Direct binding of benzoate derivatives to two chemoreceptors with Cache sensor domains in Halomonas titanicae KHS3.

Halomonas titanicae KHS3, isolated from a hydrocarbon-contaminated sea harbor in Argentina, is able to grow on aromatic hydrocarbons and displays chemotaxis toward those compounds. This behavior might contribute to the efficiency of its degradation capacity. Using high throughput screening, we identified two chemoreceptors (Htc1 and Htc2) that bind benzoate derivatives and other organic acids. Whereas Htc1 has a high affinity for benzoate (Kd 112 µM) and 2-hydroxybenzoate (Kd 83 µM), Htc2 binds 2-hydroxybenzoate with low affinity (Kd 3.25 mM), and also C3/C4 dicarboxylates. Both chemoreceptors are able to trigger a chemotactic response of E. coli cells to the specific ligands. A H. titanicae htc1 mutant has reduced chemotaxis toward benzoate, and is complemented upon expression of the corresponding receptor. Both chemoreceptors have a Cache-type sensor domain, double (Htc1) or single (Htc2), and their ability to bind aromatic compounds is reported here for the first time.

Chemosensory pathways of Halomonas titanicae KHS3 control chemotaxis behaviour and biofilm formation.

Halomonas titanicae KHS3 is a marine bacterium whose genome codes for two different chemosensory pathways. Chemosensory gene cluster 1 is very similar to the canonical Che cluster from Escherichia coli. Chemosensory cluster 2 includes a gene coding for a diguanylate cyclase with receiver domains, suggesting that it belongs to the functional group that regulates alternative cellular functions other than chemotaxis. In this work we assess the functional roles of both chemosensory pathways through approaches that include the heterologous expression of Halomonas proteins in E. coli strains and phenotypic analyses of Halomonas mutants. Our results confirm that chemosensory cluster 1 is indeed involved in chemotaxis behaviour, and only proteins from this cluster complement E. coli defects. We present evidence suggesting that chemosensory cluster 2 resembles the Wsp pathway from Pseudomonas, since the corresponding methylesterase mutant shows an increased methylation level of the cognate receptor and develops a wrinkly colony morphology correlated with an increased ability to form biofilm. Consistently, mutational interruption of this gene cluster correlates with low levels of biofilm. Our results suggest that the proteins from each pathway assemble and function independently. However, the phenotypic characteristics of the mutants show functional connections between the pathways controlled by each chemosensory system.

New Findings on Aromatic Compounds' Degradation and Their Metabolic Pathways, the Biosurfactant Production and Motility of the Halophilic Bacterium Halomonas sp. KHS3.

The study of the aromatic compounds' degrading ability by halophilic bacteria became an interesting research topic, because of the increasing use of halophiles in bioremediation of saline habitats and effluents. In this work, we focused on the study of aromatic compounds' degradation potential of Halomonas sp. KHS3, a moderately halophilic bacterium isolated from hydrocarbon-contaminated seawater of the Mar del Plata harbour. We demonstrated that H. sp. KHS3 is able to grow using different monoaromatic (salicylic acid, benzoic acid, 4-hydroxybenzoic acid, phthalate) and polyaromatic (naphthalene, fluorene, and phenanthrene) substrates. The ability to degrade benzoic acid and 4-hydroxybenzoic acid was analytically corroborated, and Monod kinetic parameters and yield coefficients for degradation were estimated. Strategies that may enhance substrate bioavailability such as surfactant production and chemotactic responses toward aromatic compounds were confirmed. Genomic sequence analysis of this strain allowed us to identify several genes putatively related to the metabolism of aromatic compounds, being the catechol and protocatechuate branches of ß-ketoadipate pathway completely represented. These features suggest that the broad-spectrum xenobiotic degrader H. sp. KHS3 could be employed as a useful biotechnological tool for the cleanup of aromatic compounds-polluted saline habitats or effluents.

Bacterial chemoreceptors of different length classes signal independently.

