Studies on the bacterial utilization of phenol and related compounds

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m m tm m mm m m tm u i m z u M n m m w m m . km m u tm

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%• %!** ftotvltjr o f tfe» Mmm& o f t i l 9«ftiv*rt&ty o f Worylimci l a §>*r%isl f u l f l lisa ant ©f r©m*h«m>■*

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Dawson who a w m tftin ln g t r y p t l a a a a soy b ro th i s @s@h a ^mnn#r aa t o avoid a«y e ^ ltu r® * # a t# ria l a d h a r la s to th # aid#® o f th a tnb®#

Tha#® w ar# i**e«b#t#d a t § C, 20 C# SO C, ST 0 , 4§ 0# SS C# SO C

sscl axa& inad f o r grcw ih ®,& la d la a to d by t u r b i d i t y i w y 14 hour# f o r s i x day#*

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sosf p lao a d a t 30 0 f o r f u r t h e r in c u b a tio n *

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o f d a t a n a l a l ^ ahath (♦ ) 4 44 4 4

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f*04O**4xla.o V?

iJCl oOU-i) " 'foF;:\:oL*J o f p o r^ m v to o on. sisec

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44*

d i s t i n c t i n i t i a l la g pha*#a*

Witti .suocimat# grown e o lla * m m m i m t a ,

iso la te sjk! pyruvate war# a lsm ltanooualy adapted ins: th a e l t r a t o .group ahewwd & la c k o f adaptation* -* O ^iifsrm tioB si o f p h # rio l* | 5r©wn e e l'li? ®n tka adapts tier*

o f other e lo a o ly roXated aromatic c#sip©UBKSa Xm aaeord w ith th.# pwm$im-B th a t whoa phoaol^grcnm c a l l s war# alm ltan oou oX y adapted t o #th#r compounds I t wt* assuaed th a t thooo oosa^ouacl# w#r« m eta b elie Intojrme&lafeoo i n phenol oxid ation *

f it h th is

la Ti#w f*p#rim #8ta war# eordweted t o determ ine hetaaees do not accum ulate to any proet ex ten t*

Another cK plaoatlon In th e p oaalb lo tendency o f -certa in eom*

pound® to s u f f i c i e n t l y e m p e r e te prm tucisg a aubmiaimal q u a n tity in th e £*edlum*

In t h i s stu dy fo r in sta n ce* i t was obserrwd th a t c a te c h o l did

not alw ays support growth o f th e orgenl«t% y e t when oxy m uptake was determ ined by the a d a p tlre eiancsBctrio te c h n iq u e c a te c h o l i t i 4 o f In i to l y u t i l is e d *

In t h e fe m u r oaoo when growth s t u d ie s w ere co n sid ered c a te *

o h o l could not be consid ered a p a s s ib le intern ed l a t e but th e f a l l a c y o f t h i s was shows, by th e l e t t e r technique* w ith r e e c r e in e l end hydreqolttone#

The same belt awl or was observed

Th# pr###*ae# o f a si«&X# hydroxyl group or a© a lip fea ti# iM # ch a in appear#d to aid in a 1louring th # r in g soApound to support good growthf h#i*#tr#r# it' must b# sgp h aslssA th a t t h i s p rop erty i s d e f i n i t e l y lim ite d by th# number o f th#s# group# and fey th# p o s it io n i s whiah th «y ar# arranged ©» th e rin g*

Inoramstnf; th# auahar o f aid # groups hod th#

t»ad#«#y to d##r#«s# th# p o s s i b i l i t y o f g r w ls ii on th # «#np#uand*

For

«9ea#pi«# phanyl * s # ta t# # whloh p«« •##■•# ©aXy on# #* # t* t# r a d to a l on th e r in g allow ed good growth o f n i l 14 organism# wh#r#a# th# a d d itio n ©f two or per# a e s ta t # r a d io * !# , a s i s p y r o g a llo l t r i a e o t a t # , r e s u lte d in growth o f o n ly 8 organisms#

