Putative Regulatory Genes Research Articles

Dehalogenimonas lykanthroporepellensBL-DC-9Tsimultaneously transcribes manyrdhAgenes during organohalide respiration with 1,2-DCA, 1,2-DCP, and 1,2,3-TCP as electron acceptors

The genome sequence of the organohalide-respiring bacterium Dehalogenimonas lykanthroporepellensBL-DC-9(T) contains numerous loci annotated as reductive dehalogenase homologous (rdh) genes based on inferred protein sequence identity with functional dehalogenases of other bacterial species. Many of these genes are truncated, lack adjacent regulatory elements, or lack cognate genes coding for membrane-anchoring proteins typical of the functionally characterized active reductive dehalogenases of organohalide-respiring bacteria. To investigate the expression patterns of the rdh genes in D. lykanthroporepellensBL-DC-9(T), oligonucleotide primers were designed to uniquely target 25 rdh genes present in the genome as well as four putative regulatory genes. RNA extracts from cultures of strain BL-DC-9(T) actively dechlorinating three different electron acceptors, 1,2-dichloroethane, 1,2-dichloropropane, and 1,2,3-trichloropropane were reverse-transcribed and subjected to PCR amplification using rdh-specific primers. Nineteen rdh gene transcripts, including 13 full-length rdhA genes, six truncated rdhA genes, and five rdhA genes having cognate rdhB genes were consistently detected during the dechlorination of all three of the polychlorinated alkanes tested. Transcripts from all four of the putative regulatory genes were also consistently detected. Results reported here expand the diversity of bacteria known to simultaneously transcribe multiple rdh genes and provide insights into the transcription factors associated with rdh gene expression.

Chronic exposure to a predator or its scent does not inhibit male–male competition in male mice lacking brain serotonin

Although it is well-known that defective signaling of the 5-HT system in the brain and stressful stimuli can cause psychological disorders, their combined effects on male–male aggression and sexual attractiveness remain unknown. Our research aimed at examining such effects using tryptophan hydroxylase 2 (Tph2) knockout male mice vs. a rat- or rat scent-based chronic stress model. Tph2+/+ and Tph2−/− male mice were placed individually into the rat home cage (rat), a cage containing soiled rat bedding (rat scent) or a cage containing fresh bedding (control) for 5 h every other day for 56 consecutive days. In Tph2+/+ male mice, rat-exposure decreased male–male aggression and sexual attractiveness of urine odor relative to either rat scent-exposure or control; and rat scent-exposure decreased aggression rather than sexual attractiveness of urine odor compared with control. However, such dose-dependent and long-lasting behavioral inhibitory effects vanished in Tph2−/− male mice. RT-PCR assay further revealed that putative regulatory genes, such as AR, ERα and GluR4 in the prefrontal cortex, and TrkB-Tc and 5-HTR1A in the hippocampus, were down-regulated at the mRNA level in either rat- or rat scent-exposed Tph2+/+ male mice, but partially in the Tph2−/− ones. Hence, we suggest that the dose-dependent and long-lasting inhibitory effects of chronic predator exposure on male–male aggression, sexual attractiveness of urine odor, and mRNA expression of central regulatory genes might be mediated through the 5-HT system in the brain of male mice.

Complex Intra-Operonic Dynamics Mediated by a Small RNA in Streptomyces coelicolor

Streptomyces are predominantly soil-dwelling bacteria that are best known for their multicellular life cycle and their prodigious metabolic capabilities. They are also renowned for their regulatory capacity and flexibility, with each species encoding >60 sigma factors, a multitude of transcription factors, and an increasing number of small regulatory RNAs. Here, we describe our characterization of a conserved small RNA (sRNA), scr4677. In the model species Streptomyces coelicolor, this sRNA is located in the intergenic region separating SCO4677 (an anti-sigma factor-encoding gene) and SCO4676 (a putative regulatory protein-encoding gene), close to the SCO4676 translation start site in an antisense orientation. There appears to be considerable genetic interplay between these different gene products, with wild type expression of scr4677 requiring function of the anti-sigma factor SCO4677, and scr4677 in turn influencing the abundance of SCO4676-associated transcripts. The scr4677-mediated effects were independent of RNase III (a double stranded RNA-specific nuclease), with RNase III having an unexpectedly positive influence on the level of SCO4676-associated transcripts. We have shown that both SCO4676 and SCO4677 affect the production of the blue-pigmented antibiotic actinorhodin under specific growth conditions, and that this activity appears to be independent of scr4677.

