Isolation and characterization of mutants of the citrus canker pathogen Xanthomonas axonopodis pv. citri that induce a distinct pattern of disease

2000 ◽  
Vol 78 (8) ◽  
pp. 1002-1009
Author(s):  
Shu Yun Tung ◽  
Tsong Teh Kuo
Keyword(s):  
Blot Hybridization ◽  
Random Mutagenesis ◽  
Southern Blot Hybridization ◽  
Citrus Canker ◽  
Distinct Pattern ◽  
Wild Type ◽  
In Planta ◽  
Xanthomonas Axonopodis ◽  
Isolation And Characterization ◽  
Indigenous Plasmids

Random mutagenesis with the transposon Tn5tac1 in Xanthomonas axonopodis pathovar citri, the causal agent of citrus canker, generated four mutants with altered pathogenicity. These mutants were classified into three groups: (i) the nonpathogenic (NP) mutants XT10 and XT122, which did not induce any visible symptoms in the host; (ii) the WS- mutant XT27, which induced a callus-like lesion but not a watersoaked lesion; and (iii) the CL- mutant XT37, which was unable to induce a callus-like eruption but did induce the formation of a watersoaked lesion around the infection site. The NP mutants failed to grow in planta, whereas the WS- and CL- mutants showed a reduced growth rate relative to that of the wild type. Co-inoculation of leaves with the WS- and CL- mutants did not result in complementation of their respective defects. The extent of extracellular accumulation of polysaccharide, protease, and amylase activities by each of the mutants was similar to that of the wild type. The extracellular activity of polygalacturonate lyase of XT27 was reduced relative to that of the wild type and other mutants. Unlike the wild type and other mutants, XT27 also required glutamic acid for growth in culture. Southern blot hybridization revealed that each of the mutants resulted from transposon insertion at a single site; the insertion sites for XT10 and XT27 were located in the chromosome, whereas those for XT37 and XT122 were located in the indigenous plasmids. These results provide evidence that bacterial genes contribute independently to the pathogenesis of citrus canker.Key words: citrus canker, pathogenicity genes, transposon mutagenesis.

scholarly journals Attacin A Gene from Tricloplusia ni Reduces Susceptibility to Xanthomonas axonopodis pv. citri in Transgenic Citrus sinensis `Hamlin'

2006 ◽  
Vol 131 (4) ◽  
pp. 530-536 ◽  
Author(s):  
Raquel L. Boscariol ◽  
Mariza Monteiro ◽  
Elizabete K. Takahashi ◽  
Sabrina M. Chabregas ◽  
Maria Lucia C. Vieira ◽  
...  
Keyword(s):  
Citrus Sinensis ◽  
Fluorescent Protein ◽  
Blot Hybridization ◽  
Plasmid Vector ◽  
Southern Blot Hybridization ◽  
Citrus Canker ◽  
Green Fluorescent Protein Gene ◽  
Xanthomonas Axonopodis ◽  
Transgenic Lines

Citrus canker, caused by Xanthomonas axonopodis Starr and Garces pv. citri (Hasse) Vauterin et al., is one of the main problems affecting citrus production. In order to obtain resistance to phytopathogenic bacteria, insect genes, coding for antimicrobial proteins, have been used in plant genetic transformation. In this study, transgenic Citrus sinensis (L.) Osb. `Hamlin' plants expressing the antimicrobial insect-derived attacin A gene (attA) were obtained by Agrobacterium tumefaciens (Smith and Towns.) Conn-mediated transformation. Initially, the cDNA clone was used to construct a binary plasmid vector (pCattA 2300). The construction included the native signal peptide (SP) responsible for directing the insect protein to the extracellular space where bacteria is supposed to accumulate in vivo. In order to investigate the native SP effectiveness in a plant model system, onion (Allium cepa L.) epidermal cells were transformed, via biobalistics, using plasmids containing the attA gene with or without SP, fused with the green fluorescent protein gene (pattA 1303 and pSPattA 1303). Fluorescence accumulation surrounding the cells was observed only in tissues transformed with the plasmid containing the gene with SP, indicating the protein secretion to the apoplast. Citrus transformation was confirmed by PCR and Southern blot hybridization analysis in 12 regenerated plants. Transcription of attA gene was detected by Northern blot analysis in all transgenic plants. Eight selected transgenic lines were propagated and inoculated with a 106 cfu/mL suspension of the pathogen X. axonopodis pv. citri. Compared to control (non-transformed plant), seven transgenic lines showed a significant reduction in susceptibility to citrus canker. The results obtained here indicate the potential use of antibacterial proteins to protect citrus from bacterial diseases.

