinases at 1 μM final concentration. After incubation at room temperature, 15-μL samples were taken at the indicated time points and the reaction stopped by adding 5 μL of 4-fold concentrated Laemmli buffer. The samples were then subjected to 15% SDS-PAGE and thin layer chromatography (TLC) followed by autoradiography as previously described (Scaramozzino et al., 2009). Briefly, for TLC analysis 2-μL samples were spotted onto polyethylenamine cellulose paper polygram cel 300 PEI (Macherey-Nagel) that had been pre-run with either 0.75 M KH2PO4 pH3.75 or 0.3 M K phosphate, pH7.4. The TLC sheet was developed with the same buffer, dried in an oven at 37 °C and exposed to a Phosphorimager screen for 24 h.

Supplementary Material

Supp Fig S1-S6 ,Table S1-S4 & App S1

Acknowledgments

ES and DIY constructed and characterised the mutant strains and undertook the gene expression experiments in Aberystwyth. The ClosTron mutagenesis tool was developed in Nottingham by JTH and NPM, who also provided the spo0A mutant. Protein expression and in vitro phosphorylation studies were carried out by AED, DIY, MY & JAH at The Scripps Research Institute and supported by grants GM19416 from the National Institute of General Medical Sciences and AI055860 from the National Institute of Allergy and Infectious Diseases, National Institutes of Health, United States Public Health Service. MIY, JAH & ES wrote the manuscript. We thank Rebecca Edwards and Carol Evans who analysed spo0A from the Spo variants of the cac0437 mutant. Finally, MY and NPM gratefully acknowledge the financial support of the UK Biotechnology and Biological Sciences Research Council (grant reference: BB/D001498/1 and BB/D522797/1).

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