What do operons enable bacteria to do

However, when tryptophan accumulates in the cell, two tryptophan molecules bind to the trp repressor molecule, which changes its shape, allowing it to bind to the trp operator. This binding of the active form of the trp repressor to the operator blocks RNA polymerase from transcribing the structural genes, stopping expression of the operon.

Thus, the actual product of the biosynthetic pathway controlled by the operon regulates the expression of the operon. Figure 2. The five structural genes needed to synthesize tryptophan in E. When tryptophan is absent, the repressor protein does not bind to the operator, and the genes are transcribed. When tryptophan is plentiful, tryptophan binds the repressor protein at the operator sequence.

This physically blocks the RNA polymerase from transcribing the tryptophan biosynthesis genes. The lac operon is an example of an inducible operon that is also subject to activation in the absence of glucose Figure 3. The lac operon encodes three structural genes necessary to acquire and process the disaccharide lactose from the environment, breaking it down into the simple sugars glucose and galactose. For the lac operon to be expressed, lactose must be present.

This makes sense for the cell because it would be energetically wasteful to create the enzymes to process lactose if lactose was not available. In the absence of lactose, the lac repressor is bound to the operator region of the lac operon, physically preventing RNA polymerase from transcribing the structural genes. However, when lactose is present, the lactose inside the cell is converted to allolactose.

Allolactose serves as an inducer molecule, binding to the repressor and changing its shape so that it is no longer able to bind to the operator DNA. Removal of the repressor in the presence of lactose allows RNA polymerase to move through the operator region and begin transcription of the lac structural genes. Figure 3. The three structural genes that are needed to degrade lactose in E. When lactose is absent, the repressor protein binds to the operator, physically blocking the RNA polymerase from transcribing the lac structural genes.

When lactose is available, a lactose molecule binds the repressor protein, preventing the repressor from binding to the operator sequence, and the genes are transcribed. Figure 4. When grown in the presence of two substrates, E. Then, enzymes needed for the metabolism of the second substrate are expressed and growth resumes, although at a slower rate.

Bacteria typically have the ability to use a variety of substrates as carbon sources. However, because glucose is usually preferable to other substrates, bacteria have mechanisms to ensure that alternative substrates are only used when glucose has been depleted.

Additionally, bacteria have mechanisms to ensure that the genes encoding enzymes for using alternative substrates are expressed only when the alternative substrate is available. In the s, Jacques Monod was the first to demonstrate the preference for certain substrates over others through his studies of E. Such studies generated diauxic growth curves, like the one shown in Figure 4. Although the preferred substrate glucose is used first, E. However, once glucose levels are depleted, growth rates slow, inducing the expression of the enzymes needed for the metabolism of the second substrate, lactose.

Notice how the growth rate in lactose is slower, as indicated by the lower steepness of the growth curve.

As a result, cAMP levels begin to rise in the cell Figure 5. Figure 5. Thus, increased cAMP levels signal glucose depletion. The lac operon also plays a role in this switch from using glucose to using lactose. The complex binds to the promoter region of the lac operon Figure 6. In the regulatory regions of these operons, a CAP binding site is located upstream of the RNA polymerase binding site in the promoter.

Binding of the CAP-cAMP complex to this site increases the binding ability of RNA polymerase to the promoter region to initiate the transcription of the structural genes. Thus, in the case of the lac operon, for transcription to occur, lactose must be present removing the lac repressor protein and glucose levels must be depleted allowing binding of an activating protein.

When glucose levels are high, there is catabolite repression of operons encoding enzymes for the metabolism of alternative substrates. See Table 1 for a summary of the regulation of the lac operon. Figure 6. In prokaryotes, there are also several higher levels of gene regulation that have the ability to control the transcription of many related operons simultaneously in response to an environmental signal. A group of operons all controlled simultaneously is called a regulon.

When sensing impending stress, prokaryotes alter the expression of a wide variety of operons to respond in coordination. They do this through the production of alarmones , which are small intracellular nucleotide derivatives.

Alarmones change which genes are expressed and stimulate the expression of specific stress-response genes. The use of alarmones to alter gene expression in response to stress appears to be important in pathogenic bacteria. On encountering host defense mechanisms and other harsh conditions during infection, many operons encoding virulence genes are upregulated in response to alarmone signaling.

Knowledge of these responses is key to being able to fully understand the infection process of many pathogens and to the development of therapies to counter this process. Although most gene expression is regulated at the level of transcription initiation in prokaryotes, there are also mechanisms to control both the completion of transcription as well as translation concurrently.

