Catabolite Repression

Background: 2-Deoxyglucose (2DG) has been used as an antimetabolite since the 1950s, however, mechanisms associated with its toxicity continue to be evaluated. Studies on 2-DG resistance in Neurospora have identified and characterized some mutants, including rco-3 (NCU02582) and exo-1 (NCU09899). Compared to yeast what is known in terms of genes and mechanisms, our knowledge of this phenomenon in filamentous fungi is still sketchy, though growing. We felt that examining, various aspects of Carbon Catabolite Repression for a certain group of dgr mutants might provide us with some insights. Methods: In the present manuscript, we have employed physiological as well as transcriptional approaches to better understand various properties associated with dgr strains vis-à-vis other known hexose transporter knockouts ∆ hgt-1 (NCU10021), ∆ hgt-2 (NCU04963), ∆ glt-1 (NCU01633) as well as glucose sensors such as ∆rco-3 (NCU02582), the dgr mutants included in this study are dgr-L1 ‘a’, dgr-L1 ‘A...

Background Plastics are an indispensable part of our daily life. However, mismanagement at their end-of-life results in severe environmental consequences. The microbial conversion of these polymers into new value-added products offers a promising alternative. In this study, we engineered the soil-bacterium Comamonas testosteroni KF-1, a natural degrader of terephthalic acid, for the conversion of the latter to the high-value product 2-pyrone-4,6-dicarboxylic acid. Results In order to convert terephthalic acid to 2-pyrone-4,6-dicarboxylic acid, we deleted the native PDC hydrolase and observed only a limited amount of product formation. To test whether this was the result of an inhibition of terephthalic acid uptake by the carbon source for growth (i.e. glycolic acid), the consumption of both carbon sources was monitored in the wild-type strain. Both carbon sources were consumed at the same time, indicating that catabolite repression was not the case. Next, we investigated if the acti...

Bruckner and Titgemeyer, 2002). Portland, Oregon 97239 However, unlike gram-negative bacteria, gram-positive 2 Lehrstuhl fu ¨r Mikrobiologie bacteria do not encode a CRP protein nor do they con-Institut fu ¨r Mikrobiologie, Biochemie und Genetik tain or respond to cAMP (Saier et al., 1995). Instead, Friedrich-Alexander Universita ¨t Erlangen-Nu ¨rnberg these bacteria employ a completely different mecha-Staudstrasse 5 nism of CCR whereby the transcription of catabolic 91058 Erlangen genes is controlled by three main components, the car-Germany bon catabolite protein A (CcpA), a master transcriptional regulator with both repressor and activator activities, the histidine-containing protein (HPr), and catabolite re-Summary sponsive element (cre) DNA sites, which are bound by CcpA (Henkin, 1996; Jones et al., 1997a; Warner and Carbon catabolite repression (CCR) is one of the most Lolkema, 2003). fundamental environmental-sensing mechanisms in CcpA has been identified in over 20 low-GC grambacteria and imparts competitive advantage by estabpositive bacteria (Kraus et al., 1998). Extensive mutagenlishing priorities in carbon metabolism. In gram-posiesis studies on cre sequences from several B. subtilis tive bacteria, the master transcription regulator of CCR promoters revealed the consensus pseudopalindromic is CcpA. CcpA is a LacI-GalR family member that emsequence, WTGNAANCGNWNNCWW (where W ϭ A or ploys, as an allosteric corepressor, the phosphopro-T and N ϭ any base; Weickert and Chambliss, 1989; tein HPr-Ser46-P, which is formed in glucose-replete Miwa et al., 2000). Approximately 10% of the B. subtilis conditions. Here we report structures of the Bacillus genome responds directly or indirectly to CcpA, undermegaterium apoCcpA and a CcpA-(HPr-Ser46-P)-DNA scoring the vital metabolic role played by CcpA (Moreno complex. These structures reveal that HPr-Ser46-P et al., 2001). The multiple genes regulated by CcpA enmediates a novel two-component allosteric DNA bindcode proteins involved in carbon metabolism and utilizaing activation mechanism that involves both rotation tion, including enzymes, transporters, and transcription of the CcpA subdomains and relocation of pivot-point factors (Moreno et al., 2001). The location of the cre residue Thr61, which leads to juxtaposition of the DNA site(s) within a given promoter appears to determine binding regions permitting "hinge" helix formation in whether CcpA functions as a repressor or activator; prothe presence of cognate DNA. The structure of the moters with upstream cre sites are activated by CcpA, CcpA-(HPr-Ser46-P)-cre complex also reveals the elewhile promoters with overlapping cre sites are regant mechanism by which CcpA family-specific interpressed (Go ¨sseringer et al.

