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Brief Note: Diet of the Rainbow Darter (Etheostoma caeruleum) in Rock Run, Clark County, Ohio
(1988-12) Stewart, Craig A.
The diet of the rainbow darter, Etheostoma caeruleum, was studied during fall and winter in Rock Run (Clark County, Ohio), a second-order tributary of the Mad River. Chironomid larvae comprised over half of the diet (by numerical percentage) during both seasons. Other important components of the diet were simuliid larvae, trichopteran larvae (Cheumatopsyche and Hydropsyche) and coleopteran larvae (Stenelmis). Both chironomid and simuliid larvae appeared in the diet of E. caeruleum in greater abundance than they appeared in the available benthic macroinvertebrate populations. Stenelmis larvae comprised a smaller portion of the diet than of the benthic macroinvertebrate population. Etheostoma caeruleum typically feeds heavily on dipteran, trichopteran, and ephemeropteran larvae, but other locally abundant prey may also be an important part of the diet. It appears that dietary selectivity of E. caeruleum may be greater during the winter than during the fall.
A note on the microdistribution of the Red-Backed Vole, Clethrionomys gapperi, in the E.N. Huyck Preserve, New York
(1991) Stewart, Craig A.
In a trapping study of small mammals on the E.N. Huyck Preserve (Rensselaerville, New York) it was found that Chlethrionomys gapperi was captured only in the immediate vicinity of abandoned stone fences. Peromyscus leucopus were common throughout the study area. Captrues of P. leucopus did not seem related to the presence or absence of abandoned stone fences. Thus, interspecific interactions seem unlikely to restrict C. gapperi to the areas near abandoned stone fences. The latter may provide essential nesting habitat or protection from predators for Red-backed Voles.
The Thermodynamics of Virus Capsid Assembly
(Elsevier, 2009) Zlotnick, Adam; Katen, Sarah
Virus capsid assembly is a critical step in the viral life cycle. The underlying basis of capsid stability is key to understanding this process. Capsid subunits interact with weak individual contact energies to form a globally stable icosahedral lattice; this structure is ideal for encapsidating the viral genome and host partners and protecting its contents upon secretion, yet the unique properties of its assembly and inter-subunit contacts allow the capsid to dissociate upon entering a new host cell. The stability of the capsid can be analyzed by treating capsid assembly as an equilibrium polymerization reaction, modified from the traditional polymer model to account for the fact that a separate nucleus is formed for each individual capsid. From the concentrations of reactants and products in an equilibrated assembly reaction, it is possible to extract the thermodynamic parameters of assembly for a wide array of icosahedral viruses using well-characterized biochemical and biophysical methods. In this chapter we describe this basic analysis and provide examples of thermodynamic assembly data for several different icosahedral viruses. These data provide new insights into the assembly mechanisms of spherical virus capsids, as well as into the biology of the viral life cycle.
Role of conserved cysteines in the alphavirus E3 protein
(American Society for Microbiology, 2009) Parrott, Megan M.; Sitarski, Sarah A.; Arnold, Randy J.; Picton, Lora K.; Hill, R. Blake; Mukhopadhyay, Suchetana
Alphavirus particles are covered by 80 glycoprotein spikes that are essential for viral entry. Spikes consist of the E2 receptor binding protein and the E1 fusion protein. Spike assembly occurs in the endoplasmic reticulum, where E1 associates with pE2, a precursor containing E3 and E2 proteins. E3 is a small, cysteine-rich, extracellular glycoprotein that mediates proper folding of pE2 and its subsequent association with E1. In addition, cleavage of E3 from the assembled spike is required to make the virus particles efficiently fusion competent. We have found that the E3 protein in Sindbis virus contains one disulfide bond between residues Cys19 and Cys25. Replacing either of these two critical cysteines resulted in mutants with attenuated titers. Replacing both cysteines with either alanine or serine resulted in double mutants that were lethal. Insertion of additional cysteines based on E3 proteins from other alphaviruses resulted in either sequential or nested disulfide bond patterns. E3 sequences that formed sequential disulfides yielded virus with near-wild-type titers, while those that contained nested disulfide bonds had attenuated activity. Our data indicate that the role of the cysteine residues in E3 is not primarily structural. We hypothesize that E3 has an enzymatic or functional role in virus assembly, and these possibilities are further discussed.
