The developmental cycle of the myxobactetium Myxococcus xanthus consists of three partially overlapping morphological stages referred to as rippling, fruiting body formation, and sporulation, all of which are absent in csgA null mutants. The CsgA gene product is an extracellular protein, referred to as the C signal, which is essential for developmental cell-cell interactions. csgA expression increases throughout development, reaching its peak during sporulation. CsgA was made limiting for development by constructing nested deletions upstream from the csgA gene, which resulted in reduced csgA expression. Successively larger deletions resulted in termination of development at earlier and earlier stages, with rippling requiring -20% maximum csgA expression, fruiting body formation requiring -30% expression, and sporulation requiring 82% expression. Conversely, artificial induction of csgA also induced development provided nutrients were limiting. These results suggest that steady increases in CsgA over the course of development entrain the natural sequence of morphological events. The csgA upstream region appears to process information concerning the levels of nutrients, peptidoglycan components, and the B signal. In the absence of nutrients, a region extending 400 bp upstream from the start site of transcription was necessary for development and maximal csgA expression. In the presence of low levels of nutrients, a region extending -930 bp upstream was essential for the same tasks. It appears that the upstream region extending from -400 to -930 stimulates csgA expression in the presence of excess carbon, nitrogen, and phosphate, thereby allowing development to go to completion.
CsgA is a cell surface protein that plays an essential role in tactile responses during Myxococcus xanthus fruiting body formation by producing the morphogenic C-signal. The primary amino acid sequence of CsgA exhibits homology with members of the short-chain alcohol dehydrogenase (SCAD) family and several lines of evidence suggest that NAD(P)+ binding is essential for biological activity. First, the predicted CsgA secondary structure based on the 3a120P-hydroxysteroid dehydrogenase crystal structure suggests that the amino-terminal portion of the protein contains an NAD(P)+ binding pocket. Second, strains with csgA alleles encoding amino acid substitutions T6A and RlOA in the NAD(P)+ binding pocket failed to develop. Third, exogenous MalE-CsgA rescues csgA development, whereas MalE-CsgA with the amino acid substitution CsgA T6A does not. Finally, csgA spore yield increased -20% when buffer containing 100 nM of MalE-CsgA was supplemented with 10 p~ of NAD+ or NADP+. Conversely, 10 p~ of NADH or NADPH delayed development for -24 hr and depressed spore levels -10%. Together, these results argue that NAD(P)' binding is critical for C-signaling. S135 and K155 are conserved amino acids in the catalytic domain of SCAD members. Strains with csgA alleles encoding the amino acid substitutions S135T or K155R failed to develop. Furthermore, a MalE-CsgA protein containing CsgA S135T was not able to restore development to csgA cells. In conclusion, a&ino acids conserved in the coenzyme binding pocket and catalytic site are essential for C-signaling.
We compared the transcriptome, proteome, and nucleotide sequences between the parent strain Escherichia coli W3110 and the L-threonine-overproducing mutant E. coli TF5015. DNA macroarrays were used to measure mRNA levels for all of the genes of E. coli, and two-dimensional gel electrophoresis was used to compare protein levels. It was observed that only 54 of 4,290 genes (1.3%) exhibited differential expression profiles. Typically, genes such as aceA, aceB, icdA, gltA, glnA, leu operon, proA, thrA, thrC, and yigJ, which are involved in the glyoxylate shunt, the tricarboxylic acid cycle, and amino acid biosynthesis (L-glutamine, L-leucine, proline, and L-threonine), were significantly upregulated, whereas the genes dadAX, hdeA, hdeB, ompF, oppA, oppB, oppF, yfiD, and many ribosomal protein genes were downregulated in TF5015 compared to W3110. The differential expression such as upregulation of thr operon and expression of yigJ would result in an accumulation of L-threonine in TF5015. Furthermore, two significant mutations, thrA345 and ilvA97, which are essential for overproduction of L-threonine, were identified in TF5015 by the sequence analysis. In particular, expression of the mutated thrABC (pATF92) in W3110 resulted in a significant incremental effect on L-threonine production. Upregulation of aceBA and downregulation of b1795, hdeAB, oppA, and yfiD seem to be linked to a low accumulation of acetate in TF5015. Such comprehensive analyses provide information regarding the regulatory mechanism of L-threonine production and the physiological consequences in the mutant stain.In recent years, the completion of the genome project on numerous organisms has accelerated the development of very powerful tools for functional genomics such as DNA arrays (6) and two-dimensional gel electrophoresis (31). Comparative analysis of the gene expression profiles has provided extensive biological information on a genome scale regarding response to stress and/or environmental change, dissection of regulatory circuitry, drug target characterization or identification, cellular response to bacterial infection, and other information for many organisms, including Escherichia coli, Bacillus subtilis, Saccharomyces cerevisiae, and human cells (1,6,22,25,38). In addition to studies of transcription levels, proteome analysis is important in the understanding of global regulatory processes in living organisms (13,14,17,19,31) since the gene expression profiles often do not directly relate to protein expression levels (28). In this sense, functional genomic techniques, along with genomic information, may enable us to unravel the global regulatory processes or complex metabolic networks in living organisms (18), consequently offering a comprehensive blueprint of the physiology of the bacterium (17,19,22,38).Amino acids have been the prominent target metabolites from microorganisms in bioindustry due to large commercial demands for flavor enhancers, animal feed, sweeteners, and therapeutic agents. Of them, L-threonine, one of the essential am...
BACKGROUND: Environmental contamination by nitroaromatic compounds such as 2,4,6-trinitrotoluene (TNT), hexahydro-1,3,5-trinitro-1,3,5-s-triazine (RDX), atrazine, and/or simazine (TRAS) generated as waste from military and agricultural activities is a serious worldwide problem. Microbiological treatment of these compounds is an attractive method because many explosives and herbicides are biodegradable and the process can be made cost-effective. We explored the feasibility of using cultures of Pseudomonas putida HK-6 for simultaneous degradation of TRAS with the aim of microbial application in wastewater treatment in bench-scale bioreactors.
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