scholarly journals PhhB, a Pseudomonas aeruginosa Homolog of Mammalian Pterin 4a-Carbinolamine Dehydratase/DCoH, Does Not Regulate Expression of Phenylalanine Hydroxylase at the Transcriptional Level

1999 ◽  
Vol 181 (9) ◽  
pp. 2789-2796 ◽  
Author(s):  
Jian Song ◽  
Tianhui Xia ◽  
Roy A. Jensen
Keyword(s):  
Escherichia Coli ◽  
Pseudomonas Aeruginosa ◽  
Phenylalanine Hydroxylase ◽  
Transcriptional Level ◽  
Gene Encoding ◽  
Catalytic Function ◽  
Genes Encoding ◽  
Catalytic Role ◽  
Regulatory Effects

ABSTRACT Pterin 4a-carbinolamine dehydratase is bifunctional in mammals. In addition to playing a catalytic role in pterin recycling in the cytoplasm, it plays a regulatory role in the nucleus, where it acts as a dimerization-cofactor component (called DCoH) for the transcriptional activator HNF-1α. A thus far unique operon in Pseudomonas aeruginosa contains a gene encoding a homolog (PhhB) of the regulatory dehydratase, together with genes encoding phenylalanine hydroxylase (PhhA) and aromatic aminotransferase (PhhC). Using complementation of tyrosine auxotrophy in Escherichia colias a functional test, we have found that the in vivo function of PhhA requires PhhB. Strikingly, mammalian DCoH was an effective substitute for PhhB, and either one was effective in trans. Surprisingly, the required presence of PhhB for complementation did not reflect a critical positive regulatory effect of phhB onphhA expression. Rather, in the absence of PhhB, PhhA was found to be extremely toxic in E. coli, probably due to the nonenzymatic formation of 7-biopterin or a similar derivative. However, bacterial PhhB does appear to exert modest regulatory effects in addition to having a catalytic function. PhhB enhances the level of PhhA two- to threefold, as was demonstrated by gene inactivation ofphhB in P. aeruginosa and by comparison of the levels of expression of PhhA in the presence and absence of PhhB inEscherichia coli. Experiments using constructs having transcriptional and translational fusions with a lacZreporter indicated that PhhB activates PhhA at the posttranscriptional level. Regulation of PhhA and PhhB is semicoordinate; both PhhA and PhhB are induced coordinately in the presence of eitherl-tyrosine or l-phenylalanine, but PhhB exhibits a significant basal level of activity that is lacking for PhhA. Immunoprecipitation and affinity chromatography showed that PhhA and PhhB form a protein-protein complex.

scholarly journals Overproduction of SecA Suppresses the Export Defect Caused by a Mutation in the Gene Encoding the Escherichia coli Export Chaperone SecB

1999 ◽  
Vol 181 (10) ◽  
pp. 3010-3017 ◽  
Author(s):  
Heather A. Cook ◽  
Carol A. Kumamoto
Keyword(s):  
Escherichia Coli ◽  
Outer Membrane Proteins ◽  
Protein A ◽  
In Vivo Studies ◽  
Wild Type ◽  
Gene Encoding ◽  
Precursor Proteins ◽  
Additional Support ◽  
Insight Into

ABSTRACT SecB is a cytosolic protein required for rapid and efficient export of particular periplasmic and outer membrane proteins inEscherichia coli. SecB promotes export by stabilizing newly synthesized precursor proteins in a nonnative conformation and by targeting the precursors to the inner membrane. Biochemical studies suggest that SecB facilitates precursor targeting by binding to the SecA protein, a component of the membrane-embedded translocation apparatus. To gain more insight into the functional interaction of SecB and SecA, in vivo, mutations in the secA locus that compensate for the export defect caused by the secBmissense mutation secBL75Q were isolated. Two suppressors were isolated, both of which led to the overproduction of wild-type SecA protein. In vivo studies demonstrated that the SecBL75Q mutant protein releases precursor proteins at a lower rate than does wild-type SecB. Increasing the level of SecA protein in the cell was found to reverse this slow-release defect, indicating that overproduction of SecA stimulates the turnover of SecBL75Q-precursor complexes. These findings lend additional support to the proposed pathway for precursor targeting in which SecB promotes targeting to the translocation apparatus by binding to the SecA protein.

