scholarly journals A Novel Gene Involved in Regulating the Flagellar Gene Cascade in Proteus mirabilis

2006 ◽  
Vol 188 (22) ◽  
pp. 7830-7839 ◽  
Author(s):  
Lindsay G. Stevenson ◽  
Philip N. Rather
Keyword(s):  
Cell Differentiation ◽  
Amino Acid ◽  
Proteus Mirabilis ◽  
Fold Increase ◽  
Single Copy ◽  
Flagellar Gene ◽  
Mrna Accumulation ◽  
Swarmer Cell ◽  
Novel Gene ◽  
Expression Of Genes

ABSTRACT In this study, we identified a transposon insertion in a novel gene, designated disA, that restored swarming motility to a putrescine-deficient speA mutant of Proteus mirabilis. A null allele in disA also increased swarming in a wild-type background. The DisA gene product was homologous to amino acid decarboxylases, and its role in regulating swarming was investigated by examining the expression of genes in the flagellar cascade. In a disA mutant background, we observed a 1.4-fold increase in the expression of flhDC, which encodes FlhD2C2, the master regulator of the flagellar gene cascade. However, the expressions of class 2 (fliA, flgM) and class 3 (flaA) genes were at least 16-fold higher in the disA background during swarmer cell differentiation. Overexpression of DisA on a high-copy-number plasmid did not significantly decrease flhDC mRNA accumulation but resulted in a complete block in mRNA accumulation for both fliA and flaA. DisA overexpression also blocked swarmer cell differentiation. The disA gene was regulated during the swarming cycle, and a single-copy disA::lacZ fusion exhibited a threefold increase in expression in swarmer cells. Given that DisA was similar to amino acid decarboxylases, a panel of decarboxylated amino acids was tested for effects similar to DisA overexpression, and phenethylamine, the product of phenylalanine decarboxylation, was capable of inhibiting both swarming and the expression of class 2 and class 3 genes in the flagellar regulon. A DisA-dependent decarboxylated amino acid may inhibit the formation of active FlhD2C2 heterotetramers or inhibit FlhD2C2 binding to DNA.

scholarly journals Regulation of the Min Cell Division Inhibition Complex by the Rcs Phosphorelay in Proteus mirabilis

2015 ◽  
Vol 197 (15) ◽  
pp. 2499-2507 ◽  
Author(s):  
Kristen E. Howery ◽  
Katy M. Clemmer ◽  
Emrah Şimşek ◽  
Minsu Kim ◽  
Philip N. Rather
Keyword(s):  
Cell Division ◽  
Cell Differentiation ◽  
Proteus Mirabilis ◽  
Cell Elongation ◽  
Response Regulator ◽  
Swarmer Cell ◽  
Content Type ◽  
Expression Of Genes ◽  
Rapid Amplification

ABSTRACTA key regulator of swarming inProteus mirabilisis the Rcs phosphorelay, which repressesflhDC, encoding the master flagellar regulator FlhD4C2. Mutants inrcsB, the response regulator in the Rcs phosphorelay, hyperswarm on solid agar and differentiate into swarmer cells in liquid, demonstrating that this system also influences the expression of genes central to differentiation. To gain a further understanding of RcsB-regulated genes involved in swarmer cell differentiation, transcriptome sequencing (RNA-Seq) was used to examine the RcsB regulon. Among the 133 genes identified,minCandminD, encoding cell division inhibitors, were identified as RcsB-activated genes. A third gene,minE, was shown to be part of an operon withminCD. To examineminCDEregulation, theminpromoter was identified by 5′ rapid amplification of cDNA ends (5′-RACE), and both transcriptionallacZfusions and quantitative real-time reverse transcriptase (qRT) PCR were used to confirm that theminCDEoperon was RcsB activated. Purified RcsB was capable of directly binding theminCpromoter region. To determine the role of RcsB-mediated activation ofminCDEin swarmer cell differentiation, a polarminCmutation was constructed. This mutant formed minicells during growth in liquid, produced shortened swarmer cells during differentiation, and exhibited decreased swarming motility.IMPORTANCEThis work describes the regulation and role of the MinCDE cell division system inP. mirabilisswarming and swarmer cell elongation. Prior to this study, the mechanisms that inhibit cell division and allow swarmer cell elongation were unknown. In addition, this work outlines for the first time the RcsB regulon inP. mirabilis. Taken together, the data presented in this study begin to address howP. mirabiliselongates upon contact with a solid surface.

