77374 Federal Register / Vol. 45, No. 227 / Friday, November^21^1^80^/^Notic^ 
of DNA derived from E. coli K-12 and 
Streptomyces coelicolor, S. 
aureofaciens, S. rimosus, S. griseus, S. 
cyaneus, and S. venezuelae using NIH- 
approved Staphylococcus aureus 
plasmids as vectors under P2 conditions. 
• " Streptomyces coelicolor, S. 
aureofaciens, S. rimosus, S. griseus, S. 
cyaneus, and S. venezuelae can be used 
as hosts for the cloning of DNA derived 
from B. subtilis, E. coli K-12, or from S. 
aureus vectors that have been approved 
for use in B. subtilis, under P2 
conditions, using as vectors any 
plasmids indigenous to these 
Streptomyces species or able to 
replicate in these hosts by natural 
biological mechanisms." 
The proposed appeared in the Federal 
Register of August 21, 1980 (45 FR 
55928). No comments were received 
concerning the proposal during the thirty 
day comment period. The RAC 
evaluated the proposal at the September 
25-26, 1980 meeting, considering and 
voting separately on each of the two 
sections proposed to be amended. 
With regard to the First section, by a 
vote of 14 in favor, none opposed, and 3 
abstentions, the RAC recommended that 
the proposed amended language be 
adopted, i.e.: 
• "Bacillus subtilis strains that do not 
carry an asporogenic mutation can be 
used as hosts specifically for the cloning 
of DNA derived from E. coli K-*12 and 
Streptomyces coelicolor S. aureofaciens, 
S. rimosus, S. griseus, S. cyaneus, and S. 
venezuelae using NIH-approved 
Staphylococcus aureus plasmids as 
vectors under P2 conditions.” 
I accept this recommendation, and 
this section of Appendix E has been so 
amended. 
The RAC then considered the second 
section proposed to be amended. During 
the discussion it was suggested that the 
language be hiodified to specify the use 
of "nonconjugative” plasmid vectors. By 
a vote of 15 in favor, none opposed, and 
2 abstentions, the RAC recommended 
approval of the following modified 
language: 
• "Streptomyces coelicolor S. 
aureofaciens, S. rimosus, S. griseus, S. 
cyaneus, and S. venezuelae can be used 
as hosts for the cloning of DNA derived 
from B. subtilis, E. coli K-12, or from S. 
aureus vectors that hqve been approved 
for use in B. subtilis, under P2 
conditions, using as vectors any 
nonconjugative plasmids indigenous to 
these Streptomyces species or able to 
replicate in these hosts by natural 
biological mechanisms." 
Subsequent to the RAC meeting, two 
comments on this amended language 
were received by the NIH. 
One commentator wrote that the 
proposal: 
"* * ‘would have the effect of reversing a 
previous decision by the RAC and increasing 
the level of containment required for 
experiments that have been permitted under 
P2 conditions for some time. Thus, the 
amendment is a ‘major action'; it does not 
seem appropriate that it be made part of the 
Guidelines in the absence of a 30-day period 
providing an opportunity for workers in the 
field and others to offer comments on it. 
"It should also be noted that the concept of 
'conjugative' versus ‘non-conjugative’ 
plasmids has been derived from enteric 
bacteria and may not apply to a wide variety 
of other bacterial species. For example, the 
mechanism for genetic exchange in 
Streptomyces species is not fully understood; 
it may be a consequence of hyphal fusion that 
permits transfer of plasmid and chromosomal 
genes. If this is true, it may never be possible 
to isolate a ‘non-conjugative‘ plasmid per 
se‘ * *” 
“Since all of the Streptomyces species that 
exchange genetic information with S. 
coelicolor are free-living non-pathogenic soil 
organisms — and since the change in the 
Guidelines originally proposed under Action 
910 involved genes from only non-pathogenic, 
well-characterized donor bacteria, I urge your 
approval of the original proposal without the 
amendment.” 