scholarly journals Structure of active chromatin: isolation and characterization of transcriptionally active chromatin from rat liver

1997 ◽  
Vol 322 (1) ◽  
pp. 273-279 ◽  
Author(s):  
Kulbhushan TIKOO ◽  
Sunita GUPTA ◽  
Q. Anwar HAMID ◽  
Vanya SHAH ◽  
Bishwanath CHATTERJEE ◽  
...  
Keyword(s):  
Rat Liver ◽  
Blot Hybridization ◽  
Southern Blot Hybridization ◽  
Micrococcal Nuclease ◽  
Active Chromatin ◽  
Chromatin Fraction ◽  
Isolation And Characterization ◽  
Bivalent Cations ◽  
Composite Gel ◽  
A Minor

Rat liver nuclei were isolated in low-ionic-strength buffer in the absence of bi- and multi-valent cations. Digestion of these nuclei by endogenous nuclease, micrococcal nuclease and DNase I revealed that a minor chromatin fraction was preferentially digested into poly- and oligo-nucleosomes. Southern blot hybridization with various active gene probes confirmed that these chromatin fragments represent coding and 5ƀ upstream regions of transcriptionally active chromatin. Active chromatin fragments were released selectively into the medium, with inactive chromatin remaining inside the nuclei, under the above ionic conditions. The inclusion of bivalent cations during the digestion of nuclei reversed the solubility behaviour of active chromatin. Rearrangement and exchange of histone H1 between chromatin fragments was prevented by using low-salt conditions in all steps in the absence of bivalent cations. All histones, including H1, were present in stoichiometric amounts in this active chromatin fraction. Active nucleosomes showed a lower electrophoretic mobility than bulk nucleosomes in an acrylamide/agarose composite gel in the absence of Mg2+, but were selectively bound to the gel in the presence of this ion.

scholarly journals Vibrio vulnificus Has the Transmembrane Transcription Activator ToxRS Stimulating the Expression of the Hemolysin GenevvhA

2000 ◽  
Vol 182 (12) ◽  
pp. 3405-3415 ◽  
Author(s):  
Shee Eun Lee ◽  
Sung Heui Shin ◽  
Soo Young Kim ◽  
Young Ran Kim ◽  
Dong Hyeon Shin ◽  
...  
Keyword(s):  
Vibrio Vulnificus ◽  
Blot Hybridization ◽  
Southern Blot Hybridization ◽  
Amino Acid Sequences ◽  
Open Reading Frames ◽  
Wild Type ◽  
Transcription Activator ◽  
Reading Frame ◽  
Type Gene ◽  
Virulence Regulator

ABSTRACT In an attempt to dissect the virulence regulatory mechanism inVibrio vulnificus, we tried to identify the V. cholerae transmembrane virulence regulator toxRS(toxRS Vc) homologs in V. vulnificus. By comparing the sequences of toxRS ofV. cholerae and V. parahaemolyticus(toxRS Vp), we designed a degenerate primer set targeting well-conserved sequences. Using the PCR product as an authentic probe for Southern blot hybridization, a 1.6-kbBglII-HindIII fragment and a 1.2-kbHindIII fragment containing two complete open reading frames and one partial open reading frame attributable totoxR Vv, toxS Vv, andhtpG Vv were cloned. ToxRVv shared 55.0 and 63.0% sequence homology with ToxRVc and ToxRVp, respectively. ToxSVv was 71.5 and 65.7% homologous to ToxSVc and ToxSVp, respectively. The amino acid sequences of ToxRSVv showed transmembrane and activity domains similar to those observed in ToxRSVc and ToxRSVp. Western blot analysis proved the expression of ToxRVv in V. vulnificus. ToxRSVv enhanced, in an Escherichia coli background, the expression of the V. vulnificushemolysin gene (vvhA) fivefold. ToxRSVv also activated the ToxRVc-regulated ctx promoter incorporated into an E. coli chromosome. AtoxR Vv null mutation decreased hemolysin production. The defect in hemolysin production could be complemented by a plasmid harboring the wild-type gene. ThetoxR Vv mutation also showed a reversed outer membrane protein expression profile in comparison to the isogenic wild-type strain. These results demonstrate that ToxRVv may regulate the virulence expression of V. vulnificus.

scholarly journals In Planta Interaction and Transreplication of Distinct Begomoviruses and their Associated Components