Since their discovery, these mechanisms have been shown to control the completion of transcription and translation of many prokaryotic operons. Because these mechanisms link the regulation of transcription and translation directly, they are specific to prokaryotes, because these processes are physically separated in eukaryotes. Beyond the transcriptional repression mechanism already discussed, attenuation also controls expression of the trp operon in E.

The trp operon regulatory region contains a leader sequence called trpL between the operator and the first structural gene, which has four stretches of RNA that can base pair with each other in different combinations. However, when an antiterminator stem-loop forms, this prevents the formation of the terminator stem-loop, so RNA polymerase can transcribe the structural genes.

Figure 7. Click to view a larger image. When tryptophan is plentiful, translation of the short leader peptide encoded by trpL proceeds, the terminator loop between regions 3 and 4 forms, and transcription terminates. When tryptophan levels are depleted, translation of the short leader peptide stalls at region 1, allowing regions 2 and 3 to form an antiterminator loop, and RNA polymerase can transcribe the structural genes of the trp operon.

A riboswitch may bind to a small intracellular molecule to stabilize certain secondary structures of the mRNA molecule. The binding of the small molecule determines which stem-loop structure forms, thus influencing the completion of mRNA synthesis and protein synthesis.

While regulation via differential operon decay was reported in multiple bacterial operons over the last few decades [ 9 — 17 ], the extent of this regulatory mechanism or its evolutionary conservation in bacteria remains unknown. In this study, we combine a set of multi-layered high-resolution RNA-seq approaches to extensively map and characterize differentially decaying operons in E. Furthermore, we find that differential decay is often dependent on translation and that stabilized operon segments are characterized by relatively higher ribosome densities than non-stabilized segments, suggesting that variation in translation efficiency guides endonuclease cleavage to ribosome depleted sections.

Taken together, our data support differential operon decay as a common and frequently conserved mode of regulation in bacteria. To examine the possible extent of differential RNA degradation in shaping operon stoichiometries, we first sought to limit our analysis to a set of well-defined E. We discarded from the set operons that were expressed at low levels or that are likely regulated by iTSSs or by internal partial transcription termination under the conditions tested in this study Methods.

For this, we comprehensively mapped the TSSs in exponentially growing E. We further excluded cases where two consecutive genes within the operon were separated by an intrinsic terminator motif a stem-loop structure followed by a uridine tract or in which substantial Rho-dependent termination was previously described [ 20 ] Methods. We then treated exponentially growing E. A-C , Differential decay in three representative E. RNA-seq coverage was normalized by the number of uniquely mapped reads in each library.

Bar graphs show average half-life calculations from three replicates with error bars representing standard deviation. Gene names are marked below the x-axis. Shown is the average ratio between the genes with error bars denoting standard deviation. It is well established mathematically and experimentally that the ratio in steady-state mRNA levels of two equally transcribed genes should equal to the ratio in their half-lives for example, if the half-life of gene A is 2 minutes and the half-life of gene B is 1 minute, then at steady-state gene A will be 2-fold overexpressed as compared to gene B [ 24 ].

Indeed, for the majority of operonic gene-pairs in which we found differential decay rates, the estimated differences in half-life closely matched the observed differences in steady-state mRNA abundance Fig 1D ; S3 Table. In about one quarter of the cases we found that the differences in decay rates did not fully match the differences observed in the mRNA level, indicating either measurement error or additional regulation by iTSSs or leaky termination in these operons S3 Table.

We measured the half-lives of the malFG and the malE segments to be 1 and 3. In all these operons, we found that the stabilized gene was invariably the substrate-binding subunit, which is typically needed in higher quantities than the channel-forming units. For example, while the stabilized maltose substrate-binding subunit gene, malE , is the first gene in the operon Fig 1A , the substrate-binding subunits of the polyamine potD and arginine artI transporters occur at the end or the middle of their operons, respectively Fig 1B and 1C.

Stabilization of the mRNA of the substrate-binding subunit results in higher steady-state mRNA levels of the respective gene, suggesting that differential decay is a common mechanism for tuning differential stoichiometries in ABC transport systems. Another transport system subject to decay-based regulation was the tatABC operon, which encodes the evolutionarily conserved twin-arginine protein export system [ 25 ] Fig 1D.

We also observed differential decay in the rpoZ-spoT containing operon, such that the segment encoding rpoZ is stabilized compared with that containing spoT. The rpoZ gene encodes the RNAP omega subunit, which binds the regulatory alarmone ppGpp [ 27 ], and SpoT is one of the major enzymes involved in the synthesis and degradation of this alarmone [ 28 ] S3 Table.

