scholarly journals Campylobacter jejuniBumSR directs a response to butyrate via sensor phosphatase activity to impact transcription and colonization

2020 ◽  
Vol 117 (21) ◽  
pp. 11715-11726 ◽  
Author(s):  
Kyle N. Goodman ◽  
Matthew J. Powers ◽  
Alexander A. Crofts ◽  
M. Stephen Trent ◽  
David R. Hendrixson
Keyword(s):  
Signal Transduction ◽  
Target Genes ◽  
Diarrheal Disease ◽  
Response Regulator ◽  
Short Chain Fatty Acids ◽  
Signal Transduction System ◽  
Genes Encoding ◽  
In Vivo Growth

Campylobacter jejunimonitors intestinal metabolites produced by the host and microbiota to initiate intestinal colonization of avian and animal hosts for commensalism and infection of humans for diarrheal disease. We previously discovered thatC. jejunihas the capacity to spatially discern different intestinal regions by sensing lactate and the short-chain fatty acids acetate and butyrate and then alter transcription of colonization factors appropriately for in vivo growth. In this study, we identified theC. jejunibutyrate-modulated regulon and discovered that the BumSR two-component signal transduction system (TCS) directs a response to butyrate by identifying mutants in a genetic screen defective for butyrate-modulated transcription. The BumSR TCS, which is important for infection of humans and optimal colonization of avian hosts, senses butyrate likely by indirect means to alter transcription of genes encoding important colonization determinants. Unlike many canonical TCSs, the predicted cytoplasmic sensor kinase BumS lacked in vitro autokinase activity, which would normally lead to phosphorylation of the cognate BumR response regulator. Instead, BumS has likely evolved mutations to naturally function as a phosphatase whose activity is influenced by exogenous butyrate to control the level of endogenous phosphorylation of BumR and its ability to alter transcription of target genes. To our knowledge, the BumSR TCS is the only bacterial signal transduction system identified so far that mediates responses to the microbiota-generated intestinal metabolite butyrate, an important factor for host intestinal health and homeostasis. Our findings suggest that butyrate sensing by this system is vital forC. jejunicolonization of multiple hosts.

scholarly journals Regulation of Sulfur Assimilation Pathways in Burkholderia cenocepacia: Identification of Transcription Factors CysB and SsuR and Their Role in Control of Target Genes

2006 ◽  
Vol 189 (5) ◽  
pp. 1675-1688 ◽  
Author(s):  
Roksana Iwanicka-Nowicka ◽  
Agata Zielak ◽  
Anne M. Cook ◽  
Mark S. Thomas ◽  
Monika M. Hryniewicz
Keyword(s):  
Target Genes ◽  
Positive Control ◽  
Transcription Unit ◽  
Burkholderia Cenocepacia ◽  
Sulfur Assimilation ◽  
Allelic Replacement ◽  
Genes Encoding ◽  
Target Promoters

ABSTRACT Two genes encoding transcriptional regulators involved in sulfur assimilation pathways in Burkholderia cenocepacia strain 715j have been identified and characterized functionally. Knockout mutations in each of the B. cenocepacia genes were constructed and introduced into the genome of 715j by allelic replacement. Studies on the utilization of various sulfur sources by 715j and the obtained mutants demonstrated that one of the B. cenocepacia regulators, designated CysB, is preferentially involved in the control of sulfate transport and reduction, while the other, designated SsuR, is required for aliphatic sulfonate utilization. Using transcriptional promoter-lacZ fusions and DNA-binding experiments, we identified several target promoters for positive control by CysB and/or SsuR—sbpp (preceding the sbp cysT cysW cysA ssuR cluster), cysIp (preceding the cysI cysD1 cysN cysH cysG cluster), cysD2p (preceding a separate cluster, cysD2 cysNC), and ssuDp (located upstream of the ssuDCB operon)—and we demonstrated overlapping functions of CysB and SsuR at particular promoters. We also demonstrated that the cysB gene is negatively controlled by both CysB and SsuR but the ssuR gene itself is not significantly regulated as a separate transcription unit. The function of B. cenocepacia CysB (in vivo and in vitro) appeared to be independent of the presence of acetylserine, the indispensable coinducer of the CysB regulators of Escherichia coli and Salmonella. The phylogenetic relationships among members of the “CysB family” in the γ and β subphyla are presented.

scholarly journals CovR Activation of the Dipeptide Permease Promoter (PdppA) in Group A Streptococcus