Discovery of a family of γ-aminobutyrate ureas via rational derepression of a silent bacterial gene cluster

Gaburedins, a family of γ-aminobutyrate (GABA)-derived ureas, have been discovered by deletion of gbnR, an arpA-like putative transcriptional repressor in Streptomyces venezuelae ATCC 10712. Comparison of metabolite profiles in the wild type and mutant strains revealed six metabolites in the mutant that are lacking from the wild type. The structure of gaburedin A was established by HRMS combined with 1- and 2-D NMR spectroscopy and was confirmed by total synthesis. The other metabolites were confirmed as congeners using HRMS, MS/MS and feeding of putative biosynthetic precursors. Two genes, gbnA and gbnB, are proposed to be involved in gaburedin biosynthesis. Consistent with this hypothesis, deletion of gbnB in the gbnR mutant abolished gaburedin production. This is the first report to disclose the discovery of novel natural products via rational deletion of a putative pathway-specific regulatory gene.

SACE_5599, a putative regulatory protein, is involved in morphological differentiation and erythromycin production in Saccharopolyspora erythraea

BackgroundErythromycin is a medically important antibiotic, biosynthesized by the actinomycete Saccharopolyspora erythraea. Genes encoding erythromycin biosynthesis are organized in a gene cluster, spanning over 60 kbp of DNA. Most often, gene clusters encoding biosynthesis of secondary metabolites contain regulatory genes. In contrast, the erythromycin gene cluster does not contain regulatory genes and regulation of its biosynthesis has therefore remained poorly understood, which has for a long time limited genetic engineering approaches for erythromycin yield improvement.ResultsWe used a comparative proteomic approach to screen for potential regulatory proteins involved in erythromycin biosynthesis. We have identified a putative regulatory protein SACE_5599 which shows significantly higher levels of expression in an erythromycin high-producing strain, compared to the wild type S. erythraea strain. SACE_5599 is a member of an uncharacterized family of putative regulatory genes, located in several actinomycete biosynthetic gene clusters. Importantly, increased expression of SACE_5599 was observed in the complex fermentation medium and at controlled bioprocess conditions, simulating a high-yield industrial fermentation process in the bioreactor. Inactivation of SACE_5599 in the high-producing strain significantly reduced erythromycin yield, in addition to drastically decreasing sporulation intensity of the SACE_5599-inactivated strains when cultivated on ABSM4 agar medium. In contrast, constitutive overexpression of SACE_5599 in the wild type NRRL23338 strain resulted in an increase of erythromycin yield by 32%. Similar yield increase was also observed when we overexpressed the bldD gene, a previously identified regulator of erythromycin biosynthesis, thereby for the first time revealing its potential for improving erythromycin biosynthesis.ConclusionsSACE_5599 is the second putative regulatory gene to be identified in S. erythraea which has positive influence on erythromycin yield. Like bldD, SACE_5599 is involved in morphological development of S. erythraea, suggesting a very close relationship between secondary metabolite biosynthesis and morphological differentiation in this organism. While the mode of action of SACE_5599 remains to be elucidated, the manipulation of this gene clearly shows potential for improvement of erythromycin production in S. erythraea in industrial setting. We have also demonstrated the applicability of the comparative proteomics approach for identifying new regulatory elements involved in biosynthesis of secondary metabolites in industrial conditions.

