Hortencia Silva-Jiménez

Bacterial Sensor Kinases: Diversity in the Recognition of Environmental Signals

Bacteria sense and respond to a wide range of physical and chemical signals. Central to sensing and responding to these signals are two-component systems, which have a sensor histidine kinase (SK) and a response regulator (RR) as basic components. Here we review the different molecular mechanisms by which these signals are integrated and modulate the phosphorylation state of SKs. Apart from the basic mechanism, which consists of signal recognition by the SK that leads to an alteration of its autokinase activity and subsequently a change in the RR phosphorylation state, a variety of alternative modes have evolved. The biochemical data available on SKs, particularly their molecular interactions with signals, nucleotides, and their cognate RRs, are also reviewed.

Analysis of solvent tolerance in Pseudomonas putida DOT‐T1E based on its genome sequence and a collection of mutants

Pseudomonas putida strains are prevalent in a variety of pristine and polluted environments. The genome of the solvent‐tolerant P. putida strain DOT‐T1E which thrives in the presence of high concentrations of monoaromatic hydrocarbons, contains a circular 6.3 Mbp chromosome and a 133 kbp plasmid. Omics information has been used to identify the genes and proteins involved in solvent tolerance in this bacterium. This strain uses a multifactorial response that involves fine‐tuning of lipid fluidity, activation of a general stress‐response system, enhanced energy generation, and induction of specific efflux pumps that extrude solvents to the medium. Local and global transcriptional regulators participate in a complex network of metabolic functions, acting as the decision makers in the response to solvents.

Responses of Pseudomonas putida to toxic aromatic carbon sources

A number of bacteria can use toxic compounds as carbon sources and have developed complex regulatory networks to protect themselves from the toxic effects of these compounds as well as to benefit from their nutritious properties. As a model system we have studied the responses of Pseudomonas putida strains to toluene. Although this compound is highly toxic, several strains are able to use it for growth. Particular emphasis was given to the responses in the context of taxis, resistance and toluene catabolism. P. putida strains analysed showed chemotactic movements towards toluene. Strain DOT-T1E was characterised by an extreme form of chemotaxis, termed hyperchemotaxis, which is mediated by the McpT chemoreceptor encoded by plasmid pGRT1. Close McpT homologs are found in a number of other plasmids encoding degradation pathways of toxic compounds. The pGRT1 plasmid harbours also the genes for the TtgGHI efflux pump which was identified as the primary determinant for the resistance of strain DOT-T1E towards toluene. Pump expression is controlled by the TtgV repressor in response to a wide range of different mono- and biaromatic compounds. Strain DOT-T1E is able to degrade toluene, benzene and ethylbenzene via the toluene dioxygenase (TOD) pathway. The expression of the pathway operon is controlled by the TodS/T two component system. The sensor kinase TodS recognizes toluene with nanomolar affinity, which in turn triggers an increase in its autophosphorylation and consequently transcriptional activation. Data suggest that transcriptional activation of the TOD pathway occurs at very low toluene concentrations whereas TtgV mediated induction of pump expression sets in as the toluene concentration further increases.

Study of the TmoS/TmoT two‐component system: towards the functional characterization of the family of TodS/TodT like systems

