MisR/MisS Two-Component Regulon in Neisseria meningitidis

ABSTRACT Two-component regulatory systems are involved in processes important for bacterial pathogenesis. Inactivation of the misR/misS system in Neisseria meningitidis results in the loss of phosphorylation of the lipooligosaccharide inner core and causes attenuation in a mouse model of meningococcal infection. One hundred seventeen (78 up-regulated and 39 down-regulated) potential regulatory targets of the MisR/MisS (MisR/S) system were identified by transcriptional profiling of the NMBmisR mutant and the parental wild-type meningococcal strain NMB. The regulatory effect was further confirmed in a subset of target genes by quantitative real-time PCR and β-galactosidase transcriptional fusion reporter assays. The MisR regulon includes genes encoding proteins necessary for protein folding in the bacterial cytoplasm and periplasm, transcriptional regulation, metabolism, iron assimilation, and type I protein transport. Mutation in the MisR/S system caused increased sensitivity to oxidative stress and also resulted in decreased susceptibility to complement-mediated killing by normal human serum. To identify the direct targets of MisR regulation, electrophoretic mobility shift assays were carried out using purified MisR-His6 protein. Among 22 genes examined, misR directly interacted with 14 promoter regions. Six promoters were further investigated by DNase I protection assays, and a MisR-binding consensus sequence was proposed. Thus, the direct regulatory targets of MisR and the minimal regulon of the meningococcal MisR/S two-component signal transduction system were characterized. These data indicate that the MisR/S system influences a wide range of biological functions in N. meningitidis either directly or via intermediate regulators.

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Myostatin regulates fiber-type composition of skeletal muscle by regulating MEF2 and MyoD gene expression

AJP Cell Physiology ◽

10.1152/ajpcell.00259.2007 ◽

2009 ◽

Vol 296 (3) ◽

pp. C525-C534 ◽

Cited By ~ 104

Author(s):

Alex Hennebry ◽

Carole Berry ◽

Victoria Siriett ◽

Paul O'Callaghan ◽

Linda Chau ◽

...

Keyword(s):

Growth Factor ◽

Target Genes ◽

Fiber Type ◽

Fiber Formation ◽

Type I ◽

Mobility Shift ◽

Enhancer Activity ◽

Fiber Type Composition ◽

Nuclear Extracts ◽

Type Composition

Myostatin (Mstn) is a secreted growth factor belonging to the tranforming growth factor (TGF)-β superfamily. Inactivation of murine Mstn by gene targeting, or natural mutation of bovine or human Mstn, induces the double muscling (DM) phenotype. In DM cattle, Mstn deficiency increases fast glycolytic (type IIB) fiber formation in the biceps femoris (BF) muscle. Using Mstn null (−/−) mice, we suggest a possible mechanism behind Mstn-mediated fiber-type diversity. Histological analysis revealed increased type IIB fibers with a concomitant decrease in type IIA and type I fibers in the Mstn−/−tibialis anterior and BF muscle. Functional electrical stimulation of Mstn−/−BF revealed increased fatigue susceptibility, supporting increased type IIB fiber content. Given the role of myocyte enhancer factor 2 (MEF2) in oxidative type I fiber formation, MEF2 levels in Mstn−/−tissue were quantified. Results revealed reduced MEF2C protein in Mstn−/−muscle and myoblast nuclear extracts. Reduced MEF2-DNA complex was also observed in electrophoretic mobility-shift assay using Mstn−/−nuclear extracts. Furthermore, reduced expression of MEF2 downstream target genes MLC1F and calcineurin were found in Mstn−/−muscle. Conversely, Mstn addition was sufficient to directly upregulate MLC promoter-enhancer activity in cultured myoblasts. Since high MyoD levels are seen in fast fibers, we analyzed MyoD levels in the muscle. In contrast to MEF2C, MyoD levels were increased in Mstn−/−muscle. Together, these results suggest that while Mstn positively regulates MEF2C levels, it negatively regulates MyoD expression in muscle. We propose that Mstn could regulate fiber-type composition by regulating the expression of MEF2C and MyoD during myogenesis.

