Borrelia burgdorferi, the Lyme disease spirochete, possesses genetically-encoded responses to doxycycline, but not to amoxicillin.

Some species of bacteria respond to antibiotic stresses by altering their transcription profiles, in order to produce proteins that provide protection against the antibiotic. Understanding these compensatory mechanisms allows for informed treatment strategies, and could lead to the development of improved therapeutics. To this end, studies were performed to determine whether Borrelia burgdorferi, the spirochetal agent of Lyme disease, also exhibits genetically-encoded responses to the commonly prescribed antibiotics doxycycline and amoxicillin. After culturing for 24 h in a sublethal concentration of doxycycline, there were significant increases in a substantial number of transcripts for proteins that are involved with translation. In contrast, incubation with a sublethal concentration of amoxicillin did not lead to significant changes in levels of any bacterial transcript. We conclude that B. burgdorferi has a mechanism(s) that detects translational inhibition by doxycycline, and increases production of mRNAs for proteins involved with translation machinery in an attempt to compensate for that stress.

The Lyme disease spirochete's BpuR DNA/RNA-binding protein is differentially expressed during the mammal-tick infectious cycle, which affects translation of the SodA superoxide dismutase.

When the Lyme disease spirochete, Borrelia burgdorferi, transfers from a feeding tick into a human or other vertebrate host, the bacterium produces vertebrate-specific proteins and represses factors needed for arthropod colonization. Previous studies determined that the B. burgdorferi BpuR protein binds to its own mRNA and autoregulates its translation, and also serves as co-repressor of erp transcription. Here, we demonstrate that B. burgdorferi controls transcription of bpuR, expressing high levels of bpuR during tick colonization but significantly less during mammalian infection. The master regulator of chromosomal replication, DnaA, was found to bind specifically to a DNA sequence that overlaps the bpuR promoter. Cultured B. burgdorferi that were genetically manipulated to produce elevated levels of BpuR exhibited altered levels of several proteins, although BpuR did not impact mRNA levels. Among these was the SodA superoxide dismutase, which is essential for mammalian infection. BpuR bound to sodA mRNA in live B. burgdorferi, and a specific BpuR-binding site was mapped 5' of the sodA open reading frame. Recognition of posttranscriptional regulation of protein levels by BpuR adds another layer to our understanding of the B. burgdorferi regulome, and provides further evidence that bacterial protein levels do not always correlate directly with mRNA levels.

Transcriptomic insights on the virulence-controlling CsrA, BadR, RpoN, and RpoS regulatory networks in the Lyme disease spirochete.

Borrelia burgdorferi, the causative agent of Lyme disease, survives in nature through a cycle that alternates between ticks and vertebrates. To facilitate this defined lifestyle, B. burgdorferi has evolved a gene regulatory network that ensures transmission between those hosts, along with specific adaptations to niches within each host. Several regulatory proteins are known to be essential for the bacterium to complete these critical tasks, but interactions between regulators had not previously been investigated in detail, due to experimental uses of different strain backgrounds and growth conditions. To address that deficit in knowledge, the transcriptomic impacts of four critical regulatory proteins were examined in a uniform strain background. Pairs of mutants and their wild-type parent were grown simultaneously under a single, specific culture condition, permitting direct comparisons between the mutant strains. Transcriptomic analyses were strand-specific, and assayed both coding and noncoding RNAs. Intersection analyses identified regulatory overlaps between regulons, including transcripts involved in carbohydrate and polyamine metabolism. In addition, it was found that transcriptional units such as ospC and dbpBA, which were previously observed to be affected by alternative sigma factors, are transcribed by RNA polymerase using the housekeeping sigma factor, RpoD.

Mechanisms of proton relay and product release by Class A ß-lactamase at ultrahigh resolution.

The ß-lactam antibiotics inhibit penicillin-binding proteins (PBPs) by forming a stable, covalent, acyl-enzyme complex. During the evolution from PBPs to Class A ß-lactamases, the ß-lactamases acquired Glu166 to activate a catalytic water and cleave the acyl-enzyme bond. Here we present three product complex crystal structures of CTX-M-14 Class A ß-lactamase with a ruthenocene-conjugated penicillin-a 0.85 Å resolution structure of E166A mutant complexed with the penilloate product, a 1.30 Å resolution complex structure of the same mutant with the penicilloate product, and a 1.18 Å resolution complex structure of S70G mutant with a penicilloate product epimer-shedding light on the catalytic mechanisms and product inhibition of PBPs and Class A ß-lactamases. The E166A-penilloate complex captured the hydrogen bonding network following the protonation of the leaving group and, for the first time, unambiguously show that the ring nitrogen donates a proton to Ser130, which in turn donates a proton to Lys73. These observations indicate that in the absence of Glu166, the equivalent lysine would be neutral in PBPs and therefore capable of serving as the general base to activate the catalytic serine. Together with previous results, this structure suggests a common proton relay network shared by Class A ß-lactamases and PBPs, from the catalytic serine to the lysine, and ultimately to the ring nitrogen. Additionally, the E166A-penicilloate complex reveals previously unseen conformational changes of key catalytic residues during the release of the product, and is the first structure to capture the hydrolyzed product in the presence of an unmutated catalytic serine. DATABASE: Structural data are available in the PDB database under the accession numbers 5TOP, 5TOY, and 5VLE.