Abstract Pathogenic Leptospira modulate their virulence via the Lvr signaling system, with the histidine kinase LvrB being a central element. LvrB is a prototype of Rec-controlled histidine kinases, which are frequently found in bacterial two-component systems, and yet whose regulatory mechanisms remain largely unknown. Here, we report full-length structures of LvrB in different states uncovering its mechanism of activation. Kinase-inactive LvrB is a symmetric homodimer, with its catalytic domains rigidly clasped onto the central helical domain. Phosphorylation of the N-terminal Rec domains induces coiled-coil formation of the central αS helices thereby breaking symmetry through liberation of the catalytic domains into a dynamic, auto-phosphorylation competent state. We further identified LvrB’s downstream effector partner LvrC, an anti-σ factor that reprograms the transcription of hundreds of virulence genes. Our findings set a mechanistic paradigm for Rec-controlled histidine kinases enabling the design of virulence inhibitors.

AbstractIn two-component systems, the information gathered by the histidine kinase (HK) is relayed to the cognate response regulator (RR). Thereby, the phosphoryl group of the autophosphorylated HK is transferred to the receiver domain of the RR to allosterically activate its effector domain. In contrast, multi-step phosphorelays comprise at least one additional Rec domain (Recinter) that is part of the HK and acts as an intermediary for phosphoryl-shuttling. While RR Rec domains have been studied extensively, little is known about Recinter domains and their potentially discriminating features. As a bona-fide Recinter domain, here we have studied the C-terminal Rec domain of the hybrid HK CckA (CckARec) from Caulobacter crescentus by X-ray crystallography and NMR spectroscopy. CckARec exhibits the canonical Rec-fold, though with a degenerated α4 helix, in which all active site residues are pre-arranged for phosphoryl-binding. BeF3- binding does not alter secondary structure nor the oligomeric state, indicating the absence of allosteric changes, the hall mark of RRs. Based on structural modeling and sequence co-variation analysis, we present a detailed picture for the intramolecular association of the CckA DHp/Rec domains and discuss the role of a FATGUY motif, a distinguishing feature of CckARec orthologs.

Bacterial second messengers c-di-GMP and (p)ppGpp have broad functional repertoires ranging from growth and cell cycle control to the regulation of biofilm formation and virulence. The recent identification of SmbA, an effector protein from Caulobacter crescentus that is jointly targeted by both signaling molecules, has opened up studies on how these global bacterial networks interact. C-di-GMP and (p)ppGpp compete for the same SmbA binding site, with a dimer of c-di-GMP inducing a conformational change that involves loop 7 of the protein that leads to downstream signaling. Here, we report a crystal structure of a partial loop 7 deletion mutant, SmbA∆loop in complex with c-di-GMP determined at 1.4 Å resolution. SmbA∆loop binds monomeric c-di-GMP indicating that loop 7 is required for c-di-GMP dimerization. Thus the complex probably represents the first step of consecutive c-di-GMP binding to form an intercalated dimer as has been observed in wild-type SmbA. Considering the prevalence of intercalated c-di-GMP molecules observed bound to proteins, the proposed mechanism may be generally applicable to protein-mediated c-di-GMP dimerization. Notably, in the crystal, SmbA∆loop forms a 2-fold symmetric dimer via isologous interactions with the two symmetric halves of c-di-GMP. Structural comparisons of SmbA∆loop with wild-type SmbA in complex with dimeric c-di-GMP or ppGpp support the idea that loop 7 is critical for SmbA function by interacting with downstream partners. Our results also underscore the flexibility of c-di-GMP, to allow binding to the symmetric SmbA∆loop dimer interface. It is envisaged that such isologous interactions of c-di-GMP could be observed in hitherto unrecognized targets.

Bacterial second messengers c-di-GMP and (p)ppGpp have broad functional repertoires ranging from growth and cell cycle control to the regulation of biofilm formation and virulence. The recent identification of SmbA, an effector protein from Caulobacter crescentus that is jointly targeted by both signaling molecules, has opened up studies on how these global bacterial networks interact. C-di-GMP and (p)ppGpp compete for the same SmbA binding site, with a dimer of the former ligand inducing a conformational change of loop 7 leading to downstream signaling. Here, we report a crystal structure of a partial loop 7 deletion mutant, SmbA Δloop in complex with c-di-GMP determined at 1.4 Å resolution. SmbA Δloop binds monomeric c-di-GMP strengthening the view that loop 7 is required for c-di-GMP dimerization. In the crystal, SmbA Δloop forms a 2-fold symmetric dimer via isologous interactions with the two symmetric halves of c-di-GMP. Structural comparisons of SmbA Δloop with wild-type SmbA in complex with dimeric c-di-GMP or ppGpp support the idea that loop 7 is critical for SmbA function by interacting with downstream partners. These results underscore the flexibility of c-di-GMP in binding to the symmetric interface between protein subunits. It is envisaged that such isologous interactions of c-di-GMP will be observed in hitherto unrecognized targets.

