Expression of glutamine metabolism-related proteins in thyroid cancer.

PURPOSE: This study aimed to investigate the expression of glutamine metabolism-related protein in tumor and stromal compartments among the histologic subtypes of thyroid cancer. RESULTS: GLS1 and GDH expression in tumor and stromal compartments were the highest in AC than in other subtypes. Tumoral ASCT2 expression was higher in MC but lower in FC (p < 0.001). In PTC, tumoral GLS1 and tumoral GDH expression was higher in the conventional type than in the follicular variant (p = 0.043 and 0.001, respectively), and in PTC with BRAF V600E mutation than in PTC without BRAF V600E mutation (p<0.001). Stromal GDH positivity was the independent factor associated with short overall survival (hazard ratio: 21.48, 95% confidence interval: 2.178-211.8, p = 0.009). METHODS: We performed tissue microarrays with 557 thyroid cancer cases (papillary thyroid carcinoma [PTC]: 344, follicular carcinoma [FC]: 112, medullary carcinoma [MC]: 70, poorly differentiated carcinoma [PDC]: 23, and anaplastic carcinoma [AC]: 8) and 152 follicular adenoma (FA) cases. We performed immunohistochemical staining of glutaminolysis-related proteins (glutaminase 1 [GLS1], glutamate dehydrogenase [GDH], and amino acid transporter-2 [ASCT-2]). CONCLUSION: Glutamine metabolism-related protein expression differed among the histologic subtypes of thyroid cancer.

Characterization of the transcriptional regulation of the tarIJKL locus involved in ribitol-containing wall teichoic acid biosynthesis in Lactobacillus plantarum.

Lactobacillus plantarum strains produce either glycerol (Gro)- or ribitol (Rbo)-backbone wall teichoic acid (WTA) (Gro-WTA and Rbo-WTA, respectively). The strain WCFS1 has been shown to be able to activate the tarIJKL locus involved in Rbo-WTA synthesis when the tagD1F1F2 locus for Gro-WTA synthesis was mutated, resulting in switching of the native Gro-WTA into Rbo-WTA. Here, we identify a regulator involved in the WTA backbone alditol switching and activation of the tarIJKL locus. Promoter reporter assays of the tarI promoter (Ptar) demonstrated its activity in the Rbo-WTA-producing mutant derivative (ΔtagF1-2) but not in the parental strain WCFS1. An electrophoresis mobility shift assay using a Ptar nucleotide fragment showed that this fragment bound to Ptar-binding protein(s) in a cell-free extract of WCFS1. Three proteins were subsequently isolated using Ptar bound to magnetic beads. These proteins were isolated efficiently from the lysate of WCFS1 but not from the lysate of its ΔtagF1-2 derivative, and were identified as redox-sensitive transcription regulator (Lp_0725), catabolite control protein A (Lp_2256) and TetR family transcriptional regulator (Lp_1153). The role of these proteins in Ptar regulation was investigated by knockout mutagenesis, showing that the Δlp_1153 mutant expressed the tarI gene at a significantly higher level, supporting its role as a repressor of the tarIJKL locus. Notably, the Δlp_1153 mutation also led to reduced expression of the tagF1 gene. These results show that Lp_1153 is a regulatory factor that plays a role in WTA alditol switching in Lb. plantarum WCFS1 and we propose to rename this gene/protein wasR/WasR, for WTA alditol switch regulator.

Efficient Production of Hydroxylated Human-Like Collagen Via the Co-Expression of Three Key Genes in Escherichia coli Origami (DE3).

Mature collagen is abundant in human bodies and very valuable for a range of industrial and medical applications. The biosynthesis of mature collagen requires post-translational modifications to increase the stability of collagen triple helix structure. By co-expressing the human-like collagen (HLC) gene with human prolyl 4-hydroxylase (P4H) and D-arabinono-1, 4-lactone oxidase (ALO) in Escherichia coli, we have constructed a prokaryotic expression system to produce the hydroxylated HLC. Then, five different media, as well as the induction conditions were investigated with regard to the soluble expression of such protein. The results indicated that the highest soluble expression level of target HLC obtained in shaking flasks was 49.55 ± 0.36 mg/L, when recombinant cells were grew in MBL medium and induced by 0.1 mM IPTG at the middle stage of exponential growth phase. By adopting the glucose feeding strategy, the expression level of target HLC can be improved up to 260 mg/L in a 10 L bench-top fermentor. Further, HPLC analyses revealed that more than 10 % of proline residues in purified HLC were successfully hydroxylated. The present work has provided a solid base for the large-scale production of hydroxylated HLC in E. coli.