The p o in t in a ls o shown. wh#n th e ring- #©**»

ta ie o d o n ly on# auXfonl# gr©«p as In b#is.*#»# *txlf#al# sold #

E«r## XZ

organism s grow w a ll on th # oosspcraxKlf fom m m r* whom a oarbosryl and a hydromyl group #r# added t o t h i s

m ating i t sulfas** 11#y l i e sold *

o n ly 3 organisms war# able to grow w a ll on i t #

th # a b i l i t y o f tha&a

organism s t o grow on ©cw^otaiefs oontalnim g a s u lfo n ic group was eeasld orod a lg a lf la u n t in f i « f o f th® appurvnt t o x i e i t y o f t h i s group and th # reported f a llu r # to support growth o f b a a io ria * The Imflneis## o f p o s it io n a l s u b s titu t io n In th# r in g was b #*t iliue** tr a to d w ith th# or#«oX# and dihydroayX eompouads# wo# #fes#irr@i th«.t in th # perm p o s itio n th # than i n # ith # r th # orth o or i»#ta p o s itio n s #

"Itli th# # r# # o l# i t gar# b « tt# r growth

This s « i# prefer#® ## fo r

para p o s it io n a l compounds was not shown in th # as® o f th # dihydromyl oompoun-#•

In th# dih ydroxyl compound# th# ortho p o s itio n (a a to u h el)

gar# b e tto r growth than e it h e r th # s?eta (r e s o r e ln o l) or th# oar# (hydro* ^ulnon#) eentpouB'-i #• Th®## r e s u l t s would In d ic a te th© h igh er s p e c i f ic nature o f th # maehanlsm In ro leed In th# d#jgradatlen o f arom atic compound# by b a cteria *

ecu T his c o n c lu sio n i s supports^ by th® ® bmnmtl® M th a t w d m r ad d ition #* #wb#tlt«tiea« ©r d # l# tle iu i of slmpl# radicals 1© th® Flag ghostly alter tb# ©'©11tty ©r th# ooapouM to support good growth ©f any iadiridwal

b a o to rittn torfcod# Th# n#a##*?lty o f ##rtai*s ton * f o r th # growlh o f th # # # crgoTism * wo# p r e v io u s ly ta b u la te d f*mI i t w i l l b* n#t#d t h o t & tp#b*#sltiai fit©#* p h * t# ©nd m sg a o sia a # » l f a t # w «r# g e n e r a lly r# # u ir# d by ©11 organ l***#

TH# © bserratloa th o t th# medium d##©id o f d l pot* •aim# phosphftt# #111 n ot «wp?#rt th# growth o f any o f th# ©rg#al*»## •# # » whoa th # pH o f th# » # d l* » wm# ad ju sted w ith sodii*!* hydrogld#* would In d io # t# th# n##d o f w ith er pho*phat«* or potassium #

®r##a# B«rb#rt and 3ubr#h®a©yaa ( I f 40)

r«p©rb#d th a t m agaosim or sukagaa### ion # w©r# req u ired fo r Marfeexyla## o stS w ity j th u s fo r th# #«*# r©##om# i t i« assumed th a t noguoaiuea Ion# nr# rooutrod by th # organ I on# l a t h i s study#

fh# marked in flo o a o # th a t

s o s t o f th,©## om its had ©a th© ©*yg«n uptak# o f ph#«©l fu r th e r suhstaa* tin t© # th# n««d o f th® « p « # ifl# d s a l t s i s th# growth o f th### organisms# f t ©on b© ©baorood from fig a r # 1 th a t © a ll# which *r« w«#h#d mod r#suspended in phosphate b u ffe r p lu s th# a s i t # ss#go##iis& siilfa b # and •od in # e h lo r ld # had * aar© ra p id r a t # ©f ©xyg#a uptak# th a n th # * # c o l l s w hich ar# washed and rosuspondod o n ly in phosphat# b u ffer#

A a la lla r

1a c r o s s # in r a t # ca r b# ©btorrod whoa th# c o n cen tra tio n o f ptieasol i s «¥##r#*##£ from ® jxM t# f i*SS# High ©#»e#titrati©R# o f phenol (B >#Sf)' and washing th© c o l l s o n ly l a phosphat# b u ffe r s o lu tio n r e s u lt in r a t# # o f ©aiygoa uptake which mate# I t appear th a t phonal i s not ©wldiaod by th# a d a p tlr# proa#*##

t h i s la

©f o*tr©ts# importon## whan t h i s t##bniqu# Is oisployad t o d #t«ra|ji# p o s s ib le m etab olic pathway##