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SirR, a Novel Iron-Dependent Repressor inStaphylococcus epidermidis

Infection and Immunity ◽

10.1128/iai.66.9.4123-4129.1998 ◽

1998 ◽

Vol 66 (9) ◽

pp. 4123-4129 ◽

Cited By ~ 63

Author(s):

Philip J. Hill ◽

Alan Cockayne ◽

Patrick Landers ◽

Julie A. Morrissey ◽

Catriona M. Sims ◽

...

Keyword(s):

Binding Site ◽

Dna Sequences ◽

Blot Analysis ◽

Target Genes ◽

Consensus Sequence ◽

Dependent Manner ◽

Chromosomal Dna ◽

Iron Starvation ◽

Mobility Shift ◽

Western Blots

ABSTRACT In Staphylococcus epidermidis and Staphylococcus aureus, a number of cell wall- and cytoplasmic membrane-associated lipoproteins are induced in response to iron starvation. To gain insights into the molecular basis of iron-dependent gene regulation in the staphylococci, we sequenced the DNA upstream of the 3-kb S. epidermidis sitABC operon, which Northern blot analysis indicates is transcriptionally regulated by the growth medium iron content. We identified two DNA sequences which are homologous to elements of the Corynebacterium diphtheriae DtxR regulon, which controls, in response to iron stress, for example, production of diphtheria toxin, siderophore, and a heme oxygenase. Upstream of thesitABC operon and divergently transcribed lies a 645-bp open reading frame (ORF), which codes for a polypeptide of approximately 25 kDa with homology to the DtxR family of metal-dependent repressor proteins. This ORF has been designated SirR (staphylococcal iron regulator repressor). Within thesitABC promoter/operator region, we also located a region of dyad symmetry overlapping the transcriptional start ofsitABC which shows high homology to the DtxR operator consensus sequence, suggesting that this region, termed the Sir box, is the SirR-binding site. The SirR protein was overexpressed, purified, and used in DNA mobility shift assays; SirR retarded the migration of a synthetic oligonucleotide based on the Sir box in a metal (Fe2+ or Mn2+)-dependent manner, providing confirmatory evidence that this motif is the SirR-binding site. Furthermore, Southern blot analysis of staphylococcal chromosomal DNA with the synthetic Sir box as a probe confirmed that there are at least five Sir boxes in the S. epidermidis genome and at least three in the genome of S. aureus, suggesting that SirR controls the expression of multiple target genes. Using a monospecific polyclonal antibody raised against SirR to probe Western blots of whole-cell lysates of S. aureus, S. carnosus,S. epidermidis, S. hominis, S. cohnii, S. lugdunensis, and S. haemolyticus, we identified an approximately 25-kDa cross-reactive protein in each of the staphylococcal species examined. Taken together, these data suggest that SirR functions as a divalent metal cation-dependent transcriptional repressor which is widespread among the staphylococci.

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Orphan nuclear receptor SHP interacts with and represses hepatocyte nuclear factor-6 (HNF-6) transactivation

Biochemical Journal ◽

10.1042/bj20071637 ◽

2008 ◽

Vol 413 (3) ◽

pp. 559-569 ◽

Cited By ~ 15

Author(s):

Yong-Soo Lee ◽

Don-Kyu Kim ◽

Yong Deuk Kim ◽

Ki Cheol Park ◽

Minho Shong ◽

...

Keyword(s):