Improvement of gougerotin and nikkomycin production by engineering their biosynthetic gene clusters

Nikkomycins and gougerotin are peptidyl nucleoside antibiotics with broad biological activities. The nikkomycin biosynthetic gene cluster comprises one pathway-specific regulatory gene (sanG) and 21 structural genes, whereas the gene cluster for gougerotin biosynthesis includes one putative regulatory gene, one major facilitator superfamily transporter gene, and 13 structural genes. In the present study, we introduced sanG driven by six different promoters into Streptomyces ansochromogenes TH322. Nikkomycin production was increased significantly with the highest increase in engineered strain harboring hrdB promoter-driven sanG. In the meantime, we replaced the native promoter of key structural genes in the gougerotin (gou) gene cluster with the hrdB promoters. The heterologous producer Streptomyces coelicolor M1146 harboring the modified gene cluster produced gougerotin up to 10-fold more than strains carrying the unmodified cluster. Therefore, genetic manipulations of genes involved in antibiotics biosynthesis with the constitutive hrdB promoter present a robust, easy-to-use system generally useful for the improvement of antibiotics production in Streptomyces.

Genes ycfR, sirA and yigG Contribute to the Surface Attachment of Salmonella enterica Typhimurium and Saintpaul to Fresh Produce

Salmonella enterica is a frequent contaminant of minimally-processed fresh produce linked to major foodborne disease outbreaks. The molecular mechanisms underlying the association of this enteric pathogen with fresh produce remain largely unexplored. In our recent study, we showed that the expression of a putative stress regulatory gene, ycfR, was significantly induced in S. enterica upon exposure to chlorine treatment, a common industrial practice for washing and decontaminating fresh produce during minimal processing. Two additional genes, sirA involved in S. enterica biofilm formation and yigG of unknown function, were also found to be differentially regulated under chlorine stress. To further characterize the roles of ycfR, sirA, and yigG in S. enterica attachment and survival on fresh produce, we constructed in-frame deletions of all three genes in two different S. enterica serovars, Typhimurium and Saintpaul, which have been implicated in previous disease outbreaks linked to fresh produce. Bacterial attachment to glass and polystyrene microtiter plates, cell aggregation and hydrophobicity, chlorine resistance, and surface attachment to intact spinach leaf and grape tomato were compared among wild-type strains, single-gene deletion mutants, and their respective complementation mutants. The results showed that deletions of ycfR, sirA, and yigG reduced bacterial attachment to glass and polystyrene as well as fresh produce surface with or without chlorine treatment in both Typhimurium and Saintpaul. Deletion of ycfR in Typhimurium significantly reduced bacterial chlorine resistance and the attachment to the plant surfaces after chlorinated water washes. Deletions of ycfR in Typhimurium and yigG in Saintpaul resulted in significant increase in cell aggregation. Our findings suggest that ycfR, sirA, and yigG collectively contribute to S. enterica surface attachment and survival during post-harvest minimal processing of fresh produce.

Organization and characterization of a biosynthetic gene cluster for bafilomycin from Streptomyces griseus DSM 2608

Streptomyces griseus DSM 2608 produces bafilomycin, an antifungal plecomacrolide antibiotic. We cloned and sequenced an 87.4-kb region, including a polyketide synthase (PKS) region, methoxymalonate genes, flavensomycinate genes, and other putative regulatory genes. The 58.5kb of PKS region consisting 12 PKS modules arranged in five different PKS genes, was assumed to be responsible for the biosynthesis of plecomacrolide backbone including 16-membered macrocyclic lactone. All the modules showed high similarities with typical type I PKS genes. However, the starting module of PKS gene was confirmed to be specific for isobutyrate by sequence comparison of an acyltransferase domain. In downstream of PKS region, the genes for methoxymalonate biosynthesis were located, among which a gene for FkbH-like protein was assumed to play an important role in the production of methoxymalonyl-CoA from glyceryl-CoA. Further the genes encoding flavensomycinyl-ACP biosynthesis for the post-PKS tailoring were also found in the upstream of PKS region. By gene disruption experiments of a dehydratase domain of module 12 and an FkbH-like protein, this gene cluster was confirmed to be involved in the biosynthesis of bafilomycin.

Genomic clustering and co-regulation of transcriptional networks in the pathogenic fungus Fusarium graminearum.

