Publications

2012
Wang, W, Wu Y, Messing J.  2012.  The mitochondrial genome of an aquatic plant, Spirodela polyrhiza. PloS one. 7:e46747. AbstractWebsite
BACKGROUND: Spirodela polyrhiza is a species of the order Alismatales, which represent the basal lineage of monocots with more ancestral features than the Poales. Its complete sequence of the mitochondrial (mt) genome could provide clues for the understanding of the evolution of mt genomes in plant. METHODS: Spirodela polyrhiza mt genome was sequenced from total genomic DNA without physical separation of chloroplast and nuclear DNA using the SOLiD platform. Using a genome copy number sensitive assembly algorithm, the mt genome was successfully assembled. Gap closure and accuracy was determined with PCR products sequenced with the dideoxy method. CONCLUSIONS: This is the most compact monocot mitochondrial genome with 228,493 bp. A total of 57 genes encode 35 known proteins, 3 ribosomal RNAs, and 19 tRNAs that recognize 15 amino acids. There are about 600 RNA editing sites predicted and three lineage specific protein-coding-gene losses. The mitochondrial genes, pseudogenes, and other hypothetical genes (ORFs) cover 71,783 bp (31.0%) of the genome. Imported plastid DNA accounts for an additional 9,295 bp (4.1%) of the mitochondrial DNA. Absence of transposable element sequences suggests that very few nuclear sequences have migrated into Spirodela mtDNA. Phylogenetic analysis of conserved protein-coding genes suggests that Spirodela shares the common ancestor with other monocots, but there is no obvious synteny between Spirodela and rice mtDNAs. After eliminating genes, introns, ORFs, and plastid-derived DNA, nearly four-fifths of the Spirodela mitochondrial genome is of unknown origin and function. Although it contains a similar chloroplast DNA content and range of RNA editing as other monocots, it is void of nuclear insertions, active gene loss, and comprises large regions of sequences of unknown origin in non-coding regions. Moreover, the lack of synteny with known mitochondrial genomic sequences shed new light on the early evolution of monocot mitochondrial genomes.
Maliga, P.  2012.  Plastid transformation in flowering plants. Genomics of Chloroplasts and Mitochondria. 35:393-414. Abstract
The plastid genome of higher plants is relatively small, 120–230-kb in size, and present in up to 10,000 copies per cell. Standard protocols for the introduction of transforming DNA employ biolistic DNA delivery or polyethylene glycol treatment. Genetically stable, transgenic plants are obtained by modification of the plastid genome by homologous recombination, followed by selection for the transformed genome copy by the expression of marker genes that protect the cells from selective agents. Commonly used selective agents are antibiotics, including spectinomycin, streptomycin, kanamycin and chloramphenicol. Selection for resistance to amino acid analogues has also been successful. The types of plastid genome manipulations include gene deletion, gene insertion, and gene replacement, facilitated by specially designed transformation vectors. Methods are also available for post-transformation removal of marker genes. The model species for plastid genetic manipulation is Nicotiana tabacum, in which most protocols have been tested. Plastid transformation is also available in several solanaceous crops (tomato, potato, eggplant) and ornamental species (petunia, Nicotianasylvestris). Significant progress has been made with Brasssicaceae including cabbage, oilseed rape and Arabidopsis. Recent additions to the crops in which plastid transformation is reproducibly obtained are lettuce, soybean and sugar beet. The monocots are a taxonomic group recalcitrant to plastid transformation; initial inroads have been made only in rice.