Elevated CO2 increases root exudation from loblolly pine (Pinus taeda L.) seedlings as an N-mediated response
(Oxford University Press, 2009-10-08) Schlesinger, William; Bernhardt, Emily; Phillips, Richard P.
The degree to which forest ecosystems provide a long-term sink for increasing atmospheric CO2 depends upon the capacity of trees to increase the availability of growth-limiting resources. It has been widely speculated that trees exposed to CO2 enrichment may increase the release of root exudates to soil as a mechanism to stimulate microbes to enhance nutrient availability. As a first test to examine how the atmospheric CO2 and nitrogen availability affect the rates of root exudation, we performed two experiments in which the exudates were collected from loblolly pine (Pinus taeda L.) seedlings that were grown in controlled growth chambers under low and high CO2 and at low and high rates of N supply. Despite the differences in experimental design between the two studies, plants grown at high CO2 were larger, and thus whole plant exudation rates were higher under elevated CO2 (P = 0.019), but the magnitude of this response depended on the N level in both studies. Seedlings increased mass-specific exudation rates in response to elevated CO2 in both experiments, but only at low N supply. Moreover, N supply had a greater impact on the exudation rates than did CO2, with mass-specific exudation rates significantly greater (98% and 69% in Experiments 1 and 2, respectively) in the seedlings grown at low N supply relative to high N supply. These results provide preliminary evidence that loblolly pines alter exudation rates in response to both CO2 concentration and N supply, and support the hypothesis that increased C allocation to root exudates may be a mechanism by which trees could delay progressive N limitation in forested ecosystems.
Virus assembly, allostery, and antivirals
(Elsevier, 2010-12-28) Mukhopadhyay, Suchetana; Zlotnick, Adam
Assembly of virus capsids and surface proteins must be regulated to ensure that the resulting complex is an infectious virion. In this review, we examine assembly of virus capsids, focusing on hepatitis B virus and bacteriophage MS2, and formation of glycoproteins in the alphaviruses. These systems are structurally and biochemically well-characterized and are simplest-case paradigms of self-assembly. Published data suggest that capsid and glycoprotein assembly is subject to allosteric regulation, that is regulation at the level of conformational change. The hypothesis that allostery is a common theme in viruses suggests that deregulation of capsid and glycoprotein assembly by small molecule effectors will be an attractive antiviral strategy, as has been demonstrated with hepatitis B virus.
Generating enveloped virus-like particles with in vitro assembled cores
(Elsevier, 2011-02-19) Mukhopadhyay, Suchetana; Cheng, Fan
Alphaviruses are comprised of a nucleocapsid core surrounded by a lipid membrane containing glycoprotein spikes. Previous work demonstrated that in vitro assembled core-like particles are similar in structure to the nucleocapsid core in the native virus. Here we demonstrate that in vitro assembled core-like particles can be inserted into viral glycoprotein-expressing cells to generate enveloped virus-like particles. These virus-like particles bud from cells like native virus, are similar in size to the native virus, and can enter cells to release the contents of the core-like particle into the cytoplasm of the cell. Virus-like particles can be used to infect cells with biological and non-biological cargoes. The generation of enveloped virus-like particles containing an in vitro core and in vivo synthesized glycoproteins has applications for gene and drug delivery, medical imaging, and also basic mechanistic studies of virus assembly.
Conspecific plant-soil feedbacks of temperate tree species in the southern Appalachians, USA
(PLOS ONE, 2012) Reinhart, K.O.; Johnson, D.; Clay, K.