Bacterial chemoreceptors sense environmental stimuli and govern cell movement by transmitting the information to the flagellar motors. The highly conserved cytoplasmic domain of chemoreceptors consists in an alpha-helical hairpin that forms in the homodimer a coiled-coil four-helix bundle. Several classes of chemoreceptors that differ in the length of the coiled-coil structure were characterized. Many bacterial species code for chemoreceptors that belong to different classes, but how these receptors are organized and function in the same cell remains an open question. E. coli cells normally code for single class chemoreceptors that form extended arrays based on trimers of dimers interconnected by the coupling protein CheW and the kinase CheA. This structure promotes effective coupling between the different receptors in the modulation of the kinase activity. In this work, we engineered functional derivatives of the Tsr chemoreceptor of E. coli that mimic receptors whose cytoplasmic domain is longer by two heptads. We found that these long Tsr receptors did not efficiently mix with the native receptors and appeared to function independently. Our results suggest that the assembly of membrane-bound receptors of different specificities into mixed clusters is dictated by the length-class to which the receptors belong, ensuring cooperative function only between receptors of the same class.

Functional and structural effects of seven-residue deletions on the coiled-coil cytoplasmic domain of a chemoreceptor.

Chemoreceptors transmit signals from the environment to the flagellar motors via a histidine kinase that controls the phosphorylation level of the effector protein CheY. The cytoplasmic domain of chemoreceptors is strongly conserved and consists of a long alpha-helical hairpin that forms, in the dimer, a coiled-coil four-helix bundle. Changes in this domain during evolution are characterized by the presence of seven-residue insertions/deletions located symmetrically with respect to the hairpin turn, suggesting that specific interactions between the helices that form the hairpin are required for function. We assessed the impact of seven-residue deletions on the signalling ability and higher-order organization of the serine chemoreceptor from Escherichia coli. Our results indicate that symmetry alterations between the two branches of the cytoplasmic hairpin seriously compromise chemoreceptor function. Shorter functional versions of Tsr with symmetrical deletions form mixed trimers of dimers when coexpressed with Tar, the aspartate receptor of E. coli. However, Tar function in those cells is impaired, suggesting that the length difference between receptors introduces non-functional distortions into the chemoreceptor cluster. This observation is reinforced by the analysis of coexpression of Tar with chemoreceptors from Rhodobacter sphaeroides that naturally belong to a shorter-length class.

A chemoreceptor from Pseudomonas putida forms active signalling complexes in Escherichia coli.

Chemoreceptors sense environmental stimuli and transmit the information to the flagellar motors via a histidine kinase that controls the phosphorylation level of the effector protein CheY. The cytoplasmic domain of chemoreceptors consists of a long α-helical hairpin that forms, in the dimer, a coiled-coil four-helix bundle. Even though the sequence and general structure of the cytoplasmic domain are strongly conserved within Eubacteria and Archaea, the total length of the α-helical hairpin is variable and defines seven classes of chemoreceptors. In this work we assessed the functional properties of a Pseudomonas receptor when assembled in signalling complexes with Escherichia coli proteins. Our results show that the foreign receptor does not confer fully chemotactic abilities upon E. coli cells, but is able to form active ternary complexes that respond to the specific stimuli by modulating the activity of the associated kinase. The observed responses are subject to adaptation, depending on the presence of the methylation enzymes CheR and/or CheB. The ability of foreign receptors to signal through signalling complexes with non-cognate proteins would allow the use of the well-studied E. coli system to reveal the detection specificity of uncharacterized chemoreceptors from other micro-organisms.

Genetic dissection of a motility-associated c-di-GMP signalling protein of Pseudomonas putida.

Lack of the Pseudomonas putida PP2258 protein or its overexpression results in defective motility on solid media. The PP2258 protein is tripartite, possessing a PAS domain linked to two domains associated with turnover of c-di-GMP - a cyclic nucleotide that controls the switch between motile and sessile lifestyles. The second messenger c-di-GMP is produced by diguanylate cyclases and degraded by phosphodiesterases containing GGDEF and EAL or HD-GYP domains respectively. It is common for enzymes involved in c-di-GMP signalling to contain two domains with potentially opposing c-di-GMP turnover activities; however, usually one is degenerate and has been adopted to serve regulatory functions. Only a few proteins have previously been found to have dual enzymatic activities - being capable of both synthesizing and hydrolysing c-di-GMP. Here, using truncated and mutant derivatives of PP2258, we show that despite a lack of complete consensus in either the GGDEF or EAL motifs, the two c-di-GMP turnover domains can function independently of each other, and that the diguanylate cyclase activity is regulated by an inhibitory I-site within its GGDEF domain. Thus, motility-associated PP2258 can be added to the short list of bifunctional c-di-GMP signalling proteins.