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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 endogen ous metabolism of the soil microor ganism N. corallina

has been studied with special reference to physiological and structural

changes in starvation cond iti 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 produced.

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 were prepared from cul tur es harvested at full growth and

resusDended in phosphate buffer containing magnesium ions . Analyse s of

total and viable cell counts were affected by cluEte rin g 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 via bility 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. Fol lowing this

drop in polysaccharide, ammonia was released at a r el atively constant

rat e and at the same time th ere was a f all in the level of cell protein.

ii .

Ther e was a fall i n the l evels o f intrac ellular free ni t rog enous c ompo und s

at th e ons e t o f st arvation but no correE�onding rel eas� o f these sub stanc es

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 o f the indi vidual c ell fractions

to the t o t al fall i n 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 el l dry wt . yi elded t rehalos e , manno s e , inosi t ol and gly c erol a s t h e

wat er solub l e c omponents. Tri glyc eri d e s were isolat ed from the total

lipids by sili c ic acid column and thin-layer chrom atogr aphy . 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 d ry 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 t h e

n ocardic acids. Trimethylsilyl d eri vati ves o f the m e 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 acid s occ u r r e d ) , and in th eir i som eri c

configurations .

iii .

Studi e s on N. corallina using b o th ligh t and el ec t ron micro scopy

showed clearly the pl eomorphic natur e o f t he o rganism . Par t s o f the

c ell surfac e were covered with fibrous ma t erial which app eared t o be

di sti nc t from the c ell wall . C ell division occurred by the f o rmati on

of septa which were g en erally associ a t ed wi t h ext ensi v e cyt oplasmic

m embrane syst ems . Polyphospha t e granul e s , 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 f r e e z e- e tch t echnique illu s t rat ed the granular nature o f t h e

cytoplasm , cell wal l and m embrane surfaces . S tarva tion o f the cells

app eared to be associ a t ed 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 , C c ) an

increase i n the si z e o f t h e polypbosph a t e granul e s 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 tiga tions wi th t his o rgani sm and t o s t ud i es

o f t h e endo genous m e t a bolism and survival chC'.rac t eri s tics o f o t h er

microbial sp eci 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 Survival

(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

29

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 reduc i ng sugar

Rib o s e

D eoxyri b o s e

RNA

Pr eparati ve Methods

Fre e z e Dri ed C e l l s

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 o f sugars in perchlori c acid soluti ons

o b t ained during RNA estimati ons

Solvent sys t ems and d e t ection of c ompon e nts

Silicic Acid Column Chroma tography

To tal unbound lipids

To tal fatty acid s

Thin-Layer Chromat ography

Prepara tion o f lhin- laye r p la t es

Applic ation o f sampl es

Solvent sys t ems

I d enti fication and isolation of components

Autorad i ography o f thin-layer plate s

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

43

44

41+

4-5

4 5

45

Chap t er 1

Chap t er 2

Chap t e r 3

Gas Chromatography

Gas c hroma tography o f m ethyl est ers o f fat ty

ac ids derived from t ri glyc erides

Silyloxy derivatives of 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

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

p age

46

4 6

46

�7

48

��

48

�9

5 1

5 1

52

5 3

53

55

55

56

56

Thin-Layer Chromato graphy o f Lipid s o f N. c orallina 57

I d enti fic ation of Tri glyc erid e s in the Lipi d s o f

N. ·c orallina

The Incorporation o f U_14

C Palmi t i c Acid into the

Tri glyc eri d e Frac t ion

Summary

58

60

62

Cha pt er It

Chap t e r 5

FATTY ACIDS I N N. CO RALLINA

Ext ra ction , Fractionation and I d en ti fi cati o n

o f Fat ty Aci ds from N. corallina

Silylation of Methyl No cardat e s

Gas Chro ma t o gra�hy o f t h e TMS D e ri va ti ve s o f

Me thyl No card a t e s

Mass Sp ect rometry o f the 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 Aci d s

Oxi dation o f No cardic Aci d s

Fract i onation o f Me t hyl No card a t e s o n Silve r Ni trat e

Impregn a t ed Pla t e s

Summary

VARIATI ONS rh" DRY WEIGHT, TOTAL AND VIABLE COUNTS AND

RESPIRATORY ACTIVITY I N STARVED SUSPENSI ONS OF

H. CORALLINA

Vari ations in Vi abl e and Total C el l C oun t s and in

p age

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

Respiration Experi men t s wi t h Ace t a t e Gro \vn C el l s 77

Respi rati on Experi men t s wi th C e l ls Incuba t ed wi th

Palmi tat e

Summary

77

78

Chapt er 6

Chap t e r 7

Chap t er 8

C HANGES IN 'lIHE LEVELS OF LIPID , C ARBOHYD RA'T'E AND

PROTEIN IN STARVED SUSPEI'SIONS OF l�. CORALLINA

Lipid C on t ent o f Cells o f N. c o ral lina

Fatty Ac id Level s in N . corallina

To t al Nit rogen , Ammonia, Pro t ein and C arbohydrat e

L e vels Rel ated to Changes in Dry Wei ght

C han ges in Level s of To tal Fat ty Acid s , Pro t eins ,

C arbohydrat e , Dry Wei ght and C lust er Viabili t y

Dur in g Endo genous Inc ubation

C han ges in Levels of To tal Fat ty Acids and C o rrelation

of Pro t ein Br eakdown wi t h Ammonia Produc tion

Correlation of Br eakdown o f Cellular C arbo hydra t e wi th

Produc tion of Ammonia

Summary

C HANGES IN THE LEVEL OF RNA IN STARVED S USPENSIO NS

OF N. CO Rll.LLIJ A

Phosphate Buffer Supernatant s

O . 2N HC 10 Frac tion '+

0 . 1N HC10,+

Frac ti on

Level s o f RNA in C el l Suspensions Incuba t ed Under

En dogenous Condi tions

Summary

STUDIES ON T:IE ULTRASTRUC TURE OF N . CORALLINA

Methods and Mat erials for El e c t ron Mi c ro scopy

Resul t s and Di sc ussion

page

79

79

8 0

8 1

8 ,+

8 5

87

8 7

8 8

90

9 1

9 2

9 3

93

9 5

C hap t er 8 General I'4orphology and Growth Charac t er i s t i c s

in Li quid Medium

C ell C o a t

Cell Wall

Cytoplasmic Membrane

Intracytoplasmic Membrane Sys t ems

Cyt oplasm

Folyph os phate Granul e s

Nuc l ear Ma t eri a l

Cell Di vision

Summary

SECTIO N III

DI SCUSSION

Growth Charac t eri s t i c s of N. c orallina

Lipid S tudi es

Changes in Viabili ty , T ot al C el l C oun t s , Dry Wei eht

and Respirat ory Ac ti vi t y in S t arved Cell

Susp ensi ons

Changes in Levels of Intrac ellular C omp on en t s During

End ogenous Metabolism

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

Met ab olism

REFEREI"fCES

APPENDI X I . C ul t ure medi a .

APPENDI X n. Derivation of formulae for es t imating

c ell viabili t y from clus t er vi abili ty and clus t er

size distrib uti on .

, page

95

97

100

100

101

102

103

105

105

106

108

108

110

112

11Lt

117