Conditions Of Catabolite Repression Research Articles

RETRACTED: Genome sequence and comparative genomics of Rhizobium sp. Td3, a novel plant growth promoting phosphate solubilizing Cajanus cajan symbiont

Rhizobium sp. Td3 is a Sesbania plant growth promoting, Cajanus cajan nodulating rhizobia. Studying its whole genome was important as it is a potent phosphate solubilizer with constitutive gluconic acid production ability through operation of the periplasmic glucose oxidation pathway even under conditions of catabolite repression. This is in contrast to the other explored phosphate solubilizers. Rhizobial isolates sequenced so far are known to lack components of the direct glucose oxidation pathway and cannot produce gluconic acid on its own. Here, we present the genome sequence of Rhizobium sp. Td3. Genome comprises of a single chromosome of size 5,606,547 bp (5.6 Mb) with no symbiotic plasmid. Rhizobium leguminosarum bv. viciae USDA2370 was the closest whole genome known. 109 genes responsible for diverse plant growth promoting activities like P solubilization, synthesis of acetoin, nitric oxide, indole-3 acetic acid, exopolysaccharide, siderophore and trehalose have been identified. Flagellar proteins, genes encoding antibiotic and metal resistance, enzymes required for combating oxidative stress as well as attachment and colonization in the plant rhizosphere are also present. Availability of genome sequence of such a versatile plant growth promoting agent will help in exploiting all the phyto-beneficial traits of Td3 for its use as a biofertilizer.

The interplay of StyR and IHF regulates substrate-dependent induction and carbon catabolite repression of styrene catabolism genes in Pseudomonas fluorescens ST

BackgroundIn Pseudomonas fluorescens ST, the promoter of the styrene catabolic operon, PstyA, is induced by styrene and is subject to catabolite repression. PstyA regulation relies on the StyS/StyR two-component system and on the IHF global regulator. The phosphorylated response regulator StyR (StyR-P) activates PstyA in inducing conditions when it binds to the high-affinity site STY2, located about -40 bp from the transcription start point. A cis-acting element upstream of STY2, named URE, contains a low-affinity StyR-P binding site (STY1), overlapping the IHF binding site. Deletion of the URE led to a decrease of promoter activity in inducing conditions and to a partial release of catabolite repression. This study was undertaken to assess the relative role played by IHF and StyR-P on the URE, and to clarify if PstyA catabolite repression could rely on the interplay of these regulators.ResultsStyR-P and IHF compete for binding to the URE region. PstyA full activity in inducing conditions is achieved when StyR-P and IHF bind to site STY2 and to the URE, respectively. Under catabolite repression conditions, StyR-P binds the STY1 site, replacing IHF at the URE region. StyR-P bound to both STY1 and STY2 sites oligomerizes, likely promoting the formation of a DNA loop that closes the promoter in a repressed conformation. We found that StyR and IHF protein levels did not change in catabolite repression conditions, implying that PstyA repression is achieved through an increase in the StyR-P/StyR ratio.ConclusionWe propose a model according to which the activity of the PstyA promoter is determined by conformational changes. An open conformation is operative in inducing conditions when StyR-P is bound to STY2 site and IHF to the URE. Under catabolite repression conditions StyR-P cellular levels would increase, displacing IHF from the URE and closing the promoter in a repressed conformation. The balance between the open and the closed promoter conformation would determine a fine modulation of the promoter activity. Since StyR and IHF protein levels do not vary in the different conditions, the key-factor regulating PstyA catabolite repression is likely the kinase activity of the StyR-cognate sensor protein StyS.

English

  Cellulose degrading organisms have been used for the conversion of cellulolytic materials into soluble sugars or solvents in several biotechnological and industrial applications. In this report, a mutant of Bacillus pumilus was obtained after chemical mutagenesis and screened for cellulase production. This mutant named BpCRI 6 was selected for its ability to produce cellulase under catabolite repression. Cellulase yield by BpCRI 6 was four times higher than that of the wild type under optimum growth conditions (pH 6.5, 25°C and Ca2+ 1mM). In shaking flask cultures, production of cellulase  by the wild type was completely repressed in the presence of 25 mM glucose, while BpCRI 6 strain still exhibited a residual cellulase production of 80 and 40% at 25 mM and 40 mM of glucose concentrations respectively. The mutant strain is stable and grows rapidly in liquid and solid media. Under conditions of catabolite repression (40 mM of glucose), the production of cellulase by this mutant is particularly significant when compared to Trichoderma reesei a well-known cellulase producer, which is under control of end-product inhibition. This is the first report of a successful catabolite repression insensitivity of cellulase production by a mutant of B. pumilus.   Key words: Cellulase, Bacillus pumilus, BpCRI 6, Catabolite repression.

