FEMS Microbiology Reviews 15 (1994) 195-215 195 ;'~, 1994 Federation of European Microbiological Societies 0168-6445/94/$15.(10 Published by Elsevier Microbial metabolism of sulfur- and phosphorus-containing xenobiotics M.A. Kertesz *, A.M. Cook and T. Leisinger Institute of Microbiolo~', Swiss Federal Institute of Technology, ETH-Zentrum, CH-8092 Ziirich. Switzerland Abstract: The enzymes involved in the microbial metabolism of many important phosphorus- or sulfur-containing xenobiotics, including organophosphate insecticides and precursors to organosulfate and organosulfonate detergents and dyestuffs have been characterized. In several instances their genes have been cloned and analysed. For phosphonate xenobiotics, the enzyme system responsible for the cleavage of the carbon-phosphorus bond has not yet been observed in vilro, though much is understood on a genetic level about phosphonate degradation. Phosphonate metabolism is regulated as part of the Pho regulon, under phosphate starvation control. For organophosphorothionate pesticides the situation is not so clear, and the mode of regulation appears to depend on whether the compounds are utilized to provide phosphorus, carbon or sulfur for cell growth. The same is true for organosulfonate metabolism, where different (and differently regulated) enzymatic pathways are involved in the utilization of sulfonates as carbon and as sulfur sources, respectively. Observations at the protein level in a number of bacteria suggest that a regulatory system is present which responds to sulfate limitation and controls the synthesis of proteins involved in providing sulfur to the cell and which may reveal analogies between the regulation of phosphorus and sulfur metabolism. Key word,s: Xenobiotics; Biodegradation: Phosphorus metabolism; Sulfur metabolism: Global regulation Introduction compounds can be metabolized to some extent by bacterial cultures [2], either by co-metabolism Xenobiotic compounds have been defined as with other substrates [3,4] or during their utiliza- "compounds that are released in any compart- tion as sources of energy or nutrients (carbon, ment of the environment by the action of man nitrogen, phosphorus or sulfur). As this review and therefore occur in a concentration in this or will show, the mode and extent of degradation of another compartment of the environment that is a xenobiotic compound by any particular organ- higher than 'natural'" [1]. Most often, these com- ism depends crucially on which of these elemen- pounds are chemicals whose synthetic nature and tal components is required by that organism for non-natural structure preclude or retard their growth. degradation by microbial species, and therefore The metabolic pathways involved in assimila- lead to their accumulation in the environment. tion of the above nutrients by bacteria are regu- However, even the most persistent xenobiotic lated not only by specific substrate induction mechanisms, but to a large extent also by global control systems [5]. Catabolite repression exerts global control on utilization of various sources of * Corresponding author. carbon; in the presence of a 'preferred' carbon SSDI 0168-6445(94)00033-U 196 source, expression of the genes involved in work was termed the SSI-stimulon (sulfate starva- metabolism of alternative compounds are re- tion-induced), due to the superficial resemblance pressed [6,7]. Global nitrogen regulation (the Ntr of the response to the synthesis of phosphate system) likewise regulates expression of a large starvation-induced (PSI) proteins during growth number of different genes, in response to ammo- with alternative phosphorus sources. However, nia levels within the cell [6]. A third mode of the mechanism of regulation and the nature of global cellular regulation is the stringent re- the factors involved are still unknown. For car- sponse, which reacts to starvation for either amino bon, nitrogen and phosphorus metabolism, cross- acids or carbon and energy sources, and is medi- talk between regulatory systems has also been ated by levels of the nucleotide ppGpp [8,9]. observed, leading to a highly complex control Global systems for regulation of phosphorus network capable of finely tuned responses to en- metabolism have also been well characterized, vironmental signals [12-15]. and have been described in a recent review [10]. In most studies of xenobiotic degradation, the The Pho regulon is governed by a two-component compounds under investigation have been sup- sensor-regulator system which in enteric bacteria plied to microorganisms exclusively as sources of controls the expression of a number of genes carbon and energy. Since bacteria require signifi- concerned with uptake and metabolism of non- cantly more carbon for growth than any other phosphate phosphorus. For sulfur, by contrast, nutrient, this leads to maximum removal of the relatively little has been reported. In Pseu- xenobiotic, an important aim in studies concen- domonas putida, Staphylococcus aureus or Es- trating on detoxification or bioremediation. Uti- cherichia coli a set of proteins is coordinately lization of xenobiotics as sources of phosphorus induced during growth with sulfur sources other and sulfur has been less well studied until now. In than cysteine or sulfate [11]; this regulatory net- this review we will concentrate on the metabolism S E t O~ // E t O~ P\O ~NO2 HOOC~NH~PO3 H2 GI yphosate Parathion S E ! O.