BoNT synthesis in C. botulinum
has been found to be a highly regulated process. Antp
genes are clustered in the botulinum locus and are transcribed as two polycistronic operons as evidenced by qRT-PCR or Northern blot analysis 
. The botR
gene, which is located in the botulinum locus in C. botulinum
A, B, C, D, F and G plays a critical role in the regulation of BoNT and ANTP synthesis. Indeed, BotR/A has been demonstrated to be an alternative sigma factor controlling the transcription of the two operons of the botulinum locus at the transition phase from the exponential to the stationary growth phase 
. However, in C. botulinum
type E, the transcription of bont
genes is also regulated during the transition phase, although botR
or a homologous gene has not been identified in the genome of this C. botulinum
. This strongly supports that botR
is not the only regulatory gene controlling the expression of toxin gene in C. botulinum
. Since at least 39 putative regulatory genes, distributed in TCS or orphan RR genes, have been identified in the genome of C. botulinum
strain Hall, we have investigated their possible role in the control of BoNT/A synthesis.
A total of 34 Hall isogenic antisense strains have been generated with the antisense mRNA method targeting 29 regulatory genes predicted to be part of TCSs and 5 putative orphan RR genes. Among the 34 Hall isogenic antisense strains, 31 retained similar growth kinetics compared to the control strain, whereas 2 strains showed a more rapid growth and one isogenic antisense strain had a significantly delayed growth (). BoNT/A was detected in a delayed manner in the culture supernatant of the isogenic antisense strain with a delayed growth kinetic (Hall/1001), but reached a similar OD than the control strain at 24 h, indicating that the repressed corresponding orphan RR (CLC_0632) is involved in global metabolism but does not directly control toxin synthesis. Interestingly, CLC_0632 shares sequence similarity with VirR (38% protein identity), which regulates the production of toxins in C. perfringens
including prefringolysin, alpha-toxin and collagenase 
. VirR belongs to a complex regulatory network controlling more than 147 genes such as genes for catalytic enzymes, transporters and energy metabolism and thus controlling multiple cellular functions 
. CLC_0632 does not have closer homologs (protein identity >60%) in other clostridia (). CLC_1105 also shows similarity to VirR (37% on protein level). However, Hall/1002 targeting CLC_1105 showed no alteration in growth kinetics and toxin production. The VirR homologs in C. botulinum
Hall probably regulate basic functions but not specifically BoNT production.
The two isogenic antisense strains with a more rapid growth (Hall/1147 and Hall/1148) showed drastic changes in the bacterial cell wall or surface structure, which is probably the reason for the observed cell lysis. Reduced toxin levels were measured in the lyzed culture supernatant of both isogenic antisense strains reflecting lower BoNT synthesis rather than an impaired secretion. The corresponding RRs (CLC_0411 and CLC_3293), members of the OmpR family (), are probably part of regulatory cascades, which control multiple functions including bacterial surface polysaccharide synthesis and/or assembly and indirectly BoNT production. Besides the HATPase_c and the HisKA domains, the SHK CLC_0411 possesses a partial VicK domain (COG5002). Interestingly, VicK of Streptococcus mutans
has been identified as a TCS sensor involved in the regulation of several virulence-associated genes affecting synthesis and adhesion to polysaccharides 
. SHK CLC_3293 shows similarity to the BaeS family (COG0642) of sensors. Interestingly, the BaeS sensor in E. coli
is involved in envelope stress response 
. Taken together, it can be hypothesized that CLC_0410/CLC_0411 and CLC_3294/CLC_3293 are TCSs involved in regulating cell surface properties, e.g. surface polysaccharide synthesis and integrity.
Interestingly, a quorum sensing system related to that of Staphylococcus aureus
and consisting of two agr
loci has been identified in the group I of C. botulinum
strains, which controls both sporulation and BoNT production. Each agr
locus seems to have a specific function, agr
-1 regulating sporulation and agr
-2 regulating BoNT synthesis 
. We have also identified homologous genes to agrA
from S. aureus
in the Hall genome. The isogenic antisense strain Hall/651 targeting the agrA
homolog was not impaired in BoNT production. Thus, the quorum sensing-dependent regulation pathway and its effects on the control of toxin production in C. botulinum
remain to be defined.
