Transcriptome analysis of cyst formation in Rhodospirillum centenum reveals large global changes in expression during cyst development
© Dong and Bauer; licensee BioMed Central. 2015
Received: 25 September 2014
Accepted: 15 January 2015
Published: 13 February 2015
Rhodospirillum centenum is a photosynthetic member of the Gram-negative Azospirillum clade members of which exhibit a complex developmental life-cycle featuring morphologically distinct cell types. Under periods of nutrient deprivation, replicative vegetative cells differentiate into metabolically dormant cysts that survive harsh environmental stresses such as desiccation. Encystment involves a multi-stage developmental process that includes the rounding of cells, production of large intracellular storage granules of poly-hydroxybutyrate (PHB) and the excretion of a protective exopolysaccharide coating that envelops dormant cysts.
To study the process of cyst development, we performed RNA-seq studies on cells that were induced to undergo cyst development. To assay for temporal changes in gene expression, RNA was extracted at 4, 24, 48, 72, 96 hours during development and subjected to deep sequence analysis. These results show that 812 genes exhibit log2 ≥ 1.5-fold changes in expression over a 96 hour cyst induction period demonstrating large global changes in gene expression during cyst development.
Notable changes in expression occurred in numerous genes involved in cell wall and lipid biosynthesis, metabolic enzymes, and numerous regulatory genes such as histidine kinases and transcription factors. Many genes involved in protein synthesis and DNA replication were also significantly reduced during late stages of cyst development. Genes previously identified by genetic screens as being critical for cyst development also exhibited changes of expression during cyst induction. This study provides the first transcriptome profile of global changes in gene expression that occur during development of cysts in a Gram-negative species.
Many bacteria survive unfavorable conditions by differentiating into metabolically quiescent resting stages, commonly referred to as cysts or spores . Production of resting cells involves major rearrangements of physiology, ultrastructure and biochemical composition [2-4]. Resting stage differentiation in prokaryotes is diverse and in some model systems is known to be regulated by complex and hierarchical signal transduction pathways .
The formation of endospores has been intensively investigated in Gram-positive species such as Bacillus subtilis, which forms endospores in response to unfavorable growth conditions [4,5]. Gram-positive spores display exceptional resistance to physical and chemical stresses such as desiccation, high temperatures (>100°C), radiation, oxidizing agents and pressure [3,6,7]. These spores arise from asymmetric cell division, which produces a prespore that becomes engulfed, matured, and then released by lysis of the mother cell. Less characterized are resting cysts produced by Gram-negative bacteria. Resting cysts produced by this group do not survive high temperatures and pressure but do provide resistance to desiccation and to moderate heat stress. The best-studied Gram-negative resting cells are myxospores synthesized by myxobacteria, a group of soil inhabiting delta proteobacteria. Myxospores from Myxococcus xanthus are produced by transforming an entire vegetative cell into a dormant cell. As is typical of cysts produced by other Gram-negative genera, myxospores are only moderately resistant to heat (up to 60°C), as well as resistant to desiccation, sonication, UV-irradiation, detergents and enzymatic digestion .
In addition to myxobacteria, there are agronomically important soil-inhabiting members of the genera Azospirillum and Azotobacter that produce desiccation and moderate heat resistant cysts [9,10]. Members of the Azospirillum clade are associated with root rhizospheres in a broad range of plants . These aerobic nitrogen fixing organisms are capable of stimulating plant growth through the donation of both bacterially synthesized fixed nitrogen and plant hormones such as indole-3-acetic, gibberellins and cytokinins . Inoculating fields and/or seeds with Azospirillum sp. are known to significantly enhance crop yields for a wide diversity of cultivars including corn and wheat . Azospirillum encystment involves morphological transitions during which cells round up to form a thick outer exopolysaccharide coat termed the exine layer . The formation of cysts also correlates with the appearance of intracellular poly-hydroxybutyrate (PHB) granules that are presumably used as energy reserves [15,16]. Once water and nutrients are available, cysts germinate by reforming vegetative cells that emerge from the exine coat. Beyond myxobacteria, Azospirillum and Azotobacter there are several Gram-negative pathogens such as Legionella pneumophila that also produce dormant cysts [17,18]. However, little is known about the biology of cyst development in pathogenic species.
Analysis of the regulation of resting cell synthesis has been extensively studied in Gram-positive organisms. In characterized model systems such as B. subtilis, the induction of endospore development is tightly regulated by a complex of well-studied hierarchical signal transduction pathways [19,20]. However, among Gram-negative organisms, there is much less known about the control of resting cell development. The best-characterized system is M. xanthus, however many molecular details on the control of myxospores development remain lacking. To further expand the understanding of resting cell development among Gram-negative organisms, we have undertaken detailed genetic and biochemical studies of cyst formation by Rhodospirillum centenum, a photosynthetic member of the Azospirillum clade. These studies have identified numerous regulatory factors that contribute to the control of encystment which include the involvement of five histidine kinases and a CtrA transcription regulator [21-26]. Cyst production is also influenced by production of cGMP and subsequent binding of cGMP by a CRP homolog . We have also shown that the timing of cyst development is regulated by a Che-like signal transduction cascade that contains a methyl-accepting chemotaxis receptor and several chemotaxis-like regulators [22,23,26]. Null mutations of these effectors fall within phenotypes that either block or activate encystment.