Variations in oxygen concentration cause differential antioxidant response and expression of related genes in Beauveria bassiana.

The entomopathogenic fungus Beauveria bassiana is widely used in pest biocontrol strategies. We evaluated both the antioxidant response mediated by compatible solutes, trehalose or mannitol, and the expression of related genes using oxygen pulses at three oxygen concentrations in solid state culture (SSC): normal atmosphere (21% O2), low oxygen (16% O2) and enriched oxygen (26% O2). Trehalose concentration decreased 75% after atmospheric modifications in the cultures, whereas mannitol synthesis was three-fold higher under the 16% O2 pulses relative to normal atmosphere (100 and 30 µg mannitol mg(-1) biomass, respectively). Confirming this result, expression of the mpd gene, coding for mannitol-1-P dehydrogenase (MPD), increased up to 1.4 times after O2 pulses. The expression of the bbrgs1 gene, encoding a regulatory G protein related to conidiation, was analysed to explain previously reported differences in conidial production. Surprisingly, expression of bbrgs1 decreased after atmospheric modification. Finally, principal component analysis (PCA) indicated that 83.39% of the variability in the data could be explained by two components. This analysis corroborated the positive correlation between mannitol concentration and mpd gene expression, as well as the negative correlation between conidial production and bbrgs1 gene expression. This study contributes to understanding of antioxidant and molecular response of B. bassiana induced under oxidant conditions.

Overexpression of bacterial mtlD gene in peanut improves drought tolerance through accumulation of mannitol.

In the changing global environmental scenarios, water scarcity and recurrent drought impose huge reductions to the peanut (Arachis hypogaea L.) crop yield. In plants, osmotic adjustments associated with efficient free radical scavenging ability during abiotic stress are important components of stress tolerance mechanisms. Mannitol, a compatible solute, is known to scavenge hydroxyl radicals generated during various abiotic stresses, thereby conferring tolerance to water-deficit stress in many plant species. However, peanut plant is not known to synthesize mannitol. Therefore, bacterial mtlD gene coding for mannitol 1-phosphate dehydrogenase under the control of constitutive promoter CaMV35S was introduced and overexpressed in the peanut cv. GG 20 using Agrobacterium tumefaciens-mediated transformation. A total of eight independent transgenic events were confirmed at molecular level by PCR, Southern blotting, and RT-PCR. Transgenic lines had increased amount of mannitol and exhibited enhanced tolerance in response to water-deficit stress. Improved performance of the mtlD transgenics was indicated by excised-leaf water loss assay and relative water content under water-deficit stress. Better performance of transgenics was due to the ability of the plants to synthesize mannitol. However, regulation of mtlD gene expression in transgenic plants remains to be elucidated.

Engineering of photosynthetic mannitol biosynthesis from CO2 in a cyanobacterium.

D-Mannitol (hereafter denoted mannitol) is used in the medical and food industry and is currently produced commercially by chemical hydrogenation of fructose or by extraction from seaweed. Here, the marine cyanobacterium Synechococcus sp. PCC 7002 was genetically modified to photosynthetically produce mannitol from CO2 as the sole carbon source. Two codon-optimized genes, mannitol-1-phosphate dehydrogenase (mtlD) from Escherichia coli and mannitol-1-phosphatase (mlp) from the protozoan chicken parasite Eimeria tenella, in combination encoding a biosynthetic pathway from fructose-6-phosphate to mannitol, were expressed in the cyanobacterium resulting in accumulation of mannitol in the cells and in the culture medium. The mannitol biosynthetic genes were expressed from a single synthetic operon inserted into the cyanobacterial chromosome by homologous recombination. The mannitol biosynthesis operon was constructed using a novel uracil-specific excision reagent (USER)-based polycistronic expression system characterized by ligase-independent, directional cloning of the protein-encoding genes such that the insertion site was regenerated after each cloning step. Genetic inactivation of glycogen biosynthesis increased the yield of mannitol presumably by redirecting the metabolic flux to mannitol under conditions where glycogen normally accumulates. A total mannitol yield equivalent to 10% of cell dry weight was obtained in cell cultures synthesizing glycogen while the yield increased to 32% of cell dry weight in cell cultures deficient in glycogen synthesis; in both cases about 75% of the mannitol was released from the cells into the culture medium by an unknown mechanism. The highest productivity was obtained in a glycogen synthase deficient culture that after 12 days showed a mannitol concentration of 1.1 g mannitol L(-1) and a production rate of 0.15 g mannitol L(-1) day(-1). This system may be useful for biosynthesis of valuable sugars and sugar derivatives from CO2 in cyanobacteria.

