Weinberg, Roger

PubMed

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NOTES
CELLULAR STRUCTURE OF LARGE SPIRILLA
K. A. BISSET AND PHYLLIS PEASE
Department of Bacteriology, University of Birmingham, Birmingham7, England
Received for publication November 17, 1959
Evidence from many different sources, and
founded upon considerations of cytochemistry,
flagellar development, and observational experi-
ment, suggests that the main growth of unicellu-
lar bacteria is at the poles. This evidence is
discussed more fully by Bisset and Pease (J. Gen.
Microbiol., 16, 382-384, 1957). In multicellu-
lar bacteria, conditions are more complex, and
the poles of all constituent cells are possible areas
of growth.
A recent paper by AW'illiams (J. lBacteriol., 78,
374-377, 1959) states that the main growth of a
"unicellular" spirillum occurred in the central
area; accordingly, this author takes issue with the
supporters of the theory of polar growth.
In our experience, large spirilla are not uni-
cellular, although it is not possible (as in the case
of most other bacteria) to discern their structure
by ordinary methods. Cross-walls can be demon-
strated by appropriate techniques (figure 1), and
may occasionally be visible in partially disrupted
organisms by electron microscopy (figure 2).
We consider that a spirillum of the configura-
tion illustrated by Williams would normally pos-
sess at least one developing cross-wall centrally;
thus the active growth of the organism at this
Figure 1. Spirillfti sp.; mordant, 10 per cent
phosphomolybdic acid aind 0.3 per cent aqueous
iodine; stain, 1 per cent methyl green. Arrows
indicate cross-walls. (X3000)
Figure 2. Spirillumiii uinduila; electron micro-
graph, gold-shadlowed, showing cross-walls.
(X7500)
point (i. e., at the coincidlence of two developing
poles of the component cells), is absolutely in
accordance with the suggestion that bacterial
growth occurs mainly at the poles of the cells.
PHAGE Ti-RESISTANT MUTANTS OF ESCHERICHIA COLI
ROGER WEINBERG'
Department of Medical Microbiology, University of Southern California, andl Los Angeles
County General Hospital,2 Los Angeles, California
Received for publication November 23, 1959
Experiments were performed to obtain informa-
tion concerning the genetic nature of the simul-
I U. S. Public Health Service Postdoctoral
Fellow CF-6931-C. Present address: Department
of Biological Sciences, University of Pittsbuirgh,
Pittsburgh 13, Pennsylvania.
2 This work was initiated at the California
Institute of Technology, Pasadena, California.
taneous mutation to phage Ti resistance and
requirement for tryptophan (Ti, try-) in Esch-
erichia coli (Anderson, Proc. Natl. Acad. Sci.
U. S., 32, 120-128, 1946; Skaar and Davidson,
Cold Spring Harbor Symposia Quant. Biol., 18,
120-121, 1956; Yanofskv and Lennox, Virology,
8, 425-447, 1959). Phages used were P1 kc (Len-
nox, Virology, 1, 190-206) and Ti (described in
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NOTES
Adams, Bacteriophages, Interscience Publishers,
New York, 1959). Bacteria used were a nonlyso-
genic Hfr Hayes derivative of E. coli strain K-12,
(Wollman et al., Cold Spring Harbor Symposia
Quant. Biol., 21, 141-162, 1956), referred to as
strain K-12, E. coli strain C, referred to as strain
C, and a derivative of strain K-12 containing the
Tl ,try+ locus (ability to synthesize tryptophan
and sensitivity to T1) of strain C, referred to as
K-12(C), which was obtained by transducing
(by means of phage P1) first the Tl,try- locus
from strain C-5 of Bertani and Six (Virology, 6,
357-381, 1959) into strain K-12, selecting for
Ti resistance, and then the Tl,try+ locus from
strain C into such a transductant, selecting for
tryptophan independence.