2011
Joyce, EF, Pedersen M, Tiong S, White-Brown SK, Paul A, Campbell SD, McKim KS.  2011.  Drosophila ATM and ATR have distinct activities in the regulation of meiotic DNA damage and repair. J Cell Biol. 195:359-67. AbstractWebsite
Ataxia telangiectasia-mutated (ATM) and ataxia telangiectasia-related (ATR) kinases are conserved regulators of cellular responses to double strand breaks (DSBs). During meiosis, however, the functions of these kinases in DSB repair and the deoxyribonucleic acid (DNA) damage checkpoint are unclear. In this paper, we show that ATM and ATR have unique roles in the repair of meiotic DSBs in Drosophila melanogaster. ATR mutant analysis indicated that it is required for checkpoint activity, whereas ATM may not be. Both kinases phosphorylate H2AV (gamma-H2AV), and, using this as a reporter for ATM/ATR activity, we found that the DSB repair response is surprisingly dynamic at the site of DNA damage. gamma-H2AV is continuously exchanged, requiring new phosphorylation at the break site until repair is completed. However, most surprising is that the number of gamma-H2AV foci is dramatically increased in the absence of ATM, but not ATR, suggesting that the number of DSBs is increased. Thus, we conclude that ATM is primarily required for the meiotic DSB repair response, which includes functions in DNA damage repair and negative feedback control over the level of programmed DSBs during meiosis.
Tanneti, NS, Landy K, Joyce EF, McKim KS.  2011.  A Pathway for Synapsis Initiation during Zygotene in Drosophila Oocytes. Curr Biol. 21:1852-7. AbstractWebsite
Formation of the synaptonemal complex (SC), or synapsis, between homologs in meiosis is essential for crossing over and chromosome segregation [1-4]. How SC assembly initiates is poorly understood but may have a critical role in ensuring synapsis between homologs and regulating double-strand break (DSB) and crossover formation. We investigated the genetic requirements for synapsis in Drosophila and found that there are three temporally and genetically distinct stages of synapsis initiation. In "early zygotene" oocytes, synapsis is only observed at the centromeres. We also found that nonhomologous centromeres are clustered during this process. In "mid-zygotene" oocytes, SC initiates at several euchromatic sites. The centromeric and first euchromatic SC initiation sites depend on the cohesion protein ORD. In "late zygotene" oocytes, SC initiates at many more sites that depend on the Kleisin-like protein C(2)M. Surprisingly, late zygotene synapsis initiation events are independent of the earlier mid-zygotene events, whereas both mid and late synapsis initiation events depend on the cohesin subunits SMC1 and SMC3. We propose that the enrichment of cohesion proteins at specific sites promotes homolog interactions and the initiation of euchromatic SC assembly independent of DSBs. Furthermore, the early euchromatic SC initiation events at mid-zygotene may be required for DSBs to be repaired as crossovers.
Cesario, J, McKim KS.  2011.  RanGTP is required for meiotic spindle organization and the initiation of embryonic development in Drosophila. J Cell Sci. 124:3797-810. AbstractWebsite
RanGTP is important for chromosome-dependent spindle assembly in Xenopus extracts. Here we report on experiments to determine the role of the Ran pathway on microtubule dynamics in Drosophila oocytes and embryos. Females expressing a dominant-negative form of Ran have fertility defects, suggesting that RanGTP is required for normal fertility. This is not, however, because of a defect in acentrosomal meiotic spindle assembly. Therefore, RanGTP does not appear to be essential or sufficient for the formation of the acentrosomal spindle. Instead, the most important function of the Ran pathway in spindle assembly appears to be in the tapering of microtubules at the spindle poles, which might be through regulation of proteins such as TACC and the HURP homolog, Mars. One consequence of this spindle organization defect is an increase in the nondisjunction of achiasmate chromosomes. However, the meiotic defects are not severe enough to cause the decreased fertility. Reductions in fertility occur because RanGTP has a role in microtubule assembly that is not directly nucleated by the chromosomes. This includes microtubules nucleated from the sperm aster, which are required for pronuclear fusion. We propose that following nuclear envelope breakdown, RanGTP is released from the nucleus and creates a cytoplasm that is activated for assembling microtubules, which is important for processes such as pronuclear fusion. Around the chromosomes, however, RanGTP might be redundant with other factors such as the chromosome passenger complex.