Evolution of vitamin B2 biosynthesis: eubacterial RibG and fungal Rib2 deaminases.

Eubacterial RibG and yeast Rib2 possess a deaminase domain for pyrimidine deamination in the second and third steps, respectively, of riboflavin biosynthesis. These enzymes are specific for ribose and ribitol, respectively. Here, the crystal structure of Bacillus subtilis RibG in complex with a deaminase product is reported at 2.56 Å resolution. Two loops move towards the product on substrate binding, resulting in interactions with the ribosyl and phosphate groups and significant conformational changes. The product carbonyl moiety is bent out of the pyrimidine ring to coordinate to the catalytic zinc ion. Such distortions in the bound substrate and product may play an essential role in enzyme catalysis. The yeast Rib2 structure was modelled and a mutational analysis was carried out in order to understand the mechanism of substrate recognition in these two enzymes. Detailed structural comparisons revealed that the two consecutive carbonyl backbones that occur prior to the PCXXC signature constitute a binding hole for the target amino group of the substrate. This amino-binding hole is essential in B. subtilis RibG and is also conserved in the RNA/DNA-editing deaminases.

Enhancement of xylitol production in glycerol kinase disrupted Candida tropicalis by co-expression of three genes involved in glycerol metabolic pathway.

Glycerol can be used as a primary carbon source by yeasts, little is known regarding glycerol metabolism in Candida tropicalis. In this study, glycerol kinase gene (gk) was disrupted from xylitol dehydrogenase gene (XYL2) knockout C. tropicalis strain BSXDH-3. The resultant gk knockout C. tropicalis strain was incapable to grow on glycerol. The cells growth on glycerol was resumed by co-expressing Scheffersomyces stipitis gcy1, 2 and 3 genes, which respectively encode NADP(+)-dependent glycerol dehydrogenase 1, 2 and 3, under the control of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) promoter. NADPH-dependent xylitol production was higher in the engineered strain, termed "GK", than in BSXDH-3. In fermentation experiments using glycerol as co-substrate with xylose, strain GK produced xylitol 0.85 and 1.28 g l(-1) h(-1) at the time periods of 16 and 24 h, respectively, which is 30 and 18 % higher at same time intervals in BSXDH-3. This is the first report of gk gene disruption and co-expression of gcy1, 2 and 3 genes for NADPH regeneration and enhanced xylitol production in C. tropicalis.

Evolution of D-lactate dehydrogenase activity from glycerol dehydrogenase and its utility for D-lactate production from lignocellulose.

Lactic acid, an attractive, renewable chemical for production of biobased plastics (polylactic acid, PLA), is currently commercially produced from food-based sources of sugar. Pure optical isomers of lactate needed for PLA are typically produced by microbial fermentation of sugars at temperatures below 40 °C. Bacillus coagulans produces L(+)-lactate as a primary fermentation product and grows optimally at 50 °C and pH 5, conditions that are optimal for activity of commercial fungal cellulases. This strain was engineered to produce D(-)-lactate by deleting the native ldh (L-lactate dehydrogenase) and alsS (acetolactate synthase) genes to impede anaerobic growth, followed by growth-based selection to isolate suppressor mutants that restored growth. One of these, strain QZ19, produced about 90 g L(-1) of optically pure D(-)-lactic acid from glucose in < 48 h. The new source of D-lactate dehydrogenase (D-LDH) activity was identified as a mutated form of glycerol dehydrogenase (GlyDH; D121N and F245S) that was produced at high levels as a result of a third mutation (insertion sequence). Although the native GlyDH had no detectable activity with pyruvate, the mutated GlyDH had a D-LDH specific activity of 0.8 µmoles min(-1) (mg protein)(-1). By using QZ19 for simultaneous saccharification and fermentation of cellulose to D-lactate (50 °C and pH 5.0), the cellulase usage could be reduced to 1/3 that required for equivalent fermentations by mesophilic lactic acid bacteria. Together, the native B. coagulans and the QZ19 derivative can be used to produce either L(+) or D(-) optical isomers of lactic acid (respectively) at high titers and yields from nonfood carbohydrates.

1,3-Propandiol production by engineered Hansenula polymorpha expressing dha genes from Klebsiella pneumoniae.

