Construction of a Vibrio splendidus Mutant Lacking the Metalloprotease Gene vsm by Use of a Novel Counterselectable Suicide Vector

ABSTRACT Vibrio splendidus is a dominant culturable Vibrio in seawater, and strains related to this species are also associated with mortality in a variety of marine animals. The determinants encoding the pathogenic properties of these strains are still poorly understood; however, the recent sequencing of the genome of V. splendidus LGP32, an oyster pathogen, provides an opportunity to decipher the basis of the virulence properties by disruption of candidate genes. We developed a novel suicide vector based on the pir-dependent R6K replicative origin, which potentially can be transferred by RP4-based conjugation to any Vibrio strain and which also carries the plasmid F toxin ccdB gene under control of the P BAD promoter. We demonstrated that this genetic system allows efficient counterselection of integrated plasmids in the presence of arabinose in both V. splendidus and Vibrio cholerae and thus permits efficient markerless allelic replacement in these species. We used this technique to construct several mutants of V. splendidus LGP32, including a derivative with a secreted metalloprotease gene, vsm, deleted. We found that this gene is essential for LGP32 extracellular product toxicity when the extracellular products are injected into oysters but is not necessary for virulence of bacteria in the oyster infection model when bacteria are injected.

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A Genetic System for Clostridium ljungdahlii: a Chassis for Autotrophic Production of Biocommodities and a Model Homoacetogen

Applied and Environmental Microbiology ◽

10.1128/aem.02891-12 ◽

2012 ◽

Vol 79 (4) ◽

pp. 1102-1109 ◽

Cited By ~ 118

Author(s):

Ching Leang ◽

Toshiyuki Ueki ◽

Kelly P. Nevin ◽

Derek R. Lovley

Keyword(s):

Ethanol Production ◽

Gene Deletion ◽

Genetic Manipulation ◽

Electron Flow ◽

Genetic System ◽

Content Type ◽

Suicide Vector ◽

Double Deletion ◽

Clostridium Ljungdahlii ◽

Genomic Studies

ABSTRACTMethods for genetic manipulation ofClostridium ljungdahliiare of interest because of the potential for production of fuels and other biocommodities from carbon dioxide via microbial electrosynthesis or more traditional modes of autotrophy with hydrogen or carbon monoxide as the electron donor. Furthermore, acetogenesis plays an important role in the global carbon cycle. Gene deletion strategies required for physiological studies ofC. ljungdahliihave not previously been demonstrated. An electroporation procedure for introducing plasmids was optimized, and four different replicative origins for plasmid propagation inC. ljungdahliiwere identified. Chromosomal gene deletion via double-crossover homologous recombination with a suicide vector was demonstrated initially with deletion of the gene for FliA, a putative sigma factor involved in flagellar biogenesis and motility inC. ljungdahlii. Deletion offliAyielded a strain that lacked flagella and was not motile. To evaluate the potential utility of gene deletions for functional genomic studies and to redirect carbon and electron flow, the genes for the putative bifunctional aldehyde/alcohol dehydrogenases,adhE1andadhE2, were deleted individually or together. Deletion ofadhE1, but notadhE2, diminished ethanol production with a corresponding carbon recovery in acetate. The double deletion mutant had a phenotype similar to that of theadhE1-deficient strain. Expression ofadhE1intranspartially restored the capacity for ethanol production. These results demonstrate the feasibility of genetic investigations of acetogen physiology and the potential for genetic manipulation ofC. ljungdahliito optimize autotrophic biocommodity production.

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Biotyping and Virulence Properties of Skin Isolates ofCandida parapsilosis

Journal of Clinical Microbiology ◽

10.1128/jcm.37.11.3481-3486.1999 ◽

1999 ◽

Vol 37 (11) ◽

pp. 3481-3486 ◽

Cited By ~ 58

Author(s):

Flavia De Bernardis ◽

Francesca Mondello ◽

Rosario San Millàn ◽

Josè Pontòn ◽

Antonio Cassone

Keyword(s):

Enzyme Activity ◽

Candida Parapsilosis ◽

Systemic Infection ◽

Infection Model ◽

Electrophoretic Karyotype ◽

Aspartyl Protease ◽

Highly Pathogenic ◽

Mucosal Disease ◽

Organ Invasion ◽

Virulence Properties

The biotype and virulence of skin isolates of Candida parapsilosis were compared with blood isolates of the same fungus. Morphotype, resistotype, and electrophoretic karyotype determinations did not reveal any special cluster with a unique or dominant pathogenic feature among all of the isolates, regardless of their source. However, all cutaneous isolates had uniformly elevated secretory aspartyl-protease (Sap) activity, more than four times higher than the enzyme activity of the blood isolates. They were also highly vaginopathic in a rat vaginitis model, being significantly more virulent than blood isolates in this infection model. In contrast, skin isolates were nonpathogenic in systemic infection of cyclophosphamide-immunodepressed mice, while some blood isolates were, in this model, highly pathogenic (median survival time, 2 days, with internal organ invasion at autopsy). Finally, skin isolates did not differ, as a whole, from blood isolates in their adherence to plastic. This property was associated with a morphotype, as defined by a colony with continuous fringe, which was present among both skin and blood isolates. While confirming the genetic heterogenicity of C. parapsilosis, our data strongly suggest that the potential of this fungus to cause mucosal disease is associated with Sap production and is substantially distinct from that of systemic invasion.