Transductions with phage P1 were carried out
essentially as described by Lennox (Virology, 1,
190-206, 1955). Since P1 grown on strain C did
not grow well on strain K-12, characters to be
transduced into strain K-12 were first trans-
ferred by transduction from strain C to a hybrid
between strain C and strain K-12 obtained in
bacterial crosses, and then from the hybrid to
strain K-12. Bacterial crosses were made using
techniques described by Weinberg (J. Bacteriol.
79, 558-563, 1960).
For isolation of Ti-resistant mutants, single
colonies of Ti-sensitive strains were picked and
streaked onto plates previously coated with Ti.
Less than half of such streaks gave any mutants
at all. From each streak which gave rise to non-
mucoid Ti-resistant mutants, one such mutant
was picked and streaked on a minimal agar plate
containing phage T5 and tryptophan crystals
on one side of the plate, and classified, after in-
cubation, as to T5 resistance and tryptophan re-
quirement. Rare mutants gave no growth on these
plates. It was not investigated, however, whether
these were caused by faulty technique or by re-
quirements for factors other than tryptophan.
The distribution of mutant types obtained
from strain K-12 (C) resembles that of the mu-
tants from strain K-12 much more than that of
the mutants from strain C (table 1). Since in
strain K-12 (C) a small chromosomal region com-
prising the locus Ti,try originated from strain C
(by transduction), it appears that the high
mutability to the mucoid, Ti-resistant type is
not controlled by factors located in such a region
of the chromosome.
Of the Ti,try- mutants obtained from K-12,
21 were crossed to two Ti-sensitive tryptophan-
TABLE 1
Recovery of various types of independently isolated,
spontaneous T'i-resistant mutants of
Escherichia coli
Per Cent of Mutant types
Obtained from Strain
Type of Ti-Resistant Mutant
C K-12 K-12 (C)
Mucoid ..................... 2 45 45
Nonmucoid:
Prototrophic, resistant to
phage T5............. 70 27 44
Prototrophic, sensitive to
phage T5 18 26 9
Requiiring tryptophan,
sensitive to phage T5. 12 2 2
Requiring tryptophan, re-
sistant to phage T5. 0 0 0
Total mutants scored 53 451 110
requiring mutants obtained from Dr. C. Yanofsky,
td-3 and T-4 (Yanofsky, J. Biol. Chem., 224,
783-791, 1957), made F by the acridine
method (Hirota et al., Nature, 180, 655, 1957),
selecting for tryptophan independence in the
crosses. Only two gave recombinants. These
two were then crossed to each other (for
this purpose the two Tl,try- markers were first
crossed into K-12 F-Sr (streptomycin-resistant)
strains, so that Hfr X F crosses could be made),
but no tryptophan-independent recombinants
were obtained.
None of the 21 Ti1,try- mutants gave rise to
tryptophan independent revertants when ap-
proximately 1010 cells were spread on minimal
agar. Thus the Tl,try- mutation is probably a
deletion in strain K-12 (Yanofsky and Lennox,
Virology 8, 425-447, 1959) as it seems to be in
strain B (Skaar and Davidson, Cold Spring Har-
bor Symposia Quant. Biol., 18, 120-121, 1956).
The results of Weinberg (J. Bacteriol., 79,
558-563, 1960) indicate that the Tl ,try- marker
is in the same order on the genetic chromosome
as is the Try- mutation, unconnected with Ti
resistance, used by Jacob and Wollman (Ann.
inst. Pasteur, 95, 497-519, 1958), as one would
expect from Skaar and Davidson's, and Yanofsky
and Lennox's findings.
