Transcriptional Activation of the Rhodobacter sphaeroides Cytochrome c2 Gene P2 Promoter by the Response Regulator PrrA

ABSTRACT Anoxygenic photosynthetic growth of Rhodobacter sphaeroides, a member of the α subclass of the class Proteobacteria, requires the response regulator PrrA. PrrA and the sensor kinase PrrB are part of a two-component signaling pathway that influences a wide range of processes under oxygen-limited conditions. In this work we characterized the pathway of transcription activation by PrrB and PrrA by purifying these proteins, analyzing them in vitro, and characterizing a mutant PrrA protein in vivo and in vitro. When purified, a soluble transmitter domain of PrrB (cPrrB) could autophosphorylate, rapidly transfer phosphate to PrrA, and stimulate dephosphorylation of phospho-PrrA. Unphosphorylated PrrA activated transcription from a target cytochrome c 2 gene (cycA) promoter, P2, which contained sequences from −73 to +22 relative to the transcription initiation site. However, phosphorylation of PrrA increased its activity since activation of cycA P2 was enhanced up to 15-fold by treatment with the low-molecular-weight phosphodonor acetyl phosphate. A mutant PrrA protein containing a single amino acid substitution in the presumed phosphoacceptor site (PrrA-D63A) was not phosphorylated in vitro but also was not able to stimulate cycA P2 transcription. PrrA-D63A also had no apparent in vivo activity, demonstrating that aspartate 63 is necessary both for the function of PrrA and for its phosphorylation-dependent activation. The cellular level of wild-type PrrA was negatively autoregulated so that less PrrA was present in the absence of oxygen, conditions in which the activities of many PrrA target genes increase. PrrA-D63A failed to repress expression of the prrA gene under anaerobic conditions, suggesting that this single amino acid change also eliminated PrrA function in vivo.

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A Point Mutation in the Rhesus Rotavirus VP4 Protein Generated through a Rotavirus Reverse Genetics System Attenuates Biliary Atresia in the Murine Model

Journal of Virology ◽

10.1128/jvi.00510-17 ◽

2017 ◽

Vol 91 (15) ◽

Cited By ~ 3

Author(s):

Sujit K. Mohanty ◽

Bryan Donnelly ◽

Phylicia Dupree ◽

Inna Lobeck ◽

Sarah Mowery ◽

...

Keyword(s):

Amino Acid ◽

Biliary Atresia ◽

Amino Acid Change ◽

Reverse Genetics ◽

Single Amino Acid ◽

Wild Type ◽

Neonatal Mice ◽

Single Amino Acid Change

ABSTRACT Rotavirus infection is one of the most common causes of diarrheal illness in humans. In neonatal mice, rhesus rotavirus (RRV) can induce biliary atresia (BA), a disease resulting in inflammatory obstruction of the extrahepatic biliary tract and intrahepatic bile ducts. We previously showed that the amino acid arginine (R) within the sequence SRL (amino acids 445 to 447) in the RRV VP4 protein is required for viral binding and entry into biliary epithelial cells. To determine if this single amino acid (R) influences the pathogenicity of the virus, we generated a recombinant virus with a single amino acid mutation at this site through a reverse genetics system. We demonstrated that the RRV mutant (RRVVP4-R446G) produced less symptomatology and replicated to lower titers both in vivo and in vitro than those seen with wild-type RRV, with reduced binding in cholangiocytes. Our results demonstrate that a single amino acid change in the RRV VP4 gene influences cholangiocyte tropism and reduces pathogenicity in mice. IMPORTANCE Rotavirus is the leading cause of diarrhea in humans. Rhesus rotavirus (RRV) can also lead to biliary atresia (a neonatal human disease) in mice. We developed a reverse genetics system to create a mutant of RRV (RRVVP4-R446G) with a single amino acid change in the VP4 protein compared to that of wild-type RRV. In vitro, the mutant virus had reduced binding and infectivity in cholangiocytes. In vivo, it produced fewer symptoms and lower mortality in neonatal mice, resulting in an attenuated form of biliary atresia.

