Gerhard Gottschalk Bacterial Metabolism Second Edition With 204 Figures 6 inger-Verlag oi w York Berlin Heidelberg Tokyo a \Chapter 9 Chemolithotrophic and Phototrophic Metabolism % ‘Chemolithotrophie and phototrophic bacteria have in common the ability to grow in mincral media, deriviag their cell carbon from CO3. The reducing power required for CO, reduction is obtained from inorganic compounds and energy is provided either by light-dependent reactions or by oxidation of inorganic compounds with oxygen or nitrate (aerobic chemolithotrophs}. The anacrobie chemolithotrophs (methanogenic, ace- Aogenie and snifidegenie hacteria) have been discussed in Chapter 8. 1, Chemolithotrophic Metabolism A. Physiological groups of aerobic chemolithotrophs As already mentioned chemolithotrophs gain enerey by oxidation of an inorganic compound Depencling on the nature of the inorganic compound oxidized, six groups of aerobic chemolithatrophs ace recognized; they are summarized in Table 9.1. The reactions carried out and their free energy changes are also given, ‘Hydrogen-oxidizing bacteria have in common that they use molecular hydrogen as energy source. In other physiological properties and mozpho- fogically, however, they are very diverse. Since they all are facultative chemolithotropbs, taxonomists have preferred to classify them with their chemoheterotrophic retatives. ‘The hydrogen-oxidizing bacteria include Pseudomonas saccharophila, P. facitis, Alcaligenes eutrophus, Nocardia© Choalidhnropie and Hh seed cons (035 -nome01/ 45 2020 Cm Ie. ab le ang LWP ew oro a 0H} HN GOT, (ssl) re (OR) Fee SON —*0% + TON, ‘wazIpRO aaLNE (TZ TO6— yO-)L0E~ OTH + WHE + FON "OH + HN Stoapo row. 2>) 8Ee— DL) per OHF — H+ Ob + 24 Puapeq uo vop—)ors6l— (yee —) Bre 4H + 10S — OM + “ORL + 6S (Lp—yyael——(e'68T-) srse— 18 702 + S euSIEG INNS (s19-) case Ig t9) Tae 203 “FoF +99 wunnegopbeons teos-eaee— iss) eae OOF +H eunoug anvoupie See. e884 ogee BupTaaLY dot 1997 steonoynyounp Jo sdno:8 jroBojorsCg “V6 ORL‘Chemolithstrapihe Metabolism 288 aucotrophica, the niteogen fiiing Xanthobacter autotrophicus and Paracoc ‘cus denitrificans. The latter is able t0 use nitrate instead of oxygen as eleciron acceptor Carbon monoxide-oxidizing bacteria ate also found in various genera, Examples are: Pyewdomonas carboxydavorans, Alcaligenes carcoxydus, and the thermophile Bacils schlegelit. All CO-oxidizing hacteria are Hkewise Hy oxidizers (not vice versat); they are taculanve chemoiitho- trophs. Sulfur oxidizers are the thiobacili, Thlomicrospira pelophite, a marine, spital organism, and Swfolobus, a shermophilic archaebacterium of irregular cell form. In addition, hlamentous gliding organisms, such as Beggiatoa and Thiothrix are able to oxidize sulfide to elemental sulfur and subsequently to sulfate, Most sulfur bacteria are obligate chemolitho- traphs. Some of them, c.g., Thiobucillus intermedius, can grow as aerobic heterotrophs. T. denitrificans can utilize nitrate instead) of oxygen as electron acceptor. “The Fe onlalaer Thiotucila erroneous sul compounds and ferrous ions alternatively as electron donors, The iron bacterium. Gulfionella probably also gains energy by oxidation of Pe 1. Be'". The frce energy change of Fe*” oxidation at low pH values is large enough 10 be coupled wy ATP yyuliesi, iti tallzr sisal at jeuteal pH and iron bacteria cannot grow at pH values above about 4. ‘The exhlation of anunwnia to ultite is carried out by Noosomonas, Nitrosospira, Nivosovibrio, and Nitrosococcuy species; they all are obli- gate chemolithotrophs and s0 arc the nitrite oxidizers Nitrobacter, Nitro- spina, and Nitrococcus, with the exception of some Nirobacter strains. Because nitrite is very toxic (0 most ergamrisiiy, the processes of witite production and nitrite oxidation are remarkable B. Energy production and generation of reducing power in H, and CO oxidizers ‘The ratio in which Hy, O,, and CO, are consumed by a growing culture of hydrogen-oxidizing bacteria is about the following 4M; 120; > 10 2H, + CO, —> (CH;0) + H,0 GH, + 20, * CO, —> GO + 50 ‘Thus, the oxidation of 4H, to water yields enough ATP to allow the synthesis to cell material ((CH;0)) from CO; and Hy TATP synthesis proceeds hy» cheminsmotic mechanism as in aerobic respiration. Cytochromes, ubiquinone, and menaquinonc have been found in membrane fractinns of hydragen-oxidiring bacteria. Differences be- tween hydrogen-oxidizing species have been encountered as tothe transfer af electrons from He ta the respiratory chain. Nocardia anace contains @206 % Cheimumotraphic and Phototraphic Metabolism soluble hydrogenase hich catalyzes the reduction ot NAD by Hy, The product, NADH, then serves as H-ddonor for the tespiratory chain, This is the exception. All other hydrogen-oxidizing bacteria studied contain a Particulate (membrane-bound) hydrogenase which feeds clectrons directly into the respiratory chain. This eueyme does not react with NAD. Very few hydrogen-oxidizing bacteria (c.g., Alculigenes eutrophus,) contain, in addition (0 the particulate enzyme a suluble hydrogenase which reduces NAD* and whieh is primarily responsible for the provision of NADH for CO, reduction. The function of the eno hydrugenases in these organisms is summarized in Fig. 9.1 ‘The uptake hydrogennscs discussed hee catalyze the oxtdation of Hy to H® according to the equation H, — 2H" + 22° and the transfer ofthe electrons to the appropriate acceptous, The enzymes of the H; oxidizers have been shown to be nickel-proteins. In addition, they contain iran.sulfur centers. The Kyy valies for H, are in the order Ur 30-40 pA, so these cuzymes are very effective in oxidizing Hy, ‘The composition of the membrane-bound and of the soluble ly drogenase of A. eutrophus and of hydrogenases from some other micro. ‘organisms is depicted in Tobie 9.2. It is obvious that the cvotutions hydrogenases belong to the group of enzymes that have a simpler composition. They consist of just one subunit; nickel ie apparently not essential. The most complex ones ute the uptake-hydrogenases that reduce NAD* or Fro, They consist of several subunits and contain a flavin. The funetion of the latter may be the one of a redox-switch from I-clectron carriers to 2-eleetron acceptors: [ron sulfur-centers reduce Ue flavin that subsequently transfers the electrons 10 NAD" oF Fre Recently, it has been shown by Friedrich and Schlegel that the genetic information for hydrogenase synthesis in.A. ewtrophus resides on a large plasmid. In other H, oxidizers this information is chromosame-eneoded, and the advantage of either location is not elear yet. ave, ah apr SS. GC Sep maptee Figure 9.1. The function of the two hydrogenases of A. eutraphus. [H. G. Scbleget, Antonie van Leeuwenhoek 42, 181-201 (1976),)a Chemolthotrophis Metals sesoiuaned iy sion jo 29qnuny “slam29 [s+ — 4p] pap=sfap Samm 20 Casatet) Sp aah apse @sr—mr yO - mores ureepaspy —_uonnyoeo noo divdone (arate) ‘sh 93r tanemumesod Wsp-ear - 00°08 unxopsimy —vonntose sh—ear BT - 0908 esouyen ymin (sp - ar wore We ede 8U - 0025 © omxyoxs oyster 90°92 OOO'TE Xz umordouomvouusrs aN 8 vat ono" or x Z sy oyncn umuanogounsoy ‘cou'g (sv - ear moe SE ~ 34 ‘ou'9s ‘St - 24z = Nhar a0e9 ayeicn mrydoune -y Sh - 9a cute SE-3d LO - 9028 ayeicn srydouna -y sus1u29 yusos §— yuo14o>—Gqulom AMaojoUN) —sordesot a ono asic) anpns-H0 PPK uReg ngns ‘sapads jruaioeg sneuea 70 seseuadospty yo uonjsodwor °2°6 