枯草杆菌耐药转运蛋白Bmr及其基因bmr的表达调控研究进展

枯草杆菌多重耐药转运蛋白Bmr是其主要的耐药外排蛋白之一,由位于基因组DNA的bmr基因编码,介导对多种抗生素、杀菌剂等药物的耐药性。bmr基因的表达受到BmrR及MtaN的转录调控,二者均属于MerR家族调节子。关于近年对多重耐药转运蛋白Bmr和调节蛋白BmrR、MtaN的结构、生理功能及作用机制等研究情况进行综述。     

Characterization of novel Brown midrib 6 mutations affecting lignin biosynthesis in sorghum

The presence of lignin reduces the quality of lignocellulosic biomass for forage materials and feedstock for biofuels. In C4 grasses,the brown midrib phenotype has been linked to mutations to genes in the monolignol biosynthesis pathway. For example,the Bmr6 gene in sorghum(Sorghum bicolor) has been previously shown to encode cinnamyl alcohol dehydrogenase(CAD),which catalyzes the final step of the monolignol biosynthesis pathway. Mutations in this gene have been shown to reduce the abundance of lignin,enhance digestibility,and improve saccharification efficiencies and ethanol yields. Nine sorghum lines harboring five different bmr6 alleles were identified in an EMS-mutagenized TILLING population. DNA sequencing of Bmr6 revealed that the majority of the mutations impacted evolutionarily conserved amino acids while three-dimensional structural modeling predicted that all of these alleles interfered with the enzyme's ability to bind with its NADPH cofactor. All of the new alleles reduced in vitro CAD activity levels and enhanced glucose yields following saccharification. Further,many of these lines were associated with higher reductions in acid detergent lignin compared to lines harboring the previously characterized bmr6-ref allele. These bmr6 lines represent new breeding tools for manipulating biomass composition to enhance forage and feedstock quality.     

Identification and Characterization of Four Missense Mutations in Brown midrib 12 (Bmr12), the Caffeic O-Methyltranferase (COMT) of Sorghum

Modifying lignin content and composition are targets to improve bioenergy crops for cellulosic conversion to biofuels. In sorghum and other C4 grasses, the brown midrib mutants have been shown to reduce lignin content and alter its composition. Bmr12 encodes the sorghum caffeic O-methyltransferase, which catalyzes the penultimate step in monolignol biosynthesis. From an EMS-mutagenized TILLING population, four bmr12 mutants were isolated. DNA sequencing identified the four missense mutations in the Bmr12 coding region, which changed evolutionarily conserved amino acids Ala71Val, Pro150Leu, Gly225Asp, and Gly325Ser. The previously characterized bmr12 mutants all contain premature stop codons. These newly identified mutants, along with the previously characterized bmr12-ref, represent the first allelic series of bmr12 mutants available in the same genetic background. The impacts of these newly identified mutations on protein accumulation, enzyme activity, Klason lignin content, lignin subunit composition, and saccharification yield were determined. Gly225Asp mutant greatly reduced protein accumulation, and Pro150Leu and Gly325Ser greatly impaired enzyme activity compared to wild type (WT). All four mutants significantly reduced Klason lignin content and altered lignin composition resulting in a significantly reduced S/G ratio relative to WT, but the overall impact of these mutations was less severe than bmr12-ref. Except for Gly325Ser, which is a hypomorphic mutant, all mutants increased the saccharification yield relative to WT. These mutants represent new tools to decrease lignin content and S/G ratio, possibly leading toward the ability to tailor lignin content and composition in the bioenergy grass sorghum.     