Currently, 1,3-propanediol (1,3-PD) is an important chemical widely used in polymer production, but its availability is being restricted owing to its expensive chemical synthesis. A methylotrophic yeast Hansenula polymorpha was engineered by expression of dhaB1, dhaB2, dhaB3, dhaB(RA1) and dhaB(RA2) encoding glycerol dehydratase complex and dhaT encoding 1,3-PD oxidoreductase from Klebsiella pneumoniae under direction of promoter of glyceraldehyde-3 phosphate dehydrogenase (GAPDH). The engineered recombinant yeast strain can produce 1,3-PD from glucose (2.4 g L(-1)) as well as glycerol (0.8 g L(-1)), which might lead to a safe and cost-effective method for industrial production of 1,3-PD from various biomass resources.

Metabolic engineering to improve ethanol production in Thermoanaerobacter mathranii.

Thermoanaerobacter mathranii can produce ethanol from lignocellulosic biomass at high temperatures, but its biotechnological exploitation will require metabolic engineering to increase its ethanol yield. With a cofactor-dependent ethanol production pathway in T. mathranii, it may become crucial to regenerate cofactor to increase the ethanol yield. Feeding the cells with a more reduced carbon source, such as mannitol, was shown to increase ethanol yield beyond that obtained with glucose and xylose. The ldh gene coding for lactate dehydrogenase was previously deleted from T. mathranii to eliminate an NADH oxidation pathway. To further facilitate NADH regeneration used for ethanol formation, a heterologous gene gldA encoding an NAD(+)-dependent glycerol dehydrogenase was expressed in T. mathranii. One of the resulting recombinant strains, T. mathranii BG1G1 (Deltaldh, P(xyl)GldA), showed increased ethanol yield in the presence of glycerol using xylose as a substrate. With an inactivated lactate pathway and expressed glycerol dehydrogenase activity, the metabolism of the cells was shifted toward the production of ethanol over acetate, hence restoring the redox balance. It was also shown that strain BG1G1 acquired the capability to utilize glycerol as an extra carbon source in the presence of xylose, and utilization of the more reduced substrate glycerol resulted in a higher ethanol yield.

[Cloning expression and purification of glycerol dehydrogenase from Klebsiella pneumoniae].

The gldA gene coding glycerol dehydrogenase (GDH) was amplified by PCR with the genomic DNA of Klebsiella pneumoniae as the template. The gldA were inserted in pMD-18T to construct the recombinant cloning vector pMD-gldA. After the DNA sequence was determined, the gldA was subcloned into expression vector pET-32a (+) to construct the recombinant expression vector pET-32gldA. Upon lactose induction, soluble GDH was over-produced by E. coli BL21 (DE3) harboring the expression construct. Recombinant GDH purified by Ni-NTA affinity chromatography showed a single band about 54 kD on SDS-PAGE gel, and the specified activity was about 188 u/mg, the purification fold is 3 times and the activity recovery is 67.5%.

Dicarbonyl/L-xylulose reductase: a potential biomarker identified by laser-capture microdissection-micro serial analysis of gene expression of human prostate adenocarcinoma.

To identify genes involved in prostate carcinogenesis, we used laser-capture microdissection-micro serial analysis of gene expression to construct libraries of paired cancer and normal cells from human tissue samples. After computational comparison of the two libraries, we identified dicarbonyl/l-xylulose reductase (DCXR), an enzyme that catalyzes alpha-dicarbonyl and l-xylulose, as being significantly up-regulated in prostate cancer cells. The specificity of DCXR up-regulation for prostate cancer tissues was confirmed by quantitative real-time reverse transcriptase-PCR, virtual Northern blot, and Western blot analyses. Furthermore, DCXR expression at the protein level was assessed using fresh-frozen tissues and a tissue microarray consisting of 46 cases of organ-confined early-stage prostate cancer and 29 cases of chemohormonally treated prostate cancer. In most normal prostate epithelial cells, DCXR was expressed at low levels and was localized predominantly in the cytoplasmic membrane. In contrast, in virtually all grades of early-stage prostate cancer and in all chemohormonally treated cases, DCXR was strikingly overexpressed and was localized predominantly in the cytoplasm and nucleus. In all samples, the stromal cells were completely devoid of DCXR expression. Based on these findings, we suggest that DCXR overexpression has the potential to be an additional useful biomarker for prostate cancer.

Evaluation of inoculum of Candida guilliermondii grown in presence of glucose on xylose reductase and xylitol dehydrogenase activities and xylitol production during batch fermentation of sugarcane bagasse hydrolysate.

