1. health and fitness
2. disease

JOURNAL OF BACTRIOLOGY, Jan. 1981, p. 358-368
0021-9193/81/010358-11$02.00/0
Vol. 145, No. 1
Introduction of Bacteriophage Mu into Bacteria of Various
Genera and Intergeneric Gene Transfer by RP4: :Mu
YOSHIKATSU MUROOKA,* NOBORU TAKIZAWA, AND TOKUYA HARADA
The Institute of Scientific and Industrial Research, Osaka University, Suita, Osaka (565), Japan
The host range of coliphage Mu was greatly expanded to various genera of
gram-negative bacteria by using the hybrid plasmid RP4::Mu cts, which is
temperature sensitive and which confers resistance to ampicillin, kanamycin, and
tetracycline. These drug resistance genes were transferred from Escherichia coli
to members of the genera Klebsiella, Enterobacter, Citrobacter, Salmonella,
Proteus, Erwinia, Serratia, Alcaligenes, Agrobacterium, Rhizobium, Pseudomonas, Acetobacter, and Bacillus. Mu phage was produced by thermal induction
from the lysogens of all these drug-resistant bacteria except Bacillus. Mu phage
and RP4 or the RP4: :Mu plasmid were used to create intergeneric recombinant
strains by transfer of some genes, including the arylsulfatase gene, between
Klebsiella aerogenes and E. coli. Thus, genetic analysis and intergeneric gene
transfer are possible in these RP4: :Mu-sensitive bacteria.
Bacteriophage Mu seems to be a potentially
useful tool for in vivo genetic engineering, because it can act as a transposable genetic element. The host range of Mu is normally restricted to certain strains of Escherichia coli,
Shigella dysenteriae, and Citrobacter freundii,
but not Salmonella typhimurium (21). A mutant
of Klebsiellapneumoniae that is sensitive to Mu
infection has also been isolated (30). However,
the insertion of Mu into broad-host-range plasmids of the P-1 incompatibility group, such as
RP4 or RK2, allow the introduction of Mu into
other strains of gram-negative bacteria (4, 7).
Unlike other temperate phages, bacteriophage
Mu inserts its DNA at many different loci in the
genome of its host bacterium, E. coli, and induces stable mutations by gene inactivation (3,
5, 35). Mu also mediates integration of circular
DNA, such as phage A DNA (3, 13), F factor (12,
39), and RP4 (10), into the host chromosome.
After induction or infection with Mu, closed
circles are formed that contain bacterial sequences covalently linked to complete Mu genomes. Mu can mediate transfer of chromosomal
markers by promoting the formation of plasmid
primes or the integration of plasmids into the
chromosome to form "intermediate Hfr" donor
strains (12, 39). This property and the finding
that the Mu genome can be expressed in new
hosts other than E. coli indicate that the Mu
genome should be very useful tool for genetic
manipulations (6, 10, 14).
Previously, we extended the host range of
coliphage P1 (25) and created intergeneric hybrid strains of enteric bacteria (26). However,
the arylsulfatase gene, atsK, from Klebsiella aer-
ogenes could not be transferred to E. coli, because the DNA homology of the ats genes of
these two bacteria is low. Nonhomologous gene
transfer between different bacteria would be
possible with a Mu prophages of a transmissible
plasmid and of the chromosome in a bacterial
strain because of recombination between the two
prophages (10, 39). In the present work, we
expanded the bacterial host range of Mu and
examined the conditions necessary for transfer
of several genes, including atsK, between taxonomically different bacteria by using RP4: :Mu
cts.
MATERIALS AND METHODS
Chemicals. Sodium ampicillin and kanamycin sulfate were obtained from Meiji Seika Co. Ltd., Tokyo,
Japan. Tetracycline hydrochloride was purchased
from Takeda Chemical Industries Co. Ltd., Osaka,
Japan. Cephalexin [7-(D-a-amino-a-phenylacetamido)-3-methyl-3-cephem-4-carboxylic acid] was
kindly provided by T. Ishimaru, Osaka University,
Suita, Osaka, Japan. [3H]tyramine hydrochloride was
purchased from the Radiochemical Centre, Amersham, England. p-Nitrophenyl sulfate, obtained from
Sigma Chemical Co., St. Louis, Mo., was recrystallized
from aqueous ethanol before use. The other compounds used were standard commercial preparations.
Bacterial strains and phage. The bacterial
strains used in this study are listed in Table 1. Most of
these strains were obtained from the Institute for
Fermentation, Jusonishino-cho, Higashi-yodogawaku, Osaka, Japan (IFO), but E. coli EG47 was obtained
from R. A. Bender, Massachusetts Institute of Technology, Cambridge, Mass., and E. coli strains JC5466
(RP4::Mu cts62) and BE228 (RP4::Mu cts6l) were
kindly provided by J. Denari6 (Institut National de la
Recherche Agronomique, Paris, France) and R. N.