BackgroundGenes for the production of a broad range of fungal secondary metabolites are frequently colinear. The prevalence of such gene clusters was systematically examined across the genome of the cereal pathogen Fusarium graminearum. The topological structure of transcriptional networks was also examined to investigate control mechanisms for mycotoxin biosynthesis and other processes.ResultsThe genes associated with transcriptional processes were identified, and the genomic location of transcription-associated proteins (TAPs) analyzed in conjunction with the locations of genes exhibiting similar expression patterns. Highly conserved TAPs reside in regions of chromosomes with very low or no recombination, contrasting with putative regulator genes. Co-expression group profiles were used to define positionally clustered genes and a number of members of these clusters encode proteins participating in secondary metabolism. Gene expression profiles suggest there is an abundance of condition-specific transcriptional regulation. Analysis of the promoter regions of co-expressed genes showed enrichment for conserved DNA-sequence motifs. Potential global transcription factors recognising these motifs contain distinct sets of DNA-binding domains (DBDs) from those present in local regulators.ConclusionsProteins associated with basal transcriptional functions are encoded by genes enriched in regions of the genome with low recombination. Systematic searches revealed dispersed and compact clusters of co-expressed genes, often containing a transcription factor, and typically containing genes involved in biosynthetic pathways. Transcriptional networks exhibit a layered structure in which the position in the hierarchy of a regulator is closely linked to the DBD structural class.

Transcriptional analysis and proteomics of the holomycin gene cluster in overproducer mutants of Streptomyces clavuligerus

Expression of the holomycin biosynthesis genes (hlm) has been studied in the wild type strain Streptomyces clavuligerus ATCC 27064 and holomycin overproducer mutants. RT-PCR transcription analysis of S. clavuligerus oppA2::aph showed a higher transcription of the hlmA, B, C, D, E, F, G, H, I and hlmL genes, a slightly lower expression for hlmK and no significant differences for the transcription of the two putative regulatory genes, hlmM and hlmJ, in relation to the wild type strain. Accordingly, protein spots corresponding to HlmD, HlmF and HlmG, which were barely detectable in the wild type strain, were present in high amounts in the holomycin overproducer S. clavuligerus oppA2::aph proteome. Transcription start point analysis of the hlm genes revealed that the annotated sequences in the databases for several hlm genes were incorrect. The hlm cluster was introduced into Streptomyces coelicolor M1154 and holomycin production by S. coelicolor M1154 [pVR-hol1] was validated by bioassays and confirmed by HPLC analysis and mass spectrometry. Heterologous holomycin production by the S. coelicolor transformant is 500-fold lower than in S. clavuligerus oppA2::aph. The transformant S. coelicolor M1154 [pVR-hol1] shows holomycin sensitivity to 100μg/ml, similar to that of the parental S. coelicolor M1154 strain, suggesting that heterologous expression in S. coelicolor might be toxic due to the lack of an holomycin resistance gene in this host strain.

A combined histology and transcriptome analysis unravels novel questions on Medicago truncatula seed coat

The seed coat is involved in the determination of seed quality traits such as seed size, seed composition, seed permeability, and hormonal regulation. Understanding seed coat structure is therefore a prerequisite to deciphering the genetic mechanisms that govern seed coat functions. By combining histological and transcriptomic data analyses, cellular and molecular events occurring during Medicago truncatula seed coat development were dissected in order to relate structure to function and pinpoint target genes potentially involved in seed coat traits controlling final seed quality traits. The analyses revealed the complexity of the seed coat transcriptome, which contains >30 000 genes. In parallel, a set of genes showing a preferential expression in seed coat that may be involved in more specific functions was identified. The study describes how seed coat anatomy and morphological changes affect final seed quality such as seed size, seed composition, seed permeability, and hormonal regulation. Putative regulator genes of different processes have been identified as potential candidates for further functional genomic studies to improve agronomical seed traits. The study also raises new questions concerning the implication of seed coat endopolyploidy in cell expansion and the participation of the seed coat in de novo abscisic acid biosynthesis at early seed filling.

