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In nitrogen-limited boreal forests, associations between feathermoss and diazotrophic cyanobacteria control nitrogen inputs and thus carbon cycling, but little is known about the molecular regulators required for initiation and maintenance of these associations. Specifically, a benefit to the cyanobacteria is not known, challenging whether the association is a nutritional mutualism. Targeted mutagenesis of the cyanobacterial alkane sulfonate monooxygenase results in an inability to colonize feathermosses by the cyanobacterium Nostoc punctiforme, suggesting a role for organic sulfur in communication or nutrition. Isotope probing paired with high-resolution imaging mass spectrometry (NanoSIMS) demonstrated bidirectional elemental transfer between partners, with carbon and sulfur both being transferred to the cyanobacteria, and nitrogen transferred to the moss. These results support the hypothesis that moss and cyanobacteria enter a mutualistic exosymbiosis with substantial bidirectional material exchange of carbon and nitrogen and potential signaling through sulfur compounds.

Functional genomics studies in minimal mycoplasma cells enable unobstructed access to some of the most fundamental processes in biology. Conventional transposon bombardment and gene knockout approaches often fail to reveal functions of genes that are essential for viability, where lethality precludes phenotypic characterization. Conditional inactivation of genes is effective for characterizing functions central to cell growth and division, but tools are limited for this purpose in mycoplasmas. Here we demonstrate systems for inducible repression of gene expression based on clustered regularly interspaced short palindromic repeats-mediated interference (CRISPRi) in Mycoplasma pneumoniae and synthetic Mycoplasma mycoides, two organisms with reduced genomes actively used in systems biology studies. In the synthetic cell, we also demonstrate inducible gene expression for the first time. Time-course data suggest rapid kinetics and reversible engagement of CRISPRi. Targeting of six selected endogenous genes with this system results in lowered transcript levels or reduced growth rates that agree with lack or shortage of data in previous transposon bombardment studies, and now produces actual cells to analyze. The ksgA gene encodes a methylase that modifies 16S rRNA, rendering it vulnerable to inhibition by the antibiotic kasugamycin. Targeting the ksgA gene with CRISPRi removes the lethal effect of kasugamycin and enables cell growth, thereby establishing specific and effective gene modulation with our system. The facile methods for conditional gene activation and inactivation in mycoplasmas open the door to systematic dissection of genetic programs at the core of cellular life. This work was supported by internal funding from the J. Craig Venter Institute to H.O.S. and C.A.H., as well as grants BIO2013-4870R and BIO2013-50176EXP from the Ministerio de Economía y Competitividad to E.Q. and J.P., respectively, and Japan Society for the Promotion of Science KAKENHI grants JP26710015, JP15KK0266, and JP26106004 to S.K.A.M.M. is a recipient of a predoctoral fellowship from the Generalitat de Catalunya (FI-DGR 2014).

Advances in synthetic biology methods to assemble and edit DNA are enabling genome engineering at a previously impracticable scale and scope. The synthesis of the Mycoplasma mycoides genome followed by its transplantation to convert a related cell into M. mycoides has transformed strain engineering. This approach exemplifies the combination of newly emerging chromosome-scale genome editing strategies that can be defined in three main steps: (1) chromosome acquisition into a microbial engineering platform, (2) alteration and improvement of the acquired chromosome, and (3) installation of the modified chromosome into the original or alternative organism. In this review, we outline recent progress in methods for acquiring chromosomes and chromosome-scale DNA molecules in the workhorse organisms Bacillus subtilis, Escherichia coli, and Saccharomyces cerevisiae. We present overviews of important genetic strategies and tools for each of the three organisms, point out their respective strengths and weaknesses, and highlight how the host systems can be used in combination to facilitate chromosome assembly or engineering. Finally, we highlight efforts for the installation of the cloned/altered chromosomes or fragments into the target organism and present remaining challenges in expanding this powerful experimental approach to a wider range of target organisms.

Bacterial conjugation utilizes a type IV secretion system and a DNA transfer mechanism to deliver DNA from one cell to another. Conjugative partners are conventionally confined to the prokaryotic domain. In a prominent exception, Agrobacterium tumefaciens type IV secretion-mediated transfer of DNA to plant cells can result in subsequent chromosomal integration. Recently, we demonstrated interdomain conjugation from Escherichia coli to the diatom Phaeodactylum tricornutum with the subsequent maintenance of an episome at chromosomal copy numbers if it contains diatom centromeres or centromere-like elements. The genes involved in the conjugation process can be separated into those encoding the type IV secretion system, also called the mating pair formation (MPF) genes, and genes involved in DNA processing called the mobilization (MOB) genes. Various protein families compose each class of conjugation genes, including common MOB types F, P, and Q and MPF types F, P, and T. The conjugative transfer from E. coli to P. tricornutum was demonstrated with a vector expressing MOBP and MTFP. Here we show that the MOBPsystem can be deleted and complemented with a MOBQ system in E.coli-diatom conjugations with subsequent episomal maintenaince. Utilization of both MOBP and MOBQ systems results in substantially higher efficiencies in E. coli-diatom conjugation. Finally, we demonstrate conjugative gene transfer between P. tricornutum and A. tumefaciens expressing a MPFT, the first demonstration of this system in diatoms,resulting in episomal maintainance or chromosomal integration, depending on the ex-conjugant. The promiscuity of MOB and MTF systems permitting prokaryote to diatom conjugative DNA transfer suggest major environmental and evolutionary importance of this process. The increased efficiency of dual MOB systems immediately improves genetic engineering in diatoms and has interesting basic cellular biology implications.