358
EXPANSION OF THE HOST RANGE OF Mu
VOL. 145, 1981
359
TABLE 1. List of bacterial strains and their characteristics
Strain
Escherichia coli
C600
EG47
BE228
JC5466
CT3
CTM4
CTMAl
CTMA2
CTMA3
CTMA4
CTMA5
Relevant characteristics or geno-
type
Source or reference
Mu5 hsdRa hsdMa thr-1 leu-6
thi-1
Mu' lac gal hsdR
thr-1 leu-6 thi-1 (RP4::Mu cts6l)
trp his recA56 rps (RP4::Mu
cts62)
thr-1 leu-6 thi-I tynb
thr-I leu-6 thi-I t7p::Mu cts62
tyn::Mu' cts62
thr-1 leu-6 thi-l trpKd tYnK+
atsK+e
thr-l leu-6 thi-1 tiPK+ tYnK+
atsK+
leu-6 thi-I
thr-strPKK
tYnK
atsK+
thr-1 leu-6 thi-1 trPK+ tYnK
atsK+
thr-1 leu-6 thi-1 trPK+ tYnK+
This laboratory (26)
Mu' wild type
Mu'
Mu'
Mu'
Mu'
Mu'
This laboratory (24)
This laboratory (26)
Mutagenesis of W70
RP4::Mu cts6l mated with K204
RP4 mated with the Mu cts61
lysogen of K204
Mutagenesis of MK9000 by Mu
IFO 3317
IFO 3318
IFO 12010
IFO 3320
This laboratory (26)
IFO 12681
IFO 3849
IFO 13501
IFO 3406
IFO 12687
IFO 3380
Mu'
IFO 3084
Mu'
Mu'
Mu'
Mu'
Mu'
Mu'
Mu'
Mu'
Mur
This laboratory (17)
This laboratory (18)
IFO 3058
IFO 12664
IFO 13337
IFO 13338
IFO 3456
This laboratory (19)
IFO 3130
Mur purB6 leu-8 metB5 nonA
nonB+
Mur leu arg nonA nonB
H. Saito (33)
attsK+
R. A. Bender (15)
R. N. Rao (30)
J. D6narie (4)
Mutagenesis of MK9000
Mutagenesis of C600 by Mu
RP4::Mu cts6l mated with
CTM4
RP4::Mu cts6l mated with
CTM4
RP4::Mu cts6l mated with
CTM4
RP4::Mu cts61 mated with
CTM4
RP4::Mu cts6l mated with
CTM4
Klebsiella aerogenes
W70
MK9000
K204
K204-1
K204-2
MKNM1
Klebsiellapneumoniae
KlebsieUapneumoniae
Enterobacter aerogenes
Enterobacter cloacae
Sabnonella typhimurium LT2
Citrobacter freundii
Proteus mirabilis
Proteus rettgeri
Serratia marcescens
Erwinia amylovora
Erwinia carotovora subsp. caroto-
Mur Pli
Mu' cys pur
cyspur (RP4::Mu cts6l)
cys pur Mu cts6l (RP4)
tyn::Mu cts6l
Mu'
Mu'
Mur
Mu'
Mur
Mur
vora
Alcaligenes (Achromobacter) liquidum
Akaligenes faecalis subsp. myxogenes
22
10C3K
Agrobacterium tumefaciens
Agrobacterium radiobacter
Rhizobium trifolii
Rhizobiumjaponicum
Pseudomonas (aeruginosa)
Pseudomonas amyloderamosa KIC
Acetobacter suboxydans
Bacillus subtilis
101
Y125
Bacillus subtilis
Bacillus macerans
Bacillus cereus
Bacillus sphaericus
Mur
Mur
Mur
Mur
H. Saito (33)
IFO 3021
IAMf 1227
IFO 3001
IFO 3341
360
MUROOKA, TAKIZAWA, AND HARADA
J. BACTERIOL.
TABLE 1-Continued
Strain
Relevant characteristics or genotype
Mu'
Mur
Mur
Mur
Source or reference
Arthrobacter snppkx
IAM 1660
Brevibacterium ammloniagenes
LAIM 1641
Corynebacterium acetophilum A51
This laboratory
Corynebacterium ethanolaminoThis laboratory
philum E17
Mur
Micrococcus luteus
IFO 12708
Mur
Mycobacterium smegmatis
IFO 3384
Mur
Staphylococcus aureus
IFO 3145
a hsdR, Host restriction activity; hsdM, host modification activity (1).
b
tyn, Tyramine oxidase defective (1, 26).
C:: indicates that Mu is inserted into the gene preceding.
trpK, t7p region of K aerogenes.
atsK, Structural gene for arylsulfatase of K. aerogenes (24).
f IAM, Institute of Applied Microbiology, University of Tokyo, Tokyo, Japan.
Rao (University of California, Davis), respectively.
Media. The rich media used were Penassay broth
(antibiotic assay medium 3; Difco Laboratories, Detroit, Mich.) and LB medium, consisting of 1%
tryptone (Difco), 0.5% yeast extract (Difco), and 0.5%
NaCl. The supplemented LB media used were as
follows: LBC, LB plus 5 mM CaCl262H20; LB plus 25
pig of kanamycin per ml and LBAKT, LB plus ampicillin (100 pug/ml), kanamycin (25 pig/ml), and tetracycline (50 pg/ml). The minimal medium used for
most bacteria was K medium, consisting of 0.5% carbon source, 0.1% nitrogen source, 0.1 M potassium
phosphate buffer (pH 7.2), 0.01% MgCl2.6H20, 1 mM
sulfur compound, and 0.001% each NaCl, MnCl2.
4H20, and FeCl3 6H20. The medium used for growth
of E. coli, S. typhimurium, Proteus mirabilis, Proteus
rettgeri, Erwinia amylovora, and Erwinia carotovora
was supplemented with 1 mM CaCl2.2H20 and thiamine (10jpg/ml). Unless otherwise mentioned, glucose,
NH4Cl, and Na2SO4 were used as the carbon, nitrogen,
and sulfur sources, respectively. Sodium glutamate
was used as the nitrogen source for Pseudomonas
amyloderamosa KIC (19). For growth of Rhizobium
strains (NH4)2HP04 was used as a nitrogen source,
and thiamine (10 pg/ml) and biotin (0.5 pg/ml) were
added to the K medium. For growth of Bacillus cereus,
CDS medium (28) was used. Amino acids (50 pg/ml)
were added when necessary.