Many tree species have seedling recruitment patterns suggesting that they are affected by non-competitive distance-dependent sources of mortality. We conducted an experiment, with landscape-level replication, to identify cases of negative distance-dependent effects and whether variation in these effects corresponded with tree recruitment patterns in the southern Appalachian Mountains region. Specifically, soil was collected from 14 sites and used as inocula in a 62 day growth chamber experiment determining whether tree seedling growth was less when interacting with soil from conspecific (like) than heterospecific (other) tree species. Tests were performed on six tree species. Three of the tree species had been previously described as having greater recruitment around conspecifics (i.e. facilitator species group) compared to the other half (i.e. inhibitor species group). We were then able to determine whether variation in negative distance-dependent effects corresponded with recruitment patterns in the field. Across the six species, none were negatively affected by soil inocula from conspecific relative to heterospecific sources. Most species (four of six) were unaffected by soil source. Two species (Prunus serotina and Tsuga canadensis) had enhanced growth in pots inoculated with soil from conspecific trees vs. heterospecifics. Species varied in their susceptibility to soil pathogens, but trends across all species revealed that species classified as inhibitors were not more negatively affected by conspecific than heterospecific soil inocula or more susceptible to pathogenic effects than facilitators. Although plant-soil biota interactions may be important for individual species and sites, it may be difficult to scale these interactions over space or levels of ecological organization. Generalizing the importance of plant-soil feedbacks or other factors across regional scales may be especially problematic for hyperdiverse temperate forests where interactions may be spatially variable.
Natal-host environmental effects on juvenile size, transmission success, and operational sex ratio in the entomopathogenic nematode Steinernema carpocapsae
(American Society of Parasitologists, 2012) Therese, M.O.; Bashey, F.
Trans-host effects can alter the ecological and evolutionary dynamics of parasite and host populations. Here, we examine whether resource limitation within a parasite's natal host affects propagule size and influences parasite fitness in a new host. To alter resource competition, we infected caterpillars with 3 doses of the nematode Steinernema carpocapsae and harvested transmission-stage juveniles either early or late in the infection. We measured the size of these juveniles, and then we examined their ability to colonize and their sex ratio upon maturity in a new host. We found a trade-off between the cumulative number and size of nematodes emerging from a host. Emerging nematode size declined significantly over the time course of the infection, but dose had no significant effects. Larger, early emerging nematodes had greater success in colonizing a new host than smaller, later emerging nematodes, independently of whether they needed to locate the host. Furthermore, although early emerging nematodes resulted in an equal sex ratio in the new host, late emerging nematodes resulted in female-biased populations. These transmission and sex-ratio effects demonstrate that conditions in the natal host can affect parasite fitness, and they suggest that trans-host effects need to be more fully integrated into our studies of parasite populations.
Global Patterns of Tissue-Specific Alternative Polyadenylation in Drosophila
(Cell Reports, 2012) Smibert, P.; Miura, P.; Westholm, J.O.; Shenker, S.; May, G.; Duff, M.O.; Zhang, D.; Eads, B.D.; Carlson, J.; Brown, J.B.; Eisman, R.C.; Andrews, J.; Kaufman, T.; Cherbas, P.; Celniker, S.E.; Graveley, B.R.; Lai, E.C.
We analyzed the usage and consequences of alternative cleavage and polyadenylation (APA) in $\textit{Drosophila melanogaster}$ by using >1 billion reads of stranded mRNA-seq across a variety of dissected tissues. Beyond demonstrating that a majority of fly transcripts are subject to APA, we observed broad trends for 3′ untranslated region (UTR) shortening in the testis and lengthening in the central nervous system (CNS); the latter included hundreds of unannotated extensions ranging up to 18 kb. Extensive northern analyses validated the accumulation of full-length neural extended transcripts, and in situ hybridization indicated their spatial restriction to the CNS. Genes encoding RNA binding proteins (RBPs) and transcription factors were preferentially subject to 3′ UTR extensions. Motif analysis indicated enrichment of miRNA and RBP sites in the neural extensions, and their termini were enriched in canonical cis elements that promote cleavage and polyadenylation. Altogether, we reveal broad tissue-specific patterns of APA in $\textit{Drosophila}$ and transcripts with unprecedented 3′ UTR length in the nervous system.