aq.288 5% Chemalitioerophue ang Pointe Metahotse ‘The composition of the respiratory chain of A. euiropis and of P. denitrificans (which is a Hz oxidizer) seems to be identical (sce Fig. 5.16). Kc is not clear yet at which site the electrons rom the particulate hydrogenase enter the respiratory chain, Since iton-sulfur proteins are located between NADH dehydrogenase and ubiquinone in die chain and since hydrogenase contains iron-sulfur centers, the enzyme could feed electrons into the chain at the ubiquinone of iron-sulfur provein level as indicated in Fig, 9.2 Whereas there are no dificulties with the provision of NADIT tur CO, reduction in Hy oxidizers that contain a soluble NAD-dependent hydrogenase these problems exist in organisms with only @ membiane- bound enzyme. They have to carry out reverse dectron transfer for NADH formation. The basis for NAD* reduction by reverse electron transfer is that the redox reactions of the respiratory chain including the proton translocation are reversible. The purtiv of He respiratory chain in Fig. 9.2 indicated hy dashed fines is oxidized when hydrogen is taken up by the ydrogewase anu fed iuto the chain, A protonmotive force is built ups itis partly used for ATP synthesis but also partly consumed to reduce the ppotliun of the chain enclosed by dashed Kines and finally to reduce NAD. ADP EES, ATH sath j— a ae NADH +E ae, NAIL denydrpsesee Y J want =" or stop rome pokea " a stiguinooe a places Figure 9.2, Tentative schome of the eloctton flow from the membrane-bound hydrogenase to the components ofthe respirktory chain. Revise clectron transfer leading to NADH formation is indicated by dashed lines.i Stee 2a “The characteristic enzyme oF the carboxydobs oxidase, cteria is carbon monoxide h is membrane-associated and oxidizes CO 19 COs. The roms released ate fe ity the respiratory chain and travel to oxygen thereby generating the protanmotive force: CO + HO CO, + 2H" + 2e DHT | 2e7 11/20, SERS HAG Carbon monoxide oxidase is a molybdoprotein; it contains the classic Mo- cofactor, In addition, iron-suifur centers and FAD are present in the protein. The tedox potential of the CO/CO; couple is very low. Neverthe- less, the coupling of CO oxidase with carriers in the membrane Seems to be such that NAD™ is reduced by reverse electron transfer as with the membrane-bound hydrogenase. Carboxydobacteria contain a branched sespiratory chain; one branch is remarkably insensitive to inhibition by ‘CO, which, of course, isa prerequisite of the ability of these organisms to grow with CO as energy source. C. Ammonia, sulfur, and ferrous ion oxidation Table 9.3 gives the redox potentials of a number of reactions relevant for chemolithotrophic metabolism. It is obvious that NAD’ reduction is very dificult for most of the chemolithotrophic organisms. AS an example we vonsider Nlrobacter species that oxidize nitrite to nitrate NOs + 1/20; —+ NOF ‘The redox potential for the couple NOZ /NOs is +0.42 V. Thus, nitrite is san extremely weak feducing agent for NAD ". the situation is illustrated in Fig. 9.3. 11 first becomes clear from this figure that nitrite is not directly ofidized with O3. Oxygen from water is imuxporated into nite. The Table 9.3, Redox potentials of veautioy important in chemolithotrophie metabolism FilV) CO + H,0> CO, + 2H +26 ~058 Hy 2H* + 267 -041 NADIE HI" NAD? + 20° +2H* <3 HESS + 2M" 4 26 =nas S11 310 50; = aH = ge~ +03 SO} + H,0 + SOP + 2H* + 26° 0.28 NH +2H,0--NO; + ath 6 10H NO; + H,0 + NO; + 2H* + 267 +042 Fol! A. Balt 4 6 0, + 4H" 4 de 24,020 4: Chomotithatniphie and Protoieuphie Metabotia, Napet erat Nose YI =e ( ao a sore ON J— sx air _7 I Figure 9-9. Frononananatacising mitre oxidase of Wimopaerer, Kf