The two‐component system TmoS/TmoT controls the expression of the toluene‐4‐monooxygenase pathway in Pseudomonas mendocina RK1 via modulation of PtmoX activity. The TmoS/TmoT system belongs to the family of TodS/TodT like proteins. The sensor kinase TmoS is a 108 kDa protein composed of seven different domains. Using isothermal titration calorimetry we show that purified TmoS binds a wide range of aromatic compounds with high affinities. Tightest ligand binding was observed for toluene (KD = 150 nM), which corresponds to the highest affinity measured between an effector and a sensor kinase. Other compounds with affinities in the nanomolar range include benzene, the 3 xylene isomers, styrene, nitrobenzene or p‐chlorotoluene. We demonstrate that only part of the ligands that bind to TmoS increase protein autophosphorylation in vitro and consequently pathway expression in vivo. These compounds are referred to as agonists. Other TmoS ligands, termed antagonists, failed to increase TmoS autophosphorylation, which resulted in their incapacity to stimulate gene expression in vivo. We also show that TmoS saturated with different agonists differs in their autokinase activities. The effector screening of gene expression showed that promoter activity of PtmoX and PtodX (controlled by the TodS/TodT system) is mediated by the same set of 22 compounds. The common structural feature of these compounds is the presence of a single aromatic ring. Among these ligands, toluene was the most potent inducer of both promoter activities. Information on the TmoS/TmoT and TodS/TodT system combined with a sequence analysis of family members permits to identify distinct features that define this protein family.

Catabolite Repression of the TodS/TodT Two-Component System and Effector-Dependent Transphosphorylation of TodT as the Basis for Toluene Dioxygenase Catabolic Pathway Control

The TodS/TodT two-component system of Pseudomonas putida regulates the expression of the toluene dioxygenase (tod) operon for the metabolism of toluene, benzene, and ethylbenzene. The sensor kinase TodS has a complex domain arrangement containing two functional modules, each harboring a sensor and an autokinase domain separated by a receiver domain. The TodT protein is the cognate response regulator that activates transcription of the toluene dioxygenase (TOD) pathway genes at the P(todX) promoter. We report in this study that the todST operon is transcribed from a main promoter and that the +1 initiation point is located 31 nucleotides upstream from the A of the first ATG codon and is preceded by a -10/-35 canonical promoter. Expression from P(todS) is under catabolite control, and in cells growing with glucose, the level of expression from this promoter is reduced, which in turn translates to low levels of the TodS/TodT regulators and results in a decrease of transcription from the P(todX) promoter. Thus, the main underlying regulatory mechanisms of the tod structural genes are at the levels of catabolite repression control from P(todS) and transcription activation, mediated by the TodT response regulator through a regulatory cascade in which the effector enhances autophosphorylation of TodS by ATP, with subsequent transphosphorylation of TodT.

The enigma of cytosolic two-component systems: a hypothesis

One-component systems (OCSs) and cytosolic two-component regulatory systems (TCSs) appear to share the same biological function, which consists in the transcriptional control in response to the cellular concentration of signal molecules. However, cytosolic TCSs as compared with OCSs represent a genetic and metabolic burden to the cell: the genetic information encoding a TCS is significantly larger than that of an OCS, two or more proteins instead of one need to be synthesized for a TCS and operation of the latter system requires the expense of ATP which is not the case for most OCSs. The evolutionary advantages of cytosolic TCSs over OCSs are thus not obvious. We hypothesize here that the increased capacity of cytosolic TCSs to respond to multiple signals is a major advantage over OCSs. Different mechanisms for the incorporation of additional signals into the regulatory circuit are discussed. The inclusion of several signals into the definition of the final regulatory response is proposed to result in a better adaptation of the host to given environmental conditions.

Characterization of molecular interactions using isothermal titration calorimetry.

Isothermal titration calorimetry (ITC) is based on a simple titration of one ligand with another and the small heat changes caused by the molecular interaction are detected. From one ITC experiment the complete set of thermodynamic parameters of binding including association and dissociation constants as well as changes in enthalpy, entropy, and free energy can be derived. Using this technique almost any type of molecular interaction can be analyzed. Both ligands are in solution, and there is no need for their chemical derivatization. There are no limits as to the choice of the analysis buffer, and the analysis temperature can be set between 4 and 80 °C. This technique has been primarily applied to study the interaction between various proteins of Pseudomonas with small molecule ligands. In addition, ITC has been used to study the binding of Pseudomonas proteins to target DNA fragments.