Genetics of germination-arrest factor (GAF) production by Pseudomonas fluorescens WH6: identification of a gene cluster essential for GAF biosynthesis

The genetic basis of the biosynthesis of the germination-arrest factor (GAF) produced by Pseudomonas fluorescens WH6, and previously identified as 4-formylaminooxyvinylglycine, has been investigated here. In addition to inhibiting the germination of a wide range of grassy weeds, GAF exhibits a selective antimicrobial activity against the bacterial plant pathogen Erwinia amylovora. We utilized the in vitro response of E. amylovora to GAF as a rapid screen for loss-of-function GAF phenotypes generated by transposon mutagenesis. A Tn5 mutant library consisting of 6364 WH6 transformants was screened in this Erwinia assay, resulting in the identification of 18 non-redundant transposon insertion sites that led to loss of GAF production in WH6, as confirmed by TLC analysis. These insertions mapped to five different genes and four intergenic regions. Three of these genes, including two putative regulatory genes (gntR and iopB homologues), were clustered in a 13 kb chromosomal region containing 13 putative ORFs. A GAF mutation identified previously as affecting an aminotransferase also maps to this region. We suggest that three of the genes in this region (a carbamoyltransferase, an aminotransferase and a formyltransferase) encode the enzymes necessary to synthesize dihydroGAF, the putative immediate precursor of GAF in a proposed GAF biosynthetic pathway. RT-qPCR analyses demonstrated that mutations in the gntR and iopB regulatory genes, as well as in a prtR homologue identified earlier as controlling GAF formation, suppressed transcription of at least two of the putative GAF biosynthetic genes (encoding the aminotransferase and formyltransferase) located in this 13 kb region.

Three pathway-specific regulators control streptolydigin biosynthesis in Streptomyces lydicus

The streptolydigin biosynthetic gene cluster from Streptomyces lydicus NRRL 2433 contains three putative regulatory genes, slgR1, slgR2 and slgY, encoding proteins belonging to TetR and LuxR transcriptional regulator families and ATP/GTP-binding proteins of DNA and RNA helicase superfamily I, respectively. Inactivation of slgR1 or slgR2 resulted in the abolition of streptolydigin production, suggesting that these proteins are pathway-specific positive regulators. In the case of the slgR1 mutant, low amounts of streptolydigin C were produced instead of streptolydigin. RT-PCR transcription analysis of streptolydigin biosynthesis genes revealed a hierarchical regulation process. SlgY was found to control the expression of the regulator slgR2. SlgR2 regulates the expression of structural genes involved in the formation of the streptolydigin bicyclic ketal moiety, incorporation and processing of 3-methylaspartate, and the regulator slgR1. On the other hand, SlgR1 controls the expression of slgE1-E2, involved in the conversion of glutamate to 3-methylaspartate, and putative glycoside hydrolases slgC1 and slgC2. Ectopic expression of slgR1, slgR2 and slgY regulatory genes in S. lydicus led to considerable increases in streptolydigin yields, 18-, 11- and 8.5-fold, respectively. Ectopic expression of slgY in an slgR1 mutant led to a 14-fold increase of streptolydigin C yields, while no effect was observed to result from expression of slgR2.

Characterization of the mbd cluster encoding the anaerobic 3‐methylbenzoyl‐CoA central pathway

The mbd cluster encoding genes of the 3-methylbenzoyl-CoA pathway involved in the anaerobic catabolism of 3-methylbenzoate and m-xylene was characterized for the first time in the denitrifying β-Proteobacterium Azoarcus sp. CIB. The mbdA gene product was identified as a 3-methylbenzoate-CoA ligase required for 3-methylbenzoate activation; its substrate spectrum was unique in activating all three methylbenzoate isomers. An inducible 3-methylbenzoyl-CoA reductase (mbdONQP gene products), displaying significant amino acid sequence similarities to known class I benzoyl-CoA reductases catalysed the ATP-dependent reduction of 3-methylbenzoyl-CoA to a methyldienoyl-CoA. The mbdW gene encodes a methyldienoyl-CoA hydratase that hydrated the methyldienoyl-CoA to a methyl-6-hydroxymonoenoyl-CoA compound. The mbd cluster also contains the genes predicted to be involved in the subsequent steps of the 3-methylbenzoyl-CoA pathway as well as the electron donor system for the reductase activity. Whereas the catabolic mbd genes are organized in two divergent inducible operons, the putative mbdR regulatory gene was transcribed separately and showed constitutive expression. The efficient expression of the mbd genes required the oxygen-dependent AcpR activator, and it was subject of carbon catabolite repression by some organic acids and amino acids. Sequence analyses suggest that the mbd gene cluster was recruited by Azoarcus sp. CIB through horizontal gene transfer.