Background Custom synthesized DNA is in high demand for synthetic biology applications. However, current technologies to produce these sequences using assembly from DNA oligonucleotides are costly and labor-intensive. The automation and reduced sample volumes afforded by microfluidic technologies could significantly decrease materials and labor costs associated with DNA synthesis. The purpose of this study was to develop a gene assembly protocol utilizing a digital microfluidic device. Toward this goal, we adapted bench-scale oligonucleotide assembly methods followed by enzymatic error correction to the Mondrian™ digital microfluidic platform. Results We optimized Gibson assembly, polymerase chain reaction (PCR), and enzymatic error correction reactions in a single protocol to assemble 12 oligonucleotides into a 339-bp double- stranded DNA sequence encoding part of the human influenza virus hemagglutinin (HA) gene. The reactions were scaled down to 0.6-1.2 μL. Initial microfluidic assembly methods were successful and had an error frequency of approximately 4 errors/kb with errors originating from the original oligonucleotide synthesis. Relative to conventional benchtop procedures, PCR optimization required additional amounts of MgCl2, Phusion polymerase, and PEG 8000 to achieve amplification of the assembly and error correction products. After one round of error correction, error frequency was reduced to an average of 1.8 errors kb− 1. Conclusion We demonstrated that DNA assembly from oligonucleotides and error correction could be completely automated on a digital microfluidic (DMF) platform. The results demonstrate that enzymatic reactions in droplets show a strong dependence on surface interactions, and successful on-chip implementation required supplementation with surfactants, molecular crowding agents, and an excess of enzyme. Enzymatic error correction of assembled fragments improved sequence fidelity by 2-fold, which was a significant improvement but somewhat lower than expected compared to bench-top assays, suggesting an additional capacity for optimization. Electronic supplementary material The online version of this article (10.1186/s12896-018-0439-9) contains supplementary material, which is available to authorized users.

Centromeres are essential for cell division and growth in all eukaryotes, and knowledge of their sequence and structure guides the development of artificial chromosomes for functional cellular biology studies. Centromeric proteins are conserved among eukaryotes; however, centromeric DNA sequences are highly variable. We combined forward and reverse genetic approaches with chromatin immunoprecipitation to identify centromeres of the model diatom Phaeodactylum tricornutum We observed 25 unique centromere sequences typically occurring once per chromosome, a finding that helps to resolve nuclear genome organization and indicates monocentric regional centromeres. Diatom centromere sequences contain low-GC content regions but lack repeats or other conserved sequence features. Native and foreign sequences with similar GC content to P. tricornutum centromeres can maintain episomes and recruit the diatom centromeric histone protein CENH3, suggesting nonnative sequences can also function as diatom centromeres. Thus, simple sequence requirements may enable DNA from foreign sources to persist in the nucleus as extrachromosomal episomes, revealing a potential mechanism for organellar and foreign DNA acquisition.

Dinitrogen (N2)-fixation by cyanobacteria in symbiosis with feathermosses is the primary pathway of biological nitrogen (N) input into boreal forests. Despite its significance, little is known about the cyanobacterial gene repertoire and regulatory rewiring needed for the establishment and maintenance of the symbiosis. To determine gene acquisitions and regulatory changes allowing cyanobacteria to form and maintain this symbiosis, we compared genomically closely related symbiotic-competent and -incompetent Nostoc strains using a proteogenomics approach and an experimental set up allowing for controlled chemical and physical contact between partners. Thirty-two gene families were found only in the genomes of symbiotic strains, including some never before associated with cyanobacterial symbiosis. We identified conserved orthologs that were differentially expressed in symbiotic strains, including protein families involved in chemotaxis and motility, NO regulation, sulfate/phosphate transport, and glycosyl-modifying and oxidative stress-mediating exoenzymes. The physical moss–cyanobacteria epiphytic symbiosis is distinct from other cyanobacteria–plant symbioses, with Nostoc retaining motility, and lacking modulation of N2-fixation, photosynthesis, GS-GOGAT cycle and heterocyst formation. The results expand our knowledge base of plant–cyanobacterial symbioses, provide a model of information and material exchange in this ecologically significant symbiosis, and suggest new currencies, namely nitric oxide and aliphatic sulfonates, may be involved in establishing and maintaining the cyanobacteria–feathermoss symbiosis.

