The exploration of yet unknown catabolic pathways, in respect to the degradation of xenobiotic compounds, in microbes and microbial communities is of great importance: (i) for the general understanding of the global cycling of matter and (ii) for a more detailed understanding of the mechanisms behind the metabolic flexibility of microbes and their adaptation to a utilization of growth substrates.
There are plenty of examples where microbes fail to provide the major ‘ecosystem service’, of an effective recycling of organic matter into, ultimately, CO2, for synthetic industrial chemicals.
A ‘success story’ in this respect is the complete degradation of linear alkylbenzenesulfonates (LAS), which are entirely synthetic bulk laundry surfactants of petrochemical origin. LAS are disposed into the environment in massive amounts without causing problems. These surfactants replaced branched-chain alkylbenzenesulfonates, which are only very poorly degraded in wastewater treatment plants (Sawyer and Ryckman 1957, Kölbener et al. 1995), and it has been shown exhaustively that LAS are completely degradable. However, the details on how LAS and their degradation intermediates, sulfophenylcarboxylates (SPCs), are degraded, remained elusive, until a defined LAS- and SPC-degrading laboratory model community had been established for experimental work (Schleheck et al. 2004a). Now, this community is genome-sequenced and their genomes have been analyzed, Parvibaculum lavamentivorans DS-1T (chapter 2), Comamonas testosteroni KF-1 (chapter 3) and Delftia acidovorans SPH-1 (chapter 4).
Access to genome-sequenced organisms is very helpful for studies of bacterial degradation pathways on a molecular level. Particularly, the application of modern proteomics greatly enhances the straightforwardness of candidate-gene calling, the production of recombinant protein in adequate amount and purity, and enzymatic confirmation of their predicted function and role in the degradative pathway. Another striking advantage of working with genome-sequenced organisms is the fact that paralogs or copies of genes for certain enzymes under consideration (e.g., for BVMO reactions) encoded in the same organism, can be identified and tested. Comparative studies in order to discriminate the genes desired (e.g. by reverse transcription-PCR) are based on these studies at a genomic level. However, the function of the identified gene products has to be checked with biochemical and enzymatic studies. Therefore, the access to the enzyme’s substrates is essential. For the substrates which are not commercially
172 CHAPTER 9 available, this issue has been addressed by chemical synthesis, e.g., for the growth substrates 3-(4-sulfophenyl)butyrate (3-C4-SPC) and sulfoquinovose (SQ). The second inducible SPC degradation pathway, for 4-(4-sulfophenyl)hexanoate (4-C6-SPC), is only now becoming experimentally accessible, because the substrate for generating active biomass and proteomics is now available in reasonable amounts and purity. Another possibility to generate rare substrates for enzyme testing is in-vitro reconstitution of the complete pathway, thus, one recombinant enzyme is used to generate the substrate for the next enzyme in the pathway, e.g., as for the intermediates of sulfoglycolysis and when starting with the available SQ and dihydroxypropanesulfonate (DHPS).
The degradation of LAS and SPCs is an intriguing example of interplay between non-specific and highly specific enzymatic attack in order to access and utilize substrates completely. At the first degradation level, P. lavamentivorans DS-1T utilizes the alkyl chains of LAS for growth but releases the inaccessible remainder, many short-chain SPCs and related compounds (Schleheck et al. 2000, Schleheck et al. 2004a, Schleheck et al. 2007). This initial, non-specific attack on the alkyl chains of LAS is a hydroxylation of the terminal carbon atom (omega-oxygenation), an oxidation of the hydroxyl-carbon to a carbonyl-carbon, and its activation as coenzyme A (CoA)-ester followed by several rounds of acetyl-CoA abstractions through fatty-acid beta-oxidations. However, the non-specific chain-shortening stops, when the 4-sulfophenyl groups on the many different SPCs become sterically hindering, thus, when specific attack is required. The CoA is recovered and the short-chain SPCs are released (Schleheck and Cook 2005). In fact, P. lavamentivorans DS-1T is also able to degrade 16 other alkyl-chain containing commercially important surfactants, as well as alkanes; the latter was found also for the other members of the genus Parvibaculum, which were, e.g., isolated from alkane contaminated environments (see chapter 2).
