FURTHER ANALYSES OF MONOPHASIC sATURE OF STRAIN S¥1061
(PSEUDO-MOWOPHASICS Ii A STRAIu OF SALMONELLA TYPHIMURIUM)
Report 1956-4

by
Tetsuo Iino (Sept. 14, 1956)

In "Report 1956-h", Fla” # 1.2 variatiouswere described ou phase 2
monophasic variant SW1061 (-:1.2) of Sal. typhimurium strain TY (i:1.2).
The transductional analyses have suggested that Hj in SW1061 is inactive,
gud Fla # 1.2 change corresponds to phase variation genotypically; that is
the change of the state of Hp:

The iaactivatiou of H, may have occurred either by a chauge of Hy
itself or by an inhibitory change of au adjuscent factor against Hy functibu.

The present report concerns about experimental tests of these alternatives.

MBTERIALS AnD METHODS,

The experimeats consist of two transductious, from Sal. typhimriun
SW10G1 (-:1.2) to Sal. heidelberg Sw1092 (Fla, r:1.2) and from Sal. abony
Sw803 (bsenx) to SW1061,.

General methods of transductious are same as described by Lederberg
and Tino (1954).

For the transduction from SW1061 th Sw1092, lysates of SW1061 were
prepared from 1.2 phase cultures. Two lysates, each of which wac prepared
from a different single colouy culture, were used for the preseut experiment.
As recipieuts, five oversight penassay broth cultures of $w1092, prepared
from siugle colonies each, were used, Fla-trausductious were screened ou
MGA-plates and their antigens were tested by slide agglutination. When
it is necessary, alteruative phases were screened by antiserum-MGA deep tubes,
Monophasics -:1.2 were confirmed both by non-swarm production ou auti-1.2

MGA deep tubes aud by segregation of motile aud nou-motile colonies ou MGA plates.
The secoud transduction SW803 -x SW1061 is the repetition of the same
experiment with the transduction in "Report 1956-h", except equall size of
the recipient culture was used as a coutrol, to compare the frequency of
i:1.2 type production between Iysate-treated and non-treated cultures.
EXPERIMENTAL RESULTS.

The results were summarized in Table 1 and Table 2, In transduction
from SW1061 to SW1092, most of the types are r:1.2 which were produced by
the transduction of Flayogze« Besides r:1.2 type, a few number of -:1.2
aud i:1.2 types were recovered. Phase 1 culture of 1:1.2 type obtained
shows the same intensity of agglutination by anti-i (react to 1/12800 dilution
of the original anti-i serum) as phase 1 culture of diphasic Sal. typhimurium
TM2, #:1.2 type does uot agelutinate even by 1/10 anti-i serum #like original
SW1061. The ratio of i:1,2 among transductionus is high exough to elimiuate
the possibility of its origin by reversion. It may be explained better by
assuming that "the factor which inactivates H, function in SW1061 is linked
closely but not identical with Hy itself",

The non-recovery of (1):1.2 type amoug i:1.2 may be explaiued by the
small size of samplixg, as x° test suggests that there is no significant
interdependence between the phase of recipient and the frequency of i:1.2
(x*=5.8, 0.2<P<0.3). A poiut remained iu question is the low frequexucy
of -:1.2 production compared to the other cases of linked transduction
of Flaiggg with H). The difference of the frequency ofthe—frequeucy of the
linked transduction Fla) 999781 between different cultures has been observed
in other cases (Report 1955-b, and unreported data 1956). Low yield of
~:1.2 type in the present experiment may also be coneidered as a case of
such diversities of the frequency of the Jinked transduction.

In transduction from SW803 to SW1061, the uumber of i:1.2 produced is
larger in the treated than in the control. However, the difference of the
frequency is not definitely siguificant statistically (x? 21.8, P<0.2).
The data may be explained as foblows: 1:1.2 type, atleast a part of which,
is produced as a result of transduction, but the frequency of reversion from
-:1.2 to i:1.2 is about the same as the frequency of i:1.2 production by
trausduction an@ it made the difference betweem the treated and the control

{nsignificant.

