Whole genome-scale assessment of gene fitness of Novosphingobium aromaticavorans during spaceflight.

In microgravity, bacteria undergo intriguing physiological adaptations. There have been few attempts to assess global bacterial physiological responses to microgravity, with most studies only focusing on a handful of individual systems. This study assessed the fitness of each gene in the genome of the aromatic compound-degrading Alphaproteobacterium Novosphingobium aromaticavorans during growth in spaceflight. This was accomplished using Comparative TnSeq, which involves culturing the same saturating transposon mutagenized library under two different conditions. To assess gene fitness, a novel comparative TnSeq analytical tool was developed, named TnDivA, that is particularly useful in leveraging biological replicates. In this approach, transposon diversity is represented numerically using a modified Shannon diversity index, which was then converted into effective transposon density. This transformation accounts for variability in read distribution between samples, such as cases where reads were dominated by only a few transposon inserts. Effective density values were analyzed using multiple statistical methods, including log2-fold change, least-squares regression analysis, and Welch's t-test. The results obtained across applied statistical methods show a difference in the number of significant genes identified. However, the functional categories of genes important to growth in microgravity showed similar patterns. Lipid metabolism and transport, energy production, transcription, translation, and secondary metabolite biosynthesis and transport were shown to have high fitness during spaceflight. This suggests that core metabolic processes, including lipid and secondary metabolism, play an important role adapting to stress and promoting growth in microgravity.

The transcriptional regulator CtrA controls gene expression in Alphaproteobacteria phages: Evidence for a lytic deferment pathway.

Pilitropic and flagellotropic phages adsorb to bacterial pili and flagella. These phages have long been used to investigate multiple aspects of bacterial physiology, such as the cell cycle control in the Caulobacterales. Targeting cellular appendages for adsorption effectively constrains the population of infectable hosts, suggesting that phages may have developed strategies to maximize their infective yield. Brevundimonas phage vB_BsubS-Delta is a recently characterized pilitropic phage infecting the Alphaproteobacterium Brevundimonas subvibrioides. Like other Caulobacterales, B. subvibrioides divides asymmetrically and its cell cycle is governed by multiple transcriptional regulators, including the master regulator CtrA. Genomic characterization of phage vB_BsubS-Delta identified the presence of a large intergenic region with an unusually high density of putative CtrA-binding sites. A systematic analysis of the positional distribution of predicted CtrA-binding sites in complete phage genomes reveals that the highly skewed distribution of CtrA-binding sites observed in vB_BsubS-Delta is an unequivocal genomic signature that extends to other pilli- and flagellotropic phages infecting the Alphaproteobacteria. Moreover, putative CtrA-binding sites in these phage genomes localize preferentially to promoter regions and have higher scores than those detected in other phage genomes. Phylogenetic and comparative genomics analyses show that this genomic signature has evolved independently in several phage lineages, suggesting that it provides an adaptive advantage to pili/flagellotropic phages infecting the Alphaproteobacteria. Experimental results demonstrate that CtrA binds to predicted CtrA-binding sites in promoter regions and that it regulates transcription of phage genes in unrelated Alphaproteobacteria-infecting phages. We propose that this focused distribution of CtrA-binding sites reflects a fundamental new aspect of phage infection, which we term lytic deferment. Under this novel paradigm, pili- and flagellotropic phages exploit the CtrA transduction pathway to monitor the host cell cycle state and synchronize lysis with the presence of infectable cells.

The Impacts of Microgravity on Bacterial Metabolism.

The inside of a space-faring vehicle provides a set of conditions unlike anything experienced by bacteria on Earth. The low-shear, diffusion-limited microenvironment with accompanying high levels of ionizing radiation create high stress in bacterial cells, and results in many physiological adaptations. This review gives an overview of the effect spaceflight in general, and real or simulated microgravity in particular, has on primary and secondary metabolism. Some broad trends in primary metabolic responses can be identified. These include increases in carbohydrate metabolism, changes in carbon substrate utilization range, and changes in amino acid metabolism that reflect increased oxidative stress. However, another important trend is that there is no universal bacterial response to microgravity, as different bacteria often have contradictory responses to the same stress. This is exemplified in many of the observed secondary metabolite responses where secondary metabolites may have increased, decreased, or unchanged production in microgravity. Different secondary metabolites in the same organism can even show drastically different production responses. Microgravity can also impact the production profile and localization of secondary metabolites. The inconsistency of bacterial responses to real or simulated microgravity underscores the importance of further research in this area to better understand how microbes can impact the people and systems aboard spacecraft.

