Many species of Proteobacteria communicate by using LuxI-LuxR–type quorum-sensing systems that produce and detect acyl-homoserine lactone (acyl-HSL) signals. Most of the known signals are ...straight-chain fatty acyl-HSLs, and evidence indicates that LuxI homologs prefer fatty acid-acyl carrier protein (ACP) over fatty acyl-CoA as the acyl substrate for signal synthesis. Two related LuxI homologs, RpaI and BtaI from Rhodopseudomonas palustris and photosynthetic stem-nodulating bradyrhizobia, direct production of the aryl-HSLs p-coumaroyl-HSL and cinnamoyl-HSL, respectively. Here we report that BjaI from the soybean symbiont Bradyrhizobium japonicum USDA110 is closely related to RpaI and BtaI and catalyzes the synthesis of isovaleryl-HSL (IV-HSL), a branched-chain fatty acyl-HSL. We show that IV-HSL induces expression of bjaI, and in this way IV-HSL functions like many other acyl-HSL quorum-sensing signals. Purified histidine-tagged BjaI was an IV-HSL synthase, which was active with isovaleryl-CoA but not detectably so with isovaleryl-ACP. This suggests that the RpaI-BtaI-BjaI subfamily of acyl-HSL synthases may use CoA- rather than ACP-linked substrates for acyl-HSL synthesis. The bjaI-linked bjaR1 gene is involved in the response to IV-HSL, and BjaR1 is sensitive to IV-HSL at concentrations as low as 10 pM. Low but sufficient levels of IV-HSL (about 5 nM) accumulate in B. japonicum culture fluid. The low levels of IV-HSL synthesis have likely contributed to the fact that the quorum-sensing signal from this bacterium has not been described elsewhere.
Summary
The environmental bacterium Legionella pneumophila is the causative agent of Legionnaires' disease, a life‐threatening pneumonia. For cell–cell communication the bacteria employ the ...autoinducer LAI‐1 (3‐hydroxypentadecane‐4‐one), which is produced and detected by the Lqs (Legionella quorum sensing) system. The system comprises the autoinducer synthase LqsA, the putative sensor kinases LqsS and LqsT, and the prototypic response regulator LqsR. Lqs‐regulated processes include L. pneumophila‐phagocyte interactions, production of extracellular filaments, and natural competence. Using biochemical approaches we show here that LqsS and LqsT are autophosphorylated by γ‐32P‐ATP at a conserved histidine residue (H200 or H204) located in their cytoplasmic histidine kinase domain. Pull‐down assays revealed that LqsS and LqsT are bound by LqsR or phospho‐LqsR. Dependent on the conserved receiver domain aspartate (D108), the response regulator prevented autophosphorylation of both sensor kinases by catalysing the dephosphorylation of phospho‐LqsS or phospho‐LqsT. Moreover, LqsR formed dimers upon phosphorylation at D108 by either acetyl‐phosphate or phospho‐LqsT. Finally, upon heterologous production in Escherichia coli, LqsT (but not LqsS) was autophosphorylated by ATP, and LqsR prevented the autophosphorylation by catalysing the dephosphorylation of phospho‐LqsT. In summary, these results indicate that phosphorylation signalling through the Legionella quorum sensing histidine kinases LqsS and LqsT converges on the response regulator LqsR.
Summary
Legionella pneumophila is an amoeba‐resistant opportunistic pathogen that performs cell–cell communication through the signalling molecule 3‐hydroxypentadecane‐4‐one (LAI‐1, Legionella ...autoinducer‐1). The lqs (Legionella quorum sensing) gene cluster encodes the LAI‐1 autoinducer synthase LqsA, the cognate sensor kinase LqsS and the response regulator LqsR. Here we show that the Lqs system includes an ‘orphan’ homologue of LqsS termed LqsT. Compared with wild‐type L. pneumophila, strains lacking lqsT or both lqsS and lqsT show increased salt resistance, greatly enhanced natural competence for DNA acquisition and impaired uptake by phagocytes. Sensitive novel single round growth assays and competition experiments using Acanthamoeba castellanii revealed that ΔlqsT and ΔlqsS‐ΔlqsT, as well as ΔlqsA and other lqs mutant strains are impaired for intracellular growth and cannot compete against wild‐type bacteria upon co‐infection. In contrast to the ΔlqsS strain, ΔlqsT does not produce extracellular filaments. The phenotypes of the ΔlqsS‐ΔlqsT strain are partially complemented by either lqsT or lqsS, but are not reversed by overexpression of lqsA, suggesting that LqsT and LqsS are the sole LAI‐1‐responsive sensor kinases in L. pneumophila. In agreement with the different phenotypes of the ΔlqsT and ΔlqsS strains, lqsT and lqsS are differentially expressed in the post‐exponential growth phase, and transcriptome studies indicated that 90% of the genes, which are downregulated in absence of lqsT, are upregulated in absence of lqsS. Reciprocally regulated genes encode components of a 133 kb genomic ‘fitness island’ or translocated effector proteins implicated in virulence. Together, these results reveal a unique organization of the L. pneumophila Lqs system comprising two partially antagonistic LAI‐1‐responsive sensor kinases, LqsT and LqsS, which regulate distinct pools of genes implicated in pathogen–host cell interactions, competence, expression of a genomic island or production of extracellular filaments.
