Federal Register / VoL 53. No. 41 / Thursday. March 1. 1990 / Notices 
7443 
be more than adequate. Self cloning 
experiments and experiments involving DNA 
clones isolated from non-pa thogenic 
organisms or dones that are known not to 
iurrade prodoction of toxic materials and 
transformed into M5al should be as harmless . 
as experiments that utilize the non- 
recombinant strain. Below we will document 
what we know of the history and the nature 
of JC oxytaca M5al. which we shall call 
“MSal" from here on. 
The earliest reference we know for MSal is 
a 1946 paper on butanediol fermentation 
(Freeman (1946], The fermentation of sucrose 
by Aerobacter asrogenes, Chemical 
Abstracts in Biochemistry 41: 389-398]. MSal 
was isolated in the 1930's by Or. Elizabeth 
McCoy at the University of Wisconsin 
(Winston Brill, personal communication]. The 
strain was originally dassified as Aerobacter 
aerogenes, (Wilson (1356]. Nitrogen Gxation 
in Aerobacter aerogenes, in Biochemistry of 
Nitrogen. AX Vitanen Homage Volume. Ann. 
Acad. Scientarium. Fennicae Ser. AH 60. 139- 
150; Mahl et aL (1985] Nitrogen fixation by 
members of the tribe Klebsiella. J. BacL 89: 
1481-1437]. The strain was distributed to 
various workers interested in.&ee living 
nitrogen fixing bacteria in the 1340‘s by Dr. 
M.J. Johnson of the University of Wisconsin 
and in the 1960's by Dr. Perry Wilson also of 
the University of Wisconsin. In 1965 the 
strain was reclassified as Klebsiella 
pneumoniae by the CDC (CDC t?2S51-63]. 
MSal was once again redassified in 1977 to 
K oxytaca (CDC Publication 78-8358]. The 
primary taxonomic difference between K 
oxytaca and K pnecmania is that K oxytaca 
produces indole while K pneumoniae does 
not. We have tested M5al for indole 
production and have confirmed that MSal 
does produce indole &om tryptophan. In 
general K oxytaca is found in the intestines 
of humans and animals, and in “botantical -■ 
and aquatic environments" {Eergey's Manual 
of Systematic Bacteriology (1383], Sneath, 
ed. Williams and Willuns, Baltimore). Thus 
K oxytaca appears to be ubiquitous. Wil j- 
type MSal is resistant to low levels of 
ampidllin’fnp to 100 ^gTinlbutitis sensitrve 
to higher levels of ampidllin and usual 
experimental levels of kanamydn. 
tetracycline, cephalosporins and 
chloramphenicol. 
Interest in MSal expanded in 1971 
(Streicher et al (1971), Transduction of 
nitrogen fixation genes in Klebsiella 
pneumoniae DNAs 63; 1174-1177). MSal was 
one of two strains of K pneumoniae that was 
shown to be sensitive to bacteriophage Pi out 
of a total of 27 strains tested. The significance 
of Pi sensitivity was that Pi is routinely used 
for generalized transduction in £ coll, an 
extremely useful genetic technique. The 
ability to transduce mutations among strains 
of K pneumoniae would greatly accelerate 
study of the genes involved in nitrogen 
fixation. Thus MSal became the strain of 
choice for studying the genetics of nitrogen 
fixation in at least four different labsrRay 
Valentine. University of California, Berkeley: 
Winston Brill University of Wisconsin; Ray 
Oixon.-Su3sex; Ethan Signer and Fred 
Ausubel MIT. The MIT lab renamed MSal as 
“KPl," which reflects Its seminal position in 
their strain collection. The MIT group then 
discovered that MSal would support growth 
of the lambdoid caliphage 424 and that MSal 
had a DNA restriction system that prevented 
efficient transfer of DNA from E. coli to 
MSal. They subsequently isolated a 
' restrictionless mutant of MSal. called 
KP5022, which became the parent of many 
other derivatives (Streicher et al (1974). 
Regulation of Nitrogen Fixation in Klebsiella 
pneumoniae, J. Bact 120: 815-821). 
MSal was then shown to be "non- 
capsulated," a trait that is common with Z. 
coli K-12, and which may account for the 
reduced pathogenicity' of £ ccli K-12 
(Shanmugam el al (1974] Bioch. Biophys. 
Acta 338; 545-533). In fact it was probably the 
non-capsulated nature of MSal that made it 
more susceptible than other Klebsiella 
strains to phages of Pi and 424. 
Winston Briirs group showed that 
bacteriophage Mu could infect MSal. The 
group then used variants of Mu to mutagenize 
and construct fusions of njfger.es to £ call 
lacZ. They rer.amed MSal as UN,' and 
generated many hundreds of derivatives, 
such as UN1290, which contains the recAS8 
allele of £ coli transduced into MSal 
(MacNeil et al (1981], Regulation of Nitrogen 
Fixation in Klebsiella pneumoniae, f. BacL 
145: 346-357; .MacNeil et al (1978] Fine 
structure mapping aiul complementation 
analysis of nif genes in Klebsiella 
pneumoniae, J. BacL 136: 253-288]. 