The omega-oxygenation of this wide range of alkyl-substrates is most likely catalyzed by cytochrome-P450 (CYP) alkane monooxygenase (MO; Schleheck and Cook 2005) and the genome analysis indicated that indeed several of these enzymes might act in concert in order to catalyze such a wide substrate range: The genome of strain DS-1 encodes, in total, ten valid candidates for this type of oxygenase (chapter 2). Whereas the LAS-oxygenases, as measured with whole cells in the oxygen electrode, appear to be active also in acetate-grown cells, hence, are constitutively expressed, some of the individual CYP-MOs appear to be 3-fold to 10-fold up regulated during growth with LAS in comparison to acetate-grown cells, as determined by transcriptional analyses (Serif 2012). However, all attempts of heterologously overexpressing
such a candidate CYP-MO system (i.e., the oxygenase component, ferredoxin and ferredoxin-reductase) in an active state did fail so far (Serif 2012).
The representatives for the complete degradation of SPCs are the specialists in specificity, Comamonas testosteroni KF-1 and Delftia acidovorans SPH-1 (Schleheck et al. 2004a). They are able to utilize only a very narrow range of the many SPCs available as potential substrates.
Clearly, the specificity lies in the yet unknown mechanisms of abstractions of the different branched carboxylate side chains of SPCs. However, only the 3-C4-SPC degradation pathway was experimentally accessible in this study, since so far only 3-C4-SPC was chemically synthesized in order to follow its inducible degradation pathway in C. testosteroni KF-1. A 3-C4-SPC degradation pathway was postulated (Schleheck et al. 2010) after the identification of degradation intermediates (4-sulfoacetophenone (SAP) and 4-sulfophenol), which allowed for the suggestion of a Baeyer-Villiger-type monooxygenase (BVMO) reaction leading to 4-sulfophenyl acetate and the subsequent hydrolytic cleavage into acetate and 4-sulfophenol.
This was confirmed in this work through the identification of the responsible BVMO gene by reverse transcription-PCR (RT-PCR), the heterologous expression of the identified gene, and the confirmation of the function of the purified enzyme. Further, the corresponding esterase, which catalyzes the hydrolytic cleavage of 4-sulfophenyl acetate, was purified to homogeneity and identified (chapter 5).
The concept of using special sets of genes and enzymes to make different growth substrates available is not to be reinvented, but rather slightly modified in order to fulfill the new duties.
For example with BVMO2, strain KF-1 uses also the successful concept of inserting an oxygen atom between a C-C bond next to a keto group, followed by the subsequent hydrolytic cleavage of an acetate moiety, as it was shown for the abstraction of the acetyl side chain of progesterone, when KF-1 thrives on that substrate. Notably, in consideration of kinetic aspects of more favorable reactions, the identified intermediate of the progesterone degradation pathway, pregna-1,4-diene-3,20-dione seems to be the ʻtrueʼ substrate for BVMO2 under physiological conditions (see chapter 6).
The same concept seems also true for short-chain aliphatic ketones (Fraaije et al. 2004, Rehdorf et al. 2007), for which one was tested (2-decanone) as substrate for all four BVMO candidates of strain KF-1. Indeed, 2-decanone is a substrate for all four BVMOs of strain KF-1, however, with differing activities. Short-chain aliphatic ketones have been reported as substrates for BVMOs, e.g., in Mycobacterium tuberculosis and in Pseudomonas putida KT2440 (Fraaije et al. 2004, Rehdorf et al. 2007), and both of these enzymes, share on the phylogenetic level, closest homology to BVMO1 of C. testosteroni KF-1 (44% and 42% amino acid identity,
174 CHAPTER 9 respectively), but less to BVMO3 (SAPMO; 30% and 32% amino acid identity, respectively), although the latter exhibits the highest specific activities with 2-decanone and ethionamide.
Further, BVMO3 is phylogenetically more closely related to the steroid BVMO of Rhodococcus rhodochrous, but accepts no steroids as substrates (see chapter 6). Hence, the phylogenetic relationships of BVMOs seem to give no indication of their substrate specificities, and there are both, rather promiscuous enzymes (BVMO3) as well as comparatively specified enzymes (BVMO2) existing, as far as the substrate range tested in this study is concerned. The physiological function for BVMO4 is still unclear; it exhibited activity with 2-decanone and ethionamide only, however, with very poor kinetic magnitudes (chapter 6).