To further an understanding of how a plethora of regulatory proteins control cyst development, we must first have an understanding of what global changes in transcription occur as cells transition from vegetative growth into the cyst developmental pathway. Once baseline changes in expression are established, we can then address how individual regulatory mutations affect these global changes in expression. Towards this goal, we have utilized RNA-seq based high-throughput DNA sequencing technology to profile and quantify gene expression changes that occur in R. centenum as they develop into cysts. Up to now, there is only one example of global transcriptome analysis of resting cell development in a Gram-negative organism. Specifically, a DNA chip array study was used to provide an analysis of large changes in gene expression that occur during myxospore development . However, there is no report of transcriptome profiling of a Gram-negative organism undergoing the cyst development pathway using high resolution RNA–seq analysis. Studies have established RNA-seq as a powerful tool for transcriptional analysis as it provides unprecedented global resolution and depth of transcription profiles [29-31]. In this report, we have performed RNA-seq analysis of R. centenum as it undergoes cyst development over a multi-step time course. This high-resolution analysis provides the first detailed understanding of global changes in expression that occur at different stages of Gram-negative cyst development. The results of this study not only show alterations in many metabolic pathways but also alterations in numerous signal transduction regulatory networks and transcription factors such as sigma subunits. These results provide an understanding of physiological changes that occur during cyst development and additionally provide a road map for the study of similar developmental processes that occur in other cyst forming Gram-negative bacteria.
Overview of the transcriptional profile and identification of differentially expressed genes by RNA-Seq
To avoid metabolic gene expression changes that would occur as a result of a simple nutrient downshift, we analyzed pair-wise comparisons between RNA extracted from CENBA at 4 hours with RNA extracted from CENBA at 24 hr, 48 hr, 72 hr and 96 hr. Genes were designated as differentially expressed genes (DEGs) with log2fold change ≥ 1.5 in at least one time point and a false-discovery-rate adjusted p-value of less than 0.05. Analysis of the resulting RNA-seq data set revealed a total of 812 DEGs, which corresponds to 19.78% of the annotated R. centenum genome.
Cell wall and cell membrane synthesis
The involvement of EPS biosynthesis in encystment is underscored by prior genetic studies on cyst development by our laboratory where we have isolated a number of EPS deficient mutants that are also defective in forming viable cysts . Among the genes genetically identified as hypo-cyst suppressors were a mini-Tn5 disruption of RC1_1410 that encodes a periplasmic polysaccharide export protein whose expression increases approximately 4-fold throughout cyst development; mini-Tn5 disruptions of RC1_2536 and RC1_2537 that code for glycoside hydrolase family proteins that are closely related to the GT1 family of glycosyltransferases which in E. coli are involved in polysaccharide chain synthesis. These two proteins increase expression ~4-fold late in encystment. A mini-Tn5 disruption was also obtained for RC1_3992 that encodes NAD-dependent epimerase/dehydratase similar to E. coli protein WcaG, a synthetase of the polysaccharide precursor GDP-L-fucose. All of above are predicted to be involved in cyst exine layer synthesis. There is also a putative operon coding for several secretion genes in COG U that are involved in exopolysaccharide synthesis (exbB, exbD and exbD1) that are down-regulated in 48 through 96 hours indicating that some aspects of EPS biosynthesis may also be ramping down in late stages of encystment (Figure 3).
When analyzing expression changes at a log2 1.5 cut off there are also numerous additional cell wall and lipid metabolism genes that exhibit lower but significant 3-4 fold changes in expression (Figure 3). Among this latter group are a cluster of seven genes involved in the synthesis of the lipid A core of the outer membrane lipopolysaccharide that is down regulated 3-4-fold in day 4 late in cyst development (RC1_ 0610, RC1_ 0612, RC1_0613, RC1_0615, RC1_0616, RC1_0617, and RC1_0618).
Collectively, these results confirm microscopic and biochemical observations that cyst development involves significant changes in cell wall and cell membrane biochemistry that are presumably needed to allow survival in harsh environmental conditions such as extreme desiccation.
These alterations in transporter gene expression confirms that there are significant alterations in cell membrane composition as cells transition to cyst forms and further demonstrate that the composition of substrates that are imported/exported are also altered during cyst development.
Carbon metabolism and energy production/storage
R. centenum cysts are also known to contain large quantities of poly-hydroxybutyrate (PHB) [16,34]. PHB is an industrially significant biopolymer that presumably functions as an important energy storage source for cyst cells and an energy source for germination back into a vegetative state. Indeed disruption of genes for PHB production, are known to lead to a defect in formation of cysts . Inspection of COG I demonstrates that there is significant increase in the expression of enzymes involved in production of poly-hydroxybutyrate (PHB) late in cyst formation. For example, five genes coding for enzymes involved in synthesis of acetoacetyl-CoA (RC1_1744, RC1_1948, RC1_3948, RC1_3030) and conversion of acetoacetyl-CoA to 3-OH-butyryl-CoA (RC1_3949) which is an immediate precursor to PHB increases 3- to 8-fold in 72 hours and 96 hours (Figure 6A). These expression results confirm biochemical data which shows that PHB production is ramped up late in cyst development.
Finally, expression of a subunit for cytochrome c oxidase (RC1_0780) that is involved in utilizing oxygen to form a membrane potential during respiration, is increased ~10 fold throughout encystment (Figure 6A). Oxidative stress proteins are also ramped up during this process (discussed below) so one possibility is that increased expression of cytochrome oxidase is a mechanism to reduce oxygen levels in developing cysts. There is also an increase in expression of two energy generating carbon monoxide dehydrogenases (RC1_1074 and RC1_1075) that convert CO and H2O - > CO2, 2H+ and 2e− (Figure 6A).
