Strict Conservation yet Non-Essential Nature of Plasmid Gene bba40 in the Lyme Disease Spirochete Borrelia burgdorferi

ABSTRACT The highly segmented genome of Borrelia burgdorferi, the tick-borne bacterium that causes Lyme disease, is composed of a linear chromosome and more than 20 co-existing endogenous plasmids. Many plasmid-borne genes are unique to B. burgdorferi and some have been shown to provide essential functions at discrete points of the infectious cycle between a tick vector and rodent host. In this study, we investigated the role of bba40, a highly conserved and differentially expressed gene on a ubiquitous linear plasmid of B. burgdorferi. In a prior genome-wide analysis, inactivation of bba40 by transposon insertion was linked with a noninfectious phenotype in mice, suggesting that conservation of the gene in the Lyme disease spirochete reflected a critical function of the encoded protein. To address this hypothesis, we moved the bba40::Tn allele into a similar wild-type background and compared the phenotypes of isogenic wild-type, mutant and complemented strains in vitro and throughout the in vivo mouse/tick infectious cycle. In contrast to the previous study, we identified no defect in the ability of the bba40 mutant to colonize the tick vector or murine host, or to be efficiently transmitted between them. We conclude that bba40 joins a growing list of unique, highly conserved, yet fully dispensable plasmid-borne genes of the Lyme disease spirochete. We infer that the experimental infectious cycle, while including the tick vector and murine host, lacks key selective forces imposed during the natural enzootic cycle. IMPORTANCE The key finding of this study contradicts our premise that the ubiquitous presence and strict sequence conservation of a unique gene in the Lyme disease spirochete, Borrelia burgdorferi, reflect a critical role in either the murine host or tick vector in which these bacteria are maintained in nature. Instead, the outcome of this investigation illustrates the inadequate nature of the experimental infectious cycle currently employed in the laboratory to fully model the enzootic cycle of the Lyme disease spirochete. This study also highlights the importance of complementation for accurate interpretation of mutant phenotypes in genetic studies of Borrelia burgdorferi.

are well executed and the conclusions are justified. Below are a few minor comments.
To some degree, the title is too general and overstated as study of a single gene is insufficient to support this statement.
Line 60, "a highly conserved and tightly regulated gene". bba40 is highly conserved but there is no evidence that it is tightly regulated. If so, how? Line 120, "data not shown", suggest include the southern blot result in Figure 1. Line 146, "ref 17", delete "ref". Line 218 "BamH1 and Kpn1" should be "BamHI and KpnI" Table 1, please define "Ti, qF, and qR" for those primers. Figure 2, In addition to comparing the expression level of bba40 to that of bba41, the expression level of bba40 between wt, the mutant, and two complemented strains should be compared too.
Reviewer #2 (Comments for the Author): It is an appropriately-designed study that addresses an interesting disparity in the infection phenotype of a Borrelia burgdorferi bba40 mutant. A few minor comments/suggestions are provided below for the authors to address.
-Line 101 -The statement about bba40 expression is vague. Please specifically state the expression pattern at the different temperatures and/or in response to different host signals (e.g., bba40 is more highly expressed at ...).
-Line 112-114 -I would like the authors to provide more details about how this result was determined. Labs that utilize this Tn library could benefit from information regarding how to screen their mutants of interest for clonality or multiple insertions. Line 124 -Also reference Table 2 when discussing the PCR screening of their mutant.
-The sum total of the murine infection data presented by the authors is convincing, but they should consider adding tissue burden data if it is available.