As the enzymatic activity of a single SpoT rapidly converts multiple ppGpp molecules and hence can affect multiple rpoZ gene products, it is likely that RpoZ protein expression levels need to be higher than that of SpoT.

Conceptually similar, we also identified differential decay in operons encoding 4 different two-component systems, where in each operon the transcription factor component was consistently more stable than the histidine-kinase enzyme [ 29 ].

Finally, we identified additional differentially decaying operons involved in cellular signaling and regulation, protein translocation across the membrane, antibiotic resistance and various metabolic processes, suggesting that differential decay plays an important role in post transcriptional regulation of many physiological processes in E.

To examine whether regulation by differential decay is evolutionarily conserved we performed an identical rifampicin-based decay assay using the human pathogenic bacterium Enterobacter aerogenes grown under the exact same conditions as E. In addition, we compared the steady-state mRNA levels of E. The observed conservation of differential decay patterns between different bacteria highlights the functional importance of this phenomenon in bacterial gene regulation. Our analyses discover many new cases where the steady-state levels of individual genes within polycistronic mRNAs are controlled via selective stabilization differential operonic decay Fig 1 ; S3 Table.

However, why some transcript regions are protected from digestion whereas others are rapidly degraded was unclear. This structure exerts its protective effect on malE following an initial endonucleolytic cleavage event that generates a functional malE fragment still physically attached to the protective element.

B-D , Differential decay in three representative E. Bar graphs show average half-life calculations with error bars representing standard deviation. The sequence at the ORF of the downstream gene therefore carries, in addition to the protein-coding information, also the information guiding the differential decay of the transcript and thus, its stoichiometry at steady state.

Interestingly, multiple protective structures can be observed in a single operon, generating complex decay patterns: for example, in the menBCE operon, we detected consecutive protective hairpins downstream of both the menB and menC genes, in correlation with the gradual decrease in stability and mRNA abundance detected in this operon Fig 2D.

However, a similar downstream stabilization pattern was previously reported in the papBA operon of uropathogenic E. The position and number of reads supporting RNase E cleavage sites in the WT strain or in the RNase E mutant are shown as dark and light orange arrows, respectively. The predicted structure and stability of the RNA sequence present immediately downstream of the RNase E cleavage site end is shown next to each gene, with blue rectangles specifying the position of the structure in the genome.

Moreover, in 7 of these 10 operons, the cleavage site occurred closely upstream of a stable RNA structure, supporting a potential papBA -like protection mechanism in these cases Fig 3B—3D. While the RNA structures described above can insulate specific transcript regions during active degradation, the endonucleolytic cleavage events that initiate differential decay must first be directed to particular operon segments. However, the guiding factors that direct initializing cleavage events are poorly understood, even in the well characterized example of the malEFG operon [ 8 , 30 , 32 ].

Ribosome densities were previously found to positively correlate with mRNA stability, presumably by physically restricting access to RNase cleavage sites [ 39 — 42 ]. Re-analyzing recently published ribosome profiling data [ 3 ], we found that in almost all differentially decaying gene-pairs the stabilized gene was covered by substantially more ribosomes than the non-stabilized genes in the operon after normalization to transcript levels , with a median of 4.

Outliers are marked as red dots and the median is marked as a horizontal line within the box. B , Illustration of the effect of the translation-initiation inhibitor kasugamycin and its hypothesized effect on ribosome densities in polycistronic transcripts. C , A scatter plot showing the change in relative decay rate of regulated gene-pairs calculated using recently published decay rates [ 22 ] for control x-axis and kasugamycin treated y-axis bacteria S8 Table.

To directly examine whether differential ribosome density is involved in guiding differential decay, we analyzed recently published data in which E. In this experiment, kasugamycin treatment is expected to result in polycistronic transcripts where all genes are equally devoid of ribosomes, providing an approach to study the contribution of differential translation to operon decay Fig 4B.

Notably, the short inhibition of translation initiation resulted in a substantial reduction in differential decay in the vast majority of the regulated operons in our set Fig 4C ; S8 Table ; Methods , providing evidence that differential decay within operons is often dependent on differences in translation efficiency.

These results suggest that differences in ribosome densities guide the endonuclease cleavage events that initiate the differential decay process within polycistronic transcripts. Combined with the results from the above sections, we chart a general model for differential decay of polycistronic transcripts in E. A , A model for stabilization of the upstream gene. B A model for stabilization of the downstream gene. The mature operon is protected by the terminator structure. The relatively higher ribosome densities over gene B guide the initial cleavage to gene A.