In addition to the development of cysts, R. centenum cells are also capable of differentiating into swim cells that have a single sheathed polar flagellum or swarm cells that have numerous unsheathed lateral flagella [34,42,43]. Swarm cell differentiation occurs when cells are grown on agar solidified growth medium or when grown at elevated temperatures (42°C-44°C) . One aspect of cyst development is the ejection of flagella from vegetative cells as they round up during early stages of encystment. Accordingly, we were surprised to observe that most lateral and polar flagellar genes do not undergo significant changes in expression as cells transition to the encystment phase. The exceptions are six flagellar structural genes that have a ~3-fold increase in expression midway in cyst development (RC1_0185, RC1_ 0186, RC1_0196, RC1_ 0197, RC1_0205, and RC1_3758) (Figure 6B). In addition, the motor genes exhibit a 3-fold decrease in expression late in cyst development. These results indicate that the loss of flagella observed during cyst development may not be a result of decreased flagella gene expression but instead may be due to an as yet defined post-transcriptional event.
R-bodies are highly insoluble protein ribbons that form distinct coiled cylindrical structures synthesized by a limited number of Gram-negative species. In several species of paramecia there are bacterial endosymbionts known as kappa particles that synthesize R-bodies which enable the host to defend themselves from predation [45-48]. Beyond kappa particles, R. centenum, the soil inhabiting bacteria Pseudomonas taeniospiralis [49,50], Pseudomonas EPS-5028 , and the free living plant pathogen Pseudomonas avenae [52,53] are examples of free living bacterial species that are known to synthesize R-bodies. Interestingly, the expression of an operon (RC1_1993 through RC1_1999) in R. centenum that contains seven R-body genes is greatly induced (up to 50-fold) during cyst development (Figure 6C). Although it remains speculative, it would be intriguing if R-body formation in R. centenum were linked to defense of cysts against predation as it is for the kappa particle endosymbionts.
Late in R. centenum cyst development there is also a 5-fold increase in expression of cvaB which codes for an ABC type transporter of the colicin V system. In other species this transporter exports an inactive colicin V precursor that is cleaved upon transport to produce the bactericidal colicin V . There is also increased expression of a norM gene early in cyst development, which in other species codes for a multidrug efflux pump.
Finally, there are also genes involved in defense against oxidative damage such as catalase (kat), dps and ohr that ramp up expression 3- to 8-fold late (96 hours) in cyst development. Expression of groS and groL that provide defense against protein mis-folding are increased 3 to 4 fold throughout cyst development (COG O) (Figure 6C).
Amino acid metabolism and translation
As is the case for DNA metabolism, there is also an apparent ramp-down of protein synthesis late in cyst development. Evidence for this conclusion is centered on analysis of COG E which has numerous amino acid metabolism genes that exhibit 3 to 5-fold reduced expression late (96 hours) in cyst development. This includes genes coding for enzymes involved in lysine, valine, leucine, isoleucine aspartate, threonine, serine, glutamate, and histidine biosynthesis (Figure 7). One exception to the noted reduction in amino acid metabolism are genes coding for enzymes involved in phenylalanine biosynthesis (hppD and phhA) which exhibit a 5- to 16-fold increase in expression late in development (48 hours through 96 hours) (Figure 6). Phenylalanine is used as a precursor for flavonoid biosynthesis which also utilizes the enzyme chalcone synthase . The chalcone synthase gene (RC1_1268) has previously been shown to ramp up expression late in cyst development . Even though they are not well characterized in bacteria, flavonoids are involved in UV filtration in plants which is intriguing given that cyst cells also show resistance to UV killing .
Beyond changes in amino acid biosynthesis, cyst development also exhibits an apparent alteration in protein translation (COG J). This includes an interesting ~3-fold increase in expression of nine ribosomal protein genes at 48 hours (rplL, rspL, rspK, rpsF, rpmJ, rplS, rpmL, rpmB, rpsG) followed by a later 3- to 5-fold decrease in expression of four tRNA synthetase genes (glyS, glyQ, pheS, pheT), five tRNA modifying genes (RC1_0127, RC1_0422, RC1_0156, RC1_3597, trmU) and the translation initiation factor IF-1 (infA) (Figure 7).
In the R. centenum genome there are 55 genes predicted to encode histidine kinases, 16 of which are hybridized to receiver domains . There are also 54 proteins that are predicted to contain receiver domain only or a response regulator receiver domain linked to a DNA-binding domain. Our RNA-seq data showed that there were 11 histidine kinases and 15 response regulators significantly up- or down- regulated during cyst development (Figure 10). The expression of six regulators involved in nitrogen and phosphate assimilation are altered such as ntrB-ntrC, fixJ, nifA, phoR, narP. With the exception of fixJ and narP, which have about 4-fold increase in expression late in development, the other nitrogen and phosphate regulators have a 3-6 fold reduction in expression late in cyst development (typically in day 4). The change in expression of numerous nitrogen regulators is notable given that cyst development can be induced during growth on rich complex medium that also contains elevated amounts of nitrogen .
Cell cycle and developmental regulator genes are another class that undergo changes in expression. Specifically, divJ and pleC that exhibit a ~4-fold reduction in expression late in development (Figure 10). There are two genes coding for homologs of the cell cycle regulator CtrA that show a 4-fold increase in expression late in cyst development. Mutational studies have shown that CtrA does indeed have a role in cyst development .
A total of 22 genes have been annotated to code for proteins harboring GGDEF, EAL and HD-GYP domains that are involved in the synthesis and degradation of cyclic di-GMP (7 with GGDEF domain, 2 with EAL domain, 7 with both GGDEF and EAL domains and 6 with HD-GYP domains) [62,63] . In this group of regulators, three diguanylate cyclases (RC1_3432, RC1_3881, RC1_2560) containing GGDEF domains are down-regulated 4- to 6-fold late in cyst development suggesting that cyclic di-GMP may also be an effector signal affecting cyst development (Figure 9).
