New Motion Analysis System for Characterization of the Chemosensory Response Kinetics of Rhodobacter sphaeroides under Different Growth Conditions

ABSTRACTWe developed a new set of software tools that enable the speed and response kinetics of large numbers of tethered bacterial cells to be rapidly measured and analyzed. The software provides precision, accuracy, and a good signal-to-noise ratio combined with ease of data handling and processing. The software was tested on the single-cell chemosensory response kinetics of large numbers ofRhodobacter sphaeroidescells grown under either aerobic or photoheterotrophic conditions and either in chemostats or in batch cultures, allowing the effects of growth conditions on responses to be accurately measured. Aerobically and photoheterotrophically grownR. sphaeroidesexhibited significantly different chemosensory response kinetics and cell-to-cell variability in their responses to 100 μM propionate. A greater proportion of the population of aerobically grown cells responded to a 100 μM step decrease in propionate; they adapted faster and showed less cell-to-cell variability than photosynthetic populations. Growth in chemostats did not significantly reduce the measured cell to cell variability but did change the adaptation kinetics for photoheterotrophically grown cells.

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A Rhodobacter sphaeroides Protein Mechanistically Similar to Escherichia coli DksA Regulates Photosynthetic Growth

mBio ◽

10.1128/mbio.01105-14 ◽

2014 ◽

Vol 5 (3) ◽

Cited By ~ 7

Author(s):

Christopher W. Lennon ◽

Kimberly C. Lemmer ◽

Jessica L. Irons ◽

Max I. Sellman ◽

Timothy J. Donohue ◽

...

Keyword(s):

Escherichia Coli ◽

Structure Function ◽

Rhodobacter Sphaeroides ◽

Fatty Acid Content ◽

Regulatory Protein ◽

Growth Conditions ◽

Globular Domain ◽

Content Type ◽

E Coli

ABSTRACTDksA is a global regulatory protein that, together with the alarmone ppGpp, is required for the “stringent response” to nutrient starvation in the gammaproteobacteriumEscherichia coliand for more moderate shifts between growth conditions. DksA modulates the expression of hundreds of genes, directly or indirectly. Mutants lacking a DksA homolog exhibit pleiotropic phenotypes in other gammaproteobacteria as well. Here we analyzed the DksA homolog RSP2654 in the more distantly relatedRhodobacter sphaeroides, an alphaproteobacterium. RSP2654 is 42% identical and similar in length toE. coliDksA but lacks the Zn finger motif of theE. coliDksA globular domain. Deletion of the RSP2654 gene results in defects in photosynthetic growth, impaired utilization of amino acids, and an increase in fatty acid content. RSP2654 complements the growth and regulatory defects of anE. colistrain lacking thedksAgene and modulates transcriptionin vitrowithE. coliRNA polymerase (RNAP) similarly toE. coliDksA. RSP2654 reduces RNAP-promoter complex stabilityin vitrowith RNAPs fromE. coliorR. sphaeroides, alone and synergistically with ppGpp, suggesting that even though it has limited sequence identity toE. coliDksA (DksAEc), it functions in a mechanistically similar manner. We therefore designate the RSP2654 protein DksARsp. Our work suggests that DksARsphas distinct and important physiological roles in alphaproteobacteria and will be useful for understanding structure-function relationships in DksA and the mechanism of synergy between DksA and ppGpp.IMPORTANCEThe role of DksA has been analyzed primarily in the gammaproteobacteria, in which it is best understood for its role in control of the synthesis of the translation apparatus and amino acid biosynthesis. Our work suggests that DksA plays distinct and important physiological roles in alphaproteobacteria, including the control of photosynthesis inRhodobacter sphaeroides. The study of DksARsp, should be useful for understanding structure-function relationships in the protein, including those that play a role in the little-understood synergy between DksA and ppGpp.