Brown midrib2 (Bmr2) encodes the major 4-coumarate:coenzyme A ligase involved in lignin biosynthesis in sorghum (Sorghum bicolor (L.) Moench)

Successful modification of plant cell-wall composition without compromising plant integrity is dependent on being able to modify the expression of specific genes, but this can be very challenging when the target genes are members of multigene families. 4-coumarate:CoA ligase (4CL) catalyzes the formation of 4-coumaroyl CoA, a precursor of both flavonoids and monolignols, and is an attractive target for transgenic down-regulation aimed at improving agro-industrial properties. Inconsistent phenotypes of transgenic plants have been attributed to variable levels of down-regulation of multiple 4CL genes. Phylogenetic analysis of the sorghum genome revealed 24 4CL(-like) proteins, five of which cluster with bona fide 4CLs from other species. Using a map-based cloning approach and analysis of two independent mutant alleles, the sorghum brown midrib2 (bmr2) locus was shown to encode 4CL. In vitro enzyme assays indicated that its preferred substrate is 4-coumarate. Missense mutations in the two bmr2 alleles result in loss of 4CL activity, probably as a result of improper folding as indicated by molecular modeling. Bmr2 is the most highly expressed 4CL in sorghum stems, leaves and roots, both at the seedling stage and in pre-flowering plants, but the products of several paralogs also display 4CL activity and compensate for some of the lost activity. The contribution of the paralogs varies between developmental stages and tissues. Gene expression assays indicated that Bmr2 is under auto-regulatory control, as reduced 4CL activity results in over-expression of the defective gene. Several 4CL paralogs are also up-regulated in response to the mutation.     

Increased stability of bmr3 mRNA results in a multidrug-resistant phenotype in Bacillus subtilis

A spontaneous mutant isolated in the presence of a high concentration of puromycin acquired a multidrug-resistant phenotype. Expression of the bmr3 gene was dramatically increased. A base substitution, T to A at the +4 position, detected in the mutant resulted in the stabilization of bmr3 mRNA.     

A candidate-gene approach to clone the sorghum <Emphasis Type="Italic">Brown midrib </Emphasis>gene encoding caffeic acid <Emphasis Type="Italic">O</Emphasis>-methyltransferase

The brown midrib (bmr) mutants of sorghum have brown vascular tissue in the leaves and stem as a result of changes in lignin composition. The bmr mutants were generated via chemical mutagenesis with diethyl sulfate (DES) and resemble the brown midrib (bm) mutants of maize. The maize and sorghum brown midrib mutants are of particular value for the comparison of lignin biosynthesis across different, yet evolutionarily related, species. Although the sorghum brown midrib mutants were first described in 1978, none of the Brown midrib genes have been cloned. We have used a candidate-gene approach to clone the first Brown midrib gene from sorghum. Based on chemical analyses of the allelic mutants bmr12, bmr18 and bmr26, we hypothesized that these mutants had reduced activity of the lignin biosynthetic enzyme caffeic acid O-methyltransferase (COMT). After a northern analysis revealed strongly reduced expression of the COMT gene, the gene was cloned from the mutants and the corresponding wild types using PCR. In all three mutants, point mutations resulting in premature stop codons were identified: bmr12, bmr18 and bmr26 are therefore mutant alleles of the gene encoding COMT. RT-PCR indicated that all three mutants express the mutant allele, but at much lower levels relative to the wild-type controls. Molecular markers were developed for each of the three mutant alleles to facilitate the use of these mutant alleles in genetic studies and breeding programs.     

A Nonsense Mutation in a Cinnamyl Alcohol Dehydrogenase Gene Is Responsible for the Sorghum brown midrib6 Phenotype