Encystment involves large temporal changes in metabolic gene expression
Recent genomic studies using high resolution RNA-seq technology have established that bacteria undergo large scale global reprogramming of gene expression to cope with environmental stresses. For example, recent research has shown that 1391 out of 3189 genes in Synechocystis sp. PCC 6803 genome are differentially expressed in response to salt stress including many genes that effect energy metabolism and protein synthesis . In cases where environmental stress leads to cell differentiation, such as spore formation, large global changes in the transcriptome are known to occur as a result of complex changes in numerous regulatory networks. For example, global transcriptome analysis of myxospore formation by Myxococcus xanthus has revealed that a total of 1486 genes out of 6687 are differentially regulated in response to glycerol-induced sporulation . A large portion involve genes in energy metabolism, protein synthesis and fate. There are also a large number of two-component regulatory systems that undergo alterations in expression pattern during myxospore formation. Lower resolution DNA array studies of B. subtilis during spore development also indicate that ~520 genes undergo significant temporal changes in expression .
In addition to changes in genes that affect cell wall/membrane composition, we have also observed changes in genes affecting carbon metabolism during cyst development. Specifically, there are numerous changes in gene expression leading to the synthesis of PHB which is known to accumulate to large levels in cyst cells (Figure 11) [10,14,16,34]. Presumably, PHB is used for energy storage for survival of dormant cyst cells. There are also alterations in the expression of enzymes that affect defense against oxidative damage.
In addition to alterations in carbon metabolism, we also observed that expression of many genes involved in the synthesis of amino acids are reduced late in cyst development (Figure 11). Cyst cells are metabolically dormant so the need for amino acid biosynthesis is likely minimal. Similarly, we have also observed reduced expression of numerous genes involved in the synthesis of nucleotides (Figure 11). There is also reduced expression of cell cycle genes such as DivJ and PleC homologs that code for proteins involved in the control of DNA replication. Again these changes in expression are not surprising as DNA replication does not occur during dormancy.
High resolution RNA Seq analysis provides clues to regulatory cascades that control encystment
There are obvious links between cell’s capabilities to sense changes in extracellular environmental conditions with concomitant large-scale adaptation of genome expression. This process involves a combination of transporters, sensors, and phosphorylation/regulatory cascades that subsequently modulate the activity of transcription factors . Our data suggests that a complex regulatory network comprised of hierarchical signal transduction pathways and transcriptional regulators are involved in controlling cyst development. Notably, we observed significant up- or down-regulation of 11 histidine kinase and 15 response regulator genes during cyst development. Indeed prior genetic studies by our laboratory have implicated the involvement of five histidine kinases in the control of cyst development [21,27]. One challenge going forward will be to determine which, if any, of the developmentally altered HK genes have direct control of cyst development or if they have indirect control. For example, many could indirectly affect cyst development by affecting metabolic pathways that when altered lead to induction of encystment.
In many studied species, altering the synthesis or activity of sigma factors can lead to changes in the expression of numerous downstream genes. Thus, temporally controlling the expression or activity of sigma factors can be a facile way of differentially programming global changes in gene expression such as what occurs during cyst development. Indeed it has been reported that bacteria with a complex lifestyle, or those that encounter diverse environmental conditions, usually possess an increased number of sigma factors . In R. centenum there is a surprisingly large number of sigma factors (19 annotated) many of which are members of the σ70 family . In regards to the role of sigma factors in dormancy, it has been shown that sporulation development in B. subtilis is primarily regulated by a cascade of sigma factor expression and degradation . It was also reported recently that when exposed to a wide range of environmental and nutritional conditions, the transcriptome of B. subtilis changed drastically and 66% of the transcriptional variance occurred in regulons controlled by various RNA polymerase sigma factors . Recent transcription analysis of myxospores formation by M. xanthus has also identified five sigma transcription factors that are significantly upregulated during glycerol induced sporulation process, of which rpoN is predicted to regulate spore formation and maturation . Regarding R. centenum, the observed temporal changes in expression pattern of several sigma factor genes as identified in this study highly suggests that the cyst development process involves the use of sigma factors to control global changes in developmental gene expression. Indeed, we have observed that disruption of RC1_1638, which codes for a σ70, expressed late in cyst development, leads to a defect in cyst formation . Clearly, additional studies on the role of the identified sigma factors is warranted and needed to understand which genes are under control of differentially expressed sigma factors and their involvement in cysts development.
In extreme environmental changes such as desiccation, some species survive by forming metabolically dormant spores or cysts. How Gram-positive species form heat and desiccation resistant spores is relatively well understood. Less well characterized are mechanisms allowing Gram-negative cells to form desiccation resistant cysts. Our high resolution RNA-seq analysis provides the first detailed understanding of temporal changes in gene expression that occur during encystment in a Gram-negative organism. Multiple changes in expression occurred in genes involved in cell wall biosynthesis and cell membrane composition, which presumably allow cysts to promote cell survival in desiccating conditions (Figure 11). Genes involved in PHB energy storage are also ramped up during development. This is contrasted by reduced expression of genes involved in protein synthesis and DNA replication. The latter is not surprising as cysts are metabolically dormant and non-replicating. Numerous changes in expression of regulatory genes are also noted including several that are known to affect cyst development. Future research on these regulatory proteins will undoubtedly help our understanding of regulatory cascades that are responsible for controlling the observed changes in gene expression that occur during cyst development.
Bacterial strains, media, and growth conditions
The wild-type R. centenum strain ATCC 51521 was cultured aerobically in nutrient rich CENS medium that has pyruvate and soytone as carbon sources or in cyst inducing minimal defined CENBA medium containing 20 mM butyrate as the sole carbon source . Liquid grown cells were incubated with shaking in an Erlenmeyer flask at 37°C or on grown in agar-solidified media at 42°C.