Construction of a prototype two-component system from the phosphorelay system TodS/TodT

Two-component systems (TCSs) play key roles in the adaptation of bacteria to environmental changes. In prototype TCSs a single phosphoryl transfer between the sensor kinase and response regulator occurs, whereas phosphorelay TCSs are characterised by a His1-Asp1-His2-Asp2 phosphorylation cascade. The TodS/TodT TCS controls the expression of a toluene degradation pathway and the TodS sensor kinase operates by a three-step internal phosphorelay. Based on TodS we report the construction of a minimal form of TodS, termed as Min-TodS, that contains only three of the seven TodS domains. Min-TodS is composed of the N-terminal PAS sensor domain as well as the C-terminal dimerisation/phosphotransfer domain and catalytic domain of TodS. We have conducted a comparative analysis of the phosphorelay TCS with its prototypal derivative. We demonstrate that Min-TodS binds effector molecules with affinities comparable with those observed for TodS. Min-TodS forms a TCS with TodT and toluene increases the amount of TodT-P. In contrast to TodS, toluene does not stimulate Min-TodS autophosphorylation. The half-life of Min-TodS-P was significantly increased as compared with TodS. Analysis of TodSD500A revealed that the hydrolysis of the acylphosphate of the receiver domain is responsible for the reduced half-life of TodS. The regulation of P(todX) expression by Min-TodS/TodT and TodS/TodT in response to different effectors are compared. The Min-TodS/TodT system was characterized by a higher basal activity but a lower magnitude of response. Data will be discussed in the context that the phosphorelay system appears to be better suited for the control of a degradation pathway for toxic compounds.

Multiple signals modulate the activity of the complex sensor kinase TodS

The reason for the existence of complex sensor kinases is little understood but thought to lie in the capacity to respond to multiple signals. The complex, seven‐domain sensor kinase TodS controls in concert with the TodT response regulator the expression of the toluene dioxygenase pathway in Pseudomonas putida F1 and DOT‐T1E. We have previously shown that some aromatic hydrocarbons stimulate TodS activity whereas others behave as antagonists. We show here that TodS responds in addition to the oxidative agent menadione. Menadione but no other oxidative agent tested inhibited TodS activity in vitro and reduced PtodX expression in vivo. The menadione signal is incorporated by a cysteine‐dependent mechanism. The mutation of the sole conserved cysteine of TodS (C320) rendered the protein insensitive to menadione. We evaluated the mutual opposing effects of toluene and menadione on TodS autophosphorylation. In the presence of toluene, menadione reduced TodS activity whereas toluene did not stimulate activity in the presence of menadione. It was shown by others that menadione increases expression of glucose metabolism genes. The opposing effects of menadione on glucose and toluene metabolism may be partially responsible for the interwoven regulation of both catabolic pathways. This work provides mechanistic detail on how complex sensor kinases integrate different types of signal molecules.

The Pseudomonas putida HskA hybrid sensor kinase controls the composition of the electron transport chain

Sensor kinases play a key role in sensing and responding to environmental and physiological signals in bacteria. In this study we characterized a previously unknown orphan hybrid sensor kinase from Pseudomonas putida, which is conserved in several Pseudomonads. Inactivation of the gene coding for this sensor kinase, which we have named HskA, modified the expression of at least 85 genes in cells growing in a complete medium. HskA showed a strong influence on the composition of the electron transport chain. In cells growing exponentially in a complete medium, the absence of HskA led to a significant reduction in the expression of the genes coding for the bc1 complex and for the CIO and Cbb3-1 terminal oxidases. In stationary phase cells, however, lack of HskA caused a higher expression of the Cyo terminal oxidase and a lower expression of the Aa3 terminal oxidase. The HskA polypeptide shows two PAS (signal-sensing) domains, a transmitter domain containing the invariant phosphorylatable histidine and an ATP binding site, and a receiver domain containing the conserved aspartate capable of transphosphorylation, but lacks an Hpt module. It is therefore a hybrid sensor kinase. Phosphorylation assays showed that purified HskA undergoes autophosphorylation in the presence of ATP.