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Department of Genetics

PROGRESS REPORT

(Research Grant No. E-2872)
IMMUNOGENETICS OF SALMONELLA

Principal Investigator: Tetsuo Iino

National Institute of Genetics, Japan

Granted Period

September 1, 1959 thru August 31, 1962

(This report was prepared at Sept. 15, 1961)
SUMMARY STATEMENT

The main progress attained during the granted period is in

the studies of the functional relationship of the genes which
control the synthesis of flagellar antigen in Salmonella.
Those genes are H, and Ho » the determinant of phase-1 and
phase-2 antigen type respectively; Vhy , a regulator of the
stability of Hy » consequently of antigenic phases; Ah, + &
controller of H, activity; and Fla genes, regulators of the
quantity of the flagellar antigen produced.

H, and Hy were found not only to determine the antigenicity
of flagella but also flagellar shape and sensitivity to certain
motility phage. They are presumed to be the primary structural
determinants of flagellar protein in phase-1 and phase~2
respectively. Antigen type mutants and H inactive mytants have
been accumulated, and genetic fine structure amlysis of H-genes
is in progress. In parallel, chemical comparative analysis of
flagellar antigen substances of those mutants is on the way. A
method to purify flagellar antigen substance by cellulose chromato-
graphy was invented.

The general rule of functional interactions between H, and
Hy (: epistasis of active Hy over H, and the reversible change
of Hy activity in phase variation) in diphasic strains was
extended to phase-? monophasic strains and an unequal recombinant
on H-genes. The epistasis and the reversible change were found
to be not antigen type specific but locus specific. The function
of Hy) and Ho are duplicate in case of the antigenic specificity
is disregarded. It was further indicated that the presence of
Vh, on the vicinity of H> is essential for the unstabilization
of the Ho. Any is a complementation unit which is closely linked
to Hy, Its function is phase specific. Fla™ mutants, which have
lost the ability to produce flagella in beth antigenic phases,
were classified into ten cistrons by complementation test. A type
of Fla” mutanis was detected which produced the cross reacting

material of flagellar antigen in their cells.
FULL STATEMENT CF PROGRESS

1, PUBLICATION AND ORAL REPORTS

a). Published
Iino, T., 1960, Subunits of Hy gene in Salmonella. Ann.
Rep. Natl. Inst. Genetics (Japan) 10:111-112.

, 1960, Unequal recombination in Salmonella.
ibid. 10:112-113.

b). Accepted for publication
Iino, T., 1961, Genetics of O-H variation in Salmonella.
Japanese Journal of Genetics

» 1961, A stabilizer of antigenic phases in
Salmonella. Genetics

; 1961, Abnormal homology of flagellar phases
in Salmonella. ibid.

» 1961, Curly flagellar mutants in Salmonella.
Journal of General Microbiology

c). Oral reports

Haruna, I. and T. Iino, Separation and purification of
antigenic substance of Salmonella flagella. 34th Meeting of
the Japanese Biochemical Society (Nov. 4, 1961, at Osaka)

Iino, T., Unequal recombination in Salmonella. 31st.
Meeting of the Genetics Society of Japan (Nov. 5, 1959, at
Osaka)

 

» Genetic aspects on the phylogeny of Salmonella
serotype. 33rd Meeting of the Bacteriological Society of
Japan (July 20, 1960, at Sapporo)

 

» Genetics of Salmonella in reference to fine
structure analysis. 32nd Meeting of the Genetics Society of
Japan (Nov. 1, 1960, at Fukuoka)
26

Iino, T., Curly flagellar mutants in Salmonella. 33rd
Meeting of the Genetics Society of Japan (Sept 3, 1961, at
Sendai) . .

Sasaki, I., Multiplication of chi~phage in H-type Salmonella.
8th Meeting of the Japanese Virolcgist (Oct. -29, 1960, at Kyoto)

 

, Host range mutation of chi-phage. 33rd Meeting
of the Genetics Society of Japan (Sept. 3, 1961, at Sendai)

, Ss Tsuji and T. Tino, Relationship between chi-
phage and H-antigen of Salmonella. 13th Meeting Kanto Branch, |
Bacteriological Society of Japan (Apr. 27, 1960, at Yokohama )

STAFF ING

Teteuo Iino, Ph.D., Researcher ( principal investigator), Sept.
1959-, Spent 75% of the period on this project.

Shigekatsu Teuji, B.Sc., Research fellow, Oct. 1959 thru Apr.
* 160, Half time.

Itiro Sasaki, M.D., Researcher (associate), Dec. 1959-, Full
time.

Hideo Hirokawa, M.Sc., Researcher” (assistant), Half tine
July 1960 thru Apr. '61, Full time May 1961-.

