Molecular Characterization of a Glucose-Inhibited Division Gene, gidA, That Regulates Cytotoxic Enterotoxin of Aeromonas hydrophila

ABSTRACT By using a mini-transposon, we obtained two mutated strains of a diarrheal isolate, SSU, of Aeromonas hydrophila that exhibited a 50 to 53% reduction in the hemolytic activity and 83 to 87% less cytotoxic activity associated with the cytotoxic enterotoxin (Act). Act is a potent virulence factor of A. hydrophila and has been shown to contribute significantly to the development of both diarrhea and septicemia in animal models. Subsequent cloning and DNA sequence analysis revealed that transposon insertion occurred at different locations in these two mutants within the same 1,890-bp open reading frame for the glucose-inhibited division gene (gidA). A similar reduction in hemolytic (46%) and cytotoxic (81%) activity of Act was noted in the gidA isogenic mutant of A. hydrophila that was generated by marker exchange mutagenesis. Northern blot analysis revealed that the transcription of the cytotoxic enterotoxin gene (act) was not altered in the gidA transposon and isogenic mutants. However, by generating a chromosomal act::alkaline phosphatase gene (phoA) reporter construct, we demonstrated significantly reduced phosphatase activity in these mutants, indicating the effect of glucose-inhibited division (GidA) protein in modulating act gene expression at the translational level. The biological effects of Act in the gidA mutants were restored by complementation. The virulence of the gidA mutants in mice was dramatically reduced compared to the those of the wild-type (WT) and complemented strains of A. hydrophila. The histopathological examination of lungs, in particular, indicated severe congestion, alveolar hemorrhage, and acute inflammatory infiltrate in the interstitial compartment and the alveolar spaces when mice were infected with the WT and complemented strains. Minimal-to-mild changes were noted in the lungs with the gidA mutants. Taken together, our data indicate for the first time that GidA regulates the most-potent virulence factor of A. hydrophila, Act.

Download Full-text

Molecular and Functional Characterization of a ToxR-Regulated Lipoprotein from a Clinical Isolate of Aeromonas hydrophila

Infection and Immunity ◽

10.1128/iai.00402-06 ◽

2006 ◽

Vol 74 (7) ◽

pp. 3742-3755 ◽

Cited By ~ 22

Author(s):

Lakshmi Pillai ◽

Jian Sha ◽

Tatiana E. Erova ◽

Amin A. Fadl ◽

Bijay K. Khajanchi ◽

...

Keyword(s):

Virulence Factor ◽

Aeromonas Hydrophila ◽

Functional Characterization ◽

Affinity Column ◽

Serum Resistance ◽

Classical Pathway ◽

Dependent Manner ◽

T7 Promoter

ABSTRACT Human diseases caused by species of Aeromonas have been classified into two major groups: septicemia and gastroenteritis. In this study, we reported the molecular and functional characterization of a new virulence factor, ToxR-regulated lipoprotein, or TagA, from a diarrheal isolate, SSU, of Aeromonas hydrophila. The tagA gene of A. hydrophila exhibited 60% identity with that of a recently identified stcE gene from Escherichia coli O157:H7, which encoded a protein (StcE) that provided serum resistance to the bacterium and prevented erythrocyte lysis by controlling classical pathway of complement activation by cleaving the complement C1-esterase inhibitor (C1-INH). We purified A. hydrophila TagA as a histidine-tagged fusion protein (rTagA) from E. coli DE3 strain using a T7 promoter-based pET30 expression vector and nickel affinity column chromatography. rTagA cleaved C1-INH in a time-dependent manner. The tagA isogenic mutant of A. hydrophila, unlike its corresponding wild-type (WT) or the complemented strain, was unable to cleave C1-INH, which is required to potentiate the C1-INH-mediated lysis of host and bacterial cells. We indeed demonstrated colocalization of C1-INH and TagA on the bacterial surface by confocal fluorescence microscopy, which ultimately resulted in increased serum resistance of the WT bacterium. Likewise, we delineated the role of TagA in contributing to the enhanced ability of C1-INH to inhibit the classical complement-mediated lysis of erythrocytes. Importantly, we provided evidence that the tagA mutant was significantly less virulent in a mouse model of infection (60%) than the WT bacterium at two 50% lethal doses, which resulted in 100% mortality within 48 h. Taken together, our data provided new information on the role of TagA as a virulence factor in bacterial pathogenesis. This is the first report of TagA characterization from any species of Aeromonas.