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Response to Reviewer comments:
We thank the reviewers for their positive comments and constructive suggestions, which we have addressed as detailed below. Cited line numbers refer to the highlighted text of the "Marked-Up Manuscript" file.
Reviewer #1 (Comments for the Author): A previous report using transposon mutagenesis showed that bba40, a plasmid-encoded conserved function unknown gene, is essential for the infectivity of Borrelia burgdorferi in mice. In this report, Kasumba et al first reconstructed this mutant and then had this mutant complemented using two different methods. Using these mutant and complemented strains, they reassessed the role of bba40 in the pathogenicity of B. burgdorferi throughout the mouse-tick infection cycle. The obtained results demonstrated that bba40, though highly conserved among Borrelia genus, is dispensable for B. burgdorferi in vitro growth and infection in both mice and ticks. In general, this manuscript is well written, the genetic studies and in vivo mice and tick infectious experiments are well executed and the conclusions are justified. Below are a few minor comments.
To some degree, the title is too general and overstated as study of a single gene is insufficient to support this statement. Response: The scope of the title has been narrowed as requested.
Line 60, "a highly conserved and tightly regulated gene". bba40 is highly conserved but there is no evidence that it is tightly regulated. If so, how? Response: "tightly regulated" has been removed from this sentence on line 60 and a more explicit description of bba40 expression has been added on lines 101-105 of the revised manuscript, as per reviewer #2's comment below.
Line 120, "data not shown", suggest include the southern blot result in Figure 1. Response: The southern blot analysis is fully consistent with the PCR data, but more difficult to appreciate. The PCR data shown in Figure 1D clearly demonstrate the structure and genomic location of bba40 alleles in wt, mutant and complemented strains. Therefore, we feel the southern blot does not merit inclusion in Figure 1 and the sentence in which it was described as "data not shown" (lines 121-122) has been deleted. Line 146,"ref 17",delete "ref". deleted Line 218 "BamH1 and Kpn1" should be "BamHI and KpnI" revised Table 1, please define "Ti, qF, and qR" for those primers. Definitions added as a footnote to Table 1 Figure 2, In addition to comparing the expression level of bba40 to that of bba41, the expression level of bba40 between wt, the mutant, and two complemented strains should be compared too. Response: We were a bit confused by this comment as Figure 2 provides the bba40 transcript levels in all strains, confirming the absence of bba40 transcript in the Tn mutant and its restoration to ~wt levels by complementation. The statistical analysis of transcript levels in Figure 2 was undertaken to assess the impact of the Tn insertion in bba40 on expression of the immediate downstream gene, bba41, which is significantly reduced and not restored in strains complemented in trans with bba40. A statistical analysis of bba40 transcript levels between strains is extraneous to this point.

Reviewer #2 (Comments for the Author):
It is an appropriately-designed study that addresses an interesting disparity in the infection phenotype of a Borrelia burgdorferi bba40 mutant. A few minor comments/suggestions are provided below for the authors to address.
-Line 101 -The statement about bba40 expression is vague. Please specifically state the expression pattern at the different temperatures and/or in response to different host signals (e.g., bba40 is more highly expressed at ...). Response: As indicated above, we have described the previously reported pattern of bba40 expression during spirochete growth at different temperatures or in response to host signals on lines 101-105 of the revised manuscript.
-Line 112-114 -I would like the authors to provide more details about how this result was determined. Labs that utilize this Tn library could benefit from information regarding how to screen their mutants of interest for clonality or multiple insertions. Response: We have added a description of screening TN mutant stocks for non-clonality or multiple insertions on lines 204-212 of the Methods Section. We did not include a description of our preliminary findings in the Results Section because we do not wish to overstate our initial data, which raised concerns but were incomplete. As described in the Results and Methods Sections, we addressed the possibility of non-clonality or a second TN insertion by moving the bba40::Tn allele into a genetically comparable, but independently derived B31 clone that we have routinely employed in genetic studies. We emphasize the critical importance of complementation in all genetic studies in B. burgdorferi, including those utilizing Tn mutants, at several points throughout the manuscript (lines 46-48, 74-76 & 166-168).
Line 124 -Also reference Table 2 when discussing the PCR screening of their mutant. Response: Table 2 referenced on line 126 of the revised manuscript.
-The sum total of the murine infection data presented by the authors is convincing, but they should consider adding tissue burden data if it is available. Response: Tissue burden data are not available, but efficient acquisition of all strains by feeding larval ticks (Table 3, Fig 4A) demonstrates biologically comparable spirochete loads in infected mouse tissues.