Recombinogenic Activity of Chimeric recA Genes (Pseudomonas aeruginosa/Escherichia coli): A Search for RecA Protein Regions Responsible for This Activity

Genetics ◽  
2001 ◽  
Vol 159 (1) ◽  
pp. 7-15
Author(s):  
Irina V Bakhlanova ◽  
Tomoko Ogawa ◽  
Vladislav A Lanzov
Keyword(s):  
Escherichia Coli ◽  
Pseudomonas Aeruginosa ◽  
Unit Length ◽  
Reca Protein ◽  
Lacz Gene ◽  
Quantitative Correlation ◽  
Key Factor ◽  
Function Expression ◽  
Linkage Frequency

Abstract In the background of weak, if any, constitutive SOS function, RecA from Pseudomonas aeruginosa (RecAPa) shows a higher frequency of recombination exchange (FRE) per DNA unit length as compared to RecA from Escherichia coli (RecAEc). To understand the molecular basis for this observation and to determine which regions of the RecAPa polypeptide are responsible for this unusual activity, we analyzed recAX chimeras between the recAEc and recAPa genes. We chose 31 previously described recombination- and repair-proficient recAX hybrids and determined their FRE calculated from linkage frequency data and constitutive SOS function expression as measured by using the lacZ gene under control of an SOS-regulated promoter. Relative to recAEc, the FRE of recAPa was 6.5 times greater; the relative alterations of FRE for recAX genes varied from ~0.6 to 9.0. No quantitative correlation between the FRE increase and constitutive SOS function was observed. Single ([L29M] or [I102D]), double ([G136N, V142I]), and multiple substitutions in related pairs of chimeric RecAX proteins significantly altered their relative FRE values. The residue content of three separate regions within the N-terminal and central but not the C-terminal protein domains within the RecA molecule also influenced the FRE values. Critical amino acids in these regions were located close to previously identified sequences that comprise the two surfaces for subunit interactions in the RecA polymer. We suggest that the intensity of the interactions between the subunits is a key factor in determining the FRE promoted by RecA in vivo.

Chromosomal Integration of Heterologous DNA in Escherichia coli with Precise Removal of Markers and Replicons Used during Construction

1999 ◽  
Vol 181 (22) ◽  
pp. 7143-7148 ◽  
Author(s):  
F. Martinez-Morales ◽  
A. C. Borges ◽  
A. Martinez ◽  
K. T. Shanmugam ◽  
L. O. Ingram
Keyword(s):  
Escherichia Coli ◽  
Helper Plasmid ◽  
Flp Recombinase ◽  
E Coli ◽  
Homologous Fragment ◽  
Genes Encoding ◽  
Dna Insertion ◽  
K 12 ◽  
Multiple Cloning Sites

ABSTRACT A set of vectors which facilitates the sequential integration of new functions into the Escherichia coli chromosome by homologous recombination has been developed. These vectors are based on plasmids described by Posfai et al. (J. Bacteriol. 179:4426–4428, 1997) which contain conditional replicons (pSC101 or R6K), a choice of three selectable markers (ampicillin, chloramphenicol, or kanamycin), and a single FRT site. The modified vectors contain twoFRT sites which bracket a modified multiple cloning region for DNA insertion. After integration, a helper plasmid expressing the flippase (FLP) recombinase allows precise in vivo excision of the replicon and the marker used for selection. Sites are also available for temporary insertion of additional functions which can be subsequently deleted with the replicon. Only the DNA inserted into the multiple cloning sites (passenger genes and homologous fragment for targeting) and a single FRT site (68 bp) remain in the chromosome after excision. The utility of these vectors was demonstrated by integrating Zymomonas mobilis genes encoding the ethanol pathway behind the native chromosomaladhE gene in strains of E. coli K-12 andE. coli B. With these vectors, a single antibiotic selection system can be used repeatedly for the successive improvement of E. coli strains with precise deletion of extraneous genes used during construction.