In t h is p a r tic u la r css® wh#n d jas! o f

51*

p h en cl wwr «*sd th# I n i t i a l il«w r a t# o f o x id a tio n would iwiftediately in d ic a te th a t phenol d egrad ation cou ld mot be in v e s tig a te d by th# ^ o * ees* o f sim ultan eou s adap tation *

Another e ta # in p o in t can be o 1 tod

fro® th# d a ta p resen ted by ®mn* (194T) to which he attem pted ta eon* psr# th# r a t# # o f oxygen uptake o f phenol and c a te c h o l*

H# at#tad t h a t

a d e f in it # la t e n t period wa# e v id e n t w ith phenol a# a su b str a te but arm# a b sen t In c a te c h o l*

th.# phenol # # iie* B treti# a employed by *hrana was 0*8

mg per ml b at a# shown In t h is study* lower o c ft o m tf s tlw is o f phenol ca u ses a more rapid r a t# o f cmctdatienf th u s tomne mmf ha## shown erro n eo u sly a l a t e n t phase i n phenol oxid ation * Om o f th# main c b je e tiv # # o f t h i s t h e s i s i s to lad Seat# to e p o s s i* b le in te r m e d ia te r o le o f th e eye loph eras# e y e le compounds*

I t la shewn

t o s t th e organism used to t h i s stu d y o x id ise d p h en ol, c itr a te * e t s * a o eitita to * is o o it r a t e * ^ -k c te g lu ta r a te and fo r n a te adapt! valy*

On th#

o th e r hard, th # o x id a tl on o f su ccin ate* fumwrate* m alet#* pyruvate and o x a la e c ta te could n et b# shewn to be under ad a p tiv e co n tro l*

According

to the o r ig in a l th eo ry o n ly th e s e ecsRpoends under a d a p tiv e c o n tr o l can be need to o b ta in evid en ce o f th e p o s s i b i l i t y th a t th# p o stu la ted eon** pound .a c tu a lly i s a ® « t*h «lie Interm ediate*

Ihus i t c l e a r l y eon be seen

t h a t one o f th # b a s ic d i f f i c u l t i e s in t h i s work i s t o o b ta in unadopted c e l l # fo r th e purpose o f d e tem d n in g th e a d a p ta tio n p a tte r s* On th# b a s is o f to e s # r e s a l t s c it r a t e * c i# * a e o » ita t# * Iso# liars to* cc- feetogltrtorake and f o n s s t s c a n se t p o s s ib ly be members o f th e r e s e t!a n ch a in in th e breakdown o f phenol*

?1o#«eer* to# p o s s i b i l i t y s t i l l e x i s t s

t o s t s u e d note* fumerabe* m alate* pyruvate and o x a la eeta h e can be members o f t h i s r e a c tio n chain*

The## nr# th # a a ly eaaelu sloB # th a t ea» b# r###fe#d e o m o r ^ ia i th# vtm pv,m &9 o f th # e y # l# as* th# b a s is ©f 1ft# fteats&ulftb#d ©ridam ##

It

i « # hawarar* im portant to m m l t t# t h a t th# f a i lu r e to ds&o&stret# th a t th# aoapoun^s war# p # * sib l# t® t#rm #4i#t#s ootiM hair# b«#n 4u# t o export* » # ist# l prooadur##

For example* i t in fen### t h a t under e o r ta in

tlo n # v&ricm* hydrogen tt#*#pbors must h# pr##*»t or p o s s ib ly «m»# #©#a*yw# or ion must b# reetorodl a f t e r th# w ashing prosed **r# fa r the r e a c tio n to b# in it ia t e d * Th# o b serv a tio n th a t ©war©##!* sr»or# a « !# jHareaol.* ph©nyi a c e t a t e , resorolxsol* hytraqulnan#* and ##t#eh.ol w#r# a l l s imttlt*n#ott#ly o x id ise d fr o * phonal grow® a #11# rat##® th® q u estio n o f whether th e## ©©ssj»©uk4® gh««Xd b# co n sid ered n eta b o H e in te r n e d i* t« # in phm & l d«^r#4#tlo»a By In sp e c tto n o f th # eh eftto a l stru ctu re* th# e r e e o ls m € phenyl o e e ta t# ennnet l e g i e e l l y b# con sid ered a s in te r r e d lo t # « | w hereas resorotjaoX* hytfroouiitos.#* and e a te e h © ! e o n b# #© # # a # 1 4 # rsd «

