Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
lZ~2~73~
THIS IS A DIVISIONAL APPLIC~TION OF COPENDING APPLICA~ION
SERIAL NO. 440,127, FILED OCTOBER 31, 1983.
BACKGROUND OF THE INVENTION
(1) Field of the Invention
The present invention relates to novel derived
microorganisms and to a method for producing the derived
microorganisms which contain foreign DNA encoding for nisin
production. In particular, the present invention relates
to derived bacteria containing foreign DNA encoding for
nisin production.
(2) Prior Art
Nisin
The Merck Index (8th Edition) at page 6375
generally characterizes nisin as a polypeptide antibiotic
produced by Streptococcus lactis, citing various publications
including Mattick, Hirsch, Nature 154, 551 (1944); Lancet
2 , 417 (1946); and 253, 5(1947); Berridge et al., Biochem.
J. 52, 529 (1952); and U.S. Pat. 2,935,503 (1960). The
chemical structure is indicated to contain 34 amino acid
residues, eight of which are rarely found in nature, including
lanthionine (two alanines bonded to sulfur at the beta-
carbons) and beta-methyllanthionine as described by Gross, J.
Am. Chem. Soc. 93, 4634 (1971). Nisin is indicated to form
crystals from ethanol and to be soluble in dilute acids.
It is stable to boiling in acid solution. The Merck Index
indicates that nisin is used in food processing and as a
preservative, especially for cheese and canned fruits and
vegetables.
A chemical structure of one fo~n of nisin has
been described by Gross and Morrell of the National Institute
of Heàlth in Chem. and Eng. News Page 18 (Sept. 24, 19i3).
The structure includes alpha-beta unsaturation in amino
acids near the terminal amino and acid groups. It
~_,
~Z2()738
-- 2
is speculated that the activity of nisin is rela-ted to
reaction of the unsaturated amino acids with the sulfhydryl
groups of enzymes in the affected microorganisms. Nisin
generally has a published molecular weight range between
about 6800 and 7500. Nisin is classed as an antibiotic
produced by N-group lactic acid producing Streptococci.
The inventors prefer the phrase "inhibitory substance" to
"antibiotic" where nisin is generated in situ in a food by
the microorganism, particularly by bacteria.
Use in Foods
Nisin in foods inhibits clostridial spoilage
which is a major problem resulting from food storage. In
addition, nisin inhibits psychotrophic bacteria which are
particularly a problem with refrigerated foods. Thus nisin
inhibits Streptococci of Groups A, B, E, F, H, K, M and N,
Staphylococci, Micrococcus, Bacillus (some species)
Clostridium, Mycobacterium, Lactobacillus, Octinomyces and
Erysipelothrix. Nisin is particularly effective where the
food has been partially heat treated. It is not affected
by the presence of foods containing blood serum or milk.
It is thus useful in settings where these substances are
present in substantial amounts. At this time nisin cannot
be added to foods in the United States but is used in many
countries elsewhere in the world; however, approval is being
sought in the United States. It should be noted that nisin
occurs naturally in fermented food products where nisin
producing strains of Streptococcus lactis are present,
particularly in milk products. Use in foods is described by
Reddy et al J. of Food Science 35, 787-791 (1970) and in
30 Reddy et al J. Food Science 40, 314-318 (1975). An assay
procedure in foods is described by Trainer, J., et al
J. Sci Fd. Agriculture 15 522-528 (1964). The Assay of
Nisin in Foods. Fowler, G. G.; Garvis, B.;
Tramer, J.~, Aplln & Barret Ltd., Yeovil, Somerset,
U.K. Technical Series. Socièty for Applied Bacteriology,
1975, No. 8 pp 91-105.
ye/~
1~2(~738
--3--
Strains
- Various strains of Streptococcus 2actis are
known to produce nisin, but S. cremoris and S. Zactis
subspecies diacetiZec~is do not. Such strains are
; - 5 described in: McClintok, H. et al. J. Dairy Research 19,
187-193 (1952); Campbell, L. L. et al Food Tech 13:462-464
(1959); Hurst J. Gen ~icrobiology 44:209-220(1966);
Mattick and Hirsch 12th Intern. Dairy Congress 2 (Sect 3)
546-550 ~1949); Campbell et al, Food Preservation by Use of
Chemicals 110-119 (about 1960); ~cClintock et al, J. Dairy
Res 19:187-193 (19S2) (French); Rayman, Applied and
Environmental Tech 41:375, 380 (1381); Scott et al Journal
of Food Science 40:115-126 (1981); and in U.S. Patent Nos.
2,935,S03; 3,093,551; 2,785,108; and 3,295,989.
