scholarly journals Inactivation of the Spirochete recA Gene Results in a Mutant with Low Viability and Irregular Nucleoid Morphology

2002 ◽  
Vol 184 (2) ◽  
pp. 452-458 ◽  
Author(s):  
Ange-Patricia Tchamedeu Kameni ◽  
Evelyne Couture-Tosi ◽  
Isabelle Saint-Girons ◽  
Mathieu Picardeau
Keyword(s):  
Type Strain ◽  
Wild Type Strain ◽  
Uv Light ◽  
Fluorescent Microscopy ◽  
Kanamycin Resistance ◽  
Reca Gene ◽  
Wild Type ◽  
Chromosomal Dna ◽  
Poor Growth ◽  
Kanamycin Resistance Cassette

ABSTRACT Recently, we have shown the first evidence for allelic exchange in Leptospira spp. By using the same methodology, the cloned recA of Leptospira biflexa was interrupted by a kanamycin resistance cassette, and the mutated allele was then introduced into the L. biflexa chromosome by homologous recombination. The recA double-crossover mutant showed poor growth in liquid media and was considerably more sensitive to DNA-damaging agents such as mitomycin C and UV light than the wild-type strain. The efficiency of plating of the recA mutant was about 10% of that of the parent strain. In addition, microscopic observation of the L. biflexa recA mutant showed cells that are more elongated than those of the wild-type strain. Fluorescent microscopy of stained cells of the L. biflexa wild-type strain revealed that chromosomal DNA is distributed throughout most of the length of the cell. In contrast, the recA mutant showed aberrant nucleoid morphologies, i.e., DNA is condensed at the midcell. Our data indicate that L. biflexa RecA plays a major role in ensuring cell viability via mechanisms such as DNA repair and, indirectly, active chromosome partitioning.

scholarly journals Influence of the Alternative σ28 Factor on Virulence and Flagellum Expression of Legionella pneumophila

2002 ◽  
Vol 70 (3) ◽  
pp. 1604-1608 ◽  
Author(s):  
Klaus Heuner ◽  
Claudia Dietrich ◽  
Carina Skriwan ◽  
Michael Steinert ◽  
Jörg Hacker
Keyword(s):  
Electron Microscopy ◽  
Western Blot ◽  
Mutant Strain ◽  
Legionella Pneumophila ◽  
Blot Analysis ◽  
Type Strain ◽  
Wild Type Strain ◽  
Kanamycin Resistance ◽  
Wild Type ◽  
Kanamycin Resistance Cassette

ABSTRACT The fliA gene of Legionella pneumophila encoding the alternative σ28 factor was inactivated by introducing a kanamycin resistance cassette. Electron microscopy and Western blot analysis revealed that the fliA mutant strain is aflagellate and expresses no flagellin. Reporter gene assays indicated that the flaA promoter is not active in the fliA mutant strain. The fliA mutant strain multiplied less effectively in coculture with amoebae than the wild-type strain and was not able to replicate in coculture with Dictyostelium discoideum.

scholarly journals Mutation in the peb1A Locus ofCampylobacter jejuni Reduces Interactions with Epithelial Cells and Intestinal Colonization of Mice

1998 ◽  
Vol 66 (3) ◽  
pp. 938-943 ◽  
Author(s):  
Zhiheng Pei ◽  
Christophe Burucoa ◽  
Bernadette Grignon ◽  
Shahida Baqar ◽  
Xiao-Zhe Huang ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Type Strain ◽  
Wild Type Strain ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Kanamycin Resistance ◽  
Intestinal Colonization ◽  
Wild Type ◽  
Cell Binding ◽  
Binding Factor

ABSTRACT Campylobacter jejuni is one of the leading causes of bacterial diarrhea throughout the world. We previously found that PEB1 is a homolog of cluster 3 binding proteins of bacterial ABC transporters and that a C. jejuni adhesin, cell-binding factor 1 (CBF1), if not identical to, contains PEB1. A single protein migrating at approximately 27 to 28 kDa was recognized by anti-CBF1 and anti-PEB1. To determine the role that the operon encoding PEB1 plays inC. jejuni adherence, peb1A, the gene encoding PEB1, was disrupted in strain 81-176 by insertion of a kanamycin resistance gene through homologous recombination. Inactivation of this operon completely abolished expression of CBF1, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting. In comparison to the wild-type strain, the mutant strain showed 50- to 100-fold less adherence to and 15-fold less invasion of epithelial cells in culture. Mouse challenge studies showed that the rate and duration of intestinal colonization by the mutant were significantly lower and shorter than with the wild-type strain. In summary, PEB1 is identical to a previously identified cell-binding factor, CBF1, in C. jejuni, and the peb1A locus plays an important role in epithelial cell interactions and in intestinal colonization in a mouse model.