The Use of TnSeq to Identify Essential Alphaproteobacterial Genes Reveals Operational Variability in Conserved Developmental and Cell Cycle Systems.

A powerful method for examining genetic fitness and function on a large scale is to couple saturating transposon mutagenesis with high-throughput sequencing (TnSeq). By mapping where transposon insertions can be tolerated in a genome, it is possible to analyze the fitness of every gene in a genome simultaneously under a given growth condition. While this technique can describe genes as essential or nonessential under those growth conditions, sufficient mutagenesis and sequencing depth can provide more subtle differences in fitness. In this paper, TnSeq was used to analyze gene fitness of two Alphaproteobacteria from different environments: the freshwater oligotroph Brevundimonas subvibrioides (Caulobacterales) and the soil plant pathogen Agrobacterium tumefaciens (Rhizobiales) for the purpose of comparing conservation of gene function.

Transcriptional rewiring of the GcrA/CcrM bacterial epigenetic regulatory system in closely related bacteria.

Transcriptional rewiring is the regulation of different target genes by orthologous regulators in different organisms. While this phenomenon has been observed, it has not been extensively studied, particularly in core regulatory systems. Several global cell cycle regulators are conserved in the Alphaproteobacteria, providing an excellent model to study this phenomenon. First characterized in Caulobacter crescentus, GcrA and CcrM compose a DNA methylation-based regulatory system that helps coordinate the complex life cycle of this organism. These regulators are well-conserved across Alphaproteobacteria, but the extent to which their regulatory targets are conserved is not known. In this study, the regulatory targets of GcrA and CcrM were analyzed by SMRT-seq, RNA-seq, and ChIP-seq technologies in the Alphaproteobacterium Brevundimonas subvibrioides, and then compared to those of its close relative C. crescentus that inhabits the same environment. Although the regulators themselves are highly conserved, the genes they regulate are vastly different. GcrA directly regulates 204 genes in C. crescentus, and though B. subvibrioides has orthologs to 147 of those genes, only 48 genes retained GcrA binding in their promoter regions. Additionally, only 12 of those 48 genes demonstrated significant transcriptional change in a gcrA mutant, suggesting extensive transcriptional rewiring between these organisms. Similarly, out of hundreds of genes CcrM regulates in each of these organisms, only 2 genes were found in common. When multiple Alphaproteobacterial genomes were analyzed bioinformatically for potential GcrA regulatory targets, the regulation of genes involved in DNA replication and cell division was well conserved across the Caulobacterales but not outside this order. This work suggests that significant transcriptional rewiring can occur in cell cycle regulatory systems even over short evolutionary distances.

Analysis of Brevundimonas subvibrioides Developmental Signaling Systems Reveals Inconsistencies between Phenotypes and c-di-GMP Levels.

