| Calcium is an important messenger that is involved in many eukaryotic cellular processes, such as the initiation of DNA synthesis, mitosis, and cell division. The intracellular free calcium ion concentration ([Ca2+]i) is tightly regulated through Ca2+ channels, Ca2+pumps, and Ca2+-binding proteins (CaBPs). At rest, [Ca2+]i is maintained at very low levels (10-7 M) compared with the concentration of Ca2+in extracellular fluid (10-3 M). This situation creates a Ca2+ concentration gradient, which is utilized by cells to transmit signals from the cell surface to the interior of the cell. Cells respond to a number of stimuli by transient changes in [Ca2+]i. Calcium fluxes activate a series of events, leading to phosphorylation reactions, activation of regulatory proteins, and cell cycle regulation.In bacteria, the role of Ca2+ in cellular activities is less clear. It has been demonstrated that bacteria tightly regulate their [Ca2+]i, with values similar to those found in eukaryotes (100 to 300 nM). Similar to eukaryotes, bacterial cells have ion channels, primary and secondary transporters, and CaBPs, which may be involved in Ca2+ homeostasis. There is evidence indicating that calcium functions as a regulator in bacteria and that changes in [Ca2+]i are critical to some bacterial cellular processes, such as sporulation of Bacillus, chemotaxis of Escherichia coli, heterocyst differentiation of cyanobacteria, and fruiting body formation in myxobacteria.The ability to measure [Ca2+]i in cells is essential in evaluating the role of calcium as an intracellular messenger. The concept that Ca2+ acts as a messenger is based on the idea that environmental signals induce changes in [Ca2+]i. Therefore, determining [Ca2+]i in response to stimuli has been crucial in establishing the role of calcium in signal transduction. Measuring [Ca2+]i in bacterial cells has been difficult because of the unique structural features of bacteria (including their size and cell wall), toxic effects of reagents and the difficulty of manipulating live bacteria. Nevertheless, the calcium-binding dye fura-2 and the photoproteins aequorin and obelin have been successfully used in bacteria. Obelin is an excellent indicator for Ca2+ because it can respond quickly to allow for following the most rapid transient changes in intracellular calcium and is less sensitive to Mg2+ than aequorin. Using molecular DNA technology to constitutively express the obelin gene in bacteria, it is possible to continuously monitor [Ca2+]i in different genera of bacteria.Myxobacteria are rod-shaped Gram-negative bacteria that are characterized by their complicated multicellular behaviors, including gliding motility, morphogenetic and development processes, and cellular social behaviors, which make them an excellent prokaryotic model for the study of cell-to-cell communication, cell differentiation, and development. It has been reported that calcium plays an important role in cellular behaviors of myxobacteria; however, the [Ca2+]i in myxobacteria has not yet been reported. S protein functions in a manner similar to calmodulin during myxospore formation in Myxococcus xanthus, but it is still not clear whether there are other CaBPs.The obelin gene was inserted into the expression vectors pET15b and pET22b, and the constructed vectors were transformed into BL21 (DE3) to express obelin in E. coli. Obelin was normally expressed in cells transformed with pET22b, and when the cells were observed using fluorescence microscopy with UV light for excitation, yellow-green fluorescence was detected.To measure [Ca2+]i in myxobacteria, the obelin gene was cloned into vectors pZJY41 and pSWU30; resultant vectors were electroporated into M. xanthus DK1622. The total RNA of transformants was isolated, and reverse transcription PCR (RT-PCR) was performed. The results showed that obelin was expressed in DK1622. Unfortunately, fluorescence was not detected when cells were exposed to UV light.Local BLASTP was performed between five published CaBP sequences of bacteria and the predicted amino acid sequences of 7514 ORFs of DK1622 using BioEdit software. The amino acid sequences of the 7514 ORFs of DK1622 were also searched using the consensus sequence of the canonical EF-hand motif. Five ORFs, MXAN 0106, MXAN 0962, MXAN 1334, MXAN 2316 and MXAN 5194, were presumed to encode CaBPs. These five genes were heterogeneously expressed in E. coli, and MXAN 0106 and MXAN 2316 were successfully expressed. These data provided the fundation for identifying the calcium-binding ability of these two proteins using a Ca2+-dependent electrophoretic motility-shift assay.
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