Differential decay of polycistronic operons enables bacteria to reshape uniform transcription into differential expression. In the current study, we took a transcriptome-wide approach to extensively map differentially decaying operons in E.

We note that this number may actually be higher as recent studies reported the existence of condition specific differential decay [ 10 , 13 ].

In addition, we find that the less stable genes in the operon are covered by fewer ribosomes per transcript and that translation plays a major role in shaping the differential decay process. While the relation between ribosomes and mRNA decay has been well-established for monocistronic mRNAs [ 39 — 41 , 44 ], our results extend this concept to multi-gene operons with differentially decaying transcript segments.

Importantly, these observations provide a potential explanation for how specific operon segments are selected for initial endonucleolytic cleavage by RNases with degenerate target motifs, a piece of the mechanism that was so far less understood Fig 5. Interestingly, stable structures at protein-coding mRNA regions were previously suggested to reduce translation efficiency [ 44 — 46 ], implying that protective structures could actually play a dual role: first, blocking translation initiation, which reduces ribosome density on the flanking gene and exposes the region to increased RNase E dependent cleavage, and second, direct protection of the stable transcript region from the decay process.

Although our differential decay models provide a potential mechanism for most of the regulated operons in our dataset, additional factors, such as trans-acting ncRNAs, have been found to play a role in shaping operon decay patterns by modulating access to rate-limiting endonuclease cleavage sites [ 10 , 13 ]. Notably, such ncRNAs enable condition-specific stoichiometric regulation in operons that are otherwise degraded uniformly.

Indeed, our analysis failed to detect differential decay in both the iscRSUA and the pldB-yigL operons, which were recently shown to be regulated by ncRNAs activated under conditions other than the ones employed in our study [ 10 , 13 ]. Thus, considering the large number of trans-acting ncRNAs and antisense RNAs in bacteria, the extent of differential decay-based regulation is likely even greater than our current estimates.

Presumably, differential decay in such organisms may rely on different principles or additional molecular signals. Escherichia coli BW, Salmonella enterica subsp. The cells were then lysed by immediately adding 1ml tri-reagent Trizol followed by vigorous vortexing for 10s until solution is cleared. The sample was incubated for min at RT until visible phase separation was observed and then centrifuged at 12,g for 10min.

Sequencing reads generated for E. TSSs were mapped as was recently described [ 19 ]. Selected time points were sampled by collecting 1. The sample was quickly vortexed and then placed on ice until all time points were collected.

The samples were centrifuged for 5min at rpm to collect cell pellets and were flash frozen. RNA-seq libraries were prepared and sequenced as described above. Transcript half-lives were calculated by fitting the decay time-course abundance measurements per gene with a delayed exponential-decay function as previously described [ 22 ]. Operon gene annotations were extracted from EcoCyc [ 18 ] S2 Table. To identify and analyze gene-pairs found within the same transcriptional unit, consecutive gene-pairs were only considered if the following criteria were met: i the intergenic region interspacing the genes was shorter than nt.

Gene-pairs in which one gene was at least 2-fold more stable and 2-fold more abundant than its consecutive neighbor gene were classified as putatively decay-regulated S3 Table. We manually accepted 4 gene-pairs displaying borderline, yet consistent signal as well as 7 differentially expressed gene-pairs in which the decay rate was not measured in our experiment usually due to lack of expression in the conditions tested, but for which differential decay could clearly be identified in a recently published dataset [ 22 ] S3 Table.

Term-seq libraries were prepared as previously described [ 33 ] and sequenced using a paired-end sequencing approach [ 48 ] S1 Table. Sites were then associated with their respective genes, requiring that the average insert length would overlap the gene coding region [ 48 ].

Heterochromatin mutants with enhanced sterigmatocystin biosynthesis show decreased trimethylation of lysine residue K9 of histone H3, whilst laeA mutants show increased trimethylation of this lysine residue and a concomitant decrease in sterigmatocystin production [ 33 ]. Deletion of the histone deacetylase HdaA gene results in early and increased expression of biosynthetic genes not only for sterigmatocystin but also for another secondary metabolic gene cluster for penicillin synthesis [ 59 ].

Treatment of Aspergillus species and other fungi with selective drugs that inhibit histone deacetylase or DNA methyltransferase gives altered secondary metabolite profiles compared to control treatments [ 59 — 61 ]. It thus appears that epigenetic processes have important functions in the regulation of secondary metabolic gene clusters. Chemical genetic techniques are now being exploited along with genetic and genomics-based approaches to identify new cryptic pathways and metabolites in diverse filamentous fungi [ 55 , 58 , 60 , 61 ].