The DivJ-DivK-PleC signaling system of Caulobacter crescentus is a signaling network that regulates polar development and the cell cycle. This system is conserved in related bacteria, including the sister genus Brevundimonas Previous studies had shown unexpected phenotypic differences between the C. crescentusdivK mutant and the analogous mutant of Brevundimonas subvibrioides, but further characterization was not performed. Here, phenotypic assays analyzing motility, adhesion, and pilus production (the latter characterized by a newly discovered bacteriophage) revealed that divJ and pleC mutants have phenotypes mostly similar to their C. crescentus homologs, but divK mutants maintain largely opposite phenotypes than expected. Suppressor mutations of the B. subvibrioides divK motility defect were involved in cyclic di-GMP (c-di-GMP) signaling, including the diguanylate cyclase dgcB, and cleD which is hypothesized to affect flagellar function in a c-di-GMP dependent fashion. However, the screen did not identify the diguanylate cyclase pleD Disruption of pleD in B. subvibrioides caused no change in divK or pleC phenotypes, but did reduce adhesion and increase motility of the divJ strain. Analysis of c-di-GMP levels in these strains revealed incongruities between c-di-GMP levels and displayed phenotypes with a notable result that suppressor mutations altered phenotypes but had little impact on c-di-GMP levels in the divK background. Conversely, when c-di-GMP levels were artificially manipulated, alterations of c-di-GMP levels in the divK strain had minimal impact on phenotypes. These results suggest that DivK performs a critical function in the integration of c-di-GMP signaling into the B. subvibrioides cell cycle.IMPORTANCE Cyclic di-GMP and associated signaling proteins are widespread in bacteria, but their role in physiology is often complex and difficult to predict through genomic level analyses. In C. crescentus, c-di-GMP has been integrated into the developmental cell cycle, but there is increasing evidence that environmental factors can impact this system as well. The research presented here suggests that the integration of these signaling networks could be more complex than previously hypothesized, which could have a bearing on the larger field of c-di-GMP signaling. In addition, this work further reveals similarities and differences in a conserved regulatory network between organisms in the same taxonomic family, and the results show that gene conservation does not necessarily imply close functional conservation in genetic pathways.

Genome-scale fitness profile of Caulobacter crescentus grown in natural freshwater.

Bacterial genomes evolve in complex ecosystems and are best understood in this natural context, but replicating such conditions in the lab is challenging. We used transposon sequencing to define the fitness consequences of gene disruption in the bacterium Caulobacter crescentus grown in natural freshwater, compared with axenic growth in common laboratory media. Gene disruptions in amino-acid and nucleotide sugar biosynthesis pathways and in metabolic substrate transport machinery impaired fitness in both lake water and defined minimal medium relative to complex peptone broth. Fitness in lake water was enhanced by insertions in genes required for flagellum biosynthesis and reduced by insertions in genes involved in biosynthesis of the holdfast surface adhesin. We further uncovered numerous hypothetical and uncharacterized genes for which disruption impaired fitness in lake water, defined minimal medium, or both. At the genome scale, the fitness profile of mutants cultivated in lake water was more similar to that in complex peptone broth than in defined minimal medium. Microfiltration of lake water did not significantly affect the terminal cell density or the fitness profile of the transposon mutant pool, suggesting that Caulobacter does not strongly interact with other microbes in this ecosystem on the measured timescale. Fitness of select mutants with defects in cell surface biosynthesis and environmental sensing were significantly more variable across days in lake water than in defined medium, presumably owing to day-to-day heterogeneity in the lake environment. This study reveals genetic interactions between Caulobacter and a natural freshwater environment, and provides a new avenue to study gene function in complex ecosystems.

Stalk formation of Brevundimonas and how it compares to Caulobacter crescentus.

The Caulobacter crescentus cell extension known as a stalk represents an unusual bacterial morphology. C. crescentus produces stalks under multiple nutrient conditions, but the length of the stalk is increased in response to phosphate starvation. However, the exact function of the stalk is not known, nor is it known how much stalk biogenesis or function is conserved with other stalked bacteria. Work presented here shows that many organisms in the Caulobacter genus and the next closest genus (Brevundimonas) generally do not synthesize stalks in the relatively-rich PYE growth medium, suggesting that the synthesis of a stalk under nutrient-rich conditions by C. crescentus may be the exception instead of the norm among its phylogenetic group. Brevundimonas subvibrioides can be induced to synthesize stalks by genetically mimicking phosphate starvation conditions, indicating stalk synthesis in this organism may be performed on an as-need basis. This mutation, however, does not appear to increase the incidence of holdfast synthesis. While B. subvibrioides stalks appear to be synthesized with the same polarity with respect to holdfast as C. crescentus stalks, evidence is presented that suggests B. subvibrioides may disassemble stalks when they are no longer needed. Many homologs of C. crescentus genes encoding stalk-associated proteins are absent in the B. subvibrioides genome, and B. subvibrioides PstA-GFP as well as C. crescentus StpX-GFP are able to enter the B. subvibrioides stalk compartment, calling into question the level of compartmentalization of the B. subvibrioides stalk. In summary, this work begins to address how much the C. crescentus model for this unusual morphological adaptation can be extended to related organisms.