In Gram-positive bacteria, the catabolite control protein A (CcpA) functions as the master transcriptional regulator of carbon catabolite repression/regulation (CCR). To effect CCR, CcpA binds a phosphoprotein, either HPr-Ser 46 -P or Crh-Ser 46 -P. Although Crh and histidine-containing protein (HPr) are structurally homologous, CcpA binds Crh-Ser 46 -P more weakly than HPr-Ser 46 -P. Moreover, Crh can form domain-swapped dimers, which have been hypothesized to be functionally relevant in CCR. To understand the molecular mechanism of Crh-Ser 46 -P regulation of CCR, we determined the structure of a CcpA-(Crh-Ser 46 -P)-DNA complex. The structure reveals that Crh-Ser 46 -P does not bind CcpA as a dimer but rather interacts with CcpA as a monomer in a manner similar to that of HPr-Ser 46 -P. The reduced affinity of Crh-Ser 46 -P for CcpA as compared with that of HPr-Ser 46 P is explained by weaker Crh-Ser 46 -P interactions in its contact region I to CcpA, which causes this region to shift away from CcpA. Nonetheless, the interface between CcpA and helix ␣2 of the second contact region (contact region II) of Crh-Ser 46 -P is maintained. This latter finding demonstrates that this contact region is necessary and sufficient to throw the allosteric switch to activate cre binding by CcpA.

Kinetics of intracellular invertase production employing a double mutant derivative of Kluyveromyces marxianus was optimized by varying different process variables in a 23-litre fermentor. The maximum volumetric rate (Q P ) and invertase yield (Y P/S ) by M15 mutant were 1222 U/(L•h) and 160 U/g of substrate utilized, respectively (2-fold more than those of parental strain) at 50 °C on the molasses (150 g/L of total fermentable sugars) at pH=5.5. Glucose or sucrose (100, 150 or 170 g/L) did not repress invertase catabolically under the optimized fermentation conditions, contrary to the previous reports on other yeasts and filamentous fungi, where catabolite repression of sugars was predominant. Invertases derived by the wild (I W ) and mutant (I M ) strains were purified employing ammonium sulphate precipitation, and then characterized by column chromatographic techniques both kinetically and thermodynamically. The acidic limb of invertases was missing and collation of pK a and the heat of ionization values indicated that carboxyl groups were involved in proton transfer during active catalysis. Ratios of K cat /K m and v max /K m indicated that I M was significantly more specific for sucrose hydrolysis. The I M exhibited stability in different buffers at pH=3.0-10.0 and temperature of 50-70 °C, as reflected by long half-lives. I M showed significantly lower values of enthalpy of activation (DH*) and entropy of activation (DS*), while Gibbs free energy (DG*) was significantly increased at higher temperatures, making the I M thermodynamically more thermostable. Thus I M could be used as a catabolite-resistant invertase for the production of fructose syrup or high gravity ethanol.

Mutants from Cellulomonas sp.Ilbc were obtained by combined treatment of UV light and N-methyl-N'-nitro-N-nitrosoguanidine. The selection criterion for the screening of catabolite-repression-resistant mutants was based on the formation of clear zones around the bacterial colonies in medium containing 0.5% Walseth cellulose and 0.5% glucose. Mutants produced not only clear zones in significantly lower times than the parent strain, but also exhibited higher specific growth rates and cellulolytic activity when grown on bagasse pith. The cellulase-derepressed character of the mutants was demonstrated by the presence of cellulolytic activity in cultures grown in the presence of high levels of glucose. These results raise the possibility of enhancing the productivity of bacterial degradation of lignocellulosic substrates for single cell protein production.

