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E. coli suyuq muhitda o'smaydi

E. coli suyuq muhitda o'smaydi



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Biz muntazam ravishda bakterial transformatsiyani va DNKni ajratib olish uchun plastinkadan suyuq muhitga subkulturani amalga oshiramiz. Bu odatda juda yaxshi ketadi va oddiy, lekin vaqti-vaqti bilan plastinkada yaxshi o'sgan koloniyalar suyuq madaniyatda o'smaydi (plastinkadagi bir xil antibiotiklar bilan). Bu bir nechta plazmidlarda uch kishi bilan sodir bo'ldi.

Masalan, men ampitsillinga qarshilik genini (uning promouteri bilan) pENTR4 plazmidida (kanamitsinga qarshilikka ega) bog'lashga harakat qilardim. Mening plastinkam va LB ikkalasida ikkita antibiotik bor edi, ammo koloniyalar 16 soatlik vaqt oralig'ida biroz loyqalanishga muvaffaq bo'ldi.

Fag bilan ifloslanish ehtimoli juda kam va bu ta'sir kiritilgan bo'lakning toksikligi bilan bog'liq bo'lishi mumkin emas, chunki qo'shimcha va vektor bir xil bakteriyalarda bir necha marta muammosiz o'stirilgan.

Yordam?

EDIT1 - Biz plastinkadagi ampitsillin o'rniga Karbenitsillinni ishlatamiz, lekin suyuq muhitda emas.

EDIT2 - Ba'zi koloniyalarni suyuqlikda o'stirishga muvaffaq bo'lganimdan so'ng, men oz miqdordagi plazmidni olishga muvaffaq bo'ldim (profil yaxshi edi; konsentratsiya = 70ng/ul). G'alati narsa shundaki, men ushbu DNKni Stbl3 bakteriyasida qayta o'zgartirganimda, menda hech qanday muammo yo'q edi. Sizningcha, bakteriyalar zahirasi javobgar bo'lishi mumkinmi?


Menimcha, buning uchun bir nechta imkoniyat bor.

  1. Koloniyalar bo'lgan plitalar 4C da juda uzoq vaqt saqlanadi (@mdperry taklif qilganidek). Bu vaqt o'tishi bilan koloniyalarning plazmidni yo'qotishiga olib keladi, chunki plastinkadagi antibiotik vaqt o'tishi bilan parchalanadi. Bu borada tadqiqot bor va men havolani baham ko'rdim. (http://homepages.bw.edu/~mbumbuli/biotech/colin/index.html).
  2. Qo'shimcha hujayralar uchun toksik va promouter etarlicha kuchli emas. Hujayralarning nobud bo'lishiga olib keladigan oqish ifodasi mavjud.
  3. Vakolatli hujayralarni va ularning saqlash holatini tekshiring, chunki bu transformatsiya samaradorligiga katta ta'sir qiladi. Qo'shimchalarsiz oddiy plazmid bilan soxta transformatsiya qiling va ularni antibiotik plastinkasiga yoying.

Escherichia coli ning dam olish suyuqligida harakatchanlikni faollashtiruvchi mutatsiyalarning tez to'planishi.

Harakatlanish genlarini ifodalash bakteriyalarda potentsial foydali, ammo qimmatli jarayondir. Qizig'i shundaki, ko'pchilik shtammlarni ajratib turadi Escherichia coli harakatchanlik genlariga ega, lekin harakatchanlik foydali bo'lgan sharoitlarda ularni faollashtirish qobiliyatini yo'qotib, bu holatlarga qanday munosabatda bo'lishlari haqida savol tug'diradi. Shtammlarning transkriptom profili orqali E. coli Bir genli nokautli Keio to'plamida biz ko'plab o'chirish shtammlarida ularning kuchsiz harakatchan ota shtammi (BW25113) darajasiga nisbatan harakatlanish genlarining keskin o'sishini payqadik. Biz ko'rsatamizki, bu harakatlanuvchi fenotipga o'tish o'chirilgan genlarning to'g'ridan-to'g'ri oqibati emas, balki uning o'rniga asosiy harakat regulyatori FlhDC ekspressiyasini oshiradigan turli ikkilamchi mutatsiyalar bilan bog'liq. Muhimi, biz bu kalitni zaif harakatchanlik orqali ko'paytirish mumkinligini aniqlaymiz E. coli shtammlarni silkitmaydigan suyuqlik muhitida bir kechada, lekin chayqaladigan suyuq muhitda emas. Bir kecha-kunduzda silkitmaydigan inkubatsiyadan so'ng alohida izolatlar yuqori oqimda aniq mutatsiyalarga ega bo'ldi. flhDC operon, shu jumladan turli kiritish ketma-ketligi (IS) elementlari va kamroq darajada nuqta mutatsiyalari. Irsiy o'zgarishlarning silkinishsiz o'sgan populyatsiyalar bo'ylab o'tish tezligi shuni ko'rsatadiki, zaif harakatlanuvchi shtammlar genetik qayta o'tkazish orqali harakatchan turmush tarziga tezda moslashishi mumkin.MUHIM Oldindan belgilangan tartibga soluvchi tarmoqlardan tashqarida zarur bo'lgan vaqtlarda gen ekspressiyasini sozlash qobiliyati organizmlarga omon qolish va raqobatlashish imkonini beradigan muhim evolyutsion jarayondir. Bu erda biz suyuqlik muhitida bir kechada silkitmasdan inkubatsiya qilinganda, asosan harakatsiz populyatsiyalar paydo bo'lishini ko'rsatamiz. Escherichia coli bakteriyalar konstitutsiyaviy harakatga ega bo'lgan mutantlarni tezda to'plashi mumkin. Bu ta'sir bitta genli nokaut kutubxonasi, Keio kollektsiyasida keng tarqalgan ikkilamchi mutatsiyalarga yordam beradi. Natijada, sinovdan o'tgan Keio shtammlarining 49/71 (69%) turli darajadagi harakatchanlikni namoyish etdi, ularning ota-ona shtammi esa yomon harakatchan. Ushbu kuzatuvlar genlar ekspressiyasining plastisitivligini, hatto oldindan mavjud tartibga solish dasturlari bo'lmagan taqdirda ham ta'kidlaydi va laboratoriya shtammlari bilan ishlash tartib-qoidalari haqida xabardorlikni oshirishi kerak. E. coli.

Kalit so‘zlar: Keio to'plamining evolyutsiyasi bayroqli gen regulyatsiyasi flagellar harakatchanligi genini tartibga solish.

Mualliflik huquqi © 2019 Amerika Mikrobiologiya Jamiyati.

Raqamlar

Keio shtammlari orasida transkriptomni qayta qurish.…

Keio shtammlari orasida transkriptomni qayta qurish. (A) gen ifodasi ma'lumotlarini ierarxik klasterlash ...

Harakatlanish genining ifodalanishini tasdiqlash...

tomonidan boshqariladigan GFP muxbiri yordamida harakat geni ifodasini tasdiqlash fliC…

Motilite genining faollashuvi o'rtasidagi bog'liqlik ...

Harakatlanish genining faollashuvi va suzish fenotipi o'rtasidagi bog'liqlik. (A) Suzish harakatchanligini tahlil qilish…

... keyin harakatlanish faollashuvining yo'qligi.

Yagona gen deletsiyalarining P1 transduktsiyasidan so'ng harakatlanish faollashuvining yo'qligi. (A) mRNK darajalari ...

Turli ikkilamchi mutatsiyalar uchun javob beradi ...

Keio kollektsiyasida harakatchanlikni faollashtirish uchun mas'ul bo'lgan turli xil ikkilamchi mutatsiyalar. (A) Mutatsiyalar…

Motillikni faollashtiruvchi mutatsiyalarning tez to'planishi ...

Dam olish suyuqligi madaniyatida harakatchanlikni faollashtiruvchi mutatsiyalarning tez to'planishi. (A) Yumshoq-agar suzish tahlili ...

Shtammlarda harakatchanlik genining faollashuvi...

Aerotaksis va kemotaksis uchun etishmayotgan shtammlarda harakatlanish genining faollashuvi. (A) Transformatsiya natijalari…


Kichik RNKlar davomida stressga javob beradi E. coli chayqaladigan idishlarda o'sish

sRNKlarning stressli o'sish sharoitlariga javoban ishtiroki haqidagi ma'lumotlarning ko'p qismi dan olingan E. coli chayqaladigan kolbada past zichlikka o'sadi. Ushbu tadqiqotlar natijasida olingan ma'lumotlar harorat, osmolyarlik, temir cheklanishi, glyukoza fosfat to'planishi, pH va kislorod kabi stressga javob beradigan bir nechta sRNKlarni aniqladi. Ushbu tadqiqotlarning barchasi faqat sRNKning stressga molekulyar javobini tushunishga qaratilgan, ammo bu sRNK ifodasini manipulyatsiya qilishning mumkin bo'lgan fiziologik oqibatlariga emas. Hozirgi vaqtda ma'lum bo'lgan ma'lumotlar 1-jadvalda jamlangan.

