scholarly journals Two Distinct Mechanisms Govern RpoS-Mediated Repression of Tick-Phase Genes during Mammalian Host Adaptation byBorrelia burgdorferi, the Lyme Disease Spirochete

mBio ◽  
2017 ◽  
Vol 8 (4) ◽  
Author(s):  
Arianna P. Grove ◽  
Dionysios Liveris ◽  
Radha Iyer ◽  
Mary Petzke ◽  
Joseph Rudman ◽  
...  
Keyword(s):  
Lyme Disease ◽  
Sigma Factor ◽  
Fluorescent Protein ◽  
Core Promoter ◽  
Host Adaptation ◽  
Mammalian Host ◽  
Alternative Sigma Factor ◽  
The Core ◽  
Rpos Mutant ◽  
Green Fluorescent

ABSTRACTThe alternative sigma factor RpoS plays a key role modulating gene expression inBorrelia burgdorferi, the Lyme disease spirochete, by transcribing mammalian host-phase genes and repressing σ70-dependent genes required within the arthropod vector. To identifycisregulatory elements involved in RpoS-dependent repression, we analyzed green fluorescent protein (GFP) transcriptional reporters containing portions of the upstream regions of the prototypical tick-phase genesospAB, theglpoperon, andbba74. As RpoS-mediated repression occurs only following mammalian host adaptation, strains containing the reporters were grown in dialysis membrane chambers (DMCs) implanted into the peritoneal cavities of rats. Wild-type spirochetes harboringospAB- andglp-gfpconstructs containing only the minimal (−35/−10) σ70promoter elements had significantly lower expression in DMCs relative to growthin vitroat 37°C; no reduction in expression occurred in a DMC-cultivated RpoS mutant harboring these constructs. In contrast, RpoS-mediated repression ofbba74required a stretch of DNA located between −165 and −82 relative to its transcriptional start site. Electrophoretic mobility shift assays employing extracts of DMC-cultivatedB. burgdorferiproduced a gel shift, whereas extracts from RpoS mutant spirochetes did not. Collectively, these data demonstrate that RpoS-mediated repression of tick-phase borrelial genes occurs by at least two distinct mechanisms. One (e.g.,ospABand theglpoperon) involves primarily sequence elements near the core promoter, while the other (e.g.,bba74) involves an RpoS-induced transacting repressor. Our results provide a genetic framework for further dissection of the essential “gatekeeper” role of RpoS throughout theB. burgdorferienzootic cycle.IMPORTANCEBorrelia burgdorferi, the Lyme disease spirochete, modulates gene expression to adapt to the distinctive environments of its mammalian host and arthropod vector during its enzootic cycle. The alternative sigma factor RpoS has been referred to as a “gatekeeper” due to its central role in regulating the reciprocal expression of mammalian host- and tick-phase genes. While RpoS-dependent transcription has been studied extensively, little is known regarding the mechanism(s) of RpoS-mediated repression. We employed a combination of green fluorescent protein transcriptional reporters along with anin vivomodel to definecisregulatory sequences responsible for RpoS-mediated repression of prototypical tick-phase genes. Repression ofospABand theglpoperon requires only sequences near their core promoters, whereas modulation ofbba74expression involves a putative RpoS-dependent repressor that binds upstream of the core promoter. Thus, Lyme disease spirochetes employ at least two different RpoS-dependent mechanisms to repress tick-phase genes within the mammal.

scholarly journals Sec1p Binds to SNARE Complexes and Concentrates at Sites of Secretion

1999 ◽  
Vol 146 (2) ◽  
pp. 333-344 ◽  
Author(s):  
Chavela M. Carr ◽  
Eric Grote ◽  
Mary Munson ◽  
Frederick M. Hughson ◽  
Peter J. Novick
Keyword(s):  
Fluorescent Protein ◽  
Snare Complex ◽  
The Other ◽  
Complex Assembly ◽  
Vesicle Docking ◽  
Neuronal Protein ◽  
The Core ◽  
Target Membrane ◽  
Green Fluorescent ◽  
And Function

Proteins of the Sec1 family have been shown to interact with target-membrane t-SNAREs that are homologous to the neuronal protein syntaxin. We demonstrate that yeast Sec1p coprecipitates not only the syntaxin homologue Ssop, but also the other two exocytic SNAREs (Sec9p and Sncp) in amounts and in proportions characteristic of SNARE complexes in yeast lysates. The interaction between Sec1p and Ssop is limited by the abundance of SNARE complexes present in sec mutants that are defective in either SNARE complex assembly or disassembly. Furthermore, the localization of green fluorescent protein (GFP)-tagged Sec1p coincides with sites of vesicle docking and fusion where SNARE complexes are believed to assemble and function. The proposal that SNARE complexes act as receptors for Sec1p is supported by the mislocalization of GFP-Sec1p in a mutant defective for SNARE complex assembly and by the robust localization of GFP-Sec1p in a mutant that fails to disassemble SNARE complexes. The results presented here place yeast Sec1p at the core of the exocytic fusion machinery, bound to SNARE complexes and localized to sites of secretion.