Do regulatory regions matter in FOXG1 duplications?

Duplications of FOXG1 gene at 14q12 have been reported in patients with infantile spasms and developmental delay of variable severity.1, 2, 3 FOXG1 encodes the forkhead protein G1, a brain-specific transcriptional repressor, regulating corticogenesis in the developing brain and neuronal stem cell self-renewal in the postnatal brain.4 Recently, Amor et al.5 reported on this journal an interstitial duplication of ∼88 kb at 14q12 in a father–son pair with hemifacial microsomia and normal neurocognitive phenotype. The duplication contains only two polypeptide-encoding genes, FOXG1 and C14orf23, suggesting that FOXG1 duplication may be benign or at least incompletely penetrant. That makes the involvement of FOXG1 duplication in the pathogenesis of the neurocognitive impairment and epilepsy controversial. As also discussed by Brunetti-Pierri et al,6 we feel that this statement needs special caution. Functional consequences of chromosomal microduplication and microdeletion rely on the final gene dosage, which is strongly influenced by the location of the breakpoint. In this context, the understanding of the contribution of regulatory sequences in gene transcription is critical to understand the relationship between CNVs and human diseases. With this purpose, the Encyclopedia of DNA Elements (ENCODE) project has recently performed a systematic analysis of transcriptional regulation in different human cell lines, providing new understanding about transcription start sites, including their relationship with specific regulatory sequences and histone modification and features of chromatin accessibility.7, 8 Interestingly, analysis of histone modifications from the ENCODE project revealed the presence of a putative regulatory element upstream FOXG1 gene between 28 188 and 28 217 kb (UCSC genome browser, NCBI Build 36/hg18) (Figure 1). This conserved region localizes about 130 kb upstream FOXG1 gene and contains histone modifications typical of enhancers of gene transcription (eg, histone H3 and Lysine 4 monomethylation) in eight different human cells lines. Analysis of regulatory potential scores, comparing frequencies of short alignment patterns between known regulatory elements and neutral DNA,9 also disclose two additional putative elements typical of cis-regulatory modules within this region (Figure 1). Moreover, it contains a DNaseI hypersensitive site (DHS). DHSs reflect genomic regions thought to be enriched for regulatory information and many DHSs reside at or near transcription start site. Notably, no other polypeptide-encoding genes or non-coding RNAs and pseudogenes are present in the region, suggesting that this regulatory element might regulate FOXG1 transcription. Analysis of duplication breakpoints previously reported on 14q12 revealed that duplications associated with an epileptic phenotype localizes uniquely upstream this regulatory element, whereas downstream duplications were identified only in the cases without seizures (Figure 1). On the basis of this finding, we suggest that FOXG1 duplication including this putative regulatory region allows the efficient transcription of the supernumerary copy of FOXG1 gene, resulting in an effective increase in FOXG1 expression and, thereby, in brain hyperexcitability. In contrast, duplications starting downstream this putative regulatory site do not allow efficient transcription of FOXG1, which may underlie the lack of neurological phenotype in the case reported by Amor et al5. Figure 1 UCSC genome browser (NCBI36/hg18) schematic view of reported 14q12 duplications encompassing FOXG1 gene. Histone modifications from the ENCODE project7 in eight cells lines (GM12878, H1-hESC, HMEC, HSMM, HUVEC, K562, NHEK and NHLF) indicate the presence ... Even if the functional relevance of this putative long-range regulatory element on FOXG1 transcription deserves to be experimentally verified, it provides an interesting clue to dissect genotype–phenotype correlation in FOXG1 microduplication and to uncover the real actual contribution of FOXG1 in the neurodevelopmental phenotype associated with 14q12 duplication. Notably, chromosome rearrangements disrupting or displacing putative cis-regulatory elements distal to FOXG1 gene in patients with severe cognitive disabilities has been also reported,10, 11 pointing out the relevance of regulatory sequences in the expression of FOXG1 gene.