A sequence-independent method to turn genomic DNA into libraries of hairpins. An efficient way to get a different adapter on each end of a DNA fragment is toclone it and digest the fragment back out. Design the vector to have unique restriction sitesoutside the unique regions you want to put on each end. After digestion, your DNA fragments will have two different "adapters" on each end. Adapted from: Wang L., Zheng J., Luo Y., Xu T., Zhang Q., Zhang L., Xu M., Wan J., Wang MB., Zhang C., Fan Y. 2013. Construction of a genomewide RNAi mutant library in rice. Plant Biotechnology Journal 11:997–1005. DOI: 10.1111/pbi.12093.

Background. The diatom Phaeodactylum tricornutum is a model photosynthetic organism. Functional genomic work in this organism has established a variety of genetic tools including RNA interference (RNAi). RNAi is a post-transcriptional regulatory process that can be utilized to knockdown expression of genes of interest in eukaryotes. RNAi has been previously demonstrated in P. tricornutum, but in practice the efficiency of inducing RNAi is low. Methods. We developed an efficient method for construction of inverted repeat hairpins based on Golden Gate DNA assembly into a Gateway entry vector. The hairpin constructs were then transferred to a variety of destination vectors through the Gateway recombination system. After recombining the hairpin into the destination vector, the resulting expression vector was mobilized into P. tricornutum using direct conjugation from E. coli. Because the hairpin expression vectors had sequences allowing for episomal maintenance in P. tricornutum, we tested whether a consistent, episomal location for hairpin expression improved RNAi induction efficiency. Results. We successfully demonstrated that RNAi could be induced using hairpin constructs expressed from an episome. After testing two different reporter targets and a variety of hairpin sequences with 3 polymerase II and 2 polymerase III promoters, we achieved a maximal RNAi induction efficiency of 25% of lines displaying knockdown of reporter activity by 50% or more. We created many useful genetic tools through this work including Gateway destination vectors for P. tricornutum expression from a variety of polymerase II and III promoters including the P. tricornutum FCPB, H4, and 49202 polymerase II promoters as well as the U6 and snRNA polymerase III promoters. We also created Gateway destination vectors that allow a cassette cloned in an entry vector to be easily recombined into a transcriptional fusion with either ShBle or, for polymerase III promoters, the green fluorescent Spinach aptamer. Such transcriptional fusions allow for linkage of expression with a marker such as bleomycin resistance or fluorescence from the Spinach aptamer to easily select or screen for lines that maintain transgene expression. Discussion. While RNAi can be used as an effective tool for P. tricornutum genetics, especially where targeted knockouts may be lethal to the cell, induction of this process remains low efficiency. Techniques resulting in higher efficiency establishment of RNAi would be of great use to the diatom genetics community and would enable this technique to be used as a forward genetic tool for discovery of novel gene function.

An easy to follow template for adding Golden Gate adapters to PCR primers.

A protocol to create a genomic library (2-5 kb insert size) to screen forgenomic regions functioning as centromeres. Uses the NEBNext Illumina library prep kit from NEB to add adapters to fragments and then the fragments can be efficiently assembled into a vector containing homology to the adapter sequences using Gibson Assembly.

A conventient extraction and measurement protocol for GUS activity from Phaeodactylum tricornutum.

Summary Diatoms are unicellular photosynthetic algae with promise for green production of fuels and other chemicals. Recent genome-editing techniques have greatly improved the potential of many eukaryotic genetic systems, including diatoms, to enable knowledge-based studies and bioengineering. Using a new technique, transcription activator-like effector nucleases (TALENs), the gene encoding the urease enzyme in the model diatom, Phaeodactylum tricornutum, was targeted for interruption. The knockout cassette was identified within the urease gene by PCR and Southern blot analyses of genomic DNA. The lack of urease protein was confirmed by Western blot analyses in mutant cell lines that were unable to grow on urea as the sole nitrogen source. Untargeted metabolomic analysis revealed a build-up of urea, arginine and ornithine in the urease knockout lines. All three intermediate metabolites are upstream of the urease reaction within the urea cycle, suggesting a disruption of the cycle despite urea production. Numerous high carbon metabolites were enriched in the mutant, implying a breakdown of cellular C and N repartitioning. The presented method improves the molecular toolkit for diatoms and clarifies the role of urease in the urea cycle.

Centromeres are essential for cell division and growth in all eukaryotes, and knowledge of their sequence and structure guides the development of artificial chromosomes for functional cellular biology studies. Centromeric proteins are conserved among eukaryotes; however, centromeric DNA sequences are highly variable. We combined forward and reverse genetic approaches with chromatin immunoprecipitation to identify centromeres of the model diatom Phaeodactylum tricornutum. Diatom centromere sequences contain low GC content regions and an abundance of long contiguous AT windows, but lack repeats or other conserved sequence features. Native and foreign sequences of similar GC content can maintain episomes and recruit the diatom centromeric histone protein CENP-A, suggesting non-native sequences can also function as diatom centromeres. Thus, simple sequence requirements enable DNA from foreign sources to incorporate into the nuclear genome repertoire as stable extra-chromosomal episomes, revealing a potential mechanism for bacterial and foreign eukaryotic DNA acquisition.

