A modern, and highly effective, experimental strategy to reveal unknown bacterial degradation pathways on the molecular level is ‘reverse genetics’, i.e., access to complete genome sequences as a reference for differential proteomic and transcriptional analyses. The latter methods can reveal and confirm inducible candidate genes and, thus, candidate enzymes that are involved in the pathway under investigation. Once such candidate genes have been identified, they can be cloned, and the candidate enzymes can be heterologously produced and purified in order to confirm their function by in vitro enzyme reaction tests in combination with modern mass-spectrometry methods for identification of intermediates. This strategy may lead even to a reconstitution of complete pathways in vitro, as has been demonstrated twice in this work (Chapters 2 and 4). Importantly, such a step-by-step, in-vitro reconstitution of a complete pathway is the only viable option to produce the intermediates/substrates for a confirmation of the respective enzymatic steps of the pathways, if none of these intermediates/substrates are available from commercial suppliers or by custom chemical synthesis.
The genome sequence of the most well-studied bacterial laboratory model organism Escherichia coli K-12 substrain MG1655 is available since 1997 (Blattner et al. 1997), and when E. coli K-12 substrain MG1655 (and other substrains) was found to be able to utilize SQ for growth, it was used to examine a first SQ degradation pathway (Chapter 2). In order to get access to a second pathway, a different SQ-degrading bacterium had been isolated directly from a sample of Lake Constance littoral sediment, Pseudomonas putida SQ1 (Denger et al.
2012), and in this work, the genome of strain SQ1 has been sequenced, annotated, and analyzed (Chapter 3). Further, with SQ being available in relevant amounts through chemical synthesis (Denger et al. 2012), it was possible to uncover two different, inducible bacterial degradation pathway for SQ on the molecular level: For both pathways, all core genes and enzymes, as well as the corresponding enzyme reactions and degradation intermediates and excretion products, were identified by reverse genetics and in-vitro reconstitution of the complete pathways, as summarized in Figure 1 and further below.
Figure 1. Comparison of the two newly identified bacterial degradation pathways for SQ.
A, Sulfoglycolytic pathway in E. coli K-12 yields DHAP for energy conservation and growth of the bacterium, and DHPS as end product, which is excreted (Chapter 2). B, SQ Entner-Doudoroff pathway in P. putida SQ1, which yields pyruvate for energy conservation and growth of the bacterium, and SL as end product, which is excreted (Chapter 4).
The first known pathway, sulfoglycolysis in E. coli K-12 (Fig. 2A; Chapter 2), operates in analogy to the glycolytic pathway for glucose-6-phosphate. SQ is isomerized to 6-deoxy-6-sulfofructose (SF) by isomerase YihS, phosphorylated to 6-deoxy-6-6-deoxy-6-sulfofructosephosphate (SFP) by kinase YihV, and cleaved into dihydroxyacetone phosphate (DHAP) and sulfolactaldehyde (SLA) by aldolase YihT. DHAP is the substrate for energy conservation and growth, and the SLA is reduced to dihydroxypropane sulfonate (DHPS) by reductase YihU and then excreted. Additionally, the existence of the intermediates SF, SFP and DHPS was confirmed by HPLC-MS/MS.