Table 1. Substrate range tested for the four BVMOs of strain KF-1 (, measurable activity; , no measurable activity; n.d., not determined)
The physiological reaction product of BVMO3, 4-sulfophenyl acetate, was shown to be hydrolytically cleaved into acetate and 4-sulfophenol (chapter 5). The latter was initially thought to be converted by a 4-sulfophenol 2-monooxygenase to 4-sulfocatechol (Schleheck et al. 2010). The cleavage of 4-sulfocatechol, leading to 3-sulfomuconate, was already shown to be performed by a modified protocatechuate 3,4-dioxygenase in Hydrogenophaga intermedia S1 (Contzen et al. 2001). This type of ring cleavage has been used as a default hypothesis for the 3-C4-SPC degradation in C. testosteroni KF-1, since there is a measurable 4-sulfocatechol dependent oxygen consumption with induced strain KF-1 whole cells, as well as with cell extracts (Schleheck et al. 2010, chapter 7). A suitable candidate that might be responsible for the ring cleavage reaction was found quickly by proteomics, whose gene product, however, did not convert 4-sulfocatechol. It turned out that hydroxyquinol is the substrate for the ring cleavage dioxygenase, and this finding rejects the initially proposed pathway via a desulfonation after a ring cleavage of 4-sulfocatechol; the 4-sulfocatechol, however, may still be an appropriate intermediate in the formation of hydroxyquinol (see chapter 7).
BVMO
Additionally, the proteomic approach identified several other induced candidates that would fit nicely into the proposed route of 3-C4-SPC degradation in C. testosteroni KF-1. Clearly, there is still a lot more work to do in order to close the gaps in understanding the complete degradation of this individual SPC, of 3-C4-SPC, on the enzymatic and genetic level. However, most of the identified genes and candidate genes are encoded in a contiguous arrangement of gene clusters that is unique in C. testosteroni KF-1, and that shows clear remnants of their recent mobilization, i.e., through the transposable elements (IS1071 elements), which are co-encoded in this genome region (see chapter 7). Overall, it seems as if the pathway for xenobiotic 3-C4-SPC has been assembled only very recently (recently in the evolutionary time scale) in C. testosteroni KF-1.
In contrast, the bacterial degradation of the natural and supposedly ‘ancient’ organosulfonate SQ has had much more time to fully shape and optimize, e.g., its pathway(s), gene organization and regulation. This is exemplified by the first SQ pathway that is defined in such detail, sulfoglycolysis in Escherichia coli K-12 (see chapter 8). However, the basic concept is the same: slight modification of the pre-existing in order to fulfill the new metabolic duties, rather than reinvention. The substrate SQ is degraded in analogy to an also very ancient pathway, the
‘normal’ glycolysis (Embden-Meyerhof-Parnas (EMP) pathway). The genes for SQ are encoded in one extra operational unit, and this gene cluster (presumed operon) is widespread in Enterobacteriaceae. Sulfoglycolysis operates with a first isomerization reaction of SQ to sulfofructose, followed by a phosphorylation yielding sulfofructosephosphate, the substrate for the subsequent aldolytic cleavage, whose products are dihydroxyacetonephosphate and sulfolactaldehyde. The latter is reduced to DHPS, which is excreted and can serve as growth substrate for other organisms, e.g. Cupriavidus pinatubonensis JMP134 (Denger et al. 2012, chapter 8). In fact, the remarkable analogy of sulfoglycolysis to the EMP pathway is not surprising, since the EMP pathway is evolutionary optimized in regard to product formation and achieved by the fewest possible reaction steps (Bräsen et al. 2014). Additionally, the EMP pathway is, at least in parts, present in all organisms (Fothergill-Gilmore and Michels 1993), and these, which are able to utilize SQ did not have to ‘reinvent the wheel’ for its catabolism.
This applies also to other options for glucose catabolism (Roy et al. 2003): For instance, the SQ-utilizing Pseudomonas putida SQ1, which was isolated from Lake Constance, excretes a different sulfonated product, 3-sulfolactate, and preliminary evidence suggests that a SQ degradation pathway analogous to the glucose dissimilation route via the Entner-Doudoroff pathway, is operative in strain SQ-1 (Ann-Katrin Felux, personal communication). Notably, the
176 CHAPTER 9 excreted 3-sulfolactate is the growth substrate for other second-tier organisms, e.g. Paracoccus pantotrophus NKNCYSA (Denger et al. 2012).