Process technological effects of deletion and amplification of hydrophobins I and II in transformants of Trichoderma reesei.

Transformants of the Trichoderma reeseistrains QM9414 and Rut-C30 were constructed in which the genes for the two major hydrophobin proteins, hydrophobins I (HFBI) and II (HFBII), were deleted or amplified by molecular biological techniques. Growth parameters and foam production of the transformant strains were compared with the corresponding properties of the parent strains by cultivation in laboratory bioreactors under conditions of catabolite repression (glucose medium) or induction of cellulolytic enzymes and other secondary metabolites (cellulose and lactose media). All the transformed strains exhibited vegetative growth properties similar to those of their parent. The Delta hfb2 (but not the Delta hfb1) transformant showed reduced tendency to foam, whereas both strains overproducing hydrophobins foamed extensively, particularly in the case of HFBII. Enzyme production on cellulose medium was unaltered in the Delta hfb2 transformant VTT D-99676, but both the Delta hfb2 and HFBII-overproducing transformants exhibited somewhat decreased enzyme production properties on lactose medium. Production of HFBI by the multi-copy transformant VTT D-98692 was almost 3-fold that of the parent strain QM9414. Overproduction of HFBII by the transformant VTT D-99745, obtained by transformation with three additional copies of the hfb2 gene under the cbh1 promoter, was over 5-fold compared to production by the parent strain Rut-C30. The Delta hfb2transformant VTT D-99676 produced a greatly increased number of spores on lactose medium compared with the parent strain, whereas the HFBII-overproducing transformant VTT D-99745 produced fewer spores.

Regulation of carbon utilization by sulfur availability in Escherichia coli and Salmonella typhimurium.

Different pleiotropic transcriptional regulators are known to function in the coordination of regulons concerned with carbon, nitrogen, sulfur, phosphorus and iron metabolism, but how expression profiles of these different regulons are coordinated with each other is not known. The basis for the effects of cysB mutations on carbon utilization in Escherichia coli and Salmonella typhimurium was examined. cysB mutations affected the utilization of some carbon sources more than others and these effects could be partially, but not completely, reversed by the inclusion of cysteine or djenkolate in the growth medium. Assays of transport systems and enzymes concerned with glucitol and alanine utilization showed that these activities were depressed in cysB mutants relative to isogenic wild-type strains, and cysteine or djenkolate present in the growth media partially restored these activities. Using transcriptional fusions to the fdo (formate dehydrogenase) and gut (glucitol) operons, it was shown that decreased expression resulted from defects at the transcriptional level. Furthermore, the effects of loss of CysB were much less pronounced under conditions of catabolite repression than in the absence of a catabolite-repressing carbon source, and cAMP largely reversed the effect of the loss of CysB. Comparable effects were seen for E. coli lacZ gene expression under the control of its own native promoter, and sulfur limitation in a cysB mutant depressed net cAMP production in a cAMP phosphodiesterase mutant. Adenylate cyclase thus appears to be responsive to sulfur deprivation. These observations may have physiological significance allowing carbon and sulfur regulon coordination during the growth of enteric bacteria in response to nutrient availability.

A novel mutation of the Bacillus subtilis hut operon that relieves both catabolite repression and amino acid repression

A mutation, designated hutCR11, which resulted in high expression of the hut operon and release of the catabolite repression and amino-acid repression of hut expression, was isolated and determined to be a T-to-G transversion at position +30 (+1 indicates the transcription-initiation site). In the hutCR11 mutant, levels of hutP mRNA were 5-fold higher than those in wild-type cells under conditions of non-induction and induction and 11-fold higher under conditions of catabolite repression and amino-acid repression. Mutation analysis showed that two types of base change (T-->A and T-->C) at position +30 did not cause high expression of the hut operon, indicating that this was specifically caused by the single base substitution (T-->G) at position +30. The base substitution of A for T at position +30 also led to partial relief of both catabolite repression and amino-acid repression. These results indicate that the nucleotide sequence at +30 is important for regulation of both catabolite repression and amino-acid repression of the hut operon.

