Complementation of mutants The construction of plasmids to complement tat and
bro2 mutant strains was achieved as follows. Plasmid DNA (pRB.TatA.5, pRB.TatB.1, pRB.TatC.2, pRB.Tat.1, pRN.Bro11, pTS.BroKK.Ec) was digested with BamHI to release the cloned M. catarrhalis genes from the vector pCC1. Gene fragments were purified from agarose gel slices using the High Pure PCR Product Purification Kit (Roche Applied Science), ligated into the BamHI site of the M. catarrhalis/Haemophilus Lazertinib research buy influenza-compatible shuttle vector pWW115 , and electroporated into H. influenzae strain DB117. Spectinomycin resistant (spcR) colonies were screened by PCR using the pWW115-specific primers P17 (5′-TACGCCCTTTTATACTGTAG-3′) and P18 (5′-AACGACAGGAGCACGATCAT-3′), which flank the BamHI cloning site, to identify clones containing inserts of the appropriate size for the tat and bro2 genes. This process produced plasmids pRB.TatA, pRB.TatB, pRB.TatC, pRB.TAT, pTS.Bro, and pTS.BroKK. The O35E.TA mutant was naturally transformed with plasmids pWW115, pRB.TatA, and pRB.TatABC. The plasmids pWW115, pRB.TatB, and pRB.TAT were introduced in the O35E.TB mutant by
Rigosertib supplier natural transformation. The tatC mutants O35E.TC and O12E.TC were naturally transformed with the vector pWW115 and plasmid pRB.TatC. The plasmids pWW115, pTS.Bro, and pTS.BroKK were electroporated into the bro-2 mutant strain O35E.Bro. The successful introduction of these plasmids into the indicated strains was verified by PCR analysis of spcR transformants with the pWW115-specific primers P17 and P18, and by restriction endonuclease analysis of plasmid DNA purified from each strain. Growth rate experiments Moraxella. catarrhalis strains were first cultured onto agar plates supplemented with appropriate antibiotics. These plate-grown bacteria were used to inoculate 500-mL sidearm flasks containing 20-mL of broth (without antibiotics) to an optical density (OD) of 50 Klett units. The cultures were then incubated with shaking (225-rpm) at a temperature
of 37°C for 7-hr. The OD of each culture was determined every 60-min using a Klett™ Colorimeter (Scienceware®). These experiments were repeated on at least three separate occasions for each strain. In some experiments, aliquots were taken out of each culture after recording the optical density, Selinexor nmr diluted, and spread onto agar Histone demethylase plates to determine the number of viable colony forming units (CFU). Carbenicillin sensitivity assays Moraxella catarrhalis strains were first cultured onto agar plates supplemented with the appropriate antibiotics. These plate-grown bacteria were used to inoculate sterile Klett tubes containing five-mL of broth (without antibiotics) to an OD of 40 Klett units. Portions of these suspensions (25 μL) were spotted onto agar medium without antibiotics as well on plates supplemented with carbenicillin, and incubated at 37°C for 48-hr to evaluate growth. Each strain was tested in this manner a minimum of three times.