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Mutational analysis of the C-terminal domain of the Rhodobacter sphaeroides response regulator PrrA

Microbiology ◽

10.1099/mic.0.28300-0 ◽

2005 ◽

Vol 151 (12) ◽

pp. 4103-4110 ◽

Cited By ~ 8

Author(s):

Denise F. Jones ◽

Rachelle A. Stenzel ◽

Timothy J. Donohue

Keyword(s):

Amino Acid ◽

Rhodobacter Sphaeroides ◽

Mutational Analysis ◽

Response Regulator ◽

Dnase I ◽

Single Amino Acid ◽

Amino Acid Sequence Similarity ◽

Acetyl Phosphate ◽

Wild Type

The Rhodobacter sphaeroides response regulator PrrA directly activates transcription of genes necessary for energy conservation at low O2 tensions and under anaerobic conditions. It is proposed that PrrA homologues contain a C-terminal DNA-binding domain (PrrA-CTD) that lacks significant amino acid sequence similarity to those found in other response regulators. To test this hypothesis, single amino acid substitutions were created at 12 residues in the PrrA-CTD. These mutant PrrA proteins were purified and tested for the ability to be phosphorylated by the low-molecular-mass phosphate donor acetyl phosphate, to activate transcription and to bind promoter DNA. Each mutant PrrA protein accepted phosphate from 32P-labelled acetyl phosphate. At micromolar concentrations of acetyl phosphate-treated wild-type PrrA, a single 20 bp region in the PrrA-dependent cycA P2 promoter was protected from DNase I digestion. Of the mutant PrrA proteins tested, only acetyl phosphate-treated PrrA-N168A and PrrA-I177A protected cycA P2 from DNase I digestion at similar protein concentrations compared to wild-type PrrA. The use of in vitro transcription assays with the PrrA-dependent cycA P2 and puc promoters showed that acetyl phosphate-treated PrrA-N168A produced transcript levels similar to that of wild-type PrrA at comparable protein concentrations. Using concentrations of acetyl phosphate-treated PrrA that are saturating for the wild-type protein, PrrA-H170A and PrrA-I177A produced <45 % as much transcript as wild-type PrrA. Under identical conditions, the remaining mutant PrrA proteins produced little or no detectable transcripts from either promoter in vitro. Explanations are presented for why these amino acid side chains in the PrrA-CTD are important for its ability to activate transcription.

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Identification of the Herbicide Binding Region of the Qв-Protein by Photoaffinity Labeling with Azidoatrazine

Zeitschrift für Naturforschung C ◽

10.1515/znc-1984-0523 ◽

1984 ◽

Vol 39 (5) ◽

pp. 425-429 ◽

Cited By ~ 18

Author(s):

Paul K. Wolber ◽

Katherine E. Steinback

Keyword(s):

Amino Acid ◽

Gel Filtration ◽

Single Amino Acid ◽

Rhodopseudomonas Capsulata ◽

Major Protein ◽

Peptide Pattern ◽

Single Amino Acid Change ◽

Spinach Thylakoids

Spinach thylakoids have been photoaffinity labeled with [14C]azidoatrazine in vitro and with specific [3H]amino acids in vivo. The radiolabeled protein has been isolated, cleaved with trypsin, and the resulting fragments have been identified by their characteristic [3H]amino acid incorporation. The peptide pattern has been interpreted using the known gene sequence for the QB-protein of spinach thylakoids, which is the major protein labeled by both the in vivo and in vitro procedures. When analyzed by gel filtration, over 80% of the 14C-activity migrated as two peaks: one as undigested protein at the void volume and the other at an apparent MW of 8.35 ± 0.76 kilodaltons. The remaining activity migrated with the included volume as free azidoatrazine. Fragments were analysed for covalent [14C]azidoatrazine binding; the tryptic peptide covalently labeled by [14C]azidoatrazine was found to be the fragment Pro-141 to Arg-225. This peptide is well removed from the site of a single amino acid change at Ser-264 implicated in triazine resistance, but includes a region suggested to be involved in Qв-binding by homology with sequences of two reaction center proteins from Rhodopseudomonas capsulata.

