Materials and Methods

Bacterial strains, plasmids and oligonucleotides - The bacterial strains and plasmids used in this study are listed in Table S1 and the oligonucleotides are listed in Table S2.

Kanamycin was used at a final concentration of 100µg/ml for selection in Escherichia coli and 50µg/ml for selection in Bartonella henselae. B. henselae Houston-1 (ATCC

49882) strain with the “rough” autoadherent colony phenotype (7) was grown on chocolate agar prepared with heart infusion agar base (Difco, Detroit, MI) supplemented with 1% bovine hemoglobin (Beckton Dickinson, Cockeysville, MD). Cultures of B. henselae were maintained at 37°C and 5% CO2 and humidity to saturation.

Recombinant DNA methods - DNA ligations, restriction endonuclease digestions, and agarose gel electrophoresis were performed according to standard techniques (8). The polymerase chain reactions (PCR) were performed using Platinum PCR SuperMix High

Fidelity (Invitrogen) and a Gradient Mastercycler® (Eppendorf). Oligonucleotides were purchased from Sigma-Genosys (Sigma). Restriction and modification enzymes were purchased from Promega. QIAprep Spin Miniprep Kit from QIAGEN was used for all plasmid extraction and QIAGEN PCR cleanup kit was used for all restriction enzymes removal and DNA gel extraction.

Construction of the vectors - The broad host range pNSGroE Brucella expression vector (GenBank accession number AY576605) (12) was used as the backbone for the construction of the new vectors. The PgroE promoter with the downstream MCS was excised from pNSGroE vector using SalI and EcoRI restriction enzymes. The kanamycin resistance gene (kan) with its own promoter that allows for the selection of bacteria on kanamycin plates was amplified from pUC4K cloning vector (Pharmacia) using primers

Kan-F and Kan-R (Table S2). The EcoRI and SpeI restriction sites were added in the forward and reverse primers respectively, for directional cloning. The ScaI and XbaI sites were designed in the forward primer after EcoRI to add those two restriction sites as an extra unique cutting sites within the MCS followed by a strong translational stop signal

(UAAU) (14). The origin of replication (rep) was amplified with its own promoter as described before (12). After restriction digestion and gel purification, the tri-ligated reaction using the Brucella abortus PgroE promoter with down stream MCS, kan gene and rep gene was done with temperature cycle ligations method (TCL) (3) using

Mastercycler® Gradient programmed indefinitely to cycle between 30s at 10 º C and 30s at 30 º C for 12 hours to form plasmid pNS2GroE.

Promoter replacement - The PgroE promoter was excised with SalI and BamHI restriction enzymes and the promoter-less pNS2 construct was used for promoter replacement. The chloramphenicol resistance gene promoter (Pch), kanamycin resistance gene promoter (Pkan), ampicillin resistance gene promoter (Pamp) were amplified as described before (11). SalI and BamHI restriction sites were designed in the forward and reverse primers for directional cloning. In the reverse primer, six histidine and one glycine residues were engineered after the translational start codon to facilitate the epitope tagging at the amino terminus. To avoid the translational error or inhibition arising from rare codon bias (4) the preferred codon usage of Bartonella spp.

(http://www.cbs.dtu.dk ) for histidine and glycine were used. After restriction digestion and purification, Pch, Pkan, and Pamp were cloned into the pNS2 construct to form pNS2Ch, pNS2Kan and pNS2Amp expression vectors respectively. The hybrid Ptrc promoter with the downstream bacteriophage gene10 translational enhancer, minicistron, reinitiation ribosome binding site and lac operator was amplified from pTrc2HisA vector (Invitrogen) as described before (10) and ligated to the pNS2 construct to form pNS2Trc expression vector. The coliphage PT5 promoter with the downstream lac operator, synthetic ribosomal binding site (RBSII) and the 6XHis-tag codon sequence was excised with XhoI and BamHI from pQE-40 expression vector (Qiagen) and ligated to the pNS2 construct to form pNS2T5 expression vector. To avoid promoter occlusion (1) and plasmid destabilization (13) and to enhance mRNA stability (6), T1 and T2 transcription terminators derived from rrnB rRNA operon of E. coli (2) were amplified from pTrcHis2A expression vector (Invitrogen) using primers rrnBT-F and rrnBT-R (Table S2) and cloned downstream of the MCS.

Expression of -galactosidase (lacZ) - In order to study the expression and activity of the cloned promoters inside Bartonella, the promoter-less E. coli -galactosidase (lacZ) gene was amplified from pRSETB/-gal as described before (12) and cloned into all six expression vectors downstream of the various promoters. The ligation was performed such that an in-frame 6XHis tagged-LacZ fusion protein resulted. The plasmid constructs expressing lacZ were transformed into Mach1™ T1 Phage-Resistant (T1 R) competent

E. coli (Invitrogen). The lacZ expression constructs were extracted from E. coli and transformed into Bartonella via electroporation as previously described (9) To determine the ability of B. henselae harboring plasmids with various promoters driving expression of the lacZ reporter gene the Miller chromogenic -galactosidase assay was performed using 2-nitrophenyl β-D-galactopyranoside (ONPG) as a substrate. B. henselae were cultured on chocolate agar plates with kanamycin for 3 days and the growth collected into PBS with the OD600 adjusted to 0.3-0.5. Results were expressed in Miller units as previously described (5).

Western blots - B. henselae were collected from plates and suspended in sample buffer. Lysates were prepared by heating for 5 min at 1000C and clearing the lysates by centrifugation. Ten µg of the cleared lysate was loaded per well of a 4-12% gradient

NuPage gel (InVitrogen, CA). Proteins were resolved and transferred to nitrocellulose membranes. The membranes were blocked with phosphate buffer saline with 1.0% bovine serum albumin and reacted with purified anti-6XHis Tag antibodies at a dilution of

1:3000 according to the recommendations (ICL, OR). The bound antibody was detected using a goat anti-rabbit IgG conjugated with horse radish peroxidase at a 1:10,000 dilution (KPL, MD) and the signal developed using the Pierce ECL Western blot substrate (Thermo Scientific, IL).

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