assumed that nitrite oxidase (a gstochrome @y-inked molyodoprotein) reduces cytochrome « transfers slactrons via eytochcomte oxidise te O,. ‘The mechaniem of proton translocation is uot fully understood. enzyme nitrite oxidase is located atthe fnner aspect of the membrane. Its a molybdo-protein and redox reactions proceed via cytochrome a;, the more electroncyative cytochrome c, and cytochrome aay {© oxygen. This ‘transport is associated with proton translocation and further with ATP sylihesis, A furge pari UF he AP, however, has ko be frvested Ih WAT" reduction. This presumably is the reason why growth of Nitrobacter is exiromely slow (loubling mes in the order of 181). To dave the Feduction of | NAD", $ molecules of nitrite have to be oxidized ‘As compured to the oxidation of nitrite to nitrate, which involves one electron transfer, the oxidation of ammonia is more complex. The first and the only defintely known intermediate in ummonia oxidation is hydroxylamine; it is formed in a monooxygenase reaction (Fig. 9.4). The ccosubstiate reyuied by this euzyine (XE in Fig, 9.4) is probably veueed eylochrome P 400, a specialized type cytochrome, ihe oxidation Of hydroxylamine to witite i catalyeed by a membrane axcosintod enzymeTr Chomolthewonhie Metabo, a Hw Figure 9.4. Scheme of ammonia oxidation to nitrite, 1, Moacoxygenase reaction, XFL is probably reduced cytochrome P46l); 2, hydroxvlamine-cytochrome ¢ ‘reductase, which is coupled to 2 terminal oxidase; 3, nitroxy\ is oxidized fo nitrite and regenerates XFi, the cosubstrate in the monooxygenase reaction. Caress eouected by daolied anrows are involved in reverse slectron iranster complex hydroxylamine-cytochrome ¢ reductase and an enzyme system that oxidizes the hypothetical intermediate, nitroxyl (NOH), further to nitrite and generates XH;. It is apparent from Fig, 9.4 that only the 2 electrons released in the valence change of the nitrogen atom from —1 to +L are fed into a respiratory chain, Agam, reverse electron wansfer is necessary for NAD” reduction. Sulfur bacteria may use sulfide, elemental sultur, of thiosulfate as electron donors. The routes by which these compounds are oxidized to sulfate are indicated in tg. 9.5. Sulfide is oxidized to polysulfide-sulturin a reaction that requires glutathione (GSH): nS?” + GSH —* GS,SH + 2ne~ Tals fs ako dhe form in whieh elemental SUI Is fed into the oxidation pathway. The action of sulfur-oxidizing enzyme then leads to sulfite. All cleetrans rclcased from the substrate run through the cytochrome sectiva ol the respiratory chain and give rise to the establishment of # proton- motive force that is used for ATP synthesis or reverse electron transfer to NAD*. The acceptor of the aerobic thiobacilli is a cytochrome ¢, and of the facultative anaerobe T. devtrficuns a flavo protein that is linked to cytochrome ¢ via cytochrome 6. Growth yields of T. denitrificans are conciderably higher than those of 7. ékiooxtdan. The reusen could be that T. denitrificans can take advantage of an additional proton extraction site between the favoprotcin and cytochrome « “Thiohaciti usually grow very well on thiotulfate, Several enzyme cysteme have heen described that foed thiosulfate into the sulfur oxidation pathway: rhodanese cleaves thiosulfate into etemental sulfur and sulfite: the. action of thiosulfate vevturing enzyme yialds sulfite and sulfide; a thiosulfate oxidizing smulti-enzyme complex recently deseribed by Kelly and coworkers gives two culfates without the accumulation of free intermediates. ‘This enzyme activity is indicated in Pig, 9.3. The relative importance of the enzymes acting an thinsulfate is not known vot22 1. Chemolihotrophic ant Pritoteuphic