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.
In the form of biofilms, bacteria exhibit more resistance to antibiotics. Biofilm formers can withstand severe conditions and the host’s defense system. Therefore, it is necessary to search for effective biofilm inhibitors. In this study, we investigated the effect of a chloroform extract of turmeric on biofilm formation against antibiotic resistant bacteria. The extract exhibited its antibiofilm effect by altering adherence, motility, extracellular polymeric substance (EPS) production and cell surface hydrophobicity; important attributes of biofilm formation. Cell attachment assays indicated that a chloroform extract resulted in a 38.9–60.2% inhibition of cell adherence to a polystyrene surface, and a 44.5–58.3% inhibition to a glass surface. Static biofilm formation assays indicated that a chloroform extract resulted in a 23–74.5% reduction in biofilm formation. The chloroform extract inhibited flagella-directed swarming and swimming motility and pilus-directed twitching motility in a dose-dependent manner. In addition to repression of motility, a chloroform extract also significantly (p < 0.05) altered the hydrophobic behavior, and bacterial strains such as K. pneumoniae and E. cloacae exhibited hydrophilic behavior after the addition of the extract, as compared with control cells. The presence of the extract also significantly (p < 0.05) increased the detachment of biofilms by a surfactant as compared with controls. Fourier transformed infrared spectroscopy (FTIR) had indicated a loss of vital functional groups of polysaccharides and proteins from the EPS of cells treated with a chloroform extract. Gas chromatography mass spectrometry (GC-MS) analysis indicated the presence of many phytochemical constituents, mainly sesquiterpenes and fatty acid groups. These results clearly suggested that turmeric could affect multiple cellular activities in biofilm formers exhibiting antibiotic resistance by modulating adherence, EPS production, motility and surface hydrophobicity.
Aspergillus luchuensis is a kuro (black) koji fungus that has been used as a starch degrader for the awamori- and shochu-making industries in Japan. In this study, we investigated the effect of ion beam irradiation on A. luchuensis RIB2601 and obtained a high starch-degrading mutant strain U1. Strain U1 showed reduced growth rate, whereas it showed higher α-amylase, glucoamylase, and α-glucosidase activities on a mycelial mass basis than the wild type (wt) strain both on agar plates and in rice koji. In addition, strain U1 showed higher N-acetylglucosamine content in the cell wall and higher sensitivity to calcofluor white, suggesting a deficiency in cell wall composition. Interestingly, produced protein showed higher expression of acid-labile α-amylase (AmyA) and glucoamylase (GlaA) in strain U1, although real-time RT-PCR indicated no significant change in the transcription of the amyA or glaA gene. These results suggested that the high amylolytic activity of strain U1 is attributable to a high AmyA and GlaA production level, but the elevated production is not due to transcriptional regulation of the corresponding genes. Furthermore, RNA-seq analysis indicated that strain U1 shows transcriptional changes in at least 604 genes related to oxidation-reduction, transport, and glucosamine-containing compound metabolic processes, which may be involved in the deficient cell wall composition of strain U1.
Microbacterium foliorum GA2, an alkali-tolerant bacterium, was randomly mutated using UV radiation and sodium azide to obtain a mutant with a higher cold-active extracellular amylolytic activity. A mutant, designated as MFSD20, was selected owing to its higher amylase activity at 20°C. Under optimized conditions, amylase production was achieved best with raw banana peels (5000 units) in solid-state fermentation (SSF). The enzyme was purified by salt precipitation and chromatographic methods and afterwards characterized biochemically. The purified enzyme showed maximal activity at temperatures between 15–25°C and at pH 8.0. Interestingly, this mutant biocatalyst (MFSD20) displays higher catalytic activity under conditions of low temperature (4°C) and high pH (10.0), in the presence of SDS (0.1 and 1%), and exhibited 85% and 50% requirement of divalent metallic ions Ca2+ and Mg2+, respectively. This mutant enzyme extract in combination with “Wheel detergent” was highly effective in the removal of tomato sauce and chocolate stains from white cotton fabric was demonstrated by ~50% additional reflectance compared with detergent alone, in a wash performance analysis at 20 ± 2°C. The features shown by mutant M. foliorum GA2 make it a promising candidate for industrial applications involving starch degradation at low temperatures.
Resistant starch is not digestible in the small intestine and is fermented by lactic acid bacteria in the large intestine into short chain fatty acids, such as acetate, propionate and butyrate, which result in several health benefits in analogy with dietary fibre components. The mode and mechanism of resistant starch degradation by lactic acid bacteria is still not understood. In the present study, we have purified α-D-glucosidase from Lactobacillus fermentum NCDC 156 by employing three sequential steps i.e. ultra filtration, DEAE-cellulose and Sephadex G-100 chromatographies. It was found to be a monomeric protein (~50 kDa). The optimum pH and temperature of this enzyme were found to be 5.5 and 37°C, respectively. Under optimised conditions with p-nitrophenyl-D-glucopyranoside as the substrate, the enzyme exhibited a Km of 0.97 mM. Its activity was inhibited by Hg2+ and oxalic acid. N-terminal blocked purified enzyme was subjected to lysyl endopeptidase digestion and the resultant peptides were subjected to BLAST analysis to understand their homology with other α-D-glucosidases from lactobacillus species.
The gene ybhA of Escherichia coli encodes a phosphatase that has an in vitro specificity to dephosphorylate pyridoxal 5ʹ-phosphate (PLP or vitamin B6), a co-factor for aminotransferases and other enzymes. In this study, we found that excess pyridoxal (PL) in a minimal medium resulted in excess PLP in vivo and growth inhibition, which was alleviated by YbhA overproduction. Conversely, the YbhA overproduction resulted in PLP shortage in vivo and the correlated reduction in growth rate, which was significantly negated by PL in the medium. In addition, the overproduction of a PL kinase, PdxK or PdxY, was inhibitory to cell growth only in the absence of the functional ybhA gene, and the growth defects were alleviated by casamino acids in the medium, which suggested that both the shortage of, and excess, PLP resulted in the disturbance of amino acid metabolism and cell growth, as revealed by a metabolome analysis.