Mao, Y, Mulvaney J, Zakaria S, Yu T, Morgan K M, Allen S, Basson AM, Francis-West P, Irvine KD.  2011.  Characterization of a Dchs1 mutant mouse reveals requirements for Dchs1-Fat4 signaling during mammalian development. Development (Cambridge, England). 138:947-57. AbstractWebsite
The Drosophila Dachsous and Fat proteins function as ligand and receptor, respectively, for an intercellular signaling pathway that regulates Hippo signaling and planar cell polarity. Although gene-targeted mutations in two mammalian Fat genes have been described, whether mammals have a Fat signaling pathway equivalent to that in Drosophila, and what its biological functions might be, have remained unclear. Here, we describe a gene-targeted mutation in a murine Dachsous homolog, Dchs1. Analysis of the phenotypes of Dchs1 mutant mice and comparisons with Fat4 mutant mice identify requirements for these genes in multiple organs, including the ear, kidney, skeleton, intestine, heart and lung. Dchs1 and Fat4 single mutants and Dchs1 Fat4 double mutants have similar phenotypes throughout the body. In some cases, these phenotypes suggest that Dchs1-Fat4 signaling influences planar cell polarity. In addition to the appearance of cysts in newborn kidneys, we also identify and characterize a requirement for Dchs1 and Fat4 in growth, branching and cell survival during early kidney development. Dchs1 and Fat4 are predominantly expressed in mesenchymal cells in multiple organs, and mutation of either gene increases protein staining for the other. Our analysis implies that Dchs1 and Fat4 function as a ligand-receptor pair during murine development, and identifies novel requirements for Dchs1-Fat4 signaling in multiple organs.
Wu, Y, Messing J.  2011.  Novel Genetic Selection System for Quantitative Trait Loci of Quality Protein Maize. Genetics. 188:1019-1022. AbstractWebsite
Quality Protein Maize combines a high-lysine trait with kernel hardness, for which a new simpler genetic selection was designed.
Bao, X, Pachikara N, Oey CB, Balakrishnan A, Westblade LF, Tan M, Chase T, Nickels BE, Fan H.  2011.  Noncoding Nucleotides and Amino Acids near the Active Site Regulate Peptide Deformylase Expression and Inhibitor Susceptibility in Chlamydia trachomatis. Microbiology. AbstractWebsite
Chlamydia trachomatis, an obligate intracellular bacterium, is a highly prevalent human pathogen. Hydroxamic acid-based matrix metalloprotease inhibitors can effectively inhibit the pathogen both in vitro and in vivo, and have exhibited therapeutic potential. Here, we provide genome sequencing data indicating that peptide deformylase (PDF) is the sole target of the inhibitors in this organism. We further report molecular mechanisms that control chlamydial PDF (cPDF) expression and inhibition efficiency. In particular, we identify the o66-dependent promoter that controls cPDF gene expression and demonstrate that point mutations in this promoter lead to resistance by increasing cPDF transcription. Furthermore, we show that substitution of two amino acids near the active site of the enzyme alters enzyme kinetics and protein stability.
Goldman, SR, Sharp JS, Vvedenskaya IO, Livny J, Dove SL, Nickels BE.  2011.  NanoRNAs Prime Transcription Initiation In Vivo. Mol Cell. 42:817-825. AbstractWebsite
It is often presumed that, in vivo, the initiation of RNA synthesis by DNA-dependent RNA polymerases occurs using NTPs alone. Here, using the model Gram-negative bacterium Pseudomonas aeruginosa, we demonstrate that depletion of the small-RNA-specific exonuclease, Oligoribonuclease, causes the accumulation of oligoribonucleotides 2 to approximately 4 nt in length, "nanoRNAs," which serve as primers for transcription initiation at a significant fraction of promoters. Widespread use of nanoRNAs to prime transcription initiation is coupled with global alterations in gene expression. Our results, obtained under conditions in which the concentration of nanoRNAs is artificially elevated, establish that small RNAs can be used to initiate transcription in vivo, challenging the idea that all cellular transcription occurs using only NTPs. Our findings further suggest that nanoRNAs could represent a distinct class of functional small RNAs that can affect gene expression through direct incorporation into a target RNA transcript rather than through a traditional antisense-based mechanism.