The second known pathway in P. putida SQ1 (Fig. 1B; Chapter 4) operates in analogy to the Entner-Doudoroff pathway for glucose-6-phosphate. SQ is oxidized in a first step to 6-deoxy-6-sulfo-gluconolactone (SGL) by SQ-dehydrogenase, hydrolyzed to 6-deoxy-6-sulfogluconate (SG) by SGL lactonase, dehydrated to the key intermediate 2-keto-3,6-dideoxy-6-sulfogluconate (KDSG) by SG dehydratase, and further cleaved into pyruvate and SLA by KDSG aldolase. Pyruvate is the substrate for energy conservation and growth, and the SLA in this case - in contrast to sulfoglycolysis - is further oxidized to sulfolactate (SL) by SLA
Remarkably, the genes for SQ degradation in E. coli K-12 and P. putida SQ1 are each encoded in different gene clusters, and are regulated independently of the corresponding glycolysis and Entner-Doudoroff pathway enzymes, respectively. The gene cluster that encodes sulfoglycolysis in E. coli K-12 is found in almost all known E. coli genomes, and among many other Enterobacteriaceae, and is therefore thought to have a significant role in bacteria in the alimentary tract of all omnivores and herbivores, as well as in some plant pathogens (as discussed in Chapter 2). Homologous gene clusters with genes for the SQ Entner-Doudoroff pathway of P. putida SQ1 were found in a wide range of other Gamma- as well as in Beta- and Alphaproteobacteria, e.g., in typical freshwater, soil and plant-rhizosphere associated bacteria, in potential pathogens and, again, in isolated gut symbionts (for details, see Chapter 4). These organisms and their postulated SQ pathways need to be examined in future studies, however, preliminary results (from a current Bachelor-thesis work) confirmed already for two Rhizobium sp. strains, for an Aeromonas sp. strain, and for two Serratia/Rahnella strains, that these organisms are indeed able to utilize SQ as a sole source of carbon and energy for growth, as already predicted based on genome information (see Fig. 5 in Chapter 4). Furthermore, most of the enzymes of the sulfoglycolysis and SQ Entner-Doudoroff pathways belong to different protein families compared to the corresponding enzymes of the analogous pathways for glucose-6-phosphate (as discussed in Chapter 4). These observations allow for the speculation that the respective “sulfo”-enzymes, and therefore the complete “sulfo”-pathways, did evolve through an independent recruitment of genes/enzymes into the pathways and, thus, by convergent evolution, and not simply by gene duplications of the respective “phospho”-pathway enzymes followed by divergent evolution.
The mutational analysis (Chapter 2) confirmed the importance of a transport system in SQ degradation in E. coli K-12, of YihO (b3876), presumably for an uptake of SQ into the cell, because SQ is a highly polar, negatively charged molecule that requires a transport system across the cell membrane (Cook et al. 1999). Correspondingly, the export of the organosulfonate degradation products DHPS and SL also requires transport systems, for which in P. putida SQ1 the co-induced candidate organosulfonate/sulfite exporter TauE (Weinitschke et al. 2007, Mayer and Cook 2009) is most likely responsible for an export of SL (see Chapter 4), and in E. coli K-12 most likely the transporter YihP for an export of DHPS (see Chapter 2). Further, both gene clusters contain each a predicted gene for aldose epimerase/mutarotases (YihR in E. coli K-12, and PpSQ1_00092 in P. putida SQ1), and these genes/enzymes appeared to be expressed/produced specifically during growth with SQ (see
Chapters 2 and 4, respectively). Mutarotases catalyze the equilibrium of sugar epimers (Thoden et al. 2003), for example in this context of α- and β-D-sulfoquinovose. In preliminary experiments not included in this thesis, the yihR gene of E. coli was cloned and the protein heterologously produced and purified, and a sugar epimerase activity of the recombinant protein was confirmed when it catalyzed the equilibration of α- and β-D-glucose, as determined by polarimetry with commercially available, freshly prepared α-D-glucose solution. However, an anomer-pure preparation of SQ is not available (the SQ from chemical synthesis is in its equilibrium already), but in future experiments, the four-enzyme reaction of the sulfoglycolysis pathway, and the five-enzyme reaction of SQ Entner-Doudoroff pathway, could be incubated either in the presence or in the absence of the predicted SQ mutarotases, which may result in a much faster conversion of SQ in the presence of a mutarotase. While such observations may confirm an involvement of mutarotases in SQ degradation for both pathways, it also needs to be defined which enzyme(s) in the SQ pathways are actually catalyzing anomer-specific reactions and, thus, may slow down the overall-conversions of SQ to DHPS or SL in the absence of a mutarotase.