The developmental stages of cells were monitored by phase contrast microscopy prior to harvesting for RNA extraction with an estimated level of encystment reaching ~20 to 40% at the 96 hr time point. Aliquots of cells were imaged as wet mounts on a Nikon E800 light microscope equipped with a 100x Plan Apo oil objective. Image capture was carried out with a Cascade: 1 K Megapixel EMCCD Camera and METAMORPH imaging software, v.4.5.
Wild-type Rhodospirillum centenum was grown aerobically overnight to stationary phase in CENS medium at 37°C. The cells were sub-cultured into CENBA medium as a 1:50 dilution inoculum. Cell morphology was monitored during a 96-hour period. Cell samples from three biological replicates at five points (4, 24, 48, 72 and 96 hours) were collected. To acquire enough total RNA from each time point, 100 ml, 50 ml, 10 ml, 10 ml and 10 ml of cell cultures were collected at 4, 24, 48, 72 and 96 hours respectively. Cell samples were homogenized using the FastPrep® Instrument (MP Biomedicals) by Lysing Matrix B in impact-resistant 2 mL tubes with RNApro™ Solution (MP Biomedicals). Total RNA samples were then extracted using FastRNA® PRO BLUE KIT (MP Biomedicals). For further cleaning-up, each total RNA sample was treated with RNeasy Mini Kit (Qiagen) following RNA clean-up protocol and eluted with 50 μl RNase-free water. TURBO DNase (Ambion) was applied to remove genomic DNA. 1.5 μl of TURBO DNase (2 Unit/ μl) and 6 μl of 10x TURBO DNase Buffer was added to 50 μl of RNA sample and incubated at 37°C for 30 min. The application and incubation of TURBO DNase was repeated once to achieve maximum DNA removal. RNA clean-up was carried out again with an RNeasy Mini Kit (Qiagen) to remove any contamination introduced by TURBO DNase buffers. Final RNA concentrations were measured by NanoDrop (Thermo Scientific). A typical OD260 to OD280 ratio of RNA samples was approximately 2.0. Further quantitation and quality control of total RNA samples were performed using a 2100 Bioanalyzer (Agilent Technologies).
Transcript isolation, library construction and RNA-sequencing
Library construction and RNA-sequencing were conducted by University of Wisconsin-Madison Biotechnology Center DNA Sequencing Facility. Briefly, total RNA was reduced of ribosomal RNA content using an EpiCentre® Ribo-Zero™ Magnetic (Bacteria) kit with a targeted 2 μg total RNA input. Illumina mRNA-Seq libraries were prepared from rRNA-depleted samples using the TruSeq™ RNA Sample Prep kit (Illumina, San Diego, CA) per the manufacturer's protocol. Adapters containing 6 nucleotide indexes were ligated to the double-stranded cDNA and all cDNA libraries were amplified with 11 PCR cycles. Single end sequencing (1x100bp) was performed on the Illumina HiSeq 2000 according to the standard Illumina protocol. The sequences have been deposited in the National Center for Biotechnology Information’s Sequence Read Archive (accession no. SRP045612).
Data analysis was carried out on GALAXY platform (http://galaxyproject.org/) [69-71]. Sequences of 100-nt in length were first trimmed using FASTQ Quality Trimmer (version 1.0.0) with a window size of 6, step size of 3 and quality score greater than 20. Trimmed sequences were mapped to the annotated 4,355,548 base pair R. centenum ATCC 51521 genome harboring 4,105 genes  using the program Bowtie (version 1.1.2). Transcripts were assembled with Reads per Kilobase of Transcripts Mapped (RPKM) values calculated using Cufflinks (v2.1.1) . Mapped RNA-Seq reads were visualized using the Integrative Genomics Viewer . Fold-change calculations for time course expression were undertaken pair-wised between samples of 4 hours in CENBA medium and sample at 24, 48, 72 and 96 hours in CENBA medium using Cuffdiff with geometric library normalization method and minimum alignment counts of 10 [74,75]. Genes were considered to exhibit significant differential changes in expression (DEG) when log2 of fold change was ≥1.5 with a false discovery rate adjusted p value of <0.05. Orthologous groups of DEGs were annotated according to eggNOG (evolutionary genealogy of genes: Non-supervised Orthologous Groups, version 4.0, http://eggnog.embl.de/version_4.0.beta/).
This study is supported by National Institutes of Health grant number GM099703 awarded to CEB.