The effect of glucose on xylose-xylitol metabolism in fermentation medium consisting of sugarcane bagasse hydrolysate was evaluated by employing an inoculum of Candida guilliermondii grown in synthetic media containing, as carbon sources, glucose (30 g/L), xylose (30 g/L), or a mixture of glucose (2 g/L) and xylose (30 g/L). The inoculum medium containing glucose promoted a 2.5-fold increase in xylose reductase activity (0.582 IU/mgprot) and a 2-fold increase in xylitol dehydrogenase activity (0.203 IU/mgprot) when compared with an inoculum-grown medium containing only xylose. The improvement in enzyme activities resulted in higher values of xylitol yield (0.56 g/g) and productivity (0.46 g/[L.h]) after 48 h of fermentation.

Enhanced tolerance to salt stress in transgenic loblolly pine simultaneously expressing two genes encoding mannitol-1-phosphate dehydrogenase and glucitol-6-phosphate dehydrogenase.

A reproducible approach to improve salt tolerance of conifers has been established by using the technology of plant genetic transformation and using loblolly pine (Pinus taeda L.) as a model plant. Mature zygotic embryos of three genotypes of loblolly pine were infected with Agrobacterium tumefaciens strain LBA 4404 harboring the plasmid pBIGM which carrying two bacterial genes encoding the mannitol-1-phosphate dehydrogenase (Mt1D, EC 1.1.1.17) and glucitol-6-phosphate dehydrogenase (GutD) (EC 1.1.1.140), respectively. Transgenic plantlets were produced on selection medium containing 15 mg l(-1) kanamycin and confirmed by polymerase chain reaction (PCR) and Southern blot analysis of genomic DNA. The Mt1D and GutD genes were expressed and translated into functional enzymes that resulted in the synthesis and accumulation of mannitol and glucitol in transgenic plants. Salt tolerance assays demonstrated that transgenic plantlets producing mannitol and glucitol had an increased ability to tolerate high salinity. These results suggested that an efficient A. tumefaciens-mediated transformation protocol for stable integration of bacterial Mt1D and GutD genes into loblolly pine has been developed and this could be useful for the future studies on engineering breeding of conifers.

Overproduction of heterologous mannitol 1-phosphatase: a key factor for engineering mannitol production by Lactococcus lactis.

To achieve high mannitol production by Lactococcus lactis, the mannitol 1-phosphatase gene of Eimeria tenella and the mannitol 1-phosphate dehydrogenase gene mtlD of Lactobacillus plantarum were cloned in the nisin-dependent L. lactis NICE overexpression system. As predicted by a kinetic L. lactis glycolysis model, increase in mannitol 1-phosphate dehydrogenase and mannitol 1-phosphatase activities resulted in increased mannitol production. Overexpression of both genes in growing cells resulted in glucose-mannitol conversions of 11, 21, and 27% by the L. lactis parental strain, a strain with reduced phosphofructokinase activity, and a lactate dehydrogenase-deficient strain, respectively. Improved induction conditions and increased substrate concentrations resulted in an even higher glucose-to-mannitol conversion of 50% by the lactate dehydrogenase-deficient L. lactis strain, close to the theoretical mannitol yield of 67%. Moreover, a clear correlation between mannitol 1-phosphatase activity and mannitol production was shown, demonstrating the usefulness of this metabolic engineering approach.

Xylose chemostat isolates of Saccharomyces cerevisiae show altered metabolite and enzyme levels compared with xylose, glucose, and ethanol metabolism of the original strain.

The efficient conversion of xylose-containing biomass hydrolysate by the ethanologenic yeast Saccharomyces cerevisiae to useful chemicals such as ethanol still remains elusive, despite significant efforts in both strain and process development. This study focused on the recovery and characterization of xylose chemostat isolates of a S. cerevisiae strain that overexpresses xylose reductase- and xylitol dehydrogenase-encoding genes from Pichia stipitis and the gene encoding the endogenous xylulokinase. The isolates were recovered from aerobic chemostat cultivations on xylose as the sole or main carbon source. Under aerobic conditions, on minimal medium with 30 g l(-1) xylose, the growth rate of the chemostat isolates was 3-fold higher than that of the original strain (0.15 h(-1) vs 0.05 h(-1)). In a detailed characterization comparing the metabolism of the isolates with the metabolism of xylose, glucose, and ethanol in the original strain, the isolates showed improved properties in the assumed bottlenecks of xylose metabolism. The xylose uptake rate was increased almost 2-fold. Activities of the key enzymes in the pentose phosphate pathway (transketolase, transaldolase) increased 2-fold while the concentrations of their substrates (pentose 5-phosphates, sedoheptulose 7-phosphate) decreased correspondingly. Under anaerobic conditions, on minimal medium with 45 g l(-1) xylose, the ethanol productivity (in terms of cell dry weight; CDW) of one of the isolates increased from 0.012 g g(-1) CDW h(-1) to 0.017 g g(-1) CDW h(-1) and the yield from 0.09 g g(-1) xylose to 0.14 g g(-1) xylose, respectively.

