scholarly journals ESCHERICHIA COLI RHO FACTOR IS INVOLVED IN LYSIS OF BACTERIOPHAGE T4-INFECTED CELLS

Genetics ◽  
1985 ◽  
Vol 111 (2) ◽  
pp. 197-218
Author(s):  
Claës H Linder ◽  
Karin Carlson
Keyword(s):  
Bacteriophage T4 ◽  
Growth Conditions ◽  
Plaque Formation ◽  
Early Gene ◽  
Multicopy Plasmid ◽  
Lysis Inhibition ◽  
Cold Sensitive ◽  
Phage Production ◽  
Hydroxylamine Mutagenesis ◽  
High Level

ABSTRACT A Rid (Rho interaction deficient) phenotype of bacteriophage T4 mutants was defined by cold-sensitive restriction (lack of plaque formation) on rho  + hosts carrying additional polar mutations in unrelated genes, coupled to suppression (plaque formation) in otherwise isogenic strains carrying either a polarity-suppressing rho or a multicopy plasmid expressing the rho  + allele. This suggests that the restriction may be due to lower levels of Rho than what is available to T4 in the suppressing strains.—Rid394×4 was isolated upon hydroxylamine mutagenesis and mapped in the t gene; other t mutants (and mot, as well as dda dexA double mutants) also showed a Rid phenotype. In liquid culture in strains that restricted plaque formation Rid394×4 showed strong lysis inhibition (a known t  - phenotype) but no prolonged phage production (another well-known t  - phenotype). This implies that when Rho is limiting the t mutant shuts off phage production at the normal time. Lysis inhibition was partially relieved, and phage production prolonged to varying extents depending on growth conditions in strains that allowed plaque formation. No significant effects on early gene expression were found. Apparently, both mutant (polarity-suppressing) and wild-type Rho can function in prolonging phage production and partially relieving lysis inhibition of Rid394×4 when present at a sufficiently high level, and Rho may play other role(s) in T4 development than in early gene regulation.

scholarly journals Overexpression of the Bacteriophage T4 motB Gene Alters H-NS Dependent Repression of Specific Host DNA

Viruses ◽  
2021 ◽  
Vol 13 (1) ◽  
pp. 84
Author(s):  
Jennifer Patterson-West ◽  
Chin-Hsien Tai ◽  
Bokyung Son ◽  
Meng-Lun Hsieh ◽  
James R. Iben ◽  
...  
Keyword(s):  
Bacteriophage T4 ◽  
Early Gene ◽  
Dna Binding Domain ◽  
Host Gene Expression ◽  
Rna Seq ◽  
Specific Host ◽  
Dnase I Footprinting ◽  
Ob Fold ◽  
Host Genes

The bacteriophage T4 early gene product MotB binds tightly but nonspecifically to DNA, copurifies with the host Nucleoid Associated Protein (NAP) H-NS in the presence of DNA and improves T4 fitness. However, the T4 transcriptome is not significantly affected by a motB knockdown. Here we have investigated the phylogeny of MotB and its predicted domains, how MotB and H-NS together interact with DNA, and how heterologous overexpression of motB impacts host gene expression. We find that motB is highly conserved among Tevenvirinae. Although the MotB sequence has no homology to proteins of known function, predicted structure homology searches suggest that MotB is composed of an N-terminal Kyprides-Onzonis-Woese (KOW) motif and a C-terminal DNA-binding domain of oligonucleotide/oligosaccharide (OB)-fold; either of which could provide MotB’s ability to bind DNA. DNase I footprinting demonstrates that MotB dramatically alters the interaction of H-NS with DNA in vitro. RNA-seq analyses indicate that expression of plasmid-borne motB up-regulates 75 host genes; no host genes are down-regulated. Approximately 1/3 of the up-regulated genes have previously been shown to be part of the H-NS regulon. Our results indicate that MotB provides a conserved function for Tevenvirinae and suggest a model in which MotB functions to alter the host transcriptome, possibly by changing the association of H-NS with the host DNA, which then leads to conditions that are more favorable for infection.

scholarly journals Glucose Signaling Pathway and Growth Conditions Regulate Gene Expression in Retrotransposon Ty2

2009 ◽  
Vol 64 (7-8) ◽  
pp. 526-532 ◽  
Author(s):  
Sezai Türkel ◽  
Özgür Bayram ◽  
Elif Arık
Keyword(s):  
Gene Expression ◽  
Growth Conditions ◽  
Glucose Sensors ◽  
Yeast Cells ◽  
Growth Stages ◽  
Regulate Gene Expression ◽  
Glucose Signaling ◽  
Yeast Cultures ◽  
Membrane Bound ◽  
High Level

