Table Of Contentcr&nss AND CYSTEINE METABOLISM BY
PEoiius m m m m a m w m m
Reino Hail Kallio
A dissertation submitted in partial fulfillm ent of the
requirements for the degree of Doctor of Philosophy
in the Department of Bacteriology in the Graduate
College of the State University of lorn
dune 1950
ProQuest Number: 10991962
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Acknowledgement
Dr* J. It* Portia* has given freely of his heavily-
burdened time in encouraging the present study*
It is a point of pride and a privilege to be con
sidered one of his scientific offspring*
ill
table or contests
INTRODUCTIOW*, * * *.------------------------------------------------------0...... * ------... 1
EOT8BSBHTAL METHODS****........................ **.................... . . . .. . . . ............12
RESULTS AMD DISCUSSION.................. . . .................................».............20
srom T .*. .................................................................... »..43
APPENDIX ............ 44
BIBLIOGRAPHY., ............ . . . . . . . . . . . . . . . . . . . .............52
iv
TABLE OF FIGURES
Figure 1* the effect of gH on. cysteine deaulfhydrase activity...... 22
Figure 2. Cystine and cysteine utilisation by Proteus vulgaris
cells grown in c y s t e i n e * . 25
Figure 3. Cystine and cysteine utilisation by cells grown in
cystine ................ .........*.. 26
Figure 1* Dissimilation of cysteine under aerobic and anaerobic
co n d itio n s.......................* ^ .......,.,.. . .. . . . . ........... 51
V
TABLE OF TABLES
Table I. Cysteine desulfhydrase activity of Proteus vulgaris cells
groisn under various conditions*••«•••«.••••••..*•*««*.••*••••»• 23
Table II* Anaerobic utilization of cysteine by Proteus vulgaris
adapted to c y s t e i n e * * * * * * * * * * * . . 28
Table III* Effects of various inhibitors on hydrogen sulfide pro
duction from cysteine by Proteus vulgaris. ....... 31
Table IV. Sodium a aide inhibition of anaerobic pyruvate utilization
v u l g a r i s . 33
Table V* Anaerobic cysteine dissim ilation by a aide-inhibited resting
cells of Proteus vulgaris.* * ...* ................................................ 35
Table VI* Anaerobic dissim ilation of cysteine by toluene treated
cells of Proteus morganil. . .................................... 37
Table VII* Effects of various inhibitors on the cysteine desulf
hydrase activity of toluene killed cells of Proteus giqrganii... 39
Table VIII* Beactivatlon of "aged", toluene killed Proteus
morganil cells toward c y s t e i n e , . , , . , 4^
Table IX. Biochemical characteristics of Proteus vulgaris and
Proteus morganii used in the present study**.....,..,...,*..... 47
Table X, Production of hydrogen sulfide from various sulfur
compounds by Proteus vulgaris and Proteus marganli*. 48
fable XI, Hydrogen sulfide production as a function of the number
Proteus vulgaris cells present...
Table XII* Gcopounds tested singly and in combination for activa
tion of cell free extracts to®/ard cystein e,...*....,.,*.,...... 50
1
The observation that a variety of heterotrophic organism produce
hydrogen sulfide and other volatile sulfur compounds from protein sub
strates is old and well established. For example, in 1089 Nenckl and
Sieber noted the production of methyl mercaptan from protein putre
faction brought about by a variety of anaerobic organisms. Rettger
(1906) and Eerier (1906) confimed this observation at least in the
case of the obligate anaerobes Clostridium novvi. Clostridium feserl
and Clostridium lentooutresoens. In later studies on protein putre
faction Rettger (1912) pointed out that putrefaction was essentially an
anaerobic process and was characterised by "the evolution of foul
smelling products which are characteristic of ordinary cadaveric de
composition* It should be noted that mercaptan is of particular
significance and that indole* skatole and hydrogen sulfide are of less
importance”. Rett gear demonstrated further that hydrogen sulfide was a
'teomcion protein decomposition product” and was not indicative of true
putrefaction. 3h the same study the putrefactive abilities of a number
of facultative anaerobes, especially the Proteus group, were investi
gated, Under completely anaerobic conditions none of the members of
the genus Proteus were capable of degrading the pure proteins used
(egg and serum albumin; and ©destin). ilhen, however, oxygen was ad
mitted into the growth medium rapid decomposition of the proteins oc
curred with the liberation of some hydrogen sulfide but no methyl mor-
captan*
2
Sperry and Hettger (1915) added to these observations by noting
that facultative anaerobes failed to multiply in the presence of pure
proteins when ammonium salts and oxygen were absent. If, however, pep
tone m s added to the culture medium rapid growth and degradation of
protein took place, as evidenced by liquefaction of the protein* No
methyl mercapt&n was produced even under these conditions* Thus it was
noted quite early that there seemed to be a fundamental difference in
protein breakdown between the obligate anaerobes and the facultative
groups* If the discovery and characterization of methionine had taken
place prior to these investigations perhaps at least part of the ex
planation fbr this difference might have been patent, but methionine
ms not discovered until 1922.