2021 ◽  
Vol 26 (01) ◽  
pp. 45-51
Author(s):  
Muhammad Naeem Sattar
Keyword(s):  
Nicotiana Benthamiana ◽  
Blot Hybridization ◽  
Low Frequency ◽  
Southern Blot Hybridization ◽  
Bipartite Begomoviruses ◽  
In Planta ◽  
Monopartite Begomovirus ◽  
Structural Resemblance ◽  
Rep Proteins ◽  
Viral Components

The studies described here were intended to examine the transreplication and interactions abilities of a widespread ToLCNDV, and an emerging begomovirus PeLCV associated with its cognate betasatellite TbLCuB. PeLCV, a monopartite begomovirus, has been characterized from many important crops, vegetables and weeds along with its associated TbLCuB. The DNA-B of bipartite ToLCNDV genome has been successfully transreplicated by the DNA-A of different bipartite begomoviruses, albeit with low frequency. Whether PeLCV can transreplicate DNA-B of ToLCNDV is unknown. To unravel this notion, both these viruses were inoculated to the model Nicotiana benthamiana plants in all possible combinations and the in planta existence of viral components were verified by PCR and Southern blot hybridization. The results demonstrated that PeLCV transreplicated and maintained ToLCNDV DNA-B. Whereas, ToLCNDV DNA-A could not transreplicate TbLCuB. Analyses of Rep proteins structure of ToLCNDV and PeLCV revealed a structural resemblance, whereas putative iteron-binding sequences of PeLCV were compatible with the Rep-binding iterons of ToLCNDV-B. The results suggested that PeLCV and ToLCNDV DNA-B can interact synergistically and can be disastrous under field conditions. © 2021 Friends Science Publishers

scholarly journals Effects of Oligopeptide Permease in Group A Streptococcal Infection

2005 ◽  
Vol 73 (5) ◽  
pp. 2881-2890 ◽  
Author(s):  
Chih-Hung Wang ◽  
Chia-Yu Lin ◽  
Yueh-Hsia Luo ◽  
Pei-Jane Tsai ◽  
Yee-Shin Lin ◽  
...  
Keyword(s):  
Type Strain ◽  
Wild Type Strain ◽  
Blot Hybridization ◽  
Southern Blot Hybridization ◽  
Wild Type ◽  
Isogenic Mutant ◽  
Transporter Family ◽  
Streptolysin O ◽  
Group A ◽  
Oligopeptide Permease

ABSTRACT The oligopeptide permease (Opp) of group A streptococci (GAS) is a membrane-associated protein and belongs to the ATP-binding cassette transporter family. It is encoded by a polycistronic operon containing oppA, oppB, oppC, oppD, and oppF. The biological function of these genes in GAS is poorly understood. In order to understand more about the effects of Opp on GAS virulence factors, an oppA isogenic mutant was constructed by using an integrative plasmid to disrupt the opp operon and confirmed by Southern blot hybridization. No transcript was detected in the oppA isogenic mutant by Northern blot analysis and reverse transcriptase PCR. The growth curve for the oppA isogenic mutant was similar to that for wild-type strain A-20. The oppA isogenic mutant not only decreased the transcription of speB, speX, and rofA but also increased the transcription of speF, sagA (streptolysin S-associated gene A), slo (streptolysin O), pel (pleotrophic effect locus), and dppA (dipeptide permease). No effects on the transcription of emm, sda, speJ, speG, rgg, and csrR were found. The phenotypes of the oppA mutant were restored by the oppA revertant and by the complementation strain. The oppA mutant caused less mortality and tissue damage than the wild-type strain when inoculated into BALB/c mice via an air pouch. Based on these data, we suggest that the opp operon plays an important role in the pathogenesis of GAS infection.

scholarly journals Cloning, primary sequence and chromosomal localization of human FMO2, a new member of the flavin-containing mono-oxygenase family

1992 ◽  
Vol 287 (1) ◽  
pp. 261-267 ◽  
Author(s):  
C T Dolphin ◽  
E A Shephard ◽  
S Povey ◽  
R L Smith ◽  
I R Phillips
Keyword(s):  
Blot Hybridization ◽  
Southern Blot Hybridization ◽  
Chromosome 1 ◽  
Cdna Clones ◽  
Amino Acid Residues ◽  
Primary Sequence ◽  
Gene Encoding ◽  
Isolation And Characterization ◽  
Distinct Member ◽  
Human Chromosome 1