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The CtrA Regulon of Rhodobacter sphaeroides Favors Adaptation to a Particular Lifestyle

Journal of Bacteriology ◽

10.1128/jb.00678-19 ◽

2020 ◽

Vol 202 (7) ◽

Author(s):

José Hernández-Valle ◽

Alejandro Sanchez-Flores ◽

Sebastian Poggio ◽

Georges Dreyfus ◽

Laura Camarena

Keyword(s):

Rhodobacter Sphaeroides ◽

Stress Responses ◽

Transcriptome Sequencing ◽

Growth Conditions ◽

Fine Tuning ◽

Rna Seq ◽

Two Component System ◽

Component System ◽

Content Type ◽

Two Component

ABSTRACT Activation of the two-component system formed by CckA, ChpT, and CtrA (kinase, phosphotransferase, and response regulator, respectively) in Rhodobacter sphaeroides does not occur under the growth conditions commonly used in the laboratory. However, it is possible to isolate a gain-of-function mutant in CckA that turns the system on. Using massive parallel transcriptome sequencing (RNA-seq), we identified 321 genes that are differentially regulated by CtrA. From these genes, 239 were positively controlled and 82 were negatively regulated. Genes encoding the Fla2 polar flagella and gas vesicle proteins are strongly activated by CtrA. Genes involved in stress responses as well as several transcriptional factors are also positively controlled, whereas the photosynthetic and CO2 fixation genes are repressed. Potential CtrA-binding sites were bioinformatically identified, leading to the proposal that at least 81 genes comprise the direct regulon. Based on our results, we ponder that the transcriptional response orchestrated by CtrA enables a lifestyle in which R. sphaeroides will effectively populate the surface layer of a water body enabled by gas vesicles and will remain responsive to chemotactic stimuli using the chemosensoring system that controls the Fla2 flagellum. Simultaneously, fine-tuning of photosynthesis and stress responses will reduce the damage caused by heat and high light intensity in this water stratum. In summary, in this bacterium CtrA has evolved to control physiological responses that allow its adaptation to a particular lifestyle instead of controlling the cell cycle as occurs in other species. IMPORTANCE Cell motility in Alphaproteobacteria is frequently controlled by the CckA, ChpT, and CtrA two-component system. Under the growth conditions commonly used in the laboratory, ctrA is transcriptionally inactive in Rhodobacter sphaeroides, and motility depends on the Fla1 flagellar system that was acquired by a horizontal transfer event. Likely, the incorporation of this flagellar system released CtrA from the strong selective pressure of being the main motility regulator, allowing this two-component system to specialize and respond to some specific conditions. Identifying the genes that are directly regulated by CtrA could help us understand the conditions in which the products of this regulon are required. Massive parallel transcriptome sequencing (RNA-seq) revealed that CtrA orchestrates an adaptive response that contributes to the colonization of a particular environmental niche.

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Quantification of purple non-sulphur phototrophic bacteria and their photosynthetic structures by means of total reflection X-ray fluorescence spectrometry (TXRF)

Journal of Analytical Atomic Spectrometry ◽

10.1039/c6ja00207b ◽

2016 ◽

Vol 31 (10) ◽

pp. 2078-2088 ◽

Cited By ~ 2

Author(s):

Joanna Fiedor ◽

Beata Ostachowicz ◽

Monika Baster ◽

Marek Lankosz ◽

Květoslava Burda

Keyword(s):

Correlation Analysis ◽

Trace Element ◽

Rhodobacter Sphaeroides ◽

Growth Conditions ◽

Phototrophic Bacteria ◽

Total Reflection ◽

Fluorescence Spectrometry ◽

Bacterial Cells ◽

X Ray

TXRF spectrometry proves useful in analysing bacterial cells and their substructures as exemplified byRhodobacter sphaeroides. Trace element profiling complemented with correlation analysis under varying growth conditions.