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Kanamycin Resistance Cassette for Genetic Manipulation of Treponema denticola

Applied and Environmental Microbiology ◽

10.1128/aem.00478-15 ◽

2015 ◽

Vol 81 (13) ◽

pp. 4329-4338 ◽

Cited By ~ 7

Author(s):

Yuebin Li ◽

John Ruby ◽

Hui Wu

Keyword(s):

Genetic Manipulation ◽

Periodontal Diseases ◽

Genetic System ◽

Kanamycin Resistance ◽

Treponema Denticola ◽

Oral Pathogen ◽

Content Type ◽

Kanamycin Resistance Cassette ◽

Allelic Replacement ◽

Kanamycin Cassette

ABSTRACTTreponema denticolahas been recognized as an important oral pathogen of the “red complex” bacterial consortium that is associated with the pathogenesis of endodontal and periodontal diseases. However, little is known about the virulence ofT. denticoladue to its recalcitrant genetic system. The difficulty in genetically manipulating oral spirochetes is partially due to the lack of antibiotic resistance cassettes that are useful for gene complementation following allelic replacement mutagenesis. In this study, a kanamycin resistance cassette was identified and developed for the genetic manipulation ofT. denticolaATCC 35405. Compared to the widely usedermF-ermAMcassette, the kanamycin cassette used in the transformation experiments gave rise to additional antibiotic-resistantT. denticolacolonies. The kanamycin cassette is effective for allelic replacement mutagenesis as demonstrated by inactivation of two open reading frames ofT. denticola, TDE1430 and TDE0911. In addition, the cassette is also functional intrans-chromosomal complementation. This was determined by functional rescue of a periplasmic flagellum (PF)-deficient mutant that had theflgEgene coding for PF hook protein inactivated. The integration of the full-lengthflgEgene into the genome of theflgEmutant rescued all of the defects associated with theflgEmutant that included the lack of PF filament and spirochetal motility. Taken together, we demonstrate that the kanamycin resistance gene is a suitable cassette for the genetic manipulation ofT. denticolathat will facilitate the characterization of virulence factors attributed to this important oral pathogen.

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Pathogenic Implication of a Fibrinogen-Binding Protein of Staphylococcus epidermidis in a Rat Model of Intravascular-Catheter-Associated Infection

Infection and Immunity ◽

10.1128/iai.01741-06 ◽

2007 ◽

Vol 75 (6) ◽

pp. 2991-2995 ◽

Cited By ~ 18

Author(s):

Beining Guo ◽

Xu Zhao ◽

Yaoguo Shi ◽

Demei Zhu ◽

Yingyuan Zhang

Keyword(s):

Staphylococcus Epidermidis ◽

Binding Protein ◽

Venous Catheter ◽

Infection Model ◽

Intravascular Catheter ◽

Erythromycin Resistance ◽

Central Venous ◽

Gene Encoding ◽

Allelic Replacement ◽

Fibrinogen Binding

ABSTRACT The involvement of Fbe, a fibrinogen-binding protein of Staphylococcus epidermidis, in the pathogenesis of catheter-associated infection was investigated. An fbe (gene encoding Fbe protein) mutant was constructed by allelic replacement, wherein an erythromycin resistance gene replaced a portion of the A region of fbe. Meanwhile, a rat central venous catheter (CVC) infection model was established to assess the importance of Fbe in the pathogenesis of CVC-associated infection due to S. epidermidis. Fbe-positive S. epidermidis strain HB was significantly more likely to cause a CVC-associated infection resulting in bacteremia and metastatic disease than its isogenic Fbe-deficient mutant (100% versus 20%, P < 0.01). These results confirm the importance of adherence associated with Fbe in the pathogenesis of CVC-associated infection caused by S. epidermidis.

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Surface Protein EF3314 Contributes to Virulence Properties of Enterococcus faecalis

The International Journal of Artificial Organs ◽

10.1177/039139880903200910 ◽

2009 ◽

Vol 32 (9) ◽

pp. 611-620 ◽

Cited By ~ 12

Author(s):

Roberta Creti ◽

Francesca Fabretti ◽

Stefanie Koch ◽

Johannes Huebner ◽

Danielle A. Garsin ◽

...

