Identification of a Cupin Protein Gene Responsible for Pathogenicity, Phage Susceptibility and LPS Synthesis of Acidovorax citrulli

Bacteriophages infecting Acidovorax citrulli, the causal agent of bacterial fruit blotch, have been proven to be effective for the prevention and control of this disease. However, the occurrence of bacteriophage-resistant bacteria is one of hurdles in phage biocontrol and the understanding of phage resistance in this bacterium is an essential step. In this study, we aim to investigate possible phage resistance of A. citrulli and relationship between phage resistance and pathogenicity, and to isolate and characterize the genes involved in these phenomena. A phage-resistant and less-virulent mutant named as AC-17-G1 was isolated among 3,264 A. citrulli Tn5 mutants through serial spot assays and plaque assays followed by pathogenicity test using seed coating method. The mutant has the integrated Tn5 in the middle of a cupin protein gene. This mutant recovered its pathogenicity and phage sensitivity by complementation with corresponding wild-type gene. Site-directed mutation of this gene from wild-type by CRISPR/Cas9 system resulted in the loss of pathogenicity and acquisition of phage resistance. The growth of AC-17-G1 in King’s B medium was much less than the wild-type, but the growth turned into normal in the medium supplemented with D-mannose 6-phosphate or D-fructose 6-phosphate indicating the cupin protein functions as a phosphomannos isomerase. Sodium dodecyl sulfa analysis of lipopolysaccharide (LPS) extracted from the mutant was smaller than that from wild-type. All these data suggest that the cupin protein is a phosphomannos isomerase involved in LPS synthesis, and LPS is an important determinant of pathogenicity and phage susceptibility of A. citrulli.

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The Budding Yeast Msh4 Protein Functions in Chromosome Synapsis and the Regulation of Crossover Distribution

Genetics ◽

10.1093/genetics/158.3.1013 ◽

2001 ◽

Vol 158 (3) ◽

pp. 1013-1025 ◽

Cited By ~ 7

Author(s):

Janet E Novak ◽

Petra B Ross-Macdonald ◽

G Shirleen Roeder

Keyword(s):

Mismatch Repair ◽

Budding Yeast ◽

Null Mutation ◽

Crossing Over ◽

Wild Type ◽

Crossover Interference ◽

Meiotic Chromosomes ◽

Chromosome Synapsis ◽

Protein Functions ◽

Or Gene

AbstractThe budding yeast MSH4 gene encodes a MutS homolog produced specifically in meiotic cells. Msh4 is not required for meiotic mismatch repair or gene conversion, but it is required for wild-type levels of crossing over. Here, we show that a msh4 null mutation substantially decreases crossover interference. With respect to the defect in interference and the level of crossing over, msh4 is similar to the zip1 mutant, which lacks a structural component of the synaptonemal complex (SC). Furthermore, epistasis tests indicate that msh4 and zip1 affect the same subset of meiotic crossovers. In the msh4 mutant, SC formation is delayed compared to wild type, and full synapsis is achieved in only about half of all nuclei. The simultaneous defects in synapsis and interference observed in msh4 (and also zip1 and ndj1/tam1) suggest a role for the SC in mediating interference. The Msh4 protein localizes to discrete foci on meiotic chromosomes and colocalizes with Zip2, a protein involved in the initiation of chromosome synapsis. Both Zip2 and Zip1 are required for the normal localization of Msh4 to chromosomes, raising the possibility that the zip1 and zip2 defects in crossing over are indirect, resulting from the failure to localize Msh4 properly.

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C/EBPα Is Critical for the Neonatal Acute-Phase Response to Inflammation

Molecular and Cellular Biology ◽

10.1128/mcb.18.12.7269 ◽

1998 ◽

Vol 18 (12) ◽

pp. 7269-7277 ◽

Cited By ~ 52

Author(s):

Bonnie L. Burgess-Beusse ◽

Gretchen J. Darlington

Keyword(s):