Harorat

Porinlarning tarjimasi va barqarorligini tartibga solishda ishtirok etadigan ikkita sRNK, MicC va MicF aniqlandi [49]. Ikkalasi ham harorat o'zgarishiga javob berish bilan bog'liqligi aniqlandi. MicC past haroratda (24 ° C) ifodalangan va o'sish orqali aniqlangan E. coli K-12 (JM109) LB va M9-glitserinli minimal muhitda turli haroratlarda statsionar va eksponensial fazalarda ODgacha.600 0,2 yoki 0,4 [25]. MicF yuqori haroratlarda (37 yoki 42 ° C) ifodalanganligi aniqlandi va o'sishi bilan aniqlandi. E. coli K-12 JA221 kechada OD ga550 0,2 ni tashkil etdi, shundan so'ng 50 °C da teng hajmdagi muhit qo'shib, o'sish harorati 37 ° C ga oshirildi [32]. Harorat o'zgarishiga javob beradigan yana bir sRNK DsrA bo'lib, bu sRNK induksiyalanganligi aniqlandi. E. coli K-12 madaniyati LB muhitida 24 °C da OD ga yetishtiriladi600 0,4–0,6 [27]. DsrA - bu tartibga soluvchi sRNK rpoS va hns mRNKlar [29]. Ushbu sRNK transkripsiya regulyatori s S ning mRNKsi bilan o'zaro ta'sir qiladi (kodlangan: rpoS) va transkripsiyaviy repressor H-NS [50], u RpoS tarjimasini faollashtiradi va inhibe qiladi. hns tarjimasi [29, 30].

Osmolyarlik

sRNK MicF, harorat o'zgarishiga javob berishdan tashqari, o'sish muhitiga 20% saxaroza qo'shib yaratilgan yuqori osmolyarlik sharoitlari bilan ham bog'liq. MicF ifodasi qachon oshdi E. coli K-12 LB, past fosfatli muhitda yoki ozuqaviy bulonda yoki 0,4% glyukoza bilan to'ldirilgan M9 muhitida o'stirildi, madaniyat yuqori osmolyarlik sharoitlariga ta'sir qilganda MicF ifodasi oshdi [26].

Osmolyarlikdagi o'zgarishlarga javob beradigan yana bir sRNK RprA bo'lib, bakteriyalar osmotik zarbaga duchor bo'lganda RpoS translatsiyasini faollashtiradigan 105nt sRNKdir [33, 34]. Eksperimentlar o'stirish orqali amalga oshirildi E. coli K-12 (MG1655) LB da statsionar fazaga (OD600 = 3,5) va 20 daqiqa davomida 0,12, 0,23, 0,46 yoki 1,0 M saxaroza qo'shiladi [34]. RprA ifodasi ham osmotik zarba bilan induktsiya qilingan E. coli K-12 MC1061 shtammi LBda OD ga yetishtirildi600 0,3 ning 0,46 M saxaroza qo'shilishi bilan [33].

Temir konsentratsiyasi

90 nt sRNK bo'lgan RyhB transkripsiyasi ommaviy axborot vositalarida temir mavjudligi bilan bog'liqligi aniqlandi. RyhB odatda temirga boy sharoitlarda repressiya qilinadi [51] va temir kontsentratsiyasi past bo'lganda ifodalanadi, shu bilan temir o'z ichiga olgan oqsillarning ekspressiyasini pasaytirib, temir iste'molini kamaytiradi va natijada hujayra ichidagi erkin Fe 3+ konsentratsiyasini oshiradi. [51, 52]. Bu ning hosilalari yordamida o'rganildi E. coli 1 mkM FeSO bilan to'ldirilgan M63 muhitida o'stirilgan K-12 MG16554 [52]. Yuqori temir kontsentratsiyasining ta'siriga kirish uchun boshqa tadqiqotlar o'sish orqali o'tkazildi E. coli K-12 MG1655 OD uchun600 = 50 mkM FeSO da 0,2% glitserin bilan to'ldirilgan LB yoki M63 muhitida 0,34 [35]. Bu shuni ko'rsatdiki, RyhB ekspressiyasining ortishi TCA sikli va nafas olish zanjiri bilan bog'liq genlarning ekspressiyasining pasayishiga olib keldi. acnA (akonitaza), sdhCDAB (suksinatdehidrogenaza), sodB (superoksid dismutaza), fumA (fumaraza) va ferritinlar bfr va ftnA [35, 36].

sRNK GadY kislotali sharoitlarga javob berish bilan bog'liq genlarning ko'payishi bilan bog'liq. GadY sRNKning uchta turi mavjud: to'liq uzunlikdagi GadY 105 nt va ikkita qayta ishlangan shakllar mos ravishda 90 va 59 nt da aniqlanadi. E. coli K-12 MC4100 va E. coli K-12 MG1655 pH 5,8 [45] da LB da chayqaladigan kolbalarda yetishtirildi. Boshqa eksperimentda GadY va uning qayta ishlangan shakllari ichida aniqlandi E. coli OD gacha o'sgan600 100 mM 2-(N-morfolino) etansulfonik kislota (MES) bilan pH 5,5 da tamponlangan LB-MES muhitida 2,0 ning [46].

GadY ning haddan tashqari ko'payishi kislota qarshilik genlarining faollashishi bilan bog'liq edi gadA, gadB, va gadC (GDS-glutamat dekarboksilaza tizimining bir qismi), bu esa o'z navbatida GadX mRNKni faollashtirib, GDS ifodasini keltirib chiqardi [45, 53, 54]. GadY, shuningdek, pH 5,8 [55] da Lizin dekarboksilaza tizimini (LDS) faollashtirishi aniqlandi. Yana ikkita sRNK RprA va DsrA kislota qarshiligi bilan bog'langan [47]. RprA va DsrA qachon ifodalangan E. coli K-12 MG1655 hujayralari 0,4% glyukoza bilan to'ldirilgan M9 muhitida o'stirildi. Madaniyat 0,4% glyukoza va 1,5 mmol/L glutamat bilan to'ldirilgan M9 minimal muhiti bilan 1:10 yoki 1:100 nisbatda suyultirildi, u konsentrlangan HCl bilan pH 2,0 yoki 3,0 ga sozlandi [47]. RprA ifodasini induktsiya qiladi rpoS kislota qarshiligidan himoya qiladi [47]. DsrA ning haddan tashqari ko'payishi induktsiya qiladi rpoS RprA sRNK ga o'xshash [48].

Glyukoza tashish va glyukoza fosfat to'planishi

Glyukoza tashuvchisi ptsG ning ekspressiyasi posttranskripsiya darajasida mRNK bilan bog'langan 227 nt sRNK SgrS tomonidan tartibga solinishi aniqlandi. ptsG [40, 56]. SgrS ta'siri qachon kuzatilgan E. coli K-12 (MG1655) 0,2% glyukoza bilan to'ldirilgan M63 minimal media agar plastinkalarida va 1% glyukoza yoki metabolizatsiyalanmagan glyukoza alfa metil glyukozid (aMG) bilan to'ldirilgan suyuq LB muhitida o'sdi [40, 57]. SgrS glyukoza-6-fosfat to'planishiga javoban ifodalanganligi aniqlandi, bu esa uning tarjimasini inhibe qiladi. ptsG mRNK va natijada glyukoza tashuvchisi IICB Glc ning pasayishiga va glyukozaning hujayralarga kirishiga olib keladi [58,59,60,61]. SgrS shuningdek, oldindan mavjud IICB Glc tashuvchilarining faoliyatini tartibga soluvchi 43 ta aminokislota polipeptid SgrTni kodlaydi [41]. Ushbu polipeptid mRNK degradatsiyasidan mustaqil ravishda glyukoza tashishni kamaytirish orqali aMG ishtirokida o'sadigan hujayralarni qutqaradi [41, 62]. Uglerod almashinuvi bilan bog'liq bo'lgan boshqa sRNKlar - CyaR va Spot42. CyaR 87 nt sRNK, past glyukoza konsentratsiyasida induktsiya qilinadi va global regulyator Crp [42] tomonidan ijobiy tartibga solinadi. Bu o'sish orqali o'rganildi E. coli Lennox bulyoni yoki 0,001% vitamin B1 va 0,2% glyukoza yoki glitserin bilan to'ldirilgan M63 minimal muhitini o'z ichiga olgan chayqaladigan idish yoki agar plastinkalarida [42]. CyaR ning haddan tashqari ko'payishi hujayralarni past glyukoza sharoitlariga moslashtirish uchun membrana oqsillari, tashuvchilar va muhim fermentlarni ifodalash uchun javobgar bo'lgan kamida 25 ta qo'shimcha genlarni pasaytirdi [42]. Spot42 a109 nt Hfq ga bog'liq bo'lgan sRNK bo'lib, u markaziy va ikkilamchi metabolizmda, redoks muvozanatida va turli afzal bo'lmagan uglerod manbalarini iste'mol qilishda ishtirok etuvchi genlarni bostiradi, bu esa sekin o'sishga olib keladi [63]. Yuqoridagi ma'lumotlar o'sish orqali olingan E. coli 10 mkg/ml tiamin, 2 mM MgSO bilan to'ldirilgan LB yoki M9 muhitidagi K-124, 0,1 mM CaCl2, va 0,2% kasamin kislotalari va turli uglerod manbalari [63].

