Identification of RNA molecules which contain 5 S ribosomal RNA and transfer RNA in an RNAase E− RNAase P− double mutant strain of Escherichia coli

ABSTRACT Escherichia coli O157:H7 carries a chromosomal msbB1 and a plasmid-encoded msbB2 gene. We characterized msbB2 function as a homologue of msbB1 by examination of wild-type organisms and mutant strains that lacked functional msbB1, msbB2, and both msbB1 and msbB2. The msbB double-mutant strain generated pentaacyl lipid A, while the single-mutant strains synthesized hexaacyl lipid A. Complementation with overexpressed msbB2 converted pentaacyl into hexaacyl lipid A in the double-mutant strain. The transcription of both msbB genes occurred simultaneously. Lack of MsbB2 activity slightly increased the microheterogeneity of the lipid A species. These results suggest that the msbB2 gene plays a role not only in the routine generation of fully hexaacylated lipid A but also in suppressing the microheterogeneity of lipid A species, the endotoxic determinant of the organism.

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Epistatic and Synergistic Interactions Between Circadian Clock Mutations in Neurospora crassa

Genetics ◽

10.1093/genetics/159.2.537 ◽

2001 ◽

Vol 159 (2) ◽

pp. 537-543

Author(s):

Louis W Morgan ◽

Jerry F Feldman

Keyword(s):

Circadian Clock ◽

Neurospora Crassa ◽

Mutant Strain ◽

Temperature Compensation ◽

Double Mutant ◽

Synergistic Interactions ◽

Long Period ◽

Double Mutant Strain ◽

Short Period ◽

Compensation Mechanisms

Abstract We identified a series of epistatic and synergistic interactions among the circadian clock mutations of Neurospora crassa that indicate possible physical interactions among the various clock components encoded by these genes. The period-6 (prd-6) mutation, a short-period temperature-sensitive clock mutation, is epistatic to both the prd-2 and prd-3 mutations. The prd-2 and prd-3 long-period mutations show a synergistic interaction in that the period length of the double mutant strain is considerably longer than predicted. In addition, the prd-2 prd-3 double mutant strain also exhibits overcompensation to changes in ambient temperature, suggesting a role in the temperature compensation machinery of the clock. The prd-2, prd-3, and prd-6 mutations also show significant interactions with the frq7 long-period mutation. These results suggest that the gene products of prd-2, prd-3, and prd-6 play an important role in both the timing and temperature compensation mechanisms of the circadian clock and may interact with the FRQ protein.

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Characterizing KAS Enzymes Using the E. Coli Double Mutant Strain CY244

Advanced Research on Plant Lipids ◽

10.1007/978-94-017-0159-4_14 ◽

2003 ◽

pp. 65-68

Author(s):

Anne Vinther Rasmussen ◽

Rie Yasuno ◽

Penny von Wettstein-Knowles

Keyword(s):

Mutant Strain ◽

Double Mutant ◽

E Coli ◽

Double Mutant Strain

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Rate of chain elongation of ribosomal RNA molecules in Escherichia coli

Journal of Molecular Biology ◽

10.1016/0022-2836(74)90084-9 ◽

1974 ◽

Vol 87 (4) ◽

pp. 775-795 ◽

Cited By ~ 8

Author(s):

Alfred H. Dougan ◽

Donald A. Glaser

Keyword(s):

Escherichia Coli ◽

Ribosomal Rna ◽

Chain Elongation ◽

Rna Molecules

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Evaluation of the effects of sdiA, a luxR homologue, on adherence and motility of Escherichia coli O157 : H7

Microbiology ◽

10.1099/mic.0.034330-0 ◽

2010 ◽

Vol 156 (5) ◽

pp. 1303-1312 ◽

Cited By ~ 37

Author(s):

Vijay K. Sharma ◽

Shawn M. D. Bearson ◽

Bradley L. Bearson

Keyword(s):