- Sadoff HL. Comparative aspects of morphogenesis in three prokaryotic genera. Annu Rev Microbiol. 1973;27:133–53.View ArticlePubMedGoogle Scholar
- Sudo SZ, Dworkin M. Comparative biology of prokaryotic resting cells. Adv Microb Physiol. 1973;9:153–224.View ArticlePubMedGoogle Scholar
- Nicholson WL, Munakata N, Horneck G, Melosh HJ, Setlow P. Resistance of Bacillus endospores to extreme terrestrial and extraterrestrial environments. Microbiol Mol Biol Rev. 2000;64(3):548–72.View ArticlePubMed CentralPubMedGoogle Scholar
- Higgins D, Dworkin J. Recent progress in Bacillus subtilis sporulation. FEMS Microbiol Rev. 2012;36(1):131–48.View ArticlePubMed CentralPubMedGoogle Scholar
- Piggot PJ, Hilbert DW. Sporulation of Bacillus subtilis. Curr Opin Microbiol. 2004;7(6):579–86.View ArticlePubMedGoogle Scholar
- Paidhungat M, Setlow B, Driks A, Setlow P. Characterization of spores of Bacillus subtilis which lack dipicolinic acid. J Bacteriol. 2000;182(19):5505–12.View ArticlePubMed CentralPubMedGoogle Scholar
- Chung JD, Stephanopoulos G, Ireton K, Grossman AD. Gene-expression in single cells of bacillus-subtilis - evidence that a threshold mechanism controls the initiation of sporulation. J Bacteriol. 1994;176(7):1977–84.PubMed CentralPubMedGoogle Scholar
- Sudo SZ, Dworkin M. Resistance of vegetative cells and microcysts of Myxococcus xanthus. J Bacteriol. 1969;98(3):883–7.PubMed CentralPubMedGoogle Scholar
- Sadoff HL. Encystment and germination in Azotobacter vinelandii. Bacteriol Rev. 1975;39(4):516–39.PubMed CentralPubMedGoogle Scholar
- Sadasivan L, Neyra CA. Flocculation in Azospirillum brasilense and Azospirillum lipoferum: exopolysaccharides and cyst formation. J Bacteriol. 1985;163(2):716–23.PubMed CentralPubMedGoogle Scholar
- Michiels K, Vanderleyden J, Vangool A. Azospirillum - plant-root associations - a review. Biol Fert Soils. 1989;8(4):356–68.View ArticleGoogle Scholar
- Tien TM, Gaskins MH, Hubbell DH. Plant growth substances produced by Azospirillum brasilense and Their Effect on the Growth of Pearl Millet (Pennisetum americanum L.). Appl Environ Microbiol. 1979;37(5):1016–24.PubMed CentralPubMedGoogle Scholar
- Okon Y, Labanderagonzalez CA. Agronomic applications of Azospirillum - an evaluation of 20 years worldwide field inoculation. Soil Biol Biochem. 1994;26(12):1591–601.View ArticleGoogle Scholar
- Berg RH, Tyler ME, Novick NJ, Vasil V, Vasil IK. Biology of azospirillum-sugarcane association: enhancement of nitrogenase activity. Appl Environ Microbiol. 1980;39(3):642–9.PubMed CentralPubMedGoogle Scholar
- Sadasivan L, Neyra CA. Cyst production and brown pigment formation in aging cultures of Azospirillum brasilense ATCC 29145. J Bacteriol. 1987;169(4):1670–7.PubMed CentralPubMedGoogle Scholar
- Berleman JE, Bauer CE. Characterization of cyst cell formation in the purple photosynthetic bacterium Rhodospirillum centenum. Microbiology. 2004;150(Pt 2):383–90.View ArticlePubMedGoogle Scholar
- Garduno RA, Faulkner G, Trevors MA, Vats N, Hoffman PS. Immunolocalization of Hsp60 in Legionella pneumophila. J Bacteriol. 1998;180(3):505–13.PubMed CentralPubMedGoogle Scholar
- Garduno RA, Quinn FD, Hoffman PS. HeLa cells as a model to study the invasiveness and biology of Legionella pneumophila. Can J Microbiol. 1998;44(5):430–40.View ArticlePubMedGoogle Scholar
- Lemon KP, Kurtser I, Wu J, Grossman AD. Control of initiation of sporulation by replication initiation genes in Bacillus subtilis. J Bacteriol. 2000;182(10):2989–91.View ArticlePubMed CentralPubMedGoogle Scholar
- Quisel JD, Grossman AD. Control of sporulation gene expression in Bacillus subtilis by the chromosome partitioning proteins Soj (ParA) and Spo0J (ParB). J Bacteriol. 2000;182(12):3446–51.View ArticlePubMed CentralPubMedGoogle Scholar
- Berleman JE, Hasselbring BM, Bauer CE. Hypercyst mutants in Rhodospirillum centenum identify regulatory loci involved in cyst cell differentiation. J Bacteriol. 2004;186(17):5834–41.View ArticlePubMed CentralPubMedGoogle Scholar
- He K, Marden JN, Quardokus EM, Bauer CE. Phosphate flow between hybrid histidine kinases CheA(3) and CheS(3) controls Rhodospirillum centenum cyst formation. PLoS Genet. 2013;9(12):e1004002.View ArticlePubMed CentralPubMedGoogle Scholar
- Berleman JE, Bauer CE. Involvement of a Che-like signal transduction cascade in regulating cyst cell development in Rhodospirillum centenum. Mol Microbiol. 2005;56(6):1457–66.View ArticlePubMedGoogle Scholar
- Bird TH, MacKrell A. A CtrA homolog affects swarming motility and encystment in Rhodospirillum centenum. Arch Microbiol. 2011;193(6):451–9.View ArticlePubMedGoogle Scholar
- Din N, Shoemaker CJ, Akin KL, Frederick C, Bird TH. Two putative histidine kinases are required for cyst formation in Rhodospirillum Centenum. Arch Microbiol. 2011;193(3):209–22.View ArticlePubMedGoogle Scholar
- He K, Bauer CE. Chemosensory signaling systems that control bacterial survival. Trends Microbiol. 2014;22(7):389–98.View ArticlePubMed CentralPubMedGoogle Scholar