Bacterial mating. Transfer frequencies were determined by mating on membrane filters as described
by De Graff et al. (9). Crosses between E. coli strain
JC5466 or BE228 and various strains of bacteria were
made as follows. Exponentially growing donors containing RP4::Mu cts (about 2 x 109 to 3 x 108 colonyforming units per ml) were mixed with an equal volume of recipient cultures in the late exponential phase
(about 2 x i09 colony-forming units per ml). The
mixture was filtered on a Toyo membrane filter (0.45pAm pore size, 25-mm diameter-, Toyoroshi Co. Ltd.,
Tokyo, Japan). Membranes were placed on the surface
of a freshly prepared Penassay broth agar plate and
incubated at 30°C for 5 h. Bacteria were then suspended in 1 ml of saline, and 0.2-ml samples of suitable
dilutions were spread on the selective medium. Donors
were counterselected by omission of an essential nu-
trient. Unless otherwise stated, transfer of RP4::Mu
cts was selected with 100 pg of ampicillin, 50 pg of
tetracycline, or 25 pug of kanamycin per ml. For selection of members of the genera Serratia, Alkaligenes,
Agrobacterium, Pseudomonas, Rhizobium, and Bacillhs, 100pug of kanamycin per ml was used. In control
experiments, donor and recipient cultures were filtered
on separate membranes and then incubated and plated
under the same conditions as for mating. Transfer
frequencies are expressed per donor cell. Most of the
transcipient cells proved to be Mu cts lysogens. Mu
cts lysogeny was checked by transferring a purified
colony to two LB plates, and incubating one at 30°C
and the other at 41°C Lysogens of Mu cts grow well at
30°C and very poorly at or above 356C. P. amyloderamosa, Alcaligenes faecalis, Agrobacterium tumefaciens, Agrobacterium radiobacter, Rhizobum trifolii,
and Rhizobium japonicum showed very poor growth
at 41°C, even when not in infected with RP4::Mu cts.
Preparation of phage lysates and titration of
Mu. A modification of the method of Razzald and
Bukhari (31) was used for preparation of Mu cts
lysates of purified lysogens. Overnight cultures of lysogens of all bacteria except Rhizobium, grown in
LBAKT medium at 30°C, were diluted 1:100 with LBC
medium and grown with reciprocal shaking to a density of about 108 bacteria per ml. Chemically defined
medium was used for R. trifolii and R. japonicum,
since they cannot grow in LB medium. MgS02 7H20
was added at a final concentration of 0.2 M, and the
cultures were incubated at 43°C for 30 min with vigorous shaking and then at 37°C until lysis was observed (about 30 to 90 min, depending on the strain
used). Lysis was detected as decrease in turbidity and
the appearance of flocculated cells at the bottom of
test tubes. After lysis, the culture was treated with
chloroform, and the debris was removed by centrifugation. Phages were stored over a few drops of chloroform.
Phage titers were determined as follows. E. coli
EG47 was grown in LBC medium containing 10 mM
MgSO4.7H20 to a density of 109 bacteria per ml.
Appropriate dilutions of the lysate (0.2 ml) were mixed
with the culture broth of strain EG47 (0.2 ml) and
incubated at 30°C for 30 min to allow adsorption.
VOL. 145, 1981
EXPANSION OF THE HOST RANGE OF Mu
361
cae and P. rettgeri also showed high efficiencies
of transfer of drug resistance genes. Enterobacter aerogenes, P. mirabilis, E. carotovora, and
Serratia marcescens showed similar orders of
efficiency of transfer. A few drug-resistant colonies of S. typhimurium LT2 were obtained, but
their number was 10i times less than that of K.
aerogenes W70, which is taxonomically related
to the genera Salmonella and Escherichia. Gen7
erally, the number of drug-resistant colonies obtained was proportional to the number of RP4::
Mu cts-carrying cells added. The members of
recipient cells used was about twice that of donor
cells.
The genera mentioned above are all Enterobacteriaceae. We also succeeded in transferring
the genes for kanamycin or tetracycline resistance or both to Alcaligenes (Achromobacter)
liquidum, A. faecalis subsp. myxogenes, A. radiobacter, A. tumefaciens, P. amyloderamosa,
RESULTS
and Acetobacter suboxydans (Gluconobacter
Transfer of genes for antibiotic resistTABLE 2. Transfer frequencies of RP4::Mu
ance to various bacteria. RP4: Mu plasmids
from E. coli strains JC5466 (RP4::Mu
plasmids
in E. coli strains were used to select Mu-sensitive cts62) and BE228
(RP4::Mu cts6l) to various genera
of
bacteria.
variants from a resistant population
of bacteriaa
RP4::Mu cts6l and RP4::Mu cts62 confer amTransfer frequencyc
picillin, kanamycin, and tetracycline resistances,
Selected
strain
Recipient
and cells are temperature sensitive when the
markerb RP4::Mu RP4::Mu
cts62
cts6l
prophage is present (4). When 3 x i05 cells of E.
coli BE228 (keu thr thi RP4: :Mu cts6l) and 2 x E. coli C600
x
2 10-4
Ap Km Tc
Ap Km Tc 3 x 10-2 1 x 10-3
108 cells of strain JC5466 (thp his RP4: :Mu cts E. coli EG47
5
x
Km Tc
10-4 6 x 10-4
62) were mated with 5 x 10' cells of E. coli EG47 K. aerogenes W70FO 3318 Ap
Ap Km Tc 2 x 10-5 2 x 10-6
pneumoniae
(RP4::Mu and antibiotic sensitive) on mem- K.
IFO
x
12010 Ap Km Tc 3 10-7 2 x 10-7
E. aerogenes
brane filters, about 9 x 10" and 2 x 105 antibiotic- E. cloacae IFO 3320
Ap Km Tc 6 x 10-6 5 x 10-6
resistant colonies, respectively, were obtained. S. typhiutrium LT2
Ap Km Tc 3 x 10" 3 x 10-9
Ap Km Tc 7 x 10-5 6 x 10-4
This indicates that about 1o-3 to 10-2 of the C. freundii IFO 12681
1 x 10-7
Ap Km Tc
mirabilis IO 3849
RP4::Mu cts plnids transferred from the do- P.