Nuclear Receptor ◽

Phosphoenolpyruvate Carboxykinase ◽

Target Genes ◽

Consensus Sequence ◽

Nuclear Factor ◽

Orphan Nuclear Receptor ◽

Hepatocyte Nuclear Factor ◽

Mobility Shift ◽

Endocrine Differentiation

SHP (small heterodimer partner; NR0B2) is an atypical orphan NR (nuclear receptor) that functions as a transcriptional co-repressor by interacting with a diverse set of NRs and transcriptional factors. HNF-6 (hepatocyte nuclear factor-6) is a key regulatory factor in pancreatic development, endocrine differentiation and the formation of the biliary tract, as well as glucose metabolism. In this study, we have investigated the function of SHP as a putative repressor of HNF-6. Using transient transfection assays, we have shown that SHP represses the transcriptional activity of HNF-6. Confocal microscopy revealed that both SHP and HNF-6 co-localize in the nuclei of cells. SHP physically interacted with HNF-6 in protein–protein association assays in vitro. EMSAs (electrophoretic mobility-shift assays) and ChIP (chromatin immunoprecipitation) assays demonstrated that SHP inhibits the DNA-binding activity of HNF-6 to an HNF-6-response element consensus sequence, and the HNF-6 target region of the endogenous G6Pase (glucose 6-phosphatase) promoter respectively. Northern blot analysis of HNF-6 target genes in cells infected with adenoviral vectors for SHP and SHP siRNAs (small inhibitory RNAs) indicated that SHP represses the expression of endogenous G6Pase and PEPCK (phosphoenolpyruvate carboxykinase). Our results suggest that HNF-6 is a novel target of SHP in the regulation of gluconeogenesis.

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Transcriptional regulation of the mouse alpha A-crystallin gene: activation dependent on a cyclic AMP-responsive element (DE1/CRE) and a Pax-6-binding site.

Molecular and Cellular Biology ◽

10.1128/mcb.15.2.653 ◽

1995 ◽

Vol 15 (2) ◽

pp. 653-660 ◽

Cited By ~ 74

Author(s):

A Cvekl ◽

F Kashanchi ◽

C M Sax ◽

J N Brady ◽

J Piatigorsky

Keyword(s):

Cyclic Amp ◽

Leukemia Virus ◽

Consensus Sequence ◽

Gene Activation ◽

Type I ◽

Loss Of Function ◽

Mobility Shift ◽

Cell Leukemia Virus Type ◽

Human T Cell ◽

Responsive Element

Two cis-acting promoter elements (-108 to -100 and -49 to -33) of the mouse alpha A-crystallin gene, which is highly expressed in the ocular lens, were studied. Here we show that DE1 (-108 to -100; 5'TGACGGTG3'), which resembles the consensus cyclic AMP (cAMP)-responsive element sequence (CRE; 5'TGACGT[A/C][A/G]3'), behaves like a functional CRE site. Transfection experiments and electrophoretic mobility shift assays (EMSAs) using site-specific mutations correlated a loss of function with deviations from the CRE consensus sequence. Results of EMSAs in the presence of antisera against CREB, delta CREB, and CREM were consistent with the binding of CREB-like proteins to the DE1 sequence. Stimulation of alpha A-crystallin promoter activity via 8-bromo-cAMP, forskolin, or human T-cell leukemia virus type I Tax1 in transfections and reduction of activity of this site in cell-free transcription tests by competition with the somatostatin CRE supported the idea that DE1 is a functional CRE. Finally, Pax-6, a member of the paired-box family of transcription factors, activated the mouse alpha A-crystallin promoter in cotransfected COP-8 fibroblasts and bound to the -59 to -29 promoter sequence in EMSAs. These data provide evidence for a synergistic role of Pax-6 and CREB-like proteins for high expression of the mouse alpha A-crystallin gene in the lens.

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A Two-Component System Plays an Important Role in the Root-Colonizing Ability of Pseudomonas fluorescens Strain WCS365

Molecular Plant-Microbe Interactions ◽

10.1094/mpmi.1998.11.1.45 ◽

1998 ◽

Vol 11 (1) ◽

pp. 45-56 ◽

Cited By ~ 83

Author(s):

Linda C. Dekkers ◽

Cees Jan P. Bloemendaal ◽

Letty A. de Weger ◽

Carel A. Wijffelman ◽

Herman P. Spaink ◽

...