[Construction of recombinant expression vector and prokaryotic expression of human epididymal sperm protein P34H].

OBJECTIVE: To acquire purified recombinant human epididymal sperm protein P34H for basic and clinical studies. METHODS: On the basis of cloning of P34H coding region, P34H fragment was subcloned into the pQE-30 expression vector. The recombinant expression vector designated pQE-30/P34H was transformed into E. coli to induce the expression of the recombinant protein P34H on the reduction of IPTG. After sonication, the recombinant protein P34H was purified from the supernatant with Ni-NTA resin under native conditions. It was identified by SDS-PAGE analysis and DNA sequencing. RESULTS: Recombinant expression vector pQE-30/P34H was correctly constructed, identified with PCR and double-enzyme digestion. And the results of SDS-PAGE analysis and DNA sequencing showed that the protein was what we had hoped to acquire. CONCLUSION: Purified recombinant P34H can be acquired successfully with the above mentioned prokaryotic expression method.

Purification and identification of an Escherichia coli beta-keto ester reductase as 2,5-diketo-D-gluconate reductase YqhE.

An NADPH-dependent enzyme that reduces ethyl 2-methylacetoacetate stereoselectively to ethyl (2R)-methyl-(3S)-hydroxybutanoate was purified 730-fold from Escherichia coli. The N-terminal amino acid sequence data obtained from the purified reductase were used to search the E. coli genome, and a single match was found at the start of the yqhE open reading frame. The YqhE protein had been identified previously by Yum et al. as a 2,5-diketo-D-gluconate reductase on the basis of sequence similarity to other bacterial homologues [Yum, D.-Y.; Lee, B.-Y.; Pan, J.-G. Appl.Environ. Microbiol. 1999, 65, 3341-3346]; however, it had not been examined for beta-keto ester reductions. Our results thus link a key enzyme in the microbial production of ascorbate with stereoselective beta-keto ester reductions, two important fields in biocatalysis. The purified YqhE reductase accepts ethyl acetoacetate and a variety of 2-substituted derivatives, and its sequence is similar to other aldose reductase superfamily members that also reduce alpha-substituted beta-keto esters to syn-(2R,3S) alcohols.

Involvement of two distinct catabolite-responsive elements in catabolite repression of the Bacillus subtilis myo-inositol (iol) operon.

The Bacillus subtilis inositol operon (iolABCDEFGHIJ) is involved in myo-inositol catabolism. Glucose repression of the iol operon induced by inositol is exerted through catabolite repression mediated by CcpA and the iol induction system mediated by IolR. In this study, we identified two iol catabolite-responsive elements (cre's), to which CcpA complexed with P-Ser-HPr or P-Ser-Crh probably binds. One is located in iolB (cre-iolB, nucleotides +2397 to +2411; +1 is the transcription initiation nucleotide), which was the only cre-iol found in the previous cre search of the B. subtilis genome using a query sequence of WTGNAANCGNWNNCW (W stands for A or T, and N stands for any base). Deletion and base substitution analysis of the iol region indicated that cre-iolB functions even if it is located far downstream of the iol promoter. Further deletion and base substitution analysis revealed another cre located between the iol promoter and the iolA gene (cre-iiolA, nucleotides +86 to +100); the prefix "i" indicates a location in the intergenic region. Both cre-iiolA and cre-iolB appeared to be recognized to almost the same extent by CcpA complexed with either P-Ser-HPr or P-Ser-Crh. Sequence alignment of the six known cre's, including cre-iiolA, which were not revealed in the previous cre search, exhibited another consensus sequence of WTGAAARCGYTTWWN (R stands for A or G, and Y stands for C or T); the right two thymines (TT) were found to be essential for the function of cre-iiolA by means of base substitution analysis. A cre search with this query sequence led to the finding of 14 additional putative cre's.