Kislorod

sRNK OxyS, OxyR regulonining ko'payishiga javoban induktsiya qilinadi, bu ta'sir qilish natijasida paydo bo'ladi. E. coli K-12, LB muhitida ODgacha yetishtiriladi600 = 0,2, H gacha2O2 [37]. OxyS rpoS ni inhibe qilish va hujayralarni mutagenezdan himoya qiluvchi FhlA (formatli vodorod liyaza faollashtiruvchisi) ifodasini tartibga solish orqali post-transkripsiyaviy ta'sir ko'rsatadi [37, 64, 65].

Kislorodning yuqori konsentratsiyasi reaktiv kislorod turlarini (ROS) hosil qilish orqali fermentlar, oqsillar va DNKga ta'sir qilish orqali hujayra shikastlanishiga olib keladi. E. coli SoxRS va OxyR regulonlarini faollashtirish orqali oksidlovchi stressdan himoyalangan [66, 67]. Qachon E. coli LB yoki aniqlangan muhitda o'stirilgan K-12 superoksid yoki redoks-sikulyatsiya qiluvchi dorilarga ta'sir qiladi, SoxR regulon faollashtiradi. soxS SoxRS regulonidan bir nechta genlarning ifodalanishini keltirib chiqaradigan transkripsiya omili sifatida ishlaydigan gen. SoxS oqsili tomonidan faollashtirilgan ba'zi genlar sodaA, acnA, fumC, micF, va zwf, akonitaza B va fumaraza A va B kabi sezgir fermentlarni kislorodga chidamli akonitaza A va fumaraza C izozimlari bilan almashtirish [68, 69].


Madaniyat vositalarini tayyorlash

Madaniyat vositasini tayyorlash | Rasm muallifi: SouravBio (microbiologynote.com)

Savdoda barcha vositalar chang shaklida mavjud. Madaniyat muhitini yoki bakteriologik muhitni tayyorlash quyidagi bosqichlar orqali amalga oshirilishi mumkin

  1. Kerakli ingredientlarni yoki to'liq suvsizlangan muhitni tegishli hajmdagi distillangan suvda eritib yuboring.
  2. Keyin pH o'lchagich yordamida muhitning pH qiymatini aniqlang va agar kerak bo'lsa, uni sozlang.
  3. Bu muhitga agar qo'shing va agar qattiq muhit kerak bo'lsa, agar uni eritish uchun qaynatib oling.
  4. Keyin vositani naychalarga yoki kolbalarga tarqating.
  5. Shundan so'ng ularni avtoklav yordamida sterilizatsiya qiling. Ba'zi issiqlikka chidamli vositalar yoki maxsus ingredientlar filtrlash yordamida sterilizatsiya qilinadi.

2-jadvalda barqaror muhitda har qanday va B ning koloniya xususiyatlari ko'rsatilgan

Eksperimental natijalarga ko'ra, A dan bakterial koloniyalar mikroskop ostida ko'rib chiqilganda, qutbli, gramm-manfiy va izolyatsiya qilingan joylashuvga ega edi. Koloniya xususiyatlaridan bahramand bo'lgan A dan koloniyalar yumaloq, rangsiz, shaffof, baland, silliq ko'rinishga ega bo'lib, kattaroq koloniyaga ega edi. Bundan tashqari, A koloniyasining tepasi tekis bo'lib, uning butun uchlari bor edi. Shuning uchun bakteriyalarning koloniya xususiyatlari, morfologiyasi va gramm tabiatiga ko'ra, A koloniyasidagi bakteriyalar E. coli bo'lishi mumkin. Boshqa tomondan, B dan bakteriyalar koloniyalari gramm-musbat, kokklar shakllangan va uzumga o'xshash tuzilishga ega edi. B dan koloniyalar yumaloq, oqartiruvchi, noaniq, qavariq, donador va punktiform oʻlchamda boʻlgan. Shunday qilib, B dan bakteriyalar koloniyalari, ehtimol, S. epidermidis edi.

Nomi: Vey Xiao Yang


Ko'rsatgan fenotipik o'zgarishlar E. coli Kosmosda madaniyatli

Bakteriyalar kosmosni tadqiq qilishda odamlarga hamroh bo'ladi, bu ularning mikrogravitatsiya muhitiga moslashishini o'rganishni muhim qiladi. Kosmosda o'sadigan bakteriyalar uchun potentsial fenotipik o'zgarishlarni o'rganish uchun, Escherichia coli Xalqaro kosmik stansiya bortida Yerdagi mos boshqaruvlar bilan o'stirildi. Kosmik muhitdagi qaram o'zgaruvchilarga dori konsentratsiyasining rolini o'rganish uchun namunalar gentamitsin sulfatning turli konsentratsiyasi bilan sinovdan o'tkazildi. Tahlillar yakuniy hujayralar soni, hujayra hajmi, hujayra konvertining qalinligi, hujayra ultrastrukturasi va madaniyat morfologiyasini baholashni o'z ichiga oladi. Kosmosda yerdan boshqarish vositalariga nisbatan oxirgi hujayralar sonining 13 barobar ortishi kuzatildi va kosmik parvoz hujayralari gentamitsin sulfatning odatda inhibitiv darajalari mavjudligida o'sishi mumkin edi. Kontrastli yorug'lik mikroskopiyasi va fokuslangan ion nurlari / skanerlash elektron mikroskopiyasi shuni ko'rsatdiki, kosmosdagi hujayralar o'rtacha 37% ni tashkil qiladi, bu esa diffuziya bilan cheklangan massa tashish rejimida molekula-hujayra o'zaro ta'siri tezligini o'zgartirishi mumkin. mikrogravitatsiyada yuzaga kelishi kutilmoqda. TEM tasvirlari Yerni nazorat qilish guruhiga nisbatan kosmosda hujayra konvertining qalinligining 25 dan 43% gacha o'sishini ko'rsatdi. Kosmik parvozlar namunalarida tashqi membrana pufakchalari kuzatilgan, ammo Yer madaniyatlarida kuzatilmagan. Vaholanki E. coli Yerdagi suspenziya kulturalari suyuq muhit bo'ylab bir hil taqsimlangan, kosmosda ular klaster hosil qilishga moyil bo'lib, atrofdagi muhit hujayralardan ko'rinadigan darajada toza bo'lib qolgan. Hujayralarni yig'ishning bu harakati boshqa kosmik parvoz tajribalarida kuzatilgan biofilm shakllanishining kuchayishi bilan bog'liq bo'lishi mumkin.

Kalit so‘zlar: agregatsiya bakterial o'sish bioastronautics hujayra konvert hujayra hajmi mikrogravitatsiya pufak.

Raqamlar

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NASA astronavti Rik Mastrakkio o'ng qo'lida GAP ushlab turganini ko'rsatdi va ...

Kosmosda, E. coli hujayralar…

Kosmosda, E. coli Hujayralar 59% uzunlikda, 83% diametrda va…

Fokuslangan ion nurlari/skanerlovchi elektron mikroskop...

Fokuslangan ion nurlari / skanerlash elektron mikroskopiyasi (FIB / SEM) 25 mkg / ml bilan sinovdan o'tgan namunalar tasviri ...

Kosmosda hujayra konvertining qalinligi ...

Kosmosda hujayra konvertining qalinligi 25 va 25 ... 43% ga oshdi.

Yupqa kesimli transmissiya elektron mikroskopi (TEM) ...

Yupqa kesimli elektron mikroskop (TEM) tasvirlari E. coli . Chapga : namuna…

Fazali kontrastli tasvirlar E.…

Fazali kontrastli tasvirlar E. coli Yerda madaniyatli (yuqori rasm) va ...


Natijalar

Etti xil genning haddan tashqari ko'payishi shtammning o'sishini tiklaydi E. coli M9/glyukozada PdxB etishmasligi

PdxB PLP sintez yo'lining ikkinchi bosqichini katalizlaydi (1-rasm). Unda bo'lgan shtamm pdfxB oʻchirilgan 37°C da minimal muhitda oʻsmaydi, chunki u PLP ni sintez qila olmaydi. Biroq, u 30 ° C da sekin o'sishi mumkin, bu sekin o'sishni ta'minlash uchun etarli PLP boshqa yo'l bilan sintezlanishi mumkinligini ko'rsatadi (Lam va Winkler, 1990). Biz ortiqcha ishlab chiqarish PdxB o'rnini bosadigan 4PE dehidrogenaza faolligini ta'minlaydigan fermentlarni aniqlash maqsadida ko'p nusxali bostirish ekranini o'tkazdik. 3276 ORFni o'z ichiga olgan ifoda kutubxonasi E. coli lekin etishmaydi pdfxB ASKA kolleksiyasidagi klonlardan ajratilgan plazmidlar yordamida yig'ilgan (Kitagava va boshqalar, 2005) va D ga kiritilganpdfxBkan shtamm (bundan keyin D deb yuritiladipdfxB kuchlanish). M9/glyukozada o'sgan 28 ta koloniyadan olingan plazmidlar tiklandi va har biri tomonidan olib boriladigan ORF ketma-ketlik bilan aniqlandi. Shunisi e'tiborga loyiqki, biz yetti xil genning haddan tashqari ifodalanishi D ning o'sishini tiklaganini aniqladikpdfxB M9/glyukoza shtammi (I-jadval, 1-rasm va II-jadvalga qarang). (Etti gendan uchtasi faqat bir marta topilganligi sababli, populyatsiyadan kam tanlab olingan va o'sishni tiklaydigan qo'shimcha genlar haligacha kashf etilgan bo'lishi mumkin.) Etti gendan uchtasi (uningB, php va yjbQ) suyuq va qattiq muhitda o'sishni ta'minlaydi, boshqalari faqat qattiq muhitda o'sishni ta'minlaydi.