DNA methyltransferases and epigenetic regulation in bacteria.

Epigenetics is a change in gene expression that is heritable without a change in DNA sequence itself. This phenomenon is well studied in eukaryotes, particularly in humans for its role in cellular differentiation, X chromosome inactivation and diseases like cancer. However, comparatively little is known about epigenetic regulation in bacteria. Bacterial epigenetics is mainly present in the form of DNA methylation where DNA methyltransferases add methyl groups to nucleotides. This review focuses on two methyltransferases well characterized for their roles in gene regulation: Dam and CcrM. Dam methyltransferase in Escherichia coli is important for expression of certain genes such as the pap operon, as well as other cellular processes like DNA replication initiation and DNA repair. In Caulobacter crescentus and other Alphaproteobacteria, the methyltransferase CcrM is cell cycle regulated and is involved in the cell-cycle-dependent regulation of several genes. The diversity of regulatory targets as well as regulatory mechanisms suggests that gene regulation by methylation could be a widespread and potent method of regulation in bacteria.

Essential Genes Predicted in the Genome of Rubrivivax gelatinosus.

UNLABELLED: Rubrivivax gelatinosus is a betaproteobacterium with impressive metabolic diversity. It is capable of phototrophy, chemotrophy, two different mechanisms of sugar metabolism, fermentation, and H2 gas production. To identify core essential genes, R. gelatinosus was subjected to saturating transposon mutagenesis and high-throughput sequencing (TnSeq) analysis using nutrient-rich, aerobic conditions. Results revealed that virtually no primary metabolic genes are essential to the organism and that genomic redundancy only explains a portion of the nonessentiality, but some biosynthetic pathways are still essential under nutrient-rich conditions. Different essentialities of different portions of the Pho regulatory pathway suggest that overexpression of the regulon is toxic and hint at a larger connection between phosphate regulation and cellular health. Lastly, various essentialities of different tRNAs hint at a more complex situation than would be expected for such a core process. These results expand upon research regarding cross-organism gene essentiality and further enrich the study of purple nonsulfur bacteria. IMPORTANCE: Microbial genomic data are increasing at a tremendous rate, but physiological characterization of those data lags far behind. One mechanism of high-throughput physiological characterization is TnSeq, which uses high-volume transposon mutagenesis and high-throughput sequencing to identify all of the essential genes in a given organism's genome. Here TnSeq was used to identify essential genes in the metabolically versatile betaproteobacterium Rubrivivax gelatinosus The results presented here add to the growing TnSeq field and also reveal important aspects of R. gelatinosus physiology, which are applicable to researchers working on metabolically flexible organisms.

Identification of essential alphaproteobacterial genes reveals operational variability in conserved developmental and cell cycle systems.

The cell cycle of Caulobacter crescentus is controlled by a complex signalling network that co-ordinates events. Genome sequencing has revealed many C. crescentus cell cycle genes are conserved in other Alphaproteobacteria, but it is not clear to what extent their function is conserved. As many cell cycle regulatory genes are essential in C. crescentus, the essential genes of two Alphaproteobacteria, Agrobacterium tumefaciens (Rhizobiales) and Brevundimonas subvibrioides (Caulobacterales), were elucidated to identify changes in cell cycle protein function over different phylogenetic distances as demonstrated by changes in essentiality. The results show the majority of conserved essential genes are involved in critical cell cycle processes. Changes in component essentiality reflect major changes in lifestyle, such as divisome components in A. tumefaciens resulting from that organism's different growth pattern. Larger variability of essentiality was observed in cell cycle regulators, suggesting regulatory mechanisms are more customizable than the processes they regulate. Examples include variability in the essentiality of divJ and divK spatial cell cycle regulators, and non-essentiality of the highly conserved and usually essential DNA methyltransferase CcrM. These results show that while essential cell functions are conserved across varying genetic distance, much of a given organism's essential gene pool is specific to that organism.