Nickels, BE, Dove SL.  2011.  NanoRNAs: A Class of Small RNAs That Can Prime Transcription Initiation in Bacteria. J Mol Biol. AbstractWebsite
It has been widely assumed that all transcription in cells occur using NTPs only (i.e., de novo). However, it has been known for several decades that both prokaryotic and eukaryotic RNA polymerases can utilize small (2 to approximately 5 nt) RNAs to prime transcription initiation in vitro, raising the possibility that small RNAs might also prime transcription initiation in vivo. A new study by Goldman et al. has now provided the first evidence that priming with so-called "nanoRNAs" (i.e., 2 to approximately 5 nt RNAs) can, in fact, occur in vivo. Furthermore, this study provides evidence that altering the extent of nanoRNA-mediated priming of transcription initiation can profoundly influence global gene expression. In this perspective, we summarize the findings of Goldman et al. and discuss the prospect that nanoRNA-mediated priming of transcription initiation represents an underappreciated aspect of gene expression in vivo.
Rauskolb, C, Pan G, Reddy BVVG, Oh H, Irvine KD.  2011.  Zyxin links fat signaling to the hippo pathway. PLoS Biology. 9:e1000624. AbstractWebsite
The Hippo signaling pathway has a conserved role in growth control and is of fundamental importance during both normal development and oncogenesis. Despite rapid progress in recent years, key steps in the pathway remain poorly understood, in part due to the incomplete identification of components. Through a genetic screen, we identified the Drosophila Zyxin family gene, Zyx102 (Zyx), as a component of the Hippo pathway. Zyx positively regulates the Hippo pathway transcriptional co-activator Yorkie, as its loss reduces Yorkie activity and organ growth. Through epistasis tests, we position the requirement for Zyx within the Fat branch of Hippo signaling, downstream of Fat and Dco, and upstream of the Yorkie kinase Warts, and we find that Zyx is required for the influence of Fat on Warts protein levels. Zyx localizes to the sub-apical membrane, with distinctive peaks of accumulation at intercellular vertices. This partially overlaps the membrane localization of the myosin Dachs, which has similar effects on Fat-Hippo signaling. Co-immunoprecipitation experiments show that Zyx can bind to Dachs and that Dachs stimulates binding of Zyx to Warts. We also extend characterization of the Ajuba LIM protein Jub and determine that although Jub and Zyx share C-terminal LIM domains, they regulate Hippo signaling in distinct ways. Our results identify a role for Zyx in the Hippo pathway and suggest a mechanism for the role of Dachs: because Fat regulates the localization of Dachs to the membrane, where it can overlap with Zyx, we propose that the regulated localization of Dachs influences downstream signaling by modulating Zyx-Warts binding. Mammalian Zyxin proteins have been implicated in linking effects of mechanical strain to cell behavior. Our identification of Zyx as a regulator of Hippo signaling thus also raises the possibility that mechanical strain could be linked to the regulation of gene expression and growth through Hippo signaling.
Miclaus, M, Xu JH, Messing J.  2011.  Differential gene expression and epiregulation of alpha zein gene copies in maize haplotypes. PLoS Genet. 7:e1002131. AbstractWebsite
Multigenic traits are very common in plants and cause diversity. Nutritional quality is such a trait, and one of its factors is the composition and relative expression of storage protein genes. In maize, they represent a medium-size gene family distributed over several chromosomes and unlinked locations. Two inbreds, B73 and BSSS53, both from the Iowa Stiff Stock Synthetic collection, have been selected to analyze allelic and non-allelic variability in these regions that span between 80-500 kb of chromosomal DNA. Genes were copied to unlinked sites before and after allotetraploidization of maize, but before transposition enlarged intergenic regions in a haplotype-specific manner. Once genes are copied, expression of donor genes is reduced relative to new copies. Epigenetic regulation seems to contribute to silencing older copies, because some of them can be reactivated when endosperm is maintained as cultured cells, indicating that copy number variation might contribute to a reserve of gene copies. Bisulfite sequencing of the promoter region also shows different methylation patterns among gene clusters as well as differences between tissues, suggesting a possible position effect on regulatory mechanisms as a result of inserting copies at unlinked locations. The observations offer a potential paradigm for how different gene families evolve and the impact this has on their expression and regulation of their members.