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Tuning the circadian period of cyanobacteria up to 6.6 days by the single amino acid substitutions in KaiC

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.2005496117 ◽

2020 ◽

Vol 117 (34) ◽

pp. 20926-20931

Author(s):

Kumiko Ito-Miwa ◽

Yoshihiko Furuike ◽

Shuji Akiyama ◽

Takao Kondo

Keyword(s):

Amino Acid ◽

Atpase Activity ◽

Temperature Compensation ◽

Period Change ◽

Single Amino Acid ◽

Circadian Period ◽

Clock Proteins ◽

Wide Range

The circadian clock of cyanobacteria consists of only three clock proteins, KaiA, KaiB, and KaiC, which generate a circadian rhythm of KaiC phosphorylation in vitro. The adenosine triphosphatase (ATPase) activity of KaiC is the source of the 24-h period and temperature compensation. Although numerous circadian mutants of KaiC have been identified, the tuning mechanism of the 24-h period remains unclear. Here, we show that the circadian period of in vitro phosphorylation rhythm of mutants at position 402 of KaiC changed dramatically, from 15 h (0.6 d) to 158 h (6.6 d). The ATPase activities of mutants at position 402 of KaiC, without KaiA and KaiB, correlated with the frequencies (1/period), indicating that KaiC structure was the source of extra period change. Despite the wide-range tunability, temperature compensation of both the circadian period and the KaiC ATPase activity of mutants at position 402 of KaiC were nearly intact. We also found that in vivo and in vitro circadian periods and the KaiC ATPase activity of mutants at position 402 of KaiC showed a correlation with the side-chain volume of the amino acid at position 402 of KaiC. Our results indicate that residue 402 is a key position of determining the circadian period of cyanobacteria, and it is possible to dramatically alter the period of KaiC while maintaining temperature compensation.

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Single amino acid changes in the mumps virus haemagglutinin–neuraminidase and polymerase proteins are associated with neuroattenuation

Journal of General Virology ◽

10.1099/vir.0.009449-0 ◽

2009 ◽

Vol 90 (7) ◽

pp. 1741-1747 ◽

Cited By ~ 11

Author(s):

Tahir H. Malik ◽

Candie Wolbert ◽

Laura Nerret ◽

Christian Sauder ◽

Steven Rubin

Keyword(s):

Amino Acid ◽

Clinical Isolate ◽

Amino Acid Change ◽

Mumps Virus ◽

Site Directed Mutagenesis ◽

Single Amino Acid ◽

Supporting Evidence ◽

L Protein ◽

Single Amino Acid Change

It has previously been shown that three amino acid changes, one each in the fusion (F; Ala/Thr-91→Thr), haemagglutinin–neuraminidase (HN; Ser-466→Asn) and polymerase (L; Ile-736→Val) proteins, are associated with attenuation of a neurovirulent clinical isolate of mumps virus (88-1961) following serial passage in vitro. Here, using full-length cDNA plasmid clones and site-directed mutagenesis, it was shown that the single amino acid change in the HN protein and to a lesser extent, the change in the L protein, resulted in neuroattenuation, as assessed in rats. The combination of both amino acid changes caused neuroattenuation of the virus to levels previously reported for the clinical isolate following attenuation in vitro. The amino acid change in the F protein, despite having a dramatic effect on protein function in vitro, was previously shown to not be involved in the observed neuroattenuation, highlighting the importance of conducting confirmatory in vivo studies. This report provides additional supporting evidence for the role of the HN protein as a virulence factor and, as far as is known, is the first report to associate an amino acid change in the L protein with mumps virus neuroattenuation.