Site-directed mutagenesis is a commonly used molecular biology technique to manipulate biological sequences, and is especially useful for studying sequence determinants of enzyme function or designing proteins with improved activity. We describe a strategy using Gibson Isothermal DNA Assembly to perform site-directed mutagenesis on large (~20 kbp) constructs that are outside the effective range of standard techniques such as QuikChange II (Agilent Technologies), but more reliable than traditional cloning using restriction enzymes and ligation.

Conjugation of episomal plasmids from bacteria to diatoms advances diatom genetic manipulation by simplifying transgene delivery and providing a stable and consistent gene expression platform. To reach its full potential, this nascent technology requires new optimized expression vectors and a deeper understanding of episome maintenance. Here, we present the development of an additional diatom vector (pPtPBR1), based on the parent plasmid pBR322, to add a plasmid maintained at medium copy number in Escherichia coli to the diatom genetic toolkit. Using this new vector, we evaluated the contribution of individual yeast DNA elements comprising the 1.4-kb tripartite CEN6-ARSH4-HIS3 sequence that enables episome maintenance in Phaeodactylum tricornutum. While various combinations of these individual elements enable efficient conjugation and high exconjugant yield in P. tricornutum, individual elements alone do not. Conjugation of episomes containing CEN6-ARSH4 and a small sequence from the low GC content 3' end of HIS3 produced the highest number of diatom exconjugant colonies, resulting in a smaller and more efficient vector design. Our findings suggest that the CEN6 and ARSH4 sequences function differently in yeast and diatoms, and that low GC content regions of greater than ~500 bp are a potential indicator of a functional diatom episome maintenance sequence. Additionally, we have developed improvements to the conjugation protocol including a high-throughput option utilizing 12-well plates and plating methods that improve exconjugant yield and reduce time and materials required for the conjugation protocol. The data presented offer additional information regarding the mechanism by which the yeast-derived sequence enables diatom episome maintenance and demonstrate options for flexible vector design.

Alteromonas macleodii Deep ecotype is a marine, heterotrophic, gammaproteobacterium isolated in the Mediterranean Sea between depths of 1000 and 3500 m. The sequenced strain was previously reported to contain a [NiFe] hydrogenase. We verified the presence of this hydrogenase in other strains of A. macleodii Deep ecotype that were previously isolated from several bathypelagic microenvironments. We developed a system for the genetic manipulation of A. macleodii Deep ecotype using conjugation and used this system to create mutant strains that lack the [NiFe] hydrogenase structural genes (hynSL). The mutants did not possess hydrogenase activity, and complementation of the mutant strain with the hynSL genes successfully restored hydrogenase activity. Both the mutant and the wild-type strains grew at the same rate in a variety of media and under different environmental conditions, indicating little effect of the hydrogenase mutation under the conditions tested.

HynSL fromAlteromonas macleodii‘deep ecotype’ (AltDE) is an oxygen-tolerant and thermostable [NiFe] hydrogenase. Its two structural genes (hynSL), encoding small and large hydrogenase subunits, are surrounded by eight genes (hynD,hupHandhypCABDFE) predicted to encode accessory proteins involved in maturation of the hydrogenase. A 13 kb fragment containing the ten structural and accessory genes along with three additional adjacent genes (orf2,cytandorf1) was cloned into an IPTG-inducible expression vector and transferred into anEscherichia colimutant strain lacking its native hydrogenases. Upon induction, HynSL from AltDE was expressed inE. coliand was active, as determined by anin vitrohydrogen evolution assay. Subsequent genetic analysis revealed thatorf2,cyt,orf1andhupHare not essential for assembling an active hydrogenase. However,hupHandorf2can enhance the activity of the heterologously expressed hydrogenase. We used this genetic system to compare maturation mechanisms between AltDE HynSL and itsThiocapsa roseopersicinahomologue. When the structural genes for theT. roseopersicinahydrogenase,hynSL, were expressed along with knownT. roseopersicinaaccessory genes (hynD, hupK, hypC1C2andhypDEF), no active hydrogenase was produced. Further, co-expression of AltDE accessory geneshypAandhypBwith the entire set of theT. roseopersicinagenes did not produce an active hydrogenase. However, co-expression of all AltDE accessory genes with theT. roseopersicinastructural genes generated an activeT. roseopersicinahydrogenase. This result demonstrates that the accessory genes from AltDE can complement their counterparts fromT. roseopersicinaand that the two hydrogenases share similar maturation mechanisms.

Nitrogenase produces hydrogen as a normal byproduct of the reduction of dinitrogen to ammonia. The Nif2 nitrogenase in Anabaena variabilis is an alternative Mo-nitrogenase and is expressed in vegetative cells grown with fructose under strictly anaerobic conditions. We report here that the V75I substitution in the α-subunit of Nif2 showed greatly impaired acetylene reduction and reduced levels of 15N2 fixation but had similar hydrogen production rates as the wild-type enzyme under argon. Another mutant containing a substitution in the α-subunit, V76I, would result in a decrease in the size of the putative gas channel of nitrogenase and, thus, was hypothesized to affect substrate selectivity of nitrogenase. However, this substitution had no effect on the enzyme selectivity, suggesting that access by gases to the active site through this putative gas channel is not limited by the increased size of the amino acid side chain in the α-subunit, V76I substitution.