We showed that three TCSs, CLC_1093/CLC_1094, CLC_1914/CLC_1913 and CLC_0661/CLC_0663 (were involved in the regulation of e BoNT/A and ANTP production. BoNT/A, NTNH and HA34 levels in the culture supernatants of the corresponding isogenic antisense strainsHall/707, Hall/707K, Hall/714, Hall/714K, Hall/1146, Hall/1146K were repressed similarly to that observed in the botR/A isogenic antisense strain (Hall/306). Expression of bont/A, ntnh, and ha34 genes was reduced throughout the 24 h growth period, as also observed in Hall/306. However, botR/A expression was not affected or even slightly increased in the three Hall isogenic antisense strains. These results argue that the corresponding TCSs, CLC_1093/CLC_1094, CLC_1914/CLC_1913 and CLC_0661/CLC_0663, control, directly or indirectly, the expression of the botulinum locus genes independently of botR/A. Other regulatory genes, notably from the AraC family, and genes encoding for transporters are distributed in the vicinity of these TCSs (). This suggests that the TCSs could be part of a complex regulatory network in response to external signals. However, the TCS might interact with other genes, which are distantly located on the genome. The biological role of the TCS CLC_0661/CLC_0663 remains to be investigated. The TCS is homologous to TCSs of the PhoP/PhoR family involved in, but not restricted to, sensing and reacting to phosphate starvation. It is homologous to CTC00411/CTC00412 of C. tetani (65% and 53% protein identity, respectively), and CPE2098/CPE2099 of C. perfringens (50 and 40% identity, respectively). In the latter organism, the system has been designated VirI/VirJ, and it has been described as “a novel two-component regulatory system involved in the shutdown of extracellular toxin production in C. perfringens”, although further data is not available (EMBL/GenBank/DDBJ databases: BAA78773.1 and BAA78774.1).
Genetic environment of the TCS genes involved in the regulation of toxin synthesis in strain Hall.
Albeit C. botulinum and C. tetani synthesize related neurotoxins, a specific regulatory network based on TCSs seems to control BoNT production in C. botulinum type A. The 5 orphan regulatory genes tested in strain Hall () seem not to be involved in the regulation of the botulinum locus genes. Interestingly, the 5 TCSs, which directly or indirectly are involved in the regulation of toxin synthesis in strain Hall, are conserved in the known genomes of C. botulinum from group I including the subtypes A2, A3, Ba4, B1 and F, but not in the genomes of group II strains such as C. botulinum type E or group IV (C. botulinum C and D). These results suggest that group I C. botulinum strains might share a regulatory network distinct from that in the other C. botulinum groups.
This raises the question about the external signals governing the toxin production via TCS regulation. The environmental factors controlling toxin gene expression in C. botulinum
are still poorly known. Carbon dioxide has been reported to stimulate toxin gene expression and toxin formation in non-proteolytic C. botulinum
strains type B and E despite a growth rate reduction 
, in contrast to proteolytic C. botulinum
type A, which seems insensitive to carbon dioxide 
. High temperature did not control the transcription of bont
gene but influences the stability of the toxin in C. botulinum
. A better understanding of the regulation of toxin synthesis in C. botulinum
would permit the development of novel strategies to prevent botulism by counteracting the toxin production in food and/or in the digestive tract.
In summary, BoNT/A and ANTP synthesis in C. botulinum strain Hall seems to be under the control of a complex regulatory network. In addition to the alternative sigma factor BotR/A, which regulates bont/A and antp transcription at the transition phase between the exponential and stationary growth, we have found that at least three TCSs also control BoNT/A and ANTP synthesis at the transcriptional level independently of BotR/A. Two other TCSs seem to retain various pleiotropic effects including the control of BoNT/A synthesis as well as the regulation of bacterial cell wall or cell surface synthesis and/or assembly and granule accumulation in the cytoplasm. Further investigations of the regulatory network controlling toxin production in C. botulinum would allow the identification of environmental factors triggering BoNT/A synthesis.