Bacterial effector proteins translocated via a type-IV secretion system (T4SS) typically harbor a C-terminal segment required for recognition by the type-IV secretion coupling protein. In the α-proteobacterial pathogen Bartonella, the signal is bipartite being composed of a BID (Bep intracellular delivery) domain and a positively charged C-terminal tail. Here, we show the crystal structure of full length Bartonella effector protein 1 (Bep1), which shows a FIC - OB - BAS(BID) domain arrangement conserved in the majority of Beps with the BID domain inserted into the newly discovered BAS parent domain. We propose that the BAS domain is necessary for the overall “boomerang”-like shape of Bep1 and that it plays a role during translocation through the T4SS.

Nucleotide-based signaling molecules (NSMs) are widespread in bacteria and eukaryotes, where they control important physiological and behavioral processes. In bacteria, NSM-based regulatory networks are highly complex, entailing large numbers of enzymes involved in the synthesis and degradation of active signaling molecules. How the converging input from multiple enzymes is transformed into robust and unambiguous cellular responses has remained unclear. Here we show that Escherichia coli converts dynamic changes of c-di-GMP into discrete binary signaling states, thereby generating heterogeneous populations with either high or low c-di-GMP. This is mediated by an ultrasensitive switch protein, PdeL, which senses the prevailing cellular concentration of the signaling molecule and couples this information to c-di-GMP degradation and transcription feedback boosting its own expression. We demonstrate that PdeL acts as a digital filter that facilitates precise developmental transitions, confers cellular memory, and generates functional heterogeneity in bacterial populations to evade phage predation. Based on our findings, we propose that bacteria apply ultrasensitive regulatory switches to convert dynamic changes of NSMs into binary signaling modes to allow robust decision-making and bet-hedging for improved overall population fitness. ### Competing Interest Statement The authors have declared no competing interest.

Proteins containing a FIC domain catalyze AMPylation and other post-translational modifications (PTMs). In bacteria, they are typically part of FicTA toxin-antitoxin modules that control conserved biochemical processes such as topoisomerase activity, but they have also repeatedly diversified into host-targeted virulence factors. Among these, Bartonella effector proteins (Beps) comprise a particularly diverse ensemble of FIC domains that subvert various host cellular functions. However, no comprehensive comparative analysis has been performed to infer molecular mechanisms underlying the biochemical and functional diversification of FIC domains in the vast Bep family. Here, we used X-ray crystallography, structural modelling, and phylogenetic analyses to unravel the expansion and diversification of Bep repertoires that evolved in parallel in three Bartonella lineages from a single ancestral FicTA toxin-antitoxin module. Our analysis is based on 99 non-redundant Bep sequences and nine crystal structures. Inferred from the conservation of the FIC signature motif that comprises the catalytic histidine and residues involved in substrate binding, about half of them represent AMP transferases. A quarter of Beps show a glutamate in a strategic position in the putative substrate binding pocket that would interfere with triphosphate-nucleotide binding but may allow binding of an AMPylated target for deAMPylation or another substrate to catalyze a distinct PTM. The β-hairpin flap that registers the modifiable target segment to the active site exhibits remarkable structural variability. The corresponding sequences form few well-defined groups that may recognize distinct target proteins. The binding of Beps to promiscuous FicA antitoxins is well conserved, indicating a role of the antitoxin to inhibit enzymatic activity or to serve as a chaperone for the FIC domain before translocation of the Bep into host cells. Taken together, our analysis indicates a remarkable functional plasticity of Beps that is mostly brought about by structural changes in the substrate pocket and the target dock. These findings may guide future structure–function analyses of the highly versatile FIC domains.

Diguanylate cyclases synthesising the bacterial second messenger c-di-GMP are found to be regulated by a variety of sensory input domains that control the activity of their catalytical GGDEF domain, but how activation proceeds mechanistically is, apart from a few examples, still largely unknown. As part of two-component systems, they are activated by cognate histidine kinases that phosphorylate their Rec input domains. DgcR from Leptospira biflexa is a constitutively dimeric prototype of this class of diguanylate cyclases. Full-length crystal structures reveal that BeF3- pseudo-phosphorylation induces a relative rotation of two rigid halves in the Rec domain. This is coupled to a reorganisation of the dimeric structure with concomitant switching of the coiled-coil linker to an alternative heptad register. Finally, the activated register allows the two substrate-loaded GGDEF domains, which are linked to the end of the coiled-coil via a localised hinge, to move into a catalytically competent dimeric arrangement. Bioinformatic analyses suggest that the binary register switch mechanism is utilised by many diguanylate cyclases with N-terminal coiled-coil linkers.

Immune cells are well equipped to eliminate invading bacteria, and one of their primary tools is the synthesis of bleach, hypochlorous acid (HOCl), the same chemical used as a household disinfectant. In this work, we present findings showing that many host-associated bacteria possess a bleach-sensing protein that allows them to adapt to the presence of this chemical in their environment.