Keyword(s):

Pseudomonas Fluorescens ◽

Consensus Sequence ◽

Integration Host Factor ◽

Amino Acid Sequences ◽

Root Tip ◽

Sensor Kinase ◽

Two Component System ◽

Component System ◽

Wide Range ◽

Two Component

We describe the characterization of a novel Tn5lacZ colonization mutant of the efficiently colonizing Pseudomonas fluorescens strain WCS365, mutant strain PCL1210, which is at least 300- to 1,000-fold impaired in colonization of the potato root tip after co-inoculation of potato stem cuttings with a 1:1 mixture of mutant and parental cells. Similarly, the mutant is also impaired in colonization of tomato, wheat, and radish, indicating that the gene involved plays a role in the ability of P. fluorescens WCS365 to colonize a wide range of plant species. A 3.1-kb DNA fragment was found to be able to complement the observed mutation. The nucleotide sequence of the region around the Tn5lacZ insertion showed three open reading frames (ORFs). The transcriptional start site was determined. The operon is preceded by an integration host factor (IHF) binding site consensus sequence whereas no clear −10 and −35 sequences are present. The deduced amino acid sequences of the first two genes of the operon, designated as colR and colS, show strong similarity with known members of two-component regulatory systems. ColR has homology with the response regulators of the OmpR-PhoB subclass whereas ColS, the product of the gene in which the mutation resides, shows similarity to the sensor kinase members of these two-component systems. Hydrophobicity plots show that this hypothetical sensor kinase has two transmembrane domains, as is also known for other sensor kinases. The product of the third ORF, Orf222, shows no homology with known proteins. Only part of the orf222 gene is present in the colonization-complementing, 3.1-kb region, and it therefore does not play a role in complementation. No experimental evidence for a role of the ColR/ ColS two-component system in the suspected colonization traits chemotaxis and transport of exudate compounds could be obtained. The function of this novel two-component system therefore remains to be elucidated. We conclude that colonization is an active process in which an environmental stimulus, through this two-component system, activates a so far unknown trait that is crucial for colonization.

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lpt6, a Gene Required for Addition of Phosphoethanolamine to Inner-Core Lipopolysaccharide of Neisseria meningitidis and Haemophilus influenzae

Journal of Bacteriology ◽

10.1128/jb.186.20.6970-6982.2004 ◽

2004 ◽

Vol 186 (20) ◽

pp. 6970-6982 ◽

Cited By ~ 33

Author(s):

J. Claire Wright ◽

Derek W. Hood ◽

Gaynor A. Randle ◽

Katherine Makepeace ◽

Andrew D. Cox ◽

...

Keyword(s):

Amino Acid ◽

Haemophilus Influenzae ◽

Neisseria Meningitidis ◽

Inner Core ◽

Amino Acid Sequences ◽

Gram Negative Bacteria ◽

Structural Studies ◽

Conserved Residues ◽

Wide Range ◽

Β Chain

ABSTRACT We previously described a gene, lpt3, required for the addition of phosphoethanolamine (PEtn) at the 3 position on the β-chain heptose (HepII) of the inner-core Neisseria meningitidis lipopolysaccharide (LPS), but it has long been recognized that the inner-core LPS of some strains possesses PEtn at the 6 position (PEtn-6) on HepII. We have now identified a gene, lpt6 (NMA0408), that is required for the addition of PEtn-6 on HepII. The lpt6 gene is located in a region previously identified as Lgt-3 and is associated with other LPS biosynthetic genes. We screened 113 strains, representing all serogroups and including disease and carriage strains, for the lpt3 and lpt6 genes and showed that 36% contained both genes, while 50% possessed lpt3 only and 12% possessed lpt6 only. The translated amino acid sequence of lpt6 has a homologue (72.5% similarity) in a product of the Haemophilus influenzae Rd genome sequence. Previous structural studies have shown that all H. influenzae strains investigated have PEtn-6 on HepII. Consistent with this, we found that, among 70 strains representing all capsular serotypes and nonencapsulated H. influenzae strains, the lpt6 homologue was invariably present. Structural analysis of LPS from H. influenzae and N. meningitidis strains where lpt6 had been insertionally inactivated revealed that PEtn-6 on HepII could not be detected. The translated amino acid sequences from the N. meningitidis and H. influenzae lpt6 genes have conserved residues across their lengths and are part of a family of proven or putative PEtn transferases present in a wide range of gram-negative bacteria.