Protein Funktsiya
PdxA Fosfohidroksitreonin dehidrogenaza/dekarboksilaza
AroB Degidrokinat sintaza
ThrB Gomoserin kinaz
YeaB a a NudL nomi bilan ham tanilgan.
Bashorat qilingan NUDIX gidrolaza
UningB Imidazol-glitserin-fosfatdegidrataz/gistidinolfosfataza
Php Prognoz qilingan metallogidrolaza
YjbQ Noma'lum

D ni to'ldiradigan etti gendan faqat ikkitasipdfxB shtamm dehidrogenaza faolligiga ega bo'lgan fermentlarni kodlaydi. 4-Hidroksi-L-treonin (4HT) dehidrogenaza (PdxA) PdxB tomonidan katalizlangan reaktsiyaning quyi oqimida dehidrogenatsiya reaktsiyasini katalizlaydi. Degidrokinat sintaza (AroB) 3-deoksi-D-arabino-geptuloson kislotasi 7-fosfatning degidrokinatga ko'p bosqichli konversiyasi paytida qattiq bog'langan NAD + kamayadi va keyin oksidlanadi murakkab reaktsiyani katalizlaydi ( Duradgor va boshqalar, 1998). Qolgan beshta fermentdan to'rttasi suvsizlanish, fosfor o'tkazish yoki gidrolitik reaktsiyalarni katalizlashi ma'lum yoki taxmin qilingan. YjbQ past darajadagi tiamin fosfat sintaza faolligini namoyish etadi (Morett va boshqalar, 2008), garchi bu uning fiziologik funktsiyasi deb hisoblanmasa ham.

Yetti xil fermentda 4PE dehidrogenaza faolligini tahlil qilish, ular haddan tashqari ishlab chiqarilganda D o'sishini tiklaydi.pdfxB glyukoza ustida kuchlanish

4PE dehidrogenaza faolligi bilan yuqori darajadagi ferment hosil bo'lishi genning haddan tashqari ifodalanishi D ni to'ldirishi mumkin bo'lgan eng aniq mexanizmdir.pdfxB kuchlanish. Biroq, ThrB, YeaB, HisB va Php uzoq muddatli inkubatsiyadan keyin ham 4PE oksidlanishini ko'rsatmadi. YjbQ ni tahlil qilib bo'lmadi, chunki eruvchan oqsilni olish qiyin edi.

PdxA 4PE ni asta-sekin oksidlaydi, a kmushuk ning 0,020±0,002 s -1 , a KM 150±13 mkM va a kmushuk/KM ning 138±19 M -1 s -1, NAD + ning quyidagi qisqarishi bilan o'lchanadi. PdxB C-2 da 4PE ni oksidlaydi. PdxA uchun normal substrat bo'lgan 4PE va 4-fosfohidroksitreonin (4PHT) tuzilmalarini taqqoslash asosida PdxA C-2 emas, balki C-3 da 4PE ni oksidlanishini kutgan edik (2A va B-rasm). Reaktsiya termodinamik jihatdan yuqoriga ko'tarilganligi va mahsulot NMR bilan tavsiflash uchun etarli konsentratsiyalarda to'planmasligi sababli, biz PdxA va ortiqcha [4R- 2 H] NADH yoki [4S mavjudligida 4PE ni NAD + bilan muvozanatlash orqali oksidlanish joyini bilvosita aniqladik. - 2 H]NADH. Teskari reaktsiyada deuteridning mahsulotga o'tkazilishi tufayli 4PEda deyteriyning paydo bo'lishi (1-sxemaga qarang) oksidlanish joyini aniqlashga imkon berdi. 4PE ni [4R-2 H]NADH va PdxA bilan inkubatsiya qilgandan so'ng, 4PE ning C-3 protonidan kelib chiqadigan signal kamayadi va C-2 va C-4 protonlari tufayli signallar o'zgaradi, chunki ular endi bo'linmaydi. C-3 dagi proton tomonidan (2E-rasm). NADH (2D-rasm) yoki [4S-2 H]NADH (2F-rasm) ishtirokida signallarning hech biri o'zgarmaydi. Shunday qilib, 4PE oksidlanishi C-2 emas, balki C-3 da sodir bo'ladi va shuning uchun PdxA ning bu faolligi PdxB faolligini almashtira olmaydi.

AroB 4PE ni ham oksidlaydi, lekin faqat bitta aylanmani amalga oshiradi (ma'lumotlar ko'rsatilmagan). AroB ning normal katalitik sikli davomida bu ajablanarli emas, qattiq bog'langan NAD + NADH ga kamayadi. Oksidlangan oraliq mahsulotdan fosfatni yo'q qilgandan so'ng, NADH gidridni substratga qaytaradi va faol joyda NAD + ni qayta tiklaydi ( Duradgor va boshqalar, 1998). 4PE ning oksidlanishi NAD + ning qattiq bog'langanligini kamaytiradi, lekin qayta oksidlanish imkoniyatini bermaydi va fermentni faolsizlantiradi. Biz yuqorida tavsiflangan yondashuv yordamida 4PE ning AroB tomonidan oksidlangan holatini aniqlay olmaymiz, chunki NADH fermentdan ajralib chiqmaydi va NAD 2 H bilan almashtirib bo'lmaydi. Biroq, bu faollik, ehtimol, fiziologik ahamiyatga ega emas, quyida tavsiflangan genetik tajribalar taklif qiladi. D ning to'ldiruvchisipdfxB ning haddan tashqari ko'payishi bilan kuchlanish aroB 4PE ni oksidlash qobiliyatiga bog'liq emas.

Ushbu natijalar D da o'sishning tiklanishini ko'rsatadipdfxB ThrB, YeaB, HisB, Php yoki PdxA (va, ehtimol, AroB) haddan tashqari ishlab chiqarilganda shtamm PdxB faolligini almashtirish bilan bog'liq emas. Shunday qilib, biz ushbu fermentlarning ortiqcha ishlab chiqarilishi PdxB yo'qligida PLP sintezlanishiga imkon beruvchi bir yoki bir nechta serendipit yo'llarni osonlashtirishi mumkinligini o'rganib chiqdik.

PLP sintez yo'lida boshqa genlar mavjud bo'lmagan shtammlarni to'ldirishga qaratilgan sa'y-harakatlar natijasida uchta tasodifiy yo'l aniqlanadi.

PLP sintez yo'lida boshqa genlar mavjud bo'lmagan shtammlardan foydalangan holda ko'p nusxali bostirish tajribalari 1-jadvalda sanab o'tilgan fermentlar tomonidan osonlashtirilgan serendipit yo'llar normal PLP sintezi yo'liga kirishini aniqlash uchun o'tkazildi. Umuman olganda, kesishish nuqtasining yuqori oqimida fermenti bo'lmagan shtammlar serendipit yo'l ishlayotganda o'sishi kerak, lekin kesishish nuqtasining quyi oqimida fermenti bo'lmagan shtammlar o'smasligi kerak. Ushbu tajribalar ko'pgina shtammlar uchun oddiy edi, ammo D ning o'sish talablari bilan murakkablashdi.serCkan shtamm (bundan keyin D deb yuritiladiserC kuchlanish). SerC ham PLP, ham serin sintezi uchun talab qilinadi. Bundan tashqari, o'chirish serC ifodasini kamaytiradi aroA, xuddi shu operon tomonidan kodlangan (Duncan and Coggins, 1986). Binobarin, DserC shtammi minimal muhitda o'sishi uchun serin, piridoksin va shikimat yo'li mahsulotlarini (Lam va Winkler, 1990) qo'shishni talab qiladi. D da PLP ishlab chiqarish uchun serendipit yo'llarining qobiliyatini tekshirishserC shtammi, biz bu qo'shimchalarning barchasini piridoksindan tashqari qo'shdik. Tekshirish sifatida biz ushbu qo'shimchalarning D ni to'ldirishga ta'sirini ko'rib chiqdikpdfxB kuchlanish. Qo'shimchalar D ni to'ldirishga ta'sir qilmaydipdfxB tomonidan pdfxB, uningB, yjbQ va php (II-jadval). uningB, php va yjbQ etishmayotgan shtammlarni to'ldira olmaydi serC, pdfxA, pdxJ yoki pdxH (2-jadval), 2-yo'l 2-okso-3-gidroksi-4-fosfobutanoat (OHPB) hosil qiladi (1-rasm). Biroq, qo'shimchalar qobiliyatini inhibe qiladi thrB, haB, pdfxA va aroB D ni to'ldirish uchunpdfxB kuchlanish. Bu ta'sir faqat serin mavjudligiga bog'liq ekanligi aniqlandi (qo'shimcha jadval III). Mumkin tushuntirish shundan iboratki, bu genlarning haddan tashqari ifodalanishi bilan osonlashtirilgan yo'llar serin biosintezidagi oraliq mahsulotlardan boshlanadi, ular serin biosintezidagi birinchi bosqichni katalizlovchi SerA ning allosterik inhibisyoni tufayli serin ishtirokida yo'qoladi (Grant va boshqalar, 1996). Biroq, serin qo'shilishi pleiotrop ta'sirga ega (Hama va boshqalar, 1990, 1991 Grant va boshqalar, 1996), shuning uchun bu nuqtada biz ushbu fenotipni oddiygina turli xil serendipit yo'llarni ajratib turadigan xususiyat sifatida ishlatishni tanlaymiz.