Overexpression of transcription factors implicated in regulation of secondary metabolic pathways offers a further route to identification of new pathways and their end-products [ 55 , 62 ]. Synthesis of secondary metabolites by filamentous fungi is also under environmental and developmental control. The signaling cascades associated with these tiers of regulation are integrated into the hierarchical regulation of expression of the gene clusters [ 32 , 33 , 63 ] Fig.

LaeA has also recently been shown to control penicillin biosynthesis, pigmentation, and sporulation in Penicillium chrysogenum [ 64 ]. Different levels of regulation of the aflatoxin gene cluster in the fungus, Aspergillus nidulans.

The genes for aflatoxin biosynthesis are clustered in a kb region and encode at least 23 co-regulated transcripts not all cluster genes are shown in this diagram.

The positive regulatory gene aflR lies within the gene cluster and is required for the transcriptional activation of most, if not all, pathway genes. LaeA is a master regulator of secondary metabolism in the Aspergilli and is required for AlfR-mediated expression of the aflatoxin biosynthetic cluster.

LaeA also controls transcription of gene clusters for other secondary metabolites and is believed to act via chromatin remodeling. Histone deacetylase HdaA performs an opposing role to LaeA and functions to repress gene expression in this region. Synthesis of secondary metabolites in the Aspergilli is linked to environmental stimuli and development. In the light asexual development sporulation is induced and genes involved in aflatoxin are not expressed.

Darkness induces sexual development with associated induction of metabolite production, mediated by the proteins VelB and VeA. Adapted from [ 33 ]. There is evidence to suggest HGT of the penicillin gene cluster from bacteria to fungi [ 65 — 67 ].

HGT of clusters between fungi may in part explain the discontinuous distribution of gene clusters for synthesis of secondary metabolites within the Ascomycetes [ 68 , 69 ].

The selfish cluster hypothesis has been put forward to explain clustering of functionally related genes in fungi [ 66 ]. However, fungal genomes are very plastic, and it is likely that the formation and maintenance of metabolic gene clusters in fungal genomes is driven by selection for optimized production of metabolites that fulfil an adaptive function. Many fungal metabolic gene clusters are located close to telomeres, a chromosomal location that would be expected to facilitate recombination, DNA inversions, partial deletions, translocations, and other genomic rearrangements [ 70 — 73 ].

Intragenic reorganization followed by vertical descent is therefore a more satisfactory explanation. Clustering may facilitate co-regulation of gene expression, although it is clearly not a prerequisite for this since expression of unlinked genes for other metabolic pathways can be readily co-regulated. As the number of complete genome sequences of filamentous fungi increases, it should become possible to elucidate and perhaps model the mechanisms that drive cluster formation and maintenance, following approaches similar to those used to study the life and death of bacterial operons.

While this section has focused on gene clusters for the synthesis of secondary metabolites in filamentous fungi, it is noteworthy that clusters of diverse virulence genes with no obvious function in metabolism have recently been identified in the corn smut fungus Ustilago maydis following completion of the full genome sequence of this organism [ 74 ]. Clusters of genes of related function are relatively unusual in S.

However, the S. These include gene clusters for utilization of specific carbon sources [e. Studies of the distribution, origin, and fate of these gene clusters have provided important insights into the mechanisms underpinning adaptation of yeasts to new ecological niches. The DAL gene cluster of S. These genes enable yeast to use allantoin a purine degradation product as a nitrogen source Fig. The DAL gene cluster is completely conserved in the four closest relatives of S.

In less closely related species, the six genes are also clustered, but there are differences in the internal arrangement of gene order and the cluster is located in a different part of the genome although probably still subtelomeric. The DAL cluster is not present in the genomes of more distantly related hemiascomycetes.

However, homologues of the six DAL genes are found scattered around the genomes of these species. The species that possess a DAL cluster form a monophyletic group. Comparative analysis of the DAL genes and clusters in the genomes of different yeast species in combination with phylogenetic information has provided compelling evidence to suggest that the DAL cluster was assembled quite recently in evolutionary terms, after the split of the sensu stricto group of yeasts from other yeasts and hemiascomycetes [ 76 ].

Six of the eight genes involved in allantoin degradation, which were previously scattered around the genome, became relocated to a single subtelomeric site in an ancestor of S. This could have occurred by gene duplication followed by loss of the gene at the original locus. These genomic rearrangements coincided with a biochemical reorganization of the purine degradation pathway, which switched to importing allantoin instead of urate.