Bacterial genome mining for novel natural product discovery

Bioinformatics analyses have identified gene clusters encoding cryptic polyketide biosynthetic pathways, not associated with the production of known metabolites, in several actinomycete genome sequences. Discovery of the metabolic products of such cryptic gene clusters promises to unearth a hitherto untapped wealth of novel bioactive compounds. However, a major obstacle to the discovery of novel natural products by genome mining is that many cryptic pathways are expressed poorly or not at all under normal laboratory conditions. The discovery of the stambomycins, a new family of 51-membered macrolides with promising anti-cancer activity, as the products of a novel type I modular polyketide synthase (PKS) system identified in the partial genome sequence of Streptomyces ambofaciens will be described. Activation of expression of the pathway by genetic manipulation of a putative pathway-specific regulatory gene was key to this discovery. The structures of these novel macrolides suggests that their biosynthesis involves novel features, including in trans hydroxylation during polyketide chain assembly to provide the hydroxyl group required for offloading of the fully-assembled polyketide chain from the PKS via macrocyclisation. Experiments aimed at probing this unusual transformation will be described.

Effects of dietary carbohydrates on rumen epithelial metabolism of nonlactating heifers

Ruminal wall metabolism was studied in nonlactating heifers by altering the carbohydrate (CHO) digestion site between rumen and intestine. The CHO digestion site was estimated from in situ and total-tract digestibility of control (CONT) diets and diets supplemented with corn (CRN), barley (BARL), or soy hulls (SOYH). Ruminal epithelial metabolism regulating gene expression, morphology, and nutrient delivery was assessed from a combination of rumen volatile fatty acid (VFA) concentration, biopsies for papilla morphology, and expression of putative metabolic regulatory genes encoding enzymes that facilitate VFA utilization. Digestible dry matter and CHO intake were 25 and 45% higher, respectively, in the supplemented diets than in CONT diets. Fiber supplementation increased the intestinal and decreased ruminal CHO digestion. Ruminal nonfiber CHO digestibility was 10% lower in CRN than with the high rumen-degradable supplement. The CONT heifers had lowest total ruminal VFA and highest acetate concentration relative to the other treatments. Total VFA concentration in BARL and CRN diets tended to be higher than in SOYH. The SOYH diet tended to reduce papilla dimension relative to CRN and BARL. The CRN diet tended to increase papilla surface area relative to BARL and SOYH. Gene expression of propionyl-coenzyme A carboxylase was higher in CRN and BARL than in SOYH diets, and tended to be higher in CRN than in BARL and SOYH diets. Lactate dehydrogenase and butyryl coenzyme A synthase gene transcripts tended to be higher in CONT than in the supplemented treatments. Thus, rumen epithelial expression of genes involved in VFA metabolism and ruminal wall-structure development are influenced by other regulatory mechanism that is not directly affected by local signals. The in situ methods used are a useful tool for differentiating ruminal from extraruminal nutrient supply.

