The model organism, Escherichia coli, contains a total of more than 4,500 genes, but the total number of RNA polymerase (RNAP) core enzyme or the transcriptase is only about 2,000 molecules per genome. The regulatory targets of RNAP are, however, modulated by changing its promoter selectivity through two-steps of protein-protein interplay with 7 species of the sigma factor in the first step, and then 300 species of the transcription factor (TF) in the second step. Scientists working in the field of prokaryotic transcription in Japan have made considerable contributions to the elucidation of genetic frameworks and regulatory modes of the genome transcription in E. coli K-12. This review summarizes the findings by this group, first focusing on three sigma factors, the stationary-phase sigma RpoS, the heat-shock sigma RpoH, and the flagellar-chemotaxis sigma RpoF, as examples. It also presents an overview of the current state of the systematic research being carried out to identify the regulatory functions of all TFs from a single and the same bacterium E. coli K-12, using the genomic SELEX and PS-TF screening systems. All these studies have been undertaken with the aim of understanding the genome regulation in E. coli K-12 as a whole.
The efficiency of hydrogen gas production by nitrogenase in bacteria has been improved by the inhibition of antagonistic activity by the uptake hydrogenase. In this study, a mutant lacking the gene coding for the uptake hydrogenase was generated from the photosynthetic beta-proteobacterium Rubrivivax gelatinosus IL144 to explore new ways of hydrogen gas production driven by light energy. The mutant cells produced 25–30% higher amounts of molecular hydrogen than the wild-type cells under nitrogen-deficient conditions under light. Furthermore, by the addition of 5 mM glutamate, the photosynthetic growth rate was greatly enhanced, and the hydrogen gas production activity reached 41.1 (mmol/l) in the mutant.
Methionine sulphoxide reductases (Msr) are able to reduce methionine sulfoxide to methionine and protect bacteria against reactive oxygen species (ROS). Many organisms express both methionine sulphoxide reductase A (MsrA), specific for methionine-S-sulfoxide and methionine sulphoxide reductase B (MsrB), active against methionine-R-sulfoxide. Corynebacterium glutamicum expresses MsrA, the function of which has been well defined; however, the function of MsrB has not been studied. Whether MsrB and MsrA play an equally important role in the antioxidant process is also poorly understood. In this study, we identified MsrB encoded by ncgl1823 in C. glutamicum, investigated its function and made a comparison with MsrA. The msrB gene showed a slight effect on utilizing methionine sulfoxide (MetO) as the sole Met source; however, the survival rates showed no sensitivity to oxidants. MsrB showed catalytic activity using thioredoxin/thioredoxin reductase (Trx/TrxR) reducing system as electron donors, but independent from the mycoredoxin 1/mycothione reductase/mycothiol (Mrx1/Mtr/MSH) system. Therefore, MsrB plays a limited role in resisting oxidative stress and it could reduce MetO to Met by the Trx/TrxR reducing system, which is useful for expanding the understanding of the functions of Msr in this important industrial microbe.
A gene (gkdA) (741 bp) encoding a putative protein of 247 amino acids was cloned from the Bacillus licheniformis SR01. The protein was expressed in Escherichia coli BL21 with a molecular mass estimated by SDS-PAGE of approximately 28.03 kDa and showed a calculating isoelectric point (pI) of 6.42. Structure analysis and function identification showed that the enzyme was a multifunctional glycosidase. Its specific activity was 0.013 U/μg. The recombinant glycosidase showed a maximum activity at 50°C and pH 7.0. It was very stable below 90°C and may have heat activation at higher temperatures. The relative residual activity was still more than 80% after 120 min at pH 5.0–10.0. The enzyme activity was inhibited by Cu2+, Fe2+, Ca2+, Mg2+, Co2+, Li+, SDS and EDTA, activated by Ca2+, and not affected by Mn2+ and K+. Under simulated stomach, and in vitro intestine, conditions, the enzyme retained 80%, and more than 100%, activity, respectively, after incubation for 90 min. The excellent properties of this enzyme, specifically its thermal stability and multifunctional abilities, give it potential application in the field of feed processing and other high-temperature processing industries.
Gordonia jacobaea is a bacterium belonging to the mycolata group characterized by its ability to produce carotenoids. Mycolic acids in the cell wall contribute to reducing the permeability of their envelopes requiring the presence of channel-forming proteins to allow the exchange of hydrophilic molecules with the surrounding medium. Identification and purification of the channel-forming proteins was accomplished by SDS-PAGE, Mass spectrometry and Mass peptide fingerprinting and the channel-forming activity was studied by reconstitution in lipid bilayers. Here, we describe for the first time the presence of a cell-wall protein from G. jacobaea with channel-forming activity. Our results suggest that this protein bears a low similarity to other hypothetical proteins from the genus Gordonia of uncharacterized functions. The channel has an average single-channel conductance of 800 pS in 1 M KCl, is moderately anion-selective, and does not show any voltage dependence for voltages between +100 and –100 mV. The channel characteristics suggest that this protein could be of relevance in the import and export of negatively charged molecules across the cell wall. This could contribute to design treatments for mycobacterial infections, as well as being of interest in biotechnology applications.
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Edited and published by : Applied Microbiology, Molecular and Cellular Biosciences Research Foundation/Center for Academic Publications Japan Produced and listed by : TERRAPUB, Center for Academic Publications Japan/Shobi Printing Co., Ltd. (-Vol.60,No12), Center for Academic Publications Japan/InternationalAcademic Printing Co., Ltd.(-Vol.54,No1)