This change circumvented the need for urate oxidase, one of several oxygen-consuming enzymes lost by yeasts that can grow vigorously in anaerobic conditions Fig. It has therefore been proposed that selection for reduced dependence on oxygen led to a switch from urate to allantoin utilization in an ancestor of the sensu stricto group of yeasts [ 76 ]. Natural sources of allantoin for yeasts are plants [ 79 ] and insect excretion [ 80 ].

The allantoin degradation pathway—an adaptation to growth under oxygen-limiting conditions in Saccharomyces cerevisiae. In the classical purine degradation pathway, xanthine is converted to urate and then to allantoin, in two successive oxidation steps catalyzed by the peroxisomal enzymes xanthine dehydrogenase XDH and urate oxidase UOX.

XDH genes are present in filamentous fungi but not in yeasts. To use purine derivatives as a nitrogen source yeasts must therefore import urate, allantoin or allantoate from outside the cell. Yeasts that lack the DAL cluster e. In contrast, S. The Dal4 and Fur4 proteins are members of a purine-related transporter family. The subsequent degradation steps involve the same DAL pathway genes in all yeasts, but in S. The reaction catalyzed by UOX requires molecular oxygen as a substrate and takes place in the peroxisome.

Biochemical reorganization of the purine degradation pathway to enable import of allantoin instead of urate eliminates the oxygen-requiring step mediated by UOX and coincided with the formation of the DAL gene cluster. This biochemical reorganization may have been driven by selection for ability to grow under conditions of oxygen limitation. Reproduced from [ 76 ]. The birth of the DAL gene cluster. The DAL gene cluster on S.

The corresponding region in K. The K. The region of the K. Adapted from [ 76 ]. The selection for formation of new metabolic gene clusters such as the DAL gene cluster is likely to be intense, driven by the need to adapt to growth under different environmental conditions.

Gene clusters that have been formed by epistatic selection are expected to be recombination cold spots and so to be in linkage disequilibrium [ 81 ], and this is indeed the case for the DAL gene cluster [ 76 ]. Epistatic selection for linkage may in addition be driven by the need to select for combinations of alleles that interact well in order to avoid the accumulation of toxic pathway intermediates within cells. For example, glyoxylate, which is an intermediate in the DAL pathway Fig.

Glyoxylate is produced by the Dal3 reaction and removed by the Dal7 reaction. The finding that Dal3 enzyme activity is reduced in a dal7 mutant is consistent with this channeling hypothesis [ 76 , 82 ]. Z Htz1 rather than with the normal histone H2A [ 83 ]. Htz1 preferentially associates with narrow regions within the promoters of genes that are normally maintained in repressed form but are strongly induced under specific growth conditions or during particular gene expression programmes.

A model has been proposed in which Hzt1 associates to specific nucleosomes in the promoters of inactive genes in order to poise, and perhaps organize, chromatin structure in a manner that is permissive to transcription initiation [ 84 ]. The genes in the DAL cluster are negatively regulated by the Hst-Sum1 system, which represses expression of mid-sporulation genes during mitotic growth Fig. Assuming that there is a selective advantage to repressing DAL gene expression when nitrogen is not limiting, there would have been an incremental selective advantage to relocating each gene into the chromatin modification HZAD domain.

Thus, one way in which alleles could interact well is by being amenable to the same type of chromatin modification [ 76 ]. Activation of expression of the DAL genes. The exchange of histone H2A for H2A. Z is mediated by the Swr1 chromatin remodeling complex [ ]. Under new selection regimes, adaptations may evolve while established functions may become less important. The GAL genes, which are required for galactose utilization, are clustered in the genomes of every yeast species in which they are present [ 75 ].

This pathway converts galactose into glucosephosphate, a substrate for glycolysis. Galactose utilization is widespread amongst yeasts and is likely to be ancestral.

However, several yeast species have lost the ability to use this carbon source. Comparisons of the genomes of galactose-utilizing and non-utilizing yeast species have revealed that three out of the four non-utilizing species examined lack any trace of the pathway except for a single gene.

However, S. Thus, whilst a newly formed functional gene cluster confers a selective advantage in a new ecological niche, rapid and irreversible gene inactivation and pathway degeneration can occur under non-selective conditions. It has been suggested for S. The loss of genes and pathways through reductive evolution has been inferred for many organisms that have adapted to pathogenic or endosymbiotic lifestyles [ 85 — 92 ].