1 x 10-6
Ap Km Tc
P. reffgeri IFO 13501
nors formed drug-resistant colonies (Table 2).
1
x 10-7 X 10-7
S. marcescens IFO 3046 Km
The effects of mating time, temperature, and E. amylovora IFO 12687 Km Tc 4 x 10-8 1 X 10-7
medium on intergeneric transfer between K. aer- E. carotovora subap. car- Km Tc 2 x 10-7 6 x 10-7
otovora IFO 3380
ogenes W70 and A. faecalis subap. myxogenes
Km Tc
2 x 10-9 2 x 10-9
A. liquidum IFO 3084
22 as recipients and E. coli JC5466 as a donor A.
8 x 10-9 4 x 10-9
faecalis subsp. myxo- Km Tc
strain were tested. The transfer frequencies were
genes 22
3 x 10-9 2 x 10A. radiobacter IFO 12664 Km Tc
maximal in 3 to 5 and 5 to 8 h when K. aerogenes
7 x 10`"
tumefaciens IFO 3068 Tc
and A. faecalis subap. myxogenes, respectively, A.
5 x 10-9d 3 x 10T9d
R. japonicum IFO 13338 Km
were mated on membrane filters placed on PenKm
2 x l10"d 1 x i0T"d
R. trifoli IFO 13337
assay broth plates. However, significant differ1 x 10-9 7 x 10`'
Km
Pseudomonas (aeruginosa) IFO 3456
ences in the frequencies were not observed beKIC Km
9 x i0-9
tween 30 and 370C. Thus, matings between the P. amyloderamosa
5 x 10-1"
suboxydans IFO 3130 Km
various bacteria and E. coli donors were carried A.
Km Tc
5 x 10-'° 3 x 10-'°
B. cereus IFO 3001
out for 5 h on membrane filters at 30°C placed
aAbout 1 x 109 to 5 x 10' recipient strain bacteria per ml
on the surface of Penassay broth agar. The
used, and mating was performed on membrane filters for
efficiencies of transfer of antibiotic resistances were
5 h at 300C.
to various strains of gram-negative bacteria were
b Ap, Ampicillin resistance; Km, kanamycin resistance; Tc,
compared (Table 2). Of the antibiotic-sensitive tetracycline resistance.
initial donor (E. coli JC5466, 2 x 1O9/ml; strain BE228,
bacteria tested, K. aerogenes, K. pneumoniae, 3 x Per
109/ml).
and C. freundii formed the largest numbers of
d The recipient strain was heated at 55°C for 6 min before
antibiotic-resistant colonies. Enterobacter cloa- mating.
Then 3 ml of top agar was added, and the mixture was
poured into LBC plates and incubated at 410C for 16
h.
Mu-induced mutations. Strains containing RP4::
Mu cts were grown overnight at 380C without shaking.
This partial induction was mutagenic for the host
chromosome and killed about 50% of the cells. Chlorate-resistant mutants were selected as described by
Boram and Abelson (3). Auxotroph mutants of K.
aerogenes and E. coli were enriched by treatment
with cephalexin (500 pg/ml) instead of penicillin. Ampicillin-resistant strains of E. coli and K. aerogenes
were killed on incubation for 2 to 4 h in K medium
containing 250 pg of cephalexin per ml. Tyramine
oxicase-negative mutants were isolated as described
previously (24).
Enzyme assays. Arylsulfatase and tyramine oxidase activities were assayed as descibed previously
(24). One unit of arylsulfatase activity was defined as
the amount causing formation of 1 nmol of p-nitrophenol per min at 300C.
c
362
MUROOKA, TAKIZAWA, AND HARADA
J. BACTERIOL.
oxydans subsp. suboxydans). Most of these
strains were already ampicillin resistant, and
some of them were tetracycline resistant. The
efficiencies of transfer of the plasmid were much
lower in these strains than in strains of the
family Enterobacteriaceae. No transconjugant
was obtained by using R. japonicum or R. trifolii
as a recipient. Since heat-sensitive restriction
has been described in a Rhizobium strain (6), we
treated the recipient Rhizobium cultures at
550C for 6 min before mating and so obtained
transconjugants at a frequency of about 10-8.
This indicates that the transfer frequency was
increased about 101 to 102 times by heat treatment, suggesting that the restriction system was
relieved by heating. These transconjugants were
unable to grow on Penassay broth or LB at
410C. Drug-resistant colonies were obtained
from B. cereus IF03001, but not from any other
strains of gram-positive bacteria tested, including several strains of Bacillus subtilis, Bacillus
macerans, and Bacillus sphaericus and members of the genera Staphylococcus, Brevibacterium, Corynebacterium, Micrococcus, and Mycobacterium. The prototrophs of the E. coli
donor strains JC5466 and BE228 were not reversed when 1011 cells were used on the selection
plates. Transconjugants obtained at low transduction frequencies were also tested for unselected markers: members of the genera Alcaligenes and Agrobacterium were tested for production of curdlan (8B-1,3-glucan) or succinoglycan-type polysaccharide by using polypeptoneyeast extract plates containing aniline blue as
indicators (27); R. trifolii and R. japonicum were
tested for production of polysaccharide in minimal medium with 4% glucose (16) and for inability to grow in anaerobic conditions with nitrate (4); P. amyloderamosa KIC was tested for
ability to grow on waxy corn starch and to
produce isoamylase (19). B. cereus was easily
distinguished from the E. coli donor strain by
its colony type and by Gram staining. These
results also show that plasnid RP4, of the P-1
incompatibility group, has a much broader host
range than reported previously (8).