The bacteria’s motivation for each catabolic pathway is the same: gorge whatever is possible, based on their metabolic capabilities and relative to opportunity. In this thesis, two examples for catabolic pathways for natural (SQ) and of xenobiotic (LAS/SPCs) organosulfonates have been examined, and in both cases, there seem to be two tiers of bacteria necessary in order to catalyze these pathways completely. Based on their metabolic capabilities, the members of the first tier can utilize only a part of the carbon, provided by LAS and SQ, and the remainder is excreted (SPCs and DHPS or 3-sulfolactate), but this opens up the opportunity for the second tier of organisms to thrive on these excreted, still sulfonated, remainder molecules. Hence, the work with defined bacterial communities as laboratory model systems mirrors, even though in a highly simplified version, the interactions between the members of the environmental microbial communities and their complex catabolic network. On the other hand, from the evolutionary perspective, it would be surprising if there had not been a strong selection for complete pathways in the same organism: Such organisms may not yet exist for the ‘young’
substrate LAS, or may not yet be available as isolated laboratory model organisms, but such organisms do exist for SQ (Martelli 1967), however, the respective strain, isolated by that time, has not been maintained in any culture collection (Cook and Denger 2002).
CHAPTER 10
Appendix
Abbreviations
°C degree Celsius
1D-PAGE one dimensional polyacrylamide gel electrophoresis 2-C4-SPC 2-(4-sulfophenyl)butyrate
2D-PAGE two dimensional polyacrylamide gel electrophoresis 2Fe-2S Rieske type iron-sulfur cluster
3-C4-SPC 3-(4-sulfophenyl)butyrate 3-C4-2en-SPC 3-(4-sulfophenyl)-Δ2-butyrate 3-C5-SPC 3-(4-sulfophenyl)pentanoate 3-C5-2en-SPC 3-(4-sulfophenyl)-Δ2-pentanoate 3-C12-LAS 3-(4-sulfophenyl)dodecane 4-C5-SPC 4-(4-sulfophenyl)pentanoate 4-C5-2en-SPC 4-(4-sulfophenyl)-Δ2-pentanoate 4-C6-SPC 4-(4-sulfophenyl)hexanoate 4-C6-2en-SPC 4-(4-sulfophenyl)-Δ2-hexanoate 5-C6-SPC 5-(4-sulfophenyl)hexanoate
Å angstrom (10-10m)
AAP 4-aminoacetophenone
ADD androsta-1,4-diene-3,17-dione ADP adenosine diphosphate
amu atomic mass unit
AP acetophenone
aph genes responsible for the meta degradation of phenol appr./approx. approximately
178 CHAPTER 10 ars genes responsible for arsenical resistance
ATP adenosine triphosphate
Au gold (aurum)
BA benzaldehyde
Bcr/CflA drug resistance transporter subfamily (including bicyclomycin resistance protein)
BenAB benzoate dioxygenase
BLASTn Basic Local Alignment Search Tool (for nucleotide sequences) BLASTp Basic Local Alignment Search Tool (for amino acid sequences) box genes responsible for CoA-dependent aerobic benzoate degradation
bp base pairs
BphC biphenyl dioxygenase
BSA bovine serum albumin
Bsh bile salt hydrolase
BVMO Baeyer-Villiger monooxygenase c centi (10-2, as prefix)
CA California
cDNA complementary deoxyribonucleic acid
CDSs Coding DNA Sequences
cf. to be compared with (confer)
CHMO cyclohexanone Baeyer-Villiger monooxygenase
cnb genes responsible for the 4-chloronitrobenzene degradation cntA gene for a cobalt nickel transporter
CoA coenzyme A
COG Clusters of Orthologous Groups
cop genes responsible for copper resistance
CRISPR clustered regularly interspaced short palindromic repeats CTAB cetyltrimethylammonium bromide
C. testosteroni Comamonas testosteroni
CuyA cysteate sulfo-lyase
CYP cytochrome-P450
CzcA heavy metal resistance protein (for cobalt, zinc and cadmium resistance)
