To date, all B. burgdorferi genome arrays have been designed based on the genome sequence of strain B31. Initially, whole genome arrays were constructed with PCR products of >1600 putative B. burgdorferi open reading frames (ORFs) and were spotted on either glass slides or nylon membranes [8,9,10]. Subsequently, 70-mer oligonucleotides spotted on glass slides were employed in order to improve the reliability of hybridization signal intensity [11,12]. In addition, several groups have employed other custom glass slide or chip designs representing the complete genome or smaller sub-arrays of selected ORFs [13,14,15]. Table 1 contains a listing of all published B. burgdorferi microarray studies and provides the array types employed.

In general, the steps for studying global gene expression changes in the B. burgdorferi transcriptome are as follows: RNA isolation, generation of labeled cDNA, array hybridization and scanning, data acquisition and analysis. In initial studies with nylon membrane arrays, cDNA was radioactively labeled with ³³P; a detailed protocol is described in Ojaimi et al. [9]. Subsequently, a variety of high density microarray designs were developed and used fluorescently labeled DNA or cDNA for hybridization. Data acquisition, normalization and statistical analysis were particular to each type of microarray employed and details can be found in the respective references in Table 1. All published microarray data were deposited either in Array Express or NCBI’s Gene Expression Omnibus (GEO) databases. In addition, the results of virtually all published microarray studies reported to date have been validated by quantitative reverse transcription polymerase chain reaction (qRT-PCR).

To identify temperature-responsive genes, Ojaimi et al. [10] compared gene expression of B. burgdorferi cells grown at 23 or 35 °C (to mimic the tick or mammalian environment, respectively). 215 genes were differentially expressed at the two temperatures; with 133 showing greater expression at 35 °C relative to 23 °C. Interestingly, 136/215 (63%) temperature-responsive genes were encoded on plasmids. Of particular note, are linear plasmid lp54 and the circular cp32 plasmids; 45% of the putative ORFs encoded on lp54 exhibited temperature-regulated expression and >20% of cp32-encoded ORFs responded to temperature shift. Transcripts known to have elevated levels during mammalian infection (e.g., those for outer surface protein C (OspC), decorin binding proteins A/B (DbpAB) and the alternative sigma factor RpoS) displayed elevated expression at 35 °C. Similarly, genes subsequently shown to be more highly expressed during the tick phase (glycerol uptake and utilization operon (glpFKD) and chbC, encoding a component of the chitobiose transporter) had significantly elevated transcript levels at 23 °C [10].

Revel et al. [8] carried out a similar analysis, but also varied the pH of the growth medium so as to mimic the environment in the unfed tick (23 °C/pH 7.5) and fed tick states (37 °C/pH 6.8). A total of 94 genes were differentially expressed between the two temperatures; 79 had higher expression at 37 °C. Among transcripts elevated at the higher temperature were those for OspC and DbpA/B, as expected. In addition, transcripts for chemotaxis and motility functions and the OppA components of the oligopeptide ABC transporter were also elevated under the “fed tick” condition. Fifteen transcripts encoded on lp54 were differentially expressed, consistent with the findings of Ojaimi et al. [10]. Interestingly, there was only limited concordance between the Ojaimi and Revel data sets. This is likely the result of methodological differences between the two studies, including different array types (membrane array vs. glass slide microarray), pH of the BSK growth medium, slightly different temperatures (35 °C vs. 37 °C) and differences in the data analysis approaches.

The paucibacillary nature of B. burgdorferi infection in mammals led Akins et al. [37] to develop an alternative approach for isolating spirochetes in the host-adapted state. The method involves cultivating B. burgdorferi in BSK medium contained within dialysis membrane chambers (DMCs) implanted in a rat peritoneal cavity [37]. Numerous studies have demonstrated that gene expression of B. burgdorferi cultivated in DMCs is markedly different from that observed for spirochetes cultivated in vitro in the same medium at 37 °C [38,39,40]. Three microarray studies have been published in which transcriptome comparisons between B. burgdorferi cultivated in vitro at 37 °C and in DMCs were reported. In Revel et al. [8], 66 genes showed altered expression between these two conditions; only 6/66 exhibited higher expression in DMCs. Surprisingly, expression of some recognized mammalian phase genes such as ospC and dbpA/B was not induced. In a subsequent study by Brooks et al. [18], a total of 125 transcripts were differentially expressed between B. burgdorferi cultivated at 37 °C in vitro and DMCs—58 transcripts were induced (including ospC) and 67 were repressed. Among the latter, only three were chromosomally-encoded and the vast majority encode putative proteins of unknown function [18]. Interestingly, there was less than 10% overlap between the Revel and Brooks’ datasets, likely the result of methodological differences between the two studies. Caimano et al. [12], also performed whole transcriptome analysis of B. burgdorferi grown at 37 °C and in DMCs. Their study was designed to identify the regulon controlled by RpoS and a direct comparison of wild-type B. burgdorferi at the two conditions was not provided. However, the results clearly demonstrate that gene expression differs substantially between in vitro- and DMC-cultivated organisms. Taken together, these findings demonstrate that temperature alone does not elicit the distinctive mammalian host modulation of B. burgdorferi gene expression, but in addition requires mammalian host-specific signals.

