No abstract

Little is known about how Borrelia burgdorferi, the Lyme disease pathogen, adapts and survives in the tick vector. We previously identified a bacterial CarD N-terminal-like (CdnL) protein, LtpA (BB0355), in B. burgdorferi that is preferably expressed at lower temperatures, which is a surrogate condition mimicking the tick portion of the enzootic cycle of B. burgdorferi. CdnL-family proteins, an emerging class of bacterial RNAP-interacting transcription factors, are essential for the viability of Mycobacterium tuberculosis and Myxococcus xanthus. Previous attempts to inactivate ltpA in B. burgdorferi have not been successful. In this study, we report the construction of a ltpA mutant in the infectious strain of B. burgdorferi, strain B31-5A4NP1. Unlike CdnL in M. tuberculosis and M. xanthus, LtpA is dispensable for the viability of B. burgdorferi. However, the ltpA mutant exhibits a reduced growth rate and a cold-sensitive phenotype. We demonstrate that LtpA positively regulates 16S rRNA expression, which contributes to the growth defects in the ltpA mutant. The ltpA mutant remains capable of infecting mice, albeit with delayed infection. Additionally, the ltpA mutant produces markedly reduced spirochetal loads in ticks and was not able to infect mice via tick infection. Overall, LtpA represents a novel regulator in the CdnL family that has an important role in the enzootic cycle of B. burgdorferi.

Borrelia burgdorferi sensu lato, the agent of Lyme disease, exists in nature through a complex enzootic life cycle that involves both ticks and mammals. The B. burgdorferi genome encodes five Oligopeptide ABC transporters (Opp) that are predicted to be involve in transport of various nutrients. Previously, it was reported that OppA5 is important for the optimal production of OspC, a major virulence factor of B. burgdorferi. In this study, possible role of another Oligopeptide ABC transporter, OppA4 in ospC expression was investigated by construction of an oppA4 deletion mutant and the complemented strain. Inactivation of oppA4 resulted an increased production of OspC, suggesting that OppA4 has a negative impact on ospC expression. Expression of ospC is controlled by Rrp2-RpoN-RpoS, the central pathway essential for mammal infection. We showed that increased ospC expression in the oppA4 mutant was due to an increased rpoS expression. We then further investigated how OppA4 negatively regulates this pathway. Two regulators, BosR and BadR, are known to positively and negatively, respectively, regulate the Rrp2-RpoN-RpoS pathway. We found that deletion of oppA4 resulted in an increased level of BosR. Previous reports showed that bosR is mainly regulated at the post-transcriptional level by other factors. However, OppA4 appears to negatively regulate bosR expression at the transcriptional level. The finding of OppA4 involved in regulation of the Rrp2-RpoN-RpoS pathway further reinforces the importance of nutritional virulence to the enzootic cycle of B. burgdorferi.

Bacteriophages (phages) are predicted to be the most ubiquitous biological entity on earth, and yet, there are still vast knowledge gaps in our understanding of phage diversity and phage–host interactions. Approximately one hundred Acinetobacter-infecting DNA viruses have been identified, and in this report, we describe eight more. We isolated two typical dsDNA lytic podoviruses (CAP1–2), five unique dsRNA lytic cystoviruses (CAP3–7), and one dsDNA lysogenic siphovirus (SLAP1), all capable of infecting the multidrug resistant isolate Acinetobacter radioresistens LH6. Using transmission electron microscopy, bacterial mutagenesis, phage infectivity assays, carbohydrate staining, mass-spectrometry, genomic sequencing, and comparative studies, we further characterized these phages. Mutation of the LH6 initiating glycosyltransferase homolog, PglC, necessary for both O-linked glycoprotein and capsular polysaccharide (CPS) biosynthesis, prevented infection by the lytic podovirus CAP1, while mutation of the pilin protein, PilA, prevented infection by CAP3, representing the lytic cystoviruses. Genome sequencing of the three dsRNA segments of the isolated cystoviruses revealed low levels of homology, but conserved synteny with the only other reported cystoviruses that infect Pseudomonas species. In Pseudomonas, the cystoviruses are known to be enveloped phages surrounding their capsids with the inner membrane from the infected host. To characterize any membrane-associated glycoconjugates in the CAP3 cystovirus, carbohydrate staining was used to identify a low molecular weight lipid-linked glycoconjugate subsequently identified by mutagenesis and mass-spectrometry as bacterial lipooligosaccharide. Together, this study demonstrates the isolation of new Acinetobacter-infecting phages and the determination of their cell receptors. Further, we describe the genomes of a new genus of Cystoviruses and perform an initial characterization of membrane-associated glycoconjugates.