Background: Geobacillus stearothermophilus secretes an extracellular xylanase (Xyn10A, XT-6) for the utilization of xylan. Results: The expression of xynA is regulated by XylR, CodY, and XynX, catabolite repression (CcpA), and quorum sensing. Conclusion: The expression of the extracellular xylanase gene (xynA) is highly regulated by many transcriptional schemes. Significance: The xynA gene is regulated by unique and not yet fully characterized mechanisms. Geobacillus stearothermophilus T-6 produces a single extracellular xylanase (Xyn10A) capable of producing short, decorated xylo-oligosaccharides from the naturally branched polysaccharide, xylan. Gel retardation assays indicated that the master negative regulator, XylR, binds specifically to xylR operators in the promoters of xylose and xylan-utilization genes. This binding is efficiently prevented in vitro by xylose, the most likely molecular inducer. Expression of the extracellular xylanase is repressed in medium containing either glucose or casamino acids, suggesting that carbon catabolite repression plays a role in regulating xynA. The global transcriptional regulator CodY was shown to bind specifically to the xynA promoter region in vitro, suggesting that CodY is a repressor of xynA. The xynA gene is located next to an uncharacterized gene, xynX, that has similarity to the NIF3 (Ngg1p interacting factor 3)-like protein family. XynX binds specifically to a 72-bp fragment in the promoter region of xynA, and the expression of xynA in a xynX null mutant appeared to be higher, indicating that XynX regulates xynA. The specific activity of the extracellular xylanase increases over 50-fold during early exponential growth, suggesting cell density regulation (quorum sensing). Addition of conditioned medium to fresh and low cell density cultures resulted in high expression of xynA, indicating that a diffusible extracellular xynA density factor is present in the medium. The xynA density factor is heat-stable, sensitive to proteases, and was partially purified using reverse phase liquid chromatography. Taken together, these results suggest that xynA is regulated by quorum-sensing at low cell densities.

Type 1 diabetes mellitus (T1D) is an endocrine disorder that affects over 1.25 million Americans. Safe, effective T1D treatments should mimic natural homeostatic functions by continually monitoring blood glucose levels (BGLs) and secreting an amount of insulin appropriate to current BGL. We envision a glucose-sensitive insulin- administration device to replace the function of damaged beta cells in T1D patients. Here, we prototyped a DNA-based glucose biosensor to eventually connect with an insulin actuator. We re-engineered the E. coli natural regulatory sequence in the lac operon, which normally increases downstream gene expression under low glucose concentrations, to increase gene expression under high glucose concentrations. Then, we assayed the function of this sensor through GFP expression. Fluorescent assays demonstrated that our construct exhibits glucose inducibility and responds actively to changing glucose concentrations from 0.007 to 0.28 mM. While our results do not sugg...

Repression of the synthesis of isocitrate lyase by glucose and/or induction of the synthesis of isocitrate lyase by acetate in Phycomyces blakesleeanus were demonstrated. Both glycerol and ethanol failed to induce isocitrate lyase activity. Furthermore, glucose appeared to cause an in vivo catabolite inactivation of the derepressed enzyme. Isocitrate lyase was inactivated both reversibly and irreversibly by glucose.

A temperature-sensitive mutant of Escherichia coli in which the synthesis of l-arabinose isomerase is blocked during growth at 42 C was found to possess the following properties. (i) The mutation occurred in the structural gene for the isomerase, gene araA. (ii) During growth at elevated temperatures the mutant accumulates a product which is a precursor to the active enzyme. (iii) The precursor produced at 42 C is slowly converted to active enzyme at 28 C in the absence of protein and ribonucleic acid synthesis. It is concluded that the mutation results in a change in the structure of isomerase which causes formation of active enzyme to be thermolabile at a step beyond the level of translation.