Effect of a ctrA promoter mutation, causing a reduction in CtrA abundance, on the cell cycle and development of Caulobacter crescentus.

BACKGROUND: Polar development during the alphaproteobacterium Caulobacter crescentus cell cycle is integrated to the point that individual mutations can have pleiotropic effects on the synthesis of polar organelles. Disruption of the genes encoding the histidine kinase PleC, or its localization factor PodJ, disrupts synthesis or functionality of pili, flagella and adhesive holdfast. However, the mechanism by which these mutations affect polar development is not well understood. The aim of this study was to identify new regulators that control multiple aspects of polar organelle development. RESULTS: To identify mutants with pleiotropic polar organelle synthesis defects, transposon mutagenesis was performed and mutants were selected based resistance to the pili-tropic bacteriophage ΦCbK. Mutants were then screened for defects in motility and holdfast production. Only a single podJ/pleC-independent mutant was isolated which had defects in all three phenotypes. Directed phage assays confirmed the phage resistance phenotype, while the strain demonstrated a similar dispersal radius as a podJ mutant in swarm agar, and treatment with a fluorescent lectin that labels the holdfast showed no staining for this mutant. The transposon had inserted into the promoter region of ctrA, a gene encoding a master transcriptional regulator of the cell cycle, disrupting native transcription but still allowing reduced transcriptional activity and protein production of this essential protein. Transcriptional fusions showed that essential genes controlled by CtrA exhibited minor to moderate changes in expression in the ctrA promoter mutant, while the pilA gene, encoding the subunit of the pilus filament, had a drastic decrease in gene expression. Introduction of a plasmid-born copy of ctrA under its native promoter complemented the phage resistance and holdfast defects, as well as a moderate cell morphology defect, but not the swarming defect. CONCLUSIONS: A mutation was identified that caused pleiotropic defects in polar organelle synthesis, and revealed the surprising result that some CtrA-dependent promoters are more sensitive to changes in CtrA concentration than others. However, the fact that no pleiotropic mutations were found in new regulators suggests that downstream signaling of PleC/PodJ is either essential, redundant, or branching such that all three phenotypes were not simultaneously affected.

The scaffolding and signalling functions of a localization factor impact polar development.

In the differentiating alphaproteobacterium Caulobacter crescentus, organelle synthesis at cell poles is critical to forming different progeny after cell division. Co-ordination of polar organelle synthesis, including pili and holdfast, and flagellum ejection, is mediated in part by the scaffolding protein PodJ. At the time of cell division, PodJ undergoes regulated processing to a short form that persists at the flagellar pole of swarmer cells. This study analyses how PodJ's role in structural and signalling protein localization impacts organelle synthesis. A PodJ mutant with an internal deletion exhibits reduced sensitivity to pili-tropic phage ΦCbK, resulting from reduced pilA gene expression, which can be linked to altered signalling protein localization. The phage sensitivity defect of a ΔpodJ mutant can be partially suppressed by ectopic pilA expression. Induction of PodJ processing, by manipulation of podJ itself or controlled perP expression, resulted in decreased pilus biogenesis and, when coupled with a podJ mutation that reduced pilA expression, led to complete loss of phage sensitivity. As a whole, the results show that PodJ's scaffolding role for structural and signalling proteins both contribute to flagellar pole organelle development.

A novel effector protein modulates response regulator activity without altering phosphorylation.

Genes positively regulated by the global transcriptional response regulator CtrA are not expressed during a life cycle stage of Caulobacter crescentus when the regulator is activated by phosphorylation. Gora et al. (2010), in this issue of Molecular Cell, have discovered a novel effector protein that prevents activation but not repression by the regulator without altering its phosphorylation.