scholarly journals Biological Characterization of Endotoxins Released from Antibiotic-Treated Pseudomonas aeruginosa and Escherichia coli

1998 ◽  
Vol 42 (5) ◽  
pp. 1015-1021 ◽  
Author(s):  
Teruo Kirikae ◽  
Fumiko Kirikae ◽  
Shinji Saito ◽  
Kaoru Tominaga ◽  
Hirohi Tamura ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Pseudomonas Aeruginosa ◽  
Lipid A ◽  
Biological Activities ◽  
Polymyxin B ◽  
Mitogen Activated Protein Kinase ◽  
No Production ◽  
Endotoxic Activity

ABSTRACT The supernatants taken from Pseudomonas aeruginosa andEscherichia coli cultures in human sera or chemically defined M9 medium in the presence of ceftazidime (CAZ) contained high levels of endotoxin, while those taken from the same cultures in the presence of imipenem (IPM) yielded a very low level of endotoxin. The biological activities of endotoxin in the supernatants were compared with those of phenol water-extracted lipopolysaccharide (LPS). The endotoxin released from the organisms as a result of CAZ treatment (CAZ-released endotoxin) contained a large amount of protein. The protein, however, lacked endotoxic activity, since the endotoxin did not show any in vivo toxic effects in LPS-hyporesponsive C3H/HeJ mice sensitized with d-(+)-galactosamine (GalN) or any activation of C3H/HeJ mouse macrophages in vitro. The activities of CAZ- and IPM-released endotoxin (as assessed by a chromogenicLimulus test) were fundamentally the same as those ofP. aeruginosa LPS, since their regression lines were parallel. The CAZ-released endotoxin was similar to purified LPS with respect to the following biological activities in LPS-responsive C3H/HeN mice and LPS-hyporesponsive C3H/HeJ mice: lethal toxicity in GalN-sensitized mice, in vitro induction of tumor necrosis factor- and NO production by macrophages, and mitogen-activated protein kinase activation in macrophages. The macrophage activation by CAZ-released endotoxin as well as LPS was mainly dependent on the presence of serum factor and CD14 antigen. Polymyxin B blocked the activity. These findings indicate that the endotoxic activity of CAZ-released endotoxin is due primarily to LPS (lipid A).

A novel peroxiredoxin of the plant Sedum lineare is a homologue of Escherichia coli bacterioferritin co-migratory protein (Bcp)

2000 ◽  
Vol 351 (1) ◽  
pp. 107-114 ◽  
Author(s):  
Wei KONG ◽  
Susumu SHIOTA ◽  
Yixin SHI ◽  
Hiroaki NAKAYAMA ◽  
Koji NAKAYAMA
Keyword(s):  
Escherichia Coli ◽  
Peroxidase Activity ◽  
Discrete Group ◽  
Sequence Data ◽  
Accession Number ◽  
Gene Encoding ◽  
Reaction Cycle ◽  
Mutant Proteins ◽  
E Coli

We cloned a gene encoding a 17-kDa protein from a cDNA library of the plant Sedum lineare and found that its deduced amino acid sequence showed similarities to those of Escherichia coli bacterioferritin co-migratory protein (Bcp) and its homologues, which comprise a discrete group associated with the peroxiredoxin (Prx) family. Studies of the recombinant 17-kDa protein produced in E. coli cells revealed that it actually had a thioredoxin-dependent peroxidase activity, the hallmark of the Prx family. PrxQ, as we now designate the 17-kDa protein, had two cysteine residues (Cys-44 and Cys-49) well conserved among proteins of the Bcp group. These two cysteines were demonstrated to be essential for the thioredoxin-dependent peroxidase activity by analysis of mutant proteins, suggesting that these residues are involved in the formation of an intramolecular disulphide bond as an intermediate in the reaction cycle. Expression of PrxQ suppressed the hypersensitivity of an E. coli bcp mutant to peroxides, indicating that it might exert an antioxidant activity in vivo. The sequence data presented have been deposited in the GenBank/EMBL/DDBJ nucleotide sequence databases under the accession number AB037598.

scholarly journals The Locus of Enterocyte Effacement-Encoded Effector Proteins All Promote Enterohemorrhagic Escherichia coli Pathogenicity in Infant Rabbits