Production of Mu phage by drug-resistant bacteria. Transconjugants were also confirmed by demonstrating that the drug-resistant
and temperature-sensitive clones isolated were
capable of releasing phage and forming plaques.
Cells lysogenic for Mu cts are temperature inducible, owing to a mutation in the repressor
gene C (3). To eliminate the few contaminating
donor cells of E. coli, we purified the drug-resistant and temperature-sensitive clones by successive single colony isolation on LB plates without antibiotics and then twice on the correspond-
ing selective plates containing ampicillin, kanamycin, and tetracycline. The Mu-phage yields
from the lysogens of E. coli JC5466, K. aerogenes MK9000, P. amyloderamosa KIC, and A.
faecalis subsp. myxogenes 22 were tested. Lysogens were cultured in LBC broth to a density
of about 1 x 108 to 5 x 108 cells per ml, heated
at 43°C for 30 min, and then shifted to 37°C
until lysis was observed. The times required for
lysis after shifting to 37°C were about 20 to 30
min for the lysogens of E. coli JC5466 and K.
aerogenes MK9000 and about 40 to 60 min for
those of P. amyloderamosa KIC and A. faecalis
subsp. myxogenes 22. In contrast, fewer phages
were produced and longer heat treatment was
required when the cultures were shifted to 30°C.
Lysis was indicated by decrease in turbidity and
the appearance of flocculated cells at the bottoms of the test tubes. When a higher density of
cells (>109 cells per ml) was used, a longer heating time was required. Unexpectedly, we found
that addition of a high concentration of MgSO4
to LB broth before heat induction stimulated
the yield of Mu phage: phage production was
maximal when cells were incubated with 0.2 to
0.4 M MgSO4 (Fig. 1), and under these conditions the number produced was 102 times greater
than that without MgSO4. Lysis was delayed
when lysogens were incubated with over 0.5 M
MgSO4. MgCl2 had a similar effect. However,
addition of CaCl2 was less effective, although it
slightly increased the phage yield. Thus, we used
LBC containing 0.2 M MgSO4 in subsequent
experiments. Cells lysogenic for Mu cts in 5-ml
cultures in test tubes (diameter, 16 mm) were
cultured with reciprocal shaking at 140 rpm during heat induction. Lysis was delayed or not
observed when cells were cultured with poor
aeration (<110 rpm).
When Mu phages from K. aerogenes W70
were seeded onto lawns of E. coli strains W3350
and EG47 (a restrictionless strain), phages
formed plaques on strain W3350 with an efficiency of about 10-1 to 10-2 of that on strain
EG47, suggesting the operation of a restrictionmodification system in strain W3350. Therefore,
E. coli EG47 was used as an indicator in assays
of Mu phage from various bacteria. The highest
efficiency of plating was obtained with 5 mM
CaC12 and MgSO4 in LB plates incubated at
410C for 6 to 16 h. Table 3 shows typical phage
yields from various lysogens obtained by thermal induction. Although the phage yield in different bacteria and in different Mu cts types
varied considerably, all the drug-resistant strains
except B. cereus produced temperature-sensitive
Mu phages. No plaques of phage from various
lysogens were seen when either Mu-resistant or
EXPANSION OF THE HOST RANGE OF Mu
VOL. 145, 1981
363
a
\a
Ur
aU0
h2
a
aV
El
MgS04 ceac. (mu)
FIG. 1. Effect ofMgSO4 concentration on thermal induction of Mu. A 20-h culture of a lysogen grown in LB
containing 100 jig of ampicillin, 25 pg of kanamycin, and 50 pg of tetracycline per ml at 28°C was diluted 1:10()
with LB and grown with reciprocal shaking at 28°C to a density of about I x lte to 6 x 1lO bacteria per ml.
Various amounts of MgSO4.7H20 were added to LB or LBC medium, and the culture was incubated at 43°C
for 30 min and then at 37°C until lysis was observed. Symbols: closed, results in LB broth with various
concentrations of MgSO4. 7H20; open, results in-LBC broth with MgS04. 7H20; 0, 0, E. coli BE228 (Mu
cts6l); A, E. coli JCB466 (Mu cts62); U, A. faecalis subsp. myxogenes 1OC3K (Mu cts62); *, 0, P. amyloderamosa
KIC (Mu cts6l); *, S. typhimurum LT2 (Mu cts6l).
Mu-lysogenic strains were used as recipients. It
was clear that these strains were capable of
producing viable Mu particles. The lysogens of
all Enterobacteriaceae except P. rettgeri
IF013501 produced higher yields of Mu phage
than did other families of bacteria. Drug-resistant strains of R. trifolii and R. japonicum produced very low numbers of phage, and no phage
were obtained with any drug-resistant and temperature-sensitive clones of B. cereus.
Stability of RP4: :Mu cts in various bacteria. To test the stability of RP4::Mu cts in
bacteria, we purified drug-resistant colonies of
K. aerogenes MK9000 obtained by conjugation
with E. coli JC5466 or BE226 and grew them
overnight at 380C. This partial induction was
mutagenic for the host chromosome, and the
frequency of mutations of chlorate resistance
(Chl') among survivors was about 5 x 1O-5. Each
Chl' colony was grown overnight in LB broth.
Then the cells were replicated on LB plates with
ampicillin, kanamycin, and tetracycline and on
two LB plates without antibiotics, one incubated
at 280C and the other incubated at 410C. About
25 of the Chlr mutants of K. aerogenes MK9000
(RP4: :Mu cts6l) carried Mu cts without RP4 or
neither RP4 nor Mu cts (Table 4). RP4::Mu
cts61 or RP4: :Mu cts62 in E. coli also segregated
to RP4 without Mu cts or was cured completely.