Various tecnnical ar~icles have appeared describ-
ing nisin producing Streptococc~3 ~acti3 strains. Included
are Geis A, et al ~pplied 2nvironmental ~icrobiology
45:205-211 (1~83): Koza'~ et al J. of Gen. .~icrobiology
- 83:295-302 (1974); Pack, et al, J. 8acteriology 149 420-425
(1982); ~cKay, L. et al Applied and Environmental
~icrobiology 40:84-91 (1980); Hurst A, J. Gen Microbiology
- 44:209-220 (1966); Scherwitz, X., et al., ~ppLied and
Environlnental ~icrobiology 45:1506-1512 (1983); Le81anc,
D., et al., J. of ~acteriology 137:878-884 (1979) and
LeBlanc D., et al in Plasmids and Transposons.
EnvironmentaL Zffects and ~aintenance Mechanisms, ~dited by
Colin Stuttard and ~enneth R. ~ozee, Ac~delnic Press, 31-41
(1980). Some o~ these publications describe the plasmids
in Streptococcus lactis and in particular a 28 ~dal plasmid
which may encode for nisin production; ho~ever, none o~ the
publications describe the transfer o~ the nisin encoding
; plasmid to non-nisin producing recipient bacteria or to
other ~icroocganis~s.
A su.~mary regarding nisin appe~ra in ~urst, A.,
Advances Applied ~icrobiology 27:85-123 (1981). This
publication describes ~hat is generally kno~n about nisin.
.
:1~2~738
--4--
A major problem described in the prior art
is the phage susceptibility of nisin producing strains of
Strep~ococcus Zec~is. Another problem is that the nisin is
expressed at relatively low levels in the naturally
; ~ 5 occurring strains of StreptococcUs tectis and the strains
are di~ficult to grow. Still another problem is that nisin
alone has some limitations of inhibitory activity against
food spoilage microorganisms.
Objects
It is therefore an object of the present
invention to provide nisin producing derived microorganisms
containing foreign DNA which encodes for nisin production.
It is further an object of the present invention to provide
a large collection of nisin producing derived micro-
; ~ 15 organisms which have unrelated phage susceptibilities and
which generate relatively large amounts of nisin. Also,
provided are nisin producing derived microorganisms which
can be adapted to become phage insensitive derivatives by
direct challenge with phage for selection of insensitive ~
clones without loss of desired functional characteristics,
particularly nisin production. Further still it is an
object of the present invQntion to provide nisin producing
derived microorganisms which can ~e combined ~ith ot-ner
inhibitory substance producing microorganisms. These and
other objects will become increasingly apparent by
reference to the following description.
General Description
The present invention broadly relates to a novel
nisin producing derived microorganism which contains
foreign DNA, either chromosomal or extrachromosomal, which
encodes for nisin production, which foreign DNA was
obtained from a nisin producing donor microorganism and
which foreign DNA was transferred to a recipient micro-
organism resulting in the nisin producing and usually nisin
resistant derived microorganism. The present invention
particularly relates to a novel nisin producing derived
1~2~)738
microorganis~ selected from a derived bacterium, yeast or
fungus.
The following definitions are used herein:
(l) the phrase "donor microorganism" means a
parental strain containing transferable DNA which encodes
- for nisin prqduction, preferably strains of Streptococcus
Zactis bacteria whic~ naturally produce nisin.
t2) The phrase "recipient microorganism" means
a parental strain which d~es not naturally produce nisin or
which produces relatively low amounts o~ nisin, preferably
a ~acterium, and may or may not be sensitive to (inhibited
by) nisin.
t3) The phrase ~derived microorganism" means
nisin producin~ strains that result from introducing
foreign D~A into a recipient microorganism, preferably from
mating a donor microorganism which is nisin producing with
a recipient microorganîsm which is non-nisin producing or
low ni~in producing to increase nisin production,
and preferably a bacteria. - -
(4) The phrase "foreign D~A" means DNA which
does nat naturally occur in the recipient bacterium. The
foreign ~A is introduced into the recipient bacterium by
the method of the pres2nt invention.
(S) The phrase "inhibitory substance" ~eans an
antimicrobial a~ent including nLsin produced by a
microorganism which prevents the gro~th of other
microorganisms.
The present invention particularly relates to a
nisin producing derived bacterium selected from
Streptococcus species, Pediococcus species, ~actobacilZus
species, Propionibacterium species, Micrococcus species,
Leuconostoc species, S~ephyZococcus species, CZostridium
species FZavobactsrium species, Brevibacterium species, E.
coZi a.~d Pseudomonas species.
The present invention further relates to a r.isin
pr~ducing derived yeast selected from Saccharomyces
species, especially S. cerevisiee; Debaryomyces species,
12Z073~ .