Finally, both gene clusters contain a gene predicted for an α-glucosidase (YihQ in E. coli, and PpSQ1_00094 in P. putida SQ1), and both of these were found to be highly expressed specifically during growth with SQ. Most likely, these enzymes catalyze a hydrolysis of SQ-glyceride, and could be essential for a hypothetical degradation of the lipid SQDG; in this case, an extracellular lipase might liberate the fatty acid residues from SQDG, and the SQ-glycerol might then be transported into the cell, where the α-glucosidase would liberate SQ.
Preliminary growth experiments and HPLC-MS analyses, which are not further described in this thesis, indicated that P. putida SQ1 is indeed able to utilize also the whole lipid SQDG as sole source of carbon and energy for growth: It was observed that in an initial growth phase, SQDG disappeared concomitant with a transient excretion of, most likely, SQ-glycerol (preliminary identification), and that within a second growth phase, the SQ-glycerol disappeared concomitant with a transient excretion of SQ, until finally SQ disappeared and SL was formed. Clearly, there is future experimental work needed to confirm the functions of the above-mentioned candidate genes for transporters, mutarotases and α-glucosidases, as well as to define the pathway, and the additional enzymes and genes involved in degradation of the sulfolipid via SQ-glyceride and SQ to SL in P. putida SQ1.
From the direct comparison of the two newly discovered SQ degradation pathways, it seems plausible that the respective pathway preferred by SQ-degrading microorganisms is dependent on their energetic situation, analogous to the glucose metabolism (Fuhrer et al. 2005,
Flamholz et al. 2013), as discussed in detail in Chapter 4. Briefly, sulfoglycolysis inclusive the SLA reductase reaction produces one ATP by substrate-level phosphorylation, but no reducing equivalents (NADH), and thus, this pathway seems to be optimal for anaerobic fermentative bacteria, which need to invest pyruvate as electron acceptor. Indeed, preliminary growth experiments and HPLC analyses, which are not further described in this thesis, indicated that E. coli K-12 is able to grow with SQ also under fermentative growth conditions concomitant with a much lower biomass yield in comparison to its aerobic growth with SQ, and that acetate and succinate (preliminary identifications) were formed. Hence, E. coli most likely catalyzes a mixed-acid fermentation with SQ, and clearly, this type of metabolism needs to be explored in future experimental work, given its high relevance for the intestinal habitats of many Enterobacteriaceae. In contrast, the opposite energetic situation is considerable for the SQ Entner-Doudoroff pathway inclusive the SLA dehydrogenase reaction: No ATP is formed directly through substrate-level phosphorylation in this pathway, but reducing equivalents (2 NADH), which can be funneled into the respiratory chain to produce ATP. Furthermore, the pyruvate formed from SQ can be respired completely, producing additional ATP. Hence, the SQ Entner-Doudoroff pathway seems to be optimal for respiring bacteria (aerobic and anaerobic). Nevertheless, the pathways as observed in E. coli K-12 and P. putida SQ1 can access only half of the carbon of SQ while a C3-organosulfonate is excreted, obviously due to a lack of DHPS/SL pathways inclusive desulfonative enzymes in these organisms.
