When appropriate, plates or broths were supplemented with erythro

When appropriate, plates or broths were supplemented with erythromycin (Erm) (5 μg/ml) and/or chloramphenicol (Cm) (5 μg/ml). Growth TPX-0005 molecular weight was monitored by measuring optical density of cultures at 600 nm (OD600) at regular time intervals. To investigate the effect of various stress agents on RpoE activity, cells were grown to mid log phase (OD600 = 0.6-0.7) and

treated for different time periods (30 min-1 h) with hydrogen peroxide (5 mM), diamide (100 mM), 0.01% SDS-0.1 mM EDTA or methylene blue (1 μM) in the presence of white light (source of singlet oxygen) [77]. Sequences from the following strains (with Genbank ID) were downloaded for comparative aligments: N.meningitidis_MC58 (AE002098); N.meningitidis_FAM18 (AM421808); N.meningitidis_053442 (Tideglusib CP000381); N.meningitidis_Z2491 (AL157959); N.gonorrhoeae_FA1090 (AE004969); N.gonorrhoeae_NCCP11945 (CP001050); N.cinerea_ATCC_14685 check details (ACDY00000000); N.flavescens_NRL30031/H210 (ACEN00000000); N.lactamica_ATCC_23970 (ACEQ00000000); N.subflava_NJ9703 (ACEO00000000); N.sicca_ATCC_29256 (ACKO00000000); N.mucosa_ATCC_25996 (ACDX00000000); Streptomyces coelicolor_A3(2)

(AL645882); Rhodobacter sphaeroides_ATCC_17025 (CP000661). Construction of ΔrpoE and ΔNMB2145 mutants of N. meningitidis N. meningitidis H44/76 knock-out mutants of rpoE (NMB2144) and NMB2145 were constructed

using the PCR-ligation-PCR method [79, 80]. All primers used in this study are listed in of Table 1. PCR products were generated with primer pairs CTsE-1/CTsE-2 and CTsE-3/CTsE4 for creating ΔrpoE and primer pairs CT-2145-1/CT2145-2 and CT-2145-3/CT-2145-4 for creating ΔNMB2145, ligated and the ligation products were reamplified with primer pairs CTsE-1/CTsE-4 (for ΔrpoE) and CT-2145-1/CT-2145-4 (for ΔNMB2145). The resulting PCR products were cloned into pCR2.1 (Invitrogen). The EcoRI digested Erm resistance cassette from pAErmC’ [81] was introduced into the created unique MfeI restriction site yielding plasmids pCR2.1-NMB2144 and pCR2.1-NM2145. The ΔrpoE and ΔNMB2145 strains were generated by natural transformation of strain H44/76 with pCR2.1-NMB2144 and pCR2.1-NMB2145 respectively, and selection for Erm resistance. Replacement of NMB2144 and NMB2145 by the Erm cassette was confirmed by PCR with primer pair CTsE-5/CTsE-6 (for ΔrpoE) and primer pair 2144-01/CT-2145-6 for ΔNMB2145. The orientation of the Erm cassette was determined by PCR using primer pair JP19/JP20 and mutant strains in which the transcriptional direction of the Erm cassette was in accordance with the transcriptional direction of the deleted genes were selected.

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