Differential regulation of mRNAs encoding for rat intestinal alkaline phosphatase

1990 ◽  
Vol 259 (1) ◽  
pp. G93-G98 ◽  
Author(s):  
R. Eliakim ◽  
S. Seetharam ◽  
C. C. Tietze ◽  
D. H. Alpers
Keyword(s):  
Alkaline Phosphatase ◽  
Amino Acid ◽  
Cdna Probe ◽  
Fold Increase ◽  
Maximal Activity ◽  
Amino Acid Residues ◽  
Intestinal Alkaline Phosphatase ◽  
Mrna Accumulation ◽  
Dihydroxyvitamin D3 ◽  
Fat Feeding

A cDNA probe encoding the entire structural region of the 62-kDa rat intestinal alkaline phosphatase from amino acid residues 1 to 531 detected multiple mRNA species (3.0, 2.7, and 2.2 kb) in rat intestinal RNA. The 3.0-kb species was most evident in duodenum but could be easily detected in jejunum using a 48-mer oligonucleotide encoding amino acid residues 492-508. This 48-mer oligonucleotide bound preferentially to the 3.0-kb mRNA, suggesting that the 2.7-kb mRNA differed in this region. To determine whether each of the mRNAs encoding rat intestinal alkaline phosphatase responded coordinately to physiological stimuli, the full-length cDNA and the 48-mer oligonucleotide were used as probes for the 2.7- and 2.2-kb and the 3.0-kb mRNAs, respectively. Intestinal mRNA concentration was measured by Northern blot analysis in acute (single feed, 17 kcal) and chronic (3 wk, 30% fat diet) fat feeding and in rachitic rats after 1,25-dihydroxyvitamin D3 therapy. There was a large increase (8- to 25-fold) in the 3.0-kb mRNA 7 h after acute fat feeding, with a much smaller increase (1.4- to 5.0-fold) in the 2.7- and 2.2-kb species. The peak in 3.0-kb mRNA accumulation correlated in time with the maximal activity of serum phosphatase activity after acute fat feeding (4- to 5-fold increase). In contrast, there was a much smaller increase in all mRNAs and in tissue and serum enzyme activity after chronic fat feeding.(ABSTRACT TRUNCATED AT 250 WORDS)

scholarly journals Characterization of a Novel Gene, wosA, Regulating FlhDC Expression in Proteus mirabilis

2008 ◽  
Vol 190 (6) ◽  
pp. 1946-1955 ◽  
Author(s):  
Janet K. Hatt ◽  
Philip N. Rather
Keyword(s):  
Cell Differentiation ◽  
Liquid Medium ◽  
Proteus Mirabilis ◽  
Solid Surfaces ◽  
Swarming Motility ◽  
Swarmer Cell ◽  
Fourfold Increase ◽  
Nonpermissive Condition

ABSTRACT In this study, we describe wosA, a Proteus mirabilis gene identified by its ability to increase swarming motility when overexpressed. At various times during the swarming cycle, the increased expression of wosA resulted in a 4- to 16-fold upregulation of the transcription of flhDC, encoding the master regulator of the flagellar cascade. In turn, the expression of flaA, encoding flagellin, was substantially increased in wosA-overexpressing strains. The overexpression of wosA also resulted in constitutive swarmer cell differentiation in liquid medium, a normally nonpermissive condition. However, in wosA-overexpressing strains, the onset of swarming was not altered. A null wosA allele resulted in a slight decrease in swarming motility. The expression of wosA was growth phase dependent during growth in liquid and on agar plates during swarmer cell differentiation. Increasing the viscosity of liquid medium by the addition of polyvinylpyrrolidone induced swarmer cell differentiation and resulted in a fourfold increase in wosA transcription. A fliL mutation that results in constitutive swarmer cell elongation also increased wosA transcription. In this study, we discuss the possible role of the wosA gene product in signal transduction from solid surfaces to induce swarmer cell differentiation, possibly via alterations in the motor switch complex. This study also suggests that despite constitutive swarmer cell differentiation in wosA-overexpressing strains, there are additional regulatory and/or environmental conditions that may control the onset of swarming migration.

Identification and characterization of a new carbonic anhydrase in the marine diatom Phaeodactylum tricornutum

2005 ◽  
Vol 83 (7) ◽  
pp. 909-916 ◽  
Author(s):  
Hisashi Harada ◽  
Yusuke Matsuda
Keyword(s):  
Amino Acid ◽  
Carbonic Anhydrase ◽  
Amino Acid Sequence ◽  
Phaeodactylum Tricornutum ◽  
Fold Increase ◽  
Marine Diatom ◽  
Porphyridium Purpureum ◽  
Mrna Accumulation ◽  
Reading Frame ◽  
Endoplasmic Reticulum Targeting