- Marden JN, Dong Q, Roychowdhury S, Berleman JE, Bauer CE. Cyclic GMP controls Rhodospirillum centenum cyst development. Mol Microbiol. 2011;79(3):600–15.View ArticlePubMed CentralPubMedGoogle Scholar
- Muller FD, Treuner-Lange A, Heider J, Huntley SM, Higgs PI. Global transcriptome analysis of spore formation in Myxococcus xanthus reveals a locus necessary for cell differentiation. BMC Genomics. 2010;11:264.View ArticlePubMed CentralPubMedGoogle Scholar
- Croucher NJ, Thomson NR. Studying bacterial transcriptomes using RNA-seq. Curr Opin Microbiol. 2010;13(5):619–24.View ArticlePubMed CentralPubMedGoogle Scholar
- Fang G, Passalacqua KD, Hocking J, Llopis PM, Gerstein M, Bergman NH, et al. Transcriptomic and phylogenetic analysis of a bacterial cell cycle reveals strong associations between gene co-expression and evolution. BMC Genomics. 2013;14:450.View ArticlePubMed CentralPubMedGoogle Scholar
- Sorek R, Cossart P. Prokaryotic transcriptomics: a new view on regulation, physiology and pathogenicity. Nat Rev Genet. 2010;11(1):9–16.View ArticlePubMedGoogle Scholar
- Favinger J, Stadtwald R, Gest H. Rhodospirillum centenum, sp. nov., a thermotolerant cyst-forming anoxygenic photosynthetic bacterium. Antonie Van Leeuwenhoek. 1989;55(3):291–6.View ArticlePubMedGoogle Scholar
- Yildiz FH, Gest H, Bauer CE. Attenuated effect of oxygen on photopigment synthesis in Rhodospirillum centenum. J Bacteriol. 1991;173(17):5502–6.PubMed CentralPubMedGoogle Scholar
- Nickens D, Fry CJ, Ragatz L, Bauer CE, Gest H. Biotype of the purple nonsulfur photosynthetic bacterium, Rhodospirillum centenum. Arch Microbiol. 1996;165(2):91–6.View ArticleGoogle Scholar
- Frost GE, Rosenberg H. Relationship between the tonB locus and iron transport in Escherichia coli. J Bacteriol. 1975;124(2):704–12.PubMed CentralPubMedGoogle Scholar
- Hantke K, Braun V. Membrane receptor dependent iron transport in Escherichia coli. FEBS Lett. 1975;49(3):301–5.View ArticlePubMedGoogle Scholar
- McIntosh MA, Earhart CF. Coordinate regulation by iron of the synthesis of phenolate compounds and three outer membrane proteins in Escherichia coli. J Bacteriol. 1977;131(1):331–9.PubMed CentralPubMedGoogle Scholar
- Noinaj N, Guillier M, Barnard TJ, Buchanan SK. TonB-dependent transporters: regulation, structure, and function. Annu Rev Microbiol. 2010;64:43–60.View ArticlePubMed CentralPubMedGoogle Scholar
- Braun V, Braun M. Iron transport and signaling in Escherichia coli. FEBS Lett. 2002;529(1):78–85.View ArticlePubMedGoogle Scholar
- Hollenstein K, Dawson RJ, Locher KP. Structure and mechanism of ABC transporter proteins. Curr Opin Struct Biol. 2007;17(4):412–8.View ArticlePubMedGoogle Scholar
- Kadouri D, Burdman S, Jurkevitch E, Okon Y. Identification and isolation of genes involved in poly(beta-hydroxybutyrate) biosynthesis in Azospirillum brasilense and characterization of a phbC mutant. Appl Environ Microbiol. 2002;68(6):2943–9.View ArticlePubMed CentralPubMedGoogle Scholar
- McClain J, Rollo DR, Rushing BG, Bauer CE. Rhodospirillum centenum utilizes separate motor and switch components to control lateral and polar flagellum rotation. J Bacteriol. 2002;184(9):2429–38.View ArticlePubMed CentralPubMedGoogle Scholar
- Ragatz L, Jiang ZY, Bauer CE, Gest H. Macroscopic phototactic behavior of the purple photosynthetic bacterium Rhodospirillum centenum. Arch Microbiol. 1995;163(1):1–6.View ArticlePubMedGoogle Scholar
- Berleman JE, Bauer CE. A che-like signal transduction cascade involved in controlling flagella biosynthesis in Rhodospirillum centenum. Mol Microbiol. 2005;55(5):1390–402.View ArticlePubMedGoogle Scholar
- Gibson I. The endosymbionts of Paramecium. CRC Crit Rev Microbiol. 1974;3(3):243–73.View ArticlePubMedGoogle Scholar
- Preer Jr JR. The killer cytoplasmic factor kappa; its rate of reproduction, the number of particles per cell, and its size. Am Nat. 1948;82(802):35–42.View ArticlePubMedGoogle Scholar
- Preer Jr JR. Microscopically visible bodies in the cytoplasm of the "killer" strains of Paramecium aurelia. Genetics. 1950;35(3):344–62.PubMed CentralPubMedGoogle Scholar
- Preer Jr JR, Preer LB, Jurand A. Kappa and other endosymbionts in Paramecium aurelia. Bacteriol Rev. 1974;38(2):113–63.PubMed CentralPubMedGoogle Scholar
- Lalucat J, Meyer O, Mayer F, Pares R, Schlegel HG. R-bodies in newly isolated free-living hydrogen-oxidizing bacteria. Arch Microbiol. 1979;121(1):9–15.View ArticleGoogle Scholar
- Lalucat J, Pares R, Schlegel HG. Pseudomonas-Taeniospiralis Sp-Nov, an R-body-containing hydrogen bacterium. Int J Syst Bacteriol. 1982;32(3):332–8.View ArticleGoogle Scholar
- Fuste MC, Simonpujol MD, Marques AM, Guinea J, Congregado F. Isolation of a new free-living bacterium containing R-bodies. J Gen Microbiol. 1986;132:2801–5.Google Scholar
- Bird B, Gibson I. Studies on R-bodies of pseudomonas-avenae. Micron Microsc Acta. 1987;18(3):187–91.View ArticleGoogle Scholar