We describe a quenching-free, 'online' ion exchange chromatography (oIEC) method for the quantitative analysis of enzymatic reactions in real-time. We show that separate quenching of the ongoing reaction performed conventionally is not required, since enzymatic reactions are interrupted upon immobilization of the reaction compounds by binding to the stationary phase of the ion exchange column. The reaction mix samples are directly injected into the column, thereby improving data consistency and allowing automation of the process. The method allows reliable and efficient acquisition of enzymatic progress curves by automatic loading of aliquots of an ongoing reaction at predefined timepoints. We demonstrate the applicability of this method for a variety of enzymatic reactions. SUBJECT: Enzymatic assays and analysis.

Small GTPases of the Ras-homology (Rho) family are conserved molecular switches that control fundamental cellular activities in eukaryotic cells. As such, they are targeted by numerous bacterial toxins and effector proteins, which have been intensively investigated regarding their biochemical activities and discrete target spectra; however, the molecular mechanism of target selectivity has remained largely elusive. Here we report a bacterial effector protein that selectively targets members of the Rac subfamily in the Rho family of small GTPases but none in the closely related Cdc42 or RhoA subfamilies. This exquisite target selectivity of the FIC domain AMP-transferase Bep1 from Bartonella rochalimae is based on electrostatic interactions with a subfamily-specific pair of residues in the nucleotide-binding G4 motif and the Rho insert helix. Residue substitutions at the identified positions in Cdc42 enable modification by Bep1, while corresponding Cdc42-like substitutions in Rac1 greatly diminish modification. Our study establishes a structural understanding of target selectivity toward Rac-subfamily GTPases and provides a highly selective tool for their functional analysis.

Bacteria adapt their growth rate to their metabolic status and environmental conditions by modulating the length of their G1 period. Here we demonstrate that a gradual increase in the concentration of the second messenger c-di-GMP determines precise gene expression during G1/S transition in Caulobacter crescentus. We show that c-di-GMP stimulates the kinase ShkA by binding to its central pseudo-receiver domain, activates the TacA transcription factor, and initiates a G1/S-specific transcription program leading to cell morphogenesis and S-phase entry. Activation of the ShkA-dependent genetic program causes c-di-GMP to reach peak levels, which triggers S-phase entry and promotes proteolysis of ShkA and TacA. Thus, a gradual increase of c-di-GMP results in precise control of ShkA-TacA activity, enabling G1/S-specific gene expression that coordinates cell cycle and morphogenesis.

The bacterial second messenger cyclic diguanylate (c-di-GMP) regulates a wide range of cellular functions from biofilm formation to growth and survival. Targeting a second-messenger network is challenging because the system involves a multitude of components with often overlapping functions. Here, we present a strategy to intercept c-di-GMP signaling pathways by directly targeting the second messenger. For this, we developed a c-di-GMP-sequestering peptide (CSP) that was derived from a CheY-like c-di-GMP effector protein. CSP binds c-di-GMP with submicromolar affinity. The elucidation of the CSP⋅c-di-GMP complex structure by NMR identified a linear c-di-GMP-binding motif, in which a self-intercalated c-di-GMP dimer is tightly bound by a network of H bonds and π-stacking interactions involving arginine and aromatic residues. Structure-based mutagenesis yielded a variant with considerably higher, low-nanomolar affinity, which subsequently was shortened to 19 residues with almost uncompromised affinity. We demonstrate that endogenously expressed CSP intercepts c-di-GMP signaling and effectively inhibits biofilm formation in Pseudomonas aeruginosa, the most widely used model for serious biofilm-associated medical implications.

Bacteria use small signalling molecules such as (p)ppGpp or c-di-GMP to tune their physiology in response to environmental changes. It remains unclear whether these regulatory networks operate independently or whether they interact to optimize bacterial growth and survival. We report that (p)ppGpp and c-di-GMP reciprocally regulate the growth of Caulobacter crescentus by converging on a single small-molecule-binding protein, SmbA. While c-di-GMP binding inhibits SmbA, (p)ppGpp competes for the same binding site to sustain SmbA activity. We demonstrate that (p)ppGpp specifically promotes Caulobacter growth on glucose, whereas c-di-GMP inhibits glucose consumption. We find that SmbA contributes to this metabolic switch and promotes growth on glucose by quenching the associated redox stress. The identification of an effector protein that acts as a central regulatory hub for two global second messengers opens up future studies on specific crosstalk between small-molecule-based regulatory networks.

Summary Ras-homology (Rho) family GTPases are conserved molecular switches controlling fundamental cellular activities in eukaryotic cells. As such, they are targeted by numerous bacterial toxins and effector proteins, which have been intensively investigated regarding their biochemical activities and discrete target spectra; however, molecular mechanisms of target selectivity have remained elusive. Here, we report a bacterial effector protein that targets all four Rac subfamily members of Rho family GTPases, but none of the closely related Cdc42 or RhoA subfamilies. This exquisite target selectivity of the FIC domain AMP-transferase Bep1 from Bartonella rochalimae is based on electrostatic interactions with a subfamily-specific pair of residues in the nucleotide-binding motif and the Rho insert helix. Residue substitutions at the identified positions in Cdc42 facilitate modification by Bep1, while corresponding Cdc42-like substitutions in Rac1 greatly diminish modification. Our study establishes a structural paradigm for target selectivity towards Rac subfamily GTPases and provides a highly selective tool for their functional analysis.