The segregated cells containing Mu cts without
RP4 released Mu phage by heat induction. Since
the Chl' mutant should contain Mu prophage,
364
MUROOKA, TAKIZAWA, AND HARADA
TABLE 3. Mu phage yield by thermal induction
from various bacterial strains carrying RP4::Mua
Plaque-forming units/
Mlb
Bacterial strain
Mu cts6l
Mu cts62
2 x 109
E. coli JC5466
E. coli BE228
2 x 109
K. aerogenes MK9000
4 x 10O'
6 x 10"
K. aerogenes W70
1 X 10l
4 x 105
K. pneumoniae IFO 3317
4 x 107
3 x 108
1 x 109
K. pneumoniae IFO 3318
2 x 109
8 X 1i0
E. aerogenes IFO 12010
2 x 10"
E. cloacae IFO 3320
3 x 107
3 x 108
S. typhimurium LT2
3 x 109
2 x 10"
8 X 107
C. freundii IFO 12681
7 x 107
5 x 107
2 x 107
P. mirabilis IFO 3849
8 X 105
P. rettgeri IFO 13501
5 x 108
S. marcescens IFO 3046
4 x 107
2 x 107
E. amylovora IFO 12687
4 x 108
1 x 109
2 x 109
E. carotovora subsp. carotovora IFO 3380
1 X 107
2 x 107
A. liquidum IFO 3084
3 x 107
2 x 107
A. faecalis subsp. myxogenes 22
1 X i06
A. faecalis subsp. myxo4 X 107
genes lC3K
5 X 107
4 x 107
A. radiobacter IFO 12664
6 x 10l
A. tumefaciens IFO 3058
1 X 103
1 X 103
R. japonicum IFO 13338
1 X 104
1 X 103
R. trifolii IFO 13337
6 X 107
4 x 107
Pseudomonas (aeroginosa)
IFO 3456
2 x 106
P. amyloderamosa KIC
5 x 106
A. suboxydans IFO 3130
a A 20-h culture of a lysogen grown in LB medium
containing ampicillin, kanamycin, and tetracycline at
280C was diluted 1:000 with LBC and grown with
reciprocal shaking to a density of about 1 x 108 to 6
x 108 bacteria per ml. After addition of MgSO4 at a
final concentration of 0.2 M, the culture was incubated
at 430C for 30 min and then at 370C until lysis was
observed. After lysis, the culture was treated with
chloroform, and the debris was removed by centrifugation.
b The phage titer was determined with E. coli EG47
as the indicator.
the RP4: :Mu cts plasmid was more unstable in
K. aerogenes than in E. coli when grown in
nonselective medium.
The stabilities of lysogens of various bacteria
stocked in LB agar for 3 to 4 weeks at 150C were
tested after growing the lysogens in LB broth for
20 h at 280C. The loss of RP4: :Mu cts plasmids
from lysogens of S. typhimurium LT2, A. faecalis subsp. myxogenes strains 22 and 1OC3K, A.
tumefaciens IFO 3058, P. amyloderamosa KIC,
and A. suboxydans IFO 3130 varied in different
strains (18 to 49%), but was not significantly
higher than that of K. aerogenes. Conuugants of
B. cereus IFO 3001 with drug resistance, proba-
J. BACTERIOL.
bly having RP4::Mu cts6l or RP4::Mu cts62,
grew poorly at 410C (whereas the drug-sensitive
strain grew well at 4100) and were very fragile
even at lower temperatures (5 to 300C). When
drug-resistant cells of B. cereus were grown in
LB broth without antibiotics, more than 99% of
the cells lost drug resistance.
Intergeneric gene transfer by RP4::Mu
cts and RP4 Mu cts. Partial heat induction of
Mu results in formnation of Mu-bacterial circular
DNA which can be integrated at random into
the bacterial chromosome or into a plasnid.
This can lead to transfer of the bacterial sequences carried by this circular DNA to any
location in either the chromosome or the plasmid (10, 39). For intergeneric transfer of various
genes, including ats, from K. aerogenes strains
to E. coli strains, we constructed RP4 Mu cts 61
in K. aerogenes by introducing RP4 without Mu
into strains carrying the Mu cts6l prophage on
the chromosome, using segregants isolated as
shown in Table 4. Anticipating that the frequency of such intergeneric transduction would
be low because of the existence of a restrictionmodification system and action of zygotic induction when the RP4 DNA carried a Mu prophage
(10), we used Mu-lysogenized E. coli C600, a
restrictionless strain, as the recipient. Thus, E.
coli C600 was lysogenized with Mu cts62. The
tyn::Mu cts, trp::Mu cts, and tyn::Mu cts trp::
Mu cts mutants were obtained by partial heat
induction at 380C for 16 h. These mutants were
isolated by enrichment with cephalexin (500 ltg/
ml) instead of treatment with penicillin. E. coli
and K. aerogenes strains carrying RP4 were
resistant to penicillin G but were sensitive to
cephalexin, which (like penicillin G) inhibits cell
TABLE 4. Segregation of RP4::Mu cts of K.
aerogenes and E. coli'
No. of colonies
Strain
K. aerogenes MK9000
Chlr
RP4 and
Mucts
56
38
Mu Neither
Mu~CtsR4csRP4
~~Muctsnor
4
1
13
(RP4::Mu cts6l)
42
1
2
11
K. aerogenes MK9000 56
(RP4::Mu ctb62)
0
2
0
56
50
E. coli BE228 (RP4::
Mu cts6l)
0
0
47
E. coli JC5466 (RP4::
56
9
Mu ctb62)
a Bacterial strains carrying RP4::Mu ct were heated at
38°C for 16 h, and Chl' mutants were isolated. The Chlr
colonies were grown in LB broth for 16 h at 300C. Single
colonies isolated on LB plates were replicated onto LB plates
containing ampicillin, kanamycin, and tetracycline and onto
two LB plates, one incubated at 28°C and the other incubated
at 41°C. Lysogens of Mu cts grew well at 28°C, but very poorly
at 41°C.