The other commentator wrote: 
"I would like to see this amended proposal 
published for public comment and put to a 
second vote at the next RAC meeting before 
you approve this action. The amended 
proposal failed to receive sufficient 
consideration of the following points. First of 
all, in no previous actions regarding 
Streptomyces species was any consideration 
given during the deliberations to establish 
levels of physical containment as to whether 
the vectors were capable of mediating 
exchange of genetic information. Therefore, 
this action by the RAC is more restrictive 
than previous actions and, in my opinion, is 
inconsistent with the intent of the RAC. In 
fact, experiments can be carried out at P2 
levels of containment in which B. subtilis, E. 
coli K-12, or S. aureus vector DNA is cloned 
into S. coelicolor hosts using vectors capable 
of self-mediated exchange in accordance 
with Appendix E of the current NIH 
Guidelines. 
"Second of all, the use of the term ‘non- 
conjugative' is inappropriate because the 
actinomycetes do not exchange genetic 
information by the process of conjugation as 
it is known for eubacteria. Rather 
actinomycetes exchange genetic information 
by heterokaryosis. In some experiments, the 
frequency of genetic exchange can be altered 
by the presence of plasmids and some 
plasmids can mediate their own transfer to 
new hosts. 
“I feel that to restrict cloning in the 
actinomycetes to using ‘non-conjugative’ or 
non-self-transmissible vectors would be 
unduly harsh and would severely restrict 
research with this valuable group of 
organisms. Considering the level of potential 
hazard (which I consider minimal) and the 
potential benefit (which I view as 
considerable), P2 containment is more than 
sufficient protection regardless of the vectors 
employed." 
Taking into consideration these 
comments, I am deleting the requirement 
for use of nonconjugative plasmids in 
these systems. If anyone wishes to 
propose a requirement for use of 
nonconjugative plasmids in these 
systems, it will be published for 
comment in the Federal Register as a 
proposed major action for consideration 
at a future RAC meeting. 
Accordingly, the relevant section of 
Appendix E has been amended to read 
as follows: 
• "Streptomyces coelicolor, S. 
aureofaciens, S. rimosus, S. griseus, S. 
cyaneus, and S. venezuelae can be used as 
hosts for the cloning of DNA derived from B. 
subtilis, E. coli K-12, or from S. aureus 
vectors that have been approved for use in B. 
subtilis, under P2 conditions, using as vectors 
any plasmids indigenous to these 
Streptomyces species or able to replicate in 
these hosts by natural biological 
mechanisms." 
VII. Request for Consideration of a 
Proposal to Clone the Tox A Gene of 
Staphylococcus Aureus 
In a memorandum dated July 29, 1980, 
Drs. Alan G. Barbour and Leonard W. 
Mayer of the Laboratory of Molecular 
Structure and Functions, Rocky 
Mountain Laboratory, National Institute 
of Allergy and Infectious Dieases, 
requested an assessment of the 
containment levels appropriate for 
cloning the Staphylococcus aureus 
pyrogenic exotoxin type A (Tox A). Drs. 
Barbour and Mayer indicated that they 
would prefer to clone the Tox A gene in 
an HV2 Bacillus subtilis host-vector 
system (since one of the activities of 
Tox A is enhancement of the toxicity of 
E. coli endotoxin) but, if no HV2 B. 
subtilis host-vector system were 
available, they requested permission to 
clone the Tox A gene in an EK2 E. coli 
K-12 host-vector system. 
The RAC took this request under 
consideration at the September 25-26, 
1980 meeting. The major determination 
for the RAC to make was whether Tox 
A is a "potent toxin” under Section I-D- 
2 of the Guidelines. It was rioted that the 
lethality of Tox A for animals is not 
great. The RAC by a vote of 17 in favor, 
none opposed, recommended that the 
cloning of the Tox A gene be permitted 
under P3 containment with either an 
HV2 B. subtilis or an EK2 E. coli K-12 
host-vector system. 
The scientists who submitted this 
request, noting that one of the activities 
of Tox A is enhancement of the toxicity 
of E. coli endotoxin, said they preferred 
to work in B. subtilis rather than E. coli 
[ 208 ] 
