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Protein ubiquitylation plays a major role in the regulation of cellular processes mainly through proteasome-dependent degradation, although it has become increasingly clear that it is also involved in other processes. In the fungus Mucor circinelloides, blue light regulates carotene biosynthesis, with this response controlled by crgA and mcwc-1c genes. CrgA shows characteristics of ubiquitin ligases and represses carotenogenesis in the dark, whereas mcwc-1c is a white collar 1-like gene required for its light induction. Another two white collar 1-like genes have been identified in M. circinelloides: mcwc-1a, which is involved in phototropism, and mcwc-1b, of unknown function. Analysis of double knockout mutants generated for crgA and every mcwc-1 gene demonstrated that crgA and mcwc-1c regulate carotenogenesis by independent pathways. It was also shown that the effect of crgA on carotenogenesis is mediated by mcwc-1b, which acts as a carotenogenesis activator. CrgA is involved in proteolysis-independent mono- and di-ubiquitylation of MCWC-1b, which results in its inactivation. Regulation of carotenogenesis in M. circinelloides by proteolysis-independent ubiquitylation suggests that this mechanism of control could be more widespread than previously thought.  相似文献   

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Interaction with host cells is essential in meningococcal pathogenesis especially at the blood-brain barrier. This step is likely to involve a common regulatory pathway allowing coordinate regulation of genes necessary for the interaction with endothelial cells. The analysis of the genomic sequence of Neisseria meningitidis Z2491 revealed the presence of many repeats. One of these, designated REP2, contains a -24/-12 type promoter and a ribosome binding site 5 to 13 bp before an ATG. In addition most of these REP2 sequences are located immediately upstream of an ORF. Among these REP2-associated genes are pilC1 and crgA, described as being involved in steps essential for the interaction of N. meningitidis with host cells. Furthermore, the REP2 sequences located upstream of pilC1 and crgA correspond to the previously identified promoters known to be induced during the initial localized adhesion of N. meningitidis with human cells. This characteristic led us to hypothesize that at least some of the REP2-associated genes were upregulated under the same circumstances as pilC1 and crgA. Quantitative PCR in real time demonstrated that the expression of 14 out of 16 REP2-associated genes were upregulated during the initial localized adhesion of N. meningitidis. Taken together, these data suggest that these repeats control a set of genes necessary for the efficient interaction of this pathogen with host cells. Subsequent mutational analysis was performed to address the role of these genes during meningococcus-cell interaction.  相似文献   

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Mucor circinelloides responds to blue light by activating the biosynthesis of carotenoids. Gene crgA acts as a repressor of this light-regulated process, as its inactivation leads to overaccumulation of carotenoids in both the dark and the light. The predicted CrgA protein contains different recognizable structural domains, including a RING-finger zinc-binding motif, several glutamine-rich regions, a putative nuclear localization signal and an isoprenylation domain. To gain insight into the specific mode of action of the CrgA protein, we sought to define the CrgA domains critical for the light regulation of carotenogenesis. For this, mutant crgA alleles harbouring missense or deletion mutations in conserved residues of those domains were generated, and their functionality was assessed by testing their ability to complement a null crgA mutation. Point mutations of the amino-terminal RING-finger domain abrogated the ability of CrgA to repress carotenogenesis in the dark, as did the deletion of a poly glutamine-rich region at the carboxyl domain of CrgA. In contrast, mutations of the isoprenylation domain only slightly affected the CrgA function in carotenogenesis. The results identify two functional domains presumably involved in protein-protein interaction in the CrgA protein and suggest a role for the ubiquitin-proteasome pathway in the light regulation of carotenogenesis in fungi.  相似文献   

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The role(s) in cell division of the Mycobacterium tuberculosis Rv0011c gene product, a homolog of the Streptomyces CrgA protein that is responsible for coordinating growth and cytokinesis in sporogenic aerial hyphae, is largely unknown. We show that an enhanced cyan fluorescent protein-M. tuberculosis CrgA (ECFP-CrgA(MT)) fusion protein is localized to the cell membrane, midcell, and cell pole regions in Mycobacterium smegmatis. Furthermore, the ECFP-CrgA(MT) fusion protein colocalized with FtsZ-enhanced yellow fluorescent protein (EYFP) in M. smegmatis. Bacterial two-hybrid assays indicated strong interactions of M. tuberculosis CrgA with FtsZ, FtsQ, and the class B penicillin-binding proteins, FtsI (PBPB) and PBPA. The midcell localization of CrgA(MT) was severely compromised under conditions of FtsZ depletion, which indicated that CrgA localizes to the midcell region after assembly of the FtsZ ring. M. tuberculosis cells with reduced CrgA levels were elongated and grew more slowly than wild-type cells, which indicated defects in cell division, whereas CrgA overproduction did not show growth defects. A M. smegmatis ΔcrgA strain exhibited a bulged cell morphology, elongated cells with a chain-like phenotype, cells with polar bulbous structures, and a modest growth defect. FtsZ and FtsI levels were not affected in cells producing altered levels of CrgA. Septal and membrane localization of GFP-FtsI was enhanced by CrgA overproduction and was diminished in a ΔcrgA strain, which indicates that one role of CrgA is to promote and/or stabilize FtsI localization. Overall, these data indicate that CrgA is a novel member of the cell division complex in mycobacteria and possibly facilitates septum formation.  相似文献   