Gen DpdfxB (JU283) DpdfxB (JU283)+qo‘shimcha. a L-Ser (1,0 mM), L-Phe (0,1 mM), L-Tyr (0,1 mM), L-Trp (0,1 mM), p-gidroksibenzoat (1 mkM), p-aminobenzoat (1 mkM) va 2,3-dihidroksibenzoat (1 mkM).
DserC (JWK0890)+qo‘shimcha. a L-Ser (1,0 mM), L-Phe (0,1 mM), L-Tyr (0,1 mM), L-Trp (0,1 mM), p-gidroksibenzoat (1 mkM), p-aminobenzoat (1 mkM) va 2,3-dihidroksibenzoat (1 mkM).
DpdfxA (JWK0051) DpdxJ (JWK2548) DpdxH (JWK1630)
pdfxB ++++ b b Koloniyalar diametri 1 mm ga 1-2 kun (++++), 3-5 kun (+++) yoki 6-8 kun (++) da yetdi. (+) 9 kundan keyin o1 mm bo'lgan koloniyalarni bildiradi.
++++
uningB ++++ ++++
yjbQ +++ +++
php ++++ ++++
thrB ++++ +
haB +++ +
pdfxA +++ ++ ++++
aroB +++ + ++
  • Deformatsiya belgilari qo'shimcha I jadvalda keltirilgan.
  • a L-Ser (1,0 mM), L-Phe (0,1 mM), L-Tyr (0,1 mM), L-Trp (0,1 mM), p-gidroksibenzoat (1 mkM), p-aminobenzoat (1 mkM) va 2,3-dihidroksibenzoat (1 mkM).
  • b Koloniyalar 1-2 kun (++++), 3-5 kun (+++) yoki 6-8 kun (++) ichida 1 mm diametrga yetdi. (+) 9 kundan keyin o1 mm bo'lgan koloniyalarni bildiradi.

haB va thrB D ni to‘ldiringpdfxB shtammi, lekin PdxA, PdxJ yoki PdxH bo'lmagan shtammlarni to'ldirmang. Ushbu ma'lumotlar shuni ko'rsatadiki, serendipit yo'l 1 normal yo'lga OHPB yoki 4-fosfohidroksitreonin (4PHT) darajasida kiradi. Noaniqlik D ning o'sishi uchun zarur bo'lgan serin tufayli yuzaga keladiserC deformatsiya D ning komplementatsiyasiga xalaqit beradipdfxB tomonidan torting haB va thrB. Shunday qilib, biz ushbu ma'lumotlardan 1-yo'lning SerC tomonidan katalizlangan reaktsiyadan oldin yoki keyin ovqatlanishini aniqlay olmaymiz. Biroq, biokimyoviy ma'lumotlar (pastga qarang) 1-yo'l 4PHT hosil qiladi.

aroB D ni to‘ldiradipdfxA shtamm, lekin etishmayotgan shtammlarni to'ldira olmaydi pdxJ yoki pdxH. Biz AroB ni PdxA bo'lmagan shtammdan tozaladik va AroBda aniqlanmaydigan 4PHT dehidrogenaza faolligi yo'qligini aniqladik (ma'lumotlar ko'rsatilmagan). Shuning uchun D ning to'ldirilishipdfxA ning haddan tashqari ifodalanishi bilan kuchlanish aroB Bu shunchaki PdxA faolligini almashtirish bilan bog'liq emas. Ushbu ma'lumotlar 3-yo'l 1-amino-propan-2-bir-3-fosfat hosil qilishini ko'rsatadi.

1-yo'lda reaktsiyalarni katalizlashi mumkin bo'lgan fermentlarni aniqlash

3-gidroksipiruvat (3HP) (Shimizu va Dempsi, 1978), glikolaldegid (Tani va Dempsi, 1973) yoki 4HT (Drewke) qo'shilishi kuzatuvlari orqali 1-yo'l uchun mumkin bo'lgan reaktsiyalar ketma-ketligi taklif etiladi. va boshqalar, 1993 ) ma'lum o'sishni qo'llab-quvvatlashi mumkin E. coli PLP yaratish qobiliyatiga ega bo'lmagan mutantlar. Izotopik etiketlash tajribalari glikolaldegiddan uglerod atomlari mavjudligini tasdiqladi (Xill va Spenser, 1973 Hill. va boshqalar, 1977), glitsin (Xill va boshqalar, 1987) va 4HT (Hill va boshqalar, 1996) va glitsindan azot atomlari (Hill va boshqalar, 1987) piridoksolga kiritilgan. Bir muncha vaqt davomida bu birikmalar PLP sintez yo'lida mumkin bo'lgan oraliq moddalar sifatida ko'rib chiqildi (Shimizu va Dempsey, 1978 Duncan and Coggins, 1986), garchi oxir-oqibat 1-rasmda ko'rsatilgan yo'l ishlab chiqilgan. Biz tasdiqladikki, bizning DpdfxB shtammi M9/glyukozada 37°C da 3HP, glikolaldegid va 4HT ishtirokida o'sadi. Bizning shtamimiz serin biosintezidagi oraliq 3PHP ishtirokida ham o'sadi (Qo'shimcha IV jadval).

Taklif etilgan 1-yo'lning birinchi bosqichi 3PHP ning fosforillanishidir (3-rasm). E. coli Ushbu reaktsiyani katalizlashi mumkin bo'lgan 75 ta ma'lum yoki taxmin qilingan fosfatazani o'z ichiga oladi. YeaB, bashorat qilingan NUDIX gidrolazasining haddan tashqari ishlab chiqarilishi D ning o'sishini tiklaydi, degan kuzatish qiziqarli imkoniyatni taklif qildi.pdfxB kuchlanish. NUDIX gidrolazalari odatda organik pirofosfatlarning gidrolizini katalizlaydi (McLennan, 2006) va shuning uchun past darajadagi fosfataza faolligiga ega bo'lishi mumkin. Biz YeaB ni eruvchan oqsilni olish uchun maltoza bog'lovchi oqsil (MalE) bilan sintez sifatida ifodaladik. MalE-YeaB termoyadroviy oqsili haqiqatan ham 3PHP fosfataza faolligiga ega (III-jadval). Bizda 3PHP cheklanganligi sababli aniqligini aniqlay olmadik KM 3PHP uchun ferment 2 mM 3PHP da toʻyingan koʻrinadi, degan xulosaga keldik. KM 3PHP uchun <2 mM bo'lishi kerak. 3PHP fosfataza faolligi MalE-dan ko'ra YeaB ga bog'liq edi, chunki MalE-LacZ termoyadroviy oqsili faollik ko'rsatmadi. Biz 3PHP ni 3HP ga aylantirish uchun ferment bo'lmagan tezligi 7,2 × 10 -10 M -1 s -1 ekanligini ta'kidlaymiz. Shunday qilib, YeaB samarasiz ferment bo'lsa ham, u stavkani fon tezligidan 4 × 10 7 marta oshiradi.

Protein Substrat kmushuk (s −1 ) KM (mM) kmushuk/KM (M −1 s −1 )
MalE–YeaB a a YeaB eruvchanligini yaxshilash uchun maltoza bog‘lovchi oqsil (MalE) bilan termoyadroviy oqsil sifatida ifodalangan.
3PHP 5.7±0.7 × 10 −5 <2 >0,028±0,003
Dxs Piruvat+ D -GAP b b (Kuzuyama va boshqalar, 2000).
345 0,096 (piruvat) 3.6 × 10 6
0,24 (D -GAP) 1.4 × 10 6
3 HP 0.026±0.001 0.050±0.008 5.2±0.4 × 10 2
SucA 3 HP 0.0139±0.0003 0.72±0.07 19.3±1.9
LtaE L -allo- treonin 3.2±0.2 0.052±0.004 6.2±0.5 × 10 4
L -treonin 1.1±0.1 4.0±0.2 2.8±0.2 × 10 2
4HT 1.44±0.03 0.027±0.002 5.3±0.4 × 10 4
ThrB Gomoserin 46±4 0.12±0.02 3.8±0.7 × 10 5
4HT 9.7±0.8 2.0±0.1 4.8±0.6 × 10 3
  • 1-yo'lda ishtirok etuvchi substratlar qalin shrift bilan ta'kidlangan.
  • a YeaB eruvchanligini yaxshilash uchun maltoza bog'lovchi oqsil (MalE) bilan termoyadroviy oqsil sifatida ifodalangan.
  • b (Kuzuyama va boshqalar, 2000).