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Analysis of Saccharomyces cerevisiae his3 transcription in vitro: biochemical support for multiple mechanisms of transcription

Molecular and Cellular Biology ◽

10.1128/mcb.10.6.2832-2839.1990 ◽

1990 ◽

Vol 10 (6) ◽

pp. 2832-2839

Author(s):

A S Ponticelli ◽

K Struhl

Keyword(s):

Saccharomyces Cerevisiae ◽

Transcriptional Activation ◽

Transcription Initiation ◽

Molecular Mechanisms ◽

Initiation Site ◽

Activator Proteins ◽

Nuclear Extracts ◽

Transcription In Vitro

The promoter region of the Saccharomyces cerevisiae his3 gene contains two TATA elements, TC and TR, that direct transcription initiation to two sites designated +1 and +13. On the basis of differences between their nucleotide sequences and their responsiveness to upstream promoter elements, it has previously been proposed that TC and TR promote transcription by different molecular mechanisms. To begin a study of his3 transcription in vitro, we used S. cerevisiae nuclear extracts together with various DNA templates and transcriptional activator proteins that have been characterized in vivo. We demonstrated accurate transcription initiation in vitro at the sites used in vivo, transcriptional activation by GCN4, and activation by a GAL4 derivative on various gal-his3 hybrid promoters. In all cases, transcription stimulation was dependent on the presence of an acidic activation region in the activator protein. In addition, analysis of promoters containing a variety of TR derivatives indicated that the level of transcription in vitro was directly related to the level achieved in vivo. The results demonstrated that the in vitro system accurately reproduced all known aspects of in vivo his3 transcription that depend on the TR element. However, in striking contrast to his3 transcription in vivo, transcription in vitro yielded approximately 20 times more of the +13 transcript than the +1 transcript. This result was not due to inability of the +1 initiation site to be efficiently utilized in vitro, but rather it reflects the lack of TC function in vitro. The results support the idea that TC and TR mediate transcription from the wild-type promoter by distinct mechanisms.

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Biochemical and genetic characterization of a yeast TFIID mutant that alters transcription in vivo and DNA binding in vitro

Molecular and Cellular Biology ◽

10.1128/mcb.12.5.2372-2382.1992 ◽

1992 ◽

Vol 12 (5) ◽

pp. 2372-2382

Author(s):

K M Arndt ◽

S L Ricupero ◽

D M Eisenmann ◽

F Winston

Keyword(s):

Amino Acid ◽

Dna Binding ◽

Direct Repeat ◽

Single Amino Acid ◽

Wild Type ◽

Dna Binding Specificity ◽

Selection For

A mutation in the gene that encodes Saccharomyces cerevisiae TFIID (SPT15), which was isolated in a selection for mutations that alter transcription in vivo, changes a single amino acid in a highly conserved region of the second direct repeat in TFIID. Among eight independent spt15 mutations, seven cause this same amino acid change, Leu-205 to Phe. The mutant TFIID protein (L205F) binds with greater affinity than that of wild-type TFIID to at least two nonconsensus TATA sites in vitro, showing that the mutant protein has altered DNA binding specificity. Site-directed mutations that change Leu-205 to five different amino acids cause five different phenotypes, demonstrating the importance of this amino acid in vivo. Virtually identical phenotypes were observed when the same amino acid changes were made at the analogous position, Leu-114, in the first repeat of TFIID. Analysis of these mutations and additional mutations in the most conserved regions of the repeats, in conjunction with our DNA binding results, suggests that these regions of the repeats play equivalent roles in TFIID function, possibly in TATA box recognition.

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Single amino acid mutations effect Zika virus replication in vitro and virulence in vivo

10.1101/2020.08.06.239392 ◽

2020 ◽

Author(s):

Nicole M. Collette ◽

Victoria H.I. Lao ◽

Dina R. Weilhammer ◽

Barbara Zingg ◽

Shoshana D. Cohen ◽

...