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On solid media, the reproductive growth of Streptomyces involves antibiotic biosynthesis coincident with the erection of filamentous aerial hyphae. Following cessation of growth of an aerial hypha, multiple septation occurs at the tip to form a chain of unigenomic spores. A gene, crgA, that coordinates several aspects of this reproductive growth is described. The gene product is representative of a well-conserved family of small actinomycete proteins with two C-terminal hydrophobic-potential membrane-spanning segments. In Streptomyces avermitilis, crgA is required for sporulation, and inactivation of the gene abolished most sporulation septation in aerial hyphae. Disruption of the orthologous gene in Streptomyces coelicolor indicates that whereas CrgA is not essential for sporulation in this species, during growth on glucose-containing media, it influences the timing of the onset of reproductive growth, with precocious erection of aerial hyphae and antibiotic production by the mutant. Moreover, CrgA subsequently acts to inhibit sporulation septation prior to growth arrest of aerial hyphae. Overexpression of CrgA in S. coelicolor, uncoupling any nutritional and growth phase-dependent regulation, results in growth of nonseptated aerial hyphae on all media tested, consistent with a role for the protein in inhibiting sporulation septation.  相似文献   

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The RING finger protein CrgA acts as a negative regulator of light-induced carotene biosynthesis in the fungus Mucor circinelloides. Sequence analysis of the crgA coding region upstream of the first AUG codon revealed the existence of an additional non-canonical RING finger domain at the most N-terminal end of the protein. The newly identified RING finger domain is required for CrgA to regulate photocarotenogenesis, as deduced from site-directed mutagenesis experiments. The role of both RING finger domains in the stability of CrgA has been investigated in a yeast system. Wild type CrgA, but not the RING finger deleted forms, is highly unstable and is stabilized by inhibition of the proteasome function, which suggests that native CrgA is degraded by the proteasome and that active RING finger domains are required for proteasome-mediated CrgA degradation. To identify the translation start of CrgA, a mutational analysis of putative initiation codons in the 5' region of the crgA gene was accomplished. We demonstrated that a GUG codon located upstream of the first AUG is the sole initiator of CrgA translation. To our knowledge, this is the first report of a naturally occurring non-AUG start codon for a RING finger regulatory protein. A combination of suboptimal translation initiation and proteasome degradation may help to maintain the low cellular levels of CrgA observed in wild type cells, which is probably required for accurate regulation of photocarotenogenesis.  相似文献   

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Mucor circinelloides responds to blue light by activating the biosynthesis of carotenoids and bending its sporangiophores towards the light source. The CrgA protein product acts as a repressor of carotene biosynthesis, as its inactivation leads to the overaccumulation of carotenoids in both the dark and the light. We show here that asexual sporulation in Mucor is also stimulated by light and that the crgA gene is involved in sporulation, given that lack of crgA function affects both carotenogenesis and the normal production of spores. A small interference RNA (siRNA) gene silencing approach was used to block the biosynthesis of carotenoids and to demonstrate that abnormal sporulation in crgA mutants is not a consequence of a defective production of carotenes. These results reveal an active role for the predicted CrgA product, a RING-finger protein, in the control of cellular light-regulated processes in Mucor.  相似文献   

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The product of the crgA gene of Streptomyces coelicolor represents a novel family of small proteins. A single orthologous gene is located close to the origin of replication of all fully sequenced actinomycete genomes and borders a conserved gene cluster implicated in cell growth and division. In S. coelicolor, CrgA is important for coordinating growth and cell division in sporogenic hyphae. In this study, we demonstrate that CrgA is an integral membrane protein whose peak expression is coordinated with the onset of development of aerial hyphae. The protein localizes to discrete foci away from growing hyphal tips. Upon overexpression, CrgA localizes to apical syncytial cells of aerial hyphae and inhibits the formation of productive cytokinetic rings of the bacterial tubulin homolog FtsZ, leading to proteolytic turnover of this major cell division determinant. In the absence of known prokaryotic cell division inhibitors in actinomycetes, CrgA may have an important conserved function influencing Z-ring formation in these bacteria.  相似文献   

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