Gene expression in the yeast retrotransposon Ty2 is regulated at transcriptional and translational levels. In this study, we have shown that the transcription of Ty2 is partially dependent on the membrane-bound glucose sensors Gpr1p and Mth1p in Saccharomyces cerevisiae. Transcription of Ty2 decreased approx. 3-fold in the gpr1, mth1 yeast mutant. Moreover, our results revealed that the transcription of Ty2 fluctuates during the growth stages of S. cerevisae. Both transcription and the frameshift rate of Ty2 rapidly dropped when the stationary stage yeast cells were inoculated into fresh medium. There was an instant activation of Ty2 transcription and a high level expression during the entire logarithmic stage of yeast growth. However, the transcription of Ty2 decreased 2-fold when the yeast cultures entered the stationary stage. The frameshift rate in Ty2 also varied depending on the growth conditions. The highest frameshift level was observed during the mid-logarithmic stage. It decreased up to 2-fold during the stationary stage. Furthermore, we have found that the frameshift rate of Ty2 diminished at least 5-fold in slowly growing yeasts. These results indicate that the transcription and the frameshift efficiency are coordinately regulated in the retrotransposon Ty2 depending on the growth conditions of S. cerevisiae.

scholarly journals The strong efficiency of the Escherichia coli gapA P1 promoter depends on a complex combination of functional determinants

2004 ◽  
Vol 383 (2) ◽  
pp. 371-382 ◽  
Author(s):  
Benoit THOUVENOT ◽  
Bruno CHARPENTIER ◽  
Christiane BRANLANT
Keyword(s):  
Escherichia Coli ◽  
Growth Conditions ◽  
Site Directed Mutagenesis ◽  
Dna Interactions ◽  
Template Strand ◽  
Chemical Probing ◽  
Upstream Promoter ◽  
Promoter Dna ◽  
High Level ◽  
Protein Pocket

The Escherichia coli multi-promoter region of the gapA gene ensures a high level of GAPDH (glyceraldehyde-3-phosphate dehydrogenase) production under various growth conditions. In the exponential phase of growth, gapA mRNAs are mainly initiated at the highly efficient gapA P1 promoter. In the present study, by using site-directed mutagenesis and chemical probing of the RPo (open complex) formed by Eσ70 (holoenzyme associated with σ70) RNAP (RNA polymerase) at promoter gapA P1, we show that this promoter is an extended −10 promoter that needs a −35 sequence for activity. The −35 sequence compensates for the presence of a suboptimal −10 hexamer. A tract of thymine residues in the spacer region, which is responsible for a DNA distortion, is also required for efficient activity. We present the first chemical probing of an RPo formed at a promoter needing both a −10 extension and a −35 sequence. It reveals a complex array of RNAP–DNA interactions. In agreement with the fact that residue A-11 in the non-template strand is flipped out in a protein pocket in previously studied RPos, the corresponding A residue in gapA P1 promoter is protected in RPo and is essential for activity. However, in contrast with some of the previous findings on RPos formed at other promoters, the −12 A:T pair is opened. Strong contacts with RNAP occur both with the −35 sequence and the TG extension, so that the σ4 and σ2 domains may simultaneously contact the promoter DNA. RNAP–DNA interactions were also detected immediately downstream of the −35 hexamer and in a more distal upstream segment, reflecting a wrapping of RNAP by the core and upstream promoter DNA. Altogether, the data reveal that promoter gapA P1 is a very efficient promoter sharing common properties with both extended −10 and non-extended −10 promoters.

scholarly journals Evidence of positive regulation of mycobacteriophage D29 early gene expression obtained from an investigation using a temperature-sensitive mutant of the phage

2020 ◽  
Vol 367 (21) ◽  
Author(s):  
Shrestha Ghosh ◽  
Rahul Shaw ◽  
Apurba Sarkar ◽  
Sujoy K Das Gupta
Keyword(s):  
Gene Expression ◽  
Proteome Analysis ◽  
Early Gene ◽  
Specific Factor ◽  
Temperature Sensitive ◽  
Expression Of Genes ◽  
Infected Cells ◽  
Ts Mutant ◽  
The Right ◽  
High Level

ABSTRACT Mycobacteriophages are phages that infect and kill Mycobacteria, several of which, Mycobacterium tuberculosis (Mtb), for example, cause the disease tuberculosis. Although genomes of many such phages have been sequenced, we have very little insight into how they express their genes in a controlled manner. To address this issue, we have raised a temperature-sensitive (ts) mutant of phage D29 that can grow at 37°C but not at 42°C and used it to perform differential gene expression and proteome analysis studies. Our analysis results indicate that expression of genes located in the right arm, considered to be early expressed, was lowered as the temperature was shifted from 37°C to 42°C. In contrast, expression of those on the left, the late genes were only marginally affected. Thus, we conclude that transcription of genes from the two arms takes place independently of each other and that a specific factor must be controlling the expression of the right arm genes. We also observe that within the right arm itself; there exists a mechanism to ensure high-level synthesis of Gp48, a thymidylate synthase X. Enhanced presence of this protein in infected cells results in delayed lysis and higher phage yields.