2006 ◽  
Vol 189 (4) ◽  
pp. 1407-1416 ◽  
Author(s):  
Asiya A. Gusa ◽  
Barbara J. Froehlich ◽  
Devak Desai ◽  
Virginia Stringer ◽  
June R. Scott
Keyword(s):  
Group A Streptococcus ◽  
Response Regulator ◽  
Activation Of Transcription ◽  
Genes Encoding ◽  
Group A ◽  
Transcription In Vitro ◽  
Dna Template ◽  
Regulated Promoters

ABSTRACT CovR, the two-component response regulator of Streptococcus pyogenes (group A streptococcus [GAS]) directly or indirectly represses about 15% of the genome, including genes encoding many virulence factors and itself. Transcriptome analyses also showed that some genes are activated by CovR. We asked whether the regulation by CovR of one of these genes, dppA, the first gene in an operon encoding a dipeptide permease, is direct or indirect. Direct regulation by CovR was suggested by the presence of five CovR consensus binding sequences (CBs) near the putative promoter. In this study, we identified the 5′ end of the dppA transcript synthesized in vivo and showed that the start of dppA transcription in vitro is the same. We found that CovR binds specifically to the dppA promoter region (PdppA) in vitro with an affinity similar to that at which it binds to other CovR-regulated promoters. Disruption of any of the five CBs by a substitution of GG for TT inhibited CovR binding to that site in vitro, and binding at two of the CBs appeared cooperative. In vivo, CovR activation of transcription was not affected by individual mutations of any of the four CBs that we could study. This suggests that the binding sites are redundant in vivo. In vitro, CovR did not activate transcription from PdppA in experiments using purified GAS RNA polymerase and either linear or supercoiled DNA template. Therefore, we propose that in vivo, CovR may interfere with the binding of a repressor of PdppA.

scholarly journals Subcellular Clustering of the Phosphorylated WspR Response Regulator Protein Stimulates Its Diguanylate Cyclase Activity

mBio ◽  
2013 ◽  
Vol 4 (3) ◽  
Author(s):  
Varisa Huangyutitham ◽  
Zehra Tüzün Güvener ◽  
Caroline S. Harwood
Keyword(s):  
Signal Transduction ◽  
Biofilm Formation ◽  
Response Regulator ◽  
Cluster Formation ◽  
Cyclase Activity ◽  
Diguanylate Cyclase ◽  
Regulator Protein ◽  
Response Regulator Protein

ABSTRACT WspR is a hybrid response regulator-diguanylate cyclase that is phosphorylated by the Wsp signal transduction complex in response to growth of Pseudomonas aeruginosa on surfaces. Active WspR produces cyclic di-GMP (c-di-GMP), which in turn stimulates biofilm formation. In previous work, we found that when activated by phosphorylation, yellow fluorescent protein (YFP)-tagged WspR forms clusters that are visible in individual cells by fluorescence microscopy. Unphosphorylated WspR is diffuse in cells and not visible. Thus, cluster formation is an assay for WspR signal transduction. To understand how and why WspR forms subcellular clusters, we analyzed cluster formation and the enzymatic activities of six single amino acid variants of WspR. In general, increased cluster formation correlated with increased in vivo and in vitro diguanylate cyclase activities of the variants. In addition, WspR specific activity was strongly concentration dependent in vitro, and the effect of the protein concentration on diguanylate cyclase activity was magnified when WspR was treated with the phosphor analog beryllium fluoride. Cluster formation appears to be an intrinsic property of phosphorylated WspR (WspR-P). These results support a model in which the formation of WspR-P subcellular clusters in vivo in response to a surface stimulus is important for potentiating the diguanylate cyclase activity of WspR. Subcellular cluster formation appears to be an additional means by which the activity of a response regulator protein can be regulated. IMPORTANCE Bacterial sensor proteins often phosphorylate cognate response regulator proteins when stimulated by an environmental signal. Phosphorylated response regulators then mediate an appropriate adaptive cellular response. About 6% of response regulator proteins have an enzymatic domain that is involved in producing or degrading cyclic di-GMP (c-di-GMP), a molecule that stimulates bacterial biofilm formation. In this work, we examined the in vivo and in vitro behavior of the response regulator-diguanylate cyclase WspR. When phosphorylated in response to a signal associated with surface growth, WspR has a tendency to form oligomers that are visible in cells as subcellular clusters. Our results show that the formation of phosphorylated WspR (WspR-P) subcellular clusters is important for potentiating the diguanylate cyclase activity of WspR-P, making it more active in c-di-GMP production. We conclude that oligomer formation visualized as subcellular clusters is an additional mechanism by which the activities of response regulator-diguanylate cyclases can be regulated.