Download Full-text

Molecular Characterization of a Flagellar Export Locus of Helicobacter pylori

Infection and Immunity ◽

10.1128/iai.67.5.2060-2070.1999 ◽

1999 ◽

Vol 67 (5) ◽

pp. 2060-2070 ◽

Cited By ~ 31

Author(s):

Steffen Porwollik ◽

Brian Noonan ◽

Paul W. O’Toole

Keyword(s):

Helicobacter Pylori ◽

Virulence Factor ◽

Housekeeping Genes ◽

Reading Frame ◽

Reverse Transcription Pcr ◽

Mutant Strains ◽

H Pylori ◽

Export Protein ◽

Successful Colonization

ABSTRACT Motility of Helicobacter species has been shown to be essential for successful colonization of the host. We have investigated the organization of a flagellar export locus in Helicobacter pylori. A 7-kb fragment of the H. pylori CCUG 17874 genome was cloned and sequenced, revealing an operon comprising an open reading frame of unknown function (ORF03), essential housekeeping genes (ileS and murB), flagellar export genes (fliI and fliQ), and a homolog to a gene implicated in virulence factor transport in other pathogens (virB11). A promoter for this operon, showing similarity to the Escherichia coli ς70 consensus, was identified by primer extension. Cotranscription of the genes in the operon was demonstrated by reverse transcription-PCR, and transcription of virB11, fliI, fliQ, andmurB was detected in human or mouse biopsies obtained from infected hosts. The genetic organization of this locus was conserved in a panel of H. pylori clinical isolates. EngineeredfliI and fliQ mutant strains were completely aflagellate and nonmotile, whereas a virB11 mutant still produced flagella. The fliI and fliQ mutant strains produced reduced levels of flagellin and the hook protein FlgE. Production of OMP4, a member of the outer membrane protein family identified in H. pylori 26695, was reduced in both thevirB11 mutant and the fliI mutant, suggesting related functions of the virulence factor export protein (VirB11) and the flagellar export component (FliI).

Download Full-text

Characterization of Mariner transposons in seven species of Rhus gall aphids

Scientific Reports ◽

10.1038/s41598-021-95843-5 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Aftab Ahmad ◽

Gabriel Luz Wallau ◽

Zhumei Ren

Keyword(s):

Transposable Elements ◽

Present Report ◽

Wide Distribution ◽

Inverted Repeats ◽

Genomic Evolution ◽

Reading Frame ◽

Jumping Genes ◽

First Time ◽

Intact Open Reading Frame

AbstractTransposable elements (TEs), also known as jumping genes, are widely spread in the genomes of insects and play a considerable role in genomic evolution. Mariner/DD34D family belongs to class II transposable elements which is widely spread in the genomes of insects and have considerable role in genomic evolution. Mariner like elements (MLEs) were searched in the genomes of seven species of Rhus gall aphids belonging to six genera. In total, 121 MLEs were detected in the genomes of the seven investigated species of Rhus gall aphids, which showed a wide distribution in both close and distant related species. The sequences of MLEs ranged from 1 to 1.4 kb in length and the structural analysis of the MLEs showed that only five copies were potentially active with intact open reading frame (ORF) and terminal inverted repeats (TIRs). Phylogenetic analysis showed that all the 121 MLE sequences belonged to four subfamilies, i.e., Mauritiana, Drosophila, Vertumana and Irritans, among which Drosophila and Vertumana subfamilies were reported in aphids for the first time. Our present report revealed the diversity and distribution of MLEs in Rhus gall aphid genomes and expanded our understandings on the characterization of transposable elements in aphid genomes, which might be useful as genetic markers and tools and would play an important role in genomic evolution and adaptation of aphids.

Download Full-text

Identification and Molecular Characterization of Novel Mycoviruses in Saccharomyces and Non-Saccharomyces Yeasts of Oenological Interest

Viruses ◽

10.3390/v14010052 ◽

2021 ◽

Vol 14 (1) ◽

pp. 52

Author(s):

Dalila Crucitti ◽

Marco Chiapello ◽

Daniele Oliva ◽

Marco Forgia ◽

Massimo Turina ◽

...