brown midrib6 (bmr6) affects phenylpropanoid metabolism, resulting in reduced lignin concentrations and altered lignin composition in sorghum (Sorghum bicolor). Recently, bmr6 plants were shown to have limited cinnamyl alcohol dehydrogenase activity (CAD; EC 1.1.1.195), the enzyme that catalyzes the conversion of hydroxycinnamoyl aldehydes (monolignals) to monolignols. A candidate gene approach was taken to identify Bmr6. Two CAD genes (Sb02g024190 and Sb04g005950) were identified in the sorghum genome based on similarity to known CAD genes and through DNA sequencing a nonsense mutation was discovered in Sb04g005950 that results in a truncated protein lacking the NADPH-binding and C-terminal catalytic domains. Immunoblotting confirmed that the Bmr6 protein was absent in protein extracts from bmr6 plants. Phylogenetic analysis indicated that Bmr6 is a member of an evolutionarily conserved group of CAD proteins, which function in lignin biosynthesis. In addition, Bmr6 is distinct from the other CAD-like proteins in sorghum, including SbCAD4 (Sb02g024190). Although both Bmr6 and SbCAD4 are expressed in sorghum internodes, an examination of enzymatic activity of recombinant Bmr6 and SbCAD4 showed that Bmr6 had 1 to 2 orders of magnitude greater activity for monolignol substrates. Modeling of Bmr6 and SbCAD4 protein structures showed differences in the amino acid composition of the active site that could explain the difference in enzyme activity. These differences include His-57, which is unique to Bmr6 and other grass CADs. In summary, Bmr6 encodes the major CAD protein involved in lignin synthesis in sorghum, and the bmr6 mutant is a null allele.Plant cell walls constitute a vast reserve of fixed carbon. Cellulose and lignin are the first and second most abundant polymers on the planet, respectively (Jung and Ni, 1998). The world community has started to look to biomass as substrates for plant-based biologically sustainable fuels, which would mitigate carbon dioxide emission and reduce petroleum dependence (Sarath et al., 2008; Schmer et al., 2008). In the current generation of biofuels, ethanol is being synthesized via the fermentation of grain starch or sugarcane juice. For the next generation of biofuels, research is being directed toward the conversion of lignocellulosic biomass into biofuels (Chang, 2007). As bioenergy technologies progress, the conversion of biomass to biofuels could involve a range of chemical, biochemical, and fermentation processes to produce biofuels; alternate biofuels, such as butanol or dimethylfuran, are also on the horizon (Ezeji et al., 2007; Roman-Leshkov et al., 2007). Most liquid biofuel production processes will likely rely on the conversion of the cell wall polysaccharides cellulose and hemicellulose into monomeric sugars.Plant cell walls consist of a complex polysaccharide moiety composed of cellulose microfibrils, composed of β-1,4-linked Glc polymers (Carpita and McCann, 2000). Connecting the cellulose microfibrils to each other is a hemicellulose network, whose structure and composition are species dependent, and which is mainly composed of glucuronoarabinoxylans in grasses (Carpita and McCann, 2000). Lignin, a nonlinear heterogeneous polymer derived from aromatic precursors, cross-links these polysaccharides, rigidifying and reinforcing the cell wall structure (Carpita and McCann, 2000). The addition of lignin polymers to the polysaccharide matrix creates a barrier that is chemically and microbially resistant.Lignin can block the liberation of sugars from the cell wall polysaccharide moieties, release compounds that can inhibit microbes used for fermenting sugars to fuels, and adhere to hydrolytic enzymes. Understanding lignin synthesis, structure, and function to increase cell wall digestibility has long been a goal for forage improvement and paper processing (Mackay et al., 1997; Jung and Ni, 1998). Recently, manipulating lignin has also become an important target for bioenergy feedstock improvement (Chen and Dixon, 2007; Li et al., 2008).Lignin is derived from the phenylpropanoid pathway and contains primarily three types of phenolic subunits: p-hydroxyphenyl, guaiacyl, and syringyl units (Dixon et al., 2001). The phenolic aldehyde precursors are reduced into their corresponding alcohols (monolignols) and subsequently transported to the cell