Masatoshi Fnomoto, M.Sc., Researcher (assistant), Apr. 1961-,
Half time. |

_ Ichiro Haruna, D.Sc., Researcher of the Institute of Virus
Research, Kyoto University, Kyoto, Japan, cooperated
with us the chemical parte of the investigation since
Jan. 1960.
3

PROGRESS IN RESEARCH

The project aims to clarify the genetic mechanism of specific
antigen (especially H-antigen) production and the antigenic
variations in Salmonella. The investigation has been focused in
the following three subjects: I). genetics of antigenic phase
variation (Iino, Sasaki, Teuji); II). genetic analysis on the
biosynthesis of flagellar antigen (Iinc, Hirokawa, Enomoto);
III). genetic fine structure analysis of H-antigen determinants
(Iino, Haruna).

IT). Genetics of antigenic phase variation

The preceding studies had shown that H}- and Ho-gene are the
determinants of H-antigen type in phase-l and phase-2 respectively
in Salmonella, and the antigenic phase variation in a diphasic
strain occurs by the oscillatory change of Ho between active and
inactive states. In other words, Hj is epistatic over H) and
changes its inherent activity reversibly: when both Hy and Ho are
in active state in a cell, the cell expresses the phase-2 antigen;
when Ho is inactivated, the H, determined phase-] antigen ts
produced. >

1

During the granted period, several new informations have been
obtained as regards antigenic phase variation.

(1). Interaction between H-genes

Phase-2 monophasic mutants of diphasic 5. typhimurium were
found to perform 0-H variation in place of phase variation:
phase-1 is O-type and phase-2 is H-type. Transductional analysis
demonstrated that both Hy and H, are inactive in phase-l and

- only H, is active in phase-2 in such mutants. These results

indicate that the function of H, and Hy is duplicate in flagellar —
synthesis in cases that the antigenic specificity is disregarded.
The inactivation of H, ia caused by mutation in a cistron, called
Ah), which is closely linked to H, but separable from it by
recombination. Hy and Ah, show at least partial complementation in
cis-trane test.
(2). A stabilizer of phase variation

Genetic amlysis of a stability controller, Vh5, of phase
variation was continued. In S. abortus-equi, an allele Vh,”
stabilizes Ho in its existing state, whether inactive or active,
and produces phase-l or phase~2 monophasic typea respectively.
Vhy is transduced linked to Hy. The stability of the antigenic
phases of the strains which carry Vh..~ is exceedingly high, and
such strains may be used as excellent meterials for the production

of specific anti-H serums.
(3). Anomalous homology of flagellar phases

An abnormal Heantigen type recombinant which alternatively
expresses phase-] antigens of both donor (i) and recipient (»)
was obtained from a transduction between 5. typhimurium and 5S.
abony. In the transductional progeny test, the duplicated
phase~-1 antigen type determinant Hy> of the recombinant behaves
as an allelic locus of phase-2 antigen type determinant Ho. The
recombinant is presumed to be originated by unequal recombination:
Hz locus is replaced by H, of the donor in the transduction.
This phenomenon suggests the phylogenical homology between Hy
and Ho: one of them might have originated by duplication and
translocation of the other: the structural differentiation
might have occurred between them thereafter. It also indicates
that the effect of Vho is not antigen type specific but locus
specific.

(4). The effect of various chemicals on phase variation

The chemical agents which can alter the frequency of phase
variation have been looked for. The chemicals include bromouracil,
fluorouracil], chloromycetin, penicillin, streptomycin, acriflavin,
formaldehyde, Na-azide and methyl green. The significant effect
of those chemicals on phase variation has not been detected.

The survey is being continued and the continuous culture apparatus
is being under construction for this purpose.
(5). Changes associated with phase variation

 

Other than the specificity of H-antigen, the following two
characters were found to be determined by Hy and Ho» and the
changes of those characters associate with antigenic phase
variation.

a). curly flagellar shape

Flagella having an wavelength of half that found in the wild
type have been called curly flagella. A curly flagellar mutant
obtained from a strain of S. typhimurium is unstable and repeatedly
dissociates curly and normal subclones. Examination of the
flagellar antigens of the normal and curly flagellar aubclones~
demonstrated that the change in flagellar shape correspond exactly
with phase variation: subclones with curly flagella are always
in phase-1 (i-antigen) and those with curly flagella are in
phase-2 (1.2-antigen).

In transduction from a normal flagellar strain to the curly
phase-] strain, transductional olones with normal flagella were
isolated. The transductional clones showed the antigen of the
donor in phase-l and that of the recipient in phase-2. From
this result it is concluded that the phase-1 curly determinant
is closely associated with the phase-l antigen type determinant,
Hy. :

Seven curly mutants were obtained from a strain of S. abortus-
equi. Transductional analysis with these strains showed that the
phase-2 curly determinant is closely associated with the phase-2
antigen type determinant H5; and the phase-1 curly determinant
with Hy. -

In cross absorption experiments with antiserum prepared
ageinst flagella of either normal or its curly mutant, no antigenic
difference between normal and curly flagella could be detected.