--6--
especially D. hansenii; ToruZopsis species; Bret~anomyces
species; Candida species; Cryptococcus species; Ktoec~era
species; ~uyveromyces species and schizosaccheromyces
species.
The present invention finally relates to the
nisin producing derived fungus selected from PeniciZZium
species, ~uco~ species, Rhizopus species, Aspergi~Zus
species, Deuteromycetes species, Ascomycetes species,
Geotrichum species and Monascus species.
The foreign D~A can be transferred to a
recipient microorganism by transformation in the manner
described in U.S. Patent Wo. 4,237,224 to Cohen and BoYer
and related patents using splicing of ~he nisin encoding
~NA in a cloning vector. The cloning vectors described in
U.S. Patent No. 4,374,200 to Ronald H. Olsen and similar
patents are useful. More conventional conjugal mating of
donor and recipient microorganisms, which is preferred, can
be used for the transfer. Transduction of the nisin encod-
ing DNA using a virus ~phage) is also possible.
The present invention also relates to the method
for engineering a nisin producing derived microorganism
which comprises transferring D~A, which encodes for nisin
production in a donor microorganism, to a recipient
microorganism, to result in a nisin producing dQrived
microorganism, wherein the D~A is foreign to the recipient
microorganism and encodes for nisin production in the
derived microorganism; and isolating the nisin producing
derived microorganism with the foreign DNA.
The present invention also relates to a nisin
containing product elaborated by the nisin producing
microorganism ~ith the foreign DNA. The nisin containing
product can contain in addition other inhibitory substances
preerably those simultaneously elaborated by the nisin
producing derived microorganism. The present invention
includes a mixture o~ nisin, as a pure chemical or a crude
nisin containing extract, mixed with a non-nisin producing
Streptococcus tectis subspecies diacet~acts or a nisin
~Z~)738
--7--
producing derived microorganism which has been selected for
resistance to nisin.
The present invention further relates to two or
more of the nisin producing derived microorganism strains
containing the foreign DNA as a mixture, which strains have
different phage susceptibilities so as to enable the pro-
duction of nisin without stringent anti-bacteriophage
precautions. The strains can be packaged mixed or
separately.
Finally the present inventibn relates to the
method for treating animals which comprises feeding the
animal the nisin producing microorganism which contains the
foreign DNA, alone or in admixture with other
mi~croorganisms.
The present invention is based upon the fact
that DNA encoding for nisin production in a donor micro-
organism can be incorporated into a recipient microorganism
as foreign DNA to provide a new nisin producing derived
microorganism. The resulting nisin producing derived micro-
organism is able to preserve foods and other materials and
to provide inhibitory substances in animals, particularly
- birds and ma~nals, to maintain or improve their health.
The nisin producing derived microorganisms containing the
foreign DNA can be combinea with non-nisin producing, nisin
insensitive bacteria or other microorganisms preferably
those -~hich can produce other inhibitory substances
structurally different from nisin.
Sometimes nisin can be encoded for in the
naturally occurring nisin producing Streptococcus Zactis by
a plasmid. Also the plasmid DNA encoding for nisin can be
incorporated into the chromosome of the recipient bacterium
by natural ligation processes, which makes the removal of
the characteristic in the derived microorganism difficult.
Thus, in the present invention, the foreign DNA after
transfer can be chrornosornal or extrachromosomal in the
derived microorganism.
~Z~ 38
--8--
DNA enccding for nisin can be separated from the
natural nisin producing Streptococcus Zactis in the form of
a cleaved fragment or plasmid. This may be accomplished by
redigestion of the nisin encoding DNA. The fragment can
then be cloned into a suitable cloning vector and
- transferred with an introduced DNA into a recipient
microorganism. The introduced foreign D~A can also be
transferred by transduction of the chromosomal DNA or
plasmid DNA using a virus. The plasmid can also be
transferred by conjugal mating of related or unrelated
genus and species. The term "transfer" is used herein in
its broadest sense to designate any means for placing the
- DN~ encoding for nisin production into a recipient
microorganism as foreign DNA. The resulting nisin
producing derived microorganism may have multiple copies of
the foreign DN~ in each cell which can greatly increase the
amount of nisin produced per cell subject to cellular
limitations as to concentration of nisin.
As is known in the art, the derived
microorganisms are provided in various preserved forms
which increase viability during storage and shipment. Such
for~s include: liquid non-concentrated or concentrated
cultur~s, frozen or non-frozen cultures and sta~ilized
dried or lyophilized cultures. The cultures usually
contain biologically pure forms of the derived
microorganisms. Amounts of the microorganism over about
106 cells per gr~m, and preferably over about 1 x 109 cells
per gram in preserved form are preerred. Generally,
concentrations over 109 cells per gram require means for
removal of liquid growth medium from the cells usually
by centrifugation or reverse osmosis. Various stabilizing
agents such as glycerol, milk powder and the like are used
for freezing and lyophilization. The derived microorganism
cells are usually grown in a growth medium including a
source of carbon, nitrogen and essential minerals. All of
this ia well known to those skilled in the art as can be
seen from the large body of patent art.