The Saccharomyces cerevisiae RTG2 gene is a regulator of aconitase expression under catabolite repression conditions.

The ACO1 gene, encoding mitochondrial aconitase of Saccharomyces cerevisiae, is required both for oxidative metabolism and for glutamate prototrophy. This gene is subject to catabolite repression; the ACOI mRNA level is further reduced when glutamate is supplied with glucose. To further explore regulation of ACOI expression, we have screened for mutations that reduce expression of an ACOI-lacZ fusion borne on a multicopy vector. We identified a gene required for wild-type expression of ACOI only under catabolite repression conditions. Sequencing of the corresponding cloned gene revealed that it is identical to RTG2 previously cloned as a pivotal gene in controlling interorganelle retrograde communication. Cells containing either the original rtg2-2 mutation or a null rtg2 allele are not petite but show a residual growth on minimum glucose medium with ammonium sulfate as the sole nitrogen source. This growth defect is partially restored by supplying aspartate or threonine, and fully with glutamate or proline supplement. Surprisingly, this phenotype is not observed on complete medium lacking either of these amino acids. In addition, a genetic analysis revealed an interaction between RTG2 and ASP5 (encoding aspartate amino transferase), thus supporting our hypothesis that RTG2 may be involved in the control of several anaplerotic pathways.

Construction of industrial baker's yeast strains able to assimilate maltose under catabolite repression conditions

Spore progeny from an industrial baker's yeast strain were mutagenized with UV and mutants resistant to 2-deoxyglucose isolated. One of these mutants (10a12–13) showed high levels of maltase (α-glucosidase) and external invertase, and assimilated maltose when growing under catabolite repression conditions. This mutant was not allelic to any of the catabolite repression mutants tested cat4, cat80, cid1, cyc8, hex2, hxk2 and tup1. Mutant 10a12–13 was crossed with appropriate strains to construct hybrids that were also able to assimilate maltose in the presence of glucose. These hybrids may be useful in fermentation processes where both glucose and maltose are present.

Construction of industrial baker's yeast strains able to assimilate maltose under catabolite repression conditions

Construction of industrial baker's yeast strains able to assimilate maltose under catabolite repression conditions

Temporal control of colicin E1 induction.

The expression of the gene encoding colicin E1, cea, was studied in Escherichia coli by using cea-lacZ gene fusions. Expression of the fusions showed the same characteristics as those of the wild-type cea gene: induction by treatments that damage DNA and regulation by the SOS response, sensitivity to catabolite repression, and a low basal level of expression, despite the presence of the fusion in a multicopy plasmid. Induction of expression by DNA-damaging treatments was found to differ from other genes involved in the SOS response (exemplified by recA), in that higher levels of DNA damage were required and expression occurred only after a pronounced delay. The delay in expression following an inducing treatment was more pronounced under conditions of catabolite repression, indicating that the cyclic AMP-cyclic AMP receptor protein complex may play a role in induction. These observations also suggest a biological rationale for the control of cea expression by the SOS response and the cyclic AMP-cyclic AMP receptor protein catabolite repression system.

In vivo DNA-binding properties of a yeast transcription activator protein.

UV light can serve as a molecular probe to identify DNA-protein interactions at nucleotide level resolution from intact yeast cells. We have used the photofootprinting technique to determine during which of three regulated states (uninduced, induced, and catabolite repressed) the transcriptional activator protein encoded by GAL4 binds to its recognition sites within the GAL1-GAL10 upstream activating sequence (UASG). GAL4 protein is bound to at least four, and probably five, related sequence blocks within UASG under both induced and uninduced states. GAL4-dependent photofootprints are lost under conditions of catabolite repression. We observed no footprint patterns unique to catabolite-repressed cells, which suggests that binding of a repressor to the UASG is not involved in this process. Photofootprints of the GAL10 TATA element are strictly correlated with transcription: uninduced, catabolite-repressed, and delta gal4 cells exhibit footprints characteristic of the inactive promoter; induced and delta gal80 cells, which express GAL10 constitutively, display footprints unique to the actively transcribed gene.

In vivo DNA-binding properties of a yeast transcription activator protein.