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Barriers to 3-Hydroxypropionate-Dependent Growth of Rhodobacter sphaeroides by Distinct Disruptions of the Ethylmalonyl Coenzyme A Pathway

Journal of Bacteriology ◽

10.1128/jb.00556-18 ◽

2018 ◽

Vol 201 (4) ◽

Cited By ~ 1

Author(s):

Steven J. Carlson ◽

Angela Fleig ◽

M. Kelsey Baron ◽

Ivan A. Berg ◽

Birgit E. Alber

Keyword(s):

Rhodobacter Sphaeroides ◽

Drug Target ◽

Coenzyme A ◽

Growth Conditions ◽

Gene Encoding ◽

Gene Deletions ◽

Content Type ◽

Growth Defects ◽

Dependent Growth

ABSTRACT Rhodobacter sphaeroides is able to use 3-hydroxypropionate as the sole carbon source through the reductive conversion of 3-hydroxypropionate to propionyl coenzyme A (propionyl-CoA). The ethylmalonyl-CoA pathway is not required in this process because a crotonyl-CoA carboxylase/reductase (Ccr)-negative mutant still grew with 3-hydroxypropionate. Much to our surprise, a mutant defective for another specific enzyme of the ethylmalonyl-CoA pathway, mesaconyl-CoA hydratase (Mch), lost its ability for 3-hydroxypropionate-dependent growth. Interestingly, the Mch-deficient mutant was rescued either by introducing an additional ccr in-frame deletion that resulted in the blockage of an earlier step in the pathway or by heterologously expressing a gene encoding a thioesterase (YciA) that can act on several CoA intermediates of the ethylmalonyl-CoA pathway. The mch mutant expressing yciA metabolized only less than half of the 3-hydroxypropionate supplied, and over 50% of that carbon was recovered in the spent medium as free acids of the key intermediates mesaconyl-CoA and methylsuccinyl-CoA. A gradual increase in growth inhibition due to the blockage of consecutive steps of the ethylmalonyl-CoA pathway by gene deletions suggests that the growth defects were due to the titration of free CoA and depletion of the CoA pool in the cell rather than to detrimental effects arising from the accumulation of a specific metabolite. Recovery of carbon in mesaconate for the wild-type strain expressing yciA demonstrated that carbon flux through the ethylmalonyl-CoA pathway occurs during 3-hydroxypropionate-dependent growth. A possible role of the ethylmalonyl-CoA pathway is proposed that functions outside its known role in providing tricarboxylic acid intermediates during acetyl-CoA assimilation. IMPORTANCE Mutant analysis is an important tool utilized in metabolic studies to understand which role a particular pathway might have under certain growth conditions for a given organism. The importance of the enzyme and of the pathway in which it participates is discretely linked to the resulting phenotype observed after mutation of the corresponding gene. This work highlights the possibility of incorrectly interpreting mutant growth results that are based on studying a single unit (gene and encoded enzyme) of a metabolic pathway rather than the pathway in its entirety. This work also hints at the possibility of using an enzyme as a drug target although the enzyme may participate in a nonessential pathway and still be detrimental to the cell when inhibited.

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Response kinetics in the complex chemotaxis signalling pathway of Rhodobacter sphaeroides

Journal of The Royal Society Interface ◽

10.1098/rsif.2012.1001 ◽

2013 ◽

Vol 10 (81) ◽

pp. 20121001 ◽

Cited By ~ 13

Author(s):

Mila Kojadinovic ◽

Judith P. Armitage ◽

Marcus J. Tindall ◽

George H. Wadhams

Keyword(s):