~p// Me O~.. p~./S S COOE (~ O:~CN MeO/ \S~ COOEt Malathion Cyano I en fos S iPr Me Diazinon Fig. 1. Some phosphorothionate (parathion, diazinon), phosphorodithionate (malathion) and phosphonate (glyphosate, cyanofen- fos) xenobiotics. Glyphosate is a herbicide, and the other compounds are insecticides. Me, methyl; Et, ethyl; iPr, isopropyl. 197 of selected phosphorus- and sulfur-containing tant to non-biological degradation in the environ- xenobiotics, and its control. ment than its analogues with N-P, S-P or O-P linkages [18]. The xenobiotic character of orga- nophosphonates is also emphasized by the rela- Phosphorus-containing xenobiotics tive rarity in nature of compounds bearing a C-P linkage [17]. The most widespread phosphonate Phosphorus-containing xenobiotics are of great xenobiotic is N-phosphonomethylglycine, better economic importance and have found extensive known as glyphosate, or under its tradename of application in recent years. They are generally Roundup (for a review, see [19]). It is a broad considered to be non-persistent, and a wide range spectrum, post-emergent herbicide acting against of them can be broken down by bacteria [16]. The virtually all annual and perennial plants, primar- phosphorus is usually present in the molecule ily by disrupting the biosynthesis of aromatic either as a phosphate ester or as a phosphonate amino acids [20]. (Fig. 1). Organophosphates act as inhibitors of acetylcholinesterase in the nervous system and Phosphonate xenobiotics - glyphosate have found application as insecticides in agricul- ture and in control of insect-borne diseases. The Glyphosate is rapidly degraded in the environ- most widely employed of these compounds are ment, and a number of bacterial species have the phosphorothionate parathion and the phos- been isolated which can break down the com- phorodithionate malathion (Fig. 1). Organophos- pound. These include a Flavobacterium species phonates possess antibacterial, antiviral and anti- [21], several Pseudomonas species [22-25] (of tumour activity, and are also used as herbicides, which the best studied is Pseudomonas sp. as detergent additives and as flame retardants PG2982 [23]), an Alcaligenes isolate [24], Bacillus [17]. They contain a direct carbon-phosphorus megaterium strain 2BLW [22], Arthrobacter sp. linkage, which is chemically and thermally very GLP-1 [26], four different Rhizobium species [27] stable and renders the molecule much more resis- and three Agrobacterium species [27,28]. Biodeg- HOOC~ Ni4/'~p O 3 H 2 /cl43 +[p] + [C2-unit] HOOC "NH H2N~PO3H2 sarcosi ne AMPA (i) glycine + [C1-unit] CH3NH 2 + [P] Fig. 2. Degradative pathways for glyphosate. Reactions (1) are catalysed in vivo by a 'C-P-lyase'. Phosphorus is utilized by the cell as inorganic phosphate. The initial phosphorus product of the lyase reaction is unknown, as are the one-carbon and two-carbon cleavage products. AMPA stands for aminomethylphosphonic acid. 198 radation has thus been found in both Gram-posi- atrocyaneus which was deposited in a culture tive and Gram-negative organisms, but not in collection prior to the introduction of the herbi- eukaryotes or cyanobactcria. cide can also metabolize the compound [30]. In Glyphosate is degraded in bacteria by two main almost all studies of glyphosate degradation, the pathways, both of which lead to breaking of the herbicide was supplied solely as a source of phos- carbon-phosphorus bond. In the first of these, phorus, and the organisms investigated were not initial cleavage of the carbon-phosphorus bond able to use it as a source of carbon or nitrogen. yields the reduced product N-methylglycine Although a mutant of Arthrobacter sp. GLP-1 (sarcosine). This pathway has been found in which was capable of utilizing both the nitrogen Arthrobacter sp. GLP-1 [26] and in Pseudomonas and the phosphorus of glyphosate has been re- sp. PG2982 [29]. The sarcosine formed has been ported, it grew extremely slowly and proved on shown by 13C/~SN-NMR studies to be further analysis to have a defective phosphate transport degraded to glycine and a C ~-unit, which is incor- system [34]. Similarly,