YeaB 3PHP ni 3HP ga aylantirish uchun samarasiz katalizator bo'lsa-da, bu bosqichdagi oqim PLP ta'minoti uchun etarli bo'lishi kerak. MalB-YeaB ning sitoplazmik kontsentratsiyasi ~45 mkM (2,2 g nam hujayradan 4,7 mg tozalangan oqsil ishlab chiqarishga asoslangan). YeaB 1 mM 3PHP da toʻyingan koʻrinadi, bu uning sitoplazmatik kontsentratsiyasi uchun uning bevosita kashshofi 3-fosfogliseratning kontsentratsiyasiga asoslangan, u 1,5 mM da mavjud. E. coli glyukoza asosida o'sadi (Bennett va boshqalar, 2009). Shunday qilib, oqim taxminan bo'ladi kmushuk [E]=(4,8 × 10 −5 s −1 ) (45 mkM)=7,8 mkM h −1 ( Bennett va boshqalar, 2009). Qo'shimcha ma'lumotlarda biz D ni ko'rsatadigan hisob-kitobni tasvirlaymizpdfxB MalB–YeaB haddan tashqari ifodalanganda shtamm PLP ni ~5,5 mM h -1 tezlikda sintez qiladi. Thus, the rate of conversion of 3PHP to 3HP by YeaB is comparable to the rate of synthesis of PLP.

We note that there is precedence for the ability of such an inefficient enzyme to supply sufficient flux to replace a critical metabolic enzyme. Patrick and Matsumura (2008) have shown that overexpression of a promiscuous enzyme (I198V glutamine 5-phosphoribosyl-1-pyrophosphate amidotransferase (PurF)) substitutes for a lack of phosphoribosylanthranilate isomerase (TrpF), which is required for tryptophan synthesis. The kcat/KM for the promiscuous TrpF activity of I198V PurF is estimated to be 0.012 M −1 s −1 ( Patrick and Matsumura, 2008 ) the kcat/KM for the 3PHP phosphatase activity of YeaB (>0.024 M −1 s −1 ) is greater than this.

If YeaB is the only enzyme that converts 3PHP to 3HP, a ΔpdxB ΔyeaB strain should not grow on minimal medium supplemented with 3PHP. However, this strain does grow slowly, producing colonies <1 mm in diameter after 9 days, on M9/glucose (Supplementary Table IV). These results suggest that at least one other enzyme can generate 3HP when 3PHP is present at high levels. As the ΔpdxB strain grows after 4 days when YeaB is overproduced, we conclude that overproduction of YeaB is the most efficient way to siphon material from the serine biosynthesis pathway into pathway 1, but YeaB is not the only enzyme that can produce 3HP from 3PHP.

3HP is proposed to be converted to glycolaldehyde in pathway 1. This conversion might occur by either of two routes. Direct decarboxylation of α-keto acids is catalyzed by enzymes that contain thiamine pyrophosphate (TPP). Alternatively, glycolaldehyde might be formed by initial isomerization of 3HP to the β-keto acid tartronate semialdehyde, followed by decarboxylation of tartronate semialdehyde. To search for enzymes in E. coli that catalyze decarboxylation of 3HP by either route, we attempted to measure acceleration of glycolaldehyde production from 3HP by crude extracts of E. coli K-12 BW25113 in the presence of NADH and alcohol dehydrogenase, which converts glycolaldehyde to ethylene glycol ( Barngrover va boshqalar, 1981 ). We were unable to detect any acceleration of the conversion of 3HP to glycolaldehyde. As we could not rule out inefficient catalysis that fails to rise significantly above the background rate, we examined the catalytic abilities of specific candidate enzymes that might convert 3HP to glycolaldehyde.

E. coli has 12 enzymes that are known or predicted to utilize TPP. We purified 10 of these: 1-deoxyxylulose-5-phosphate synthase (Dxs), pyruvate oxidase, transketolase A, transketolase B, glyoxalate carboligase and the predicted oxalyl CoA decarboxylase, as well as the TPP-dependent catalytic components of the multisubunit enzymes pyruvate dehydrogenase, α-ketoglutarate dehydrogenase and both isozymes of acetohydroxy acid synthase. YdbK and 2-succinyl-6-hydroxy-2,4-cyclohexadiene-1-carboxylate synthase (MenD) could not be expressed in soluble form. Only Dxs and SucA, the TPP-dependent catalytic component of the α-ketoglutarate dehydrogenase complex, had detectable 3HP decarboxylase activity (Table III).

To determine whether Dxs, SucA, YdbK or MenD is required for operation of pathway 1, we constructed double-knockout strains lacking pdxB and each of the genes encoding these enzymes. We also constructed a ΔpdxB Δdxs ΔsucA strain to determine whether glycolaldehyde formation is due to a combination of the activities of Dxs and SucA. Each of these strains grew on M9/glucose when YeaB or ThrB was overproduced at 37°C (data not shown). These results suggest that TPP-containing enzymes are not responsible for the formation of glycolaldehyde in pathway 1.

The E. coli genome encodes a hydroxypyruvate isomerase (Hyi) that converts 3HP to tartronate semialdehyde. This enzyme is expressed only when cells are grown on glyoxylate ( Ashiuchi and Misono, 1999 ). We ruled out the possibility that Hyi has a role in pathway 1 by constructing a ΔpdxB Δhyi strain. This strain grows on glucose when either yeaB yoki thrB is overexpressed or when 3HP is supplied in the medium (data not shown).

We next considered the possibility that conversion of 3HP to glycolaldehyde in pathway 1 might be non-enzymatic. We measured the rate of formation of glycolaldehyde from 3HP by 1 H-NMR. 3HP is converted to glycolaldehyde, glycolic acid and erythrulose (by reaction with glycolaldehyde, followed by decarboxylation) (Figure 4). A control experiment showed that glycolaldehyde decomposed under these conditions to unidentified products with a first-order rate constant of 0.0062±0.0003 h −1 . The kinetic model shown below was used for numerical simulation of the rate constants for appearance of glycolaldehyde (k1=0.0153±0.0003 h −1 ), erythrulose (k2=0.0092±0.0005 mM −1 h −1 ) and glycolic acid (k3=0.0069±0.0003 h −1 ), assuming that k4=0.0062 h −1 , using DynaFit ( Kuzmič, 1996 ). Notably, addition of 0.1 mM TPP had no effect on the rate of formation of glycolaldehyde. Thus, glycolaldehyde formation must occur through the initial formation of tartronate semialdehyde. Decarboxylation of β-keto acids such as tartronate semialdehyde can be accelerated by metal ions ( Hedrick and Sallach, 1961 ). However, addition of Mg ++ or Ca ++ (1 mM) had no effect on the rate of glycolaldehyde formation. Addition of Cu ++ , Co ++ , Ni ++ , Zn ++ or Mn ++ (1 mM) actually decreased the rate of glycolaldehyde formation, while concomitantly increasing the rate of glycolate formation.

The observed rate of conversion of 3HP to glycolaldehyde appears to be sufficient to supply a significant portion of the glycolaldehyde needed for the production of PLP. We estimate that the ΔpdxB strain synthesizes PLP at a rate of 5.5 μM h −1 when yeaB is overexpressed (Supplementary information). We estimated above that the rate of production of 3HP from 3PHP should be ∼8 μM h −1 . At steady state, the rates of production and depletion of 3HP are balanced. As formation of erythrulose is negligible unless the glycolaldehyde concentration exceeds 1 mM, which is unlikely, we can calculate the steady state concentration of 3HP by assuming that the rates of formation and disappearance of 3HP are equal at the steady state. Using the relationship 8 μM h −1 =(k1+k3) [3HP]ss, we calculate that [3HP]ss is ∼360 μM. If the concentration of 3HP is 360 μM, the rate of formation of glycolaldehyde will be 5.5 μM h −1 . Glycolaldehyde will also be formed during conversion of 7,8-dihydro- D -neopterin to 6-hydroxymethyl-7,8-dihydropterin by FolB in the folate biosynthesis pathway. If folate is synthesized at a rate comparable with that of PLP, this reaction could supply glycolaldehyde at ∼5 μM h −1 . Although the amount of glycolaldehyde produced by FolB is clearly not sufficient to supply pathway 1 in the ΔpdxB strain, the combined generation of glycolaldehyde from 3HP and 7,8-dihydro- D -neopterin may well be sufficient when yeaB is overexpressed, even if some glycolaldehyde is lost by diffusion through the membrane.