Summary
The causative agent of Legionnaires' disease, Legionella pneumophila, colonizes amoebae and biofilms in the environment. The opportunistic pathogen employs the Lqs (Legionella quorum sensing) ...system and the signalling molecule LAI‐1 (Legionella autoinducer‐1) to regulate virulence, motility, natural competence and expression of a 133 kb genomic “fitness island”, including a putative novel regulator. Here, we show that the regulator termed LvbR is an LqsS‐regulated transcription factor that binds to the promoter of lpg1056/hnox1 (encoding an inhibitor of the diguanylate cyclase Lpg1057), and thus, regulates proteins involved in c‐di‐GMP metabolism. LvbR determines biofilm architecture, since L. pneumophila lacking lvbR accumulates less sessile biomass and forms homogeneous mat‐like structures, while the parental strain develops more compact bacterial aggregates. Comparative transcriptomics of sessile and planktonic ΔlvbR or ΔlqsR mutant strains revealed concerted (virulence, fitness island, metabolism) and reciprocally (motility) regulated genes in biofilm and broth respectively. Moreover, ΔlvbR is hyper‐competent for DNA uptake, defective for phagocyte infection, outcompeted by the parental strain in amoebae co‐infections and impaired for cell migration inhibition. Taken together, our results indicate that L. pneumophila LvbR is a novel pleiotropic transcription factor, which links the Lqs and c‐di‐GMP regulatory networks to control biofilm architecture and pathogen–host cell interactions.
Summary
The environmental bacterium
L
egionella pneumophila
is the causative agent of Legionnaires' disease, a life‐threatening pneumonia. For cell–cell communication the bacteria employ the ...autoinducer
LAI
‐1 (3‐hydroxypentadecane‐4‐one), which is produced and detected by the
Lqs
(
L
egionella
quorum sensing) system. The system comprises the autoinducer synthase
LqsA
, the putative sensor kinases
LqsS
and
LqsT
, and the prototypic response regulator
LqsR
.
Lqs
‐regulated processes include
L
. pneumophila
‐phagocyte interactions, production of extracellular filaments, and natural competence. Using biochemical approaches we show here that
LqsS
and
LqsT
are autophosphorylated by γ‐
32
P
‐
ATP
at a conserved histidine residue (
H
200
or
H
204
) located in their cytoplasmic histidine kinase domain. Pull‐down assays revealed that
LqsS
and
LqsT
are bound by
LqsR
or phospho‐
LqsR
. Dependent on the conserved receiver domain aspartate (
D
108
), the response regulator prevented autophosphorylation of both sensor kinases by catalysing the dephosphorylation of phospho‐
LqsS
or phospho‐
LqsT
. Moreover,
LqsR
formed dimers upon phosphorylation at
D
108
by either acetyl‐phosphate or phospho‐
LqsT
. Finally, upon heterologous production in
E
scherichia coli
,
LqsT
(but not
LqsS
) was autophosphorylated by
ATP
, and
LqsR
prevented the autophosphorylation by catalysing the dephosphorylation of phospho‐
LqsT
. In summary, these results indicate that phosphorylation signalling through the
L
egionella
quorum sensing histidine kinases
LqsS
and
LqsT
converges on the response regulator
LqsR
.
Summary The causative agent of Legionnaires' disease, Legionella pneumophila, employs the autoinducer compound LAI-1 (3-hydroxypentadecane-4-one) for cell-cell communication. LAI-1 is produced and ...detected by the Lqs (Legionella quorum sensing) system, comprising the autoinducer synthase LqsA, the sensor kinases LqsS and LqsT, as well as the response regulator LqsR. Lqs-regulated processes include pathogen-host interactions, production of extracellular filaments and natural competence for DNA uptake. Here we show that synthetic LAI-1 promotes the motility of L. pneumophila by signalling through LqsS/LqsT and LqsR. Upon addition of LAI-1, autophosphorylation of LqsS/LqsT by gamma-32P-ATP was inhibited in a dose-dependent manner. In contrast, the Vibrio cholerae autoinducer CAI-1 (3-hydroxytridecane-4-one) promoted the phosphorylation of LqsS (but not LqsT). LAI-1 did neither affect the stability of phospho-LqsS or phospho-LqsT, nor the dephosphorylation by LqsR. Transcriptome analysis of L. pneumophila treated with LAI-1 revealed that the compound positively regulates a number of genes, including the non-coding RNAsrsmY and rsmZ, and negatively regulates the RNA-binding global regulator crsA. Accordingly, LAI-1 controls the switch from the replicative to the transmissive growth phase of L. pneumophila. In summary, the findings indicate that LAI-1 regulates motility and the biphasic life style of L. pneumophila through LqsS- and LqsT-dependent phosphorylation signalling.