12~0738
_g _
SPECIFIC DESCRIPTION
The present invention particularly relates to a
biologically pure nisin and lactic acid producing derived
bacterium which is not sensitive to nisin and containing
S plasmid derived DNA, encoding for nisin production from a
donor nisin producins Streptococcus Zec~is wherein the DNA
has been transferred to a nisin sensitive recipient
bacterium resulting in the nisin and lactic acid producing
derived bacterium. The reci~ient bacterium is pre~erably
o Streptococcus tactis subspecies diacetiZact~s. The donor
bacterium for S. Iect~s is preferably S~reptococcus Z~ct~s
ATCC 11,454 which transfers the nisin trait only to
isogenic strains as can be seen from Table 1. Sucrose
utilization also is transferred with the nisin trait. A
related known nisin producing strain is NRRL-B-15,470 (NIRD
1404) which could be used. The derived donors are S.
Z~ctis , ~RRL-B-15,459 and NRRL-B-15,460 which can transfer
the nisin, sucrose trait intergenetically. All of the
nisin producing derived microorg~nisms which are
transconjugants can potentially be used as donors of the
nisin trait. The reason may be that the plasmid encoding
for nisin and sucrose may have been modified in the primary
transfer. The intergen2tic recipient b~cterium is
preferaoly Streptococcus lactis subspecies diac~Iactis
NRRL-B-15,005, NRRL-B-15,006, NR~L-B-15,018, N~RL-B-12,070
(DRC3, cit~,lac+), and N~RL-B-12071 tDRC3, lac+, cit-),
- and S~ Zectis NR~L-B-15,454, NRRL-B-15,452, NRRL-B-15,455,
NRRL-8-15,456, NR~L-B-15,457. The resultant nisin
producing derived bacterium with the foreign DNA is
3Q preferably selected from Streptococcus Zectis subs~ecies
d~ace~izec~s~RRL-s-ls~464~ (and related transconjugants from
-- similar matings 15, 465, 15,466 and lS,467), 15,468 and
15,469 and isogenic bacterial derivatives thereof as
described more Cully hereinafter with the appropriate
antibiotic resistance mar~er~s) removed by selection.
- The present invention Qarticularly relates to a
biologically pure nisin and lactic acid producing bacterium
1220738
.
--10--
which is not sensitive to nisin and which i5 derived by
conjugal mating of a nisin producing Streptococcus Zactis
donor bacterium containing a plasmid, which codes for the
nisin productîon in the donor bacterium, with a non~nisin
pr~ducing and nisin sensitive lactic acid producing
recipient bacterium. The conjugal mating results in the
nisin and lactic acid producing derived bacterium wherein,
in some instances, the original plasmid from the donor
encoding for nisin production can not be removed as a
plasmid from the nisin and lactic acid producing derived
bacterium because of recombination with the host chromosome
of the recipient bacterium. The recipient bacterium can be
inhibited by nisin prior to the transfer. The plasmid
which encodes for nisin production (and sucrose
fermentation) from the donor bacterium preferably measures
about 29 Mdal in length and may exist as 29 or altered form
i.e. reduced or enlarged or recombined with host chromosome
DNA in the derived bacterium.
The present invention particularly relates to`
the method for producing a nisin and lactic acid producing
derived bacterium which is not sensitive to nisin and which
comprises: con~ugally mating a nisin producing
Streptococcus Zactis donor bacterium containing a plasmid
which codes for the nisin production in the donor bacterium
with a non-nisin producing and nisin sensitive lactic acid
producing recipient bacterium, and isolating the nisin and
lactic acid producing derived bacterium produced by the
- mating.
- Further, the present invention relates to an
improvement in a method of preserving a material such as a
food by incorpDrating a lactic acid producing derived
bacterium into the material which comprises incorporating
in the material a nisin and lactic acid producing derived
bacterium which is not sensitive to nisin and containing
DNA derived from a plasmid which codes for nisin production
from a nisin producing donor S~reptocOcCUS tac~s and ~hich
has been transferred to a nisin sensitive recipient
~22~738
bacterium to produce the nisin and lactic acid producing
derived bacterium. The foreign DNA encodes for nisin
production and is either integrated into the chromosome or
extrachromosomal in the nisin producing derived bacterium.