Protein Expression ◽

Rhodobacter Sphaeroides ◽

Bacterial Chemotaxis ◽

Growth Conditions ◽

Weber’S Law ◽

Expression Levels ◽

Weber's Law ◽

Chemosensory Pathway ◽

Cell Assays ◽

Chemosensory Response

Chemotaxis is one of the best-characterized signalling systems in biology. It is the mechanism by which bacteria move towards optimal environments and is implicated in biofilm formation, pathogenesis and symbiosis. The properties of the bacterial chemosensory response have been described in detail for the single chemosensory pathway of Escherichia coli . We have characterized the properties of the chemosensory response of Rhodobacter sphaeroides , an α-proteobacterium with multiple chemotaxis pathways, under two growth conditions allowing the effects of protein expression levels and cell architecture to be investigated. Using tethered cell assays, we measured the responses of the system to step changes in concentration of the attractant propionate and show that, independently of the growth conditions, R. sphaeroides is chemotactic over at least five orders of magnitude and has a sensing profile following Weber's Law. Mathematical modelling also shows that, as E. coli , R. sphaeroides is capable of showing fold-change detection (FCD). Our results indicate that general features of bacterial chemotaxis such as the range and sensitivity of detection, adaptation times, adherence to Weber's Law and the presence of FCD may be integral features of chemotaxis systems in general, regardless of network complexity, protein expression levels and cellular architecture across different species.

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The Flagellar Set Fla2 in Rhodobacter sphaeroides Is Controlled by the CckA Pathway and Is Repressed by Organic Acids and the Expression of Fla1

Journal of Bacteriology ◽

10.1128/jb.02429-14 ◽

2014 ◽

Vol 197 (5) ◽

pp. 833-847 ◽

Cited By ~ 12

Author(s):

Benjamín Vega-Baray ◽

Clelia Domenzain ◽

Anet Rivera ◽

Rocío Alfaro-López ◽

Elidet Gómez-César ◽

...

Keyword(s):

Organic Acids ◽

Rhodobacter Sphaeroides ◽

Phylogenetic Analyses ◽

Carbon Sources ◽

Growth Conditions ◽

Kinase Domain ◽

Content Type ◽

Endogenous Genes ◽

Number Of Cells

Rhodobacter sphaeroideshas two different sets of flagellar genes. Under the growth conditions commonly used in the laboratory, the expression of thefla1set is constitutive, whereas thefla2genes are not expressed. Phylogenetic analyses have previously shown that thefla1genes were acquired by horizontal transfer from a gammaproteobacterium and that thefla2genes are endogenous genes of this alphaproteobacterium. In this work, we characterized a set of mutants that were selected for swimming using the Fla2 flagella in the absence of the Fla1 flagellum (Fla2+strains). We determined that these strains have a single missense mutation in the histidine kinase domain of CckA. The expression of these mutant alleles in a Fla1−strain allowedfla2-dependent motility without selection. Motility of the Fla2+strains is also dependent on ChpT and CtrA. The mutant versions of CckA showed an increased autophosphorylation activityin vitro. Interestingly, we found thatcckAis transcriptionally repressed by the presence of organic acids, suggesting that the availability of carbon sources could be a part of the signal that turns on this flagellar set. Evidence is presented showing that reactivation offla1gene expression in the Fla2+background strongly reduces the number of cells with Fla2 flagella.

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Regulation of Cell Division in Bacteria by Monitoring Genome Integrity and DNA Replication Status

Journal of Bacteriology ◽

10.1128/jb.00408-19 ◽

2019 ◽

Vol 202 (2) ◽

Cited By ~ 5

Author(s):

Peter E. Burby ◽

Lyle A. Simmons

Keyword(s):

Cell Cycle ◽

Dna Damage ◽

Cell Division ◽

Dna Replication ◽

Cell Cycle Progression ◽

Genome Instability ◽

Sos Response ◽

Bacterial Cells ◽

Cycle Progression ◽

Content Type

ABSTRACT All organisms regulate cell cycle progression by coordinating cell division with DNA replication status. In eukaryotes, DNA damage or problems with replication fork progression induce the DNA damage response (DDR), causing cyclin-dependent kinases to remain active, preventing further cell cycle progression until replication and repair are complete. In bacteria, cell division is coordinated with chromosome segregation, preventing cell division ring formation over the nucleoid in a process termed nucleoid occlusion. In addition to nucleoid occlusion, bacteria induce the SOS response after replication forks encounter DNA damage or impediments that slow or block their progression. During SOS induction, Escherichia coli expresses a cytoplasmic protein, SulA, that inhibits cell division by directly binding FtsZ. After the SOS response is turned off, SulA is degraded by Lon protease, allowing for cell division to resume. Recently, it has become clear that SulA is restricted to bacteria closely related to E. coli and that most bacteria enforce the DNA damage checkpoint by expressing a small integral membrane protein. Resumption of cell division is then mediated by membrane-bound proteases that cleave the cell division inhibitor. Further, many bacterial cells have mechanisms to inhibit cell division that are regulated independently from the canonical LexA-mediated SOS response. In this review, we discuss several pathways used by bacteria to prevent cell division from occurring when genome instability is detected or before the chromosome has been fully replicated and segregated.