UV light can serve as a molecular probe to identify DNA-protein interactions at nucleotide level resolution from intact yeast cells. We have used the photofootprinting technique to determine during which of three regulated states (uninduced, induced, and catabolite repressed) the transcriptional activator protein encoded by GAL4 binds to its recognition sites within the GAL1-GAL10 upstream activating sequence (UASG). GAL4 protein is bound to at least four, and probably five, related sequence blocks within UASG under both induced and uninduced states. GAL4-dependent photofootprints are lost under conditions of catabolite repression. We observed no footprint patterns unique to catabolite-repressed cells, which suggests that binding of a repressor to the UASG is not involved in this process. Photofootprints of the GAL10 TATA element are strictly correlated with transcription: uninduced, catabolite-repressed, and delta gal4 cells exhibit footprints characteristic of the inactive promoter; induced and delta gal80 cells, which express GAL10 constitutively, display footprints unique to the actively transcribed gene.

Yeast shuttle and integrative vectors with multiple cloning sites suitable for construction of lacZ fusions

We report yeast/ Escherichia coli shuttle vectors suitable for fusing yeast promoter and coding sequences to the lacZgene of E. coli. The vectors contain a region of multiple unique restriction sites including EcoRI, KpnI, SmaI, BamHI, XbaI, SalI, PstI, SphI and HindIII. The region with the unique cloning sites has been introduced in both orientations with respect to lacZ and occurs proximal to the eighth codon of the gene. All the restriction sites have been phased to three different reading frames. Two series of vectors have been constructed. The first series (YEp) has two origins of replication ( ori), i.e., of the yeast 2μ. circle and of the ColEl plasmid of E. coli, and can therefore replicate autonomously in both organisms. These shuttle vectors also have the Ap R gene of E. coli and either the yeast LEU2 or URA3 genes to allow for selection of both E. coli and yeast transformants. The second series of vectors (YIp) are identical in all respects to the YEp vectors except that they lack the 2μ. ori. The YIp vectors can be used to integrate lacZ fusions into yeast chromosomal DNA. None of the vectors express β-galactosidase (βGal) in yeast or E. coli in the absence of inserted yeast promoter sequences. The 5′-nontranslated sequences and parts of the coding sequences of various yeast genes have been cloned into representative lacZ fusion vectors. In-frame gene fusions can be detected by βGal activity when either yeast or E. coli clones are plated on media containing XGal indicator. Quantitative determinations of promoter activity were made by colorimetric assay of βGal activity in whole cells. Fusion of the yeast CYC1 gene to lacZ in one of the vectors allowed detection of regulated expression of this gene when cells were grown under conditions of catabolite repression or derepression.

Influence of carbon catabolite repression on the G1 arrest of Saccharomyces cerevisiae MATa cells by alpha factor.

Cells of the yeast Saccharomyces cerevisiae of the a mating type were arrested at the G1 phase of the cell division cycle after treatment with alpha factor in a culture medium containing a high concentration (2%, w/v, or higher) of a catabolite-repressing sugar. In media containing either a lower concentration of sugar or a non-fermentable carbon source, the extent of G1 arrest induced by the pheromone was reduced or became undetectable. Under catabolite-derepressing conditions alpha factor was inactivated by a cells at a higher rate than that found in repressing media. These results indicate the existence of a close correlation between the action of alpha factor on a cells and conditions of catabolite repression or derepression. A joint mechanism of action of alpha factor and catabolite-repressing carbon sources on a cells is postulated.

Insensitivity of the Escherichia coli K-12 adenylate cyclase to mannose under the conditions of catabolite repression

Insensitivity of the Escherichia coli K-12 adenylate cyclase to mannose under the conditions of catabolite repression

Insensitivity of theEscherichia coliK-12 adenylate cyclase to mannose under the conditions of catabolite repression

Insensitivity of the<i>Escherichia coli</i>K-12 adenylate cyclase to mannose under the conditions of catabolite repression

Induction and catabolite repression of L-rhamnose dehydrogenase in Pullularia pullulans

The growth of Pullularia pullulans on L-rhamnose (6-deoxy-L-mannose) as the sole carbon source induces the synthesis of L-rhamnose dehydrogenase, a nicotinamide adenine dinucleotide-dependent enzyme that catalyzes the oxidation of the deoxy sugar to L-rhamnonolactone. The enzyme induction is inhibited by cycloheximide, suggesting de novo synthesis. The presence of d-glucose (0.2%) or D-galactose (0.2%) simultaneously with the inducer in the induction medium produced 50% repression of dehydrogenase synthesis, but no effect was detected with D-fructose and D-mannose at the same concentration. High levels of D-glucose (2%), under maximal catabolite repression conditions, produced a complete inhibition of enzyme synthesis.