The materials can include all sorts of microbially
degradable products for agricultural, industrial, food and
other uses including silage ! cutting oil and foods. The
foods include fresh meat, comminuted, fermented and
non-fermented meat; fresh chicken, fish, milk and other
dairy products, commercially prepared salads, vegetables,
dressings, sauces and other foods subject to spoilage
during refrigerated storage.
Also, the present ;nvention particularly relates
to an improved preserved material produced by incorporating
into the material a lactic acid producing derived bacterium
or a product produced by the derived ba~terium, which
comprises (1) a material containing a nisin and lactic acid
producing derived bacterium, which is not sensitive to
nisin and containing D~A derivea from a plasmid which
encodes for nisin production from a nisin producing Strèp~o-
coccus Zactis donor bacterium which has been transferred to
a nisin sensitive reci~ient bacterium to produce the nisin
and lactic acid derived bacterium or (2) incorporàting in
the material a nisin containing product of such derived
-25 bacterium or ~3) incorporating in the material a nisin
containing product of such derived bacterium along with a
microorganism which produces other inhibitory substance(s)
but does not produce nisin or (4) incorporating in the
material a nisin containing product oE such derived
bacterium along with a microorganism which produces other
inhibitory substances in addition to nisin.
~ inally the present invention particularly
relates to a nisin containing product produced by the
derived bacterium previously described containing in
addition an inhibitory substance ~rom S~rep~ococcus t~c~is
subspecies diaceti Zacits.
.
1~2~)~38
-12-
Detailed Descriotion
E~PLE 1
In order to determine if sucrose utilization and
nisin production could be transferred by conjugation, S.
S t~cti~ ATCC-11454 tutilize~ sucrose and produces nisin) was
used as the donor strain in a mating experiment where S.
Iactis subsp. ~iecetilactis and S. tact~s (neither strain
utilizes sucrose and neither strain produce nisin) were the
recipient strains. The mating conditions were as described
by Gonzalez and Runka, (Gonzalez, Carlos F., and Blair S.
Kunka. ~ppl. Environ. Microbiol. 46:128-132 (1983~) except
that the mating filters were not overlayed with agar.
Instead, the filter with the cells facing the agar surface
were placed in a BBL~ - Anaerobic Jar with C02/H2
atmosphere or 3 to 4 hours. Transconjugants were selected
for the chromosomal resistance of the recipient and ability
to utilize sucrose or resistance to nisin ~1000 units/ml).
An isogenic strain of S. Z~ctis ATCC 11,454 was
constructed. Strain s. ~ectis ATCC-11,454 tutilizes
lactose and sucrose, and produces nisin) was temperature
cured of a resident 2g Mdal plasmid by exposure to an
elevated growth temperature (40C). This cured strain
utiLized lacto~e but did not utilize sucrose and did not
produce nisin. Subsequently, this strain was subjected to
an additional heat curing at 40C to remove an additional
resident 32 Mdal plasmid to result in constructed strain S.
Zectis ATCC 11,454, which was lactose negati~e ~Lac~),
sucrose negative (Suc~) and did not produce nisin (Nis-).
~ This constructed strain was designated as S. tactis
NRRL-B-15,453 (Lac~, Suc~, Nis~). NRRL-B-15,453, then was
exposed to increasing concentrations of streptomycin to
obtain a chromosomaL mutation for the antibiotic at a
concentration of 1000 g/ml. This NRRL-8-Lst453 Smr
(streptomycin resistant) strain was designated as
3S NRRL-8-15,454. Strain NRRL-B-15,454 was then used as a
recipi~nt ;n the conjugal mating experiments.
12253738
--13--
Table 1 describes the experiments and the
results. In these experiments, transfer of sucrose
utilization and nisin production only was observed with the
isogenic recipient S. Zact*s NRRL-B-15,454. Transfer of
sucrose utilization and nisin production intragenerically
from S. Zac~is NRRL-B-15,~54 to S. Zactis NRRL-B-15,452 and
S. Iactis subsp. diecetiZect~s NRRL-B-15,006-Smr was not
observed.
Table 1
' Intraspecies Coniuqal ~atinqs
Transfer frequencies
DonorReciPients per donor
5. Iactis ATCC 11454 S. tac~is NO
NRRL-B-15452
~ S, Zectis 1 x 10-5
- NRRL-B-15454
S. Zectis subsp.
diacetiZectis ~0
NRRL-B-15006-Smr
NO - Not Detected
Sl-nr -,~esistance to streptomycin at concentration of 1000
g/ml .
.~ating conditions as described,by Gonzalez and
Kunka, 1983 (Appl. Environ. Microbiol. 46:81-89). See
ExampLe,l in text for exception.