scholarly journals Analysis of Promoter Elements Involved in the Transcriptional Initiation of RpoS-Dependent Borrelia burgdorferi Genes

2004 ◽  
Vol 186 (21) ◽  
pp. 7390-7402 ◽  
Author(s):  
Christian H. Eggers ◽  
Melissa J. Caimano ◽  
Justin D. Radolf
Keyword(s):  
Borrelia Burgdorferi ◽  
Sigma Factor ◽  
Fluorescent Protein ◽  
Core Promoter ◽  
Expression Patterns ◽  
Inducible Promoter ◽  
Transcriptional Initiation ◽  
E Coli ◽  
Promoter Recognition ◽  
Maximal Expression

ABSTRACT Borrelia burgdorferi, the causative agent of Lyme disease, encodes an RpoS ortholog (RpoSBb) that controls the temperature-inducible differential expression of at least some of the spirochete's lipoprotein genes, including ospC and dbpBA. To begin to dissect the determinants of RpoSBb recognition of, and selectivity for, its dependent promoters, we linked a green fluorescent protein reporter to the promoter regions of several B. burgdorferi genes with well-characterized expression patterns. Consistent with the expression patterns of the native genes/proteins in B. burgdorferi strain 297, we found that expression of the ospC, dbpBA, and ospF reporters in the spirochete was RpoSBb dependent, while the ospE and flaB reporters were RpoSBb independent. To compare promoter recognition by RpoSBb with that of the prototype RpoS (RpoSEc), we also introduced our panel of constructs into Escherichia coli. In this surrogate, maximal expression from the ospC, dbpBA, and ospF promoters clearly required RpoS, although in the absence of RpoSEc the ospF promoter was weakly recognized by another E. coli sigma factor. Furthermore, RpoSBb under the control of an inducible promoter was able to complement an E. coli rpoS mutant, although RpoSEc and RpoSBb each initiated greater activity from their own dependent promoters than they did from those of the heterologous sigma factor. Genetic analysis of the ospC promoter demonstrated that (i) the T(−14) in the presumptive −10 region plays an important role in sigma factor recognition in both organisms but is not as critical for transcriptional initiation by RpoSBb as it is for RpoSEc; (ii) the nucleotide at the −15 position determines RpoS or σ70 selectivity in E. coli but does not serve the same function in B. burgdorferi; and (iii) the 110-bp region upstream of the core promoter is not required for RpoSEc- or RpoSBb-dependent activity in E. coli but is required for maximal expression from this promoter in B. burgdorferi. Taken together, the results of our studies suggest that the B. burgdorferi and E. coli RpoS proteins are able to catalyze transcription from RpoS-dependent promoters of either organism, but at least some of the nucleotide elements involved in transcriptional initiation and sigma factor selection in B. burgdorferi play a different role than has been described for E. coli.

scholarly journals Small subunits of RNA polymerase: localization, levels and implications for core enzyme composition

Microbiology ◽  
2010 ◽  
Vol 156 (12) ◽  
pp. 3532-3543 ◽  
Author(s):  
Geoff P. Doherty ◽  
Mark J. Fogg ◽  
Anthony J. Wilkinson ◽  
Peter J. Lewis
Keyword(s):  
Fluorescent Protein ◽  
Gram Negative Bacteria ◽  
Gram Positive ◽  
Gram Negative ◽  
Gram Positive Bacteria ◽  
The Core ◽  
Bacterial Rna ◽  
Green Fluorescent

Bacterial RNA polymerases (RNAPs) contain several small auxiliary subunits known to co-purify with the core α, β and β′ subunits. The ω subunit is conserved between Gram-positive and Gram-negative bacteria, while the δ subunit is conserved within, but restricted to, Gram-positive bacteria. Although various functions have been assigned to these subunits via in vitro assays, very little is known about their in vivo roles. In this work we constructed a pair of vectors to investigate the subcellular localization of the δ and ω subunits in Bacillus subtilis with respect to the core RNAP. We found these subunits to be closely associated with RNAP involved in transcribing both mRNA and rRNA operons. Quantification of these subunits revealed δ to be present at equimolar levels with RNAP and ω to be present at around half the level of core RNAP. For comparison, the localization and quantification of RNAP β′ and ω subunits in Escherichia coli was also investigated. Similar to B. subtilis, β′ and ω closely associated with the nucleoid and formed subnucleoid regions of high green fluorescent protein intensity, but, unlike ω in B. subtilis, ω levels in E. coli were close to parity with those of β′. These results indicate that δ is likely to be an integral RNAP subunit in Gram-positives, whereas ω levels differ substantially between Gram-positives and -negatives. The ω subunit may be required for RNAP assembly and subsequently be turned over at different rates or it may play roles in Gram-negative bacteria that are performed by other factors in Gram-positives.