wall (Fig. 1), where laccases and peroxidases catalyze lignin polymerization through the formation of monolignol radicals (Boerjan et al., 2003). Therefore, most research efforts to manipulate lignin have focused on biosynthesis of the monolignols. Most of the enzymes involved in monolignol synthesis have been cloned and characterized in Arabidopsis (Arabidopsis thaliana) and other dicot species, using both mutagenic and transgenic approaches to study the impact of these gene products on dicot cell walls (Anterola and Lewis, 2002). However, there are significant differences in the architecture, polysaccharide composition, and phenylpropanoid composition of grass cell walls compared with those of dicots (Carpita and McCann, 2000; Vogel and Jung, 2001). For example, grasses contain significant amounts of p-coumaric acid and ferulic acid that are cross-linked to cell wall polysaccharides through ester and ether linkages in addition to their presence in lignin (Grabber et al., 1991; Boerjan et al., 2003). Because many of the proposed dedicated bioenergy crops are grasses, there is a need to identify and understand the function of the gene products involved in lignin biosynthesis in these species (Vermerris et al., 2007; Li et al., 2008; Sarath et al., 2008).Open in a separate windowFigure 1.The CAD enzyme and its role in the monolignol biosynthetic pathway. A, CAD catalyzes the conversion of cinnamyl aldehydes to alcohols using NADPH as its cofactor. p-Coumaryl aldehyde and alcohol, R₁ and R₂ = H; caffeoyl aldehyde and alcohol, R₁ and R₂ = OH; coniferyl aldehyde and alcohol, R₁ = H and R₂ = OCH₃; sinapyl aldehyde and alcohol, R₁ and R₂ = OCH₃. B, A simplified model of the lignin biosynthetic pathway where CAD catalyzes the final step in monolignol biosynthesis.The brown midrib phenotype has been useful for identifying mutants affecting lignin synthesis in grasses because it is a visible phenotype. Spontaneous brown midrib mutants were first discovered in maize (Zea mays; Jorgenson, 1931) and were subsequently generated in sorghum (Sorghum bicolor) using diethyl sulfate mutagenesis (Porter et al., 1978). Brown midrib mutants in maize, sorghum, and pearl millet (Pennisetum glaucum) have increased forage digestibility for livestock (Cherney et al., 1990; Akin et al., 1993; Jung et al., 1998; Oliver et al., 2004). In maize and sorghum, there are at least four brown midrib loci in their respective genomes (Jorgenson, 1931; Porter et al., 1978; Gupta, 1995). The genes encoding bm3 in maize and bmr12 in sorghum are the only loci cloned to date, and both encode highly similar caffeic acid O-methyl transferases (Vignols et al., 1995; Bout and Vermerris, 2003). A second brown midrib locus associated with reduced cinnamyl alcohol dehydrogenase (CAD) activity has been identified both in maize (bm1; Halpin et al., 1998) and sorghum (bmr6; Bucholtz et al., 1980; Pillonel et al., 1991). CAD is a member of the alcohol dehydrogenase superfamily of proteins that catalyzes the conversion of the hydroxycinnamoyl aldehydes into alcohols prior to their incorporation into lignin polymers (Fig. 1). Reduced CAD activity results in increased digestibility on dry weight basis, altered cell wall architecture, reduced lignin level, and the incorporation of phenolic aldehydes into lignin in sorghum and maize (Pillonel et al., 1991; Provan et al., 1997; Halpin et al., 1998; Marita et al., 2003; Shi et al., 2006; Palmer et al., 2008). The reduced CAD activity in bm1 has been genetically mapped to a region of the maize genome that contained a CAD gene, ZmCAD2 (Halpin et al., 1998), but a mutation was not identified. However, it has recently been shown that bm1 down-regulated the expression of several lignin biosynthetic genes, suggesting its gene product may be a regulatory protein (Shi et al., 2006; Guillaumie et al., 2007).To identify the mutation responsible for the bmr6 phenotype and to characterize how bmr6 impacts the lignin biosynthetic pathway, a candidate gene approach was taken. Here, we describe the cloning and characterization of Bmr6 and a related protein, SbCAD4. The identification and characterization of Bmr6 has revealed the major monolignol CAD protein in the grasses, which is likely to aid the development of new strategies to increase conversion of sorghum and other grass feedstocks to biofuels.     