As an alternative to DMC cultivation, Tokarz et al. [19], examined the combined effect of temperature and blood so as to simulate the environmental changes B. burgdorferi encounter as they transit from tick vector to a mammalian host. Spirochetes were incubated in the presence or absence of 6% human blood for 48 h and transcriptomes were compared. A total of 154 transcripts were differentially expressed in the presence of blood (75 induced and 79 repressed relative to no addition of blood); greater than two-thirds of the regulated transcripts are plasmid-encoded. Among the induced transcripts were those for OspC and DbpA, as expected, transcripts encoding for chemotaxis and motility functions and for two transcriptional regulators, RpoS and BosR.

Given the induction of RpoS by incubation with human blood, it was of interest to compare the list of differentially expressed genes during blood co-incubation to that of RpoS-regulated genes [12]. Thirty-nine genes were found in common; 36/39 are activated by RpoS during in vitro cultivation at 37 °C and during co-incubation with blood. Further, 40 genes that were differentially expressed in the presence of human blood were also RpoS-regulated during growth in DMCs (28 induced, 12 repressed). This analysis indicates that RpoS is induced during the nymphal blood meal and controls a regulon required for tick-to-mammal transmission and during mammalian infection and is supported by additional microarray studies discussed below.

Livengood et al. [14], performed global transcriptome analysis of B. burgdorferi following a 20-h incubation with human neuroglial cells. A total of 72 B. burgdorferi transcripts were differentially expressed in the neuroglial cells relative to in vitro-cultivated spirochetes; the levels of 58 were induced and 14 were repressed. 63/72 differentially expressed genes are located on either the chromosome or plasmid lp54. Numerous genes involved in motility/chemotaxis were induced in neuroglial cells, as was ospC. Among the transcripts with decreased expression was glpK, which has been shown in other studies to be repressed in the mammalian environment [41,33].

Iyer et al. [33], characterized and compared the transcriptional profiles of B. burgdorferi during acquisition (fed larvae), transmission (fed nymphs) and in a mammalian host-like environment (DMCs). This analysis required the introduction of a pre-amplification step prior to array hybridization in order to enrich for B. burgdorferi RNA [33]. A core transcriptome consisting of 397 genes was expressed under all experimental conditions and is likely required for spirochetal survival in nature. The three in vivo transcriptomes differ substantially among each other, as well as to that obtained from organisms cultivated in vitro at 37 °C indicating that spirochetes respond to a variety of host-specific signals. Among the key findings were the differential expression of genes encoding lipoproteins, transporters and enzymes in several metabolic pathways including the oxidative branch of the pentose phosphate pathway, glycerophospholipid biosynthesis and isoprenoid biosynthesis. Alterations in gene expression for chemotaxis/motility proteins were also noted suggesting that the chemotaxis/motility apparatus may vary in the tick and mammalian environments. This was the first report describing B. burgdorferi global gene expression profiles from in vivo samples containing limited copies of pathogen. The findings provide the necessary transcriptional framework for delineating B. burgdorferi regulatory pathways that operate throughout the enzootic cycle.

Caimano et al. [12], performed a comparative microarray analysis of B. burgdorferi strain 297 wild-type and an rpoS mutant cultivated either in vitro following temperature-shift to 37 °C or within DMCs. The expression of 110 genes was affected by the absence of RpoS during in vitro growth; all had higher expression in the wild type strain implying that their transcription is at least partially dependent on RpoS. No transcripts were found to be significantly repressed. 137 genes had altered expression in spirochetes cultivated under mammalian host-like conditions (i.e., in DMCs); 103 transcripts had significantly elevated levels in wild type relative to the RpoS mutant and 44 of these were also higher in vitro. Importantly, in contrast to in vitro-grown spirochetes, 34 genes had higher expression in mutant B. burgdorferi cultivated in DMCs demonstrating that host-specific signals are required for RpoS-dependent repression. Significantly, a number of genes in this group (ospA, bba62, glp operon) have been shown to have higher transcript levels in ticks [40,33].

Norgard and co-workers generated individual mutants in rrp2, rpoN and rpoS in a strain 297 background and compared gene expression between wild-type and mutant strains in vitro [24]. They identified 98 genes that were regulated in common by either Rrp2, RpoN or RpoS; 97 exhibited higher expression in wild type and only one (bba62) had lower expression. The substantial overlap between genes regulated by RpoS and RpoN provides evidence that the two alternative sigma factors form a congruous pathway and that RpoN regulates B. burgdorferi gene expression through RpoS [44,45]. Importantly, two-thirds (68/98) of the genes were similarly regulated by RpoS in the study by Caimano et al. [12]. It is noteworthy that transcription of an additional 106 genes was affected in the rrp2 mutant. This implies that Rrp2 controls expression of a regulon unrelated to the RpoS response.