scholarly journals The Sodium-Driven Flagellar Motor Controls Exopolysaccharide Expression in Vibrio cholerae

2004 ◽  
Vol 186 (15) ◽  
pp. 4864-4874 ◽  
Author(s):  
Crystal M. Lauriano ◽  
Chandradipa Ghosh ◽  
Nidia E. Correa ◽  
Karl E. Klose
Keyword(s):  
Vibrio Cholerae ◽  
Gene Transcription ◽  
Biofilm Formation ◽  
Diarrheal Disease ◽  
Response Regulator ◽  
Gene Clusters ◽  
Signaling Cascade ◽  
Genes Encoding ◽  
Life Threatening

ABSTRACT Vibrio cholerae causes the life-threatening diarrheal disease cholera. This organism persists in aquatic environments in areas of endemicity, and it is believed that the ability of the bacteria to form biofilms in the environment contributes to their persistence. Expression of an exopolysaccharide (EPS), encoded by two vps gene clusters, is essential for biofilm formation and causes a rugose colonial phenotype. We previously reported that the lack of a flagellum induces V. cholerae EPS expression. To uncover the signaling pathway that links the lack of a flagellum to EPS expression, we introduced into a rugose flaA strain second-site mutations that would cause reversion back to the smooth phenotype. Interestingly, mutation of the genes encoding the sodium-driven motor (mot) in a nonflagellated strain reduces EPS expression, biofilm formation, and vps gene transcription, as does the addition of phenamil, which specifically inhibits the sodium-driven motor. Mutation of vpsR, which encodes a response regulator, also reduces EPS expression, biofilm formation, and vps gene transcription in nonflagellated cells. Complementation of a vpsR strain with a constitutive vpsR allele likely to mimic the phosphorylated state (D59E) restores EPS expression and biofilm formation, while complementation with an allele predicted to remain unphosphorylated (D59A) does not. Our results demonstrate the involvement of the sodium-driven motor and suggest the involvement of phospho-VpsR in the signaling cascade that induces EPS expression. A nonflagellated strain expressing EPS is defective for intestinal colonization in the suckling mouse model of cholera and expresses reduced amounts of cholera toxin and toxin-coregulated pili in vitro. Wild-type levels of virulence factor expression and colonization could be restored by a second mutation within the vps gene cluster that eliminated EPS biosynthesis. These results demonstrate a complex relationship between the flagellum-dependent EPS signaling cascade and virulence.

scholarly journals Regulation of σB by an Anti- and an Anti-Anti-Sigma Factor in Streptomyces coelicolor in Response to Osmotic Stress

2004 ◽  
Vol 186 (24) ◽  
pp. 8490-8498 ◽  
Author(s):  
Eun-Jin Lee ◽  
You-Hee Cho ◽  
Hyo-Sub Kim ◽  
Bo-Eun Ahn ◽  
Jung-Hye Roe
Keyword(s):  
Osmotic Stress ◽  
Sigma Factor ◽  
Target Genes ◽  
Sequence Similarity ◽  
Streptomyces Coelicolor ◽  
Dependent Manner ◽  
Its Gene ◽  
Genes Encoding

ABSTRACT σB, a homolog of stress-responsive σB of Bacillus subtilis, controls both osmoprotection and differentiation in Streptomyces coelicolor A3 (2). Its gene is preceded by rsbA and rsbB genes encoding homologs of an anti-sigma factor, RsbW, and its antagonist, RsbV, of B. subtilis, respectively. Purified RsbA bound to σB and prevented σB-directed transcription from the sigBp1 promoter in vitro. An rsbA-null mutant exhibited contrasting behavior to the sigB mutant, with elevated sigBp1 transcription, no actinorhodin production, and precocious aerial mycelial formation, reflecting enhanced activity of σB in vivo. Despite sequence similarity to RsbV, RsbB lacks the conserved phosphorylatable serine residue and its gene disruption produced no distinct phenotype. RsbV (SCO7325) from a putative six-gene operon (rsbV-rsbR-rsbS-rsbT-rsbU1-rsbU) was strongly induced by osmotic stress in a σB-dependent manner. It antagonized the inhibitory action of RsbA on σB-directed transcription and was phosphorylated by RsbA in vitro. These results support the hypothesis that the rapid induction of σB target genes by osmotic stress results from modulation of σB activity by the kinase-anti-sigma factor RsbA and its phosphorylatable antagonist RsbV, which function by a partner-switching mechanism. Amplified induction could result from a rapid increase in the synthesis of both σB and its inhibitor antagonist.