Biogenesis of mitochondria: The effects of catabolite repression on the in vitro phospholipid synthetic capacities of subcellular fractions of respiratory-competent and respiratory-deficient strains of Saccharomyces cerevisiae

A respiratory-competent wild-type strain and a nuclear isogenic, mitochondrial DNA-less, petite mutant strain of Saccharomyces cerevisiae were grown under conditions of catabolite repression in batch cultures and under conditions of catabolite derepression in chemostat cultures. Subcellular fractions were isolated and the capacity of these fractions to incorporate sn-[2- 3H]glycerol 3-phosphate into phospholipids was studied. Neither catabolite repression nor loss of mitochondrial DNA appreciably altered the total in vitro lipid synthesized by mitochondrial fractions during the incubation. Mitochondria isolated from catabolite-derepressed wild-type and petite cells had approximately the same specific activity in vitro for the synthesis of phosphatidylinositol. phosphatidic acid, phosphatidylethanolamine, phosphatidylserine, and neutral lipids. Mitochondria isolated from the petite cells retained the capacity to synthesize phosphatidylglycerol and diphosphatidylglycerol, although the synthesis of these phospholipids was far less extensive than that by the mitochondria isolated from the wild-type cells. In both cases, mitochondria prepared from catabolite-repressed cells synthesized a greater proportion of phosphatidylserine than did mitochondria from catabolite-derepressed cells. The proportions of phospholipid species synthesized in vitro by the microsomal fractions studied were not grossly affected by catabolite repression or loss of mitochondrial DNA.

Effects of galU mutation on flagellar formation in Escherichia coli

Two mutants of Escherichia coli strictly deficient in uridine-diphosphoglucose pyrophosphorylase activity (galU) were found to have very small numbers of flagellar filaments and hooks. In these mutants, both the rate of flagellin (flagellar protein) synthesis and the amount of messenger ribonucleic acid specific for flagellin were considerably lower than in the parental strains. Motile revertants from the galU mutants were isolated and were found to carry a suppressor mutation, which was mapped in the flaH cistron. These strains formed swarms under conditions of catabolite repression; their intracellular concentration of cyclic adenosine 5'-monophosphate was the same as that in the parental strains. These results suggest that the outer membrane affects flagellar formation through the flaH gene product.

Temperate Bacteriophage Which Causes the Production of a New Major Outer Membrane Protein by Escherichia coli

Under most conditions of growth, the most abundant protein in the outer membrane of most strains of Escherichia coli is a protein designated as "protein 1" or "matrix protein". In E. coli B, this protein has been shown to be a single polypeptide with a molecular mass of 36,500 and it may account for more than 50% of the total outer membrane protein. E. coli K-12 contains a very similar, although probably not identical, species of protein 1. Some pathogenic E. coli strains contain very little protein 1 and, in its place, make a protein designated as protein 2 which migrates faster on alkaline polyacrylamide gels containing sodium dodecyl sulfate and which gives a different spectrum of CNBr peptides. An E. coli K-12 strain which had been mated with a pathogenic strain was found to produce protein 2, and a temperate bacteriophage was isolated from this K-12 strain after induction with UV light. This phage, designated as PA-2, is similar in morphology and several other properties to phage lambda. When strains of E. coli K-12 are lysogenized by phage PA-2, they produce protein 2 and very little protein 1. Adsorption to lysogenic strains grown under conditions where they produce little protein 1 and primarily protein 2 is greatly reduced as compared to non-lysogenic strains which produce only protein 1. However, when cultures are grown under conditions of catabolite repression, protein 2 is reduced and protein 1 is increased, and lysogenic and non-lysogenic cultures grown under these conditions exhibit the same rate of adsorption. Phage PA-2 does not adsorb to E. coli B, which appears to have a slightly different protein 1 from K-12. These results suggest that protein 1 is the receptor for PA-2, and that protein 2 is made to reduce the superinfection of lysogens.