- Wells B, Horne RW. The ultrastructure of pseudomonas-avenae.2. Intracellular refractile (R-Body) Structure. Micron Microsc Acta. 1983;14(4):329–44.View ArticleGoogle Scholar
- Gilson L, Mahanty HK, Kolter R. Genetic-analysis of an Mdr-like export system - the secretion of colicin-V. Embo J. 1990;9(12):3875–84.PubMed CentralPubMedGoogle Scholar
- Hahlbrock K, Scheel D. Physiology and molecular-biology of phenylpropanoid metabolism. Ann Rev Plant Phys. 1989;40:347–69.View ArticleGoogle Scholar
- Iwashina T. Flavonoid function and activity to plants and other organisms. Uchu Seibutsu Kagaku. 2003;17(1):24–44.PubMedGoogle Scholar
- Reisenauer A, Quon K, Shapiro L. The CtrA response regulator mediates temporal control of gene expression during the Caulobacter cell cycle. J Bacteriol. 1999;181(8):2430–9.PubMed CentralPubMedGoogle Scholar
- Wortinger M, Sackett MJ, Brun YV. CtrA mediates a DNA replication checkpoint that prevents cell division in Caulobacter crescentus. Embo J. 2000;19(17):4503–12.View ArticlePubMed CentralPubMedGoogle Scholar
- Feklistov A, Sharon BD, Darst SA, Gross CA. Bacterial sigma factors: a historical, structural, and genomic perspective. Annu Rev Microbiol. 2014;68:357–76.View ArticlePubMedGoogle Scholar
- Souza BM, Castro TL, Carvalho RD, Seyffert N, Silva A, Miyoshi A, et al. Sigma factors of gram-positive bacteria: a focus on and the CMNR group. Virulence. 2014;5(5):587–600.View ArticlePubMedGoogle Scholar
- Lu YK, Marden J, Han M, Swingley WD, Mastrian SD, Chowdhury SR, et al. Metabolic flexibility revealed in the genome of the cyst-forming alpha-1 proteobacterium Rhodospirillum centenum. BMC Genomics. 2010;11:325.View ArticlePubMed CentralPubMedGoogle Scholar
- Romling U, Galperin MY, Gomelsky M. Cyclic di-GMP: the first 25 years of a universal bacterial second messenger. Microbiol Mol Biol Rev. 2013;77(1):1–52.View ArticlePubMed CentralPubMedGoogle Scholar
- Ryan RP. Cyclic di-GMP signalling and the regulation of bacterial virulence. Microbiology. 2013;159(Pt 7):1286–97.View ArticlePubMed CentralPubMedGoogle Scholar
- Qiao J, Huang S, Te R, Wang J, Chen L, Zhang W. Integrated proteomic and transcriptomic analysis reveals novel genes and regulatory mechanisms involved in salt stress responses in Synechocystis sp. PCC 6803. Appl Microbiol Biotechnol. 2013;97(18):8253–64.View ArticlePubMedGoogle Scholar
- Fawcett P, Eichenberger P, Losick R, Youngman P. The transcriptional profile of early to middle sporulation in Bacillus subtilis. Proc Natl Acad Sci U S A. 2000;97(14):8063–8.View ArticlePubMed CentralPubMedGoogle Scholar
- Kroos L. The Bacillus and Myxococcus developmental networks and their transcriptional regulators. Annu Rev Genet. 2007;41:13–39.View ArticlePubMedGoogle Scholar
- Kroos L, Zhang B, Ichikawa H, Yu YT. Control of sigma factor activity during Bacillus subtilis sporulation. Mol Microbiol. 1999;31(5):1285–94.View ArticlePubMedGoogle Scholar
- Nicolas P, Mader U, Dervyn E, Rochat T, Leduc A, Pigeonneau N, et al. Condition-dependent transcriptome reveals high-level regulatory architecture in bacillus subtilis. Science. 2012;335(6072):1103–6.View ArticlePubMedGoogle Scholar
- Blankenberg D, Von Kuster G, Coraor N, Ananda G, Lazarus R, Mangan M, et al. A web-based genome analysis tool for experimentalists. Current protocols in molecular biology/edited by Frederick M Ausubel [et al] 2010, Chapter 19:Unit 19 10 11-21.Google Scholar
- Giardine B, Riemer C, Hardison RC, Burhans R, Elnitski L, Shah P, et al. Galaxy: a platform for interactive large-scale genome analysis. Genome Res. 2005;15(10):1451–5.View ArticlePubMed CentralPubMedGoogle Scholar
- Goecks J, Nekrutenko A, Taylor J, Galaxy T. Galaxy: a comprehensive approach for supporting accessible, reproducible, and transparent computational research in the life sciences. Genome Biol. 2010;11(8):R86.View ArticlePubMed CentralPubMedGoogle Scholar
- Trapnell C, Williams BA, Pertea G, Mortazavi A, Kwan G, van Baren MJ, et al. Transcript assembly and quantification by RNA-Seq reveals unannotated transcripts and isoform switching during cell differentiation. Nat Biotechnol. 2010;28(5):511–5.View ArticlePubMed CentralPubMedGoogle Scholar
- Robinson JT, Thorvaldsdottir H, Winckler W, Guttman M, Lander ES, Getz G, et al. Integrative genomics viewer. Nat Biotechnol. 2011;29(1):24–6.View ArticlePubMed CentralPubMedGoogle Scholar
- Trapnell C, Hendrickson DG, Sauvageau M, Goff L, Rinn JL, Pachter L. Differential analysis of gene regulation at transcript resolution with RNA-seq. Nat Biotechnol. 2013;31(1):46–53.View ArticlePubMedGoogle Scholar
- Trapnell C, Roberts A, Goff L, Pertea G, Kim D, Kelley DR, et al. Differential gene and transcript expression analysis of RNA-seq experiments with TopHat and Cufflinks. Nat Protoc. 2012;7(3):562–78.View ArticlePubMed CentralPubMedGoogle Scholar
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