The opportunistic pathogen strain PAO1 is able to use a variety of organic pollutants as growth substrates, including the anionic detergent sodium dodecyl sulfate (SDS) and long-chain alkanes. While the enzymes initiating SDS and alkane degradation are well known, the subsequent enzymatic steps for degradation of the derived primary long-chain alcohols have not yet been identified. By evaluating genes specifically induced during growth with SDS, a gene cluster encoding a putative alcohol dehydrogenase (PA0364/LaoA), a probable inner membrane protein (PA0365/LaoB), and a presumable aldehyde dehydrogenase (PA0366/LaoC) was identified and designated the Lao (ong-chain-lcohol/ldehyde-xidation) system. Growth experiments with deletion mutants with SDS, 1-dodecanol, and alkanes revealed that LaoA and LaoB are involved in the degradation of primary long-chain alcohols. Moreover, detection of 1-dodecanol oxidation in cell extracts by activity staining revealed an interdependency of LaoA and LaoB for efficient 1-dodecanol oxidation. An analysis yielded no well-characterized homologue proteins for LaoA and LaoB. Furthermore, a gene adjacent to the gene cluster encodes a putative transcriptional regulator (PA0367/LaoR). A deletion mutant exhibited constitutive expression of LaoA and LaoB, indicating that LaoR is a repressor for the expression of Taken together, these results showed that the proteins LaoA and LaoB constitute a novel oxidation system for long-chain alcohols derived from pollutants. The versatile and highly adaptive bacterium a is able to colonize a variety of habitats, including anthropogenic environments, where it is often challenged with toxic compounds. Its ability to degrade such compounds and to use them as growth substrates can significantly enhance spreading of this opportunistic pathogen in hygienic settings, such as clinics or water distribution systems. Thus, knowledge about the metabolism of can contribute to novel approaches for preventing its growth and reducing nosocomial infections. As the Lao system is important for the degradation of two different classes of pollutants, the identification of these novel enzymes can be a useful contribution for developing effective antibacterial strategies.
BackgroundThe aminoglycoside antibiotic gentamicin was supposed to induce a crosstalk between the Cpx- and the Arc-two-component systems (TCS). Here, we investigated the physical interaction of the respective TCS components and compared the results with their respective gene expression and protein abundance. The findings were interpreted in relation to the global proteome profile upon gentamicin treatment.ResultsWe observed specific interaction between CpxA and ArcA upon treatment with the aminoglycoside gentamicin using Membrane-Strep-tagged protein interaction experiments (mSPINE). This interaction was neither accompanied by detectable phosphorylation of ArcA nor by activation of the Arc system via CpxA. Furthermore, no changes in absolute amounts of the Cpx- and Arc-TCS could be determined with the sensitive single reaction monitoring (SRM) in presence of gentamicin. Nevertheless, upon applying shotgun mass spectrometry analysis after treatment with gentamicin, we observed a reduction of ArcA ~ P-dependent protein synthesis and a significant Cpx-dependent alteration in the global proteome profile of E. coli.ConclusionsThis study points to the importance of the Cpx-TCS within the complex regulatory network in the E. coli response to aminoglycoside-caused stress.Electronic supplementary materialThe online version of this article (10.1186/s12866-017-1100-9) contains supplementary material, which is available to authorized users.