2005 ◽  
Vol 73 (3) ◽  
pp. 1466-1474 ◽  
Author(s):  
Jennifer M. Ritchie ◽  
Matthew K. Waldor
Keyword(s):  
Escherichia Coli ◽  
Effector Proteins ◽  
Enterohemorrhagic Escherichia Coli ◽  
Lesion Formation ◽  
Specific Effects ◽  
Genes Encoding ◽  
Organ Specific ◽  
Rabbit Intestine ◽  
Enterocyte Effacement

ABSTRACT The genes encoding the enterohemorrhagic Escherichia coli (EHEC) type III secretion system (TTSS) and five effector proteins secreted by the TTSS are located on the locus of enterocyte effacement (LEE) pathogenicity island. Deletion of tir, which encodes one of these effector proteins, results in a profound reduction (∼10,000-fold) in EHEC colonization of the infant rabbit intestine, but the in vivo phenotypes of other LEE genes are unknown. Here, we constructed in-frame deletions in escN, the putative ATPase component of the TTSS, and the genes encoding the four other LEE-encoded effector proteins, EspH, Map, EspF, and EspG, to investigate the contributions of the TTSS and the translocated effector proteins to EHEC pathogenicity in infant rabbits. We found that the TTSS is required for EHEC colonization and attaching and effacing (A/E) lesion formation in the rabbit intestine. Deletion of escN reduced EHEC recovery from the rabbit intestine by ∼10,000-fold. Although EspH, Map, EspF, and EspG were not required for A/E lesion formation in the rabbit intestine or in HeLa cells, these effector proteins promote EHEC colonization. Colonization by the espH and espF mutants was reduced throughout the intestine. In contrast, colonization by the map and espG mutants was reduced only in the small intestine, indicating that Map and EspG have organ-specific effects. EspF appears to down-regulate the host response to EHEC, since we observed increased accumulation of polymorphonuclear leukocytes in the colonic mucosa of rabbits infected with the EHEC espF mutant. Thus, all the known LEE-encoded effector proteins influence EHEC pathogenicity.

scholarly journals Effects of Cordycepin in Cordyceps militaris during Its Infection to Silkworm Larvae

2021 ◽  
Vol 9 (4) ◽  
pp. 681
Author(s):  
Tatsuya Kato ◽  
Konomi Nishimura ◽  
Ahmad Suparmin ◽  
Kazuho Ikeo ◽  
Enoch Y. Park
Keyword(s):  
Cordyceps Militaris ◽  
Silkworm Larvae ◽  
Gene Encoding ◽  
Biosynthesis Gene Cluster ◽  
Expression Of Genes ◽  
Cuticular Proteins ◽  
Genes Encoding ◽  
Fat Bodies ◽  
In Vivo Growth

Cordyceps militaris produces cordycepin, a secondary metabolite that exhibits numerous bioactive properties. However, cordycepin pharmacology in vivo is not yet understood. In this study, the roles of cordycepin in C. militaris during its infection were investigated. After the injection of conidia, C. militaris NBRC100741 killed silkworm larvae more rapidly than NBRC103752. At 96 and 120 h, Cmcns genes (Cmcns1–4), which are part of the cordycepin biosynthesis gene cluster, were expressed in fat bodies and cuticles. Thus, cordycepin may be produced in the infection of silkworm larvae. Further, cordycepin enhanced pathogenicity toward silkworm larvae of Metarhizium anisopliae and Beauveria bassiana, that are also entomopathogenic fungi and do not produce cordycepin. In addition, by RNA-seq analysis, the increased expression of the gene encoding a lipoprotein 30K-8 (Bmlp20, KWMTBOMO11934) and decreased expression of genes encoding cuticular proteins (KWMTBOMO13140, KWMTBOMO13167) and a serine protease inhibitor (serpin29, KWMTBOMO08927) were observed when cordycepin was injected into silkworm larvae. This result suggests that cordycepin may aid the in vivo growth of C. militaris in silkworm larvae by the influence of the expression of some genes in silkworm larvae.

scholarly journals GadY, a Small-RNA Regulator of Acid Response Genes in Escherichia coli