Dna Binding ◽

Acute Phase ◽

Acute Phase Response ◽

Northern Blot Analysis ◽

Protein Gene ◽

Phase Response ◽

Wild Type ◽

Neonatal Mice ◽

Enhancer Binding Protein

ABSTRACT Members of the C/EBP (CCAAT/enhancer binding protein) family of transcription factors play important roles in mediating the acute-phase response (APR), an inflammatory process resulting from infection and/or tissue damage. Among the C/EBP family of proteins, C/EBPβ and -δ were thought to be the primary mediators of the APR. The function of C/EBPα in the APR has not been fully characterized to date. Here, we investigate the role of C/EBPα in the APR by using neonatal mice that lack C/EBPα expression. Northern blot analysis of acute-phase protein gene expression in neonatal mice treated with purified bacterial lipopolysaccharide or recombinant interleukin 1β as an inflammation stimulus showed a strong APR in wild-type mice, but a response in C/EBPα null animals was completely lacking. The C/EBPα knockout and wild-type mice demonstrated elevations in C/EBPβ and -δ mRNA expression and DNA binding as well as increased DNA binding of NF-κB, all of which are known to be important in the APR. Null mice, however, failed to activate STAT3 binding in response to lipopolysaccharide. Our results provide the first evidence that C/EBPα is absolutely required for the APR in neonatal mice, is involved in STAT3 regulation, and cannot be compensated for by other C/EBP family members.

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The Aberrant Gene-End Transcription Signal of the Matrix M Gene of Human Parainfluenza Virus Type 3 Downregulates Fusion F Protein Expression and the F-Specific Antibody ResponseIn Vivo

Journal of Virology ◽

10.1128/jvi.03148-14 ◽

2015 ◽

Vol 89 (6) ◽

pp. 3318-3331 ◽

Cited By ~ 2

Author(s):

Matthias Lingemann ◽

Sonja Surman ◽

Emérito Amaro-Carambot ◽

Anne Schaap-Nutt ◽

Peter L. Collins ◽

...

Keyword(s):

Antibody Response ◽

Virus Replication ◽

Protein Gene ◽

Parainfluenza Virus ◽

Wild Type ◽

M Gene ◽

F Protein ◽

F Gene ◽

Human Parainfluenza Virus ◽

Type 3

ABSTRACTHuman parainfluenza virus type 3 (HPIV3), a paramyxovirus, is a major viral cause of severe lower respiratory tract disease in infants and children. The gene-end (GE) transcription signal of the HPIV3 matrix (M) protein gene is identical to those of the nucleoprotein and phosphoprotein genes except that it contains an apparent 8-nucleotide insert. This was associated with an increased synthesis of a readthrough transcript of the M gene and the downstream fusion (F) protein gene. We hypothesized that this insert may function to downregulate the expression of F protein by interfering with termination/reinitiation at the M-F gene junction, thus promoting the production of M-F readthrough mRNA at the expense of monocistronic F mRNA. To test this hypothesis, two similar recombinant HPIV3 viruses from which this insert in the M-GE signal was removed were generated. The M-GE mutants exhibited a reduction in M-F readthrough mRNA and an increase in monocistronic F mRNA. This resulted in a substantial increase in F protein synthesis in infected cells as well as enhanced incorporation of F protein into virions. The efficiency of mutant virus replication was similar to that of wild-type (wt) HPIV3 bothin vitroandin vivo. However, the F-protein-specific serum antibody response in hamsters was increased for the mutants compared to wt HPIV3. This study identifies a previously undescribed viral mechanism for attenuating the host adaptive immune response. Repairing the M-GE signal should provide a means to increase the antibody response to a live attenuated HPIV3 vaccine without affecting viral replication and attenuation.IMPORTANCEThe HPIV3 M-GE signal was previously shown to contain an apparent 8-nucleotide insert that was associated with increased synthesis of a readthrough mRNA of the M gene and the downstream F gene. However, whether this had any significant effect on the synthesis of monocistronic F mRNA or F protein, virus replication, virion morphogenesis, and immunogenicity was unknown. Here, we show that the removal of this insert shifts F gene transcription from readthrough M-F mRNA to monocistronic F mRNA. This resulted in a substantial increase in the amount of F protein expressed in the cell and packaged in the virus particle. This did not affect virus replication but increased the F-specific antibody response in hamsters. Thus, in wild-type HPIV3, the aberrant M-GE signal operates a previously undescribed mechanism that reduces the expression of a major neutralization and protective antigen, resulting in reduced immunogenicity. This has implications for the design of live attenuated HPIV3 vaccines; specifically, the antibody response against F can be elevated by “repairing” the M-GE signal to achieve higher-level F antigen expression, with no effect on attenuation.

