Fungi influence nutrient cycling in terrestrial ecosystems, as they are major regulators of decomposition and soil respiration. However, little is known about the substrate preferences of individual fungal species outside of laboratory culture studies. If active fungi differ in their substrate preferences in situ, then changes in fungal diversity due to global change may dramatically influence nutrient cycling in ecosystems. To test the responses of individual fungal taxa to specific substrates, we used a nucleotide-analogue procedure in the boreal forest of Alaska (USA). Specifically, we added four organic N compounds commonly found in plant litter (arginine, glutamate, lignocellulose, and tannin-protein) to litterbags filled with decomposed leaf litter (black spruce and aspen) and assessed the responses of active fungal species using qPCR (quantitative polymerase chain reaction), oligonucleotide fingerprinting of rRNA genes, and sequencing. We also compared the sequences from our experiment with a concurrent warming experiment to see if active fungi that targeted more recalcitrant compounds would respond more positively to soil warming. We found that individual fungal taxa responded differently to substrate additions and that active fungal communities were different across litter types (spruce vs. aspen). Active fungi that targeted lignocellulose also responded positively to experimental warming. Additionally, resource-use patterns in different fungal taxa were genetically correlated, suggesting that it may be possible to predict the ecological function of active fungal communities based on genetic information. Together, these results imply that fungi are functionally diverse and that reductions in fungal diversity may have consequences for ecosystem functioning.

Transcription-generated DNA supercoiling plays a decisive role in a promoter relay mechanism for the coordinated expression of genes in the Salmonella typhimurium ilvIH-leuO-leuABCD gene cluster. A similar mechanism also operates to control expression of the genes in the Escherichia coli ilvIH-leuO-leuABCD gene cluster. However, the mechanism underlying the DNA supercoiling effect remained elusive. A bacterial gene silencer AT8 was found to be important for the repression state of the leuO gene as part of the promoter relay mechanism. In this communication, we demonstrated that the gene silencer AT8 is a nucleation site for recruiting histone-like nucleoid structuring protein to form a cis-spreading nucleoprotein filament that is responsible for silencing of the leuO gene. With a DNA geometric similarity rather than a DNA sequence specificity, the E. coli gene silencer EAT6 was capable of replacing the histone-like nucleoid structuring protein nucleation function of the S. typhimurium gene silencer AT8 for the leuO gene silencing. The interchangeability between DNA geometrical elements for supporting the silencing activity in the region is consistent with a previous finding that a neighboring transcription activity determines the outcome of the gene silencing activity. The geometric requirement, which was revealed for this silencing activity, explains the decisive role of transcription-generated DNA supercoiling found in the promoter relay mechanism.DNA supercoiling has been known to play important roles in transcriptional regulation (1-7). By using a bacterial transcription regulation model system, we have demonstrated that transcription-generated DNA supercoiling is a crucial driven force that triggers the sequential activation of genes in the Salmonella typhimurium ilvIH-leuO-leuABCD gene cluster (8 -12). This rather complex sequential gene activation process was named the promoter relay mechanism (11, 12). The exact molecular detail that underlies the effect of transcription-generated DNA supercoiling on the sequential activation of genes at this locus remains unclear. The direct DNA supercoiling effect on activating promoters of genes in this region has been ruled out. Instead, the effect appears to mediate through cis-elements within the locus control regions (LCRs 1 illustrated in Fig. 1) located between genes in the ilvIH-leuO-leuABCD gene cluster (8).Although not ruling out the possible involvement of other cis-acting elements in the transcription regulation, we have identified two cis-elements in the LCR-I that are important for the promoter relay mechanism as follows: a bacterial gene silencer, termed AT8; and a LeuO protein-binding site, termed AT7 (13, 14). The bacterial gene silencer AT8-mediated transcriptional silencing is integral to the gene expression regulation and is responsible for the repressed state of the leuO gene. LeuO protein-mediated derepression, which relieves the repression of leuO gene, is also a crucial part of the promoter relay mechanism. Transcription-generated DNA supercoili...