2004 ◽  
Vol 186 (20) ◽  
pp. 6698-6705 ◽  
Author(s):  
Jason A. Opdyke ◽  
Ju-Gyeong Kang ◽  
Gisela Storz
Keyword(s):  
Escherichia Coli ◽  
Stationary Phase ◽  
Small Rna ◽  
Acid Resistance ◽  
Base Pairs ◽  
Gene Encoding ◽  
Mrna Accumulation ◽  
Long Form ◽  
Genes Encoding ◽  
Opposite Strand

ABSTRACT A previous bioinformatics-based search for small RNAs in Escherichia coli identified a novel RNA named IS183. The gene encoding this small RNA is located between and on the opposite strand of genes encoding two transcriptional regulators of the acid response, gadX (yhiX) and gadW (yhiW). Given that IS183 is encoded in the gad gene cluster and because of its role in regulating acid response genes reported here, this RNA has been renamed GadY. We show that GadY exists in three forms, a long form consisting of 105 nucleotides and two processed forms, consisting of 90 and 59 nucleotides. The expression of this small RNA is highly induced during stationary phase in a manner that is dependent on the alternative sigma factor σS. Overexpression of the three GadY RNA forms resulted in increased levels of the mRNA encoding the GadX transcriptional activator, which in turn caused increased levels of the GadA and GadB glutamate decarboxylases. A promoter mutation which abolished gadY expression resulted in a reduction in the amount of gadX mRNA during stationary phase. The gadY gene was shown to overlap the 3′ end of the gadX gene, and this overlap region was found to be necessary for the GadY-dependent accumulation of gadX mRNA. We suggest that during stationary phase, GadY forms base pairs with the 3′-untranslated region of the gadX mRNA and confers increased stability, allowing for gadX mRNA accumulation and the increased expression of downstream acid resistance genes.

scholarly journals The Study of Antibacterial Properties of Anbarnasara Smoke on Multi-Drug Resistant Bacteria Isolated From Urinary Infection in Pregnant Women

2020 ◽  
Vol 8 (4) ◽  
pp. 110-115
Author(s):  
Afsaneh Molamirzaei ◽  
Maryam Allahdadian ◽  
Monir Doudi
Keyword(s):  
Escherichia Coli ◽  
Staphylococcus Aureus ◽  
Pseudomonas Aeruginosa ◽  
Pregnant Women ◽  
Urinary Infection ◽  
Antibacterial Properties ◽  
Resistant Bacteria ◽  
Klebsiella Pneumonia ◽  
Drug Resistant

Background: Using smoke from burning donkey dung has been popular in the treatment of many diseases in Iran. Objective: This study aimed to investigating the antimicrobial properties of donkey dung smoke on multi-drug resistant (MDR) bacteria isolated from urinary infection. Materials and Methods: First, 300 and 200 urine samples were collected from pregnant and non-pregnant women in Isfahan, Iran. Then in each group, 100 bacterial isolates including Escherichia coli, Klebsiella pneumonia, Proteus vulgaris, Staphylococcus epidermidis, Staphylococcus aureus, Pseudomonas aeruginosa, and Staphylococcus saprophyticus were isolated. Antibiotic resistant protocol was determined by antibiogram test. Donkey dung was sterilized, disintegrated, and heated. The smokes were concentrated in n-hexane solvent (65%) and were collected after evaporation of the solvent. Finally, the antibacterial activities of the concentrations of 0.25, 0.5 and 1 mg/mL of the smokes were detected using disk diffusion and macrodilution methods. Results: The most abundant MDR isolates causing urinary infections in pregnant and non-pregnant women was Escherichia coli. The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of donkey dung smoke on MDR isolates from pregnant women were 0.25 mg/mL and 0.5 mg/mL, respectively. In the case of MDR isolates in non-pregnant women, the MIC of the smoke on Escherichia coli, Pseudomonas aeruginosa, and Staphylococcus aureus was 0.25 mg/mL, and the MBC on these isolates was 0.5 mg/mL. Conclusion: The smokes from donkey dung investigated in the present study have suitable potentials for controlling the infections after In vivo analysis.

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