Transfer frequency is expressed as the number of
nisin resistant and nisin producing colonies per donor
colony forming units (CFU). Donor CFU were determined
be~ore mating. Transconjugants were selected for the
chromosomal r~sistance of the recipient and ability to
utilize sucrose or resistance to nisin (1000 units/ml),.
EX~MPLE 2
S. Zactis ATCC 11,454 was exposed to increasing
concentrations of riEamycin to obtain a riEamycin resistant
'35 (Rifr) mutant (resistant to 400 ~g/mll. The mutant was
used as a recipient in a conjugal mating experiment with a
~2~)738
-14-
S. sanguis V683 strain which contains the conjugative
plasmid pIP501. The resultant transconjugant tS. Zact*s
ATCC 11,454 Rifr (pIP501)) was isolated and was designated
as S. Zectis NRRL-s-l5~4s8~ S. Zac~is NRRL~B-15,458 along
with S . Zactis ATCC 11,454 wer~ used as donors in isogenic
and intrageneric matings to determine conjugal transfer of
the nisin genes. The mating-conditions for the experiments
were as described in Example 1 above.
Table 2 describes xesults of matings in which
strain S. tactis NRRL-B-15,458 was used as a donor and S.
Zactis NRRL-B-15,452, S. Zectis NRRL-B-15,454 (an isogenic
strain of ~. ~actis ATCC 11,454) and S. tec~is subsp.
diacetitac~is NRRL-B-15,006-Smr were used as recipients.
Transer of plasmid pIP501 was observed to occur in two S.
Zactis strains, while no transfer was detected to S. Zectis
subsp. diacetiZactis. Transfer of sucrose utilization and
nisin production was observed only to isogenic strain S.
Zectis NRRL-B-15,454 and was at a frequency of 1.4 x 10-3.
Table 2
Conjuqal Transfer of Nisin Genes
- Transfer frequency
Donor Reci~ient D~r donor
5. Zacti,s S. ~actis pIPS01 Nisin
NRRL-B-15458 NRRL-B-15452 2 x 10Z NO
5. Zactis
NRRL-8-15454
(isogenic
strain) 2.7 x 10-51.4 x 10-3
~ S. ~actis subsp.
diacetiZactis
NRRL-B-15006-Smr NO NO
.
NO - Not Detected
Mating conditions as described in Example 1
When S. Zact*s NRRL-B-15,458 was ;mated with S.
Zactis ~RRL-B-15,454 tisogenic strain) two dif~erent
phenotypes of transconjugants were obtained. One ehenotype
- ~2V738
--15--
was lactose negative, sucrose positive an~ produced nisin.
This isolate was designated as s. Zectis NRRL-B-15,459.
The other transconjugant was lactose negative, sucrose
~ositive, produced nisin and contained plasmid pIP501.
This isolate was designated as S. Zactis NRRL-B-15,460.
These derived strains, unlike the donor strain,
were streptomycin resistant, rifamycin sensitive and did
not produce acid from lactose. However, unlike the
recipient parental strain the derived strains were now
nisin producers and were able to u~ilizè sucrose a3 a
source of carbon to produce acid. There~ore, only isogenic
transfer of the sucrose utilizing nisin producing trait was
observed by strains S. Zactis ATCC 11,454 and its pIPS01
derivative S. Zactis NRRL-B-15,458.
EXAMPLE 3
S. Zactis NRRL-B-15,459 was tested for ability
to transfer nisin production to S. Zectis NRRL-B-15,452.
Recipient strain NRRL-B-15,452 was negative for production
of nisin, sensitive to nisin and unable to utilize sucrose
as a source of carbon to produce acid. The mating
conditions were as described in Example 1 and results are
described in Table 3.
Table 3
- - Intrageneric Transfer of Nisin and Sucrose
Utilization Genes
Nis+, Suc~
Transconjugants
Donor Reci~ient ~er donor
S. Zactis S. Zactis 6 x 10-5
NRRL-B-15459 NRRL-B-15452
.
Conditions as- described in Table 1
Suc~ = Sucrose positive; able to utilize sucrose to produce
acid.
Nis~ = Nisin positive; able to produce nisin.
S. 1,actis NRRL-B-15,459 was able to transfer nisin
i~2(~738
--16--
production, resistance and the ability to utilize s~crose
to a non-isogenic strain of 5. Z~ct~s NRRL-B-lS, 452 .
EXAMPLE 4
S. Iectis NRRL-B-15,460 was tested for ability
to transfer nisin production to S~ Zec~s NRRL-B-15,452, S.