From an evolutionary perspective, it is reasonable to expect that bacteria would acquire genes and enzymes to completely catabolize SQ to CO2 and sulfate, hence, that a pathway for degradation of SQ to DHPS or SL, and a pathway for a complete degradation of the DHPS/SL formed, would assemble in one single organism. However, all SQ-degrading bacteria tested in pure cultures thus far are unable to catalyze a desulfonation reaction (Roy et al. 2003, Denger et al. 2012, Denger et al. 2014), while the DHPS and SL mineralizing bacteria known thus far are all unable to utilize SQ (see Denger et al. 2012). For these SQ-utilizing bacterial strains that were obtained by classical enrichment cultures, the observation may be explained by the cultivation conditions used, in that the fastest growing, fastest SQ-utilizing bacterial strains were selected whilst these organisms, which are able to utilize SQ completely (if they do exist), were lost during the enrichment process. Further, the SQ-utilizing bacteria in their environmental, real-life conditions might indeed be exposed to, and degrade, the substrates SQDG, and not only SQ. In this case, the organisms might acquire sufficient carbon for growth from the fatty acid (e.g., twoC16 units), glycerol (C3), and SQ (C3) moieties, so that
there might be no significant selection for an acquisition of also DHPS/SL utilization in these organisms in relation to the additional cost for synthesizing these enzymes. Finally, phototrophs themselves might be able to effectively metabolize SQDG internally via SQ to DHPS/SL, which are excreted, as was shown recently for diatoms and DHPS in the open ocean (Durham et al. 2015), and/or that there are other relevant environmental sources of DHPS and SL, so that DHPS/SL utilization is a trait in bacteria that is much more independent from SQ utilization than expected.
It is also tempting to speculate whether other, yet unknown strategies exist for a degradation of SQ, for example, a pathway in analogy to the pentose-phosphate pathway. In addition, it is considerable that SQ might be desulfonated in an initial reaction step, by a hypothetical SQ-desulfurase or SQ-oxygenase: The sulfite would be excreted (most likely in form of sulfate) while the sulfonate-free sugar moiety could be metabolized via standard pathways (e.g., through normal glycolysis). Moreover, such a hypothetical SQ-desulfurase/oxygenase might be relevant in organisms that utilize SQ as an alternative sulfur source during sulfur/sulfate-limited growth: Until now, it was not possible to set up sulfur-sulfur/sulfate-limited enrichment cultures with SQ, because the SQ preparations from chemical synthesis (Denger et al. 2012) are contaminated with sodium sulfate. However, with an improved synthesis and/or purification of SQ, it will certainly be possible to address also this question: Importantly, sulfur-limited enrichment cultures with SQ might select also for these organisms that are able to degrade SQ completely, inclusive desulfonation (see above).
Despite the environmental importance of the microbial SQDG, SQ and DHPS/SL degradation pathways for closing the sulfur cycle in all habitats where SQDG and SQ is produced (as has been discussed extensively in Chapters 2 and 4), it is also important to further consider and experimentally access the degradation of SQDG, SQ and DHPS/SL in the alimentary tract of herbivores and omnivores, hence, by the gut microbial communities under anoxic conditions.
Many Enterobacteria and most of E. coli strains (see Chapter 2), and also other bacteria isolated from the human gut (see Chapter 4), have at least the genetic potential to degrade SQ that is introduced to the gut by a vegetable diet (Benson and Shibuya 1961, Norman et al.
1996, Kuriyama et al. 2005), and it has been speculated whether SQ as an analogue of glucose-6-phosphate could have effects (positive or negative?) on the sugar metabolism of the host (Sacoman et al. 2012). Moreover, the anaerobic utilization of the DHPS/SL that might be produced from SQ in the gut, might involve the formation of sulfide instead of sulfate (i.e., when the sulfite from DHPS/SL is used as an electron acceptor under anoxic conditions), and a sulfide production in the gut is connected to numerous inflammatory bowel diseases (e.g.
Loubinoux et al. 2002, Attene-Ramos et al. 2006, Wallace et al. 2009), even though it remains unclear if the presence of sulfide/hydrogen sulfide has a pro- or contra-inflammatory effect in these diseases. Hence, the degradation of SQ and of DHPS/SL by the gut microbial community might also have biomedical implications.
In conclusion, it was shown that SQ can be metabolized by two different catabolic pathways, sulfoglycolysis, as demonstrated in E. coli K-12, and SQ Entner-Doudoroff pathway, as demonstrated in P. putida SQ1. The different degradation strategies seem to reflect the energetic situation (anoxic/oxic) and thus the respective natural habitat of the bacteria, however in both pathways only a part of the provided carbon can be utilized and the remainder C3 moiety (DHPS or SL) is excreted. These findings significantly contribute to our detailed understanding of the global cycling of sulfur in nature, and suggest a significant role of these pathways in the gut. Therefore, these results open up a variety of new questions (as discussed above), which can be addressed in the future.