Escherichia Coli ◽

Epithelial Cells ◽

Mutant Strain ◽

Regulatory Networks ◽

Double Mutant ◽

Negative Regulator ◽

Wild Type ◽

Reverse Transcription Pcr ◽

Expression Of Genes ◽

Genes Encoding

Quorum-sensing (QS) signalling pathways are important regulatory networks for controlling the expression of genes promoting adherence of enterohaemorrhagic Escherichia coli (EHEC) O157 : H7 to epithelial cells. A recent study has shown that EHEC O157 : H7 encodes a luxR homologue, called sdiA, which upon overexpression reduces the expression of genes encoding flagellar and locus of enterocyte effacement (LEE) proteins, thus negatively impacting on the motility and intimate adherence phenotypes, respectively. Here, we show that the deletion of sdiA from EHEC O157 : H7 strain 86-24, and from a hha (a negative regulator of ler) mutant of this strain, enhanced bacterial adherence to HEp-2 epithelial cells of the sdiA mutant strains relative to the strains containing a wild-type copy of sdiA. Quantitative reverse transcription PCR showed that the expression of LEE-encoded genes ler, espA and eae in strains with the sdiA deletions was not significantly different from that of the strains wild-type for sdiA. Similarly, no additional increases in the expression of LEE genes were observed in a sdiA hha double mutant strain relative to that observed in the hha deletion mutant. While the expression of fliC, which encodes flagellin, was enhanced in the sdiA mutant strain, the expression of fliC was reduced by several fold in the hha mutant strain, irrespective of the presence or absence of sdiA, indicating that the genes sdiA and hha exert opposing effects on the expression of fliC. The strains with deletions in sdiA or hha showed enhanced expression of csgA, encoding curlin of the curli fimbriae, with the expression of csgA highest in the sdiA hha double mutant, suggesting an additive effect of these two gene deletions on the expression of csgA. No significant differences were observed in the expression of the genes lpfA and fimA of the operons encoding long polar and type 1 fimbriae in the sdiA mutant strain. These data indicate that SdiA has no significant effect on the expression of LEE genes, but that it appears to act as a strong repressor of genes encoding flagella and curli fimbriae, and the alleviation of the SdiA-mediated repression of these genes in an EHEC O157 : H7 sdiA mutant strain contributes to enhanced bacterial motility and increased adherence to HEp-2 epithelial cells.

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Lack of the Bacterial Phytochrome Protein Decreases Deinococcus radiodurans Resistance to Mitomycin C

Frontiers in Microbiology ◽

10.3389/fmicb.2021.659233 ◽

2021 ◽

Vol 12 ◽

Author(s):

Jong-Hyun Jung ◽

Soyoung Jeong ◽

Seonghun Im ◽

Min-Kyu Kim ◽

Ho Seong Seo ◽

...

Keyword(s):

Dna Damage ◽

Cell Survival ◽

Mitomycin C ◽

Mutant Strain ◽

Double Mutant ◽

Deinococcus Radiodurans ◽

Red Light ◽

Double Mutant Strain ◽

Significant Difference

Deinococcus radiodurans known for its extraordinary resistance to ionizing radiation contains bacterial phytochrome (BphP), a member of the family of red/far-red light-sensing proteins. In this study, we constructed a bphP mutant strain (ΔbphP) to investigate the role of D. radiodurans BphP (DrBphP) in the DNA damage response. When cells were incubated under light and dark conditions following exposure to DNA damaging agents, such as γ- and UV-radiation and mitomycin C (MMC), no significant difference in cell survival was observed between the wild-type D. radiodurans strain (WT) and ΔbphP. However, when continuously exposed to MMC under light conditions, the WT strain notably exhibited increased survival compared to cells grown in the dark. The increased survival was not observed in the ΔbphP strain. These results are indicative of the protective role of light-activated DrBphP in the presence of MMC. Site-directed mutagenesis revealed that the conserved amino acids Cys-24 and His-532 involved in chromophore binding and signal transduction, respectively, were essential for the protective function of DrBphP. Inactivation of the cognate response regulator (RR; DrBphR) of DrBphP increased MMC resistance in the dark. In trans complementation of the bphP bphR double mutant strain (ΔbphPR) with DrBphR decreased MMC resistance. Considering that DrBphP acts as a light-activated phosphatase that dephosphorylates DrBphR, it appears that phosphorylated DrBphR exerts a negative effect on cell survival in the presence of MMC. DrBphP overexpression resulted in an increase in MMC resistance of ΔbphPR, implying that other RRs might be involved in the DrBphP-mediated signaling pathway. A mutant lacking the dr_0781 gene (Δdr_0781) demonstrated the same MMC phenotype as ΔbphR. Survival was further increased in the bphR dr_0781 double mutant strain compared to each single mutant ΔbphR or Δdr_0781, suggesting that DR_0781 is also involved in the DrBphP-dependent MMC sensitivity. This study uncovered a previously unknown phenomenon of red/far-red light-dependent DNA damage survival mediated by BphP by identifying the conditions under which DrBphP exhibits a fitness advantage.