Zactis subsp. di~cet~Zactis- NRRL-B-15,455 and NRRL-B-15,456,
Recipient strains NRRL-s-15,452, NRRL-B-15,455 and
NRRL-B-15,456 are negative for production of nisin,
sensitive to nisin and unable to utilize sucrose as a
source of carbon to produce acid.
The mating conditions were as described in
Example 1 and the results are described in Table 4.
lZ~(~73~ -
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1~22~)~38
--18--
S . tact~s NRRL-B-15,460 wa~ able to transfer
nisin production, nisin resistance and ability to utiLize
sucrose to a non-isogenic strain of S. ~ac~is NRRL-8-15452
and to S ~actis subsp. d~acetiZactis NRRL-B-15455.
Many transconjugant, derived strains were
isolated from the conjugal mating~ described in Examples 3
and 4. Derived S. tact*s NRRL-8-ls46l is typical of a
transconjugant of S tect*sNRRL-B-15452. Derived S.
Zactis subsp. diecet*Zec~*s NRRL-B-15469 is typical of a
transconjugant of S. Zactis subsp. diecet*Zactis
~ WRRL-B-154S6.
Table 5 summarizes phenotypic charactistics of
transconjugant strains resulting from matings described in
Example 4. Transconjugants obtained from each mating were
analyzed for selected and unselected markers. Analysis of
plasmid content of transconjugants confirmed them as
recipient types. Additionally transconjugants were tested
for sensitivity to their respective recipient homospecific
phages. The transconjugants were lysed by their
homosQecific phages while donor controls shawed no
sensitivity.
lZi~V738
--19--
Table ~
~henotypic characteristics of Parental Recipient
Strain and Derived Stra ns of Table 4 Matinq~
_PhenotYpic Characteristics
Nisin Nisin Sucrose
Strain ~ status _ Production Resistance Utilizatlon
~ Zactis Parental
NRRL-B-15452 Recipient - - -
~ Zactis
10 ~RRL-B-15461 Derived ~ +
S Zactis
subsp.
diacetiZac~is Parental
NRRL-B-15455 Recipient
15 ~ Zactis subsp.
diacetiZactis
NRRL-B-15464 Derived + + +
S Zactis subsp,
diacetiZactis Parental
20 NRRL-B-15456 Recipient
5 Zactis subsp.
diacetiZact3s
NRRL-B-15469 Derived + + +
Nisin production - Assay (see text)
Nisin resistance - Growth on medium containing Nisaplin~ at
1000 ~g/ml.
Sucrose - utilization - medium BM containing sucrose at .5%
(Gonzalez and Kunka Appl. and Environ. Microbiol. 46:
81-8g, 1983).
EXAMPLE ~j
S tactis subsp- diacetilactis NRRL-B-15464, a
transconjugant, derived strain from a first mating
subsequently was used as a donor in an isogenic mating with
S.tactis subsp.diacetitactis NRRL-B-15018 Smr
(Streptomycin resistant) and S. Zactis subsp. diacetitactis
-NRRL-B-15005 Fusr (fusidic acid resistant).
Table 6 describes the results of the mating
experiments. Transfer of sucrose utilization and nisin
production traits were observed in the two isogenic
V~38
-20-
recipient strains. Transconjugants from each mating were
examined or their plasmid content. Additionally, analyses
for non-selected traits were conducted. Transconjugants
`were confirmed as recipient types.
.
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~2ZS~73~3
-22--
EXAMPLE 6
Production of nisin by strain S. tactis subsp.
~iaceti2actis NRRL-B-15464 in comminuted meat incubated at
10 C .
S To determine production of nisin by individual
colonies of test strains, the colonies were replicated in
petri dishes containing peptonized mil~ agar which had
previously been flooded with an 18 hour culture of S.
cremor~s ATCC 1~365. Strain ATCC 14365 is a nisin
sensitive strain used to assay nisin production ~J. Gen.
Microbiol. 4:71; 1950). After 18 hours of incubation, the
appearance of zones of inhibition is indicative of nisin
production. The method is that of Rozak et al J. Gen.
Microbiol. 83:295-302 (1974).
The nisin content of meat was determined by "The
Quantitative Agar Diffusion Assay used for the estimation
and differentiation of nisin in food" as described by
Fow~er et al. (The ~ssay of ~isin in Foods. Fowler, G.G.,
Jarvis, B., Tramer, J. Aplin ~ Barret Ltd., Yeo~il,
Somerset, UR, Technical Series, Society for Applied
Bacteriology, 1975, No. 8, pp. 91-105.
A standard curve was prepared by adding a Xno~n
concentration of purified nisin to meat. The meat sample
was then treated by the method of Tramer 2t al. The food
- 25 extract wa~ assayed by the agar dif~usion method and a log
nisin concentration vs. zone diameter size curve was
plotted. S. cremoris ATCC 14365 was used as the indicator
organism. ~dditionally, strain S. ~ectis ATCC 11454, a
nisin producing and therefore nisin insensitive, also was
used as an indicator organism.