Structure of the DNA-binding domain of the response regulator SaeR fromStaphylococcus aureus

2015 ◽  
Vol 71 (8) ◽  
pp. 1768-1776 ◽  
Author(s):  
Xiaojiao Fan ◽  
Xu Zhang ◽  
Yuwei Zhu ◽  
Liwen Niu ◽  
Maikun Teng ◽  
...  
Keyword(s):  
Dna Binding ◽  
Target Genes ◽  
Response Regulator ◽  
Regulatory System ◽  
Binding Domain ◽  
Dna Binding Domain ◽  
Promoter Regions ◽  
Winged Helix

The SaeR/S two-component regulatory system is essential for controlling the expression of many virulence factors inStaphylococcus aureus. SaeR, a member of the OmpR/PhoB family, is a response regulator with an N-terminal regulatory domain and a C-terminal DNA-binding domain. In order to elucidate how SaeR binds to the promoter regions of target genes, the crystal structure of the DNA-binding domain of SaeR (SaeRDBD) was solved at 2.5 Å resolution. The structure reveals that SaeRDBDexists as a monomer and has the canonical winged helix–turn–helix module. EMSA experiments suggested that full-length SaeR can bind to the P1 promoter and that the binding affinity is higher than that of its C-terminal DNA-binding domain. Five key residues on the winged helix–turn–helix module were verified to be important for binding to the P1 promoterin vitroand for the physiological function of SaeRin vivo.

scholarly journals Genetic and Biochemical Analyses of BvgA Interaction with the Secondary Binding Region of the fhaPromoter of Bordetella pertussis

2001 ◽  
Vol 183 (2) ◽  
pp. 536-544 ◽  
Author(s):  
Philip E. Boucher ◽  
Mei-Shin Yang ◽  
Deanna M. Schmidt ◽  
Scott Stibitz
Keyword(s):  
Bordetella Pertussis ◽  
Response Regulator ◽  
Sequence Specificity ◽  
Binding Region ◽  
Dna Sequence Specificity ◽  
Genes Encoding ◽  
Two Component Signal Transduction ◽  
Biochemical Analyses

ABSTRACT The BvgA-BvgS two-component signal transduction system regulates expression of virulence factors in Bordetella pertussis. The BvgA response regulator activates transcription by binding to target promoters, which include those for the genes encoding filamentous hemagglutinin (fha) and pertussis toxin (ptx). We have previously shown that at both promoters the phosphorylated form of BvgA binds multiple high- and low-affinity sites. Specifically, at the fha promoter, we proposed that there may be high- and a low-affinity binding sites for the BvgA dimer. In our present investigation, we used DNA binding analyses and in vitro and in vivo assays of promoters with substitutions and deletions to support and extend this hypothesis. Our observations indicate that (i) binding of BvgA∼P to a primary (high-affinity) site and a secondary binding region (lower affinity) is cooperative, (ii) although both the primary binding site and the secondary binding region are required for full activity of the wild-type (undeleted) promoter, deletion of two helical turns within the secondary binding region can produce a fully active or hyperactive promoter, and (iii) BvgA binding to the secondary binding region shows limited DNA sequence specificity.

HOXB4 Engages in Signaling Pathways Associated with HSC Self Renewal.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 1344-1344
Author(s):  
Bernhard Schiedlmeier ◽  
Ana Santos ◽  
Hannes Klump ◽  
Ana Ribeiro ◽  
Herbert Auer ◽  
...  
Keyword(s):  
Gene Expression ◽  
Signal Transduction ◽  
Signaling Pathways ◽  
Target Genes ◽  
Embryoid Bodies ◽  
Gene Products ◽  
Tnf Receptor ◽  
Self Renewal