Th# ia ja e d ta i# o xygon

uptake in th# © r eso ls and $h#®y! e o e ta t# •* * b##t be ex p la in ed on th# b a s is o f two a©f*e#pt#*

3»# i* th a t a oonsaen in term ed ia te assist#- in #

r e v e r s ib le r e a c tio n bets#**** th# o x id a tio n o f phonal .and th # o x id a tio n o f © resole end fshe&yl a e e ts te *

Th# oth er e x p la n a tio n i s t h a t phono!

i s «m in te rm e d ia te i® th e o x id a tio n o f e r s s o le and phenyl the s lo e # s im ila r it y i s ^trmtuQrt #cm##s th© phenol grows s a i l s to o x id is e s r e s o l s and phenyl a#©t*bs sim u ltan eou sly*

and

c M P tm n mmmmmn A ttend&rd lin e a r graph was obtained fo r th e c o lo r t e e t r io e # tln * » t l e n o f phonal In c u ltu r e wHidie#

s te d le # w ith four bnenm e ee e te # of

^yooplonn f e l l e d t o in d ie e t c «ny growth on ^i«no2«

B m m xm o f th in nisi

nth or reetoti# th e emp!i*#l# o f th e e h e r e e te r l t o t le a o f the genu# Mfreo* p lan e we# qu eetleaed* Fourteen orgenie®# e#pebIn e f u t i l i s i n g phenol, wore stu d ied fo r t h e i r a b i l i t y t o grow on. i f eh essicel compound#*

$ i* te e » eempoittaio

^supported $h# grow th o f e l l M «p #el#»# w h i l e 4S eewpeuade dM not s u p p o r t th# growth o f any o f th e orgenl«s*e#

Phenol u t i l i s a t i o n by th # 14 organ I#®#* no determ ined c o lo r ime tar 1~ w U y * w#« c o r r e la te d w ith #» Im reee© o f growth (tu r b id it y ) am! a d e c re a se o f pw fro® 7*4 t o §♦#*

A ll o r g a n ise s r e ta in e d th # a b i l i t y t o

u t i l i s e phenol # ft # r 1Z0 tr a n sfe r # on a m n ^ h t s s l o o a to in la g stadium* On th# b«#l# o f growth on th # 8? ecngpeiiia'is* th # fo llo w in g generiw* 11 ##t I cm# m y be madet

Saturated r in g ecetpotmd#* 41 phenyl eon pounds

or p h en o lic rin g eewpoan4s hewing #. oh lo r e , n ltr o or amine group attach ed do not «wpporh r i s i b l e growth under t e s t condition® ! lik e w is e , in c r e a sin g th e nvmbmr of #1.4# group# tended to decree## th e p o s s i b i l i t y o f ftrtmth os th e casgxnuid# th e r in g we# observed*

th e in flu e n c e o f p o s itio n a l s u b s tit u tio n ©a

w ith th e crew els* th e pars p o s it io n e l lowed

b e tte r growth. then e it h e r t h e orth o or wet# p e titio n *

However* w ith

th e dlhydrcotyl eosapeund# th e orth o pool t i e # supported b e tte r growth then e it h e r o f th e oth er two p o sitio n # *

F ire o f th e 14 organ! w&# were

§ 4 *

eaj>*ble

o f g ro w in g

in

a il

of

the e«w$Hmeiste

o f th #

c y o l opheraee

e y ele*

She eeaeatla.1 s a l t requlreaeH te were determ ined b y obaervtng t h e g ro w th resp on se i n » e d ie which v a ried i n o o ® p o « it io n #

I t w o# noted th a t

pheephete# were e s s e n t i a l f o r a l l o r g a n ! ana and wagne sites was e s s e n t ia l f o r th e g ro w th o f a l l but o n # ep eeiee#

Ferri© c h lo r id e wee not essen-*

t i a l but p r w ld e d fo r b e t t e r growth o f t s t r a in s whereas c a lc 1use eh lo r id * we# - noted to be p a r t i a l l y i n h i b i t o r y In ®o«t oases# Of t h e 14: o r g a n i s e s em p lo y ed i n t h i s s tu d y * S -wore e l e e s I f l e d th e

in

m s &ehr oetebee t e r $ b i n th e p m s M le r o e e e e ttS i end ©so in th e

gmmm A s m c I s o f Bieehem istry* ifimle n»*» ij*8tttfu —.-.iwriiMiww *wawww -n tr nm n iwnfijf up-~n>—1 3* rrimrnr ■TTTffnm