scholarly journals Utilization of Fluorescent Microspheres and a Green Fluorescent Protein-Marked Strain for Assessment of Microbiological Contamination of Permafrost and Ground Ice Core Samples from the Canadian High Arctic

2005 ◽  
Vol 71 (2) ◽  
pp. 1035-1041 ◽  
Author(s):  
D. F. Juck ◽  
G. Whissell ◽  
B. Steven ◽  
W. Pollard ◽  
C. P. McKay ◽  
...  
Keyword(s):  
Green Fluorescent Protein ◽  
Fluorescent Protein ◽  
High Arctic ◽  
Microbiological Contamination ◽  
Canadian High Arctic ◽  
Fluorescent Microspheres ◽  
Ground Ice ◽  
The Core ◽  
Core Samples ◽  
Green Fluorescent

ABSTRACT Fluorescent microspheres were applied in a novel fashion during subsurface drilling of permafrost and ground ice in the Canadian High Arctic to monitor the exogenous microbiological contamination of core samples obtained during the drilling process. Prior to each drill run, a concentrated fluorescent microsphere (0.5-μm diameter) solution was applied to the interior surfaces of the drill bit, core catcher, and core tube and allowed to dry. Macroscopic examination in the field demonstrated reliable transfer of the microspheres to core samples, while detailed microscopic examination revealed penetration levels of less than 1 cm from the core exterior. To monitor for microbial contamination during downstream processing of the permafrost and ground ice cores, a Pseudomonas strain expressing the green fluorescent protein (GFP) was painted on the core exterior prior to processing. Contamination of the processed core interiors with the GFP-expressing strain was not detected by culturing the samples or by PCR to detect the gfp marker gene. These methodologies were quick, were easy to apply, and should help to monitor the exogenous microbiological contamination of pristine permafrost and ground ice samples for downstream culture-dependent and culture-independent microbial analyses.

scholarly journals Alternate Sigma Factor RpoS Is Required for the In Vivo-Specific Repression of Borrelia burgdorferi Plasmid lp54-Borne ospA and lp6.6 Genes

2005 ◽  
Vol 187 (22) ◽  
pp. 7845-7852 ◽  
Author(s):  
Melissa J. Caimano ◽  
Christian H. Eggers ◽  
Cynthia A. Gonzalez ◽  
Justin D. Radolf
Keyword(s):  
Borrelia Burgdorferi ◽  
Sigma Factor ◽  
Mammalian Host ◽  
Alternative Sigma Factor ◽  
Tick Feeding ◽  
Accessory Factor ◽  
Repressor Molecule ◽  
Specific Virulence ◽  
Sigma Factor Rpos

ABSTRACT While numerous positively regulated loci have been characterized during the enzootic cycle of Borrelia burgdorferi, very little is known about the mechanism(s) involved in the repression of borrelial loci either during tick feeding or within the mammalian host. Here, we report that the alternative sigma factor RpoS is required for the in vivo-specific repression of at least two RpoD-dependent B. burgdorferi loci, ospA and lp6.6. The downregulation of ospA and Ip6.6 appears to require either a repressor molecule whose expression is RpoS dependent or an accessory factor which enables RpoS to directly interact with the ospA and Ip6.6 promoter elements, thereby blocking transcription by RpoD. The central role for RpoS during the earliest stages of host adaptation suggests that tick feeding imparts signals to spirochetes that trigger the RpoS-dependent repression, as well as expression, of in vivo-specific virulence factors critical for the tick-to-mammalian host transition.

scholarly journals An lp17-encoded small non-coding RNA with a potential regulatory role in mammalian host adaptation by the Lyme disease spirochete

2020 ◽  
Author(s):  
Michael A. Crowley ◽  
Troy Bankhead
Keyword(s):  
Lyme Disease ◽  
Borrelia Burgdorferi ◽  
Intergenic Region ◽  
Critical Role ◽  
Protein Translation ◽  
The United States ◽  
Host Adaptation ◽  
Mammalian Host ◽  
Intergenic Sequence ◽  
Bacterial Gene