D attenuance
Da Dalton
D. acidovorans Delftia acidovorans
dca genes responsible for dichloroaniline degradation
DEAE diethylethanolamine
del genes responsible for delftibaction production DFG Deutsche Forschungsgemeinschaft
DHAP dihydroxyacetone phosphate DHPS 2,3-dihydroxypropanesulfonate DNA deoxyribonucleic acid
DOE Department of Energy
DSM(Z) Deutsche Sammlung von Mikroorganismen (und Zellkulturen)
DTT dithiothreitol
DUF domain of unknown function
EC Enzyme Commission number
E. coli Escherichia coli
e.g. for example (exempli gratia) EHEC enterohemorrhagic Escherichia coli ELSD evaporative light scattering detector
EMP Embden-Meyerhof-Parnas
EmrB/QacA drug resistance transporter subfamily (including multiple drug resistance efflux pump)
ESI electrospray ionisation
est gene encoding an esterase
et al. and others (et alii)
etfA electron transfer flavoprotein alpha subunit
180 CHAPTER 10
eV electron volt
FAD flavin adenine dinucleotide Fba fructose bisphosphate aldolase
Fe iron (ferrum)
FMN flavin mononucleotide
for forward
fruA fructose PTS permease fruA subunit fruB fructose PTS permease fruB subunit
g grams
g earth´s standard acceleration GAP glyceraldehyde-3-phosphate
GC gas chromatography
GC content guanine-cytosine content
GC skew (guanine – cytosine)/(guanine + cytosine) gen. nov. new genus (genus novum)
gltA gene encoding a citrate synthase
GOLD Genomes OnLine Database
h hours
HAP 4-hydroxyacetophenone
HAPMO 4-hydroxyacetophenone Baeyer-Villiger monooxygenase
His histidine
HMP Human Microbiome Project
HpaB 4-hydroxyphenylacetate 3-monooxygenase HPAc 4-hydroxyphenyl acetate
HPLC high-performance liquid chromatography
HPP 4-hydroxypropiophenone
HR high resolution
ID identifier
IDA Inferred from Direct Assay
i.e. that is (id est)
IEF isoelectric focusing
IMG Integrated Microbial Genomes
IN Indiana
iph genes responsible for the isophthalate degradation IPTG isopropyl-β-D-thiogalactopyranoside
IS insertion sequence
ivaA gene responsible for the demethylation of isovanillate
JGI Joint Genome Institute
k kilo (103, as prefix)
kcat turnover rate
KEGG Kyoto Encyclopedia of Genes and Genomes
Km Michaelis constant
KoRS-CB Konstanz Research School Chemical Biology KshA 3-ketosteroid 9-monooxygenase subunit A
l liter/litre
LABs linear alkylbenzenes
LAS linear alkylbenzenesulfonate
LB lysogeny broth
LigB extradiol ring cleavage dioxygenase class III protein subunit B µ micro (10-6, as prefix)
µ specific growth rate
m meter
m milli (10-3, as prefix)
M molar
M mega (106, as prefix)
MacA macrolide export protein
MD Maryland
MEGA Molecular Evolutionary Genetics Analysis
182 CHAPTER 10 mer genes responsible for mercury resistance
MES 2-(N-morpholino)ethanesulfonic acid MFP membrane fusion protein
MFS major facilitator superfamily
(M-H)- deprotonated molecule ion measured in negative mode
mhp genes responsible for the 3-(3-hydroxyphenyl)propionic acid degradation MhpA 3-(3-hydroxyphenyl)propionate hydroxylase
MIGS minimum information about a genome sequence
min minute
MMLV moloney murine leukemia virus
mnb genes responsible for the 3-nitrobenzoate degradation
MO monooxygenase
MOPS 3-morpholinopropane-1-sulfonic acid
MS mass spectrometry
MS-MS tandem mass spectrometry
Mr relative mass
mRNA messenger ribonucleic acid
m/z mass-to-charge ratio
n nano (10-9, as prefix)
NAD+ nicotinamide adenine dinucleotide (oxidized) NADH nicotinamide adenine dinucleotide (reduced)
NADP+ nicotinamide adenine dinucleotide phosphate (oxidized) NADPH nicotinamide adenine dinucleotide phosphate (reduced)