Keyword(s):

Amino Acids ◽

Enterococcus Faecalis ◽

Polyclonal Antibodies ◽

Gc Content ◽

Surface Protein ◽

Animal Origin ◽

Infection Model ◽

Group B Streptococci ◽

Group B ◽

Virulence Properties

Purpose Identification of putative new virulence factors as additional targets for therapeutic approaches alternative to antibiotic treatment of multi-resistant enterococcal infections. Methods The EF3314 gene, coding for a putative surface-exposed antigen, was identified by the analysis of the Enterococcus faecalis V583 genome for LPXTG-motif cell wall anchor surface protein genes. A non-polar EF3314 gene deletion mutant in the E. faecalis 12030 human clinical isolate was obtained. The wild type and the isogenic mutant strain were investigated for biofilm formation, adherence to Hela cells, survival in human macrophages and a Caenorhabditis elegans infection model. The aminoterminal portion of the EF3314 protein was overexpressed in E. coli to obtain mouse polyclonal antibodies for use in Western blotting and immunolocalization experiments. Results The EF3314 gene has an unusually high GC content (46.88% vs. an average of 37.5% in the E. faecalis chromosome) and encodes a protein of 1744 amino acids that presents a series of 14 imperfect repeats of 90 amino acids covering almost the entire length of the protein. Its global organization is similar to the alpha-like protein family of group B streptococci, enterococcal surface protein Esp and bio film associated protein Bap from S. aureus. The EF3314 gene was always present and specific for E. faecalis strains of human, food and animal origin. Differences in size depended on variable numbers of repeats in the repetitive region. Conclusions EF3314 is a newly described, surface exposed protein that contributes to the virulence properties of E. faecalis.

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Generation and Characterization of a Defined Mutant of Streptococcus suis Lacking Suilysin

Infection and Immunity ◽

10.1128/iai.69.4.2732-2735.2001 ◽

2001 ◽

Vol 69 (4) ◽

pp. 2732-2735 ◽

Cited By ~ 57

Author(s):

Andrew G. Allen ◽

Steven Bolitho ◽

Heather Lindsay ◽

Shahid Khan ◽

Clare Bryant ◽

...

Keyword(s):

Parental Strain ◽

Porcine Model ◽

Systemic Infection ◽

Streptococcus Suis ◽

Infection Model ◽

Gene Encoding ◽

European Isolate ◽

Mouse Infection Model ◽

Allelic Replacement

ABSTRACT A defined allelic-replacement mutant of the sly gene, encoding a thiol-activated cytolysin, from a European isolate ofStreptococcus suis serotype 2 was generated and characterized. Unlike the parental strain, it is nonhemolytic, noncytotoxic for cultured macrophage-like cells, avirulent in a mouse infection model, yet only slightly attenuated in a porcine model of systemic infection.

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Bacillus cereus Fur regulates iron metabolism and is required for full virulence

Microbiology ◽

10.1099/mic.0.27744-0 ◽

2005 ◽

Vol 151 (2) ◽

pp. 569-577 ◽

Cited By ~ 46

Author(s):

Duncan R. Harvie ◽

Susana Vílchez ◽

James R. Steggles ◽

David J. Ellar

Keyword(s):

Bacillus Cereus ◽

Kanamycin Resistance ◽

Infection Model ◽

Predicted Amino Acid Sequence ◽

Dependent Manner ◽

Genome Data ◽

Kanamycin Resistance Cassette ◽

Allelic Replacement ◽

Cloned Gene ◽

Null Strain

A homologue of the Bacillus subtilis fur gene was identified in Bacillus cereus and characterized. The predicted amino acid sequence of the cloned gene was found to be highly similar to other members of the Fur family of transcriptional regulators. The B. cereus fur gene was shown to partially complement an Escherichia coli fur mutant. Purified B. cereus Fur bound specifically to a 19 bp DNA sequence homologous to the B. subtilis Fur box in a metal-dependent manner. Analysis of the available B. cereus genome data identified a number of genes which contain predicted Fur box sequences in the promoter region. Many of these genes are predicted to play a role in bacterial iron uptake and metabolism, but several have also been implicated as having a role in virulence. Fur and iron regulation of a siderophore biosynthesis operon was confirmed in a β-galactosidase assay. A B. cereus fur null strain was constructed by allelic replacement of the chromosomal gene with a copy disrupted with a kanamycin resistance cassette. The Δfur mutant was found to constitutively express siderophores, to accumulate iron intracellularly to a level approximately threefold greater than the wild-type, and to be hypersensitive to hydrogen peroxide. In an insect infection model, the virulence of the fur null strain was found to be significantly attenuated, highlighting the essential role played by Fur in the virulence of this pathogen.

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Isolation and Characterization of a sigBDeletion Mutant of Staphylococcus aureus

Infection and Immunity ◽

10.1128/iai.67.7.3667-3669.1999 ◽

1999 ◽

Vol 67 (7) ◽

pp. 3667-3669 ◽

Cited By ~ 75

Author(s):

R. O. Nicholas ◽

Tong Li ◽

D. McDevitt ◽

Andrea Marra ◽

S. Sucoloski ◽

...

Keyword(s):

Staphylococcus Aureus ◽

Sigma Factor ◽

No Reduction ◽

Infection Model ◽

Factor B ◽

Allelic Replacement ◽

Isolation And Characterization ◽

Sigma Factor B

ABSTRACT The sigB gene of Staphylococcus aureus, coding for the alternate sigma factor B, has been deleted by allelic replacement mutagenesis. The mutant grew as well as the parent in vitro, although it was deficient in clumping factor, coagulase, and pigment. In two murine and one rat infection model the mutant showed no reduction in virulence.

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