Nitrogen-fixing cyanobacteria such as Anabaena variabilis ATCC 29413 use an uptake hydrogenase, encoded by hupSL , to recycle hydrogen gas that is produced as an obligate by-product of nitrogen fixation. The regulation of hupSL in A. variabilis is likely to differ from that of the closely related Anabaena sp. strain PCC 7120 because A. variabilis lacks the excision element-mediated regulation that characterizes hupSL regulation in strain PCC 7120. An analysis of the hupSL transcript in a nitrogenase mutant of A. variabilis that does not produce any detectable hydrogen indicated that neither nitrogen fixation nor hydrogen gas was required for the induction of hupSL . Furthermore, exogenous addition of hydrogen gas did not stimulate hupSL transcription. Transcriptional reporter constructs indicated that the accumulation of hupSL transcript after nitrogen step-down was restricted primarily to the microaerobic heterocysts. Anoxic conditions were not sufficient to induce hupSL transcription. The induction of hupSL after nitrogen step-down was reduced in a mutant in the global nitrogen regulator NtcA, but was not reduced in a mutant unable to form heterocysts. A consensus NtcA-binding site was identified upstream of hupSL , and NtcA was found to bind to this region. Thus, while neither hydrogen gas nor anoxia controlled the expression of hupSL , its expression was controlled by NtcA. Heterocyst differentiation was not required for hupSL induction in response to nitrogen step-down, but heterocyst-localized cues may add an additional level of regulation to hupSL .

Eukaryotic microalgae hold great promise for the bioproduction of fuels and higher value chemicals. However, compared with model genetic organisms such as Escherichia coli and Saccharomyces cerevisiae, characterization of the complex biology and biochemistry of algae and strain improvement has been hampered by the inefficient genetic tools. To date, many algal species are transformable only via particle bombardment, and the introduced DNA is integrated randomly into the nuclear genome. Here we describe the first nuclear episomal vector for diatoms and a plasmid delivery method via conjugation from Escherichia coli to the diatoms Phaeodactylum tricornutum and Thalassiosira pseudonana. We identify a yeast-derived sequence that enables stable episome replication in these diatoms even in the absence of antibiotic selection and show that episomes are maintained as closed circles at copy number equivalent to native chromosomes. This highly efficient genetic system facilitates high-throughput functional characterization of algal genes and accelerates molecular phytoplankton research., Algae hold great promise for biofuel and chemical production but their use as model systems is hampered by the absence of suitable genetic tools. Here Karas et al. present a nuclear episomal vector for diatoms that is maintained in the absence of antibiotics, and a plasmid delivery method via conjugation with E. coli.

Photobiological hydrogen production is an attractive, carbon-neutral means to convert solar energy to hydrogen. We build on previous research improving the Alteromonas macleodii “Deep Ecotype” [NiFe] hydrogenase, and report progress towards creating an artificial electron transfer pathway to supply the hydrogenase with electrons necessary for hydrogen production. Ferredoxin is the first soluble electron transfer mediator to receive high-energy electrons from photosystem I, and bears an electron with sufficient potential to efficiently reduce protons. Thus, we engineered a hydrogenase-ferredoxin fusion that also contained several other modifications. In addition to the C-terminal ferredoxin fusion, we truncated the C-terminus of the hydrogenase small subunit, identified as the available terminus closer to the electron transfer region. We also neutralized an anionic patch surrounding the interface Fe-S cluster to improve transfer kinetics with the negatively charged ferredoxin. Initial screening showed the enzyme tolerated both truncation and charge neutralization on the small subunit ferredoxin-binding face. While the enzyme activity was relatively unchanged using the substrate methyl viologen, we observed a marked improvement from both the ferredoxin fusion and surface modification using only dithionite as an electron donor. Combining ferredoxin fusion and surface charge modification showed progressively improved activity in an in vitro assay with purified enzyme.

Cold and freezing damage to plants can be mitigated by inducible factors during an acclimation period. DEA1 is a circadian-regulated tomato (Solanum lycopersicum) gene with sequence similarity to EARLI1, an Arabidopsis thaliana gene that confers cold protection. To investigate whether DEA1 was responsive to environmental variables such as cold, cold-treated tomatoes were analyzed for DEA1 expression. DEA1 transcript accumulated in response to cold, and the rapidity of the cold-induced transcript accumulation was regulated by the circadian rhythm. To test whether DEA1 could protect cells from freezing damage, we transformed the yeast, Pichia pastoris, with an inducible DEA1 construct. Yeast cells transformed with the gene survived freezing at a significantly higher rate than control strains and a strain expressing the LacZ gene. Transgenic tomato plants over-expressing or knocking down DEA1 transcript levels did not have an altered phenotype with respect to cold- or pathogen-susceptibility relative to control plants.