NAP 4-nitroacetophenone
NAS non-traceable Author Statement NAST Nearest Alignment Space Termination
nbz genes responsible for the nitrobenzene degradation NCBI National Center for Biotechnology Information
NCIMB National Collection of Industrial, Food and Marine Bacteria
NC-IUBMB Nomenclature Committee of the International Union of Biochemistry and Molecular Biology
Ni nickel
NMR Nuclear magnetic resonance
no. number
NP nonylphenol
NpdB hydroxyquinol dioxygenase in the para-nitrophenol catabolism NpdC maleylacetate reductase in the para-nitrophenol catabolism NTA nitrilotriacetic acid
OD580 optical density at a wavelength of 580 nanometers OmpL outer membrane protein L
ORF open reading frame
ORNL Oak Ridge National Laboratory p pico (10-12, as prefix)
Pa Pascal
PA phenylacetone
paa genes responsible for aerobic phenylacetate degradation
Paa phenylacetate
PAGE polyacrylamide gel electrophoresis PAH polycyclic aromatic hydrocarbons
PAMO phenylacetone Baeyer-Villiger monooxygenase parAB partitioning proteins (act in chromosome segregation) pbr genes responsible for cadmium/lead resistance
PCBs polychlorinated biphenyls PCR polymerase chain reaction PdeA phosphodiesterase
PDD pregna-1,4-diene-3,20-dione Pfam protein families database
Pfk phosphofructokinase
184 CHAPTER 10 PF-MS peptide fingerprinting-mass spectrometry
Pgi phosphoglucose isomerase
pH pondus Hydrogenium
PhaR polyhydroxyalkanoate synthesis repressor PhaZ polyhydroxyalkanoate depolymerase
PHB polyhydroxybutyrate
PhbC polyhydroxybutyrate synthase
phc genes responsible for phenol catabolism
Pil pilus assembly protein
P. lavamentivorans Parvibaculum lavamentivorans
pmd genes responsible for the protocatechuate meta degradation PmdAB Protocatechuate 4,5-dioxygenase
PnpF maleylacetate reductase in the para-nitrophenol catabolism PnpG hydroxyquinol dioxygenase in the para-nitrophenol catabolism PPE carboxylester hydrolase in Pseudomonas putida
P. putida Pseudomonas putida PQQ pyrroloquinoline quinone
PRIAM profils pour l'Identification automatique du métabolisme
Pta phosphotransacetylase
PTS phosphotransferase system
Q11 ubiquinone 11
RDP Ribosomal Database Project
rev reverse
Rfam RNA families database RNaseP ribonuclease P (a ribozyme)
RNA ribonucleic acid
RND efflux transporter superfamily (resistance-nodulation-cell division)
rpm revolutions per minute
rRNA ribosomal ribonucleic acid
RT reverse transcription
s second
SAP 4-sulfoacetophenone
SAPMO 4-sulfoacetophenone Baeyer-Villiger monooxygenase SAS secondary alkanesulfonates
SDR short-chain dehydrogenase/reductase SDS sodium dodecyl sulfate
SEM scanning electron microscope SEST steroid esterase
SF 6-deoxy-6-sulfofructose
SFP 6-deoxy-6-sulfofructose-1-phosphate
sil genes responsible for silver/copper resistance SLA 3-sulfolactaldehyde
SOD superoxide dismutase
SorAB sulfite dehydrogenase (SorA, catalytic unit; SorB, cytochrome c) SorT sulfite dehydrogenase
sp. species
SP 4-sulfophenol
SPAc 4-sulfophenyl acetate SPCs sulfophenylcarboxylates sp. nov. new species (species nova)
SQ sulfoquinovose
SQDG sulfoquinovosyldiacylglycerol
STMO steroid Baeyer-Villiger monooxygenase sucD succinate CoA ligase alpha subunit SuyAB 3-sulfolactate sulfo-lyase
Taq Thermus aquaticus
TAS Traceable Author Statement
tau genes attributed to taurine uptake and degradation
186 CHAPTER 10 Tau proteins attributed to taurine uptake and degradation
186 CHAPTER 10 Tau proteins attributed to taurine uptake and degradation