Analysis of model replication origins in Drosophila reveals new aspects of the chromatin landscape and its relationship to origin activity and the prereplicative complex
(The American Society for Cell Biology, 2012) Liu, J.; Mconnell, K.; Dixon, M.; Calvi, B.R.
Epigenetic regulation exerts a major influence on origins of DNA replication during development. The mechanisms for this regulation, however, are poorly defined. We showed previously that acetylation of nucleosomes regulates the origins that mediate developmental gene amplification during Drosophila oogenesis. Here we show that developmental activation of these origins is associated with acetylation of multiple histone lysines. Although these modifications are not unique to origin loci, we find that the level of acetylation is higher at the active origins and quantitatively correlated with the number of times these origins initiate replication. All of these acetylation marks were developmentally dynamic, rapidly increasing with origin activation and rapidly declining when the origins shut off and neighboring promoters turn on. Fine-scale analysis of the origins revealed that both hyperacetylation of nucleosomes and binding of the origin recognition complex (ORC) occur in a broad domain and that acetylation is highest on nucleosomes adjacent to one side of the major site of replication initiation. It was surprising to find that acetylation of some lysines depends on binding of ORC to the origin, suggesting that multiple histone acetyltransferases may be recruited during origin licensing. Our results reveal new insights into the origin epigenetic landscape and lead us to propose a chromatin switch model to explain the coordination of origin and promoter activity during development.
TAL Effector-Nucleotide Targeter (TALE-NT) 2.0: tools for TAL effector design and target prediction
(Oxford University Press, 2012) Doyle, E.L.; Booher, N.J.; Standage, D.S.; Voytas, D.F.; Brendel, V.P.; Vandyk, J.K.; Bogdanove, A.J.
Transcription activator-like (TAL) effectors are repeat-containing proteins used by plant pathogenic bacteria to manipulate host gene expression. Repeats are polymorphic and individually specify single nucleotides in the DNA target, with some degeneracy. A TAL effector-nucleotide binding code that links repeat type to specified nucleotide enables prediction of genomic binding sites for TAL effectors and customization of TAL effectors for use in DNA targeting, in particular as custom transcription factors for engineered gene regulation and as site-specific nucleases for genome editing. We have developed a suite of web-based tools called TAL Effector-Nucleotide Targeter 2.0 (TALE-NT 2.0; https://boglab.plp.iastate.edu/) that enables design of custom TAL effector repeat arrays for desired targets and prediction of TAL effector binding sites, ranked by likelihood, in a genome, promoterome or other sequence of interest. Search parameters can be set by the user to work with any TAL effector or TAL effector nuclease architecture. Applications range from designing highly specific DNA targeting tools and identifying potential off-target sites to predicting effector targets important in plant disease.
Functional Consequences of Subunit Diversity in RNA Polymerases II and V
(Elsevier (Cell Press), 2012) Tan, E.H.; Blevins, T.; Ream, T.S.; Pikaard, C.S.
Multisubunit RNA polymerases IV and V (Pol IV and Pol V) evolved as specialized forms of Pol II that mediate RNA-directed DNA methylation (RdDM) and transcriptional silencing of transposons, viruses, and endogenous repeats in plants. Among the subunits common to Arabidopsis thaliana Pols II, IV, and V are 93% identical alternative ninth subunits, NRP(B/D/E)9a and NRP(B/D/E)9b. The 9a and 9b subunit variants are incompletely redundant with respect to Pol II; whereas double mutants are embryo lethal, single mutants are viable, yet phenotypically distinct. Likewise, 9a or 9b can associate with Pols IV or V but RNA-directed DNA methylation is impaired only in 9b mutants. Based on genetic and molecular tests, we attribute the defect in RdDM to impaired Pol V function. Collectively, our results reveal a role for the ninth subunit in RNA silencing and demonstrate that subunit diversity generates functionally distinct subtypes of RNA polymerases II and V.