Since cystine had been discovered in 1899 by Corner and cysteine
lay Embdon in 1901, it seemed clear to the earlier workers that the
hydrogen sulfide and methyl mercaptan observed must arise from these
two stalfur-containing protein components. Wohlgemuth (1904.), for
example, claimed the production of methyl mercaptan and ethyl sulfide
from cystine decomposed by bacterial action. These claims were vigor
ously denied by Burger (1914) who found hydrogen sulfide but no other
sulfide or mercaptan produced from cystine by 23 species of bacteria*
Caapek (1920) suggested the course of the reaction was a preliminary
reduction of cystine to cysteine followed by a t^rdrolytic deaminations
HSOHgCH (Nil?) GOGH + H20 V HSCH^CHGHCOOH + NH3
The ^-thiolactlc acid formed was then dacarboxylated and oxidised to
3
thloglycollic acid*
H3GH2GiiGBGG0H + 02 ------> HSCH^COOH ♦ C02 *h2o
The thioglycollic acid m s subsequently decarboxylated to methyl mer-
captan.
Betake (1924) agreed with the above reaction sequence but was Jin-
able apparently to offer mutch experimental proof for his view* The dis~
slm ilatlve pathway of cystine proposed fey Wohlgemuth and Kotake was
strongly questioned fey other investigators who were unable to find methyl
mereaptan as an end product of cystine breakdown* As a matter of fact,
as early as 1905 faoi had disagreed with the proposed scheme inasmuch
as h© found that hydrogen sulfide (but never methyl mercapt&n) ms fomed
from thiolactic or thioglycollie acid by bacterial action. Methyl raer-
captan was produced from cystine only when a fermentable carbohydrate was
ppesentf for ©stable, in the case of Escherichia coil methyl mercaptan
ms formed in the presence of glucose, lactose, sucrose or dulcltol*
Since in no case did methyl mercaptan appear to be a major metabolic end-
product it ms concluded that its appearance in the presence of a fer
mentable sugar was the result of secondary reactions*
By 1915 the ability of raany microorganisms to produce hydrogen sul
fide from protein, peptones or cystine had been well established and the
reaction was being utilised widely as a physiological aid in identifying
bacteria* Jordan and Victorson (1917) used nutrient agar to which had
been added lead acetate as a means of partially differentiating members
of the species of Salmonella* Tamer (1918) used hydrogen sulfide pro
duction to help distinguish certain yeasts, and more recently the re
4
action has been a llied to differentiating between spec lea in the genus
Brucella (Huddleson and Abell, 1927)* A great many studies have been
sad© with respect to making hydrogen sulfide production by bacteria
more easily discernable—among these may be mentioned those of Zobell
and Feliham {1934)Stekol and Rename ier (1942) , and Hansmoier and
Stekol (1942)* There is little point in discussing or ©3±ending this
list for though these investigations contributed valuable adjuncts to
the armamentarium of the diagnostic bacteriologist they are purely
qualitative in nature and contribute little or nothing to an under
standing of the mechanism of the reaction whereby hydrogen sulfide is
liberated from cystine or cysteine*
Almy and Jamas (1926) developed relatively specific and sensitive
methods for the estimation of hydrogen sulfide and mercaptans and car
ried out quantitative studies on the liberation of these compounds from
various peptone media by Escherichia coli, Proteus vulgaris and Salmon
ella aertrveke* Apart from the fact that various peptones differed in
the amounts of hydrogen sulfide liberated it was established that no
mercaptan appeared and that the hydrogen sulfide produced was directly
proportional to the cystine content of the peptone being used* When
cystine was added to a peptone medium the sulfur could quantitatively
be recovered as hydrogen sulfide* It was also noted that a large pro
portion of the hydrogen sulfide liberated from a given medium was pro
duced in a comparatively short time in the early incubation period*-6
to 12 hours following Inoculation* Ho attempt was made to ascertain
the other metabolic products produced froa cystine* However, it is