Meteo ' aw 12, — sts shoals 2, ae lo te “Re aos one on ae yO Z aes ae 5 aso} aoe sob Figure 9.5. Routes of the oxidation of sulfur corspounds in chen ‘Oxidation of sutnde to porysuldde sulfur [Sf 2, conversion of elemental so polysulie sulfur 3, thiosllate-oniizing multi enzsme comple; 4, sulfur oxidase; Sy suite oxidase; 6, APS reductra: 7, ADP rulfurylse; 8, olectronsaze transl. Red to oxygen Wa Components of the respiratory chain ‘Two enzyme systems have been found to catalyze the oxidation of sulfite tw sulfate. Sulhie oxidase is a membrane-bound molybde-protein and transfers electrons to cytochrome ¢. In addition, the enzyme APS reduc: tase is present in many sulfur bacteria. Here also a cytochrome funetions as electron acceptor. The APS formed is further converted to sulfate and ADP. Tho oxidation of sulfite to sulfate via APS is accompanied by substrate level phosphorylation (AMP —> ADP). The oxidation of Fe? to Fe? by eaygen is taken advantage of by Thiobacillus ferrooxidans. ‘The cedox potential of Fe"t/Pe)* is Eb = £790 mV And wary rine to the one of oxygen T ferroovidens however, lives at pHT values of about 2 and uses iron oxidation primarily as 4 proton-scavanging reaction insicle the cell. This is apparent from Fig. 9.6, ‘The intracellular pH of T. ferrooxidans is about 6 and the organisms have a ‘ApH of 4at their disposal. However, acidification by protons flowing ia via the ATTY synthase hus to be prevented. This fs done by Fe?” oxidation ax indicated. T. ferrooxidans and T. thiooxidans are of special importance in leaching of metal ores. Pyritic ores are penetrated by aerated solutions containing minerals and the organisms. Sulfur is then oxidized to sulfuric acid. Iron pyrite is oxidized to ferric sulfate and sulfuric acid and‘Chexnotihotrophie Metaboli a te we ate Se ake Se ape 1y20,# aa 10 igure 9.6, intacelar 1° consumption im T; ferrooxidans. 1, Rustieyanin, & high-potential copper protein; 2, cytochrome oxidase iton-copper pyrite to copper sulfate and ferric sulfate: 8° + 1.30, +f0 — 1504 2eS, + 7.50; + HzO —* Feg(S04)s + 180, 2CuFeS; 4 850; + H50, — Fe,(S0,), + 2CuSO, + 0 ‘Fluus, a copper sulfate solution is resulting from which copper can be isolated. The basis of uranium leaching is that the insoluble uranium Mioxide reacts with the ferrie sulfate produced by the thiobacilli to the soluble uranyl cation: UO; + Fex($O4), — (UO,)SO, + 2Fe8O4 Leaching proseasea worl at pI values of 2.3: thoy oun be porformed because of the remarkable acid tolerance of the thiobacilli. D. Facultative and obligate chemolithotrophs Te has been mentioned that all hydrogen-oxidizing bacteria studied so far are facultative chemolithotraphs Thay grow necohieally with sugars and amino and organic acids, some of them even with purines and pyrimidines, Nevertheless, chomolithotrophic metabolism ie somowhat dominant in these organisins, The induction of catabolic enzymes for organic substrateet ‘ Chomolithotraphic and Photolrophic Metabolism utilization is thus repressed in a H,/O,-atmosphere. An example is given in Fig. 97. Alvaligenes eurrnphus grows aerobically on fructose and employs the Entner-Doudoroff pathway for fructose degradation, Induc tion af the enzymes of the Entnet—Doudorolf pathway is repressed in a H,/Oy-atmosphere, and the organisms do not grow under such conditions. "The mechanism of this repression is not known: i€ ay be related to that of catabolite repression. In & H,/O,atmosphere ATP is readily available, and the synthesis of catabolic enzymes is kept repressed irrespective of the presence or absence of CO, With some substrates, however, mixotrophic growth is observed; succinate, for instance, is utilized together with Hy and 