Abstract Ectopic expression of the transcription factor HOXB4 has been shown to mediate expansion of adult hematopoietic stem cells (HSCs) in vitro and in vivo, and to generate embryonic stem cell (ESC) derived HSCs capable to reconstitute lethally irradiated mice. To identify target genes of HOXB4 in adult HSCs and progenitor cells proliferating in vitro, we transduced murine cells with a retroviral construct that coexpresses EGFP and an inducible form of HOXB4 (HOXB4ER). Upon addition of 4-hydroxytamoxifen (TMX), HOXB4ER translocates from the cytoplasma to the nucleus, thereby being capable to modulate gene expression. Transduced cell populations were expanded for 14 days in the presense of TMX. Gene expression profiles were obtained from FACS-sorted HOXB4ER+ LSK (lineage neagtive, Sca1+, ckit+ ) cells after culture with and without TMX. As a control, profiling was performed with LSK cells expressing unmodified active HOXB4 ± TMX. On the Genechip Mouse Genome 430 2.0 Array (Affymetrix) we observed 110 characterized gene products to be differentially expressed 4 hours after inactivation of HOXB4. Globally, the Gene Ontology categories “signal transduction”, “intracellular signaling cascade”, “Wnt receptor signaling” and “cell differentiation” were significantly over-represented among the group of up-regulated HOXB4 target genes. The list of down-regulated genes revealed a significant overrepresentation of the GO categories “regulation of cell cycle”, “regulation of apoptosis” and “regulation of DNA-dependent transcription”. Importantly, differential expression of genes involved in several signaling pathways known to either stimulate or inhibit HSC self renewal (WNT, Notch, FGF, TGFβ/BMP and TNFα) was observed as well as a high degree of concordance with HSC-specific genes discovered in previous microarray reports. TNF receptor 2 was down-regulated by HOXB4 and an inhibitor of TNF receptor 1, BRE (brain and reproductive organ expressed protein) was up-regulated. This suggested that HOXB4-expressing HSCs may still be capable of undergoing self renewal cell divisions in vitro, even in the presence of TNFα. Thus, LSK cells were transduced with the inducible HOXB4ER vector or with a control vector expressing a truncated form of human CD34 (tCD34), pooled in a 1:1 ratio, and cultivated for 7 days in serum-free medium with and without TNFα ± TMX. In the absence of TNFα and TMX, in vitro cultured HOXB4ER+ as well as tCD34+ cells both maintained high levels of multilineage reconstitution activity in vivo. However, in the presence of TNFα without HOXB4 induction, the ability of reconstitution was almost completely lost. In contrast, in the presence of TNFα, high levels of multilineage reconstitution were achieved with TMX- induced HOXB4ER cells, but not so with tCD34+ control cells. Hence, HOXB4 renders long-term repopulating HSCs insensitive to the negative effects of TNFα. We also analyzed gene expression changes in a murine ES cells containing a tetracycline-inducible HOXB4 gene. Over 700 characterized genes were differentially regulated 48 hours after doxycycline-mediated HOXB4 induction in day 6 embryoid bodies (EB). Remarkably, HOXB4 engaged in the same aforementioned signal transduction pathways during EB differentiation, in part by targeting identical gene products. In summary, our data and functional studies reveal that HOXB4 changes the response of ESC and HSC to extrinsic cues regulating self renewal and differentiation.

scholarly journals Division of labor in honey bee gut microbiota for plant polysaccharide digestion

2019 ◽  
Vol 116 (51) ◽  
pp. 25909-25916 ◽  
Author(s):  
Hao Zheng ◽  
Julie Perreau ◽  
J. Elijah Powell ◽  
Benfeng Han ◽  
Zijing Zhang ◽  
...  
Keyword(s):  
Gut Microbiota ◽  
Honey Bee ◽  
Short Chain Fatty Acids ◽  
Gene Expressions ◽  
Plant Polysaccharide ◽  
Genes Encoding ◽  
Host Nutrition ◽  
Different Strains

Bees acquire carbohydrates from nectar and lipids; and amino acids from pollen, which also contains polysaccharides including cellulose, hemicellulose, and pectin. These potential energy sources could be degraded and fermented through microbial enzymatic activity, resulting in short chain fatty acids available to hosts. However, the contributions of individual microbiota members to polysaccharide digestion have remained unclear. Through analysis of bacterial isolate genomes and a metagenome of the honey bee gut microbiota, we identify thatBifidobacteriumandGilliamellaare the principal degraders of hemicellulose and pectin. BothBifidobacteriumandGilliamellashow extensive strain-level diversity in gene repertoires linked to polysaccharide digestion. Strains from honey bees possess more such genes than strains from bumble bees. InBifidobacterium, genes encoding carbohydrate-active enzymes are colocated within loci devoted to polysaccharide utilization, as inBacteroidesfrom the human gut. Carbohydrate-active enzyme-encoding gene expressions are up-regulated in response to particular hemicelluloses both in vitro and in vivo. Metabolomic analyses document that bees experimentally colonized by different strains generate distinctive gut metabolomic profiles, with enrichment for specific monosaccharides, corresponding to predictions from genomic data. The other 3 core gut species clusters (Snodgrassellaand 2Lactobacillusclusters) possess few or no genes for polysaccharide digestion. Together, these findings indicate that strain composition within individual hosts determines the metabolic capabilities and potentially affects host nutrition. Furthermore, the niche specialization revealed by our study may promote overall community stability in the gut microbiomes of bees.

Sign in / Sign up

Export Citation Format

Share Document