Cryptocephal, the Drosophila melanogaster ATF4, Is a Specific Coactivator for Ecdysone Receptor Isoform B2
(PLoS Genetics, 2012) Gauthier, S.A.; VanHaaften, E.; Cherbas, L.; Cherbas, P.; Hewes, R.S.
The ecdysone receptor is a heterodimer of two nuclear receptors, the Ecdysone receptor (EcR) and Ultraspiracle (USP). In $\textit{Drosophila melanogaster}$, three EcR isoforms share common DNA and ligand-binding domains, but these proteins differ in their most N-terminal regions and, consequently, in the activation domains (AF1s) contained therein. The transcriptional coactivators for these domains, which impart unique transcriptional regulatory properties to the EcR isoforms, are unknown. Activating transcription factor 4 (ATF4) is a basic-leucine zipper transcription factor that plays a central role in the stress response of mammals. Here we show that Cryptocephal (CRC), the $\textit{Drosophila}$ homolog of ATF4, is an ecdysone receptor coactivator that is specific for isoform B2. CRC interacts with EcR-B2 to promote ecdysone-dependent expression of ecdysis-triggering hormone (ETH), an essential regulator of insect molting behavior. We propose that this interaction explains some of the differences in transcriptional properties that are displayed by the EcR isoforms, and similar interactions may underlie the differential activities of other nuclear receptors with distinct AF1-coactivators.
Tissue Damage Disrupts Developmental Progression and Ecdysteroid Biosynthesis in Drosophila
(PLOS ONE, 2012) Hackney, J.F.; Zolali-Meybodi, O.; Cherbas, P.
In humans, chronic inflammation, severe injury, infection and disease can result in changes in steroid hormone titers and delayed onset of puberty; however the pathway by which this occurs remains largely unknown. Similarly, in insects injury to specific tissues can result in a global developmental delay (e.g. prolonged larval/pupal stages) often associated with decreased levels of ecdysone – a steroid hormone that regulates developmental transitions in insects. We use $\textit{Drosophila melanogaster}$ as a model to examine the pathway by which tissue injury disrupts developmental progression. Imaginal disc damage inflicted early in larval development triggers developmental delays while the effects are minimized in older larvae. We find that the switch in injury response (e.g. delay/no delay) is coincident with the mid-3rd instar transition – a developmental time-point that is characterized by widespread changes in gene expression and marks the initial steps of metamorphosis. Finally, we show that developmental delays induced by tissue damage are associated with decreased expression of genes involved in ecdysteroid synthesis and signaling.
Primary and Secondary siRNAs in Geminivirus-induced Gene Silencing
(Public Library of Science, 2012) Aregger, M.; Borah, B.K.; Seguin, J.; Rajeswaran, R.; Gubaeva, E.G.; Zvereva, A.S.; Windels, D.; Vazquez, F.; Blevins, T.; Farinelli, L.; Pooggin, M.M.