Direct cell-to-cell transfer of genomes from bacteria to yeast facilitates genome engineering for bacteria that are not amenable to genetic manipulation by allowing instead for the utilization of the powerful yeast genetic tools. Here we describe a protocol for transferring whole genomes from bacterial cells to yeast spheroplasts without any DNA purification process. The method is dependent on the treatment of the bacterial and yeast cellular mixture with PEG, which induces cell fusion, engulfment, aggregation or lysis. Over 80% of the bacterial genomes transferred in this way are complete, on the basis of structural and functional tests. Excluding the time required for preparing starting cultures and for incubating cells to form final colonies, the protocol can be completed in 3 h.

Plants are often simultaneously challenged by pathogens and insects capable of triggering an array of responses that may be beneficial or detrimental to the plant. The efficacy of resistance mechanisms can be strongly influenced by the mix of signals generated by biotic stress as well as abiotic stress such as drought, nutrient limitation or high soil salinity. An understanding of their biochemical nature, and knowledge of the specificity and compatibility of the signaling systems that regulate the expression of inducible responses could optimize the utilization of these responses in crop protection. Signaling conflicts and synergies occur during a plant's response to pathogens and insect herbivores, and much of the research on defense signaling has focused on salicylate- and jasmonate-mediated responses. We will review our results using tomato (Lycopersicon esculentum) in greenhouse and field studies that illustrate a trade-off between salicylate- and jasmonate-mediated signaling, and discuss research on strategies to minimize the trade-off that can occur following the application of chemical elicitors of resistance. In addition, there is evidence of another signaling system that mediates endogenous levels of ceramide in the plant. This signal is associated with programmed cell death and protection of tomato against the fungal pathogen Alternaria alternata f. sp. lycopersici.

The ability to assemble large pieces of prokaryotic DNA by yeast recombination has great application in synthetic biology, but cloning large pieces of high G+C prokaryotic DNA in yeast can be challenging. Additional considerations in cloning large pieces of high G+C DNA in yeast may be related to toxic genes, to the size of the DNA, or to the absence of yeast origins of replication within the sequence. As an example of our ability to clone high G+C DNA in yeast, we chose to work with Synechococcus elongatus PCC 7942, which has an average G+C content of 55%. We determined that no regions of the chromosome are toxic to yeast and that S. elongatus DNA fragments over ~200 kb are not stably maintained. DNA constructs with a total size under 200 kb could be readily assembled, even with 62 kb of overlapping sequence between pieces. Addition of yeast origins of replication throughout allowed us to increase the total size of DNA that could be assembled to at least 454 kb. Thus, cloning strategies utilizing yeast recombination with large, high G+C prokaryotic sequences should include yeast origins of replication as a part of the design process.

Background Photosynthetic microorganisms that directly channel solar energy to the production of molecular hydrogen are a potential future biofuel system. Building such a system requires installation of a hydrogenase in the photosynthetic organism that is both tolerant to oxygen and capable of hydrogen production. Toward this end, we have identified the [NiFe] hydrogenase from the marine bacterium Alteromonas macleodii “Deep ecotype” that is able to be heterologously expressed in cyanobacteria and has tolerance to partial oxygen. The A. macleodii enzyme shares sequence similarity with the uptake hydrogenases that favor hydrogen uptake activity over hydrogen evolution. To improve hydrogen evolution from the A. macleodii hydrogenase, we examined the three Fe-S clusters found in the small subunit of many [NiFe] uptake hydrogenases that presumably act as a molecular wire to guide electrons to or from the active site of the enzyme. Studies by others altering the medial cluster of a Desulfovibrio fructosovorans hydrogenase from 3Fe-4S to 4Fe-4S resulted in two-fold improved hydrogen evolution activity. Results We adopted a strategy of screening for improved hydrogenase constructs using an Escherichia coli expression system before testing in slower growing cyanobacteria. From the A. macleodii enzyme, we created a mutation in the gene encoding the hydrogenase small subunit that in other systems is known to convert the 3Fe-4S medial cluster to 4Fe-4S. The medial cluster substitution did not improve the hydrogen evolution activity of our hydrogenase. However, modifying both the medial cluster and the ligation of the distal Fe-S cluster improved in vitro hydrogen evolution activity relative to the wild type hydrogenase by three- to four-fold. Other properties of the enzyme including thermostability and tolerance to partial oxygen did not appear to be affected by the substitutions. Conclusions Our results show that substitution of amino acids altering the ligation of Fe-S clusters in the A. macleodii [NiFe] uptake hydrogenase resulted in increased hydrogen evolution activity. This activity can be recapitulated in multiple host systems and with purified protein. These results validate the approach of using an E. coli-cyanobacteria shuttle system for enzyme expression and improvement.

