PMC

A) The whole genome B.burgdorferi pie chart was created using data from The Comprehensive Microbial Resource database (http://cmr.jcvi.org/cgi-bin/CMR/shared/GetNumAndPercentGenesInARole.cgi). B) The pie chart showing proteins downregulated (total 90 proteins) in hrpA mutant clones was created using the data in . C) The pie chart showing proteins upregulated (total 97 proteins) in hrpA mutant clones was created using the data in .

The sequence and location of conserved motifs of the DEAH-box family aligned with B. burgdorferi HrpA are shown. Amino acids in the DEAH-box consensus that are conserved at least 80% are shown in capital letters, and small letters represent the amino acids with 50–70% conservation. For further details regarding the DEAH-box motifs see , . * denotes either T or S at three positions in the consensus sequence. The regions highlighted in yellow are perfect matches between the B. burgdorferi HrpA protein and the consensus sequence and the numbers below the boxes represents the position of the conserved motifs in HrpA.

Whole cell protein extracts are shown for A) wild-type B. burgdorferi B31 clone 5A4 and B) hrpA mutant strain GCB1165. Whole cell protein extracts were separated on non-linear pH 3–10 IPG strips in the first dimension and 12% SDS-PAGE gels in the second dimension. Differentially expressed protein spots are circled and numbered. Select proteins were identified by trypsin digestion followed by LC MS/MS analysis of spots from silver stained gels. shows relative fold differences in expression.

A) Gene disruption strategy. The infectious B. burgdorferi strain B31, clone 5A4 (B31-5A4) was transformed with a knockout plasmid carrying a 1 kb gentamicin cassette (blue) that replaced the central 500 bp of the hrpA gene (yellow) as described in . The two possible outcomes of recombination events with the target gene are shown: allelic exchange would result in gene disruption while integrative recombination of the knockout plasmid would result in merodiploid formation. The position of PCR primers used for construct verification are shown by arrows on the schematic. B) PCR verification of the hrpA disruption. Each gene disruption was subjected to four PCR analyses. Panel 1) The presence of the gentamicin resistance cassette was confirmed as shown. The shuttle vector pBSV2G served as the positive control (c⁺) for amplification of the gent cassette (lane 5). Panel 2) The portion of hrpA expected to be deleted in a gene disruption was not detected in hrpA2, 3 or 4 (lanes 7, 8 and 9). Lane 10 was a negative control (c⁻) that lacked DNA template. Panel 3) The size of the hrpA gene was compared in the three mutant strains. The expected 2.1 kb gene disruption products were observed (lanes 12, 13 and 14) in comparison to the 1.5 kb product from the wild-type hrpA gene (lane 11). Lane 15 was a negative control (c⁻) that lacked DNA template. Panel 4) Confirmation of the correct insertion site was performed using combinations of the target gene primers and primers internal to the gentamicin cassette to amplify the hrpA boundaries. The left boundary in the hrpA knockout clones displayed the expected 1.4 kb product (lanes 18, 20 and 22) and the right boundary showed the expected product of approximately 1.3 kb (lanes 19, 21 and 23). A 100 bp ladder on the left side of is relevant to the two left panels, and a 1 kb ladder on the right side applies to the two right panels (M). The schematic in part A of the figure is modified from .