In plants, RNA silencing-based antiviral defense is mediated by Dicer-like (DCL) proteins producing short interfering (si)RNAs. In $\textit{Arabidopsis}$ infected with the bipartite circular DNA geminivirus $\textit{Cabbage leaf curl virus}$ (CaLCuV), four distinct DCLs produce 21, 22 and 24 nt viral siRNAs. Using deep sequencing and blot hybridization, we found that viral siRNAs of each size-class densely cover the entire viral genome sequences in both polarities, but highly abundant siRNAs correspond primarily to the leftward and rightward transcription units. Double-stranded RNA precursors of viral siRNAs can potentially be generated by host RDR-dependent RNA polymerase (RDR). However, genetic evidence revealed that CaLCuV siRNA biogenesis does not require RDR1, RDR2, or RDR6. By contrast, CaLCuV derivatives engineered to target 30 nt sequences of a $\textit{GFP}$ transgene by primary viral siRNAs trigger RDR6-dependent production of secondary siRNAs. Viral siRNAs targeting upstream of the $\textit{GFP}$ stop codon induce secondary siRNAs almost exclusively from sequences downstream of the target site. Conversely, viral siRNAs targeting the $\textit{GFP}$ 3′-untranslated region (UTR) induce secondary siRNAs mostly upstream of the target site. RDR6-dependent siRNA production is not necessary for robust $\textit{GFP}$ silencing, except when viral siRNAs targeted $\textit{GFP}$ 5′-UTR. Furthermore, viral siRNAs targeting the transgene enhancer region cause $\textit{GFP}$ silencing without secondary siRNA production. We conclude that the majority of viral siRNAs accumulating during geminiviral infection are RDR1/2/6-independent primary siRNAs. Double-stranded RNA precursors of these siRNAs are likely generated by bidirectional readthrough transcription of circular viral DNA by RNA polymerase II. Unlike transgenic mRNA, geminiviral mRNAs appear to be poor templates for RDR-dependent production of secondary siRNAs.
ParsEval: Parallel comparison and analysis of gene structure annotations
(BioMed Central Ltd., 2012) Standage, D.S.; Brendel, V.P.
Background: Accurate gene structure annotation is a fundamental but somewhat elusive goal of genome projects, as witnessed by the fact that (model) genomes typically undergo several cycles of re-annotation. In many cases, it is not only different versions of annotations that need to be compared but also different sources of annotation of the same genome, derived from distinct gene prediction workflows. Such comparisons are of interest to annotation providers, prediction software developers, and end-users, who all need to assess what is common and what is different among distinct annotation sources. We developed ParsEval, a software application for pairwise comparison of sets of gene structure annotations. ParsEval calculates several statistics that highlight the similarities and differences between the two sets of annotations provided. These statistics are presented in an aggregate summary report, with additional details provided as individual reports specific to non-overlapping, gene-model-centric genomic loci. Genome browser styled graphics embedded in these reports help visualize the genomic context of the annotations. Output from ParsEval is both easily read and parsed, enabling systematic identification of problematic gene models for subsequent focused analysis. Results: ParsEval is capable of analyzing annotations for large eukaryotic genomes on typical desktop or laptop hardware. In comparison to existing methods, ParsEval exhibits a considerable performance improvement, both in terms of runtime and memory consumption. Reports from ParsEval can provide relevant biological insights into the gene structure annotations being compared. Conclusions: Implemented in C, ParsEval provides the quickest and most feature-rich solution for genome annotation comparison to date. The source code is freely available (under an ISC license) at http://parseval.sourceforge.net/.
Emerging Perspectives on the Mechanisms, Regulation, and Distribution of Light Color Acclimation in Cyanobacteria
(Molecular Plant, 2012-01) Gutu, Andrian; Kehoe, David
ABSTRACT Chromatic acclimation (CA) provides many cyanobacteria with the ability to tailor the properties of their lightharvesting antennae to the spectral distribution of ambient light. CA was originally discovered as a result of its dramatic cellular phenotype in red and green light. However, discoveries over the past decade have revealed that many pairs of light colors, ranging from blue to infrared, can trigger CA responses. The capacity to undergo CA is widespread geographically, occurs in most habitats around the world, and is found within all major cyanobacterial groups. In addition, many other cellular activities have been found to be under CA control, resulting in distinct physiological and morphological states for cells under different light-color conditions. Several types of CA appear to be the result of convergent evolution, where different strategies are used to achieve the final goal of optimizing light-harvesting antenna composition to maximize photon capture. The regulation of CA has been found to occur primarily at the level of RNA abundance. The CA-regulatory pathways uncovered thus far are two-component systems that use phytochrome-class photoreceptors with sensor-kinase domains to control response regulators that function as transcription factors. However, there is also at least one CAregulatory pathway that operates at the post-transcriptional level. It is becoming increasingly clear that large numbers of cyanobacterial species have the capacity to acclimate to a wide variety of light colors through the use of a range of different CA processes.