We have previously reported the cloning of cDNAs for a flavin-containing mono-oxygenase (FMO) of man, designated FMO1 [Dolphin, Shephard, Povey, Palmer, Ziegler, Ayesh, Smith & Phillips (1991) J. Biol. Chem. 266, 12379-12385], that is the orthologue of pig and rabbit hepatic FMOs. We now describe the isolation and characterization of cDNA clones for a second human FMO, which we have designated FMO2. The polypeptide encoded by the cDNAs is 558 amino acid residues long, has a calculated M(r) of 63337, and contains putative FAD- and NADP-binding sites that align exactly with those described in other mammalian FMOs. Human FMO2 has 51-53% primary sequence identity with human FMO1, rabbit pulmonary FMO and rabbit liver FMO form 2, and thus represents a fourth, distinct, member of the mammalian FMO family. The corresponding mRNA is present in low abundance in adult human liver. Southern blot hybridization with single-exon probes demonstrated that human FMO2 and FMO1 are the products of single genes. The gene encoding FMO2 (designated FMO2) was mapped, by the polymerase chain reaction, to human chromosome 1, the same chromosome on which FMO1 is located.

scholarly journals Isolation and Characterization of Methanobacterium thermoautotrophicum ΔH Mutants Unable To Grow under Hydrogen-Deprived Conditions

1998 ◽  
Vol 180 (10) ◽  
pp. 2676-2681 ◽  
Author(s):  
Jeroen L. A. Pennings ◽  
Jan T. Keltjens ◽  
Godfried D. Vogels
Keyword(s):  
Type Strain ◽  
Wild Type Strain ◽  
Random Mutagenesis ◽  
Methanobacterium Thermoautotrophicum ◽  
Mrna Levels ◽  
Wild Type ◽  
Physiological Characterization ◽  
Isolation And Characterization ◽  
Hydrogen Supply ◽  
In The Wild

ABSTRACT By using random mutagenesis and enrichment by chemostat culturing, we have developed mutants of Methanobacterium thermoautotrophicum that were unable to grow under hydrogen-deprived conditions. Physiological characterization showed that these mutants had poorer growth rates and growth yields than the wild-type strain. The mRNA levels of several key enzymes were lower than those in the wild-type strain. A fed-batch study showed that the expression levels were related to the hydrogen supply. In one mutant strain, expression of both methyl coenzyme M reductase isoenzyme I and coenzyme F420-dependent 5,10-methylenetetrahydromethanopterin dehydrogenase was impaired. The strain was also unable to form factor F390, lending support to the hypothesis that the factor functions in regulation of methanogenesis in response to changes in the availability of hydrogen.

scholarly journals Novel Nucleoside Analogue MCC-478 (LY582563) Is Effective against Wild-Type or Lamivudine-Resistant Hepatitis B Virus

2002 ◽  
Vol 46 (8) ◽  
pp. 2602-2605 ◽  
Author(s):  
Suzane Kioko Ono-Nita ◽  
Naoya Kato ◽  
Yasushi Shiratori ◽  
Flair José Carrilho ◽  
Masao Omata
Keyword(s):  
Hepatitis B Virus ◽  
Hepatitis B ◽  
Nucleoside Analogue ◽  
Blot Hybridization ◽  
Southern Blot Hybridization ◽  
Hbv Dna ◽  
Wild Type ◽  
Human Hepatoma Cells ◽  
B Virus ◽  
Resistant Mutants

ABSTRACT The emergence of resistant hepatitis B virus (HBV) with the L528M mutation and/or the M552V and M552I mutations in the polymerase gene following long-term lamivudine treatment is becoming an important clinical problem. The aim of this study was to investigate the susceptibility of wild-type and lamivudine-resistant HBV to MCC-478 (LY582563), a novel nucleoside analogue derivative of phosphonomethoxyethyl purine. The susceptibility of wild-type HBV and lamivudine-resistant mutants (M552I, M552V, and L528M/M552V) to MCC-478 was examined by transient transfection of full-length HBV DNA into human hepatoma cells. HBV DNA replication was monitored by Southern blot hybridization, and the effective concentration required to reduce replication by 50% (EC50) was determined. The replicative intermediates of wild-type and lamivudine-resistant mutants were progressively diminished by treatment with increasing doses of MCC-478. The MCC-478 EC50s were 0.027 μM for wild-type HBV (about 20 times more efficient than lamivudine), 2.6 μM for M552I, 3.3 μM for M552V, and 2.0 μM for L528M/M552V. Wild-type HBV and lamivudine-resistant mutants are susceptible to MCC-478. MCC-478 appears to be a candidate for the treatment of HBV infection and exhibits potent activity against lamivudine-resistant HBV.

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