The next step in pathway 1 is formation of 4HT, which is not a recognized metabolite in E. coli. 4HT can be formed by aldol condensation of glycine and glycolaldehyde ( Dempsey, 1987 ). The E. coli genome encodes an enzyme annotated as low-specificity threonine aldolase (LtaE). The physiological function of this enzyme is unknown it cleaves L -allo-threonine most efficiently among known substrates ( Liu va boshqalar, 1998 ), but L -allo-threonine is also not a recognized metabolite in E. coli. We assayed the rate of cleavage of L -allo-threonine, L -threonine and 4HT by LtaE (Table III). (The aldolase reaction is the reverse of the aldol condensation proposed in the latent pathway, but aldol condensation reactions are generally reversible.) Notably, cleavage of 4HT is nearly as efficient as cleavage of L -allo-threonine. We examined the products formed by the LtaE-catalyzed aldol condensation of glycolaldehyde and glycine by NMR (Supplementary Figure 2). The reaction forms 4HT and 4-hydroxy- L -allo-threonine (4-allo-HT) in a ratio of 1.0:0.7. (LtaE does not cleave the D -isomers of threonine and allo-threonine ( Liu va boshqalar, 1998 ), so we do not expect formation of 4-hydroxy- D -threonine and 4-hydroxy- D -allo-threonine.) These data show that LtaE catalyzes the aldol condensation of glycine and glycolaldehyde, but the stereochemistry of the reaction is not well controlled, and a substantial amount of the allo-isomer is produced. However, this does not necessarily divert material from pathway 1. As the aldol condensation is reversible and the equilibrium favors the substrates, material sequestered in 4-allo-HT will ultimately be drawn into the pathway as 4HT is converted to downstream products, as long as 4-allo-HT is not converted to any other products. The most obvious enzyme that might utilize 4-allo-HT is ThrB, which phosphorylates 4HT. However, ThrB does not phosphorylate 4-allo-HT (Supplementary Figure 3).

We confirmed that LtaE is responsible for the formation of 4HT by pathway 1 in vivo by constructing a ΔpdxB ΔltaE strain. This strain can grow on M9/glucose in the presence of pyridoxine, but does not grow when intermediates upstream of the proposed aldol condensation (3PHP, 3HP or glycolaldehyde) are supplied (Supplementary Table IV). It can, however, grow when 4HT is supplied. Furthermore, overproduction of YeaB does not complement the ΔpdxB ΔltaE strain (data not shown), confirming that LtaE and YeaB participate in the same pathway.

The last step in the proposed pathway 1 is phosphorylation of 4HT. We suspected that homoserine kinase (ThrB) might catalyze this reaction since it phosphorylates homoserine, which is structurally similar to 4HT. Furthermore, ThrB was previously shown to be required for the growth of a ΔpdxB strain on glucose at 37°C in the presence of glycolaldehyde or 4HT ( Zhao and Winkler, 1996a ). ThrB does indeed turn over 4HT, but with an efficiency nearly two orders of magnitude lower than that for homoserine (Table III). We confirmed that ThrB is responsible for phosphorylation of 4HT in vivo by constructing a ΔpdxB ΔthrB strain. This strain can grow on glucose in the presence of pyridoxine, but not when intermediates upstream of the proposed phosphorylation reaction are supplied (Supplementary Table IV).

Confirmation that pathway 1 diverts material from serine biosynthesis

We carried out two experiments to test the proposition that YeaB diverts 3PHP from serine biosynthesis into serendipitous pathway 1 in vivo. First, we generated a ΔpdxB ΔserA strain that cannot make 3PHP. Pathway 1 should not operate in this strain even if yeaB is overexpressed. As predicted, the ΔpdxB ΔserA strain cannot grow on M9/glucose supplemented with serine when yeaB is overexpressed, although it can grow when pdxB is overexpressed (see Table IV). Because addition of serine can cause inhibition of ThrA ( Hama va boshqalar, 1990 , 1991 ) and thus impair synthesis of threonine and methionine, we also assessed the growth of the ΔpdxB ΔserA strain on M9/glucose supplemented with both serine and homoserine. Under these conditions as well, the ΔpdxB ΔserA strain cannot grow when yeaB is overexpressed. These results are consistent with the hypothesis that the lack of growth is due to a lack of 3PHP, but do not rule out an additional unanticipated inhibitory effect of serine on pathway 1. To address this possibility, we constructed a ΔpdxB strain carrying an allele of serA that encodes a serine-insensitive enzyme (designated serA′) ( Grant va boshqalar, 2005 ). As SerA′ is insensitive to feedback inhibition by serine, addition of serine does not shut off flux through the serine biosynthesis pathway. Thus, we predict that this strain should grow on M9/glucose supplemented with serine and homoserine when yeaB is overexpressed if there is no other inhibitory effect of serine. Indeed, complementation of the ΔpdxB serA′ strain by yeaB is unaffected by the addition of serine and homoserine (Table IV). Together with the demonstration that YeaB catalyzes conversion of 3PHP to 3HP in vitro, these results provide strong support for the proposition that serendipitous pathway 1 diverts 3PHP from the serine biosynthesis pathway.

Supplements a a L-serine concentration was 1mM and L-homoserine concentration was 50 μM.
Strain and overexpressed gene
ΔpdxB (JU283) ΔpdxB ΔserA (JU425) ΔpdxB serA’ (JU430)
pdxB yeaB pdxB yeaB pdxB yeaB
Yo'q ++++ b b Colonies reached 1mm diameter in 1–2 days (++++), 3–5 days (+++) or 6–8 days (++). (+) indicates colonies that were o1mm after 9 days.
+++ ++++ ++
L -serine ++++ + +++ +++
L -serine+ L -homoserine ++++ + +++ ++++ ++
  • Strain designations are defined in Supplementary Table 1.
  • a L-serine concentration was 1mM and L-homoserine concentration was 50 μM.
  • b Colonies reached 1mm diameter in 1–2 days (++++), 3–5 days (+++) or 6–8 days (++). (+) indicates colonies that were o1mm after 9 days.

Complementation by PdxA depends on pathway 1

Our observation that overproduction of PdxA complements the ΔpdxB strain is curious. As described above, PdxA oxidizes 4PE at C-3 and therefore cannot substitute for PdxB. Overexpression of pdxA does not restore growth of the ΔpdxB ΔltaE strain on M9/glucose (data not shown), suggesting that complementation by pdxA depends on the presence of LtaE and therefore on pathway 1. We suspect that overproduction of PdxA increases the rate of consumption of 4PHT formed by the inefficient serendipitous pathway, effectively pulling material through the pathway by catalyzing the thermodynamically favorable conversion of 4PHT to 1-amino-propan-2-one-3-phosphate.

Pathway 1 can be elevated to physiological significance at 37°C by spontaneous mutations

Pathway 1 appears to operate even without the overproduction of YeaB or ThrB during growth on minimal medium at 30°C. Previous workers reported that a ΔpdxB strain grows slowly on glucose with mucoid morphology at 30°C, but not at 37°C ( Lam and Winkler, 1990 ). Our ΔpdxB strain, which differs somewhat from the strain used by Lam and Winkler (1990 ), also shows slow growth on M9/glucose at 30°C. As neither the ΔpdxB ΔltaE strain nor the ΔpdxB ΔthrB strain grows at 30°C (data not shown), pathway 1 can apparently generate sufficient PLP for slow growth at 30°C in the absence of PdxB.

Pseudorevertants of the ΔpdxB strain that grow on M9/glucose at 37°C arise frequently. We have deleted ltaE in one of these pseudorevertants (JK19). Deletion of ltaE slows growth at 37°C substantially small mucoid colonies can be seen after incubation for 5 days, whereas colonies of the pseudorevertant JK19 are much larger (Figure 5). (The variation in colony size seen in Figure 5A was observed repeatedly, even when plates were streaked from a single colony, suggesting that colony phenotype is influenced by some type of phase variation.) These results suggest that pathway 1 contributes significantly to PLP synthesis in pseudorevertant JK19, although is apparently not the only source of PLP. Efforts to identify the mutations responsible for the newly acquired ability of this pseudorevertant to grow at 37°C on M9/glucose are in progress.


Johndoeanonymous

Ampicillin Resistance in E. coli During DNA Transformation

The lab “DNA Transformation-Ampicillin Resistance” tested four different variables (“B1 Amp,” “B1 No Amp,” “B2 Amp,” and “B2 No Amp) , to show how a plasmid with a resistance gene to the antibiotic ampicillin can be used to place the resistance gene into an able strand of bacteria. Both microcentrifuge tubes (labeled “B1” and “B2”) contained the E. coli bacteria and calcium chloride solution. Microcentrifuge tube “B1” received a drop of a solution, which has a gene that is resistant to ampicillin. In the results, “B2 Amp” had no bacterial growth, “B1 Amp” had growth in the form of multiple small colonies, and “B2 No Amp” and “B1 No Amp” both had regular bacterial growth. These results explain how DNA transformation works, and how it can be used in medical science.

Ampicillin: A semisynthetic penicillin used to treat various infections.

Cell Suspension: Cells in culture in moving or shaking liquid medium, often used to describe suspension cultures of single cells and cell aggregates.

E. coli: A bacillus Escherichia coli a bacillus normally found in the human gastrointestinal tract and existing as numerous strains, some of which are responsible for diarrheal diseases. Other strains have been used experimentally in molecular biology.

Resistant: impervious to the action of corrosive substances.

Nonresistant: Not resistant, especially to a disease or an environmental factor, such as heat or moisture.

Transformation: genetic modification of a bacterium by incorporation of free DNA from another ruptured bacterial cell compare.

Agar: A moist support medium used to grow bacteria.