Molecular characterization of allelic variation in spontaneous brown midrib mutants of sorghum (Sorghum bicolor (L.) Moench)

Modification of lignin composition and content are important to enhance the saccharification potential of lignocellulosic biomass. Brown midrib (bmr) mutants with altered lignin and enhanced glucose yields are a valuable resource for modification of the lignin biosynthetic pathway in sorghum (Sorghum bicolor (L.) Moench). Of the 38 bmr mutants reported in sorghum, some have been classified into four independent groups, namely bmr2, bmr6, bmr12 and bmr19, based on the allelic test, and a few have been characterized at the molecular level. The bmr2, bmr6 and bmr12 groups have mutations that impair 4-coumarate:coenzyme A ligase (4CL), cinnamyl alcohol dehydrogenase (CAD2) and caffeic O-methyltransferase (COMT), respectively. The molecular basis of bmr19 is unknown. In the present study, four spontaneous bmr mutants of sorghum were analyzed for allelic variation at two candidate gene loci. cDNAs of CAD2 and COMT genes were cloned and sequenced from these mutants. Sequence analysis revealed that two of these mutants, IS23789 and IS23253, share a new allele of CAD2. These mutants have a G-to-C transversion at position 3699 of the genomic sequence that leads to glycine-to-arginine (G191R) substitution in the CAD2 protein sequence. This mutation lies in the highly conserved glycine-rich motif ¹⁸⁸G(X)GGV(L)G¹⁹³ that participates in the binding of the pyrophosphate group of NADP⁺ cofactor and hence might impair the activity of CAD2. Phloroglucinol staining of midribs of these mutants also showed a dark wine-red color that is characteristic of the bmr6 group. These two mutants can be distinguished by an intron length polymorphic marker developed based on the COMT gene sequence in this study. Mutant IS23549, which has also been assigned to the bmr6 group, was found to have another new allele with alanine-to-valine (A164V) substitution in CAD2. Alanine-164 is highly conserved among MDR proteins in plants and hence may be necessary for the activity of the enzyme. In mutant IS11861, there was no mutation that led to a change in amino acid in CAD2, while a threonine-to-serine (T302S) substitution was found in COMT. This single nucleotide polymorphism (SNP) at position 2645 in the COMT gene was converted into a cleaved amplified polymorphic sequence marker that can be used for its identification. In addition, additional SNP- and/or indel-based markers were developed, which can be used for exploiting these alleles in the molecular breeding of sorghum for dedicated bioenergy feedstock.     

Mutations affecting substrate specificity of the Bacillus subtilis multidrug transporter Bmr.

The Bacillus subtilis multidrug transporter Bmr, a member of the major facilitator superfamily of transporters, causes the efflux of a number of structurally unrelated toxic compounds from cells. We have shown previously that the activity of Bmr can be inhibited by the plant alkaloid reserpine. Here we demonstrate that various substitutions of residues Phe143 and Phe306 of Bmr not only reduce its sensitivity to reserpine inhibition but also significantly change its substrate specificity. Cross-resistance profiles of bacteria expressing mutant forms of the transporter differ from each other and from the cross-resistance profile of cells expressing wild-type Bmr. This result strongly suggests that Bmr interacts with its transported drugs directly, with residues Phe143 and Phe306 likely to be involved in substrate recognition.     

Paradoxical Enhancement of the Activity of a Bacterial Multidrug Transporter Caused by Substitutions of a Conserved Residue

Substitution of threonine or serine for the evolutionary conserved intramembrane proline P³⁴⁷ of the Bacillus subtilis multidrug transporter Bmr significantly increases the toxin-effluxing activity of Bmr without affecting its abundance in the cell. In cocultivation experiments, we demonstrate that although the mutant T³⁴⁷ Bmr is advantageous to cells growing in the presence of a toxin, the wild-type P³⁴⁷ Bmr is advantageous under the conditions of nutritional limitation. This may explain why Bmr has evolved the way it did, that is, with proline at position 347. These observations provide a basis for speculating that the evolution of Bmr has been determined by its presently unidentified natural function rather than by its ability to expel diverse toxins from the cell.     

