The Regulatory System of the Sty Operon

The two-component regulatory system StyS/StyR of the sty-operon from the strains Pseudomonas putida CA-3, Pseudomonas sp. Y2, and Pseudomonas fluo-rescens ST was investigated in detail (Velasco et al. 1998; Santos et al. 2000; O'Leary et al. 2001, 2002a; Leoni et al. 2003). With respect to the conserved operon organization among pseudomonads (Fig. 3.3a, b), the described features of this regulatory system are supposed to occur and interact similarly in all functional sty-operons of pseudomonads.

The gene products of styS and styR, which are located proximate upstream to styABCD (Figs. 3.3 and 3.4), both show similarities to several two-component transduction systems from prokaryotes and eukaryotes (Reizer and Saier 1997). In fact, they were found to positively affect sty-operon transcription (Fig. 3.4) and both genes are most likely expressed in a transcription-coupled fashion (O'Leary et al. 2001). On amino acid level, StyS exhibits similarity to sensor kinase proteins, especially to TodS, TutC, and TutS which regulate toluene catabolism in Pseudomonas spp. and Thaurea sp. (Coschigano and Young 1997; Lau et al. 1997; Leuthner and Heider 1998). A similar two-component regulator was reported for the biphenyl degradation pathway of Rhodococcus sp. M5 (Labbe et al. 1997). Close relationship of StyS to the above regulator proteins could also be demonstrated by the ability of styrene to act as an inducer of toluene degradation (Cho et al. 2000; Mosqueda and Ramos 2000). The sensor kinase StyS consists of five functionally different domains: input-1, histidine kinase-1 (HK-1), receiver, input-2, and histidine kinase-2 (HK-2) (Fig. 3.4). Both input domains contain typical motives of PAS-sensing domains (these are signaling modules for changes of oxygen level, redox potential, light, and small ligand concentrations), and are

Stye Mechanism

Fig. 3.4 Structure and mechanism of the styrene-inducible regulatory system of the sty-operon and a detailed view on the complex styA-promoter region (adapted from O'Leary et al. 2002b). The regulatory system (StyS/StyR with conserved motifs, sty-operon with indicated promoter sites PstySR and PstyABCD, and styA-promoter region) and its effect on the transcription of styrene-catabolic genes based upon data obtained from styrene-utilizing Pseudomonas fluores-cens ST. Styrene is sensed by the input 1 domain of StyS and changing oxygen concentrations might be also sensed by another input domain of StyS. Afterwards one of the two histidine kinase domains gets activated by phosphorylation of a histidine residue (bold 'H') and the phosphoryl group is subsequently transferred onto an aspartate residue (bold 'D') of the receiver domain of StyR. This activated response regulator StyR can then bind by its C-terminal DNA-binding domain to one of the three STY-sites of the promoter region. In the presence of styrene as the sole source of carbon, activated StyR binds to STY2 and RNA polymerase and then binds to the promoter region (underlined) and initiates transcription of styABCD. The complete styA-promoter region is outlined, numbered according to the transcriptional start site (+1, bold 'A'), and the following elements between StyR-stop and StyA-start codon (bold faced) are indicated: the three palindromic sequences STY1, STY2, STY3 (arrows), the consensus sequence 5'-WAT-CAANNNNTTR-3' (complementary encoded to styABCD) for binding of the integration host factor IHF (gray box), a directed repeat sequence 5'-CTGCTTC-3' at the beginning (boxed), two 5'-ATTTTTA-3' motifs (dotted lines), and the potential ribosome binding site (black box)

expected to sense traces of styrene or degradation intermediates, perhaps as a consequence of an altered redox potential of the cell (Coschigano and Young 1997; Lau et al. 1997; Velasco et al. 1998; Santos et al. 2000). The two histidine kinase domains H/N/G1/F/G2 are highly conserved and harbor a characteristic kinase amino acid motif including a single histidine residue for the phosphoryl group transfer towards the response regulator StyR (Coschigano and Young 1997; Grebe and Stock 1999; Mosqueda and Ramos 2000). Slight differences in their amino acid sequence allow their classification into the kinase superfamilies 1a (HK-1) and 4 (HK-2). The receiver domain belongs to the RA2-receiver superfamily and contains a conserved amino acid motif D/D/S/K typical for bacterial response regulators (Grebe and Stock 1999).

The second half of the sty-operon regulatory system is represented by StyR. Like StyS, this protein shows high similarity to two-component regulators, especially those ones of toluene catabolism like TodT and TudC (Lau et al. 1997; Leuthner and Heider 1998). StyR comprises two different domains, an N-terminal regulatory (or receiver) domain (pos. 1 to 127) and a C-terminal DNA-binding domain (pos. 142 to 208), which are joined by a 34-amino acid long Q-linker (O'Leary et al. 2002b). The receiver domain belongs to the RA4-receiver subfamily and harbors the conserved amino acid motif (D/)D/D/T/K (Baikalov et al. 1996; Grebe and Stock 1999), whereas the highly conserved C-terminus shows similarities to family-3 response regulators (Reizer and Saier 1997; Velasco et al. 1998). Thus, StyR belongs to the FixJ/NarL-subfamily of response regulators.