Keyword(s):

Amino Acid ◽

Zika Virus ◽

Model Systems ◽

Single Amino Acid ◽

Replication Rate ◽

Adult Mice ◽

Naturally Occurring ◽

Viral Virulence

AbstractThe 2014-2016 Zika virus (ZIKV) epidemic in the Americas resulted in large deposits of next-generation sequencing data from clinical samples. This resource was mined to identify emerging mutations and trends in mutations as the outbreak progressed over time. Information on transmission dynamics, prevalence and persistence of intra-host mutants, and the position of a mutation on a protein were then used to prioritize 544 reported mutations based on their ability to impact ZIKV phenotype. Using this criteria, six mutants (representing naturally occurring mutations) were generated as synthetic infectious clones using a 2015 Puerto Rican epidemic strain PRVABC59 as the parental backbone. The phenotypes of these naturally occurring variants were examined using both cell culture and murine model systems. Mutants had distinct phenotypes, including changes in replication rate, embryo death, and decreased head size. In particular, a NS2B mutant previously detected during in vivo studies in rhesus macaques was found to cause lethal infections in adult mice, abortions in pregnant females, and increased viral genome copies in both brain tissue and blood of female mice. Additionally, mutants with changes in the region of NS3 that interfaces with NS5 during replication displayed reduced replication in the blood of adult mice. This analytical pathway, integrating both bioinformatic and wet lab experiments, provides a foundation for understanding how naturally occurring single mutations affect disease outcome and can be used to predict the of severity of future ZIKV outbreaks.Author summaryTo determine if naturally occurring individual mutations in the Zika virus epidemic genotype effect viral virulence or replication rate in vitro or in vivo, we generated an infectious clone representing the epidemic genotype of stain Puerto Rico, 2015. Using this clone, six mutants were created by changing nucleotides in the genome to cause one to two amino acid substitutions in the encoded proteins. The six mutants we generated represent mutations that differentiated the early epidemic genotype from genotypes that were either ancestral or that occurred later in the epidemic. We assayed each mutant for changes in growth rate, and for virulence in adult mice and pregnant mice. Three of the mutants caused catastrophic embryo effects including increased embryonic death or significant decrease in head diameter. Three other mutants that had mutations in a genome region associated with replication resulted in changes in in vitro and in vivo replication rates. These results illustrate the potential impact of individual mutations in viral phenotype.

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The Diverse Roles of the Global Transcriptional Regulator PhoP in the Lifecycle of Yersinia pestis

Pathogens ◽

10.3390/pathogens9121039 ◽

2020 ◽

Vol 9 (12) ◽

pp. 1039

Author(s):

Hana S. Fukuto ◽

Gloria I. Viboud ◽

Viveka Vadyvaloo

Keyword(s):

Yersinia Pestis ◽

Current Knowledge ◽

Response Regulator ◽

Expression Profiles ◽

Critical Role ◽

Gene Expression Profiles ◽

Acanthamoeba Castellanii ◽

Wide Range

Yersinia pestis, the causative agent of plague, has a complex infectious cycle that alternates between mammalian hosts (rodents and humans) and insect vectors (fleas). Consequently, it must adapt to a wide range of host environments to achieve successful propagation. Y. pestis PhoP is a response regulator of the PhoP/PhoQ two-component signal transduction system that plays a critical role in the pathogen’s adaptation to hostile conditions. PhoP is activated in response to various host-associated stress signals detected by the sensor kinase PhoQ and mediates changes in global gene expression profiles that lead to cellular responses. Y. pestis PhoP is required for resistance to antimicrobial peptides, as well as growth under low Mg2+ and other stress conditions, and controls a number of metabolic pathways, including an alternate carbon catabolism. Loss of phoP function in Y. pestis causes severe defects in survival inside mammalian macrophages and neutrophils in vitro, and a mild attenuation in murine plague models in vivo, suggesting its role in pathogenesis. A Y. pestisphoP mutant also exhibits reduced ability to form biofilm and to block fleas in vivo, indicating that the gene is also important for establishing a transmissible infection in this vector. Additionally, phoP promotes the survival of Y. pestis inside the soil-dwelling amoeba Acanthamoeba castellanii, a potential reservoir while the pathogen is quiescent. In this review, we summarize our current knowledge on the mechanisms of PhoP-mediated gene regulation in Y. pestis and examine the significance of the roles played by the PhoP regulon at each stage of the Y. pestis life cycle.

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