Attempts to obtain recombination by transduction between the
curly flagellar determinants in each phases have been unsuccessful;
this suggests that the mutant sites of the curly types are very
closely linked or identical in each phases.
b). sensitivity to a motility phage, chi

" Chisphage has been known to attack motile Salmonella except
those which have g-antigenic flagella. Mutante of S. typhi-
murium were found which are resistant against chi-phage in motile
phase-2 (1,2-antigen), while they retain the sensitivity in phase-
1 (i-antigen). A host range mutant of chi-phage can attack both
the flagella-less and paralyzed mutants obtained from the original
strain of the resistant mutants. The resistant phenotype in phase-
2 of the mutant strains seems to be expressed by interaction of
Hp gene and some other genetic factor(s): when the H,'-? of a
resistant mutant is replaced with another Ho allele, for example
Hp°™* of S. abortus-equi by transduction, the transductional
clones become sensitive in phase-2 as well as in phase-1}; while .
1.2-antigen type of transductional clones obtained by transduction
of the reverse direction remained sensitive. The more detailed

genetic analysis is in progress.

The association of these mutant characters with H-genes may
indicate that Hy and H> are the primary structural determinants
of flagellar protein in phase-1 and phase~2 respectively; a
mutant in Hj or Hy may cause an altered configuration of flagellar
protein, resulting in a change of antigenic type, flagellar shape
or a sensitivity to the motility phage, or it may cause the failure
of flagellar morphogenesis.

II). Genetic analysis on the biosynthesis of flagellar antigen
(1). Complementation between Fla™ genes

Fla genes are regulators of flagellar synthesis. They do not
determine or modify the specificity of flagellar antigen but
regulate the amount of flagelle produced.

Complementation analysis was carried out extensively on Fla”.
mutants obtained from S. typhimurium and S. abortus-equi. The
production of trails in transduction between Fla" mutants was
used as the criterium of complementation. 53 of them were classi-

fied into ten groups (cistrons). In two of these groups, partial

 
complementation was observed. The parallelism of the complementation
map and the linkage map based on the recombination frequency is

under inveatigation.

(2). Production of the substance cross reacting with flagellar

antigen in Fla~ mutants

The antigenicity of twenty one Fla” mutants, which are highly
stable and represent all the complementation units, were examined.
They all gave negative result in agglutination test with anti-H
serum; while,when they were injected to rabbi tse two of them caused
to produce anti-H sera. The antigenic aubstarice was isclated from
the sonicated cella of these two mutants by means of cellulose
chromatography (ref. III). The chemical comparative analysis of
the H-antigen substance of the Flat wild strain and the eross

reacting material found in these fla” mutants is in progress.
(3). Test of syntrophism between Fla~ mutants

The possibility of syntrophic recovery of flagellar production
was examined with mixed cultures of two complementary Fla” mutants.
None of the combinations tested recovered the ability of flagellar

production,
(4). Temperature mutants on flagellar production

TM2 strain of S. typhimurium can grow at 42C, but cannot
produce flagella at the temperature higher than 40C. Two temperature
mutants which can produce flagella at 40C thru 45C as well as below
40C were isolated from TM2. The temperature resistant character
was not transduced linked to any known Fla genes. Detailed analysis

on the nature of the temperature resistance is under investigation.

III). Genetic fine structure analysis of H-antigen determinants
(1). Genetic amlysis

Ho inactive mutants and the partial antigen type mutants of Hj1l-2
and Ho®2™ alleles have been accumulated from a strain of S. abortus-

equi SL23 (anx-antigen) and its transductional derivative SJ25

7s (A.2aamtigna). Seetsolia nutrient-gelatine-ager aedius supplé- |

é

 
te
mented with anti-enx (or -1.2) serum was used for the selection:

an Ho inactive mitant clone produces a swarm with flagellar antigen-
aon the selective medium. The recombination amlysis with these
mutants is on the way of accumulation of genetic data. Curly
flagellar mutants and the mutants resistant to chi-phage (reported
in I.5) were also adopted for the analysis.

(2). Isolation and purification of flagellar antigen substance

In parallel with the genetic fine structure analysis of Ho gene,
chemical studies of flagellar antigen substance was started.

The method to purify flagellar antigen substance by cellulose
chromatography was invented. The outline of the procedure is as
follows: sonicate the saline suspension of the bacterial cells for
1 minute at 20 kc; the suspension was centrifuged and the oell free
supernatant was acidified (pH 3-5) by 1/10 N HCl and centrifuged;
the supernatant was dialysed and adsorbed to DEAE cellulose column
with 0.005 M borate buffer; the adsorbed materials were fractionated
by gradient elution with changing pH and salt concentration; antigeni-
city of each fraction was examined by gel-diffusion test; flagellar
antigen substance was isolated to a fraction. The flagellar antigen
substances of the enx-type flagella, 1.2-type flagella and curly
mutant flagella came to the same fraction of the chromatogran,

The advantage of this method to the previous one, in which
flagella were purified first by differential centrifugation, is —
that we can detect flagellar substance in bacterial cells before
it is organized to the visible filamentous organelle. By the appli-
cation of this method flagellar antigen substance produced by some
flagella-less mutants was isolated (reported in II.2)