Transfer of genomes into yeast facilitates genome engineering for genetically intractable organisms, but this process has been hampered by the need for cumbersome isolation of intact genomes before transfer. Here we demonstrate direct cell-to-cell transfer of bacterial genomes as large as 1.8 megabases (Mb) into yeast under conditions that promote cell fusion. Moreover, we discovered that removal of restriction endonucleases from donor bacteria resulted in the enhancement of genome transfer.

Alteromonas macleodii Deep ecotype is a marine, heterotrophic, gammaproteobacterium isolated in the Mediterranean Sea between depths of 1000 and 3500 m. The sequenced strain was previously reported to contain a [NiFe] hydrogenase. We verified the presence of this hydrogenase in other strains of A. macleodii Deep ecotype that were previously isolated from several bathypelagic microenvironments. We developed a system for the genetic manipulation of A. macleodii Deep ecotype using conjugation and used this system to create mutant strains that lack the [NiFe] hydrogenase structural genes (hynSL). The mutants did not possess hydrogenase activity, and complementation of the mutant strain with the hynSL genes successfully restored hydrogenase activity. Both the mutant and the wild-type strains grew at the same rate in a variety of media and under different environmental conditions, indicating little effect of the hydrogenase mutation under the conditions tested.

Hydrogenases are enzymes involved in the bioproduction of hydrogen, a clean alternative energy source whose combustion generates water as the only end product. In this article we identified and characterized a [NiFe] hydrogenase from the marine bacterium Alteromonas macleodii “deep ecotype” with unusual stability toward oxygen and high temperature. The A. macleodii hydrogenase (HynSL) can catalyze both H 2 evolution and H 2 uptake reactions. HynSL was expressed in A. macleodii under aerobic conditions and reached the maximum activity when the cells entered the late exponential phase. The higher level of hydrogenase activity was accompanied by a greater abundance of the HynSL protein in the late-log or stationary phase. The addition of nickel to the growth medium significantly enhanced the hydrogenase activity. Ni treatment affected the level of the protein, but not the mRNA, indicating that the effect of Ni was exerted at the posttranscriptional level. Hydrogenase activity was distributed ∼30% in the membrane fraction and ∼70% in the cytoplasmic fraction. Thus, HynSL appears to be loosely membrane-bound. Partially purified A. macleodii hydrogenase demonstrated extraordinary stability. It retained 84% of its activity after exposure to 80°C for 2 h. After exposure to air for 45 days at 4°C, it retained nearly 100% of its activity when assayed under anaerobic conditions. Its catalytic activity in the presence of O 2 was evaluated by the hydrogen-deuterium (H-D) exchange assay. In 1% O 2 , 20.4% of its H-D exchange activity was retained. The great stability of HynSL makes it a potential candidate for biotechnological applications.

A cDNA clone of unknown function, DEA1, was isolated from arachidonic acid-treated tomato (Solanum lycopersicum) leaves by differential display PCR. The gene, DEA1, is expressed in response to the programmed cell death-inducing arachidonic acid within 8 h following treatment of a tomato leaflet, 16 h prior to the development of visible cell death. DEA1 transcript levels were also affected by the late blight pathogen ,Phytophthora infestans. To gain further insight into the transcriptional regulation of DEA1, the promoter region was cloned by inverse PCR and was found to contain putative stress-, signaling-, and circadian-response elements. DEA1 is highly expressed in roots, stems, and leaves, but not in flowers. Leaf expression of DEA1 is regulated by circadian rhythms during long days with the peak occurring at midday and the low point midway through the dark period. During short days, the rhythm is lost and DEA1 expression becomes constitutive. The predicted DEA1 protein has a conserved domain shared by the eight-cysteine motif superfamily of protease inhibitors, α-amylase inhibitors, seed storage proteins, and lipid transfer proteins. A DEA1-green fluorescent protein fusion protein localized to the plasma membrane in protoplasts and plasmolysis experiments, suggesting that the native protein is associated with the plasmalemma in intact cells.

Background In order to understand the effects of FeS cluster attachment in [NiFe] hydrogenase, we undertook a study to substitute all 12 amino acid positions normally ligating the three FeS clusters in the hydrogenase small subunit. Using the hydrogenase from Alteromonas macleodii “deep ecotype” as a model, we substituted one of four amino acids (Asp, His, Asn, Gln) at each of the 12 ligating positions because these amino acids are alternative coordinating residues in otherwise conserved-cysteine positions found in a broad survey of NiFe hydrogenase sequences. We also hoped to discover an enzyme with elevated hydrogen evolution activity relative to a previously reported “G1” (H230C/P285C) improved enzyme in which the medial FeS cluster Pro and the distal FeS cluster His were each substituted for Cys. Results Among all the substitutions screened, aspartic acid substitutions were generally well-tolerated, and examination suggests that the observed deficiency in enzyme activity may be largely due to misprocessing of the small subunit of the enzyme. Alignment of hydrogenase sequences from sequence databases revealed many rare substitutions; the five substitutions present in databases that we tested all exhibited measurable hydrogen evolution activity. Select substitutions were purified and tested, supporting the results of the screening assay. Analysis of these results confirms the importance of small subunit processing. Normalizing activity to quantity of mature small subunit, indicative of total enzyme maturation, weakly suggests an improvement over the “G1” enzyme. Conclusions We have comprehensively screened 48 amino acid substitutions of the hydrogenase from A. macleodii “deep ecotype”, to understand non-canonical ligations of amino acids to FeS clusters and to improve hydrogen evolution activity of this class of hydrogenase. Our studies show that non-canonical ligations can be functional and also suggests a new limiting factor in the production of active enzyme.