scholarly journals Osmoregulated periplasmic glucans of Salmonella enterica serovar Typhimurium are required for optimal virulence in mice

Microbiology ◽  
2009 ◽  
Vol 155 (1) ◽  
pp. 229-237 ◽  
Author(s):  
Arvind A. Bhagwat ◽  
Won Jun ◽  
Liu Liu ◽  
Porteen Kannan ◽  
Mahesh Dharne ◽  
...  
Keyword(s):  
Salmonella Enterica ◽  
Type Strain ◽  
Wild Type Strain ◽  
Gene Cassette ◽  
Growth Conditions ◽  
Kanamycin Resistance ◽  
Wild Type ◽  
Serovar Typhimurium ◽  
Resistance Gene Cassette ◽  
Reduced Rate

We purified osmoregulated periplasmic glucans (OPGs) from Salmonella enterica serovar Typhimurium and found them to be composed of 100 % glucose with 2-linked glucose as the most abundant residue, with terminal glucose, 2,3-linked and 2,6-linked glucose also present in high quantities. The two structural genes for OPG biosynthesis, opgG and opgH, form a bicistronic operon, and insertion of a kanamycin resistance gene cassette into this operon resulted in a strain devoid of OPGs. The opgGH mutant strain was impaired in motility and growth under low osmolarity conditions. The opgGH mutation also resulted in a 2 log increase in the LD50 in mice compared to the wild-type strain SL1344. Inability to synthesize OPGs had no significant impact on the organism's lipopolysaccharide pattern or its ability to survive antimicrobial peptides-, detergent-, pH- and nutrient-stress conditions. We observed that the opgGH-defective strain respired at a reduced rate under acidic growth conditions (pH 5.0) and had lower ATP levels compared to the wild-type strain. These data indicate that OPGs of S. Typhimurium contribute towards mouse virulence as well as growth and motility under low osmolarity growth conditions.

scholarly journals ATP-Binding Cassette Transport System Involved in Regulation of Morphological Differentiation in Response to Glucose in Streptomyces griseus

2002 ◽  
Vol 184 (1) ◽  
pp. 91-103 ◽  
Author(s):  
Jeong-Woo Seo ◽  
Yasuo Ohnishi ◽  
Aiko Hirata ◽  
Sueharu Horinouchi
Keyword(s):  
Transport System ◽  
Type Strain ◽  
Wild Type Strain ◽  
Streptomyces Griseus ◽  
Aerial Mycelium ◽  
Transcriptional Analysis ◽  
Atp Binding ◽  
Wild Type ◽  
Chromosomal Dna ◽  
Atp Binding Cassette

ABSTRACT Streptomyces griseus NP4, which was derived by UV mutagenesis from strain IFO13350, showed a bald and wrinkled colony morphology in response to glucose. Mutant NP4 formed ectopic septa at intervals along substrate hyphae, and each of the compartments developed into a spore which was indistinguishable from an aerial spore in size, shape, and thickness of the spore wall and in susceptibility to lysozyme and heat. The ectopic spores of NP4 formed in liquid medium differed from “submerged spores” in lysozyme sensitivity. Shotgun cloning experiments with a library of the chromosomal DNA of the parental strain and mutant NP4 as the host gave rise to DNA fragments giving two different phenotypes; one complementing the bald phenotype of the host, and the other causing much severe wrinkled morphology in the host. Subcloning identified a gene (dasR) encoding a transcriptional repressor belonging to the GntR family that was responsible for the reversal of the bald phenotype and a gene (dasA) encoding a lipoprotein probably serving as a substrate-binding protein in an ATP-binding cassette (ABC) transport system that was responsible for the severe wrinkled morphology. These genes were adjacent but divergently encoded. Two genes, named dasB and dasC, encoding a membrane-spanning protein were present downstream of dasA, which suggested that dasRABC comprises a gene cluster for an ABC transporter, probably for sugar import. dasR was transcribed actively during vegetative growth, and dasA was transcribed just after commencement of aerial hypha formation and during sporulation, indicating that both were developmentally regulated. Transcriptional analysis and direct sequencing of dasRA in mutant NP4 suggested a defect of this mutant in the regulatory system to control the expression of these genes. Introduction of multicopies of dasA into the wild-type strain caused ectopic septation in very young substrate hyphae after only 1 day of growth and subsequent sporulation in response to glucose. The ectopic spores of the wild type had a thinner wall than those of mutant NP4, in agreement with the observation that the former was sensitive to lysozyme and heat. Disruption of the chromosomal dasA or dasR in the wild-type strain resulted in growth as substrate mycelium, suggesting an additional role of these genes in aerial mycelium formation. The ectopic septation and sporulation in mutant NP4 and the wild-type strain carrying multicopies of dasA were independent of a microbial hormone, A-factor (2-isocapryloyl-3R-hydroxymethyl-γ-butyrolactone), that acts as a master switch of aerial mycelium formation and secondary metabolism.