Several studies have demonstrated that StyS-StyR plays a key role within styrene degradation and affects positive as well as negative the expression of catabolic genes (Fig. 3.4) (Panke et al. 1998; Velasco et al. 1998; Santos et al. 2000, 2002; O'Leary et al. 2001, 2002a, b; Leoni et al. 2003, 2005). The presence of styrene is indispensable for the transcription of styA and the complete functional sty-operon. In most cases, transcripts of the regulatory proteins were found only in presence of styrene (O'Connor et al. 1995; O'Leary et al. 2001). Various carbon sources like phenylacetic acid, glutamate, glucose, and citrate, were found to repress the transcription of catabolic genes even when styrene is present in the culture medium (O'Connor et al. 1995; Santos et al. 2000; O'Leary et al. 2001). Only styrene induces significantly the upper route and phenylacetic acid or its metabolites do not, even at presence of phenylacetic acid in the medium, the sty-operon transcription is repressed (O'Leary et al. 2001). Thus, the upper (sty-operon) and lower (phenyla-cetic acid degradation genes) pathway of styrene metabolism are likely to be regulated separately.

Based on identified regulatory elements, the following regulation mechanism was postulated. Styrene is sensed by the sensory input domain of StyS and as a result, one of the two histidine kinase domains is activated by a kinase-catalyzed phosphory-lation of conserved histidine residue (H). This phosphoryl group is then transferred onto a conserved aspartic acid residue (D) of the receiver domain of StyR. It was demonstrated that phosphorylated and thus activated StyR binds co-operatively to a palindromic sequence STY2 (Fig. 3.4) of the styA promoter region (PstyABCD), leading to highly attracted binding of RNA polymerase to a conserved sequence of the promoter region (5'-TGTTAGCTT-3'). In that case, StyR controls gene transcription of the upper styrene degradation route as an activator, but after translation of catabolic genes, high amounts of phosphorylated StyR may accumulate and act then as a repressor of transcription. The latter effect is caused by binding of activated StyR to a negative regulatory site STY3 within the styA promoter region. Another regulatory sequence STY1, located upstream to STY2 and STY3, might affect transcription of catabolic genes positively (presence of styrene) or negatively (presence of glucose or other carbon sources). Additionally, it was demonstrated that an integration host factor (IHF, a small heterodimeric protein) affects styABCD transcription due to binding to a consensus sequence in the styA-promoter region (5'-WATCAANNNNTTR-3', complementary encoded to styABCD) (Fig. 3.4) (Leoni et al. 2005). A positive role for PstyABCD regulation is expected.

Biochemical characterization of the wild-type styrene monooxygenase StyA/ StyB from Pseudomonas sp. VLB120 indicated that the expression level of the oxygenase subunit StyA exceeds by far that of the NADH:FAD oxidoreductase StyB (Otto et al. 2004). This behavior is similar to a 4-hydroxyphenylacetate 3-monooxygenase (Louie et al. 2003) and probably accounts for the fact that StyB has a much higher specific activity than StyA. Moreover, StyA should not be limited by a molar deficit of StyB since reducing equivalents (FADH2) are transferred mainly by diffusion and a general necessity of StyA/StyB contact is not given (Kantz et al. 2005; Otto et al. 2004). Proteome analysis of Pseudomonas putida CA-3 pointed to similar results and indicated that StyA and StyD are by far the most abundant proteins of the upper sty-operon which exceed StyB and StyC for at least one order of magnitude (Nikodinovic-Runic et al. 2009).

The regulatory elements styS-styR from pseudomonads are the only ones hitherto found to be involved in styrene catabolism. Similar elements are lacking in close neighborhood of all other (putative) styrene monooxygenases identified from metagenomes and different Actinobacteria (Fig. 3.3c). However, a distant localization of related two-component regulatory systems cannot be excluded. Interestingly, two other regulatory elements were found in the neighborhood of (hypothetical) styrene monooxygenases from Actinobacteria: a PaaX- and an AraC-like regulator. The first one is typical for phenylacetic acid degradation which suggests a dependency to styrene degradation. The latter one belongs to the diverse group of AraC/XylS-family transcriptional regulators (Gallegos et al. 1997) frequently involved in the regulatory machinery of aromatic hydrocarbon degradation. A functional link of both elements to SMO regulation is still missing.

Continue reading here: Genetic Localization of Single Styrene Monooxygenases in Other Organisms

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