Correction: Metabolic and taxonomic insights into the Gram-negative natural rubber degrading bacterium Steroidobacter cummioxidans sp. nov., strain 35Y.

[This corrects the article DOI: 10.1371/journal.pone.0197448.].

Metabolic and taxonomic insights into the Gram-negative natural rubber degrading bacterium Steroidobacter cummioxidans sp. nov., strain 35Y.

The pathway of rubber (poly [cis-1,4-isoprene]) catabolism is well documented for Gram-positive rubber degraders but only little information exists for Gram-negative species. The first documented potent rubber degrading Gram-negative strain is Xanthomonas sp. strain 35Y that uses extracellular rubber oxygenases for the initial cleavage of the polyisoprene molecule. However, neither the exact phylogenetic position of Xanthomonas sp. strain 35Y nor the catabolic pathway of the primary polyisoprene cleavage products have been investigated. In this contribution, we started to address both these issues by a comprehensive taxonomic characterization and by the analysis of the draft genome sequence of strain 35Y. Evaluation of the 16S rRNA gene sequence pointed to a borderline taxonomic position of strain 35Y as a novel species of the genus Steroidobacter. Further, substantial differences in the genotypic properties of strain 35Y and the members of the genus Steroidobacter, including average nucleotide identity (ANI) and in silico DNA-DNA hybridization (DDH), resolved the taxonomic position of strain 35Y and suggested its positioning as a novel species of the genus Steroidobacter. This was further confirmed by comparative analysis of physiological and biochemical features of strain 35Y with other members of the genus Steroidobacter. Thus, we conclude that strain 35Y represents a novel species of the genus Steroidobacter, for which we propose the designation Steroidobacter cummioxidans sp. nov., strain 35YT. A comprehensive analysis of the draft genome of S. cummioxidans strain 35Y revealed similarities but also substantial differences to rubber degrading Gram-positive counterparts. In particular, the putative transporters for the uptake of polyisoprene cleavage products differ from Gram-positive rubber degrading species. The draft genome sequence of S. cummioxidans strain 35Y will be useful for researchers to experimentally verify the predicted similarities and differences in the pathways of polyisoprene catabolism in Gram-positive and Gram-negative rubber degrading species.

Prokaryotic squalene-hopene cyclases can be converted to citronellal cyclases by single amino acid exchange.

Squalene-hopene cyclases (SHCs) are prokaryotic enzymes that catalyse the cyclisation of the linear precursor squalene to pentacyclic hopene. Recently, we discovered that a SHC cloned from Zymomonas mobilis (ZMO-1548 gene product) has the unique property to cyclise the monoterpenoid citronellal to isopulegol. In this study, we performed saturation mutagenesis of three amino acids of the catalytic centre of ZMO-1548 (F428, F486 and W555), which had been previously identified to interact with enzyme-bound substrate. Replacement of F428 by tyrosine increased hopene formation from squalene, but isopulegol-forming activity was strongly reduced or abolished in all muteins of position 428. W555 was essential for hopene formation; however, three muteins (W555Y, W428F or W555T) revealed enhanced cyclisation efficiency with citronellal. The residue at position 486 turned out to be the most important for isopulegol-forming activity. While the presence of phenylalanine or tyrosine favoured cyclisation activity with squalene, several small and/or hydrophobic residues such as cysteine, alanine or isoleucine and others reduced activity with squalene but greatly enhanced isopulegol formation from citronellal. Replacement of the conserved aromatic residue corresponding to F486 to cysteine in other SHCs cloned from Z. mobilis (ZMO-0872), Alicyclobacillus acidocaldarius (SHC(Aac)), Acetobacter pasteurianus (SHC(Apa)), Streptomyces coelicolor (SHC(Sco)) and Bradyrhizobium japonicum (SHC(Bja)) resulted in more or less strong isopulegol-forming activities from citronellal. In conclusion, many SHCs can be converted to citronellal cyclases by mutagenesis of the active centre thus broadening the applicability of this interesting class of biocatalyst.

Novel redox-sensing modules: accessory protein- and nucleic acid-mediated signaling.

SIGNIFICANCE: Organisms have evolved both enzymatic and nonenzymatic pathways to prevent oxidative damage to essential macromolecules, including proteins and nucleic acids. Pathways modulated by different protein-based sensory and regulatory modules ensure a rapid and appropriate response. RECENT ADVANCES: In contrast to classical two-component systems that possess internal sensory and regulatory modules, an accessory protein-dependent redox-signaling system has been recently characterized in bacteria. This system senses extracellular iron-mediated oxidative stress signals via an extracellularly located protein (HbpS). In vivo and in vitro studies allowed the elucidation of molecular mechanisms governing this system. Moreover, recent studies show that nucleic acids may also participate in redox-signaling during antioxidative stress response. CRITICAL ISSUES: Research for novel redox-signaling systems is often focused on known types of sensory and regulatory modules. It is also often considered that the oxidative attack of macromolecules, leading to modification and degradation processes, is the final step during oxidative stress. However, recent studies have demonstrated that oxidatively modified macromolecules can be intermediary states in the process of redox-signaling. FUTURE DIRECTIONS: Analyses of adjacent regions of genes encoding for known sensory and regulatory modules can identify potential accessory modules that may increase the complexity of sensing systems. Despite the fact that the involvement of DNA-mediated signaling in the modulation of one bacterial regulator protein has been analyzed in detail, further studies are necessary to identify additional regulators. Given the role of DNA in oxidative-stress response, it is tempting to hypothesize that RNA modules may also mediate redox-signaling.

Activation-independent cyclization of monoterpenoids.

The biosynthesis of cyclic monoterpenes (C(10)) generally requires the cyclization of an activated linear precursor (geranyldiphosphate) by specific terpene cyclases. Cyclic triterpenes (C(30)), on the other hand, originate from the linear precursor squalene by the action of squalene-hopene cyclases (SHCs) or oxidosqualene cyclases (OSCs). Here, we report a novel terpene cyclase from Zymomonas mobilis (ZMO1548-Shc) with the unique capability to cyclize citronellal to isopulegol. To our knowledge, ZMO1548-Shc is the first biocatalyst with diphosphate-independent monoterpenoid cyclase activity. A combinatorial approach using site-directed mutagenesis and modeling of the active site with a bound substrate revealed that the cyclization of citronellal proceeds via a different mechanism than that of the cyclization of squalene.

Squalene-hopene cyclases.

Hopanoids and sterols are members of a large group of cyclic triterpenoic compounds that have important functions in many prokaryotic and eukaryotic organisms. They are biochemically synthesized from linear precursors (squalene, 2,3-oxidosqualene) in only one enzymatic step that is catalyzed by squalene-hopene cyclase (SHC) or oxidosqualene cyclase (OSC). SHCs and OSCs are related in amino acid sequences and probably are derived from a common ancestor. The SHC reaction requires the formation of five ring structures, 13 covalent bonds, and nine stereo centers and therefore is one of the most complex one-step enzymatic reactions. We summarize the knowledge of the properties of triterpene cyclases and details of the reaction mechanism of Alicyclobacillus acidocaldarius SHC. Properties of other SHCs are included.