Using this approach, we have constructed the first Afipia mutants, with insertions in two genes responsible for flagella biosynthesis. Furthermore, we demonstrate the suitability of the pBBR1MCS2 broad-host-range plasmid as a vector system

for the study of Afipia. All chemicals were of reagent grade and purchased from Sigma-Aldrich (Taufkirchen, Germany) or Roth (Karlsruhe), unless specified differently. EZ∷Tn〈KAN-2〉Tnp Transposome Kit and type I restriction inhibitor were from Epicentre (Madison, WI). The antibodies CSD11 directed against the flagellin of Afipia (courtesy of Mr William Bibb, formerly Division of Bacterial and Mycotic Diseases, Centers for Disease Control and Prevention, Atlanta, GA) and a rabbit antiserum raised see more to a mixture of formaldehyde-fixed Fulvestrant clinical trial A. felis, Afipia broomeae, Afipia clevelandensis, Afipia genospecies 1, 2 and 3 were used in this study. Polyclonal anti-Bartonella bacilliformis flagellae was from Dr Michael Minnick, University of Montana, Missoula (Scherer et al., 1993). Afipia felis type strain (ATCC 53690)(English et al., 1988; Brenner et

al., 1991), Afipia birgiae (CIP 106344) (La Scola et al., 2002) and A. genospecies 2 (ATCC 49722) were used in all experiments and grown on buffered charcoal yeast extract (BCYE) agar (18 g L−1) plates buffered with 2 g ACES L−1. Liquid medium used the same formulation, but charcoal and agar were omitted (modified BYE medium). Cultivation was under aerobic conditions at 30 °C stationary or in a rotatory shaker at 200 r.p.m.

Escherichia coli DH5α was used for propagation of plasmids pSC301 and pBBR1MCS-2 and was grown in Luria broth (15 g agar L−1). Luria broth/10 g L−1 agar plates were supplemented with 200 μg kanamycin sulphate mL−1 where specified. Cultivation was under aerobic conditions Vasopressin Receptor at 37 °C stationary or in a rotatory shaker at 200 r.p.m. Plasmid pBBR1mCS-2 was a kind gift of Dr Michael E. Kovac (Baldwin Wallace College, Berea, OH). To construct plasmid pBBR1MCS2-GFP, the GFP gene was removed from pSC301 (Cowley & Av-Gay, 2001) using XbaI und PvuI and overhangs were filled or digested with Klenow fragment to produce blunt ends. pBBR1MCS-2 (Kovach et al., 1995) was digested with EcoRV and ligated with the GFP fragment using T4 ligase. Transposone mutagenesis was performed using the EZ∷Tn〈KAN-2〉Tnp Transposome Kit from Epicentre. Afipia bacilli were grown in BYE broth at 30 °C to an OD600 nm of 0.2 and centrifuged for 5 min at 2500 g. The resulting pellet was rinsed three times with ice-cold 10% glycerol in phosphate-buffered saline (PBS) and bacteria were diluted to 1 × 1010 mL−1. For each electroporation sample, 100 μL was used in an Eppendorf cuvette with 0.1 cm diameter. One microlitre transposome and 1 μL type I restriction inhibitor were added and a pulse of 2,2 kV was given with an Micro Pulser Elektroporator (BioRad, München, Germany).