Allelic Association, Chemical Characterization and Saccharification Properties of brown midrib Mutants of Sorghum (Sorghum bicolor (L.) Moench)

Genetic improvement of biomass crops can significantly reduce the overall cost of biomass-to-ethanol conversion. The conversion of cellulose to monomeric sugar units is affected by lignin content and composition. Sorghum has attracted the attention of the scientific and industrial community as a promising source of biomass for bioenergy due to its great yield potential and tolerance to stresses. The brown midrib (bmr) mutants of sorghum are characterized by brown vascular tissue associated with altered lignin content. Twenty-eight bmr mutants have been identified since the late 1970s, but the allelic relationships have not been fully established, and the function of only one of the Bmr loci has been unequivocally established. In this study, we combined genetic and chemical approaches to establish that there are mutations at least four independent bmr loci, represented by the bmr2, bmr6, bmr12 and bmr19 groups. Since each allelic group presents unique staining characteristics, rapid classification of emerging bmr lines into the existing groups can be achieved using phloroglucinol-HCl as a histochemical stain. In addition, pyrolysis-gas chromatography-mass spectrometry, enabled the characterization of changes in subunit lignin composition in each of the allelic groups, to help predict the genes underlying the mutations. Enzymatic saccharification of stover from plants representing each allelic bmr group demonstrated that lignin changes in lines belonging to the bmr2, bmr6 and bmr12 groups can increase glucose yields, up to 25% compared to wild-type isolines. In order to expedite the selection of the bmr mutant alleles in breeding populations, we have developed molecular markers specific for bmr7 and bmr25, two novel mutant alleles of the gene encoding caffeic acid O-methyl transferase. Based on the results from this study, we propose to rename the bmr mutants in a manner that reflects the number of independent loci.     

高梁肉桂酸羟化酶基因SbC4H1降低拟南芥的木质素合成

肉桂酸羟化酶（C4H）是苯丙烷代谢通路的关键酶,其活性和含量直接影响木质素合成的效率。本文研究通过高粱bmr突变体的抑制差减杂交筛选、克隆到了一个C4H基因sbC4H。半定量RT-PCR发现,SbC4H1在多个bmr突变体中上调表达。将SbC4H1-GFP融合基因转化拟南芥原生质体进行瞬时表达,发现SbC4H1表达产物蛋白定位于细胞质。SbC4H1在拟南芥中的异源表达明显降低其茎的木质素含量,并且下调了拟南芥4CL1、FSH和HCT质素合成基因的表达。这些结果表明,高粱SbC4H1抑制了拟南芥木质素的合成。     

Effect of lime pretreatment of brown midrib sorghums

The effect of lime pretreatment of brown midrib sorghums on enzymatic saccharification was investigated. Under most of the pretreatment conditions, the saccharification yields of bmrs were higher than those of the normal counterparts. This result suggests that bmr is useful to reduce pretreatment costs, because the amount of lime necessary for the pretreatment of biomass can reduced by using bmr mutants.     

Litter decomposition of three lignin-deficient mutants of Sorghum bicolor during spring thaw

Decomposition of plant litter during the freeze-thaw season has recently gained attention as having a significant role in nutrient cycling in many cold ecosystems. However, few studies have examined decomposition of crop remnants during the freeze-thaw season in an agronomic setting when microbial activity is presumably low. We examined decomposition of four cultivars of sorghum (Sorghum bicolor) leaves in a field in Southern Minnesota, USA using the litterbag method. Three of the four cultivars we examined expressed the brown midrib (bmr) mutation which have altered/reduced levels of lignin in their secondary cell walls compared to the wild-type (WT). Litter was buried in the fall and harvested during the spring thaw. After 160 d the bmr mutants lost 57–62% of their initial mass, compared to 51% in the WT. Mass loss agreed with presumed initial litter quality, as the bmr litter had higher initial N, and holocellulose:lignin and lower lignin, C:N and lignin:N values compared to the WT. The increased decomposition of the bmr cultivars appears to be related to increased loss of hemicellulose and holocellulose (cellulose+hemicellulose) or higher initial N concentrations. Alterations in cell-wall deposition in the bmr cultivars may increase accessibility of microbial cell-wall degrading enzymes that accelerate mass loss. Our results demonstrate that alterations in initial lignin chemistry may influence decomposition of sorghum litter in an agronomic setting.     

Mapping malting quality and yield characteristics in a north American two-rowed malting barley × wild barley advanced backcross population

Molecular Breeding - The brown midrib (bmr) phenotype is a recessive trait of sorghum (Sorghum bicolor L. Moench) that results in overall lignin reduction and is associated with enhanced ruminant...