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Identification of pilin pools in the membranes of Pseudomonas aeruginosa

Journal of Bacteriology ◽

10.1128/jb.152.2.687-691.1982 ◽

1982 ◽

Vol 152 (2) ◽

pp. 687-691

Author(s):

T H Watts ◽

E A Worobec ◽

W Paranchych

Keyword(s):

Staphylococcus Aureus ◽

Pseudomonas Aeruginosa ◽

Gel Electrophoresis ◽

Type Strain ◽

Wild Type Strain ◽

Polyacrylamide Gel Electrophoresis ◽

Protein A ◽

Sodium Dodecyl ◽

Wild Type ◽

Outer Membranes

The proteins of purified inner and outer membranes obtained from Pseudomonas aeruginosa strains PAK and PAK/2Pfs were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis, transferred to nitrocellulose, and treated with antiserum raised against pure pili. Bound antipilus antibodies were visualized by reaction with 125I-labeled protein A from Staphylococcus aureus. The results showed that there are pools of pilin in both the inner and outer membranes of P. aeruginosa and that the pool size in the multipiliated strain is comparable with that of the wild-type strain.

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Cloning, Sequencing, and Characterization of the SdeAB Multidrug Efflux Pump of Serratia marcescens

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.49.4.1495-1501.2005 ◽

2005 ◽

Vol 49 (4) ◽

pp. 1495-1501 ◽

Cited By ~ 31

Author(s):

Ayush Kumar ◽

Elizabeth A. Worobec

Keyword(s):

Serratia Marcescens ◽

Type Strain ◽

Wild Type Strain ◽

Genomic Library ◽

Efflux Pump ◽

Sodium Dodecyl ◽

Wild Type ◽

Diverse Range ◽

Mutant Strains ◽

Encoding Genes

ABSTRACT Serratia marcescens is an important nosocomial agent known for causing various infections in immunocompromised individuals. Resistance of this organism to a broad spectrum of antibiotics makes the treatment of infections very difficult. This study was undertaken to identify multidrug resistance efflux pumps in S. marcescens. Three mutant strains of S. marcescens were isolated in vitro by the serial passaging of a wild-type strain in culture medium supplemented with ciprofloxacin, norfloxacin, or ofloxacin. Fluoroquinolone accumulation assays were performed to detect the presence of a proton gradient-dependent efflux mechanism. Two of the mutant strains were found to be effluxing norfloxacin, ciprofloxacin, and ofloxacin, while the third was found to efflux only ofloxacin. A genomic library of S. marcescens wild-type strain UOC-67 was constructed and screened for RND pump-encoding genes by using DNA probes for two putative RND pump-encoding genes. Two different loci were identified: sdeAB, encoding an MFP and an RND pump, and sdeCDE, encoding an MFP and two different RND pumps. Northern blot analysis revealed overexpression of sdeB in two mutant strains effluxing fluoroquinolones. Analysis of the sdeAB and sdeCDE loci in Escherichia coli strain AG102MB, deficient in the RND pump (AcrB), revealed that gene products of sdeAB are responsible for the efflux of a diverse range of substrates that includes ciprofloxacin, norfloxacin, ofloxacin, chloramphenicol, sodium dodecyl sulfate, ethidium bromide, and n-hexane, while those of sdeCDE did not result in any change in susceptibilities to any of these agents.

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Expressing activator protein Ap36 inBacillus thuringiensisand the function of the recombined strain on disease resistance

Chinese Journal of Agricultural Biotechnology ◽

10.1017/s1479236208002234 ◽

2008 ◽

Vol 5 (2) ◽

pp. 121-126

Author(s):

Peng Dong-Hai ◽

Zhou Chen-Fei ◽

Qiu De-Wen ◽

Zhou Kang ◽

Ruan Li-Fang ◽

...