Keyword(s):

Biological Effects ◽

Single Infection ◽

Mixed Infections ◽

Wine Yeasts ◽

Viral Origin ◽

Related Sequence ◽

Natural Hosts ◽

First Time ◽

Saccharomyces Yeasts

Wine yeasts can be natural hosts for dsRNA, ssRNA viruses and retrotransposon elements. In this study, high-throughput RNA sequencing combined with bioinformatic analyses unveiled the virome associated to 16 Saccharomyces cerevisiae and 8 non-Saccharomyces strains of oenological interest. Results showed the presence of six viruses and two satellite dsRNAs from four different families, two of which—Partitiviridae and Mitoviridae—were not reported before in yeasts, as well as two ORFan contigs of viral origin. According to phylogenetic analysis, four new putative mycoviruses distributed in Totivirus, Cryspovirus, and Mitovirus genera were identified. The majority of commercial S. cerevisiae strains were confirmed to be the host for helper L-A type totiviruses and satellite M dsRNAs associated with the killer phenotype, both in single and mixed infections with L-BC totiviruses, and two viral sequences belonging to a new cryspovirus putative species discovered here for the first time. Moreover, single infection by a narnavirus 20S-related sequence was also found in one S. cerevisiae strain. Considering the non-Saccharomyces yeasts, Starmerella bacillaris hosted four RNAs of viral origin—two clustering in Totivirus and Mitovirus genera, and two ORFans with putative satellite behavior. This study confirmed the infection of wine yeasts by viruses associated with useful technological characteristics and demonstrated the presence of complex mixed infections with unpredictable biological effects.

Download Full-text

Characterization of Moraxella(Branhamella) catarrhalis lbpB, lbpA, and Lactoferrin Receptor orf3 Isogenic Mutants

Infection and Immunity ◽

10.1128/iai.67.3.1517-1520.1999 ◽

1999 ◽

Vol 67 (3) ◽

pp. 1517-1520 ◽

Cited By ~ 32

Author(s):

Robert A. Bonnah ◽

Henry Wong ◽

Sheena M. Loosmore ◽

Anthony B. Schryvers

Keyword(s):

Moraxella Catarrhalis ◽

Open Reading Frame ◽

Reading Frame ◽

Orf3 Protein ◽

Lactoferrin Receptor ◽

The Family ◽

Specific Receptors ◽

Isogenic Mutants ◽

Branhamella Catarrhalis

ABSTRACT Pathogenic members of the family Neisseriaceae produce specific receptors to acquire iron from their host’s lactoferrin and transferrin. Recently, putative Moraxella catarrhalislactoferrin receptor genes and a third open reading frame (lbpB, lbpA, and orf3) were cloned and sequenced. We describe the preliminary characterization of isogenic mutants deficient in LbpB, LbpA, or Orf3 protein.

Download Full-text

Isolation of Polymyxin B-Susceptible Mutants ofBurkholderia pseudomallei and Molecular Characterization of Genetic Loci Involved in Polymyxin B Resistance

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.43.11.2648 ◽

1999 ◽

Vol 43 (11) ◽

pp. 2648-2656 ◽

Cited By ~ 52

Author(s):

Mary N. Burtnick ◽

Donald E. Woods

Keyword(s):

Genetic Locus ◽

Glucose Dehydrogenase ◽

Polymyxin B ◽

Genetic Loci ◽

Gram Negative ◽

Reading Frame ◽

Resistance Phenotype ◽

Transposon Insertion ◽

Killing Action ◽

Isogenic Mutants

ABSTRACT Burkholderia pseudomallei is a gram-negative bacterium that causes the disease known as melioidosis. This pathogen is endemic to Southeast Asia and northern Australia and is particularly problematic in northeastern Thailand. It has been previously reported that B. pseudomallei is resistant to the killing action of cationic antimicrobial peptides, including human neutrophil peptide, protamine sulfate, poly-l-lysine, magainins, and polymyxins. Recently, we have also found that the virulent clinical isolate B. pseudomallei 1026b is capable of replicating in media containing polymyxin B at concentrations of >100 mg/ml. In order to identify genetic loci that are associated with this particular resistance phenotype, we employed a Tn5-OT182 mutagenesis system in coordination with a replica plating screen to isolate polymyxin B-susceptible mutants. Of the 17,000 Tn5-OT182 mutants screened via this approach, five polymyxin B-susceptible mutants were obtained. Three of these mutants harbored Tn5-OT182 insertions within a genetic locus demonstrating strong homology to the lytB gene present in other gram-negative bacteria. Of the remaining two mutants, one contained a transposon insertion in a locus involved in lipopolysaccharide core biosynthesis (waaF), while the other contained an insertion in an open reading frame homologous to UDP-glucose dehydrogenase genes. Isogenic mutants were also constructed via allelic exchange and used in complementation analysis studies to further characterize the relative importance of each of the various genetic loci with respect to the polymyxin B resistance phenotype exhibited by B. pseudomallei 1026b.