scholarly journals Mycobacterium smegmatis d-Alanine Racemase Mutants Are Not Dependent on d-Alanine for Growth

2002 ◽  
Vol 46 (1) ◽  
pp. 47-54 ◽  
Author(s):  
Ofelia Chacon ◽  
Zhengyu Feng ◽  
N. Beth Harris ◽  
Nancy E. Cáceres ◽  
L. Garry Adams ◽  
...  
Keyword(s):  
Cell Wall ◽  
Mutant Strain ◽  
Mycobacterium Smegmatis ◽  
Type Strain ◽  
Wild Type Strain ◽  
Kanamycin Resistance ◽  
Alanine Racemase ◽  
Wild Type ◽  
Peptidoglycan Biosynthesis ◽  
Insertional Inactivation

ABSTRACT Mycobacterium smegmatis is a fast-growing nonpathogenic species particularly useful in studying basic cellular processes of relevance to pathogenic mycobacteria. This study focused on the d-alanine racemase gene (alrA), which is involved in the synthesis of d-alanine, a basic component of peptidoglycan that forms the backbone of the cell wall. M. smegmatis alrA null mutants were generated by homologous recombination using a kanamycin resistance marker for insertional inactivation. Mutants were selected on Middlebrook medium supplemented with 50 mM d-alanine and 20 μg of kanamycin per ml. These mutants were also able to grow in standard and minimal media without d-alanine, giving rise to colonies with a drier appearance and more-raised borders than the wild-type strain. The viability of the mutants and independence of d-alanine for growth indicate that inactivation of alrA does not impose an auxotrophic requirement for d-alanine, suggesting the existence of a new pathway of d-alanine biosynthesis in M. smegmatis. Biochemical analysis demonstrated the absence of any detectable d-alanine racemase activity in the mutant strains. In addition, the alrA mutants displayed hypersusceptibility to the antimycobacterial agent d-cycloserine. The MIC of d-cycloserine for the mutant strain was 2.56 μg/ml, 30-fold less than that for the wild-type strain. Furthermore, this hypersusceptibility was confirmed by the bactericidal action of d-cycloserine on broth cultures. The kinetic of killing for the mutant strain followed the same pattern as that for the wild-type strain, but at a 30-fold-lower drug concentration. This effect does not involve a change in the permeability of the cell wall by this drug and is consistent with the identification of d-alanine racemase as a target of d-cycloserine. This outcome is of importance for the design of novel antituberculosis drugs targeting peptidoglycan biosynthesis in mycobacteria.

scholarly journals High-Pressure-Induced Sublethal Injuries of Food Pathogens—Microscopic Assessment

Foods ◽  
2021 ◽  
Vol 10 (12) ◽  
pp. 2940
Author(s):  
Justyna Nasiłowska ◽  
Aleksandra Kocot ◽  
Paulina Natalia Osuchowska ◽  
Barbara Sokołowska
Keyword(s):  
Electron Microscopy ◽  
Type Strain ◽  
Wild Type Strain ◽  
Physiological State ◽  
Fluorescent Microscopy ◽  
Food Preservation ◽  
Epifluorescence Microscopy ◽  
Wild Type ◽  
E Coli ◽  
Percentage Ratio

High Hydrostatic Pressure (HHP) technology is considered an alternative method of food preservation. Nevertheless, the current dogma is that HHP might be insufficient to preserve food lastingly against some pathogens. Incompletely damaged cells can resuscitate under favorable conditions, and they may proliferate in food during storage. This study was undertaken to characterize the extent of sublethal injuries induced by HHP (300–500 MPa) on Escherichia coli and Listeria inncua strains. The morphological changes were evaluated using microscopy methods such as Scanning Electron Microscopy (SEM), Transmission Electron Microscopy (TEM), and Epifluorescence Microscopy (EFM). The overall assessment of the physiological state of tested bacteria through TEM and SEM showed that the action of pressure on the structure of the bacterial membrane was almost minor or unnoticeable, beyond the L. innocua wild-type strain. However, alterations were observed in subcellular structures such as the cytoplasm and nucleoid for both L. innocua and E. coli strains. More significant changes after the HHP of internal structures were reported in the case of wild-type strains isolated from raw juice. Extreme condensation of the cytoplasm was observed, while the outline of cells was intact. The percentage ratio between alive and injured cells in the population was assessed by fluorescent microscopy. The results of HHP-treated samples showed a heterogeneous population, and red cell aggregates were observed. The percentage ratio of live and dead cells (L/D) in the L. innocua collection strain population was higher than in the case of the wild-type strain (69%/31% and 55%/45%, respectively). In turn, E. coli populations were characterized with a similar L/D ratio. Half of the cells in the populations were distinguished as visibly fluorescing red. The results obtained in this study confirmed sublethal HHP reaction on pathogens cells.