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The ColRS Two-Component System Regulates Membrane Functions and Protects Pseudomonas putida against Phenol

Journal of Bacteriology ◽

10.1128/jb.01262-06 ◽

2006 ◽

Vol 188 (23) ◽

pp. 8109-8117 ◽

Cited By ~ 38

Author(s):

Paula Ann Kivistik ◽

Marta Putrinš ◽

Külliki Püvi ◽

Heili Ilves ◽

Maia Kivisaar ◽

...

Keyword(s):

Pseudomonas Putida ◽

Target Genes ◽

Type I ◽

Two Component System ◽

Component System ◽

Two Component ◽

Encoding Gene ◽

Phenol Tolerance

ABSTRACT As reported, the two-component system ColRS is involved in two completely different processes. It facilitates the root colonization ability of Pseudomonas fluorescens and is necessary for the Tn4652 transposition-dependent accumulation of phenol-utilizing mutants in Pseudomonas putida. To determine the role of the ColRS system in P. putida, we searched for target genes of response regulator ColR by use of a promoter library. Promoter screening was performed on phenol plates to mimic the conditions under which the effect of ColR on transposition was detected. The library screen revealed the porin-encoding gene oprQ and the alginate biosynthesis gene algD occurring under negative control of ColR. Binding of ColR to the promoter regions of oprQ and algD in vitro confirmed its direct involvement in regulation of these genes. Additionally, the porin-encoding gene ompA PP0773 and the type I pilus gene csuB were also identified in the promoter screen. However, it turned out that ompA PP0773 and csuB were actually affected by phenol and that the influence of ColR on these promoters was indirect. Namely, our results show that ColR is involved in phenol tolerance of P. putida. Phenol MIC measurement demonstrated that a colR mutant strain did not tolerate elevated phenol concentrations. Our data suggest that increased phenol susceptibility is also the reason for inhibition of transposition of Tn4652 in phenol-starving colR mutant bacteria. Thus, the current study revealed the role of the ColRS two-component system in regulation of membrane functionality, particularly in phenol tolerance of P. putida.

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Genomic SELEX Search for Target Promoters under the Control of the PhoQP-RstBA Signal Relay Cascade

Journal of Bacteriology ◽

10.1128/jb.00319-07 ◽

2007 ◽

Vol 189 (13) ◽

pp. 4791-4799 ◽

Cited By ~ 94

Author(s):

Hiroshi Ogasawara ◽

Akiko Hasegawa ◽

Emi Kanda ◽

Takenori Miki ◽

Kaneyoshi Yamamoto ◽

...

Keyword(s):

Promoter Activity ◽

Consensus Sequence ◽

Regulatory System ◽

Regulatory Function ◽

Mobility Shift ◽

Promoter Assay ◽

Signal Relay ◽

Two Component ◽

Genomic Selex

ABSTRACT RstBA, a two-component regulatory system of Escherichia coli with an unidentified regulatory function, is under the control of a Mg2+-sensing PhoQP two-component system. In order to identify the network of transcription regulation downstream of RstBA, we isolated a set of RstA-binding sequences from the E. coli genome by using the genomic SELEX system. A gel mobility shift assay indicated the binding of RstA to two SELEX DNA fragments, one including the promoter region of asr (acid shock RNA) and another including the promoter for csgD (a regulator of the curli operon). Using a DNase I footprinting assay, we determined the RstA-binding sites (RstA boxes) with the consensus sequence TACATNTNGTTACA. Transcription of the asr gene was induced 10- to 60-fold either in low-pH (pH 4.5) LB medium or in low-phosphate minimal medium as detected by promoter assay. The acid-induced in vivo transcription of asr was reduced after the deletion of rstA. In vivo transcription of the asr promoter was observed only in the presence of RstA. In agreement with the PhoQP-RstBA network, the addition of Mg2+ led to a severe reduction of the asr promoter activity, and the disruption of phoP also reduced the asr promoter activity, albeit to a lesser extent. These observations altogether indicate that RstA is an activator of asr transcription. In contrast, transcription of csgD was repressed by overexpression of RstA, indicating that RstA is a repressor for csgD. With these data taken together, we conclude that the expression of both asr and csgD is under the direct control of the PhoQP-RstBA signal relay cascade.