Twist, KA, Husnain SI, Franke JD, Jain D, Campbell EA, Nickels BE, Thomas MS, Darst SA, Westblade LF.  2011.  A novel method for the production of in vivo-assembled, recombinant Escherichia coli RNA polymerase lacking the alpha C-terminal domain. Protein Sci. 20:986-95. AbstractWebsite
The biochemical characterization of the bacterial transcription cycle has been greatly facilitated by the production and characterization of targeted RNA polymerase (RNAP) mutants. Traditionally, RNAP preparations containing mutant subunits have been produced by reconstitution of denatured RNAP subunits, a process that is undesirable for biophysical and structural studies. Although schemes that afford the production of in vivo-assembled, recombinant RNAP containing amino acid substitutions, insertions, or deletions in either the monomeric beta or beta' subunits have been developed, there is no such system for the production of in vivo-assembled, recombinant RNAP with mutations in the homodimeric alpha-subunits. Here, we demonstrate a strategy to generate in vivo-assembled, recombinant RNAP preparations free of the alpha C-terminal domain. Furthermore, we describe a modification of this approach that would permit the purification of in vivo-assembled, recombinant RNAP containing any alpha-subunit variant, including those variants that are lethal. Finally, we propose that these related approaches can be extended to generate in vivo-assembled, recombinant variants of other protein complexes containing homomultimers for biochemical, biophysical, and structural analyses.
Tungsuchat-Huang, T, Slivinski KM, Sinagawa-Garcia SR, Maliga P.  2011.  Visual spectinomycin resistance (aadA(au)) gene for facile identification of transplastomic sectors in tobacco leaves. Plant Mol. Biol.. 76:453-61. AbstractWebsite
Identification of a genetically stable Nicotiana tabacum (tobacco) plant with a uniform population of transformed plastid genomes (ptDNA) takes two cycles of plant regeneration from chimeric leaves and analysis of multiple shoots by Southern probing in each cycle. Visual detection of transgenic sectors facilitates identification of transformed shoots in the greenhouse, complementing repeated cycles of blind purification in culture. In addition, it provides a tool to monitor the maintenance of transplastomic state. Our current visual marker system requires two genes: the aurea bar (bar(au)) gene that confers a golden leaf phenotype and a spectinomycin resistance (aadA) gene that is necessary for the introduction of the bar(au) gene in the plastid genome. We developed a novel aadA gene that fulfills both functions: it is a conventional selectable aadA gene in culture, and allows detection of transplastomic sectors in the greenhouse by leaf color. Common causes of pigment deficiency in leaves are mutations in photosynthetic genes, which affect chlorophyll accumulation. We use a different approach to achieve pigment deficiency: post-transcriptional interference with the expression of the clpP1 plastid gene by aurea aadA(au) transgene. This interference produces plants with reduced growth and a distinct color, but maintains a wild-type gene set and the capacity for photosynthesis. Importantly, when the aurea gene is removed, green pigmentation and normal growth rate are restored. Because the aurea plants are viable, the new aadA(au) genes are useful to query rare events in large populations and for in planta manipulation of the plastid genome.
Oh, H, Irvine KD.  2011.  Cooperative Regulation of Growth by Yorkie and Mad through bantam. Developmental Cell. 20:109-22. AbstractWebsite
The Dpp and Fat-Hippo signaling pathways both regulate growth in Drosophila. Dpp is a BMP family ligand and acts via a Smad family DNA-binding transcription factor, Mad. Fat-Hippo signaling acts via a non-DNA-binding transcriptional coactivator protein, Yorkie. Here, we show that these pathways are directly interlinked. They act synergistically to promote growth, in part via regulation of the microRNA gene bantam, and their ability to promote growth is mutually dependent. Yorkie and Mad physically bind each other, and we identify a 410 bp minimal enhancer of bantam that responds to Yorkie:Mad in vivo and in cultured cells, and show that both Yorkie and Mad associate with this enhancer in vivo. Our results indicate that in promoting the growth of Drosophila tissues, Fat-Hippo and Dpp signaling contribute distinct subunits of a shared transcriptional activation complex, Yorkie:Mad.