DNA transformation refers to “the uptake and expression of foreign DNA in a living cell. Originally defined as an inherited alteration of the phenotype of the transformed cell, see stable and transient”. This meaning taking DNA from one cell, and introducing it into another cell, that may give different phenotypic (physical) outcomes. In this lab, a plasmid DNA was given to the microcentrifuge tube “B1” which gave it a gene that is resistant to ampicillin. This means that the results should show “B1” having some sort of bacterial growth on the petri dishes. It is apparent that the transformation occurred if the plate labeled “B1 Amp” had growth in the form of colonies, which means that some of the bacterial DNA took up the plasmid DNA that was resistant to ampicillin, therefor making some of the bacteria resistant as well. The bacteria on the “B1” plate will show DNA transformation because the plasmid DNA will give the bacteria DNA a gene for resistance to the ampicillin.

To complete this experiment, the following supplies will be needed: four petri dishes containing agar, two microcentrifuge tubes labeled B1 and B2, four sterile toothpicks, four sterile paperclips, ice, two pipettes, and access to an incubator (needed to heat the petri dishes to 37°C for 24 hours).

Step 1: Use a sterile toothpick to add a very small (about the size of this 0) colony of E. coli into two microcentrifuge tubes (the ones labeled B1 and B2) along with two drops of calcium chloride (which helps the bacterial DNA accept the plasmid DNA). Gently mix the E. coli with the calcium chloride solution until it appears milky. Firmly close both microcentrifuge tubes and safely dispose of the toothpicks that were used into a container to be destroyed(not the trash).

Step 2: Place the microcentrifuge tubes into a tub of ice and wait approximately fifteen minutes. (Do not freeze the tubes, the chilled calcium chloride within the tubes are the correct conditions for DNA uptake.)

Step 1: Gently finger flick the microcentrifuge tubes to suspend the cells.

Step 2: Open the tube labeled “B1” and use the sterile pipette to place one drop of solution from the “P” tube into the “B1” tube. DO NOT add anything to the “B2” tube. (The plasmid DNA from the “P” tube that was added to “B1” has a gene resistance to the antibiotic ampicillin.)

Step 3: Place both tubes in ice again for fifteen minutes. (The cells are kept cold to prevent them from growing within the tubes while the plasmids are being absorbed.)

Step 4: Take both tubes out of the ice and put them into a 42°C water bath immediately for 90 seconds. (The temperature change causes the cells to readily absorb the plasmid DNA, that was placed into tube B1.)

Step 5: Use a sterile pipette to put five drops of a sterile nutrient broth into both tube “B1” and “B2”. Close the tubes tightly, and mix the solutions by tipping them gently. (The bacteria are now provided nutrients to help them recover from the calcium chloride and heatshock treatments.)

Step 6: Label the bottom of the four petri dishes with “B1 No Amp,” “B2 No Amp,” “B1 Amp,” and “B2 Amp.” Also, be sure to properly identify the person who is doing the experiment.

Step 7: Using another sterile pipette, place three drops of cell suspension from the tube labeled “B1” onto the two petri dishes labeled “B1”. Then use a sterile paperclip to gently and evenly spread the suspension across the agar, be sure not to touch the part of the paperclip that comes into contact with the agar, so as not to compromise the containment of the E. coli. Do the same thing for the cell suspension in tube “B2,” onto the petri dishes labeled “B2”.

Step 8: Incubate the four petri dishes upside down for at least 24 hours at 37°C.

Step 9: Look over the results of the transformation on each of the four petri dishes.

The results of the lab are as follows:

B2 Amp B1 Amp B1 No Amp B2 No Amp

“B2 Amp” (Amp standing for ampicillin) shows no bacterial growth. This is because the bacteria are killed because they had no resistance to the antibiotic ampicillin. The ampicillin that was introduced to the bacteria killed every cell leaving none to reproduce and grow on the petri dish. The plate labeled “B1 Amp” showed some bacterial growth in the form of many little clusters, called colonies. The growth that is shown is from the bacteria that took up plasmids (that were added in step 2), and then became resistant to ampicillin. Since not all of the bacterium were resistant, only some grew, showing the colonies you see in the above photo labeled “B1 Amp”. On both plates “B1 No Amp” and “B2 No Amp” the bacteria grew normally. The antibiotic was not present therefore both the resistant and nonresistant bacteria grew normally.

The result of the experiment did prove that the hypothesis was correct. The procedure went as expected, and no unexpected results were encountered. The data that was collected from the results explain how DNA transformation works and supports the hypothesis. Some of variables that were in the experiment could have produced different results, are incorrect temperatures, inaccurate mixing methods, contamination of the petri dishes or the tools (such as the toothpicks, paperclips, or pipettes), or a breach of containment of the E. coli bacteria. The results directly support DNA transformation in that the plate labeled “B1 Amp” grew in colonies, stating that some of the bacterial DNA had the resistance gene and some did not, supporting the hypothesis in that DNA transformation gave the bacteria the ability to grow because of the gene in the plasmid DNA. The experiment went successfully and proves the hypothesis to be correct.

In this lab, DNA transformation was proved. The results depict a transformation occurring with the bacteria DNA and the plasma DNA where the bacteria DNA receives a gene from the plasmid DNA that is resistant to ampicillin. The plate “B1 Amp” shows growth in colonies where some of the bacteria was resistant and some was not. The bacteria that was not resistant, simply did not receive the resistant gene through transformation.

I would like to thank the Iowa State University for the clearly outlined lab instructions and equipment. I would also like to recognize my father, Alex Hefflefinger, for reading over this lab report and helping me along the way.


E. coli Strain ALS1059

From the laboratory of Mark A. Eiteman, PhD, University of Georgia.

Product Type: Bacteria Name: ALS1059 Cell Type: Bacteria Organism: Escherichia coli Strain: ALS1059 Genotype: Hfr zbi::Tn10 poxB1 &Delta(aceEF) rpsLpps-4 pfl-1 ldhA::Kan arcA726::(FRT) atpFH::Cam Antibiotic Resistance: Kanamycin, chloramphenicol Growth Conditions: TYA medium, 10g/L tryptone, 5g/L NaCl, 1g/L yeast extract, 1.36 g/L sodium acetate trihydrate Na(CH3COO)?3H2O, pH adjusted to 7.0, aerobic 37C Format: Lyophilized culture Storage: Room temperature as lyophilized culture Shipped: Ambient temperature

Protocol Notes

  1. Inject 1 mL of sterile DI water into vial and gently mix.
  2. Use small portions of vial (liquid) contents to inoculate a sterile, liquid culture to an initial OD of 0.03-0.1. For this liquid culture, use the specified growth medium with or without an antibiotic as appropriate.
  3. When culture has visibly grown in liquid medium, plate on solid (Agar) growth medium and incubate at 37C. Maintain strain.
Strain Phenotype
ALS974 Accumulates lactic acid
ALS929 Accumulates pyruvic acid
ALS1392 Does not metabolize arabinose, glucose nor xylose
ALS1391 Does not metabolize arabinose nor xylose
ALS1371 Does not metabolize arabinose nor glucose
ALS1370 Does not metabolize xylose nor glucose
ALS1074 Does not metabolize xylose but can accumulate lactic acid from glucose
ALS1073 Does metabolize glucose but can accumulate lactic acid from xylose
ALS1060 Does not metabolize xylose nor glucose
ALS1059 Accumulates pyruvic acid
ALS1058 Does not metabolize glucose
ALS1048 Does not metabolize glucose
ALS1038 Does not metabolize xylose
ALS1054 Accumulates pyruvic acid
ALS1122 Does not metabolize xylose nor glucose
  1. Y. Zhu, M. A. Eiteman, R. Altman, E. Altman, “High glycolytic flux improves pyruvate production by a metabolically engineered Escherichia coli strain,” Applied and Environmental Microbiology, 74(21):6649-6655 (2008) doi: 10.1128/AEM.01610-08
  2. A. Tomar, M. A. Eiteman, E. Altman, “The effect of acetate pathway mutations on the production of pyruvate in Escherichia coli,” Applied Microbiology and Biotechnology, 62, 76-82 (2003) doi: 10.1007/s00253-003-1234-6
  3. G. N. Vemuri, M. A. Eiteman, E. Altman, "Succinate production in dual-phase Escherichia coli fermentations depends on the time of transition from aerobic to anaerobic conditions," Journal of Industrial Microbiology and Biotechnology, 28(6), 325-332 (2002) doi:10.1038/sj.jim.7000250
  4. Y. Zhu, M. A. Eiteman, S. A. Lee, E. Altman, “Conversion of glycerol to pyruvate by Escherichia coli using acetate- and acetate/glucose-limited fed-batch processes,” Journal of Industrial Microbiology and Biotechnology, 37:307-312 (2010) doi: 10.10007/s10295-009-0675-z
  5. Y. Zhu, M. A. Eiteman, E. Altman, “Indirect monitoring of acetate exhaustion and cell recycle improve lactate production by non-growing Escherichia coli,” Biotechnology Letters, 30:1943-1946 (2008) doi: 10.1007/s10529-008-9775-5
  6. US Patent Numbers 7,749,740, 8,278,076 and 8,652,825.

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