CHAPTER 6 Appendix
Abbreviations
°C degree Celsius
µ micro (10-6, as prefix)
µ specific growth rate
1D-PAGE one dimensional polyacrylamide gel electrophoresis 2D-PAGE two dimensional polyacrylamide gel electrophoresis 2Fe-2S Rieske type iron-sulfur cluster
Å angstrom (10-10m)
ABC transporter ATP-binding cassette transporter
ADP adenosine diphosphate
appr./approx. approximately
ATP adenosine triphosphate
BLASTn Basic Local Alignment Search Tool (for nucleotide sequences) BLASTp Basic Local Alignment Search Tool (for amino acid sequences)
bp base pairs
BSA bovine serum albumin
c centi (10-2, as prefix)
cDNA complementary deoxyribonucleic acid
cf. to be compared with (confer)
CoA coenzyme A
COG Clusters of Orthologous Groups
CRISPR clustered regularly interspaced short palindromic repeats
CTAB cetyltrimethylammonium bromide
CuyA cysteate sulfo-lyase
Da Dalton
DAG diacylglycerol
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
E. coli Escherichia coli
e.g. for example (exempli gratia)
EC Enzyme Commission number
EHEC enterohemorrhagic Escherichia coli
ELSD evaporative light scattering detector
EMP Embden-Meyerhof-Parnas
fruA fructose PTS permease fruA subunit fruB fructose PTS permease fruB subunit
g earth´s standard acceleration
g grams
GAP glyceraldehyde-3-phosphate
GC content guanine-cytosine content
GC skew (guanine – cytosine)/(guanine + cytosine)
GC gas chromatography
ID identifier
KEGG Kyoto Encyclopedia of Genes and Genomes
Km Michaelis constant MIGS minimum information about a genome sequence
min minute
NAD+ nicotinamide adenine dinucleotide (oxidized) NADH nicotinamide adenine dinucleotide (reduced)
NADP+ nicotinamide adenine dinucleotide phosphate (oxidized) NADPH nicotinamide adenine dinucleotide phosphate (reduced) NCBI National Center for Biotechnology Information
NCIMB National Collection of Industrial, Food and Marine Bacteria
NC-IUBMB Nomenclature Committee of the International Union of
SEM scanning electron microscope
SF 6-deoxy-6-sulfofructose
SFP 6-deoxy-6-sulfofructose-1-phosphate
SLA 3-sulfolactaldehyde
SOD superoxide dismutase
SorAB sulfite dehydrogenase (SorA, catalytic unit; SorB, cytochrome c)
SorT sulfite dehydrogenase
tau genes attributed to taurine uptake and degradation Tau proteins attributed to taurine uptake and degradation
TIC total-ion chromatogram
TOF time of flight
Tpi triose phosphate isomerase
TRBA Technische Regeln für Biologische Arbeitsstoffe
Tris tris(hydroxymethyl)amino methane
w weight
Xsc sulfoacetaldehyde acetyltransferase
YihO sulfoquinovose transporter
YihP putative DHPS exporter
yihQ gene encoding an alpha-glucosidase
YihQ sulfolipid alpha-glucosidase
YihR epimerase
yihR gene encoding an epimerase
yihS gene encoding an isomerase
YihS sulfoquinovose isomerase
yihT gene encoding an aldolase
YihT SFP aldolase
yihU gene encoding a dehydrogenase/reductase
YihU SLA reductase
yihV gene encoding sugar kinase
YihV SF kinase
YihW putative repressor
YSF Young Scholar Fund of the University of Konstanz zntA gene attributed to a heavy metal translocating ATPase