Deighan, P, Pukhrambam C, Nickels BE, Hochschild A.  2011.  Initial transcribed region sequences influence the composition and functional properties of the bacterial elongation complex. Genes Dev. 25:77-88. AbstractWebsite
The bacterial RNA polymerase (RNAP) holoenzyme consists of a catalytic core enzyme (alpha(2)betabeta'omega) in complex with a sigma factor that is essential for promoter recognition and transcription initiation. During early elongation, the stability of interactions between sigma and the remainder of the transcription complex decreases. Nevertheless, there is no mechanistic requirement for release of sigma upon the transition to elongation. Furthermore, sigma can remain associated with RNAP during transcription elongation and influence regulatory events that occur during transcription elongation. Here we demonstrate that promoter-like DNA sequence elements within the initial transcribed region that are known to induce early elongation pausing through sequence-specific interactions with sigma also function to increase the sigma content of downstream elongation complexes. Our findings establish sigma-dependent pausing as a mechanism by which initial transcribed region sequences can influence the composition and functional properties of the transcription elongation complex over distances of at least 700 base pairs.
Sun, G, Irvine KD.  2011.  Regulation of Hippo signaling by Jun kinase signaling during compensatory cell proliferation and regeneration, and in neoplastic tumors. Developmental biology. 350:139-51. AbstractWebsite
When cells undergo apoptosis, they can stimulate the proliferation of nearby cells, a process referred to as compensatory cell proliferation. The stimulation of proliferation in response to tissue damage or removal is also central to epimorphic regeneration. The Hippo signaling pathway has emerged as an important regulator of growth during normal development and oncogenesis from Drosophila to humans. Here we show that induction of apoptosis in the Drosophila wing imaginal disc stimulates activation of the Hippo pathway transcription factor Yorkie in surviving and nearby cells, and that Yorkie is required for the ability of the wing to regenerate after genetic ablation of the wing primordia. Induction of apoptosis activates Yorkie through the Jun kinase pathway, and direct activation of Jun kinase signaling also promotes Yorkie activation in the wing disc. We also show that depletion of neoplastic tumor suppressor genes, including lethal giant larvae and discs large, or activation of aPKC, activates Yorkie through Jun kinase signaling, and that Jun kinase activation is necessary, but not sufficient, for the disruption of apical-basal polarity associated with loss of lethal giant larvae. Our observations identify Jnk signaling as a modulator of Hippo pathway activity in wing imaginal discs, and implicate Yorkie activation in compensatory cell proliferation and disc regeneration.
Miclaus, M, Wu Y, Xu JH, Dooner HK, Messing J.  2011.  The maize high-lysine mutant opaque7 is defective in an acyl-CoA synthetase-like protein. Genetics. 189:1271-80. AbstractWebsite
Maize (Zea mays) has a large class of seed mutants with opaque or nonvitreous endosperms that could improve the nutritional quality of our food supply. The phenotype of some of them appears to be linked to the improper formation of protein bodies (PBs) where zein storage proteins are deposited. Although a number of genes affecting endosperm vitreousness have been isolated, it has been difficult to clone opaque7 (o7), mainly because of its low penetrance in many genetic backgrounds. The o7-reference (o7-ref) mutant arose spontaneously in a W22 inbred, but is poorly expressed in other lines. We report here the isolation of o7 with a combination of map-based cloning and transposon tagging. We first identified an o7 candidate gene by map-based cloning. The putative o7-ref allele has a 12-bp in-frame deletion of codons 350-353 in a 528-codon-long acyl-CoA synthetase-like gene (ACS). We then confirmed this candidate gene by generating another mutant allele from a transposon-tagging experiment using the Activator/Dissociation (Ac/Ds) system in a W22 background. The second allele, isolated from approximately 1 million gametes, presented a 2-kb Ds insertion that resembles the single Ds component of double-Ds, McClintock's original Dissociation element, at codon 496 of the ACS gene. PBs exhibited striking membrane invaginations in the o7-ref allele and a severe number reduction in the Ds-insertion mutant, respectively. We propose a model in which the ACS enzyme plays a key role in membrane biogenesis, by taking part in protein acylation, and that altered PBs render the seed nonvitreous.