These capabilities may be lost either because they are no longer under selection neutral or because of a deleterious effect on fitness in a new niche [ 75 , 93 — 95 ]. Genes for metabolic pathways in plants are generally not clustered, at least for the majority of the pathways that have been characterized in detail to date. However, several examples of functional gene clusters for plant metabolic pathways have recently emerged. These are the cyclic hydroxamic acid DIBOA pathway in maize [ 96 — 98 ], triterpene biosynthetic gene clusters in oat [ 99 , ] and Arabidopsis [ ] the avenacin and thalianol gene clusters, respectively , and the diterpenoid momilactone cluster in rice [ , ].

These gene clusters all appear to have been assembled from plant genes by gene duplication, acquisition of new function, and genome reorganization and are not likely to be a consequence of horizontal gene transfer from microbes. The existence of these clusters, of which at least three are implicated in plant defense [ 98 , 99 , — ], implies that plant genomes are able to assemble functional gene clusters that confer an adaptive advantage.

The selection for rapid and recent formation of such metabolic gene clusters is likely to be intense, driven by the need to adapt to growth under different environmental conditions, and implies remarkable genome plasticity. The benzoxazinoids are defense-related compounds that occur constitutively as glucosides in certain members of the Gramineae and in some dicots. In the Poaceae, the production of benzoxazinoids is developmentally regulated with highest levels being found in the roots and shoots of young seedlings.

Induction of benzoxazinoid accumulation has also been reported in response to cis -jasmone treatment [ ]. The complete molecular pathway for benzoxazinoid biosynthesis has been elucidated in maize reviewed in [ 98 ]. Bx1 is likely to have been recruited from primary metabolism either directly or indirectly by duplication of the maize gene encoding TSA.

The glucosyltransferases BX8 and 9 catalyse glucosylation of benzoxazinoids. All the Bx genes with the exception of Bx9 are linked within 6 cM of Bx1 on maize chromosome 4 [ 97 , ]. The distribution of benzoxazinoids across the Gramineae is sporadic. Maize, wheat, rye, and certain wild barley species are capable of the synthesis of these compounds while oats, rice, and cultivated barley varieties are not [ , ]. The Bx gene cluster is believed to be of ancient origin.

Wheat and rye have undergone a shared genomic event that has led to the splitting of the Bx gene cluster into two parts that are located on different chromosomes. This can be explained by a reciprocal translocation in the ancestor of wheat and rye [ ]. Bx-deficient variants of a diploid accession of wild wheat Triticum boeoticum have recently been identified.

Molecular characterization suggests that Bx deficiency in these accessions arose by disintegration of the Bx1 coding sequence, followed by degeneration and loss of all five Bx biosynthetic genes examined [ ].

Barley species that do not produce benzoxazinoids have also lost all Bx genes [ , ]. The precise physical distances between all of the genes within the Bx cluster are not known. However, in maize, Bx1 and Bx2 genes are 2. In hexaploid wheat, the Bx3 and Bx4 genes are 7—11 kb apart within the three genomes [ ]. Although several of the Bx genes are in close physical proximity this gene cluster appears to be less tightly linked than the other examples that have been considered so far in this review.

Interestingly, barley lines that produce benzoxazinoids do not synthesize gramine, a defense compound that is also derived from the tryptophan pathway. Conversely, gramine-accumulating barley species are deficient in benzoxazinoids. This has led to the suggestion that the biosynthetic pathways for these two different classes of defense compound are mutually exclusive, possibly due to competition for common substrates [ ].

Outside the Poaceae, benzoxazinoids in particular DIBOA and its glucoside are found in certain isolated eudicot species belonging to the orders Ranunculales e. Comparison of the BX1 enzymes of grasses and benzoxazinone-producing eudicots indicates that these enzymes do not share a common monophyletic origin.

Furthermore, the CYP71C family of CYPs to which BX belong is not represented in the model eudicot, thale cress Arabidopsis thaliana , and all members of this family described to date originate from the Poaceae. It therefore seems likely that the ability to synthesize benzoxazinones has evolved independently in grasses and eudicots. Investigation of triterpene biosynthesis in plants has led to the discovery of two other examples of operon-like metabolic gene clusters, namely the avenacin gene cluster in oat Avena species and the thalianol gene cluster in A.

Avenacins are antimicrobial triterpene glycosides that confer broad spectrum disease resistance to soil-borne pathogens [ , ]. Analysis of the genes and enzymes for avenacin synthesis has revealed that the pathway has evolved recently, since the divergence of oats from other cereals and grasses [ 99 , , , , ].

Transferal of genes for the synthesis of antimicrobial triterpenes into cereals such as wheat holds potential for crop improvement but first requires the necessary genes and enzymes to be characterized.