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Clinical cases of meningococcal infection caused by Neisseria meningitidis serogroup W

Rossiyskiy Vestnik Perinatologii i Pediatrii (Russian Bulletin of Perinatology and Pediatrics) ◽

10.21508/1027-4065-2019-64-5-114-122 ◽

2019 ◽

Vol 64 (5) ◽

pp. 114-122

Author(s):

N. V. Skripchenko ◽

V. E. Karev ◽

K. V. Markova ◽

A. A. Vilnits ◽

N. F. Pulman

Keyword(s):

Neisseria Meningitidis ◽

Fatal Outcome ◽

Clinical Manifestations ◽

Meningococcal Infection ◽

Hemorrhagic Infarction ◽

Heart Damage ◽

Clinical Observations ◽

Wide Range ◽

Significant Disease ◽

The Brain

Meningococcal infection remains a socially significant disease due to a wide range of clinical manifestations, severe course with high mortality, reaching 40-80% for certain forms. In recent years we observe a change in the serogroup picture of meningococcal infection, in particular, an increase in the proportion of meningococcal serogroup W, characterized by polymorphism of clinical manifestations. The authors present two their own clinical observations of a generalized form of meningococcal infection with a severe course in adolescents caused by Neisseria meningitidis of W serogroup with fatal outcome. They describe the peculiarities of the cases, namely, inconsistency of the clinical picture at the beginning of the disease and post-mortem changes. The article describes heart damage in the form of focal purulent septic myocarditis and purulent foci of destruction in the brain stem, hemorrhagic infarction of the choroid plexus with hemotamponade of the lateral ventricles of the brain, the predominance of septicopia over septicemia. The revealed changes are likely to reflect clinical and morphological features of meningococcal infection caused by N. meningitidis of serogroup W.

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Identification of Direct Transcriptional Target Genes of ExoS/ChvI Two-Component Signaling in Sinorhizobium meliloti

Journal of Bacteriology ◽

10.1128/jb.00734-09 ◽

2009 ◽

Vol 191 (22) ◽

pp. 6833-6842 ◽

Cited By ~ 26

Author(s):

Esther J. Chen ◽

Robert F. Fisher ◽

Virginia M. Perovich ◽

Erich A. Sabio ◽

Sharon R. Long

Keyword(s):

Sinorhizobium Meliloti ◽

Target Genes ◽

Transcriptional Profiling ◽

Nutrient Utilization ◽

Wild Type ◽

Mobility Shift ◽

Gain Of Function ◽

Free Living ◽

Two Component ◽

Transcriptional Target

ABSTRACT The Sinorhizobium meliloti ExoS/ChvI two-component signaling pathway is required for the development of a nitrogen-fixing symbiosis between S. meliloti and its plant hosts. ExoS/ChvI also has important roles in regulating succinoglycan production, biofilm formation, motility, nutrient utilization, and the viability of free-living bacteria. Previous microarray experiments with an exoS96::Tn5 mutant indicated that ExoS/ChvI influences the expression of a few hundred genes, complicating the investigation of which downstream genes respond directly or indirectly to ExoS/ChvI regulation. To focus our study of ExoS/ChvI transcriptional target genes, we performed transcriptional profiling with chvI gain-of-function and reduced-function strains. The chvI gain-of-function strain that we used contains a dominant gain-of-function chvI allele in addition to wild-type chvI. We identified genes that, relative to their expression level in the wild type, are both upregulated in the chvI gain-of-function strain and downregulated in the reduced-function strain or vice versa. Guided by this focused set of genes, we performed gel mobility shift assays and demonstrated that ChvI directly binds the intergenic regions upstream of ropB1, SMb21440, and SMc01580. Furthermore, DNase I footprint analysis of the region upstream of SMc01580 identified a specific DNA sequence bound by ChvI and allowed the discovery of a possible motif for ChvI binding. Our results provide insight into the mechanism of how ExoS/ChvI regulates its downstream targets and lay a foundation for studying this conserved pathway with critical roles in free-living and symbiotic bacteria.

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