Wu, Y, Messing J.  2011.  Novel genetic selection system for quantitative trait loci of quality protein maize. Genetics. 188:1019-22. AbstractWebsite
Quality protein maize combines a high-lysine trait with kernel hardness, for which a new, simpler genetic selection was designed.
Maliga, P, Bock R.  2011.  Plastid biotechnology: food, fuel, and medicine for the 21st century. Plant Physiol.. 155:1501-10.Website
Amundsen, K, Rotter D, Li H M, Messing J, Jung G, Belanger F, Warnke S.  2011.  Miniature Inverted-Repeat Transposable Element Identification and Genetic Marker Development in Agrostis. Crop Sci.. 51:854-861.Website
Xiao, Y, Wei X, Ebright R, Wall D.  2011.  Antibiotic production by myxobacteria plays a role in predation.. Journal of bacteriology. 193(18):4626-33. Abstract
Myxobacteria are predatory and are prolific producers of secondary metabolites. Here, we tested a hypothesized role that secondary metabolite antibiotics function as weapons in predation. To test this, a Myxococcus xanthus Δta1 mutant, blocked in antibiotic TA (myxovirescin) production, was constructed. This TA(-) mutant was defective in producing a zone of inhibition (ZOI) against Escherichia coli. This shows that TA is the major M. xanthus-diffusible antibacterial agent against E. coli. Correspondingly, the TA(-) mutant was defective in E. coli killing. Separately, an engineered E. coli strain resistant to TA was shown to be resistant toward predation. Exogenous addition of spectinomycin, a bacteriostatic antibiotic, rescued the predation defect of the TA(-) mutant. In contrast, against Micrococcus luteus the TA(-) mutant exhibited no defect in ZOI or killing. Thus, TA plays a selective role on prey species. To extend these studies to other myxobacteria, the role of antibiotic corallopyronin production in predation was tested and also found to be required for Corallococcus coralloides killing on E. coli. Next, a role of TA production in myxobacterial fitness was assessed by measuring swarm expansion. Here, the TA(-) mutant had a specific swarm rate reduction on prey lawns, and thus reduced fitness, compared to an isogenic TA(+) strain. Based on these observations, we conclude that myxobacterial antibiotic production can function as a predatory weapon. To our knowledge, this is the first report to directly show a link between secondary metabolite production and predation.
Kuznedelov, K, Semenova E, Knappe TA, Mukhamedyarov D, Srivastava A, Chatterjee S, Ebright RH, Marahiel MA, Severinov K.  2011.  The Antibacterial Threaded-lasso Peptide Capistruin Inhibits Bacterial RNA Polymerase.. Journal of molecular biology. 412(5):842-8. Abstract
Capistruin, a ribosomally synthesized, post-translationally modified peptide produced by Burkholderia thailandensis E264, efficiently inhibits growth of Burkholderia and closely related Pseudomonas strains. The functional target of capistruin is not known. Capistruin is a threaded-lasso peptide (lariat peptide) consisting of an N-terminal ring of nine amino acids and a C-terminal tail of 10 amino acids threaded through the ring. The structure of capistruin is similar to that of microcin J25 (MccJ25), a threaded-lasso antibacterial peptide that is produced by some strains of Escherichia coli and targets DNA-dependent RNA polymerase (RNAP). Here, we show that capistruin, like MccJ25, inhibits wild type E. coli RNAP but not mutant, MccJ25-resistant, E. coli RNAP. We show further that an E. coli strain resistant to MccJ25, as a result of a mutation in an RNAP subunit gene, exhibits resistance to capistruin. The results indicate that the structural similarity of capistruin and MccJ25 reflects functional similarity and suggest that the functional target of capistruin, and possibly other threaded-lasso peptides, is bacterial RNAP.
