Endogenous metabolism of Nocardia corallina : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Biochemistry at Massey University

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ENDOGENOUS IvliITABOLI.Si'1 OF NOCARDIA CORALLINA

A thesiR presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Biochemistry

at Massey University.

John Gray Robertson

1968

,_ .

ACKNO'hLEDG EHEN T S

I wish to thank Profes�or R.D. Batt for his supervision and for creating the opportunity for carrying out this research.

I also wish to thank:-

The Department of Scientific and Industrial Research for granting me leave on full salary.

Nrs Pamela Lyttleton (!,assey University), Mr. le 1. 'vVilliamson (Plant �hemistry Division, D.S.I.R.) and Mr. A.S. Craig for preparing the electron micrographs.

Dr. R. Hodges (tviassey University) for mass spectrometric analyses.

Dr. N.A. Doughty (Canterbury University) for deriving the formulae for deter�ining cell viability.

Dr. Ruth Gordon (Rutgers, U.S.A.) for confirming the identity of N. corallina.

Members of the staff of Plant Chemistry Division, especially

Dr. R.T.J. Clarke, and Mr. T.D. Thomas (Dairy Research Institute) for

'-

advice and helpful discussions.

i-jiss Karen Sole for technical assistanc e.

Miss Margaret Soulsby for photographs, Miss Wendy Humphries for assistance with statistical analyses and especially Miss Cynthia Owen, the D.S.I.R. librarian.

Mrs Gail Peterson for typing, and Dr. P.J. Peterson for checking the final script.

Finally I wish to express my gratitude to my wife for her patience and assistance throuGhout the course of the study.

i.

ABSTRACT

The endogenous metabolism of the soil microorganism N. corallina

has been studied with special reference to physiological _and structural changes in starv^{ation conditi}ons.

When N . corallina was grown on the surface of nutrient agar growth was characterised by the development of branched hyphae 8-12 f. ^long, while in liGuid medium bacilli approximately � f. ^{long were pro}d^uced.

Clumping of cells in liquid medium ,Jas reduced by growinG the organism in cleated flasks on a ^{rotary shaker.}

For studies of endogenous metabolism and survival, suspensions of

N . ^{corallina w}e^{re prepared} from cultures harvested at full growth and resusDended in phosphate buffer containing magnesium ions. Analyses of total and viable cell counts were affected by cluEtering of cells and

detergents were used to reduce the size of clusters. The cell viability,

was estimated using formulae, from the cluster viability and cluster size distribution which were determined using the slide culture technique.

The v^{iability of st}arved cells fell from 99% to 90% over a period of

7 ^days and subsequently to 50% after a further period of 13 days.

A ^{rise in total} cell count of 1;.% was recorded over a ₅ day period

of starvation. During the first 48 ^hr. of starvation the bacterial dry wt. fell by 30-ttO%, and at the same time the initial QO ^of

2

approximately 10 fell to e value of appro�imately one. T:"e initial fall in dry _wt. was due largely to a decrease in the level of cell polysacchari2e from 25% to

5-10%

^of the cell dry wt. Following this drop in polysaccharide, ammonia was released at a relatively constant

rat e and at ^{the same time t}h^{ere was} a fall in the level of cell protein.

ii .

Ther e was a fall in the l evels o f intrac ellular free ni t rog enous c ompound s at th e ons e t o f st arvation but no correE�onding rel eas� o f these sub stances into the sup ernatant occ urred . Ribonu c l ei c acid appear ed to b e b roken do�n during starvation. The cont ribution of the indi vidual c ell fractions to the t o t al fall in dry wt . on prolonged starvat ion were; polysaccharid e , ltO%; pro t ein, 25%; RNA, 6% and total fatty acid s , 'J% . The d ecrease in viability of starved o rganisms coul d not be directly c or relat ed with the utilization o f any of th ese c ellul ar c omponent s.

Hydrolysis o f t h e total unbound lipid which constitut ed 15% o f t h e c ell dry wt . yi elded t rehalos e , manno se , inosi t ol and glyc erol a s t h e wat er solub l e c omponents. Tri glyc erides were isolat ed from the total

lipids by sili c ic acid column and thin-layer chromatography . Evi d enc e from thin-l ayer chromatograms indicat ed that t ri glyc erides were not major c onsti tuents o f the to t al lipid s . Incub ation o f N . corallina wi t h

u-14C-palmit at e r esul t e d in a l arg e propo rtion o f t h e radio activity being inc orporated into t h e t ri glyc eri d e s .

Total f atty acid s const i tu t ed approximat ely 12% o f th e cell dry wt.

and c on t ained ³ frac tions: (a) C10-C20 fat ty acids , ( b) nocardic acids and ( c ) a minor unid enti fi ed fraction whi c h was mo r e polar than the nocardic acids. Trimethylsilyl d eri vati ves o f the me thyl noc ardat e s w e r e separa t ed by gas c hromat ography on t h e basis o f mol e c ul ar wt . Mass spectromet ry o f methyl n o c ardates and TMS d eri vati ves , indicat ed t hat the s t ructures of the nocardic acids could vary in ³ ways : in c arbon numb er from"C}CC48' in d e gr ee o f saturation ( saturat ed , mono­

unsaturat ed and diunsaturat ed acids occurred ) , and in th eir i som eri c configurations .

iii .