Three Ti-resistant, T5-sensitive, tryptophan-
independent mutants isolated independently
from strain K-12 were crossed to one of the K-12
F Ti ,try- SI strains mentioned above, and
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NOTES
tryptophan-independent recombinants were se-
lected in the presence of streptomycin. The 19
recombinants tested from each of the three
crosses were resistant to Ti. When strain K-12
was used as the male, selecting in the same way,
the 19 recombinants tested were Tl,try+. This
indicates close linkage between the Ti locus and
the Tl ,try locus in strain K-12, giving further
support to the idea that the T1 ,try- mutation
in K-12 is a simultaneous deletion of the Ti
locus, and of some or all of the loci controlling
tryptophan synthesis.
INDIGENOUS MARINE BACTERIOPHAGES'
R. SPENCER
Humber Laboratory, Department of Scientific and Industrial Research, Hull, England
Received for publication November 24, 1959
The presence in sea water beyond the littoral
zone of bacteriophages active against marine
bacteria has not been conclusively demonstrated
despite their possible importance in marine micro-
biology. Although ZoBell (Marine Microbiology,
1946) isolated phages from sea water of the
littoral zone, he was unsuccessful with water
taken beyond. The phages isolated by Kriss and
Rukina (Rept. U. S. S. R. Acad. Sci., 57, 833,
1947) from the Black Sea were active against
such typically terrestrial species as Bacillus
subtilis and Micrococcus albus (Kriss et al., Trans.
biol. Sta. Sebastopol, 7, 50, 1949, and Kriss,
personal communication) and may thus have been
adventitious, whereas the phage isolated by Smith
and Krueger (J. Gen. Physiol., 38, 161, 1954)
against a marine vibrio was not strictly marine in
origin. Consequently, attempts were made to
isolate phages from sea water taken well beyond
the littoral zone active against typically marine
bacteria.
The sea water samples were taken from the
North Sea, some 10 miles off Aberdeen, Scotland,
and the marine bacteria consisted of two groups,
strains of Photobacterium phosphoreum isolated
from marine fish, and strains of several species
isolated from a further sample of sea water. After
preliminary experiments, two methods of isola-
tion were used in parallel, one direct and one in-
direct, each with incubation at both 20 and 0 C.
The direct method consisted of layering on a
nutrient agar base a mixture of 10 ml of 1.5 per
cent nutrient agar, 10 ml of sea water previously
membrane filtered to remove interfering bacteria,
1 The work described in this paper was carried
out as part of the program of the Department of
Scientific and Industrial Research.
and 2 ml of a dense suspension of the appropriate
bacterial culture. After incubation, the presence
of any phage active against that particular cul-
ture was detectable by plaque formation. Sam-
ples (600 ml) of sea water at both 20 and 0 C were
normally examined at a time against a total of 40
strains of bacteria.
In the indirect method, 300 ml of sea water
were mixed with 100 ml of quadruple strength
nutrient broth and 8-ml amounts of broth cul--
ture of four different bacterial strains. After in-
cubation, the bacteria were removed by eitheI
filtration or by an adaption of the chloroform
technique of Fredericq (Compt. rend. soc. biol.,
144, 295, 1950) involving carbon tetrachloride,
and the bacteria-free preparation tested for
lytic action against the appropriate bacterial
strains by the above layer technique. Samples
(3 L) of sea water at both 20 and 0 C were nor-
mally examined at a time, again with 40 strains
of bacteria.
In all, 6 L of sea water were examined by the
direct method and almost 40 L by the indirect
method, and seven phages were isolated. Four of
the phages were detected by the parallel direct
and indirect methods at both 20 and 0 C; three
were present in samples in a concentration of 1
to 5 particles per 10 ml, and one in a concentra-
tion of approximately 100 particles per 10 ml.
The remaining three phages were detected by the
indirect method.
One phage was active against a strain of Photo-
bacterium phosphoreum, three against unidenti-
fied nonpigmented Pseudomonas species, two
against an unidentified Flavobacterium, and one
against an organism provisionally classified as a
Cytophaga species.
614 [VOL. 79
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Phage T1-resistant mutants of Escherichia coli

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Phage T1-resistant mutants of Escherichia coli

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