AbstractThe bacterial agent of Lyme disease, Borrelia burgdorferi, relies on an intricate gene regulatory network to transit between the disparate Ixodes tick vector and mammalian host environments. We recently reported that a B. burgdorferi mutant lacking an intergenic region of lp17 displayed attenuated murine tissue colonization and pathogenesis due to altered antigen expression. In this study, a more detailed characterization of the putative regulatory factor encoded by the region was pursued through genetic complementation of the mutant with variants of the intergenic sequence. In cis complemented strains featuring mutations aimed at eliminating potential BBD07 protein translation were capable of full tissue colonization, suggesting that the region encodes an sRNA. In trans complementation resulted in elevated transcription levels and was found to completely abolish infectivity in both immunocompetent and immunodeficient mice. Quantitative analysis of transcription of the putative sRNA by wild type B. burgdorferi showed it to be highly induced during murine infection. Lastly, targeted deletion of this region resulted in significant changes to the transcriptome, including genes with potential roles in transmission and host adaptation. The findings reported herein strongly suggest that this lp17 intergenic region encodes for an sRNA with a critical role in the gene regulation required for adaptation and persistence of the pathogen in the mammalian host.Author SummaryLyme disease continues to emerge as a devastating infection that afflicts hundreds of thousands of people annually in the United States and abroad, highlighting the need for new approaches and targets for intervention. Successful development of these therapies relies heavily on an improved understanding of the biology of the causative agent, Borrelia burgdorferi. This is particularly true for the critical points in the life cycle of the pathogen where it must transition between ticks and mammals. Variation in the levels of bacterial gene expression is the lynchpin of this transition and is known to be driven partly by the activity of regulatory molecules known as small non-coding RNAs (sRNAs). In this work, we characterize one of these sRNAs by providing experimental evidence that the transcribed product does not code for a protein, by testing the effects of its overproduction on infectivity, and by interrogating whether its activity causes changes in expression levels of genes at the level of transcription. The findings of this study provide further evidence that regulatory sRNA activity is critical for transmission and optimal infectivity of B. burgdorferi and contribute to the recently growing effort to attribute specific roles to these important molecules in the context of Lyme disease.

Development and validation of fourLeishmaniaspecies constitutively expressing GFP protein. A model for drug discovery and disease pathogenesis studies

Parasitology ◽  
2013 ◽  
Vol 141 (4) ◽  
pp. 501-510 ◽  
Author(s):  
ASHA PARBHU PATEL ◽  
ANDREW DEACON ◽  
GIULIA GETTI
Keyword(s):  
Leishmania Major ◽  
Fluorescent Protein ◽  
Protein A ◽  
Host Cells ◽  
Mammalian Host ◽  
Disease Pathogenesis ◽  
Compound Libraries ◽  
Green Fluorescent

SUMMARYGreen fluorescent protein (GFP)-parasite transfectants have been widely used as a tool for studying disease pathogenesis in several protozoan models and their application in drug screening assays has increased rapidly. In the past decade, the expression of GFP has been established in severalLeishmaniaspecies, mostly forin vitrostudies. The current work reports generation of four transgenic parasites constitutively expressing GFP (Leishmania mexicana, Leishmania aethiopica, Leishmania tropicaandLeishmania major) and their validation as a representative model of infection. This is the first report where stable expression of GFP has been achieved inL. aethiopicaandL. tropica. Integration of GFP was accomplished through homologous recombination of the expression construct, pRib1.2αNEOαGFP downstream of the 18S rRNA promoter in all species. A homogeneous and high level expression of GFP was detected in both the promastigote and the intracellular amastigote stages. All transgenic species showed the same growth pattern, ability to infect mammalian host cells and sensitivity to reference drugs as their wild type counterparts. All four transgenicLeishmaniaare confirmed as models forin vitroand possiblyin vivoinfections and represent an ideal tool for medium throughput testing of compound libraries.

scholarly journals Sigma Factor Genes sigC, sigE, and sigG Are Upregulated in Heterocysts of the Cyanobacterium Anabaena sp. Strain PCC 7120

2007 ◽  
Vol 189 (22) ◽  
pp. 8392-8396 ◽  
Author(s):  
M. Ramona Aldea ◽  
Rodrigo A. Mella-Herrera ◽  
James W. Golden
Keyword(s):  
Sigma Factor ◽  
Fluorescent Protein ◽  
Developmental Regulation ◽  
Time Lapse ◽  
Green Fluorescent Protein Fluorescence ◽  
Protein Fluorescence ◽  
Cyanobacterium Anabaena ◽  
Reporter Strains ◽  
Green Fluorescent ◽  
Pcc 7120

ABSTRACT We used gfp transcriptional fusions to investigate the regulation of eight sigma factor genes during heterocyst development in the cyanobacterium Anabaena sp. strain PCC 7120. Reporter strains containing gfp fusions with the upstream regions of sigB2, sigD, sigI, and sigJ did not show developmental regulation. Time-lapse microscopy of sigC, sigE, and sigG reporter strains showed increased green fluorescent protein fluorescence in differentiating cells at 4 h, 16 h, and 9 h, respectively, after nitrogen step down.

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