We have previously demonstrated that sequential activation of the bacterial ilvIH-leuO-leuABCD gene cluster involves a promoter-relay mechanism. In the current study, we show that the final activation of the leuABCD operon is through a transcriptional derepression mechanism. The leuABCD operon is transcriptionally repressed by the presence of a 318-base pair AT-rich upstream element. LeuO is required for derepressing the repressed leuABCD operon. Deletion analysis of the repressive effect of the 318-bp element has led to the identification of a 72-bp AT-rich (78% A؉T) DNA sequence element, AT4, which is capable of silencing a number of unrelated promoters in addition to the leuABCD promoter. AT4-mediated gene silencing is orientation-independent and occurs within a distance of 300 base pairs. Furthermore, an increased gene-silencing effect was observed with a tandemly repeated AT4 dimer. The possible mechanism of AT4-mediated gene silencing in bacteria is discussed.The leu-500 mutation is an A to G transition in the Ϫ10 region of the promoter of the Salmonella typhimurium leuABCD operon (1). The transcriptional activity of the mutant promoter is DNA supercoiling-dependent (2). The mechanism whereby the leu-500 promoter (pleu-500) is activated in the topA mutants is intriguing (3-7). Previous studies using a plasmid system have demonstrated that activation of plasmidborne pleu-500 in topA mutants requires an upstream transcriptional activity transcribing away from pleu-500 (8 -11). This notion has been confirmed in a recent study using the chromosomal setting (12). Transcriptional activation of the ilvIH promoter (pilvIH) located 1.9 kilobases upstream of pleu-500 was shown to be responsible for pleu-500 activation (5). Transcription-driven DNA supercoiling (13) has been suggested to play a role in this long-range promoter-promoter interaction.The intervening promoter that relays the distant interaction between pilvIH and pleu-500 is the leuO promoter (pleuO). In addition to transcriptional activity from pleuO, the leuO gene product, LeuO, is also required to provide a trans-acting function for activation of pleu-500 (6). It appears that the functional pleuO (or other replaced promoter) and LeuO are coupled in activating pleu-500. The molecular basis for pleu-500 activation by the combined action of pleuO and LeuO is still a mystery.There is a stretch of 434 base pairs (bp) 1 that is AT-rich DNA flanked by the divergently arrayed leuO and leuABCD (14). Besides the promoter sequences of the flanking genes, the function of the remaining 318-bp AT-rich (69% AϩT) DNA is unknown (illustrated in Fig. 1). By monitoring pleu-500 activation, we found that the 318-bp AT-rich intervening DNA appears to repress the short-range interaction (11) between the two flanking promoters. Interestingly, LeuO relieves the repression. The repressive effect of the AT-rich intervening DNA on the short-range promoter-promoter interaction (pleuO and pleu-500) could potentially be due to anchoring of the AT-rich DNA to a large mass, which re...

Advanced-generation multiparent populations (MPPs) are a valuable tool for dissecting complex traits, having more power than genome-wide association studies to detect rare variants and higher resolution than F2 linkage mapping. To extend the advantages of MPPs in budding yeast, we describe the creation and characterization of two outbred MPPs derived from 18 genetically diverse founding strains. We carried out de novo assemblies of the genomes of the 18 founder strains, such that virtually all variation segregating between these strains is known, and represented those assemblies as Santa Cruz Genome Browser tracks. We discovered complex patterns of structural variation segregating among the founders, including a large deletion within the vacuolar ATPase VMA1, several different deletions within the osmosensor MSB2, a series of deletions and insertions at PRM7 and the adjacent BSC1, as well as copy number variation at the dehydrogenase ALD2. Resequenced haploid recombinant clones from the two MPPs have a median unrecombined block size of 66 kb, demonstrating that the population is highly recombined. We pool-sequenced the two MPPs to 3270× and 2226× coverage and demonstrated that we can accurately estimate local haplotype frequencies using pooled data. We further downsampled the pool-sequenced data to ∼20–40× and showed that local haplotype frequency estimates remained accurate, with median error rates 0.8 and 0.6% at 20× and 40×, respectively. Haplotypes frequencies are estimated much more accurately than SNP frequencies obtained directly from the same data. Deep sequencing of the two populations revealed that 10 or more founders are present at a detectable frequency for > 98% of the genome, validating the utility of this resource for the exploration of the role of standing variation in the architecture of complex traits.