EXPANSION OF THE HOST RANGE OF Mu
VOL. 145, 1981
division (34). K. aerogenes strains carrying a
thermoinducible Mu prophage on the chromosome and RP4 or RP4::Mu cts61 in the cytoplasm were mated with polyauxotrophic E. coli
recipients with or without Mu prophage. Transductants which had lost one of their auxotrophic
requirements were selected. These clones were
expected to contain an RP4 or RP4::Mu cts
plasmid that had gained the corresponding wildtype allele from the donor. The donor cells were
preincubated at 38°C.
As expected from the random integration of
Mu and the plasmid after phage induction, all
the markers were transferred at relatively high
frequencies (Table 5). Strain K204-1 (RP4::Mu
cts6l) showed higher efficiencies of transfer of
the trp and tyn-ats genes than did strain K2042 (RP4) (Mu cts) as donor. This may be due to
differences in the populations of random Mupromoted Hfr formation in the donor cells reultng from recombination between homologous Mu regions, with consequent mobilization
of the chromosome (39). However, at least part
of the transfer was due to the formation of RP4prime episomes, which transferred the thr, leu,
tip, tyn, and ats genes after induction of Mu cts
prophages in the chromosome. When E. coli
strain CT3, without Mu prophage in the chromosome, was used as a recipient, no Tyn+ Ats+
recombinants were detected, but Thr+ Leu+ intergeneric recombinants were obtained at a low
frequency. These observations suggest that Mu
integrations into the tip and tyn genes increase
the efficiency of intergeneric gene translocation
TABLE 5. Intergeneric gene transfer by RP4::Mu
cts and RP Mu cts from K. aerogenes to E. colia
Donor strain
None
Gene transfer
frequency (per
Recipient strain (geno- 10' donors)
type)
thr trp tzyn
E. coli CT3 (eu thr
1
0
tyn)
E. coli CTM4 (eu thr 1
2 0
trp::Mu cts62 tyn::
Mu cts62)
K. aerogenes K204-1 E. coli CT3 (leu thr
0
5
(RP4::Mu cts61)
tyn)
E. coli CTM4 (leu thr 35 249 125
tqp::Mu cts62 tyn::
Mu cts62)
K. aerogenes K204-2 E. coli CT3 (ku thr
0
8
(RP4) (Mu cts6l)
tyn)
E. coli CTM4 (ku thr 33 195 31
trp::Mu cts62 tyn::
Mu cts62)
a Donor strains were incubated at 38°C for 16 h. About 1
x 10' donor bacteria were mixed with 2 x 109 recipients, and
the mixtire was filtered on a membrane and then incubated
at 30°C for 5 h.
365
by homologous recombination between Mu
DNAs and that zygotic induction is prevented
by Mu cts prophages in recipient cells.
Regulation of arylsulfatase synthesis in
intergeneric recombinants of enteric bacteria. The expressions of the tynK and atsK
genes from K. aerogenes in E. coli were investigated. K. aerogenes strains MK9000 (wild
type) and MKNM1 (tyn::Mu cts6l), E. coli
strains C600 and CTM4 (tyn::Mu cts62 tip: :Mu
cts62), and the intergeneric recombinant strains
isolated as shown in Table 5 were grown with
various sulfur sources and with or without tyramine. Strains CTMA1, CTMA2, CTMA3,
CTMA4, and CTMA5 were derived from E. coli
strains CTM1, CTM2, CTM3, CTM4, and
CTM5, respectively, which were isolated independently as tyn and tip strains, like CTM4, by
Mu integration into these genes of strain C600.
The cells were harvested during the exponential
phase of growth and assayed for arylsulfatase.
In the K. aerogenes mutant strain MKNM1, the
repression of arylsulfatase by inorganic sulfate
or cysteine was not relieved by addition of tyramine (Table 6). Since the tyn gene in strain
MKNM1 is inactivated by insertion of Mu
phage, this observation confirms that derepression of arylsulfatase synthesis is due to the synthesis of tyramine oxidase (24). Moreover, no
activity was observed in E. coli strains C600 and
CTM4 even in the presence of atsz, as shown
previously (26, 38). The cells of recombinants
containing the tynK and atsK genes in E. coli
grown with sodium sulfate or cysteine as a sulfur
source had a lower level of arylsulfatase, and
this repression of arylsulfatase synthesis was
derepressed by addition of tyramine, whereas,
as in K. aerogenes MK9000, arylsulfatase was
synthesized in E. coli when the recombinant
cells were grown with methionine or taurine as
the sole source of sulfur. These results show that
the regulation system for arylsulfatase synthesis
in E. coli is similar to that in K. aerogenes.
DISCUSSION
K. aerogenes, which is taxonomically related
to E. coli and to S. typhimurium, has a number
of metabolic abilities not found in the latter two
strains; in particular, it is capable of metabolizing arylsulfate ester (24), pentitol (32), histidine
(29), and pullulan (2, 23). However, in most
bacteria our understanding of these interesting
properties is limited by lack of a suitable gene
transfer system.
Most of the bacterial strains used here are
important in agriculture or in industrial microbiology: K. aerogenes produces pullulanase (2,
23); some strains of K. pneumoniae (30), R.