Keyword(s):

Gel Electrophoresis ◽

Recombinant Strain ◽

Phenylalanine Ammonia Lyase ◽

Protein Gene ◽

Polyacrylamide Gel Electrophoresis ◽

Sodium Dodecyl ◽

Protein Elicitor ◽

Sds Page ◽

Rot Disease ◽

Derivative Strain

AbstractThe geneap36encoding a protein elicitor fromAlternariasp. was fused downstream of theslh(S-layer homology) motif ofBacillus thuringiensisS-layer protein genectc. The recombinant gene was then transferred intoB. thuringiensisplasmid-free derivative strain BMB171. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) showed that the SLH–Ap36 fusion protein was expressed inB. thuringiensisBMB171. After tomato (Lycopersicum esculentum) leaves were treated for 90 min with the recombinant strain cultured at 28°C for 24 h, the activity of peroxidase and the amount of proline of tomato leaves were increased to 57.14% and 131.59%, respectively, compared to the control, and after the tomato leaves were treated with the cultured recombinant strain for 4 days, the activity of phenylalanine ammonia lyase was also higher than that in the control. Furthermore, tubers of treated potato (Solanum tuberosum) plants showed higher resistance to rot disease caused byErwinia corotovoraSCG1 compared to the control treatments.

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Molecular Pathogenesis of Type I Congenital Plasminogen Deficiency: Expression of Recombinant Human Mutant Plasminogens in Mammalian Cells

Blood ◽

10.1182/blood.v89.1.183.183_183_190 ◽

1997 ◽

Vol 89 (1) ◽

pp. 183-190 ◽

Cited By ~ 1

Author(s):

Hiroyuki Azuma ◽

Nobuaki Mima ◽

Mitsuo Shirakawa ◽

Kazumasa Miyamoto ◽

Hiroshi Yamaguchi ◽

...

Keyword(s):

Sodium Dodecyl ◽

Resistant Mutant ◽

Molecular Pathogenesis ◽

Mutant Protein ◽

Immunocytochemical Staining ◽

Type I ◽

Wild Type ◽

Transport Pathway ◽

Stable Transfectants ◽

Plasminogen Deficiency

We previously reported the genetic abnormality in a Japanese family with type I congenital plasminogen deficiency caused by a Ser572 to Pro572 mutation. To characterize the molecular pathogenesis of the disease in this family, we expressed recombinant human wild-type and mutant (rS572P) plasminogens in COS-1 cells. Activation-resistant wild-type and mutant plasminogen stable transfectants in CHO-K1 cells also were established. Transient transfection and metabolic labeling experiments followed by immunoprecipitation analysis showed that the mutant plasminogen was secreted from COS-1 cells in reduced amounts, compared with the wild type. Endo H digestion of the wild-type and mutant plasminogen showed no shift in their migrations on sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis, indicating that both contain complex type oligosaccharide structures and could therefore be secreted. Furthermore, the secretion of activation-resistant mutant plasminogen was significantly reduced. Pulse-chase experiments and Northern blot analysis showed that the impaired secretion of the mutant plasminogen was the consequence of the accumulation of the mutant protein inside the cells but not of reduced plasminogen mRNA. Immunocytochemical staining of stable transfectants also revealed that CHO-K1 cells expressing the activation-resistant mutant plasminogen stained mainly in the perinuclear area, suggesting delayed processing of the mutant protein in the intracellular transport pathway. We conclude that the impaired secretion of mutant plasminogen, due to intracellular accumulation, is the molecular pathogenesis of type I congenital plasminogen deficiency caused by a Ser572 to Pro572 mutation.

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Evolutionary dynamics of phage resistance in bacterial biofilms

10.1101/552265 ◽

2019 ◽

Cited By ~ 5

Author(s):

Matthew Simmons ◽

Matthew C. Bond ◽

Knut Drescher ◽

Vanni Bucci ◽

Carey D. Nadell

Keyword(s):