Summary
The causative agent of Legionnaires' disease, Legionella pneumophila, employs the autoinducer compound LAI‐1 (3‐hydroxypentadecane‐4‐one) for cell–cell communication. LAI‐1 is produced and ...detected by the Lqs (Legionella quorum sensing) system, comprising the autoinducer synthase LqsA, the sensor kinases LqsS and LqsT, as well as the response regulator LqsR. Lqs‐regulated processes include pathogen–host interactions, production of extracellular filaments and natural competence for DNA uptake. Here we show that synthetic LAI‐1 promotes the motility of L. pneumophila by signalling through LqsS/LqsT and LqsR. Upon addition of LAI‐1, autophosphorylation of LqsS/LqsT by γ‐32P‐ATP was inhibited in a dose‐dependent manner. In contrast, the Vibrio cholerae autoinducer CAI‐1 (3‐hydroxytridecane‐4‐one) promoted the phosphorylation of LqsS (but not LqsT). LAI‐1 did neither affect the stability of phospho‐LqsS or phospho‐LqsT, nor the dephosphorylation by LqsR. Transcriptome analysis of L. pneumophila treated with LAI‐1 revealed that the compound positively regulates a number of genes, including the non‐coding RNAs rsmY and rsmZ, and negatively regulates the RNA‐binding global regulator crsA. Accordingly, LAI‐1 controls the switch from the replicative to the transmissive growth phase of L. pneumophila. In summary, the findings indicate that LAI‐1 regulates motility and the biphasic life style of L. pneumophila through LqsS‐ and LqsT‐dependent phosphorylation signalling.
The environmental bacterium Legionella pneumophila causes a severe pneumonia termed Legionnaires′ disease. For cell‐to‐cell communication, the pathogen uses the small signaling molecule LAI‐1, which is produced and detected by the Lqs (Legionella quorum sensing) system. We show that synthetic LAI‐1 is recognized by the membrane‐bound sensor kinases LqsS and LqsT, which transfer the phosphorylation signal to the response regulator LqsR. Thus, LAI‐1 regulates gene expression and motility of L. pneumophila through Lqs‐dependent signal transduction.
Summary
L
egionella pneumophila
is an amoeba‐resistant opportunistic pathogen that performs cell–cell communication through the signalling molecule 3‐hydroxypentadecane‐4‐one (
LAI
‐1,
L
egionella
...autoinducer‐1). The
lqs
(
L
egionella
quorum sensing) gene cluster encodes the
LAI
‐1 autoinducer synthase
LqsA
, the cognate sensor kinase
LqsS
and the response regulator
LqsR
. Here we show that the
Lqs
system includes an ‘orphan’ homologue of
LqsS
termed
LqsT
. Compared with wild‐type
L
. pneumophila
, strains lacking
lqsT
or both
lqsS
and
lqsT
show increased salt resistance, greatly enhanced natural competence for
DNA
acquisition and impaired uptake by phagocytes. Sensitive novel single round growth assays and competition experiments using
A
canthamoeba castellanii
revealed that Δ
lqsT
and Δ
lqsS
‐Δ
lqsT
, as well as Δ
lqsA
and other
lqs
mutant strains are impaired for intracellular growth and cannot compete against wild‐type bacteria upon co‐infection. In contrast to the Δ
lqsS
strain, Δ
lqsT
does not produce extracellular filaments. The phenotypes of the Δ
lqsS
‐Δ
lqsT
strain are partially complemented by either
lqsT
or
lqsS
, but are not reversed by overexpression of
lqsA
, suggesting that
LqsT
and
LqsS
are the sole
LAI
‐1‐responsive sensor kinases in
L
. pneumophila
. In agreement with the different phenotypes of the Δ
lqsT
and Δ
lqsS
strains,
lqsT
and
lqsS
are differentially expressed in the post‐exponential growth phase, and transcriptome studies indicated that 90% of the genes, which are downregulated in absence of
lqsT
, are upregulated in absence of
lqsS
. Reciprocally regulated genes encode components of a 133 kb genomic ‘fitness island’ or translocated effector proteins implicated in virulence. Together, these results reveal a unique organization of the
L
. pneumophila
Lqs
system comprising two partially antagonistic
LAI
‐1‐responsive sensor kinases,
LqsT
and
LqsS
, which regulate distinct pools of genes implicated in pathogen–host cell interactions, competence, expression of a genomic island or production of extracellular filaments.
Legionella pneumophila infects and replicates in environmental protozoa and metazoan macrophages within a specific vacuole. The infection of phagocytes by L. pneumophila can be assessed by an agar ...plating assay or by fluorescence microscopy. Here, we describe the analysis of Legionella infection by automated flow cytometry using wild-type and mutant bacteria that constitutively produce the green fluorescent protein (GFP). Advantages of the flow cytometry technique include (1) a software-assisted multiple parameter analysis of Legionella infections in real-time at distinct stages of the infection cycle, (2) the simultaneous and fast acquisition of a high number of data points, and (3) a characterization of the infecting bacteria in parallel with the infected host cells.
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