scholarly journals Identification of Novel Adhesins from Group B Streptococci by Use of Phage Display Reveals that C5a Peptidase Mediates Fibronectin Binding

2002 ◽  
Vol 70 (6) ◽  
pp. 2869-2876 ◽  
Author(s):  
Christiane Beckmann ◽  
Joshua D. Waggoner ◽  
Theresa O. Harris ◽  
Glen S. Tamura ◽  
Craig E. Rubens
Keyword(s):  
Phage Display ◽  
Type Strain ◽  
Wild Type Strain ◽  
Host Cells ◽  
Dependent Manner ◽  
Wild Type ◽  
Chromosomal Dna ◽  
Group B Streptococci ◽  
A Genome ◽  
Group B

ABSTRACT Group B streptococci (GBS) are a major cause of pneumonia, sepsis, and meningitis in newborns and infants. GBS initiate infection of the lung by colonizing mucosal surfaces of the respiratory tract; adherence of the bacteria to host cells is presumed to be the initial step in and prerequisite for successful colonization (G. S. Tamura, J. M. Kuypers, S. Smith, H. Raff, and C. E. Rubens, Infect. Immun. 62:2450-2458, 1994). We have performed a genome-wide screen to identify novel genes of GBS that mediate adherence to fibronectin. A shotgun phage display library was constructed from chromosomal DNA of a serotype Ia GBS strain and affinity selected on immobilized fibronectin. DNA sequence analysis of different clones identified 19 genes with homology to known bacterial adhesin genes, virulence genes, genes involved in transport or metabolic processes, and genes with yet-unknown function. One of the isolated phagemid clones showed significant homology to the gene (scpB) for the GBS C5a peptidase, a surface-associated serine protease that specifically cleaves the complement component C5a, a chemotaxin for polymorphonuclear leukocytes. In this work we have demonstrated that affinity-purified recombinant ScpB and a peptide ScpB fragment (ScpB-PDF), similar to the peptide identified in the phagemid, bound fibronectin in a concentration-dependent manner. Adherence assays to fibronectin were performed, comparing an isogenic scpB mutant to the wild-type strain. Approximately 50% less binding was observed with the mutant than with the wild-type strain. The mutant phenotype could be fully restored by in trans complementation of the mutant with the cloned wild-type scpB gene, providing further evidence for the role of ScpB in fibronectin adherence. Our results suggest that C5a peptidase is a bifunctional protein, which enzymatically cleaves C5a and mediates adherence to fibronectin. Since binding of fibronectin has been implicated in attachment and invasion of eukaryotic cells by streptococci, our results may imply a second important role for this surface protein in the pathogenesis of GBS infections.

Growth properties of afolAnull mutant ofEscherichia coliK12

1999 ◽  
Vol 45 (3) ◽  
pp. 191-200 ◽  
Author(s):  
Muriel B Herrington ◽  
Neema T Chirwa
Keyword(s):  
Escherichia Coli ◽  
Dihydrofolate Reductase ◽  
Type Strain ◽  
Wild Type Strain ◽  
De Novo ◽  
Kanamycin Resistance ◽  
Wild Type ◽  
Coding Region ◽  
Growth Properties ◽  
One Carbon Metabolism

In Escherichia coli, dihydrofolate reductase is required for both the de novo synthesis of tetrahydrofolate and the recycling of dihydrofolate produced during the synthesis of thymidylate. The coding region of the dihydrofolate reductase gene, folA, was replaced with a kanamycin resistance determinant. Unlike earlier deletions, this mutation did not disrupt flanking genes. When the mutation was transferred into a wild-type strain and a thymidine- (thy) requiring strain, the resulting strains were viable but slow growing on rich medium. Both synthesized less folate than their parents, as judged by the incorporation of radioactive para-aminobenzoic acid. The derivative of the wild-type strain did not grow on any defined minimal media tested. In contrast, the derivative of the thy-requiring strain grew slowly on minimal medium with thy but exhibited auxotrophies on some combinations of supplements. These results suggest that when folates are limited, they can be distributed appropriately to folate-dependent biosynthetic reactions only under some conditions. Key words: dihydrofolate reductase, Escherichia coli, biosynthesis, folates, one-carbon metabolism.