Evolutionary Dynamics ◽

Physiological State ◽

Computational Simulation ◽

Phage Infection ◽

Resistant Bacteria ◽

Phage Resistance ◽

Natural Environments ◽

Biofilm Growth ◽

Single Strain ◽

Negative Frequency Dependent Selection

AbstractInteractions among bacteria and their viral predators, the bacteriophages, are likely among the most common ecological phenomena on Earth. The constant threat of phage infection to bacterial hosts, and the imperative of achieving infection on the part of phages, drives an evolutionary contest in which phage-resistant bacteria emerge, often followed by phages with new routes of infection. This process has received abundant theoretical and experimental attention for decades and forms an important basis for molecular genetics and theoretical ecology and evolution. However, at present, we know very little about the nature of phage-bacteria interaction – and the evolution of phage resistance – inside the surface-bound communities that microbes usually occupy in natural environments. These communities, termed biofilms, are encased in a matrix of secreted polymers produced by their microbial residents. Biofilms are spatially constrained such that interactions become limited to neighbors or near-neighbors; diffusion of solutes and particulates is reduced; and there is pronounced heterogeneity in nutrient access and therefore physiological state. These factors can dramatically impact the way phage infections proceed even in simple, single-strain biofilms, but we still know little of their effect on phage resistance evolutionary dynamics. Here we explore this problem using a computational simulation framework customized for implementing phage infection inside multi-strain biofilms. Our simulations predict that it is far easier for phage-susceptible and phage-resistant bacteria to coexist inside biofilms relative to planktonic culture, where phages and hosts are well-mixed. We characterize the negative frequency dependent selection that underlies this coexistence, and we then test and confirm this prediction using an experimental model of biofilm growth measured with confocal microscopy at single-cell and single-phage resolution.

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Transformation-deficient mutants of Bacillus subtilis impaired in competence-specific nuclease activities

Journal of Bacteriology ◽

10.1128/jb.152.1.166-174.1982 ◽

1982 ◽

Vol 152 (1) ◽

pp. 166-174

Author(s):

J A Mulder ◽

G Venema

Keyword(s):

Molecular Weight ◽

Bacillus Subtilis ◽

Sodium Dodecyl Sulfate ◽

Sodium Dodecyl ◽

Magnesium Ions ◽

Polyacrylamide Gels ◽

Wild Type ◽

Molecular Weights ◽

Defective Mutant ◽

Mutant Strains

A comparison of the nucleolytic activities in competent and physiologically low-competent wild-type cultures of Bacillus subtilis in DNA-containing sodium dodecyl sulfate-polyacrylamide gels revealed the existence of three competence-associated nuclease activities with apparent molecular weights of 13,000, 15,000, and 26,000. The three activities, which were dependent on manganese or magnesium ions, were specifically present in the competent fraction of a competent culture. The competence-associated nucleolytic activities of eight transformation-defective mutant strains were assayed, resulting in the following three classes of mutants: (i) four strains which, according to this assay, were not impaired in any of the nucleolytic activities mentioned above; (ii) one strain which was strongly impaired in the 13,000- and 26,000-molecular-weight activities, but showed a considerable level of the 15,000-molecular-weight activity; and (iii) three strains which were severely impaired in all three activities. The results indicated that the 26,000-molecular-weight activity was a dimer of the 13,000-molecular-weight activity and that this nuclease was involved in the entry of DNA.

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Novel Catalatic Proteins of Bakers' Yeast. I. An Atypical Catalase

Canadian Journal of Biochemistry ◽

10.1139/o73-208 ◽

1973 ◽

Vol 51 (11) ◽

pp. 1551-1555 ◽

Cited By ~ 35

Author(s):

Tony C. M. Seah ◽

A. R. Bhatti ◽

J. G. Kaplan

Keyword(s):

Molecular Weight ◽

Gel Electrophoresis ◽

Specific Activity ◽

Polyacrylamide Gel Electrophoresis ◽

Sodium Dodecyl ◽

Native Protein ◽

Wild Type ◽

Major Band ◽

Subunit Molecular Weight ◽

Purification And Properties

At any stage of growth of a wild-type bakers' yeast, some 20% of the catalatic activity of crude extracts is not precipitable by means of antibody prepared against the typical catalase (catalase T), whose purification and properties have been previously described. Some of this catalatic activity is due to the presence of an atypical catalase (catalase A), a heme protein, with a molecular weight estimated as 170 000 – 190 000, considerably lower than that of the usual catalases (225 000 – 250 000). Preparations of catalase A were found to be homogeneous in the analytical ultracentrifuge and in polyacrylamide gel electrophoresis. Its subunit molecular weight, determined from its iron content, was 46 500, virtually the same as that of the major band obtained in gel electrophoresis in the presence of sodium dodecyl sulfate, suggesting that the native protein is tetrameric. Its specific activity is in the range of those reported for other typical catalases.

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