Studi es on N. corallina using both ligh t and el ec t ron micro scopy showed clearly the pl eomorphic natur e o f the o rganism. Par t s o f the c ell surface were covered with fibrous ma t erial which app eared to be

di stinct from the c ell wall . C ell division occurred by the fo rmati on of septa which were g en erally associ a t ed wi th ext ensi ve cyt oplasmic

m embrane syst ems . Polyphospha t e granules , ribo somes and ei ther poly- ribosom es o r glycogen granul es app e ared in t h e cytoplasm during gr?wth. U s e o f t h e freeze- e tch t echnique illustrat ed the granular nature of t h e cytoplasm , cell wal l and membrane surfaces . S tarva tion o f the cells

app eared to be associated wit h (a) a thickening of the c ell wall , (b) an increas e o f t h e amount o f figrous mat erial per cell , Cc) an

increase in the si z e o f the polypbosph a t e granules and (d) the di sapp earanc e o f large cyt oplasmic granul es .

Possibl e implications o f t h e presen t findings hav e be en con sid ered in relati on t o previous inves tigations wi th t his organi sm and t o s t ud i es o f t h e endo genous metabolism and survival chC'.rac t eri s tics o f ot h er

microbial speci es.

Chapt,er 1

Chanter 2

Chapter 1

CONTENTS

SECTION I

INTRODUC'rION

Endogenous Meta'coli srn of IV1icroorganisms wi th Speciel Reference to Nocardia corallina

The Genus Nocardia

The Species N. corallina

The Chemical Composition of Nocardia Species Endogenous Metabolism and bActerial S urvival

(a) The effect of the environ�ent on the chemical composition of mic roorganisms (b) Studies relating endogenous metabolism to

survival caracity

(c) Factors affecting the survival of

microorganisms

(d) The functional importance of endogenous meta�olism

THE _AIN OF THE PRESENT INVESTIGATION

!vI,STHODS

Organism

SECTION _II EXP�RIl"tENT AL

Bacteriological Procedures

Growth in Liquid Medium Special Growth Flasks

rage 1

1

Lt

6 9 1 1 12

17

19

2 1

23 23 23 23 2't

Chapter 1 Preparation and Incubation of Cell Suspensions Total Cell Counts

Viable Counts

Formulae for Estimating Cell Viability from Cluster Viability

Photography of Cells on Agar Slides

Analytical Methods Spectrophotometric Equipment

Dry Weight

Respiratory Quotients

Oxygen Partial Pressure Estimations Total Unbound Lipids

'rriglyc erides

Ester determination Glycerol determination Alkaline hydrolysis Total Fatty Acids

Alkaline hydrolysis of total cells Acid hydrolysis of total cells Estimation by weight

Estimation by chromate oxidation Total Nitrogen

Protein

C�llular protein Protein in solution Amino Acids

Estimation

Extraction of intracellular amino acids

page

2'5 26 26

28 2 9 2 9 29 2 9 31

31 32 32 32 32 33 33 33 33 33

34- 31+

34 34 35

Chap t er 1 Arnmonia

Total Carbohydra t e s To tal hexo s e

To tal reducing sugar Ribose

Deoxyri b o s e

RNA

Pr eparati ve Methods Fre e z e Dri ed Cells

Organi c Solvents

Po t a ssium Palmi t a t e Soluti ons Methylation of Fat t y Acids Silylati on of Hyd roxy Esters

C hromatography

Pap er Chromatography o f Polyols and Sugars Pr eparat ion o f s amples

Analysis of sugars in perchlori c acid soluti ons obtained during RNA estimati ons

Solvent sys t ems and d e t ection of c ompone nts Silicic Acid Column Chroma tography

To tal unbound lipids To tal fatty acid s Thin-Layer Chromat ography

Preparation o f lhin-laye r pla t es Applic ation o f sampl es

Solvent sys t ems

Identi fication and isolation of components Autoradiography of thin-layer plates

page

37 37 37 37

38 39 39 ,+0

1+0 40

41 Lt1 41 42 1+2 4-2

42 ,+2

43 4 3 4 3 44

41+

4-5

4 5 45

C h ap t er 1

Chap t er 2

C h ap t e r 3

Gas Chroma t o gr aphy

Ga s c hroma t o graphy o f m e thyl e s t ers o f fat ty ac id s derived from t ri glyc eri d e s

Si lylo xy derivatives o f met hyl no c a rd at e s Pyrolysis o f methyl noc ardat es