Carrieri, D, Ananyev GM, Lenz O, Bryant DA, Dismukes CG.  2011.  Contribution of a sodium ion gradient to energy conservation during fermentation in the cyanobacterium Arthrospira (Spirulina) maxima CS-328.. Applied and environmental microbiology. 77(20):7185-94. Abstract
Sodium gradients in cyanobacteria play an important role in energy storage under photoautotrophic conditions but have not been well studied during autofermentative metabolism under the dark, anoxic conditions widely used to produce precursors to fuels. Here we demonstrate significant stress-induced acceleration of autofermentation of photosynthetically generated carbohydrates (glycogen and sugars) to form excreted organic acids, alcohols, and hydrogen gas by the halophilic, alkalophilic cyanobacterium Arthrospira (Spirulina) maxima CS-328. When suspended in potassium versus sodium phosphate buffers at the start of autofermentation to remove the sodium ion gradient, photoautotrophically grown cells catabolized more intracellular carbohydrates while producing 67% higher yields of hydrogen, acetate, and ethanol (and significant amounts of lactate) as fermentative products. A comparable acceleration of fermentative carbohydrate catabolism occurred upon dissipating the sodium gradient via addition of the sodium-channel blocker quinidine or the sodium-ionophore monensin but not upon dissipating the proton gradient with the proton-ionophore dinitrophenol (DNP). The data demonstrate that intracellular energy is stored via a sodium gradient during autofermentative metabolism and that, when this gradient is blocked, the blockage is compensated by increased energy conversion via carbohydrate catabolism.
Meuser, JE, Boyd ES, Ananyev GM, Karns D, Radakovits R, Narayana Murthy UM, Ghirardi ML, Dismukes CG, Peters JW, Posewitz MC.  2011.  Evolutionary significance of an algal gene encoding an [FeFe]-hydrogenase with F-domain homology and hydrogenase activity in Chlorella variabilis NC64A.. Planta. 234(4):829-43. Abstract
[FeFe]-hydrogenases (HYDA) link the production of molecular H(2) to anaerobic metabolism in many green algae. Similar to Chlamydomonas reinhardtii, Chlorella variabilis NC64A (Trebouxiophyceae, Chlorophyta) exhibits [FeFe]-hydrogenase (HYDA) activity during anoxia. In contrast to C. reinhardtii and other chlorophycean algae, which contain hydrogenases with only the HYDA active site (H-cluster), C. variabilis NC64A is the only known green alga containing HYDA genes encoding accessory FeS cluster-binding domains (F-cluster). cDNA sequencing confirmed the presence of F-cluster HYDA1 mRNA transcripts, and identified deviations from the in silico splicing models. We show that HYDA activity in C. variabilis NC64A is coupled to anoxic photosynthetic electron transport (PSII linked, as well as PSII-independent) and dark fermentation. We also show that the in vivo H(2)-photoproduction activity observed is as O(2) sensitive as in C. reinhardtii. The two C. variabilis NC64A HYDA sequences are similar to homologs found in more deeply branching bacteria (Thermotogales), diatoms, and heterotrophic flagellates, suggesting that an F-cluster HYDA is the ancestral enzyme in algae. Phylogenetic analysis indicates that the algal HYDA H-cluster domains are monophyletic, suggesting that they share a common origin, and evolved from a single ancestral F-cluster HYDA. Furthermore, phylogenetic reconstruction indicates that the multiple algal HYDA paralogs are the result of gene duplication events that occurred independently within each algal lineage. Collectively, comparative genomic, physiological, and phylogenetic analyses of the C. variabilis NC64A hydrogenase has provided new insights into the molecular evolution and diversity of algal [FeFe]-hydrogenases.