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Revealing the Metabolic Alterations during Biofilm Development of Burkholderia cenocepacia Based on Genome-Scale Metabolic Modeling

Metabolites ◽

10.3390/metabo11040221 ◽

2021 ◽

Vol 11 (4) ◽

pp. 221

Author(s):

Ozlem Altay ◽

Cheng Zhang ◽

Hasan Turkez ◽

Jens Nielsen ◽

Mathias Uhlén ◽

...

Keyword(s):

Systems Biology ◽

Alternative Pathway ◽

Growth Conditions ◽

Pentose Phosphate ◽

Burkholderia Cenocepacia ◽

Bacterial Cells ◽

Metabolic Remodeling ◽

Transcriptomics Data ◽

Biofilm Development ◽

Genome Scale

Burkholderia cenocepacia is among the important pathogens isolated from cystic fibrosis (CF) patients. It has attracted considerable attention because of its capacity to evade host immune defenses during chronic infection. Advances in systems biology methodologies have led to the emergence of methods that integrate experimental transcriptomics data and genome-scale metabolic models (GEMs). Here, we integrated transcriptomics data of bacterial cells grown on exponential and biofilm conditions into a manually curated GEM of B. cenocepacia. We observed substantial differences in pathway response to different growth conditions and alternative pathway susceptibility to extracellular nutrient availability. For instance, we found that blockage of the reactions was vital through the lipid biosynthesis pathways in the exponential phase and the absence of microenvironmental lysine and tryptophan are essential for survival. During biofilm development, bacteria mostly had conserved lipid metabolism but altered pathway activities associated with several amino acids and pentose phosphate pathways. Furthermore, conversion of serine to pyruvate and 2,5-dioxopentanoate synthesis are also identified as potential targets for metabolic remodeling during biofilm development. Altogether, our integrative systems biology analysis revealed the interactions between the bacteria and its microenvironment and enabled the discovery of antimicrobial targets for biofilm-related diseases.

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Spo0A Suppresses sin Locus Expression in Clostridioides difficile

mSphere ◽

10.1128/msphere.00963-20 ◽

2020 ◽

Vol 5 (6) ◽

Author(s):

Babita Adhikari Dhungel ◽

Revathi Govind

Keyword(s):

Diarrheal Disease ◽

Toxin Production ◽

The United States ◽

Upstream Region ◽

Pcr Analysis ◽

Bacterial Cells ◽

Master Regulator ◽

Content Type ◽

Clostridioides Difficile

ABSTRACT Clostridioides difficile is the leading cause of nosocomial infection and is the causative agent of antibiotic-associated diarrhea. The severity of the disease is directly associated with toxin production, and spores are responsible for the transmission and persistence of the organism. Previously, we characterized sin locus regulators SinR and SinR′ (we renamed it SinI), where SinR is the regulator of toxin production and sporulation. The SinI regulator acts as its antagonist. In Bacillus subtilis, Spo0A, the master regulator of sporulation, controls SinR by regulating the expression of its antagonist, sinI. However, the role of Spo0A in the expression of sinR and sinI in C. difficile had not yet been reported. In this study, we tested spo0A mutants in three different C. difficile strains, R20291, UK1, and JIR8094, to understand the role of Spo0A in sin locus expression. Western blot analysis revealed that spo0A mutants had increased SinR levels. Quantitative reverse transcription-PCR (qRT-PCR) analysis of its expression further supported these data. By carrying out genetic and biochemical assays, we show that Spo0A can bind to the upstream region of this locus to regulates its expression. This study provides vital information that Spo0A regulates the sin locus, which controls critical pathogenic traits such as sporulation, toxin production, and motility in C. difficile. IMPORTANCE Clostridioides difficile is the leading cause of antibiotic-associated diarrheal disease in the United States. During infection, C. difficile spores germinate, and the vegetative bacterial cells produce toxins that damage host tissue. In C. difficile, the sin locus is known to regulate both sporulation and toxin production. In this study, we show that Spo0A, the master regulator of sporulation, controls sin locus expression. Results from our study suggest that Spo0A directly regulates the expression of this locus by binding to its upstream DNA region. This observation adds new detail to the gene regulatory network that connects sporulation and toxin production in this pathogen.

