Generalized transduction of serotype 1/2 and serotype 4b strains of Listeria monocytogenes

Abstract

SummaryThis is the ®rst report of generalized transduction in the Gram-positive, food-borne pathogen Listeria monocytogenes. Bacteriophages were isolated from the environment and from lysogens, or were obtained from other laboratories. Of the 59 bacteriophages tested, 34 proved to be capable of transduction. We exploited the ability of L. monocytogenes to grow at room temperature and isolated bacteriophages that were incapable of growth at 378C. Transductions at this temperature therefore eliminated transductant killing and lysogeny, as did inclusion of citrate and the use of a low multiplicity of infection. Transducing bacteriophages were found for each of the well-characterized L. monocytogenes strains: EGD, 10403, Mack (serotype1/2a), L028 (serotype 1/2c), Scott A (serotype 4b) and strains from the Jalisco and Halifax, Nova Scotia outbreaks (serotype 4b). P35 (fLMUP35) is a particularly useful generalized transducing bacteriophage with a wide host range (75% of all serotype 1/2 strains tested). Its disadvantages are that it is small and transduction is relatively infrequent. U153(fCU-SI153/95) is larger than P35 and transduction frequency increased 100-fold, but it has a very narrow host range. We demonstrated interstrain transduction and used transduction to test linkage between transposon insertions and mutant phenotypes in a variety of strains.

Listeria monocytogenes Is Resistant to Lysozyme through the Regulation, Not the Acquisition, of Cell Wall-Modifying Enzymes

“…This fragment was introduced into L. monocytogenes by using pPL2 or pAM401 as described previously (16,17). For lists of all of the strains and primers used in this study, see Tables S2 and S3 in the supplemental material, respectively. Transductions were performed by using U153 phage as previously described (18). Briefly, 10 7 phage were grown in the donor strain, incubated with 10 8 recipient bacteria, and selected for on BHI-erythromycin plates.…”

The Virulence Regulator PrfA Promotes Biofilm Formation byListeria monocytogenes

“…All mutants containing prfA::Tn917-LTV3 were constructed by the transduction of the transposon insertion in prfA from DP-L1075 (12) using L. monocytogenes phage P35 as described previously (15,17). Mutants with genes expressed from constructs integrated at the neutral locus tRNA Arg were constructed by using vectors derived from pPL2 (21).…”

Construction, Characterization, and Use of TwoListeria monocytogenesSite-Specific Phage Integration Vectors

“…More than 320 listeriophages have been isolated (summarized in reference 30), and many have restricted host ranges (19). It was unclear whether there was a biological barrier to U153 integrase gene function in host strains that do not support U153 infection.…”

“…Candidates that did not form plaques were then tested by spotting 10 l of culture on a lawn of Mack-4R to detect plaque formation. If this second test was negative, whether the candidate could support plaque formation by the phage from the parent 10403S strain (10403 [19]) was tested. Curing was confirmed molecularly by PCR with the comK-attBBЈ-specific primer pair PL60 and PL61 (sequences follow) for the absence of a phage at comK-attBBЈ.…”

“…Recently, the complete genome sequence and characterization of the phage attachment site of listeriophage A118 were reported (29), the genome sequence of the PSA prophage was completed (E. Sattelberger, M. Zimmer, R. Calendar, R. B. Inman, S. Scherer, and M. J. Loessner, submitted for publication), and a survey of listeriophage host ranges was conducted (19). These studies facilitated the development of two phage-based integration vectors, the first such vectors for use in L. monocytogenes.…”