Two additional publications reported on the RpoS, RpoN and Rrp2 regulons. Boardman et al. [23] generated an rrp2 mutant in an infectious strain B31 background. They observed 144 genes with altered expression in the mutant. Due to strain variation, the overlap among the two Rrp2 gene sets was only 42%. Fisher et al. [25], studied the RpoS and RpoN regulons using mutants in each of these alternative sigma factors. Curiously, there is <20% concordance between these datasets and those of Caimano et al. [12], Ouyang et al. [24] and Boardman et al. [23] probably the result of methodological differences.

To generate a composite picture of the Rrp2-RpoN-RpoS regulatory circuit, we merged the datasets from Caimano et al. [12], Ouyang et al. [24], and Boardman et al. [23]. The composite gene list contained 395 genes. This gene set (referred as rrp2-RpoN-RpoS) is provided in Table S1 and was used for further analysis as described below.

Borrelia oxidative stress regulator (BosR; BB0647) was initially thought to mediate the oxidative stress response in B. burgdorferi [46,47]. Subsequently, it was shown to be required for transcription of RpoS [22,48,49]. Two groups have generated bosR mutants and performed microarray-based comparative transcriptome analyses. Hyde et al. [21], employed a BosR point mutant that was sensitive to oxidative stress and an insertional disruption of this point mutant that restored resistance to oxidative stress. Due to the unusual nature of the strains used (both comparison strains were mutants and no wild-type strain was included), this study is not considered further. Ouyang et al. [22], inactivated bosR in an infectious B31 strain background. They found the BosR regulon to encompass 199 genes, 137 of which were induced. These induced genes included rpoS and ospC, as was expected, and nearly two-thirds (87/137) were also part of the RpoS regulon described by these investigators [24].

The genes regulated by the Rrp2-RpoN-RpoS cascade were compared to those in the BosR regulon as shown in Figure 1. The Venn diagram reveals a substantial overlap of Rrp2-RpoN-RpoS activated genes to those activated by BosR (125/137; 91%). This finding supports the accumulating evidence that both RpoS and BosR are required for modulation of gene expression during mammalian infection [22,49,50,51]. For further analysis (see below), the Rrp2-RpoN-RpoS and BosR regulated genes were merged to generate an Rrp2-RpoN-RpoS-BosR regulon consisting of 451 genes. The additional 56 genes that are regulated by BosR only are given in Table S2. This gene set should represent B. burgdorferi genes that are differentially regulated during late nymphal tick feeding, tick-to-mammal transmission and during mammalian infection.

Three groups have reported the construction of rrp1 mutants and studied their gene expression profiles compared to those of the parent strain. Rogers et al. [26], generated an rrp1 mutant in a non-infectious strain background that also lacked many plasmids. They identified 140 transcripts with altered abundance, 131 of which had higher expression in the wild-type than the mutant. These included products of the glp operon, bba74 and spoVG which have been shown to be repressed by RpoS in DMCs and expressed at higher levels in ticks [12,33,40]. Subsequently, He et al. [27] and Caimano et al. [42] employed mutants that were generated in an infectious strain B31 background; both mutants can infect mice but cannot survive in ticks. He et al. [27] found that 120 genes had altered expression; 99 had higher transcript levels in the wild type strain. Although Caimano et al. [42], used RNA-seq, these two datasets were merged to generate a composite rrp1 regulon. The regulon contained a total of 297 genes (222 induced and 75 repressed) (Table S3).

Regulation of differential gene expression during the enzootic cycle is mediated primarily by RpoS and Rrp1. RpoS is responsible for modulating gene expression during spirochetal transmission from the tick vector to the mammalian host and during mammalian infection; BosR also plays a role in these processes. Rrp1 mediates changes in tick phase gene expression and regulates protective responses during the tick blood meal [4,27,42]. The interplay of the two regulatory circuits controlling RpoS and Rrp1 activity is thus critical to the adaptation and survival of B. burgdorferi in the vector and host milieus. The overlap between these two regulatory cascades has not been well characterized. In order to gain further insight into the linkage between these pathways, we compared the genes comprising the Rrp2-RpoN-RpoS-BosR regulon (Tables S1 and S2) with those comprising the Hk1-Rrp1 regulon (Table S3). The resulting Venn diagram is presented in Figure 2. 140 genes were regulated by both pathways; 83 genes were activated by both Hk1-Rrp1 and Rrp2-RpoN-RpoS-BosR and three genes were commonly repressed by both pathways. In addition, 42 genes were activated by Hk1-Rrp1 but repressed by Rrp2-RpoN-RpoS-BosR and 12 genes were induced by the RpoS pathway but repressed by Rrp1. Interestingly, the 42 genes induced by Rrp1 and repressed by RpoS include the glp operon, spoVG, bba62 and bba74. These genes are known to have significantly higher expression in ticks than in the mammalian host [33,40,41,42]. These findings are consistent with the model that Rrp1 controls a regulon whose members are required during the tick phase of the B. burgdorferi life cycle [4,42].