Bacteria using toxic chemicals, such as detergents, as growth substrates face the challenge of exposing themselves to cell-damaging effects that require protection mechanisms, which demand energy delivered from catabolism of the toxic compound. Thus, adaptations are necessary for ensuring the rapid onset of substrate degradation and the integrity of the cells. Pseudomonas aeruginosa strain PAO1 can use the toxic detergent sodium dodecyl sulfate (SDS) as a growth substrate and employs, among others, cell aggregation as a protection mechanism. The degradation itself is also a protection mechanism and has to be rapidly induced upon contact to SDS. In this study, gene regulation of the enzymes initiating SDS degradation in strain PAO1 was studied. The gene and an atypical DNA-binding site of the LysR-type regulator SdsB1 were identified and shown to activate expression of the alkylsulfatase SdsA1 initiating SDS degradation. Further degradation of the resulting 1-dodecanol is catalyzed by enzymes encoded by laoCBA, which were shown to form an operon. Expression of this operon is regulated by the TetR-type repressor LaoR. Studies with purified LaoR identified its DNA-binding site and 1-dodecanoyl coenzyme A as the ligand causing detachment of LaoR from the DNA. Transcriptional studies revealed that the sulfate ester detergent sodium lauryl ether sulfate (SLES) induced expression of sdsA1 and the lao operon. Growth experiments revealed an essential involvement of the alkylsulfatase SdsA1 for SLES degradation. This study revealed that the genes for the enzymes initiating the degradation of toxic sulfate-ester detergents are induced stepwise by a positive and a negative regulator in P. aeruginosa strain PAO1. IMPORTANCE Bacterial degradation of toxic compounds is important not only for bioremediation but also for the colonization of hostile anthropogenic environments in which biocides are being used. This study with Pseudomonas aeruginosa expands our knowledge of gene regulation of the enzymes initiating degradation of sulfate ester detergents, which occurs in many hygiene and household products and, consequently, also in wastewater. As an opportunistic pathogen, P. aeruginosa causes severe hygienic problems because of its pronounced biocide resistance and its metabolic versatility, often combined with its pronounced biofilm formation. Knowledge about the regulation of detergent degradation, especially regarding the ligands of DNA-binding regulators, may lead to the rational development of specific inhibitors for restricting growth and biofilm formation of P. aeruginosa in hygienic settings. In addition, it may also contribute to optimizing bioremediation strategies not only for detergents but also for alkanes, which when degraded merge with sulfate ester degradation at the level of long-chain alcohols.
The opportunistic pathogen Pseudomonas aeruginosa can utilize unusual carbon sources like sodium dodecyl sulfate (SDS) and alkanes. Whereas the initiating enzymatic steps of the corresponding degradation pathways have been characterized in detail, the oxidation of the emerging long-chain alcohols found little attention. Recently, the genes for the Lao ( l ong-chain a lcohol/ a ldehyde o xidation) system were discovered to be involved in the oxidation of long-chain alcohols derived from SDS and alkane degradation. In the Lao-system, LaoA is predicted to contain the catalytic site, however, according to genetic studies, efficient long-chain alcohol oxidation additionally required the Tat-dependent protein LaoB. In the present study, the Lao-system was further characterized. In vivo analysis revealed that the Lao-system complements the substrate spectrum of the well-described Exa-system, which is required for growth with ethanol and other short-chain alcohols. Mutational analysis revealed that the Tat-site of LaoB was required for long-chain alcohol oxidation activity strongly suggesting a periplasmic localization of the process. Purified LaoA was only fully active when co-purified with LaoB. Interestingly, in vitro activity of the purified LaoAB-complex also depended on the presence of the Tat-site. The co-purified LaoAB-complex contained a flavin co-factor and preferentially oxidized a range of saturated, unbranched primary alcohols. Furthermore, the LaoAB-complex could reduce cytochrome c 550 type redox carriers like ExaB, a subunit of the Exa alcohol dehydrogenase system. LaoAB-complex activity was stimulated by rhamnolipids in vitro . In summary, LaoAB constitutes an unprecedented protein complex with specific properties apparently required for oxidizing long-chain alcohols. IMPORTANCE Pseudomonas aeruginosa is a major threat to public health. Its ability to thrive in clinical settings, water distribution systems or even jet fuel tanks is linked to detoxification and degradation of diverse hydrophobic substrates that are metabolized via alcohol intermediates. Our study illustrates a novel flavoprotein long-chain alcohol dehydrogenase consisting of a facultative two-subunit complex, which is unique among related enzymes while the homologs of the corresponding genes are found in numerous bacterial genomes. Understanding the involved catalytic and compartmentalization processes is of great interest for biotechnological and hygiene research as it may be a potential starting point for rationally designing novel antibacterial substances with high specificity against this opportunistic pathogen.
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