Transcriptional regulation and increased production of asukamycin in engineered Streptomyces nodosus subsp. asukaensis strains

Asukamycin, a member of the manumycin family of antibiotics, exhibits strong antibacterial, antifungal, and antineoplastic activities. However, its production in the wild-type strain of Streptomyces nodosus subsp. asukaensis ATCC 29757 is relatively low. Recently, the biosynthetic gene cluster for asukamycin was identified in the producing organism, and among the 36 genes reported in the cluster, six (asuR1-asuR6) were proposed to encode proteins that function as transcriptional regulators. In order to investigate their involvement in asukamycin biosynthesis and to engineer mutant strains of S. nodosus that are able to produce large amounts of asukamycin, we carried out in vivo gene inactivation, transcriptional analysis of the biosynthetic genes in the mutants, and gene duplication in the producing strain of S. nodosus. The results show that two of the putative regulatory genes (asuR1 and asuR5) are critical for asukamycin biosynthesis, whereas others regulate the pathway at various levels. Overexpression of a gene cassette harboring asuR1, asuR2, asuR3, and asuR4 in S. nodosus resulted in changes in morphology of the producing strain and an approximately 14-fold increase of asukamycin production. However, overexpression of the individual genes did not give a comparable cumulative level of asukamycin production, suggesting that some, if not all, of the gene products act synergistically to regulate the biosynthesis of this antibiotic.

Importance and regulation of inositol biosynthesis during growth and differentiation of Streptomyces

Unusually among bacteria, actinobacteria possess myo-inositol 1-phosphate synthase (mIPS). In the developmentally complex Streptomyces coelicolor, the mIPS-encoding gene (inoA) is cotranscribed with a putative regulatory gene (inoR). The inoRA transcript was more abundant in an inoR in-frame deletion mutant, and InoR formed different complexes in vitro with an extensive region around the inoRA promoter. Binding was relieved by adding glucose 6-phosphate. Thus, InoR is a metabolite-sensitive autorepressor that influences inoA expression, and hence the level of inositol, by controlling transcription from P(inoRA) . Disruption of inoA resulted in inositol-dependent growth and development, with full phenotypic correction at 0.1 mM inositol: at lower inositol concentrations differentiation was arrested at intermediate stages. This pattern may partly reflect increased demand for membrane phospholipids during sporulation septation. A corresponding sharp upregulation of inoRA transcription coincident with sporulation was dependent on a developmental regulator, WhiI. A truncated form of WhiI could bind two sites downstream of P(inoRA) , and one of the WhiI-binding sites overlapped the InoR-binding site. The combined action of a metabolic regulator and a developmental regulator at the simple P(inoRA) promoter is a previously undescribed strategy for the differential provision of developmentally appropriate levels of a substance required during the formation of spore chains.

Roles of fkbN in Positive Regulation and tcs7 in Negative Regulation of FK506 Biosynthesis in Streptomyces sp. Strain KCTC 11604BP

FK506 is an important 23-member polyketide macrolide with immunosuppressant activity. Its entire biosynthetic gene cluster was previously cloned from Streptomyces sp. strain KCTC 11604BP, and sequence analysis identified three putative regulatory genes, tcs2, tcs7, and fkbN, which encode proteins with high similarity to the AsnC family transcriptional regulators, LysR-type transcriptional regulators, and LAL family transcriptional regulators, respectively. Overexpression and in-frame deletion of tcs2 did not affect the production of FK506 or co-occurring FK520 compared to results for the wild-type strain, suggesting that tcs2 is not involved in their biosynthesis. fkbN overexpression improved the levels of FK506 and FK520 production by approximately 2.0-fold, and a deletion of fkbN caused the complete loss of FK506 and FK520 production. Although the overexpression of tcs7 decreased the levels of FK506 and FK520 production slightly, a deletion of tcs7 caused 1.9-fold and 1.5-fold increases in FK506 and FK520 production, respectively. Finally, fkbN overexpression in the tcs7 deletion strain resulted in a 4.0-fold (21 mg liter(-1)) increase in FK506 production compared to that by the wild-type strain. This suggests that fkbN encodes a positive regulatory protein essential for FK506/FK520 biosynthesis and that the gene product of tcs7 negatively regulates their biosynthesis, demonstrating the potential of exploiting this information for strain improvement. Semiquantitative reverse transcription-PCR (RT-PCR) analyses of the transcription levels of the FK506 biosynthetic genes in the wild-type and mutant strains proved that most of the FK506 biosynthetic genes are regulated by fkbN in a positive manner and negatively by tcs7.