Download Full-text

Characterization of Mariner transposons in Rhus gall aphids (Hemiptera: Aphididae: Eriosomatinae)

10.21203/rs.3.rs-432617/v1 ◽

2021 ◽

Author(s):

Aftab Ahmad ◽

Gabriel Luz Wallau ◽

Zhumei Ren

Keyword(s):

Transposable Elements ◽

Present Report ◽

Wide Distribution ◽

Genomic Evolution ◽

Reading Frame ◽

Jumping Genes ◽

The One ◽

First Time ◽

Intact Open Reading Frame

Abstract Background: Transposable elements (TEs), also known as jumping genes, are widely spread in the genomes of insects and play a considerable role in genomic evolution. Mariner family belongs to class II transposable elements, were searched in the genomes of seven species of Rhus gall aphids belonging to six genera. Mariner-like elements were characterized for the first time in Rhus gall aphids and classified in to respective subfamilies.Results: In total, one hundred twenty-one MLEs were detected in the genomes of the seven investigated species of Rhus gall aphids, which showed a wide distribution of MLEs in both close and distant related species. The sequences of MLEs ranged from 1kb to 1.4kb in length and the structural analysis of the MLEs showed that only five copies were potentially active with intact open reading frame (ORF) while the remaining were classified as inactive MLEs according to absence of single intact ORF or terminal inverted repeats (TIRs). Based on the MLEs in Rhus gall aphids as well as the well characterized MLEs in other organisms from GenBank, the phylogenetic analysis showed that all the one hundred twenty-one MLE sequences belonged to four subfamilies, i.e., thirty from Maurutiana subfamily, twenty-six from Drosophila subfamily, thirty-three from Vertumana subfamily and thirty-two from Irritans subfamily, among which Drosophila and Vertumana subfamilies were reported in aphids for the first time. Moreover, the phylogenetic relationship suggested possible horizontal transfer events of MLEs between aphids and other insects.Conclusion: Our present report revealed the diversity and distribution of MLEs in Rhus gall aphid genomes sequenced by shotgun genome skimming method. This study further expanded our understandings on the characterization of transposable elements in aphid genomes, which might be useful as genetic markers and tools and would play an important role in genomic evolution and adaptation of aphids.

Download Full-text

Sterol 14α-demethylase activity in Streptomyces coelicolor A3(2) is associated with an unusual member of the CYP51 gene family

Biochemical Journal ◽

10.1042/bj20011380 ◽

2002 ◽

Vol 364 (2) ◽

pp. 555-562 ◽

Cited By ~ 21

Author(s):

David C. LAMB ◽

Kay FOWLER ◽

Tobias KIESER ◽

Nigel MANNING ◽

Larissa M. PODUST ◽

...

Keyword(s):

Streptomyces Coelicolor ◽

Open Reading Frame ◽

Reading Frame ◽

E Coli ◽

Transposon Insertion ◽

Cell Extracts ◽

C Terminus ◽

Demethylase Activity ◽

First Time

The annotation of the genome sequence of Streptomyces coelicolor A3(2) revealed a cytochrome P450 (CYP) resembling various sterol 14α-demethylases (CYP51). The putative CYP open reading frame (SC7E4.20) was cloned with a tetrahistidine tag appended to the C-terminus and expressed in Escherichia coli. Protein purified to electrophoretic homogeneity was observed to bind the 14-methylated sterols lanosterol and 24-methylene-24,25-dihydrolanosterol (24-MDL). Reconstitution experiments with E. coli reductase partners confirmed activity in 14α-demethylation for 24-MDL, but not lanosterol. An S. coelicolor A3(2) mutant containing a transposon insertion in the CYP51 gene, which will abolish synthesis of the functional haemoprotein, was isolated as a viable strain, the first time a CYP51 has been identified as non-essential. The role of this CYP in bacteria is intriguing. No sterol product was detected in non-saponifiable cell extracts of the parent S. coelicolor A3(2) strain or of the mutant. S. coelicolor A3(2) CYP51 contains very few of the conserved CYP51 residues and, even though it can catalyse 14α-demethylation, it probably has another function in Streptomyces. We propose that it is a member of a new CYP51 subfamily.