366
MUROOKA, TAKIZAWA, AND HARADA
J. BACTERIOL.
TABLE 6. Regulation of arylsulfatase synthesis in intergeneric recombinants between K. aerogenes and E.
coli
Arylsulfatase activity (U/mg of cels)& with:
Strain (relevant genotype')
Na2SO4
Cysteine
Taurine
Methio- Na2SO4 + Cysteine
nine
tyramine
minera
K. aerogenes
4.11
4.63
0.12
9.8
10.9
0.13
MK9000 (wild type)
0.13
0.11
7.6
11.2
0.09
0.11
MKNM1 (tyn::Mu cts6l)
E. coli
0.01
0.01
0.01
0.01
0.01
0.01
C600 (tynE+ atsE+)
0.01
0.01
0.01
0.01
0.01
0.01
CTM4 (tyn::Mu cts62 trp::Mu cts62
atsE+)
3.20
0.15
0.17
3.36
10.5
10.3
CTMAl (tynK tpK atsKx)
0.07
0.05
3.73
1.50
5.22
3.35
CTMA2 (tynK+ trpxK+ atsK+)
0.11
1.38
0.04
7.24
6.20
9.06
CTMA3 (tynK trpK atsK )
0.09
0.08
5.78
7.74
3.54
5.34
CTMA4 (tynK trpK( atsx )
0.34
0.30
8.60
10.0
9.71
5.07
CTMA5 (tynK trpK1 atsKx)
a K, Genes from K. aerogenes; E, genes from E. coli.
b The cells were grown in xylose-NH4CI medium with the various sulfur sources indicated in the presence or
absence of 3 mM tyrarnine. The cells were harvested and assayed when the density of the culture had reached
about 100 to 150 Klett units. One unit of arylsulfatase activity was defined as the amount causing formation of
1 nmol of p-nitrophenol per min at 300C. Values are averages for three independent experiments.
japonacum, and R. trifolii have genes for nitrogen fixation; a strain of S. marcescens is used to
produce amino acids (22); some strains of Erwinia are important vascular pathogens in
plants (7); A. faecalis subsp. myxogenes 1OC3K
produces the gel-forming 8i-1,3-glucan curdlan
(18); strain 22 produces succinoglycan (17); A.
radiobacter also produces succinoglycan-type
polysaccharide (16); A. tumefaciens makes
crown gall tumors in dicotyledonous plants (11);
P. amyloderamosa KIC produces isoamylase,
which in combination with ,B-amylase is used to
make maltose (19); and A. suboxydans is used
industrially to produce vinegar. Although members of these genera are resistant to Mu infection, Mu was readily introduced into several
strains by using the hybrid plasmid RP4::Mu
cts. Transconjugants of these bacteria maintained this plasmid, but about 20 to 50% of the
Mu-infected population lost the RP4::Mu cts
plasmid during transfer or stock in an unselective medium. All the plasmid markers were expressed in these bacteria, and in most respects
the transconjugants behaved as normal Mu cts
lysogens. Mu phage was produced from these
lysogens by heat induction and formed plaques
on E. coli strains EG47 and C600, but not on Mu
lysogens.
Previously, we extended the host range of
coliphage P1 to members of the genera used
here (25) and to the strains of enteric bacteria
reported by Goldberg et al. (15) and created
intergeneric hybrid strains of enteric bacteria by
transfer of the leucine and tyramine oxidase
genes between K. aerogenes, E. coli, and S.
typhimurium (26). However, a low transduction
frequency was observed when the donor and
recipient organisms were from different bacterial
genera, owing to lack of DNA homology between
the selected genes (26, 36). Moreover, the optimal multiplicity of infection was 0.1 to 0.5 for
transduction between members of the same
strain, whereas it was 5 to 10 for intergeneric
transduction (26). The yields of phage P1 were
low in most bacteria other than typical enteric
bacteria (25). In this work, we tested suitable
conditions for obtaining a high yield of Mu phage
and found that addition of an appropriate concentration of MgSO4 (0.2 to 0.4 M) to the medium stimulated the production of Mu ets phage.
The arylsulfatase gene from K. aerogenes
could not be transferred to E. coli by using P1
clrlOOKM, because the DNA homology of the
ats genes of the two bacteria is low (26, 38). In
this report, we succeeded in transferring the atsK
gene to E. coli by intergeneric mating, using
RP4 or RP4: :Mu cts and Mu cts in a recipient
strain of E. coli. Mu may mediate in transfer of
ats or other genes by promoting the formation
of RP4-primes or the integration of RP4: :Mu cts
into the chromosome to form intermediate Hfr
donor strains, as proposed by several workers
(10, 39). In intergeneric recombination of ats,
homologous recombination probably occurred
between Mu DNAs which had been inserted
into the chromosomes of both the donor and
recipient strains near the ats gene. Thus, Mu
could be used to transfer special genes between
VOL. 145, 1981
EXPANSION OF THE HOST RANGE OF Mu
the strains of gram-negative bacteria listed here
as well as between K. pneumoniae M5al (30),
Erwinia stewartii (7), Pseudomonas solanacearum (4), A. tumefaciens (37), and Rhizobium
meliloti (4), reported by other workers. Unexpectedly, we also succeeded in transferring RP4:
:Mu cts to B. cereus IF03001, and the strain
showed kanamycin-resistance and temperature
sensitivity. Since the drug-resistant strain of B.
cereus did not produce Mu phage and was very
unstable, there may be some barriers to expression of RP4: :Mu cts in gram-positive bacteria.
When the donor and recipient organisms were
from different groups of bacteria, a low transfer
fiequency was observed, probably owing to zygotic induction of Mu in new strains and to a
restriction-modification system. It should be
possible to isolate mutants deficient in a restriction system. Heat treatment of recipient strains
may also help to reduce restriction of transfer of
different DNAs, as shown in the Rhizobium
strains used here and in R. meliloti 2011 (10)
and C. freundii (9).
ACKNOWLEDGMENTS
We thank J. D6narie of the Institut National de la Recherche Agronomique, Paris, France, and to R. N. Rao of the
University of California, Davis, for generous gifts of E. coli
strains carrying the RP4::Mu cts plasmid and also T. Ishimaru
of Osaka University for kindly providing cephalexin.
This work was supported by grant 456079 to Y. M. from
the Ministry of Education, Science and Culture of Japan.
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