Highly purified nisin (obtained from Aplin &
Barrett, Ltd.) was added to meat samples ana nisin
concentrations were calculated using a standard curve.
The inoculum of test organism was prepared by
growing ~RRL-B-15464 in 500 ml ~17 broth (Terzaghi and
Sandine Appl. Microbiol. 29:807-813, 1975) for 18 hours.
Cells were ~btained and washed twice by centrifugation at
~,C î22~3~3
-23-
10,000 RPM at 10C for lO minutes and resuspended in
Standard Methods phosphate buffer (E. H. Marth, (Ed.)
Standard Methods for Examination ~f Dairy Products, 14th
ed. p. 62, Amer. Public ~ealth Assoc. Washington, D. C.
Cell suspension was adjusted spectophotometrically to
approximately 1.5 x 109 colony forming units~ml. Actual
number was determined to be i.7 x 109 CFU/ml. A fresh
picnic (pork) (skin on bone in) was purchased from a local
- meat supply company. The s~in was asceptically removed and
the meat comminuted in a sterilized meat chopper. The meat
then was ap~ortioned into 50 and 100 g quantities and
placed in sterile containers. Containers were set up as
ollows:
- A. Eleven containers with 100 g meat and
glucose added to a final concentration of 0.5~ by weight.
For analysis, these samples were split into two aliquots.
One aliquot was assayed without further treatment. The
remaining aliquot was used to construct a standard curve by
adding a kno-~n amount of purified nisin (100-200 units/g of
meat).
B. Three containers with 50 g of meat glucose
to a final concentration of 0.S~ by weight of meat and 100
units of added nisin per gram of meat. This sam~le was
assayed daily for nisin.
2S C. Eleven containers with 100 g of meat,
glucose to a final concentration fo 0.5% by weight of meat
and strain S. ~actis NRRL-B-15464 to a final concentration
of 1.7 x 107 CFU/g of meat. Results are shown on Table 7.
~Z073~
--24--
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3 0 h Z U o z o ~--1 ~ ~ t~7 'r ~ d' ~r . ~
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~Z~738
~25-
EXAMPLE 7
Nisin content in milk was determined by the
method used for meat described in Example 6.
The inoculum of test organisms was prepared by
growing microorganisms in M17 broth for 18 hours. Cells
were obtained and washed twice by centrifugation at 10,000
rpm at 10C/10 minutes and resuspended in Standard Methods
phosphate buffer pH7.2. Suspension was adjusted
spectrophotometrically to 1.5 x 108 colony forming units/ml.
Non-fat dry mil~ (10% w/v) was steamed for 30 minutes
cooled and glucose added to a final concentration of 0.5%
by weight of milk. Fifty ml of milk-glucose was inoculated
at a rate of approximately 106 CFU/ml and incubated at 32C.
Samples were assayed daily.
Nisin assay protocol: (1) 30 ml of sample was
assayed (2) the p~ was adjusted to 2 with 5N HCl (3) The
sample was boiled for 5 minutes ~4) Chilled (5) The sample
was centrifuged 10,000 rpm for 10 minutes at 10C and (4)
Supernatant was assayed by Quantitative Agar Diffusion
~ethod (see above). The assay organism was ATCC 14365
which is sensitive to nisin. ATCC 11454 is insensitive to
nisin and a nisin producing strain. A standard curve was
constructed on a daily basis by adding nisin to a mil~
samole at 100 units/ml. The results are shown on l'able 8.
738
~26--
a~
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~
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. ' _~
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o I o ' o o - o o C~ o
a) ~d ~ I
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.
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i2'~738
-27-
The derived micoorganisms, particularly the
derived bacteria, can produce nisin ~and possibly other
substances which inhibit microorganisms) at refrigeration
temperatures which is a distinct advantage in the-
S preservation of foods. This is shown by the results inTable 7 where control meat without a derived microorganism
started to spoil at day 3 and became putrid by day 7 as
compared to meat with a derived microorganism which did not
even start to spoil until day 10.
The preferred Streptococcus 2actis subspecies
diece~tactis ~sNRRL-B-15469 with the fusidic acid resistance
marker removed by selection and S. Iactis subspecies diaceti-
Za~tis NRRL-B-15464 with the rifamycin resistance marker
removed by selection. Neither of these derived strains are
15 capable of utilizing lactose.
The reference to "NRRL" herein is to the
National Regional Research Laboratory in Peoria, Illinois.
The designated strains are freely available to those
requesting them by reference to the strain name and number.
~0