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Autophagy-Related Protein ATG8 Has a Noncanonical Function for Apicoplast Inheritance in Toxoplasma gondii

mBio ◽

10.1128/mbio.01446-15 ◽

2015 ◽

Vol 6 (6) ◽

Cited By ~ 43

Author(s):

Maude F. Lévêque ◽

Laurence Berry ◽

Michael J. Cipriano ◽

Hoa-Mai Nguyen ◽

Boris Striepen ◽

...

Keyword(s):

Toxoplasma Gondii ◽

Growth Conditions ◽

Model Organisms ◽

Secondary Endosymbiosis ◽

Related Protein ◽

Content Type ◽

Superresolution Microscopy ◽

Animal Pathogens ◽

Cellular Components ◽

Catabolic Process

ABSTRACT Autophagy is a catabolic process widely conserved among eukaryotes that permits the rapid degradation of unwanted proteins and organelles through the lysosomal pathway. This mechanism involves the formation of a double-membrane structure called the autophagosome that sequesters cellular components to be degraded. To orchestrate this process, yeasts and animals rely on a conserved set of autophagy-related proteins (ATGs). Key among these factors is ATG8, a cytoplasmic protein that is recruited to nascent autophagosomal membranes upon the induction of autophagy. Toxoplasma gondii is a potentially harmful human pathogen in which only a subset of ATGs appears to be present. Although this eukaryotic parasite seems able to generate autophagosomes upon stresses such as nutrient starvation, the full functionality and biological relevance of a canonical autophagy pathway are as yet unclear. Intriguingly, in T. gondii, ATG8 localizes to the apicoplast under normal intracellular growth conditions. The apicoplast is a nonphotosynthetic plastid enclosed by four membranes resulting from a secondary endosymbiosis. Using superresolution microscopy and biochemical techniques, we show that TgATG8 localizes to the outermost membrane of this organelle. We investigated the unusual function of TgATG8 at the apicoplast by generating a conditional knockdown mutant. Depletion of TgATG8 led to rapid loss of the organelle and subsequent intracellular replication defects, indicating that the protein is essential for maintaining apicoplast homeostasis and thus for survival of the tachyzoite stage. More precisely, loss of TgATG8 led to abnormal segregation of the apicoplast into the progeny because of a loss of physical interactions of the organelle with the centrosomes. IMPORTANCE By definition, autophagy is a catabolic process that leads to the digestion and recycling of eukaryotic cellular components. The molecular machinery of autophagy was identified mainly in model organisms such as yeasts but remains poorly characterized in phylogenetically distant apicomplexan parasites. We have uncovered an unusual function for autophagy-related protein ATG8 in Toxoplasma gondii: TgATG8 is crucial for normal replication of the parasite inside its host cell. Seemingly unrelated to the catabolic autophagy process, TgATG8 associates with the outer membrane of the nonphotosynthetic plastid harbored by the parasite called the apicoplast, and there it plays an important role in the centrosome-driven inheritance of the organelle during cell division. This not only reveals an unexpected function for an autophagy-related protein but also sheds new light on the division process of an organelle that is vital to a group of important human and animal pathogens.

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