Bacteriophages cause serious problems in biotechnology laboratories. Both prophage induction and phage infection are dangerous for ongoing and subsequent cultivations. Unfortunately, many commonly used E. coli strains harbour lambdoid prophages. We were looking for simple procedures which could alleviate the problems caused by lambdoid bacteriophages.
Although effects of bacterial growth rate on phage lytic development have been investigated previously [7,8,9], the results of these studies were used in basic research rather than in biotechnological applications. Here we report that deleterious effects of spontaneous lambdoid prophage induction are significantly decreased at low growth rates of lysogenic bacteria relative to high growth rates. These differences could be caused either by a lower frequency of prophage induction in slowly growing cells or by a less efficient lytic development after excision of the phage DNA from the host chromosome under conditions supporting low bacterial growth rates. In fact, low burst sizes of λ phage in slowly growing bacteria have already been reported [7,8,9]. Nevertheless, irrespective of the specific mechanism of the observed phenomenon, it is clear that the number of phages per cell in cultures of slowly growing lysogenic bacteria was below the detection limit (Table 1). Thus, cultivation of lysogenic bacteria in bioreactors at low growth rates should prevent contamination of the culture with induced phages.
We demonstrated that overproduction of the cI repressor from a multicopy plasmid efficiently reduces the risk of lambdoid prophage induction. Furthermore, overproduction of cI in non-lysogenic strains should make bacteria resistant to infection by a corresponding lambdoid phage due to repression of phage early promoters, necessary for lytic development. Interestingly, even moderate overproduction of cI was also inhibitory for lysogenization, most probably due to indirect impairment of the int gene expression .
Lambdoid prophage induction can be provoked by factors stimulating the SOS response [1,2]. One could imagine that certain conditions in bioreactors stimulate the SOS response. The SOS-induced RecA* protein triggers self-cleavage of the cI repressor, causing prophage induction . To avoid this problem, one can clone an ind- allele of cI (e.g. cI857), whose product, the cI857 protein, is resistant to RecA*-mediated self-cleavage.
Obviously, for achieving both large amounts of a desired product from a cloned gene and prevention of lambdoid prophage induction, one should often use a pair of compatible plasmids, one bearing a recombinant gene and second overexpressing the cI gene. However, this should not be a serious problem as many vectors bearing different origins of replication (thus being compatible) are currently available .
Glucose is the most commonly used carbon source in biotechnological cultivations of E. coli. However, in accordance with previous reports [1,2,11], we demonstrated that replacement of glucose with another carbon source results in a significant increase in the efficiency of lysogenization after infection of the host cells by λ. Therefore, we propose to replace glucose with glycerol in the culture medium when lambdoid phage contamination is plausible.
In conclusion, we have demonstrated that relatively low growth rates of bacteria and replacement of glucose with glycerol in the medium should significantly reduce deleterious effects of lambdoid prophage induction in E. coli cultures. These simple procedures should alleviate problems with lamdoid phages contamination in bacterial cultures. Moreover, spontaneous induction of lambdoid prophage is impaired by efficient overproduction of the cI repressor from a multicopy plasmid. If induction of the SOS response during the cultivation is possible, we recommend to overexpress an ind- allele of the cI gene.