- PlcRa is a quorum sensing transcriptional regulator in Bacillus cereus that is activated by the heptapeptide PapRa7 during the transition to stationary phase. - PlcRa upregulates genes involved in cysteine synthesis, oxidative stress resistance, and peptide transport while downregulating phage genes and the Hbl enterotoxin. - PlcRa helps B. cereus adapt to poor sulfur conditions and oxidative stress early in stationary phase by controlling the regulator AbrB2 which in turn regulates genes for cysteine metabolism from methionine.

1. Manuel García Ulloa Gámiz

2. Quorum Sensing

3. RNPP Family
• Regulatory quorum sensors found
exclusively in Gram-positive bacteria with
a low G+C content.
– Rap - Bacillus cereus.
– NprR - B. subtilis.
– PrgX - Enteroccocus faecalis.
– PlcR - B. cereus / thuringensis.
• All these RNPP regulators are activated
through a secreted signaling peptide.

4. 5 Tetratricopeptide
(34 aminoacid)
repeats (TPRs)
Peptide binding
domain
Helix-turn-helix
(HTH) DNA-
binding domain
PlcR (receptor)
+
RapR (ligand)
• The PlcR/PapR quorum sensing system is activated during the
transition phase in most members of the B. cereus group.
• This system controls the expression of genes encoding exported
virulence factors, including degradative enzymes, enterotoxins and
hemolysins.
Linker helix
homodimer

5. Extracellular processing
PlcR
PapR7
PapR
PlcR + PapR7
OppABCDF
PlcR/PapR system
Stationary PhaseStationary Phase

6. BLAST!
• B. cereus ATCC 14579 genome contains
3 PlcR paralogs:
– BC0988 (PlcRa).
– BC1158 (PlcRb), previously named PlcR2 in
B. anthracis.
– BC2443 (PlcRc).
*A small ORF, BC0989,
encoding a putative peptide with
a potential signal sequence is
located upstream from the
plcRa gene. We
named this gene papRa.

7. PlcR vs PlcRa analysis
• PlcR – PlcRa:
– Sequence identity: 29%
– Sequence similarity: 51%
– Protein identity: 85%
*Identity is the degree of correspondence between two sub-sequences
(no gaps between the sequences). An identity of 25% or higher
implies similarity of function, while 18-25% implies similarity of
structure or function.
*Similarity is the degree of resemblance between two sequences when
they are compared. This is dependant on their identity.
Genome comparisons revealed genes for PlcRa (identity 94%) in
B. thuringiensis BMB171, serovar chinensis CT-43, HD-789, HD-
771 strains and B. cereus B4264 and G9842 strains.

10. CHARMm (Chemistry
at HARvard
Macromolecular
Mechanics)
PapR vs PapRa

11. Analysis of PlcRa
expression
• PplcRa’-lacZ transcriptional
fusion in the low-copy-
number plasmid
pHT304–18Z.
PlcRa expression is
transiently activated
early in the
stationary phase.

12. Identification of PlcRa-controlled genes
• Comparison between the transcriptomes of the
plcRa mutant and wild-type B. cereus ATCC
14579 strains during early stationary phase.
– 117 genes were differentially expressed:
• 49 overexpressed in wild-type strain.
• 68 underexpressed in wild-type strain.
– 4 main categories of overexpressed genes:
• Sulphur metabolism (20).
• Oxidative stress responses (5).
• Peptide transport (4).
• Iron metabolism (5).

13. Autoinducer-2
Cysteine
biosynthesis
Cysteine
transport
Methionine
biosynthesis

14. Major regulators previously
known in B. subtilis.
1)O2 H2O2
2) H2O2 H2O
Stress resistance
known in B. anthracis.
Uncharacterized
oligopeptide
permease
system
Iron transport/metabolism

16. Downregulated genes
• Sigma factor SigB, its associated regulatory proteins
RbsV, RbsW and RbsP and six SigB controlled proteins.
• 31 prophage proteins encoded by the genes of two
prophages, phBC6A52 and phBC6A51.
– Thought to be involved in DNA repair and structural
maintenance of chromosome.
– **First report of a bacterial regulator controlling the expression of
numerous phage genes in this strain.
• Three gene operon encoding the components of the Hbl
enterotoxin, a virulence determinant thought to be
involved in diarrheal disease.

17. Transcriptional Control of the
BC2444 (abrB2) Gene by
PlcRa (PlcRa
characterization)
Wild-type
PlcRa mutant + PlcRa
PlcRa mutant
PlcRa controls AbrB2
expression.
PabrB2’-lacZ

18. PapRa7 appears to
be PlcRa’s ligand.
PlcRa only functions
in the presence of
PapRa7.

19. PapRa7 effect over PlcrRa in vivo
PapRa7 activates PlcRa activates AbrB2
activates lacZ.
Xylose activates PapRa7 activates PlcRa
activates AbrB2 activates lacZ.
PapRa7 is
PlcRa’s ligand

20. Effect of AbrB2 over yrrT
(cysteine biosynthesis) in
vivo
PlcRa/xylose AbrB2 yrrT
*Expression of abrB2:
inducible by PlcrRa/xylose.
*Expression of yrrT:
inducible by AbrB2.
*Expression of lacZ:
inducible by yrrT.
PlcRa controls yrrT expression
mediated by AbrB2
•pHT1618KVPxyl-abrB2
•pHT304yrrT’-lacZ

21. Sensibility of Wild Type, plcRa mutant and plcRa
mutant + PlcRa to peroxide and diamide
PlcRa helps resisting oxidative stress

22. Cysteine adition effect
No H2O2
H2O2
Cysteine plays an
important role in
oxidative stress
response.

23. Conclusions/Discussions
• PlcRa is a new member of the RNPP family of transcriptional
regulators in the B. cereus group.
– Folding similar to PlcR.
– DNA-binding domain and five TPR motifs putatively involved in the
peptide binding.
– Signaling heptapeptide: PapRa7.
• PlcRa/PapRa is probably a new quorum sensing system in B.
cereus.
– Activates at stationary phase.
– Upregulates the transcription of genes involved in regulation, cysteine
synthesis and oxidative stress resistance.
– Downregulates the expression of numerous phage genes and this
regulation may be indirect or direct.
PlcRa+PapRa7 AbrB2 yrrT (cysteine biosynthesis from methionine)

24. Conlcusions/Discussions
– **Further investigations are required to determine the molecular
mechanisms by which AbrB2 regulates the set of genes encoding
proteins involved in cysteine metabolism.
• PlcRa helps in daptation to poor sulfur source conditions and
oxidative environment early in stationary phase.
– plcRa inactivation had no significant effect on sporulation capacity in
many common laboratory conditions.
– Involvement of PlcRa in stress responses may ensure bacterial survival
during the transition state preceding the initiation of sporulation.
• Results demonstrate the existence of regulatory connections
between cysteine metabolism and the oxidative stress responses at
the onset of stationary phase in the B. cereus ATCC 14579 strain.