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Role of Hsl7 in Morphology and Pathogenicity and Its Interaction with Other Signaling Components in the Plant Pathogen Ustilago maydis

Eukaryotic Cell ◽

10.1128/ec.00237-10 ◽

2011 ◽

Vol 10 (7) ◽

pp. 869-883 ◽

Cited By ~ 4

Author(s):

C. Ben Lovely ◽

Kavita Burman Aulakh ◽

Michael H. Perlin

Keyword(s):

Ammonium Sulfate ◽

Mutant Strain ◽

Double Mutant ◽

Ustilago Maydis ◽

Cell Length ◽

Phytopathogenic Fungus ◽

Nitrogen Base ◽

Content Type ◽

Dimorphic Transition ◽

Double Mutant Strain

ABSTRACTThe phytopathogenic fungusUstilago maydisundergoes a dimorphic transition in response to mating pheromone, host, and environmental cues. On a solid medium deficient in ammonium (SLAD [0.17% yeast nitrogen base without ammonium sulfate or amino acids, 2% dextrose, 50 μM ammonium sulfate]),U. maydisproduces a filamentous colony morphology, while in liquid SLAD, the cells do not form filaments. The p21-activated protein kinases (PAKs) play a substantial role in regulating the dimorphic transition in fungi. The PAK-like Ste20 homologue Smu1 is required for a normal response to pheromone, via upregulation of pheromone expression, and virulence, and its disruption affects both processes. Our experiments suggest that Smu1 also regulates cell length and the filamentous response on solid SLAD medium. Yeast two-hybrid analysis suggested an Hsl7 homologue as a potential interacting partner of Smu1, and a unique open reading frame for such an arginine methyltransferase was detected in theU. maydisgenome sequence. Hsl7 regulates cell length and the filamentous response to solid SLAD in a fashion opposite to that of Smu1, but neither overexpression nor disruption ofhsl7attenuates virulence. Simultaneous disruption ofhsl7and overexpression ofsmu1lead to a hyperfilamentous response on solid SLAD. Moreover, only this double mutant strain forms filaments in liquid SLAD. The double mutant strain was also significantly reduced in virulence. A similar filamentous response in both solid and liquid SLAD was observed in strains lacking another PAK-like protein kinase involved in cytokinesis and polar growth, Cla4. Our data suggest that Hsl7 may regulate cell cycle progression, while both Smu1 and Cla4 appear to be involved in the filamentous response inU. maydis.

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Concurrent modifications of the C-terminus and side ring of thiostrepton and their synergistic effects with respect to improving antibacterial activities

Organic Chemistry Frontiers ◽

10.1039/c5qo00433k ◽

2016 ◽

Vol 3 (4) ◽

pp. 496-500 ◽

Cited By ~ 11

Author(s):

Shoufeng Wang ◽

Qingfei Zheng ◽

Jianfeng Wang ◽

Dandan Chen ◽

Yunsong Yu ◽

...

Keyword(s):

Mutant Strain ◽

Double Mutant ◽

Synergistic Effects ◽

Antibacterial Activities ◽

Ring Structure ◽

Quinaldic Acid ◽

C Terminus ◽

Double Mutant Strain

Five new C-terminally methylated TSR derivatives that varied in side-ring structure were obtained via the chemical feeding of quinaldic acid analogs to a double-mutant strain ΔtsrB/T.

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Mycobacterium tuberculosis CwsA Interacts with CrgA and Wag31, and the CrgA-CwsA Complex Is Involved in Peptidoglycan Synthesis and Cell Shape Determination

Journal of Bacteriology ◽

10.1128/jb.01005-12 ◽

2012 ◽

Vol 194 (23) ◽

pp. 6398-6409 ◽

Cited By ~ 45

Author(s):

P. Plocinski ◽

N. Arora ◽

K. Sarva ◽

E. Blaszczyk ◽

H. Qin ◽

...