During the 1970's there was much work at 
the University of Sussex and elsewhere on 
the enzymology of nitrogen fixation. Large 
amounts of nitrogenase enzyme were 
required, and since the genetic woric was 
..being done in MSal and its derivatives. MSal 
. became the organism of choice for producing 
. nitrogenase. MSal was grown routinely in 
1000 liter fennentors, and kilograms 
quantities of cell pastes were routinely . 
worked up. using no special precautions 
(Eady et ^ (1372] Bioebem. J. 128: 655-875). 
"In fact Ihey reported injecting live MSal into 
rabbits for the purpose of raising antibodies 
against intact cells. No pathogenic effects 
were observed (see Appendix L page 4). 
Appendix I also documents the successful 
KUal declassification petitions of the 
Postgate lab at Sussex to the Genetic 
Manipulation Advisory Committee. UJC They 
obtained permission to perform various MSal 
recombinant experiments under conditions of 
good microbiological practice. Thus MSal has 
been used in several labs, both genetic and 
biochemical since 1948. No barznful effects of 
MSal have been reported from any of the 
labs. 
Finally, starting in the 1970‘s, many 
recombinant DNA experiments hav'e been 
done with MSal. In particular, all of the genes 
- involved in nitrogen fixation and many of the 
genes involved in regulation of nitrogen 
metabolism of MSal have been clcned into £ 
coli K-12 (for examples, ace Dixon et al 
(1976) Construction of a P plasmid carrying 
.nitrogen fixation genes from £/ie6s;e//o - 
pneumoniae. Nature 260; 288-271; Cannon et 
al (1986] The nucleotide sequence of the nif 
:* gene of Klebsiella pneumoniae, Nuc. Acids. 
- Res. 16: 11379]. 
The current NTH guidelines for 
recombinant DNA work (Federal Register 
Volume 51, no. 88, May 7. 1986) are 
contradictory with respect to Klebsiella. On 
one hand, the genxia Klebsiella is considered 
to be a natural DNA exchanger with El-coli, 
and so any cloning between £ coli and 
Klebsiella in either direction is exempt (p. 
16967). On the other hand. Klebsiella — all 
species and serotypes — is listed as a Class 2 
pathogen, and as such, cloning into Klebsiella 
requires BL2 containment (paragraph IU-B-1- 
a. p. 16960) and cloning recombinant DNA 
fram Klebsiella into non-pathogenic 
prokaryotes (Le. £ call K-12] also requires 
BL2 containment (paragraph m-B-Z-a. p. 
16380). We request that the status of 
Klebsiella be clarified, particularly in the 
case of K. oxytocc strain MSal. Specifically, 
we propose that the following classes of 
experiments and fermentations of the 
resulting organism be exempted from the 
guidelines: 
(1] All self cloning experiments involving 
DNA from MSal and any of its derivatives. 
(2] All experiments involving clones of 
MSal DNA into £ coli K-12. 
In addition, vye propose that the following 
classes of experiments be given BLl stanm: 
(1) All experiments involving clones of £ 
coli K-12 DNA into MSal. 
(2) All e.xperiments involving well defined 
clones from nonpathogenic organisms or 
clones known not to contain DNA that 
encodes production of material toxic to 
vertebrates into MSal. 
We feel that the history of safe use of MSal 
and the ubiquitous distribution of K oxytaca 
justify these containment conditions. 
Tnis proposal was published for 
comment in the Federal Register of 
September 1, 1989 (54 FR 36638). 
The RAC considered this amendment 
at the October 6, 1989. meeting. 
The RAC voted to appove this 
amendment by vote of 14 in favor, none 
opposed, and one abstention. The NIH 
Guidelines will be revised to read in 
Appendix A, Sublist A No. 6, as follows: 
6. Genus Klebsiella pncluding oxytoca). 
The NIH Guidelines will be revised to 
read in Appendix as follows: 
Klebsiella — all species except oxytocc. 
I accept these recommendations and 
Appendix A Sublist A and App endix 
B-I-B-1 have been modified 
accordingly. 
EL Points to Consider for Protocols for 
the Transfer of Recombinent DNA into 
the Genome of Human Subjects 
On September 29. 1986, the RAC 
adopted the Points to Consider in the 
Design and Submission of Human 
Somatic-Cell Gene Therapy Protocols, 
which was prepared by the Human 
.•/Gene Therapy Subcommittee. 
At the January 30, 1989. meeting, the 
RAC endorsed a proposal to form a 
subcommittee to update and report to 
the Human Gene TTierapy Subcommittee 
recommendations to amend the Points to 
Consider. The Points to Censider 
Recombinant DNA Research, Volume 14 
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