Pseudomonas putida KT2440 as part of a biological safety measure

Following the amendment to the Genetic Engineering Safety Ordinance (GenTSV), it was necessary for the continued existence of already recognized biological safety measures to be confirmed by the ZKBS in accordance with Section 7 (5) GenTSV. According to Section 8 (1) GenTSV (2019), the requirements for the use of a recipient organism as part of a biological safety measure are met if

1. a scientific description and a taxonomic classification of the recipient organism are available,
2. the reproduction of the recipient organism is only possible under conditions that are rarely or never encountered outside genetic engineering facilities,
3. the recipient organism is not pathogenic to humans, animals or plants and does not have any environmentally hazardous properties and
4. the recipient organism only undergoes minimal horizontal gene exchange with other species. P. putida KT2440 was recognized as a suitable recipient organism for biological safety measures in 1990 when the GenTSV came into force and has since proven safe for use in genetic engineering facilities. In the current ZKBS statement, the suitability of P. putida KT2440 as part of biological safety measures is now being re-examined and evaluated based on the criteria listed above. P. putida is a gram-negative, non-sporulating, chemoorganotrophic, aerobic rod and belongs to the Pseudomonadaceae family. The bacterium is a globally occurring saprophytic inhabitant of soils, particularly the rhizosphere of plants. P. putida KT2440 was established in the laboratory in 1981 from the strain P. putida mt-2, which in turn was isolated from garden soil in Japan in 1960. The genome of P. putida KT2440 has been completely sequenced several times over the past decades, demonstrating that spontaneous mutations occur in the strain as a result of long-term cultivation. P. putida KT2440 is well characterized and has long been used in a variety of biotechnology applications.
   P. putida has so far only rarely appeared as an opportunistic human pathogen. Furthermore, isolated cases of infections in fish in aquaculture have been reported. Therefore, P. putida was classified in risk group 2 according to Section 6 in conjunction with Section 5 Paragraph 1 of the GenTSV. P. putida KT2440, on the other hand, is assigned to risk group 1 by the ZKBS because this strain lacks most of the virulence factor genes found in pathogenic P. putida isolates or in the pathogenic, closely related species P. aeruginosa. Numerous studies with soil and water samples have shown that P. putida KT2440 cannot spread outside of genetic engineering facilities and that P. putida KT2440 does not establish permanently in the environment. Experimental data further showed that P. putida KT2440 is unable to actively take up DNA from the environment. Furthermore, P. putida KT2440 does not produce infectious prophages and does not possess conjugative plasmids that could enable gene transfer to other bacteria. The ZKBS declares that P. putida KT2440 meets the requirements for recognition as a recipient organism for biosafety measures according to Section 8, Paragraph 1 of the GenTSV. Relevant information on the suitability of individual P. putida KT2440-derived strains as recipient strains for biosafety measures can be found in the ZKBS database "Recipient Strains for Biosafety Measures."

The current ZKBS statement on P. putida KT2440 can be found at File number Az. 45270_c can be retrieved.