02 Several hydrogen-oxidizing bacteria (e.g., Alcaligenes eutrophas) prow on fotmate. It is oxidized by formate dehydrogenase to CO,, which is assimilated as under chemolithotrophic conditions. ATP is formed by oxidation of NADH in the respiratory chain, ‘Among the nittifiers facultative chemolithotrophs are rare. Some Nitro- bacter strains grow vety slowly with acetate. In this connection, however, should be mentioned that there is a process called “heterotrophic nitrifica- tion.” It is carried out by a number of Arthobacter and Alcaligenes strains and others. These organisms produce nitrate by unknown mechanisms. ‘The occurrence of heterotrophic nitrification in various soils has been demonstrated: Apparently, ofganic nitrogen-containing compounds serve as substrate; the process is very diflerent trom the ehemotithotrophic process of nitrification, ‘Quite a few sulfur oxidizers are facultative chemolithotrophs or even heterotrophs. T. novellus, T. intermedius, and Sulfolobus acidocaldarius are able to grow with organic substrates as well as with sulfur and COs. T. perometabolis is a chemolithoheterotroph that is unable to grow auto- Wee or Figure 9.7. Aerobic growth of Alealigenes eutrophus on feuctose and repression by Uz # Og. [@. Gottachall, Déseleme Z. 244, 260 279 (1965).]Asiimilstion of COs 25 trophically. With species such us 7. novellas or T. versutus true mixotrophic gfowih has beca demonstrated: simultaneous utilization of glucose or acetate and thiosulfate + CO>. A number of thiobacili, however, are ‘obligate chemolithotrophs. They include T. thioparus, 7. neapotitanus, T. thigoxidans, and T, dentrifcans. The question is, what is the basis of ‘obligate chemolithotrophy? Several explanations can be envisaged. 1, The organisms might be impermeable to organic compounds. However, the uptake of a number of these compounds sind their incorporation into cellular material has been demonstrated for severat obligate ‘chemolithoteophs. 2, ‘The formation of ATTP from organic substrates might be impaired or impossible. This hypothesis has been advanced by Stanier and collabar- ators and, in fact, itis very plausible. Chemolithotrophs do not require ‘a complete tricarboxylic acid eyele, and they have been shown 40 lack eroxoglutarate dehydrogenase (as da the incomplete oxidizers and several strict anaerobes). Thus, NADH for the respiratory chain cannot be generated as normally in aerobic heterotrophs. In addition, the sespiratory chains of obligate chemolithotrophs might not be suited to accept electrons from NADH, as we have scen that the entry of electrons from the inorganic substrates occurs at the level of cytochromes. 3, Ammonia oxidizers and suitur bacteria might sufier from a shortage of ammonia and reduced sulfur compounds under heterotrophic condi- ions. If, for instance, Nirosomonas is incubated with an organic substrate in a mineral medium under air, ammonia is preferentially ‘oxidized to nitrite and there 1s no N source left for biosynthetic purposes, ‘The reason for obtigate chemolithotrophy might not be the same in all ‘organisms possessing this property, However, impaired energy metabolism and or shortage of ammonia or reduced sulfur compounds olfer plausible explanations. I. Assimilation of CO. Chemolithatraphs nse the ATP and the reducing power produced by ‘oxidation of inorganic substrates to reduce CO, and to convert it to cell miaterial. Since COs functions as sole source of carhon in these organisms, they are often called C-autotrophs. The mechanism of CO, fixation employed by chemolithotraphs is the Calvin eyele, the same mechanism that occurs in green plants and was discovered by Calvin, Benson, and Bassham in green algae.