No abstract

To understand the coordination of gene expression in the Salmonella typhimurium ilvIH-leuO-leuABCD gene cluster, we had previously identified a 72-bp AT-rich (78% A؉T) DNA sequence element, AT4, which was capable of silencing transcription in a promoter nonspecific manner. LeuO protein provided in trans relieved (derepressed) AT4-mediated gene silencing (transcriptional repression), but underlying mechanisms remained unclear. In the present communication, the 72-bp DNA sequence element is further dissected into two functional elements, AT7 and AT8. LeuO binds to the 25-bp AT7, which lies closest to the leuO promoter in the AT4 DNA. After deletion of the AT7 DNA sequence responsible for LeuO binding from AT4, the remaining 47-bp AT-rich (85% A؉T) DNA sequence, termed AT8, retains the full bi-directional gene-silencing activity, which is no longer relieved by LeuO. LeuO-mediated transcriptional derepression is restored when the LeuO binding site, AT7, is placed within close proximity to the gene silencer AT8. As a pair of functionally coupled transcription elements, the presence of an equal copy number of AT7 and AT8 within proximity is important for the transcription control. The characterization provides clues for future elucidation of the molecular details whereby LeuO negates the gene-silencing activity.The suppression of the leu-500 mutation in Salmonella typhimurium topA Ϫ strains (1) has been a paradigmatic phenomenon for explaining the importance of the effect of DNA supercoiling on transcriptional initiation (2). In our investigation of the mechanism that underlies this topA Ϫ genetic backgrounddependent phenomenon, we found an unprecedented long range (1.9 kbp) interaction between ilvIH and leu-500 promoters (3). Since then, we have found a promoter relay mechanism (4) responsible for the sequential activation of genes in the 1.9-kbp ilvIH-leuO-leuABCD region (illustrated below in Fig. 1). Recent studies demonstrated that this novel promoter relay mechanism is likely to be one of the bacterial stress responses in normal cell physiology rather than just a special phenomenon in S. typhimurium topA Ϫ mutants (5-7 and reviewed in Refs. 8 and 9). The promoter relay mechanism not only provides the explanation for the long range interaction between ilvIH and leu-500 promoters (3), it also reveals the interesting possibility that transcription-driven DNA supercoiling (10,11) may play roles in the transcriptional regulation processes that control the expression of the three functionally related genes in a sequential manner (illustrated in Fig. 1 below). A similar effect of transcription-driven DNA supercoiling on gene expression regulation has also been found in the ilvYC operon of Escherichia coli (12-14). Transcription-driven DNA supercoiling is indispensable in these processes, but the supercoiling itself alone is insufficient for triggering the gene expression coordination, because deleting part of the DNA sequence from the 1.9-kbp intervening region also abolished the promoter relay mechanism (3). These res...

The recently identified role of LeuO in the regulation of transcription has prompted us to search for the specific function(s) of LeuO in bacterial physiology. The cryptic nature of expression of leuO has previously limited such analysis. A conditional leuO expression was found when bacteria enter stationary phase and was shown to be guanosine 3,5-bispyrophosphate-dependent. Multiple physiological events, including the stringent response, are induced upon the increase of the bacterial stress signal, guanosine 3,5-bispyrophosphate. In this study, we tested whether LeuO was directly involved in the bacterial stringent response. LeuO was shown to be indispensable for growth resumption following a 2-h growth arrest caused by starvation for branched-chain amino acids in an E. coli K-12 relA1 strain. This result supports a functional role for LeuO in the bacterial stringent response.

AbstractAdvanced generation multi-parent populations (MPPs) are a valuable tool for dissecting complex traits, having more power than GWAS to detect rare variants, and higher resolution than F2 linkage mapping. To extend the advantages of MPPs in budding yeast, we describe the creation and characterization of two outbred MPPs derived from eighteen genetically diverse founding strains. We carried out de novo assemblies of the genomes of the eighteen founder strains, such that virtually all variation segregating between these strains is known and represent those assemblies as Santa Cruz Genome Browser tracks. We discover complex patterns of structural variation segregating amongst the founders, including a large deletion within the vacuolar ATPase VMA1, several different deletions within the osmosensor MSB2, a series of deletions and insertions at PRM7 and the adjacent BSC1, as well as copy number variation at the dehydrogenase ALD2. Resequenced haploid recombinant clones from the two MPPs have a median unrecombined block size of 66kb, demonstrating the population are highly recombined. We pool sequenced the two MPPs to 3270X and 2226X coverage and demonstrate that we can accurately estimate local haplotype frequencies using pooled data. We further down-sampled the poolseq data to ~20-40X and show that local haplotype frequency estimates remain accurate, with median error rate 0.8% and 0.6% at 20X and 40X, respectively. Haplotypes frequencies are estimated much more accurately than SNP frequencies obtained directly from the same data. Deep sequencing of the two populations revealed that ten or more founders are present at a detectable frequency for over 98% of the genome, validating the utility of this resource for the exploration of the role of standing variation in the architecture of complex traits.