Download Full-text

Identification and Characterization of a Gene on Rhizobium meliloti pSyma, syrB, That Negatively Affects syrM Expression

Molecular Plant-Microbe Interactions ◽

10.1094/mpmi.1997.10.5.550 ◽

1997 ◽

Vol 10 (5) ◽

pp. 550-559 ◽

Cited By ~ 20

Author(s):

Melanie J. Barnett ◽

Sharon R. Long

Keyword(s):

Rhizobium Meliloti ◽

Large Deletion ◽

Physical Analysis ◽

Reading Frame ◽

Transposon Insertion ◽

Gusa Gene ◽

Dna Fragment ◽

Identification And Characterization ◽

Insertion Mutants

The Rhizobium meliloti SyrM protein activates transcription of nodD3 and syrA. Regulation of syrM is complex and may involve as yet undiscovered genes. Here we report the isolation of insertion mutants showing increased expression of a syrM-gusA gene fusion. Characterization of one mutant strain, designated SYR-B, revealed a mutation consisting of a transposon insertion linked to a large deletion. The corresponding wild-type DNA was cloned as a 5.3-kb BamHI fragment. Genetic and physical analysis of this DNA demonstrated that an open reading frame (ORF) near one end of the fragment, encoding the 16.5-kDa SyrB protein, is responsible for the repression of syrM activity. Results of complementation experiments with the 5.3-kb BamHI DNA led us to hypothesize that other genes within this DNA fragment interfere with the expression or activity of SyrB. Our analysis showed that the region upstream of syrB contains three ORFs. One ORF is similar to the Ros repressor of Agrobacterium tumefaciens and the MucR repressor of R. meliloti.

Download Full-text

Canatoxin, a toxic protein from jack beans (Canavalia ensiformis), is a variant form of urease (EC 3.5.1.5): biological effects of urease independent of its ureolytic activity

Biochemical Journal ◽

10.1042/bj3600217 ◽

2001 ◽

Vol 360 (1) ◽

pp. 217-224 ◽

Cited By ~ 26

Author(s):

Cristian FOLLMER ◽

Grace B. S. BARCELLOS ◽

Russolina B. ZINGALI ◽

Olga L. T. MACHADO ◽

Elias W. ALVES ◽

...

Keyword(s):

Blood Platelets ◽

Biological Effects ◽

Enzymic Activity ◽

Variant Form ◽

Native Form ◽

Toxic Protein ◽

Canavalia Ensiformis ◽

Covalently Linked ◽

First Time

Canatoxin is a toxic protein from Canavalia ensiformis seeds, lethal to mice (LD50 = 2mg/kg) and insects. Further characterization of canatoxin showed that its main native form (184kDa) is a non-covalently linked dimer of a 95kDa polypeptide containing zinc and nickel. Partial sequencing of internal peptides indicated homology with urease (EC 3.5.1.5) from the same seed. Canatoxin has approx. 30% of urease's activity for urea, and Km of 2–7mM. The proteins differ in their affinities for metal ions and were separated by affinity chromatography on a Zn2+ matrix. Similar to canatoxin, urease activates blood platelets and interacts with glycoconjugates. In contrast with canatoxin, no lethality was seen in mice injected with urease (10mg/kg). Pretreatment with p-hydroxymercuribenzoate irreversibly abolished the ureolytic activity of both proteins. On the other hand, p-hydroxymercuribenzoate-treated canatoxin was still lethal to mice, and both treated proteins were fully active in promoting platelet aggregation and binding to glycoconjugates. Taken together, our data indicate that canatoxin is a variant form of urease. Moreover, we show for the first time that these proteins display several biological effects that are unrelated to their enzymic activity for urea.

Download Full-text