Synthesis of avenacins is developmentally regulated and occurs in the epidermal cells of the root meristem. The major avenacin, A-1, has strong fluorescence under ultra-violet light and can be readily visualized in these cells. This fluorescence, which is an extremely unusual property amongst triterpenes, has enabled isolation of over 90 avenacin-deficient mutants using a simple screen for reduced root fluorescence [ , ].

This mutant collection has facilitated gene cloning and pathway elucidation. Sad1 encodes an oxidosqualene cyclase enzyme that catalyses the first committed step in the avenacin pathway [ 99 , ], while Sad2 encodes a second early pathway enzyme—a novel cytochrome P enzyme belonging to the newly described monocot-specific CYP51H subfamily [ ].

Sterols and avenacins are both synthesized from the mevalonate pathway [ ]. While the genes for sterol synthesis are generally regarded as being constitutively expressed throughout the plant, the expression of Sad1 , Sad2 , and other cloned genes for avenacin biosynthesis is tightly regulated and is restricted to the epidermal cells of the root meristem [ 99 , , ].

Recruitment of Sad1 and Sad2 from the sterol pathway by gene duplication has therefore involved a change in expression pattern as well as neofunctionalisation. A third gene has recently been cloned and shown to encode a serine carboxypeptidase-like acyltransferase that is required for avenacin acylation.

Four other loci that are required for avenacin synthesis also co-segregate with these cloned genes, indicating that most of the genes for the pathway are likely to be clustered [ 99 ]. Since avenacins confer broad spectrum disease resistance, the gene cluster is likely to have arisen through strong epistatic selection for maintenance and co-inheritance of this gene collective.

In addition, interference with the integrity of the gene cluster can in some cases lead to the accumulation of toxic intermediates, with detrimental consequences for plant growth, so providing further selection for cluster maintenance [ ]. Gene clustering may also facilitate co-ordinate regulation of gene expression at the level of chromatin [ 2 ].

The thalianol gene cluster in A. The BAHD acyltransferase gene is predicted to be part of the cluster based on its location and expression pattern, but an acylated downstream product has not as yet been identified. In the related crucifer, A. This may be indicative of paralogy rather than orthology [ ]. Alternatively it may indicate that the BAHD acyltransferase genes are not under strong selection and so are divergent.

The thalianol gene cluster in Arabidopsis. The A. The organization of the equivalent region from the related crucifer, A. Adapted from Ref. The genes within the A. As is the case for the avenacin pathway, tight regulation of the pathway appears to be critical since accumulation of thalianol pathway intermediates can impact on plant growth and development.

There are superficial similarities between the avenacin and thalianol gene clusters in that they are both required for triterpene synthesis and contain genes for oxidosqualene cyclases, CYPs, and acyltransferases. However, phylogenetic analysis indicates that the genes within these clusters are monocot and eudicot specific, respectively, and that the assembly of these clusters has occurred recently and independently in the two plant lineages [ ]. This suggests that selection pressure may act during the formation of certain plant metabolic pathways to drive gene clustering, and that triterpene pathways are predisposed to such clustering.

A third example of a gene cluster for synthesis of terpenes in plants has been reported from rice, in this case for synthesis of diterpene defense compounds known as momilactones [ , ].

Momilactones were originally identified as dormancy factors from rice seed husks and are also constitutively secreted from the roots of rice seedlings. In rice cell suspension cultures and in leaves, expression of the rice momilactone genes can be co-ordinately induced in response to challenge with pathogens, elicitor treatment, or exposure to UV irradiation [ , ]. Synthesis of momilactones is initiated by terpene synthases that are distinct from the oxidosqualene cyclases that catalyze the first committed step in triterpene synthesis.

These genes are all co-ordinately induced in response to treatment with a chitin oligosaccharide elicitor. Analysis of the promoters of the genes within this cluster has revealed the presence of potential recognition sites for WRKY and basic leucine zipper bZIP transcription factors, proteins that are associated with activation of defense responses.

Gene clustering has been suggested to facilitate efficient coordinated expression of the momilactone gene cluster in response to elicitation [ ]. Global gene expression analysis has revealed extensive clustering of non-homologous genes that are co-ordinately expressed in eukaryotes, including in animals for reviews, see [ 2 , , ]. These groups of genes may be expressed during development, or in certain tissues and diseased states, and have been reported in studies of Drosophila , nematode, mouse, and humans.

Such co-expression domains may therefore be an important source for the discovery of new functional gene clusters in animals and other eukaryotes. However, more research is needed before we can fully understand the functional significance of co-expression domains [ ].

Of the known functional gene clusters in animals, the best characterized is the major histocompatability complex MHC , which encodes proteins involved in innate and adaptive immunity.