scholarly journals Crystal structure of bacteriophage T4 Spackle as determined by native SAD phasing

2020 ◽  
Vol 76 (9) ◽  
pp. 899-904
Author(s):  
Ke Shi ◽  
Fredy Kurniawan ◽  
Surajit Banerjee ◽  
Nicholas H. Moeller ◽  
Hideki Aihara
Keyword(s):  
Crystal Structure ◽  
Bacteriophage T4 ◽  
Early Gene ◽  
Size Exclusion ◽  
Amino Terminal ◽  
Intramolecular Disulfide Bond ◽  
Exclusion Chromatography ◽  
T4 Bacteriophage ◽  
Sad Phasing ◽  
T4 Phage

The crystal structure of a bacteriophage T4 early gene product, Spackle, was determined by native sulfur single-wavelength anomalous diffraction (SAD) phasing using synchrotron radiation and was refined to 1.52 Å resolution. The structure shows that Spackle consists of a bundle of five α-helices, forming a relatively flat disc-like overall shape. Although Spackle forms a dimer in the crystal, size-exclusion chromatography with multi-angle light scattering shows that it is monomeric in solution. Mass spectrometry confirms that purified mature Spackle lacks the amino-terminal signal peptide and contains an intramolecular disulfide bond, consistent with its proposed role in the periplasm of T4 phage-infected Escherichia coli cells. The surface electrostatic potential of Spackle shows a strikingly bipolar charge distribution, suggesting a possible mode of membrane association and inhibition of the tail lysozyme activity in T4 bacteriophage superinfection exclusion.

scholarly journals Multicopy Integration of Heterologous Genes, Using the Lactococcal Group II Intron Targeted to Bacterial Insertion Sequences

2006 ◽  
Vol 72 (9) ◽  
pp. 6088-6093 ◽  
Author(s):  
Helen Rawsthorne ◽  
Kevin N. Turner ◽  
David A. Mills
Keyword(s):  
Fluorescent Protein ◽  
Group Ii Intron ◽  
Multicopy Plasmid ◽  
Bacterial Genomes ◽  
Gfp Expression ◽  
High Frequencies ◽  
Multicopy Integration ◽  
Group Ii ◽  
Green Fluorescent ◽  
High Level

ABSTRACT Group II introns are mobile genetic elements that can be redirected to invade specific genes. Here we describe the use of the lactococcal group II intron, Ll.ltrB, to achieve multicopy delivery of heterologous genes into the genome of Lactococcus lactis IL1403-UCD without the need for selectable markers. Ll.ltrB was retargeted to invade three transposase genes, the tra gene found in IS904 (tra904), tra981, and tra983, of which 9, 10, and 14 copies, respectively, were present in IL1403-UCD. Intron invasion of tra904, tra981, and tra983 allele groups occurred at high frequencies, and individual segregants possessed anywhere from one to nine copies of intron in the respective tra alleles. To achieve multicopy delivery of a heterologous gene, a green fluorescent protein (GFP) marker was cloned into the tra904-targeted Ll.ltrB, and the resultant intron (Ll.ltrB::GFP) was induced to invade the L. lactis tra904 alleles. Segregants possessing Ll.ltrB::GFP in three, four, five, six, seven, and eight copies in different tra904 alleles were obtained. In general, increasing the chromosomal copy number of Ll.ltrB::GFP resulted in strains expressing successively higher levels of GFP. However, strains possessing the same number of Ll.ltrB::GFP copies within different sets of tra904 alleles exhibited differential GFP expression, and segregants possessing seven or eight copies of Ll.ltrB::GFP grew poorly upon induction, suggesting that GFP expression from certain combinations of alleles was detrimental. The highest level of GFP expression was observed from a specific six-copy variant that produced GFP at a level analogous to that obtained with a multicopy plasmid. In addition, the high level of GFP expression was stable for over 120 generations. This work demonstrates that stable multicopy integration of heterologous genes can be readily achieved in bacterial genomes with group II intron delivery by targeting repeated elements.

scholarly journals A Vibrio cholerae LysR Homolog, AphB, Cooperates with AphA at the tcpPH Promoter To Activate Expression of the ToxR Virulence Cascade

1999 ◽  
Vol 181 (14) ◽  
pp. 4250-4256 ◽  
Author(s):  
Gabriela Kovacikova ◽  
Karen Skorupski
Keyword(s):  
Vibrio Cholerae ◽  
Virulence Genes ◽  
Virulence Gene ◽  
Growth Conditions ◽  
Culture Conditions ◽  
Virulence Gene Expression ◽  
Environmental Stimuli ◽  
High Level ◽  
El Tor