A cDNA encoding a new isoenzyme of β-type carbonic anhydrase (CA; EC 4.2.1.1) in the marine diatom Phaeodactylum tricornutum Bohlin has been cloned. The cDNA contained an open reading frame of 819 bp, which encodes a polypeptide of 273 amino acids. This gene, which is designated as ptca2, was found to be highly homologous (83% at the nucleotide level) to the previously isolated intracellular β-CA gene from Phaeodactylum tricornutum (ptca1). Comparison of the deduced amino acid sequence of ptca2 with β-CAs from other sources demonstrated that PtCA2 possesses the completely conserved zinc coordination residues of β-CA. The N-terminus 19 amino acid sequence of PtCA2 was predicted to be an endoplasmic reticulum-targeting signal, suggesting localization of the protein in an organelle or in the periplasmic space. Quantitative analysis of mRNA accumulation of ptca2 using real-time polymerase chain reaction revealed a significant level of mRNA accumulation even under 5% CO2 and a 3.5-fold increase in accumulation upon acclimation of the diatom to air. This indicates that ptca2 belongs to a constitutive class of enzyme that responds only weakly to the ambient CO2 concentration. The sequences of both ptca1 and ptca2 were shown to be grouped into a phylogeny that is composed of mixture of sequences from the eucarya and procarya domains, including sequences from the red alga Porphyridium purpureum, the green alga Coccomyxa, the red mold Neurospora crassa, and the yeast Saccharomyces cerevisiae.Key words: carbonic anhydrase, marine diatom, inorganic carbon concentrating mechanism (CCM), Phaeodactylum tricornutum.

scholarly journals The Ability of Proteus mirabilis To Sense Surfaces and Regulate Virulence Gene Expression Involves FliL, a Flagellar Basal Body Protein

2005 ◽  
Vol 187 (19) ◽  
pp. 6789-6803 ◽  
Author(s):  
Robert Belas ◽  
Rooge Suvanasuthi
Keyword(s):  
Gene Expression ◽  
Cell Differentiation ◽  
Basal Body ◽  
Proteus Mirabilis ◽  
Virulence Gene ◽  
Wild Type ◽  
Swarmer Cell ◽  
Virulence Gene Expression ◽  
Flagellar Motors ◽  
Flagellar Rotation

ABSTRACT Proteus mirabilis is a urinary tract pathogen that differentiates from a short swimmer cell to an elongated, highly flagellated swarmer cell. Swarmer cell differentiation parallels an increased expression of several virulence factors, suggesting that both processes are controlled by the same signal. The molecular nature of this signal is not known but is hypothesized to involve the inhibition of flagellar rotation. In this study, data are presented supporting the idea that conditions inhibiting flagellar rotation induce swarmer cell differentiation and implicating a rotating flagellar filament as critical to the sensing mechanism. Mutations in three genes, fliL, fliF, and fliG, encoding components of the flagellar basal body, result in the inappropriate development of swarmer cells in noninducing liquid media or hyperelongated swarmer cells on agar media. The fliL mutation was studied in detail. FliL− mutants are nonmotile and fail to synthesize flagellin, while complementation of fliL restores wild-type cell elongation but not motility. Overexpression of fliL + in wild-type cells prevents swarmer cell differentiation and motility, a result also observed when P. mirabilis fliL + was expressed in Escherichia coli. These results suggest that FliL plays a role in swarmer cell differentiation and implicate FliL as critical to transduction of the signal inducing swarmer cell differentiation and virulence gene expression. In concert with this idea, defects in fliL up-regulate the expression of two virulence genes, zapA and hpmB. These results support the hypothesis that P. mirabilis ascertains its location in the environment or host by assessing the status of its flagellar motors, which in turn control swarmer cell gene expression.

scholarly journals Production of Phenylpyruvic Acid by Engineered L-Amino Acid Deaminase from Proteus Mirabilis

2021 ◽  
Author(s):  
Jia Liu ◽  
Cong Gao ◽  
Bin Yang ◽  
Wei Song ◽  
Guipeng Hu ◽  
...  
Keyword(s):  
Amino Acid ◽  
Proteus Mirabilis ◽  
Conformational Dynamics ◽  
Catalytic Efficiency ◽  
Fold Increase ◽  
Product Inhibition ◽  
Phenylpyruvic Acid ◽  
E Coli ◽  
Fold Reduction ◽  
Release Product

Abstract Objectives This study aimed to develop an efficient enzymatic strategy for the industrial production of phenylpyruvate (PPA) from L-phenylpyruvic acid (L-Phe). Results L-amino acid deaminase from Proteus mirabilis (L-pmAAD) was expressed in E. coli BL21(DE3) and modified to release product inhibition by employing conformational dynamics engineering. Based on structural analysis, two residues (E145A/L341A) were identified for reducing interactions between the product and enzyme and increasing flexibility of the protein, thereby facilitating the product release. The mutant M2E145A/E341A exhibited a 3.84-fold reduction in product inhibition and a 1.35-fold increase in catalytic efficiency in comparison to the wild type. Finally, 81.2 g/L PPA production with a conversion of 99.6% was obtained in a 5-L bioreactor. Conclusion The engineered catalyst can significantly reduce product inhibition and facilitate the effective industrial synthesis of PPA.

Sign in / Sign up

Export Citation Format

Share Document