In nature, bacteria live in multicellular and multispecies communities. Microbial species can sense the density and composition of their community through chemical cues using a process called quorum sensing (QS). The marine pathogen Vibrio cholerae is found in communities on the chitinous shells of crustaceans in its aquatic reservoir. V. cholerae interactions with chitin are critical for the survival, evolution, and waterborne transmission of this pathogen. Here, we show that V. cholerae uses QS to regulate the expression of one locus required for V. cholerae-chitin interactions. ABSTRACT The marine facultative pathogen Vibrio cholerae forms complex multicellular communities on the chitinous shells of crustacean zooplankton in its aquatic reservoir. V. cholerae-chitin interactions are critical for the growth, evolution, and waterborne transmission of cholera. This is due, in part, to chitin-induced changes in gene expression in this pathogen. Here, we sought to identify factors tha...

The tetrapyrrolic chlorophyllc atabolites (or phyllobilins, PBs) were analyzed in yellow fall leaveso ft he grape Chardonnay,acommon Vitis vinifera white wine cultivar.T he major fractionsi nl eaf extractso fV. vinifera,t entatively assigned to PBs, were isolated andt heir structures elucidated. The dominant fractioni sadioxobilin-typen on-fluorescent Chl-cataboliteo fapreviously observedt ype. Twol ess polar fluorescent PBs were characterized as an ovel dioxobilintype fluorescent Chl-catabolite with ab icyclo-1',6'-glycosyl architecture, and its new fluorescent formyloxobilin-type analogue. The discovery of persistenth ypermodified fluorescent PBs with the architectureo fb icyclo-[17.3.1]-PBs (bcPBs), suggestst he activity of an unknown enzyme that forges the 20-memberedm acroring at the tetrapyrrolic core of af luorescent PB. bcPBsm ay play specificp hysiological roles in grapevine plants and represent endogenous anti-infective agents, as found similarly for other organicb icyclo-[n.3.1]-1',6'-glycosyl derivatives.

Glycolysis breakdowns glucose into essential building blocks and ATP/NAD(P)H for the cell, occupying a central role in its growth and bio-production. Among glycolytic pathways, the Entner Doudoroff pathway (EDP) is a more thermodynamically favorable pathway with fewer enzymatic steps than either the Embden-Meyerhof-Parnas pathway (EMPP) or the oxidative pentose phosphate pathway (OPPP). However, Escherichia coli do not use their native EDP for glucose metabolism. Overexpression of edd and eda in E. coli to enhance EDP activity resulted in only a small shift in the flux directed through the EDP (~20 % of glycolysis flux). Disrupting the EMPP by phosphofructokinase I (pfkA) knockout increased flux through OPPP (~60 % of glycolysis flux) and the native EDP (~14 % of glycolysis flux), while overexpressing edd and eda in this ΔpfkA mutant directed ~70 % of glycolytic flux through the EDP. The downregulation of EMPP via the pfkA deletion significantly decreased the growth rate, while EDP ...

One glucokinase-deficient mutant (glkl) of Penicillium chrysogenum AS-P-78 was isolated after germ tube-emitting spores were mutated with nitrosoguanidine and selected for growth on lactose-containing medium in the presence of inhibitory concentrations of D-2-deoxyglucose (3 mM). Penicillin biosynthesis was greatly reduced (55%) in D-glucose-grown cultures of the parental strain, but this sugar had no repressive effect on the rate of penicillin biosynthesis in the mutant glkl. This mutant was deficient in ATP-dependent glucokinase and showed a greatly reduced uptake of D-glucose. The parental strain P. chrysogenum AS-P-78 showed in vitro ATP-dependent phosphorylating activities of D-glucose, D-2-deoxyglucose, and D-galactose. The glkl mutant was deficient in the in vitro phosphorylation of D-glucose and D-2-deoxyglucose but retained a normal D-galactose-phosphorylating activity. D-Glucose repressed both ,-galactosidase and isopenicillin-Nsynthase but not acyl coenzyme A:6-aminopenicillanic acid acyltransferase in the parental strain. The glucokinase-deficient mutant was simultaneously derepressed in carbon catabolite regulation of I-galactosidase and isopenicillin-N-synthase, suggesting that a common regulatory mechanism is involved in carbon catabolite regulation of both sugar utilization and penicillin biosynthesis.