Xl1 blue mrf

The E. coli strains XL1-Blue MRF’ and DH5α were purchased from Agilent technologies, INC (Santa Clara, CA, USA) and, Thermo Fisher Scientific, INC, respectively.
The wild strain of Corynebacterium pseudotuberculosis CBO 28033, which was used for library construction, was isolated aseptically from an external abscess from sheep by needle aspiration of closed lymph nodes. Identity was confirmed by biochemical tests (API CORYNE, Biomerieux, Marcy l’Etoile, France) for C. pseudotuberculosis biovar ovis.

The H. volcanii strain H26 [26 (link)] was used in this study as a wild-type for construction of the 16 deletion mutants. It contains a deletion in the pyrE2 gene, which enables two selection steps during mutant construction, and, thereby, facilitates and accelerates the procedure. It was grown in a complex medium with 2.1 M NaCl at 42 °C and good aeration (220 rpm) [27 (link)].
The Escherichia coli strain XL1-blue MRF’ (Agilent Technologies, Waldbronn, Germany) was used for cloning and was grown in standard media [28 ].

E. coli transformed with pSEX81 was grown at 37 °C in 2YTAG medium until the OD₆₀₀ reached 0.4, infected with KM13 helper phage with a multiplicity of infection of 20 (MOI = 20) at 37 °C for 30 min, and further incubated with shaking for 30 min. E. coli was collected by centrifugation at 4000 × g for 10 min and incubated in 2YTAK (16 g/l tryptone, 10 g/l yeast extract, 5 g/l NaCl, 100 μg/ml ampicillin, and 50 μg/ml kanamycin) medium at 30 °C with shaking for 16 h. The culture was centrifuged at 5000 × g for 10 min, and the supernatant was mixed with PEG/NaCl solution (20% (w/v) polyethylene glycol 6000 and 2.5 M NaCl) at the ratio of 4:1 (v/v) and incubated on ice for 1 h. The scFv-phages were pelleted by centrifugation at 10,000 × g for 30 min at 4 °C and resuspended in PBS. The scFv-phages were again precipitated using a PEG/NaCl solution and resuspended in PBS, and the bacterial debris was removed by centrifugation at 15,000 × g for 5 min. To titer the phage library, E. coli XL1-blue MRF’ (Agilent) at OD₆₀₀ = 0.4 were infected with a serial dilution of the phage library and incubated overnight on a 2YTAG agar plate at 37 °C. The grown colonies were counted to calculate the phage titer.

The strain H. volcanii H26 was obtained from Thorsten Allers (Nottingham, United Kingdom), it is a pyrE deletion strain lacking the plasmid pHV2. The deletion of the dhfr (dihydrofolate reductase) gene HVO_1279 has been described previously (Maurer et al., 2018 (link)). The deletion strains of genes encoding translation initiation factors have been generated in a previous study (Gäbel et al., 2013 (link)). Multi cycle PCRs were used to confirm that all mutants were still homozygous. The sequences of oligonucleotides are listed in Supplementary Table S1. In a few cases the mutants could not be regrown from permanent cultures, therefore, they were regenerated as described (Gäbel et al., 2013 (link)) using the oligonucleotides listed in Supplementary Table S1. All overproduction strains have been generated in this study (see below).
Haloferax volcanii strains were grown in complex medium with 50μg/ml uracil as previously described (Dambeck and Soppa, 2008 (link)). The cultures were grown in Erlenmeyer flasks at 42°C with shaking at 250rpm. Growth was either measured spectroscopically at 600nm or cells were counted using a Neubauer counting chamber.
The E. coli strain XL1-blue MRF’ (Agilent Technologies, Waldbronn, Germany) was used for cloning. It was grown in complex SOB medium under standard conditions (Hanahan, 1983 (link)).

Escherichia coli strains XL1-Blue MRF’ and BL21-Gold(DE3) (Agilent Technologies, Santa Clara, CA, USA) were used for cloning of A. actinomycetemcomitans-derived genes encoding different depolymerase enzymes and expression of recombinant proteins. BL21-Gold(DE3) harboring a plasmid construct coding for the S. aureus phage endolysin LysK in a pET-21a backbone (EMD Biosciences, San Diego, CA, USA) served as expression strain for recombinant production of a C-terminally 6× His-tagged version of LysK [21 (link)]. E. coli strains were cultured at 37 °C in Luria-Bertani (LB) medium supplemented with ampicillin (100 µg/mL; for BL21-Gold(DE3)) or ampicillin and tetracycline (30 µg/mL; for XL1-Blue MRF’) for plasmid selection. S. aureus and A. actinomycetemcomitans strains used in this work are listed in Table 1. S. aureus was routinely cultured aerobically in tryptic soy broth (TSB; Biolife, Milan, Italy) at 37 °C, and A. actinomycetemcomitans was grown on Difco^TM Columbia Blood Agar plates (BD, Allschwil, Switzerland) at 37 °C in the presence of 10% CO₂.

The H. volcanii strain H26 was used as a parent strain and wildtype for this study (Allers et al., 2004 (link)). It is a pyrE2 deletion mutant, which enables the easy generation of deletion mutants. The deletion mutant of the sRNA132 gene has been described before (Jaschinski et al., 2014 (link)), generation of the remaining deletion mutants is described below.
The H. volcanii strains were grown in complex medium with the optimal NaCl concentration of 2.2 M as described (Jantzer et al., 2011 (link)). In short, 30 ml cultures were grown in 100 ml Erlenmeyer flasks at 42°C with good aeration (250 rpm). Growth was monitored spectroscopically at 600 nm or with a counting chamber.
The Escherichia coli strain XL1-blue MRF’ (Agilent Technologies, Waldbronn, Germany) was used for cloning, it was grown under standard conditions (Green and Sambrook, 2012 ).