ABSTRACT We describe here a new member of the LysR family of transcriptional regulators, AphB, which is required for activation of the Vibrio cholerae ToxR virulence cascade. AphB activates the transcription of the tcpPH operon in response to environmental stimuli, and this process requires cooperation with a second protein, AphA. The expression of neither aphA or aphB is strongly regulated by environmental stimuli, raising the possibility that the activities of the proteins themselves may be influenced under various conditions. Strains of the El Tor biotype of V. choleraetypically exhibit lower expression of ToxR-regulated virulence genes in vitro than classical strains and require specialized culture conditions (AKI medium) to induce high-level expression. We show here that expression of aphB from the tac promoter in El Tor biotype strains dramatically increases virulence gene expression to levels similar to those observed in classical strains under all growth conditions examined. These results suggest that AphB plays a role in the differential regulation of virulence genes between the two disease-causing biotypes.

scholarly journals Poaceae Orthologs of Rice OsSGL, DUF1645 Domain-Containing Genes, Positively Regulate Drought Tolerance, Grain Length and Weight in Rice

2020 ◽  
Author(s):  
Kai Liu ◽  
Mingjuan Li ◽  
Bin Zhang ◽  
Yanchun Cui ◽  
Xuming Yin ◽  
...  
Keyword(s):  
Drought Stress ◽  
Stress Tolerance ◽  
Molecular Mechanisms ◽  
Normal Growth ◽  
Growth Conditions ◽  
Growth Stages ◽  
Grain Length ◽  
Drought Stress Tolerance ◽  
High Level ◽  
Seed Setting Percentage

Abstract BackgroundGrain yield is a polygenic trait influenced by environmental and genetic interactions at all growth stages of the cereal plant. However, the molecular mechanisms responsible for coordinating the trade-off or cross-talk between these traits remain elusive.ResultsWe characterized the hitherto unknown function of four STRESS_tolerance and GRAIN_LENGTH (OsSGL) Poaceae ortholog genes, all encoding DUF1645 domain-containing proteins, in simultaneous regulation of grain length, grain weight, and drought stress-tolerance in rice. In normal growth conditions, the four ortholog genes were mainly expressed in the developing roots and panicles of the corresponding species. Over-expressing or heterologous high-level expressing Poaceae OsSGL ortholog genes conferred remarkably increased grain length, weight, and seed setting percentage, as well as significantly improved drought-stress tolerance in transgenic rice. Microscopical analysis also showed that the transgene expression promoted cell division and development. RNA-seq and qRT-PCR analyses revealed 73.8% (18,711) overlapped DEGs in all transgenic plants. Moreover, GO and KEGG analyses of different comparisons revealed that the key DEGs participating in drought stress-response belonged to hormone (especially auxin and cytokinin) pathways, and signaling processes were apparently affected in the young panicles. ConclusionTogether, these results suggest the four OsSGL orthologs perform a conserved function in regulating stress-tolerance and cell growth by acting via a hormone biosynthesis and signaling pathway. It may also induce a strategy for tailor-made crop yield improvement.

scholarly journals Escherichia coli RcsA, a Positive Activator of Colanic Acid Capsular Polysaccharide Synthesis, Functions To Activate Its Own Expression

1999 ◽  
Vol 181 (2) ◽  
pp. 577-584 ◽  
Author(s):  
Wolfgang Ebel ◽  
Janine E. Trempy
Keyword(s):  
Escherichia Coli ◽  
Capsular Polysaccharide ◽  
Sequence Motif ◽  
Multicopy Plasmid ◽  
Transcriptional Fusion ◽  
High Level Expression ◽  
E Coli ◽  
Polysaccharide Synthesis ◽  
Positive Regulators ◽  
High Level

ABSTRACT Capsule (cps) gene expression in Escherichia coli is controlled by a complex network of regulators. Transcription of the cps operon is controlled by at least two positive regulators, RcsA and RcsB. We show here that RcsA functions to activate its own expression, as seen by the 100-fold-increased expression of arcsA::lacZ transcriptional fusion in strains with high levels of RcsA protein, either due to a mutation inlon or due to overexpression of RcsA from a multicopy plasmid. Expression of the rcsA::lacZfusion is increased by but not dependent on the presence of RcsB. In addition, the effects of H-NS and RcsB on the expression ofrcsA are independent of each other. A sequence motif, conserved between the E. coli cps promoter and theErwinia amylovora ams promoter and previously shown to be the RcsA-RcsB binding site, was identified in the rcsApromoter region and shown to be required for high-level expression ofrcsA.

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