Multiple Copies of flhDC in Paraburkholderia unamae Regulate Flagellar Gene Expression, Motility, and Biofilm Formation.

FlhDC is a heterohexameric complex that acts as a master regulator of flagellar biosynthesis genes in numerous bacteria. Previous studies have identified a single flhDC operon encoding this complex. However, we found that two flhDC loci are present throughout Paraburkholderia, and two additional flhC copies are also present in Paraburkholderia unamae. Systematic deletion analysis in P. unamae of the different flhDC copies showed that one of the operons, flhDC1, plays the predominant role, with deletion of its genes resulting in a severe inhibition of motility and biofilm formation. Expression analysis using promoter-lacZ fusions and real-time quantitative PCR support the primary role of flhDC1 in flagellar gene regulation, with flhDC2 a secondary contributor. Phylogenetic analysis shows the presence of the flhDC1 and flhDC2 operons throughout Paraburkholderia. In contrast, Burkholderia and other bacteria only carry the copy syntenous with flhDC2. The variations in impact each copy of flhDC has on downstream processes indicate that regulation of FlhDC in P. unamae, and likely other Paraburkholderia species, is regulated at least in part by the presence of multiple copies of these genes. IMPORTANCE Motility is important in the colonization of plant roots by beneficial and pathogenic bacteria, with flagella playing essential roles in host cell adhesion, entrance, and biofilm formation. Flagellar biosynthesis is energetically expensive. Its complex regulation by the FlhDC master regulator is well studied in peritrichous flagella expressing enterics. We report the unique presence throughout Paraburkholderia of multiple copies of flhDC. In P. unamae, the flhDC1 copy showed higher expression and a greater effect on swim motility, flagellar development, and regulation of downstream genes, than the flhDC2 copy that is syntenous to flhDC in Escherichia coli and pathogenic Burkholderia spp. The flhDC genes have evolved differently in these plant-growth-promoting bacteria, giving an additional layer of complexity in gene regulation by FlhDC.

Pulsed electromagnetic field (PEMF) transiently stimulates the rate of mineralization in a 3-dimensional ring culture model of osteogenesis.

Pulsed Electromagnetic Field (PEMF) has shown efficacy in bone repair and yet the optimum characteristics of this modality and its molecular mechanism remain unclear. To determine the effects of timing of PEMF treatment, we present a novel three-dimensional culture model of osteogenesis that demonstrates strong de novo generation of collagen and mineral matrix and exhibits stimulation by PEMF in multiple stages over 62 days of culture. Mouse postnatal day 2 calvarial pre-osteoblasts were cast within and around Teflon rings by polymerization of fibrinogen and cultured suspended without contact with tissue culture plastic. Ring constructs were exposed to PEMF for 4h/day for the entire culture (Daily), or just during Day1-Day10, Day11-Day 27, or Day28-Day63 and cultured without PEMF for the preceding or remaining days, and compared to no-PEMF controls. PEMF was conducted as HF Physio, 40.85 kHz frequency with a 67 ms burst period and an amplitude of 1.19 mT. Osteogenesis was kinetically monitored by repeated fluorescence measurements of continuously present Alizarin Red S (ARS) and periodically confirmed by micro-CT. PEMF treatment induced early-onset and statistically significant transient stimulation (~4-fold) of the mineralization rate when PEMF was applied Daily, or during D1-D10 and D11-D27. Stimulation was apparent but not significant between D28-D63 by ARS but was significant at D63 by micro-CT. PEMF also shifted the micro-CT density profiles to higher densities in each PEMF treatment group. Ring culture generated tissue with a mineral:matrix ratio of 2.0 by thermogravimetric analysis (80% of the calvaria control), and the deposited crystal structure was 50% hydroxyapatite by X-ray diffraction (63% of the calvaria and femur controls), independent of PEMF. These results were consistent with backscatter, secondary electron, and elemental analysis by scanning electron microscopy. Thus, in a defined, strong osteogenic environment, PEMF applied at different times was capable of further stimulation of osteogenesis with the potential to enhance bone repair.