Bodo Philipp

Anaerobic Degradation of Phenolic Compounds

Mononuclear aromatic compounds are degraded anaerobically through three main pathways, the benzoyl-CoA pathway, the resorcinol pathway, and the phloroglucinol pathway. Various modification reactions channel a broad variety of mononuclear aromatics including aromatic hydrocarbons into either one of these three pathways. Recently, a further pathway was discovered with hydroxyhydroquinone as central intermediate through which especially nitrate-reducing bacteria degrade phenolic compounds and some hydroxylated benzoates. Comparison of the various strategies taken for the degradation of aromatics in the absence of oxygen demonstrates that the biochemistry of breakdown of these compounds is determined largely by the overall reaction energetics and, more precisely, by the redox potentials of the electron acceptor systems used. Nitrate reducers differ in their strategies significantly from those used by sulfate-reducing or fermenting bacteria.

Bacterial degradation of bile salts

Bile salts are surface-active steroid compounds. Their main physiological function is aiding the digestion of lipophilic nutrients in intestinal tracts of vertebrates. Many bacteria are capable of transforming and degrading bile salts in the digestive tract and in the environment. Bacterial bile salt transformation and degradation is of high ecological relevance and also essential for the biotechnological production of steroid drugs. While biotechnological aspects have been reviewed many times, the physiological, biochemical and genetic aspects of bacterial bile salt transformation have been neglected. This review provides an overview of the reaction sequence of bile salt degradation and on the respective enzymes and genes exemplified with the degradation pathway of the bile salt cholate. The physiological adaptations for coping with the toxic effects of bile salts, recent biotechnological applications and ecological aspects of bacterial bile salt metabolism are also addressed. As the pathway for bile salt degradation merges with metabolic pathways for bacterial transformation of other steroids, such as testosterone and cholesterol, this review provides helpful background information for metabolic engineering of steroid-transforming bacteria in general.

Respiration of 2,4,6-Trinitrotoluene by Pseudomonas sp. Strain JLR11

Under anoxic conditions Pseudomonas sp. strain JLR11 can use 2,4, 6-trinitrotoluene (TNT) as the sole N source, releasing nitrite from the aromatic ring and subsequently reducing it to ammonium and incorporating it into C skeletons. This study shows that TNT can also be used as a terminal electron acceptor in respiratory chains under anoxic conditions by Pseudomonas sp. strain JLR11. TNT-dependent proton translocation coupled to the reduction of TNT to aminonitrotoluenes has been observed in TNT-grown cells. This extrusion did not occur in nitrate-grown cells or in anaerobic TNT-grown cells treated with cyanide, a respiratory chain inhibitor. We have shown that in a membrane fraction prepared from Pseudomonas sp. strain JLR11 grown on TNT under anaerobic conditions, the synthesis of ATP was coupled to the oxidation of molecular hydrogen and to the reduction of TNT. This phosphorylation was uncoupled by gramicidin. Respiration by Pseudomonas sp. strain JLR11 is potentially useful for the biotreatment of TNT in polluted waters and soils, particularly in phytorhizoremediation, in which bacterial cells are transported to the deepest root zones, which are poor in oxygen.

SiaA and SiaD are essential for inducing autoaggregation as a specific response to detergent stress in Pseudomonas aeruginosa.

Cell aggregation is a stress response and serves as a survival strategy for Pseudomonas aeruginosa strain PAO1 during growth with the toxic detergent Na-dodecylsulfate (SDS). This process involves the psl operon and is linked to c-di-GMP signalling. The induction of cell aggregation in response to SDS was studied. Transposon and site-directed mutagenesis revealed that the cupA-operon and the co-transcribed genes siaA (PA0172) and siaD (PA0169) were essential for SDS-induced aggregation. While siaA encodes a putative membrane protein with a HAMP and a PP2C-like phosphatase domain, siaD encodes a putative diguanylate cyclase involved in the biosynthesis of c-di-GMP. Complementation studies uncovered that the loss of SDS-induced aggregation in the formerly isolated spontaneous mutant strain N was caused by a non-functional siaA allele. DNA-microarray analysis of SDS-grown cells revealed consistent activation of eight genes, including cupA1, with known or presumptive important functions in cell aggregation in the parent strain compared with non-aggregating siaA and siaD mutants. A siaAD-dependent increase of cupA1 mRNA levels in SDS-grown cells was also shown by Northern blots. These results clearly demonstrate that SiaAD are essential for inducing cell aggregation as a specific response to SDS and suggest that they are responsible for perceiving and transducing SDS-related stress.