Non-growing Rhodopseudomonas palustris increases the hydrogen gas yield from acetate by shifting from the glyoxylate shunt to the tricarboxylic acid cycle
(Journal of Biological Chemistry, 2014) McKinlay, James B.; Oda, Yasuhiro; Rühl, Martin; Posto, Amanda L.; Sauer, Uwe; Harwood, Caroline S.
When starved for nitrogen, non-growing cells of the photosynthetic bacterium Rhodopseudomonas palustris continue to metabolize acetate and produce H$_2$, an important industrial chemical and potential biofuel. The enzyme nitrogenase catalyzes H$_2$ formation. The highest H$_2$ yields are obtained when cells are deprived of N$_2$ and thus use available electrons to synthesize H$_2$ as the exclusive product of nitrogenase. To understand how R. palustris responds metabolically to increase H$_2$ yields when it is starved for N$_2$, and thus not growing, we tracked changes in biomass composition and global transcript levels. In addition to a 3.5-fold higher H$_2$ yield by non-growing cells we also observed an accumulation of polyhydroxybutyrate to over 30% of the dry cell weight. The transcriptome of R. palustris showed down-regulation of biosynthetic processes and up-regulation of nitrogen scavenging mechanisms in response to N$_2$ starvation but gene expression changes did not point to metabolic activities that could generate the reductant necessary to explain the high H$_2$ yield. We therefore tracked $^{13}$C-labeled acetate through central metabolic pathways. We found that non-growing cells shifted their metabolism to use the tricarboxylic acid cycle to metabolize acetate in contrast to growing cells, which used the glyoxylate cycle exclusively. This shift enabled cells to more fully oxidize acetate, providing the necessary reducing power to explain the high H$_2$ yield.
Calvin cycle mutants of photoheterotrophic purple non-sulfur bacteria fail to grow due to an electron imbalance rather than toxic metabolite accumulation
(Journal of Bacteriology, 2014-01) Gordon, Gina C.; McKinlay, James B.
Purple nonsulfur bacteria grow photoheterotrophically by using light for energy and organic compounds for carbon and electrons. Disrupting the activity of the CO$_2$-fixing Calvin cycle enzyme, ribulose 1,5-bisphosphate carboxylase (RubisCO), prevents photoheterotrophic growth unless an electron acceptor is provided or if cells can dispose of electrons as H$_2$. Such observations led to the long-standing model wherein the Calvin cycle is necessary during photoheterotrophic growth to maintain a pool of oxidized electron carriers. This model was recently challenged with an alternative model wherein disrupting RubisCO activity prevents photoheterotrophic growth due to the accumulation of toxic ribulose-1,5-bisphosphate (RuBP) (D. Wang, Y. Zhang, E. L. Pohlmann, J. Li, and G. P. Roberts, J. Bacteriol. 193:3293-3303, 2011, http://dx.doi.org/10.1128/JB.00265-11). Here, we confirm that RuBP accumulation can impede the growth of Rhodospirillum rubrum (Rs. rubrum) and Rhodopseudomonas palustris (Rp. palustris) RubisCO-deficient (ΔRubisCO) mutants under conditions where electron carrier oxidation is coupled to H$_2$ production. However, we also demonstrate that Rs. rubrum and Rp. palustris Calvin cycle phosphoribulokinase mutants that cannot produce RuBP cannot grow photoheterotrophically on succinate unless an electron acceptor is provided or H$_2$ production is permitted. Thus, the Calvin cycle is still needed to oxidize electron carriers even in the absence of toxic RuBP. Surprisingly, Calvin cycle mutants of Rs. rubrum, but not of Rp. palustris, grew photoheterotrophically on malate without electron acceptors or H$_2$ production. The mechanism by which Rs. rubrum grows under these conditions remains to be elucidated.

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