Getting in the loop: regulation of development in Caulobacter crescentus.

Caulobacter crescentus is an aquatic Gram-negative alphaproteobacterium that undergoes multiple changes in cell shape, organelle production, subcellular distribution of proteins, and intracellular signaling throughout its life cycle. Over 40 years of research has been dedicated to this organism and its developmental life cycles. Here we review a portion of many developmental processes, with particular emphasis on how multiple processes are integrated and coordinated both spatially and temporally. While much has been discovered about Caulobacter crescentus development, areas of potential future research are also highlighted.

Spatial organization of Myxococcus xanthus during fruiting body formation.

Microcinematography was used to examine fruiting body development of Myxococcus xanthus. Wild-type cells progress through three distinct phases: a quiescent phase with some motility but little aggregation (0 to 8 h), a period of vigorous motility leading to raised fruiting bodies (8 to 16 h), and a period of maturation during which sporulation is initiated (16 to 48 h). Fruiting bodies are extended vertically in a series of tiers, each involving the addition of a cell monolayer on top of the uppermost layer. A pilA (MXAN_5783) mutant produced less extracellular matrix material and thus allowed closer examination of tiered aggregate formation. A csgA (MXAN_1294) mutant exhibited no quiescent phase, aberrant aggregation in phase 2, and disintegration of the fruiting bodies in the third phase.

Proteins associated with the Myxococcus xanthus extracellular matrix.

Fruiting body formation of Myxococcus xanthus, like biofilm formation of many other organisms, involves the production of an extracellular matrix (ECM). While the polysaccharide component has been studied, the protein component has been largely unexplored. Proteins associated with the ECM were solubilized from purified ECM by boiling with sodium dodecyl sulfate and were identified by liquid chromatography-tandem mass spectrometry of tryptic fragments. The ECM is enriched in proteins of novel function; putative functions were assigned for only 5 of the 21 proteins. Thirteen putative ECM proteins had lipoprotein secretion signals. The genes for many ECM proteins were disrupted in the wild-type (WT), fibA, and pilA backgrounds. Disruption of the MXAN4860 gene had no effect in the WT or fibA background but in the pilA background resulted in a 24-h delay in aggregation and sporulation compared to its parent. The results of this study show that the M. xanthus ECM proteome is diverse and novel.

Novel lipids in Myxococcus xanthus and their role in chemotaxis.

Organisms that colonize solid surfaces, like Myxococcus xanthus, use novel signalling systems to organize multicellular behaviour. Phosphatidylethanolamine (PE) containing the fatty acid 16:1omega5 (Delta11) elicits a chemotactic response. The phenomenon was examined by observing the effects of PE species with varying fatty acid pairings. Wild-type M. xanthus contains 17 different PE species under vegetative conditions and 19 at the midpoint of development; 13 of the 17 have an unsaturated fatty acid at the sn-1 position, a novelty among Proteobacteria. Myxococcus xanthus has two glycerol-3-phosphate acyltransferase (PlsB) homologues which add the sn-1 fatty acid. Each produces PE with 16:1 at the sn-1 position and supports growth and fruiting body development. Deletion of plsB1 (MXAN3288) results in more dramatic changes in PE species distribution than deletion of plsB2 (MXAN1675). PlsB2 has a putative N-terminal eukaryotic fatty acid reductase domain and may support both ether lipid synthesis and PE synthesis. Disruption of a single sn-2 acyltransferase homologue (PlsC, of which M. xanthus contains five) results in minor changes in membrane PE. Derivatization of purified PE extracts with dimethyldisulfide was used to determine the position of the double bonds in unsaturated fatty acids. The results suggest that Delta5 and Delta11 desaturases may create the double bonds after synthesis of the fatty acid. Phosphatidylethanolamine enriched for 16:1 at the sn-1 position stimulates chemotaxis more strongly than PE with 16:1 enriched at the sn-2 position. It appears that the deployment of a rare fatty acid (16:1omega5) at an unusual position (sn-1) has facilitated the evolution of a novel cell signal.