Cell aggregation of Pseudomonas aeruginosa strain PAO1 as an energy-dependent stress response during growth with sodium dodecyl sulfate.

Pseudomonas aeruginosa strain PAO1 grew with the detergent sodium dodecyl sulfate (SDS). The growth started with the formation of macroscopic cell aggregates which consisted of respiring cells embedded in an extracellular matrix composed of acidic polysaccharides and DNA. Damaged and uncultivable cells accumulated in these aggregates compared to those cells that remained suspended. We investigated the response of suspended cells to SDS under different conditions. At high energy supply, the cells responded with a decrease in optical density and in viable counts, release of protein and DNA, and formation of macroscopic aggregates. This response was not observed if the energy supply was reduced by inhibiting respiration with KCN, or if cells not induced for SDS degradation were exposed to SDS. Exposure to SDS caused cell lysis without aggregation if cells were completely deprived of energy, either by applying anoxic conditions, by addition of CCCP, or by addition of KCN to a mutant defective in cyanide-insensitive respiration. Aggregated cells showed a more than 100-fold higher survival rate after exposure to SDS plus CCCP than suspended cells. Our results demonstrate that cell aggregation is an energy-dependent response of P. aeruginosa to detergent stress which might serve as a survival strategy during growth with SDS.

Parasitic growth of Pseudomonas aeruginosa in co-culture with the chitinolytic bacterium Aeromonas hydrophila

Polymer-degrading bacteria face exploitation by opportunistic bacteria that grow with the degradation products without investing energy into production of extracellular hydrolytic enzymes. This scenario was investigated with a co-culture of Aeromonas hydrophila and Pseudomonas aeruginosa with chitin as carbon, nitrogen and energy source. In single cultures, A. hydrophila could grow with chitin, while P. aeruginosa could not. Co-cultures with both strains had a biphasic course. In the first phase, P. aeruginosa grew along with A. hydrophila without affecting it. The second phase was initiated by a rapid inactivation of and a massive acetate release by A. hydrophila. Both processes coincided and were dependent on quorum sensing-regulated production of secondary metabolites by P. aeruginosa. Among these the redox-active phenazine compound pyocyanin caused the release of acetate by A. hydrophila by blocking the citric acid cycle through inhibition of aconitase. Thus, A. hydrophila was forced into an incomplete oxidation of chitin with acetate as end-product, which supported substantial growth of P. aeruginosa in the second phase of the co-culture. In conclusion, P. aeruginosa could profit from a substrate that was originally not bioavailable to it by influencing the metabolism and viability of A. hydrophila in a parasitic way.

Azoarcus anaerobius sp. nov., a resorcinol- degrading, strictly anaerobic, denitrifying bacterium

A strictly anaerobic, nitrate-reducing bacterium, strain LuFRes1, was isolated using resorcinol as sole source of carbon and energy. The strain reduced nitrate to dinitrogen gas and was not able to use oxygen as an alternative electron acceptor. Cells were catalase-negative but superoxide-dismutase-positive. Resorcinol was completely oxidized to CO2. 16S rRNA sequence analysis revealed a high similarity with sequences of Azoarcus evansii and Azoarcus tolulyticus. Strain LuFRes1T (= DSM 12081T) is described as a new species of the genus Azoarcus, Azoarcus anaerobius.