DISCUSSION.
The production of mouophasics from diphasic salmonella may occur by

(1). deficiency or inactivation of Hy,
(2). deficiency or inactivation of Hj, or
(3). stabilization of H, state.
The second and the third cases have already been mentioned (Lederberg and
Iino, 1956). The moxophasics of SW1061, reported in this paper, belongs
to the first case. A remarkable feature of (1) is that flagellated cells
appear mixed with non-flagellated cells and interchange each other just as
phase variation, contraty with (2) and (3) in which cultures are composed
of only flagellated cells of single phase.

Phase variation is defined genotypically asaa change of Ho state,
The genotypic change of flagellation in SW1061 is also explained by the
change of H2 state, differed from phase variation only in a point that
SW1061 has inactivated H, instead of active H,. Iu other words, SW1061 is
mouophasic serotypically but diphasic genotypically. The term pseudo-
mouophasics is applied to such tyte.

The difference of motility between phase 1 and phase 2 of the same

a
strain is uot,rare eveat (Edwards et al. 1954). One of euch cases reported
by Seligmaun et al. (1945) aud Edwards et al. (1954) is quite similar
with the case reported here, except phase 1 is uot completely non-motile
but poorly motile and faiutly agglutiuable by anti-1. SW1061 may be
understood as an extreme type of such cases, although transducttoual
analysis has uot been done except SW1061,

The inactivation of Hq in SW1061 is not caused by the change of
H, itself but the change of a factor closely liaxed to it. The function
of the factoe is not antigen type specific but phase specific. Siuce it
suppresses the production of flagel’a only iu phase 1, the factor is
symbolized by Flani: and its allele in diphasic strain by Flay: Flap)
links most closely to Hy among the factors on which the linkage with Hy
has been observed, It is interesting that the markers linxed to Hi:
since discovered, are all "Fla", and among them the most closely liuked

One is specially related to q fuuction.

SUMMARY.

1). Monophasic strain SW1061 of Sal. typhimurium perform Fla = 1.2
change in its culture,

2). Transductional analyses indicated that H, in Sy1061 is inactive,
and the change Fla = 1.2 occures by the change of Hy state, correspond
tp phase variation,

3). The inactivation of Hq is not caused by the change of H, itself
but by other factor Fla, which is linked closely to Hj.
REFERENCES,

1954,
Edwards, P, R. et al., Studies on Grouf G of the Gems Salmonella, J. Bact.,
67 2346-349,

Iino, T, Report 1955~-b, Effects of phase difference on the Heantigen traus-

duction in Salmonella diphasic straiuas.
Tino, T. Report 1956-h, Trausductional analysis of monophasic nature of
strain SW1061.

Lederberg, J. and Iino, T, 1956, Phase variation in Salmonella. Gemetics,
in press,

Seligmann, E.etw al., 1945, A heretofore undescribed phase variation in

Sabmonella, Proc. Soc. Exptl. Biol. Med., 58:48-—50,
Table 1.

Transduction from SW1061 (-:1.2) to SW1092 (Fla, r:1.2).

Flajgg2 are used as selective marker,

 

 

Lysate no. of douor I T If II II
Total
Clone no. of reeipient 1 2 3 4 5
(r): 1.2 0 78 32 2 40 152
Antigen type r :(1.2) 110 4O 6 31 2 189
of (i): 1.2 0 0 0 0 0 0
tranusductions i :(1.2) 3 1 0 2 ) 6
(-)3 1.2 0 5 2 1 1 9
Total 113 124 40 36 43 356
Phase of recipient 1 1&2 2 1 2 1&2
Table 2.

Transduction from SW803(b:enz) to SW1061(-:1.2).

Transductious were selected by anti-b anil —-enx.

 

Antigen type
(-) :enx

b :(1.2)

4 :(1.2)

Total

wumber of swarms

Treated
84
95

183

Control

0

0