Keyword(s):

Cell Wall ◽

Mycobacterium Tuberculosis ◽

Mutant Strain ◽

Cell Shape ◽

Double Mutant ◽

Bacterial Cell Division ◽

Cell Wall Synthesis ◽

Content Type ◽

Ring Assembly ◽

Double Mutant Strain

ABSTRACTBacterial cell division and cell wall synthesis are highly coordinated processes involving multiple proteins. Here, we show that Rv0008c, a novel small membrane protein fromMycobacterium tuberculosis, localizes to the poles and on membranes and shows an overall punctate localization throughout the cell. Furthermore, Rv0008c interacts with two proteins, CrgA and Wag31, implicated in peptidoglycan (PG) synthesis in mycobacteria. Deletion of the Rv0008c homolog inM. smegmatis, MSMEG_0023, caused bulged cell poles, formation of rounded cells, and defects in polar localization of Wag31 and cell wall synthesis, with cell wall synthesis measured by the incorporation of the [14C]N-acetylglucosamine cell wall precursor. TheM. smegmatisMSMEG_0023crgAdouble mutant strain showed severe defects in growth, viability, cell wall synthesis, cell shape, and the localization of the FtsZ, FtsI, and Wag31 proteins. The double mutant strain also exhibited increased autolytic activity in the presence of detergents. Because CrgA and Wag31 proteins interact with FtsI individually, we believe that regulated cell wall synthesis and cell shape maintenance require the concerted actions of the CrgA, Rv0008c, FtsI, and Wag31 proteins. We propose that, together, CrgA and Rv0008c, renamed CwsA forcellwall synthesis and cellshape proteinA, play crucial roles in septal and polar PG synthesis and help coordinate these processes with the FtsZ-ring assembly in mycobacteria.

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The control of ribonucleic acid synthesis in bacteria. Steady-state content of messenger ribonucleic acid in Escherichia coli M.R.E. 600

Biochemical Journal ◽

10.1042/bj1200279 ◽

1970 ◽

Vol 120 (2) ◽

pp. 279-288 ◽

Cited By ~ 9

Author(s):

W. J. H. Gray ◽

J. E. M. Midgley

Keyword(s):

Escherichia Coli ◽

Ribosomal Rna ◽

Ribonucleic Acid ◽

Messenger Rna ◽

Acid Synthesis ◽

Growth Conditions ◽

Transfer Rna ◽

Average Lifetime ◽

Messenger Ribonucleic Acid ◽

Wide Range

1. The technique of DNA–RNA hybridization was used to follow changes in the amount and average lifetime of unstable messenger RNA in Escherichia coli M.R.E. 600 over a wide range of different growth conditions. The method of analysis was based on the kinetics of incorporation of exogenous labelled nucleic acid bases into the RNA of steadily growing cultures, as described by Bolton & McCarthy (1962). 2. The ratio of the average lifetime of messenger RNA to the mean generation time of E. coli cultures was constant over the temperature range 25–45°C in a given medium, but the constant varied with the nature of the growth medium. For cultures growing in sodium lactate–salts or glucose–salts media the ratio was 0.046±0.005 and in enriched broth it was 0.087±0.009. Measurements of the amounts of transfer RNA, ribosomal RNA and messenger RNA were also made. The results confirmed earlier reports that the ratio of the amount of messenger RNA to the amount of ribosomes in the cells is virtually constant. On the other hand, the ratio of the amount of transfer RNA to the amount of ribosomal RNA decreased with increasing growth rate at a given temperature. 3. In cultures at temperatures higher than necessary for optimum rates of growth the average lifetime of messenger RNA lengthened in harmony with the increased time required for cell division. It seems that suboptimum growth rates at higher temperatures cannot be explained simply as a combination of increased rates of synthesis and breakdown of messenger RNA with a grossly decreased efficiency of translation. The absolute rate of messenger RNA synthesis was lowered, and its amount in the cells was typical of all other cultures grown at lower temperatures in the same medium. 4. The rate of entry of exogenous labelled uracil into unstable messenger RNA and stable ribosomal RNA was constant in all media at all temperatures in the approximate ratio 1:2. In media supporting a lower rate of growth, e.g. lactate–salts or glucose–salts media, the messenger RNA fraction constituted 2.2±0.3% of the total cellular RNA. In enriched broth 3.6±0.3% of the total RNA was messenger.

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