Biochemical and Genetic Investigation of Initial Reactions in Aerobic Degradation of the Bile Acid Cholate in Pseudomonas sp. Strain Chol1

Bile acids are surface-active steroid compounds with toxic effects for bacteria. Recently, the isolation and characterization of a bacterium, Pseudomonas sp. strain Chol1, growing with bile acids as the carbon and energy source was reported. In this study, initial reactions of the aerobic degradation pathway for the bile acid cholate were investigated on the biochemical and genetic level in strain Chol1. These reactions comprised A-ring oxidation, activation with coenzyme A (CoA), and beta-oxidation of the acyl side chain with the C(19)-steroid dihydroxyandrostadienedione as the end product. A-ring oxidizing enzyme activities leading to Delta(1,4)-3-ketocholyl-CoA were detected in cell extracts and confirmed by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Cholate activation with CoA was demonstrated in cell extracts and confirmed with a chemically synthesized standard by LC-MS/MS. A transposon mutant with a block in oxidation of the acyl side chain accumulated a steroid compound in culture supernatants which was identified as 7alpha,12alpha-dihydroxy-3-oxopregna-1,4-diene-20-carboxylate (DHOPDC) by nuclear magnetic resonance spectroscopy. The interrupted gene was identified as encoding a putative acyl-CoA-dehydrogenase (ACAD). DHOPDC activation with CoA in cell extracts of strain Chol1 was detected by LC-MS/MS. The growth defect of the transposon mutant could be complemented by the wild-type ACAD gene located on the plasmid pBBR1MCS-5. Based on these results, the initiating reactions of the cholate degradation pathway leading from cholate to dihydroxyandrostadienedione could be reconstructed. In addition, the first bacterial gene encoding an enzyme for a specific reaction step in side chain degradation of steroid compounds was identified, and it showed a high degree of similarity to genes in other steroid-degrading bacteria.

Discovery of Two Classes of Potent Glycomimetic Inhibitors of Pseudomonas aeruginosa LecB with Distinct Binding Modes

The treatment of infections due to the opportunistic pathogen Pseudomonas aeruginosa is often difficult, as a consequence of bacterial biofilm formation. Such a protective environment shields the bacterium from host defense and antibiotic treatment and secures its survival. One crucial factor for maintenance of the biofilm architecture is the carbohydrate-binding lectin LecB. Here, we report the identification of potent mannose-based LecB inhibitors from a screening of four series of mannosides in a novel competitive binding assay for LecB. Cinnamide and sulfonamide derivatives are inhibitors of bacterial adhesion with up to a 20-fold increase in affinity to LecB compared to the natural ligand methyl mannoside. Because many lectins of the host require terminal saccharides (e.g., fucosides), such capped structures as reported here may offer a beneficial selectivity profile for the pathogenic lectin. Both classes of compounds show distinct binding modes at the protein, offering the advantage of a simultaneous development of two new lead structures as anti-pseudomonadal drugs with an anti-virulence mode of action.

Different strategies in anaerobic biodegradation of aromatic compounds: nitrate reducers versus strict anaerobes.

Mononuclear aromatic compounds are degraded anaerobically through pathways that are basically different from those used in the presence of oxygen. Whereas aerobic degradation destabilizes the aromatic π-electron system by oxidative steps through oxygenase reactions, anaerobic degradation is most often initiated by a reductive attack. The benzoyl-CoA pathway is the most important metabolic route in this context, and a broad variety of mononuclear aromatics, including phenol, cresols, toluene, xylenes and ethylbenzene, are channelled into this pathway through various modification reactions. Multifunctional phenolic compounds are metabolized via the reductive resorcinol pathway, the oxidative resorcinol pathway with hydroxyhydroquinone as key intermediate, and the phloroglucinol pathway. Comparison of the various pathways used for modification and degradation of aromatics in the absence of oxygen indicates that the strategies of breakdown of these compounds are largely determined by the redox potentials of the electron acceptors used, and by the overall reaction energetics. Consequently, nitrate reducers quite often use strategies for primary attack on aromatic compounds that differ from those used by sulfate-reducing, iron-reducing or fermenting bacteria.