GROWTH CHARACTERISTICS OF ^{N .} C ORALLINA

TRIGLYCERIDES IN N. CORALLINA

Ext rac tion and Frac tionation o f To t al Lipi d s

p age 46

4 6 46

�7

48

��

48

�9 51

5 1 52

5 3 53 5 5 55

56 56

Thin-Layer Chromato graphy o f Lipi d s o f N. c o r allina 57 I d en t i fi c at i on of Tri glyc erid e s in the Lipi d s o f

N. ·c orallina

The I n c o rp o ration o f U_14C Palmi t i c Acid into the Tri glyc eri d e Frac t ion

Summary

58

60 62

C ha pt er It

Chap t e r 5

FATTY AC IDS I N N. CO RALLINA

Ext ra ction , Fractionation and I d en ti fi cati o n o f Fat t y Aci ds from ^N. coral lina

Silylation of Methyl No cardat e s

Gas Chro ma t o gra�hy o f t h e T MS D e ri va ti ve s o f Me thyl No card a t e s

Mas s Sp ect rom e t ry o f t h e TMS D e ri va t i ve s o f Me thyl No card a t e s

Pyrolysis o f No cardi c A ci d s Oxi dat i on o f No cardic Aci d s

Fract i onation o f Me t hy l No card a t e s o n S i l ve r Ni t rat e Impregn a t ed Pla t e s

Summary

VARIA TI ONS rh" DRY WEIGHT, TOTAL AND VIABLE C OUNTS AND

RESPIRATORY ACTIVITY I N STARVED SUSPENSI ONS OF

H. CORALLINA

Vari a t i ons in Vi abl e and ^Total C e l l C oun t s and in

p ag e

6,+

67

67

68 69

70

7 1 72

73

Dry Wei ght 71t

Respi ra tion Rat es f o r C el l Sus pens ions o f N . corallina ⁷⁶ Respi rat i on Experi men t s wi t h Ace t a t e Gro \vn C el l s ⁷⁷ Re spi rati on Experi men t s wi t h C e l ls I n cuba t ed wi th

Palmi t a t e

Summary

77 78

Chapt er 6

Chap t e r 7

Chap t er 8

C HANGES IN 'lIHE LEVELS ^OF L IPID , C ARBOHYD RA'T'E AND PROTEIN IN STARVED SUSPEI'SIONS OF l�. CORALLINA Li pid C on t en t o f C e l l s o f ^N. c o ral lina

F a t t y Ac id Level s in N . corallina

To t al Nit rogen , Ammonia, Pro t ein and C arbohydrat e L e vels Rel a t ed t o Changes in Dry Wei ght C han ge s in Le vel s of To t al Fat ty Acid s , Pro t eins ,

C arbohydrat e , Dry Wei ght and C lust er Viab i li t y Dur in g End o genous Inc u bation

C han ges in Levels of To tal Fat ty Acids and C o rrelation of Pro t ein Br eakdown wi t h Ammoni a Produc tion

Correlation of Br eakdown o f C e l lu l ar C ar bo hydra t e wi t h Produc tion o f Ammonia

Summar y

C HANGES IN THE LEVEL OF RNA IN STARVED S USPENSIO NS OF N. CO Rll.LLIJ A

Pho s ph a t e Bu f f er Supernatan t s O.2N HC 10 Frac t i on

'+

0.1N HC10,+ Fra c ti on

Level s o f RNA in C el l Suspensions Inc u ba t ed Under

En d o genous C o n di tions Summary

STUDIES ON T:IE ULTRASTRUC TURE OF N. CORALLINA Me thods and Mat erials fo r El e c t ron Mi c ro s copy Resul t s and Di sc u ssion

page

79 79 8 0

8 1

8 ,+

8 5

87 8 7 8 8 90

9 1 9 2

9 3 9 3 9 5

C hap t e r 8 G ene ral I'4or ph ology and Growth C h arac t er i s t i c s in Li quid Me dium

C el l C o a t Cel l Wal l

C y t opl asmic Memb rane

I n t r a c y t oplasmic Memb r ane Sys t ems Cyt opl asm

Folyph os ph a t e Granul e s Nuc l e ar Ma t eri a l

Cell Di vi s i on

Summary

SECTIO N II I

DI SCUSSION

Growth C h arac t eri s t i c s of N. c oral lin a Lipid S tudi es

Changes in Vi abili ty , T ot al C el l C oun t s , Dry Wei eh t and R e s pirat ory Ac ti vi t y in S t arved Cell

S u sp ensi on s

Changes in Levels of I n t rac ellular C omp on en t s During End ogen ous Metabol i sm

C orrel a t i on Bet we en C el l Survi val and End ogen ou s

Me t ab ol i sm REFEREI"fC ES

APPENDI X I . C ul t ure medi a .

APPENDI X n. Deri v a t i on of formulae for es t ima ting c el l viabi li t y f r om c l us t e r vi abili ty and clus t er s i z e d i s t r ib uti on .

, p age

95 97 100 100 101 102 103 105 105 106

108 108 110

112

11Lt

117