Oxygen-tolerant [NiFe] hydrogenases may be used in future photobiological hydrogen production systems once the enzymes can be heterologously expressed in host organisms of interest. To achieve heterologous expression of [NiFe] hydrogenases in cyanobacteria, the two hydrogenase structural genes from Alteromonas macleodii Deep ecotype (AltDE), hynS and hynL, along with the surrounding genes in the gene operon of HynSL were cloned in a vector with an IPTG-inducible promoter and introduced into Synechococcus elongatus PCC7942. The hydrogenase protein was expressed at the correct size upon induction with IPTG. The heterologously-expressed HynSL hydrogenase was active when tested by in vitro H(2) evolution assay, indicating the correct assembly of the catalytic center in the cyanobacterial host. Using a similar expression system, the hydrogenase structural genes from Thiocapsa roseopersicina (hynSL) and the entire set of known accessory genes were transferred to S. elongatus. A protein of the correct size was expressed but had no activity. However, when the 11 accessory genes from AltDE were co-expressed with hynSL, the T. roseopersicina hydrogenase was found to be active by in vitro assay. This is the first report of active, heterologously-expressed [NiFe] hydrogenases in cyanobacteria.

Summary Background The cyanobacterial circadian program exerts genome-wide control of gene expression. KaiC undergoes rhythms of phosphorylation that are regulated by interactions with KaiA and KaiB. The phosphorylation status of KaiC is thought to mediate global transcription via output factors SasA, CikA, LabA, RpaA, and RpaB. Overexpression of kaiC has been reported to globally repress gene expression. Results Here, we show that the positive circadian component KaiA upregulates "subjective dusk" genes and that its overexpression deactivates rhythmic gene expression without significantly affecting growth rates in constant light. We analyze the global patterns of expression that are regulated by KaiA versus KaiC and find in contrast to the previous report of KaiC repression that there is a "yin-yang" regulation of gene expression whereby kaiA overexpression activates "dusk genes" and represses "dawn genes," whereas kaiC overexpression complementarily activates dawn genes and represses dusk genes. Moreover, continuous induction of kaiA latched KaiABC-regulated gene expression to provide constitutively increased transcript levels of diverse endogenous and heterologous genes that are expressed in the predominant subjective dusk phase. In addition to analyzing KaiA regulation of endogenous gene expression, we apply these insights to the expression of heterologous proteins whose products are of potential value, namely human proinsulin, foreign luciferase, and exogenous hydrogenase. Conclusions Both KaiC and KaiA complementarily contribute to the regulation of circadian gene expression via yin-yang switching. Circadian patterns can be reprogrammed by overexpression of kaiA or kaiC to constitutively enhance gene expression, and this reprogramming can improve 24/7 production of heterologous proteins that are useful as pharmaceuticals or biofuels.

Background Synthetic genomic approaches offer unique opportunities to use powerful yeast and Escherichia coli genetic systems to assemble and modify chromosome-sized molecules before returning the modified DNA to the target host. For example, the entire 1 Mb Mycoplasma mycoides chromosome can be stably maintained and manipulated in yeast before being transplanted back into recipient cells. We have previously demonstrated that cloning in yeast of large (> ~ 150 kb), high G + C (55%) prokaryotic DNA fragments was improved by addition of yeast replication origins every ~100 kb. Conversely, low G + C DNA is stable (up to at least 1.8 Mb) without adding supplemental yeast origins. It has not been previously tested whether addition of yeast replication origins similarly improves the yeast-based cloning of large (>150 kb) eukaryotic DNA with moderate G + C content. The model diatom Phaeodactylum tricornutum has an average G + C content of 48% and a 27.4 Mb genome sequence that has been assembled into chromosome-sized scaffolds making it an ideal test case for assembly and maintenance of eukaryotic chromosomes in yeast. Results We present a modified chromosome assembly technique in which eukaryotic chromosomes as large as ~500 kb can be assembled from cloned ~100 kb fragments. We used this technique to clone fragments spanning P. tricornutum chromosomes 25 and 26 and to assemble these fragments into single, chromosome-sized molecules. We found that addition of yeast replication origins improved the cloning, assembly, and maintenance of the large chromosomes in yeast. Furthermore, purification of the fragments to be assembled by electroelution greatly increased assembly efficiency. Conclusions Entire eukaryotic chromosomes can be successfully cloned, maintained, and manipulated in yeast. These results highlight the improvement in assembly and maintenance afforded by including yeast replication origins in eukaryotic DNA with moderate G + C content (48%). They also highlight the increased efficiency of assembly that can be achieved by purifying fragments before assembly.