Faeysiilloii Co## m

F# 1$4£ Th# #* 1 4 o tto » o f bo&tolo tucui r o lo to d oobotoiiooo by o o r to ln f&yoofeootorlo# J* Biol* ©host* 145s 3&S«»3$&+ Brood* £ •$ •* ?*tarr*y# a « l lito h o n o * A*p* 1S4# Jjog&y** S t e m ! o f •&otsrgit*sottvo 3ofit*>r1olpgy» $th M lliwuui F H lIEI ro* tta d d la * w*

1014

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*f* Agr* Sol* #*

41?«4S1*

Ctofcolcmrofcl* *1* r;* oa& ©teorsyaski* 1* lf?4S Tho broofcdmm o f phoaolo ond rolotoA ©■oKgKna®*# fey b o # to r i•* J* f3on* ttleroM ol® St £$1.~ES8* D oor ox* d o «Song* I*F * 4 o a * lit© r o llo o t iO B r o o o o s j Fiiwfis i s ! Dahoo*

J*

1940

Boot* H#v* 4 i

M J i r n i e t o t d o koaniii vmn h ot Htino*>

Th# w lo p t iv o jn r o d u tito ii o f o n o y o o o b y b o o t o r io *

14*19*

f’ssrev#* A* A* AM# ffiyisto I s g lo a t dot* 00 phono!*©*!! i *!**& thoratop&lllo boo to r lo* tflkrobiolor* to 191 4 6T -4T 7* Abot* Cho&# Abut* 4$ ( l ) * E7© (1049) • ‘ — ~ R f « i i x# C* 1047 O x id a tio n o f p h o n o l o o l b o » *o lo * « y b o e to r ia * lloohoss# 4 t» 5T2«»$&£*

Evane* ^ * £ * # Hosdlifty* %A#€#* »nd H o p p o ld , F*C# 1C40 in d a l© r o o o t lo n * $l»ofe«!*# J# 3111 dOTn&ll#

by

s o il

Th© try p to p h on ooo**

! ^ t I iT | 0*t** Ardora* F*# notf lo e k o tt* 7*f* I f 11 Th# o x id a tio n o f phenol by oortalxi b o o to r i* in puro out turn* Proo* - ** $#©* Ojoxdoa) B §3* 149-1£©* 0 * 1949 Dotor*! n o tio n o f phenol in b io lo g ic ^ o to r lo l* C lin* «M* 34 § ETfWSSt*

Orant* C* % « i i Zobo 11* v* L# tuurlno bootorlo*

IM i

J* Lob*

O xidation o f hydrosarboao by

Pro©* Poo# tfeep* Slot* fe#d* ©It

f®S»f67*

^r»y# ?V?#K# msd Thorrtsr# F#y* lit© © oil boo t o r i# th o t doooBspo®# m r tm tn oromoti# o©r-p«rands* Contr# f* Abt* II* 75i 74»S©#

m . Owwwa# B# 8#rb «rtt !>*# «?ss! #ttbf*lMs#ny*»# 7# 1040 Oa th # i s o l a t io n •**1 proport*## of earboxylft*## «?* Biol# Choai© 148» 78£*f©0# Oroon* B* S** Xj#oir1#9 w* f ## amtS Auorback, ?# H* 1940 Sto&ioa o s th# ©yelofshor*## »yfflt«iR# J# Biol* Chora# 1721 SffMIOI* P*A* 19250 Th# jlotteotlaxi o f m m t #l#r.t#« 1# .Boot* 19* 2Z$~22P*

H*ua«©R#

prodoietiott

h y boot ©rim in

lott'oia* F» c« 1900 r o r r * l* tf# it o f th # oK l4*tlen o f pH#**©I s and- of' fO* f.w rl##© i\* J* £* #ad S u r f e d , '«* A*

1M ?

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th # o eo w m M * #

o f a tr tc a rb o x y li© *#40 ©pol# i n A#ot©b##t«r © g ills # ttt«*4 170* 9 1 3 ~ ftl#

«J* liia l*

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