scholarly journals Long-Chain Aldehyde Dehydrogenase That Participates in n-Alkane Utilization and Wax Ester Synthesis in Acinetobacter sp. Strain M-1

2000 ◽  
Vol 66 (8) ◽  
pp. 3481-3486 ◽  
Author(s):  
Takeru Ishige ◽  
Akio Tani ◽  
Yasuyoshi Sakai ◽  
Nobuo Kato
Keyword(s):  
Amino Acid ◽  
Aldehyde Dehydrogenase ◽  
Type Strain ◽  
Wild Type Strain ◽  
Wax Ester ◽  
Wild Type ◽  
Chromosomal Dna ◽  
Ester Formation ◽  
Long Chain ◽  
Chain Aldehyde

ABSTRACT A long-chain aldehyde dehydrogenase, Ald1, was found in a soluble fraction of Acinetobacter sp. strain M-1 cells grown onn-hexadecane as a sole carbon source. The gene (ald1) was cloned from the chromosomal DNA of the bacterium. The open reading frame of ald1 was 1,512 bp long, corresponding to a protein of 503 amino acid residues (molecular mass, 55,496 Da), and the deduced amino acid sequence showed high similarity to those of various aldehyde dehydrogenases. Theald1 gene was stably expressed in Escherichia coli, and the gene product (recombinant Ald1 [rAld1]) was purified to apparent homogeneity by gel electrophoresis. rAld1 showed enzyme activity toward n-alkanals (C4 to C14), with a preference for longer carbon chains within the tested range; the highest activity was obtained with tetradecanal. Theald1 gene was disrupted by homologous recombination on theAcinetobacter genome. Although the ald1disruptant (ald1Δ) strain still had the ability to grow on n-hexadecane to some extent, its aldehyde dehydrogenase activity toward n-tetradecanal was reduced to half the level of the wild-type strain. Under nitrogen-limiting conditions, the accumulation of intracellular wax esters in the ald1Δ strain became much lower than that in the wild-type strain. These and other results imply that a soluble long-chain aldehyde dehydrogenase indeed plays important roles both in growth on n-alkane and in wax ester formation in Acinetobacter sp. strain M-1.

scholarly journals Relationship of plcR-Regulated Factors to BacillusEndophthalmitis Virulence

2003 ◽  
Vol 71 (6) ◽  
pp. 3116-3124 ◽  
Author(s):  
Michelle C. Callegan ◽  
Scott T. Kane ◽  
D. Clay Cochran ◽  
Michael S. Gilmore ◽  
Myriam Gominet ◽  
...  
Keyword(s):  
Inflammatory Cell ◽  
Wild Type Strain ◽  
Kanamycin Resistance ◽  
Retinal Function ◽  
Deficient Mutant ◽  
Wild Type ◽  
Global Regulator ◽  
Kanamycin Resistance Cassette ◽  
Insertional Inactivation ◽  
Relationship Of

ABSTRACT The explosive, destructive course of Bacillus endophthalmitis has been attributed to the production of toxins during infection. In this study we analyzed the contribution of toxins controlled by the global regulator plcR to the pathogenesis of experimental Bacillus endophthalmitis. Isogenic plcR-deficient mutants of Bacillus cereus and Bacillus thuringiensis were constructed by insertional inactivation of plcR by the kanamycin resistance cassette, aphA3. Rabbit eyes were injected intravitreally with approximately 100 CFU of wild-type B. cereus or B. thuringiensis or a plcR-deficient mutant. The evolution of endophthalmitis resulting from each plcR-deficient mutant was considerably slower than that caused by each wild-type strain. Retinal function was not eliminated until 42 h postinfection in rabbits with endophthalmitis caused by the plcR-deficient mutants, whereas wild-type infections resulted in a complete loss of retinal function within 18 h. The intraocular inflammatory cell influx and retinal destruction in plcR-deficient endophthalmitis approached the severity observed in wild-ype infections, but not until 36 h postinfection. Gross and histological examinations of eyes infected with plcR mutants demonstrated that the anterior and posterior segment changes were muted compared to the changes observed in eyes infected with the wild types. The loss of plcR-regulated factors significantly attenuated the severity of Bacillus endophthalmitis. The results therefore suggest that plcR may represent a target for which adjunct therapies could be designed for the prevention of blindness during Bacillus endophthalmitis.

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