Transformation of vegetative cells of Bacillus anthracis with plasmid DNA

Quinn, Conrad P.; Dancer, B. N.

doi:10.1099/00221287-136-7-1211

Cited by 32 publications

(20 citation statements)

References 28 publications

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“…All B. anthracis strains used in this study are derived from the Sterne strain 34F2 and are listed in Table 1. Electroporation of B. anthracis was performed as described previously (38). Transformants were selected on LB-1% glucose plates containing the appropriate antibiotics: 5 g/ml erythromycin (Fisher), 50 g/ml kanamycin sulfate (Jersey Lab Supply), or 10 g/ml tetracycline (Jersey Lab Supply).…”

Section: Methodsmentioning

confidence: 99%

Roles of Germination-Specific Lytic Enzymes CwlJ and SleB in Bacillus anthracis

2009

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The structural characteristics of a spore enable it to withstand stresses that typically kill a vegetative cell. Spores remain dormant until small molecule signals induce them to germinate into vegetative bacilli. Germination requires degradation of the thick cortical peptidoglycan by germination-specific lytic enzymes (GSLEs). Bacillus anthracis has four putative GSLEs, based upon sequence similarities with enzymes in other species: SleB, CwlJ1, CwlJ2, and SleL. In this study, the roles of SleB, CwlJ1, and CwlJ2 were examined. The expression levels of all three genes peak 3.5 h into sporulation. Genetic analysis revealed that, similar to other known GSLEs, none of these gene products are individually required for growth, sporulation, or triggering of germination. However, later germination events are affected in spores lacking CwlJ1 or SleB. Compared to the wild type, germinating spores without CwlJ1 suffer a delay in optical density loss and cortex peptidoglycan release. The absence of SleB also causes a delay in cortex fragment release. A double mutant lacking both SleB and CwlJ1 is completely blocked in cortex hydrolysis and progresses through outgrowth to produce colonies at a frequency 1,000-fold lower than that of the wild-type strain. A null mutation eliminating CwlJ2 has no effect on germination. High-performance liquid chromatography and mass spectroscopy analysis revealed that SleB is required for lytic transglycosylase activity. CwlJ1 also clearly participates in cortex hydrolysis, but its specific mode of action remains unclear. Understanding the lytic germination activities that naturally diminish spore resistance can lead to methods for prematurely inducing them, thus simplifying the process of treating contaminated sites.

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Section: Methodsmentioning

confidence: 99%

Roles of Germination-Specific Lytic Enzymes CwlJ and SleB in Bacillus anthracis

2009

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“…Plasmid constructs were verified after each step by restriction-enzyme digestion and sequencing. pDPV350 was prepared from E. coli INV110 (dam dcm) (Invitrogen) and was electroporated into B. anthracis Sterne as described previously (41). To select for the integration of the plasmid into the B. anthracis chromosome, a broth culture of the resulting strain was shifted to 39°C.…”

Section: Methodsmentioning

confidence: 99%

The Bacillus anthracis SleL (YaaH) Protein Is an N -Acetylglucosaminidase Involved in Spore Cortex Depolymerization

Lambert

Popham

2008

J Bacteriol

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Bacillus anthracis spores, the infectious agents of anthrax, are notoriously difficult to remove from contaminated areas because they are resistant to many eradication methods. These resistance properties are due to the spore's dehydration and dormancy and to the multiple protective layers surrounding the spore core, one of which is the cortex. In order for B. anthracis spores to germinate and resume growth, the cortex peptidoglycan must be depolymerized. This study reports on analyses of sleL (yaaH), which encodes a cortex-lytic enzyme. The inactivation of sleL does not affect vegetative growth, spore viability, or the initial stages of germination, including dipicolinic acid release. However, mutant spores exhibit a slight delay in the loss of optical density compared to that of wild-type spores. Mutants also retain more diaminopimelic acid and N-acetylmuramic acid during germination than wild-type spores, suggesting that the cortex peptidoglycan is not being hydrolyzed as rapidly. This finding is supported by high-pressure liquid chromatography analysis of the peptidoglycan structure used to confirm that SleL acts as an N-acetylglucosaminidase. When sleL is inactivated, the cortex peptidoglycan is not depolymerized into small muropeptides but instead is retained within the spore as large fragments. In the absence of the sleL-encoded N-acetylglucosaminidase, other cortex-lytic enzymes break down the cortex peptidoglycan sufficiently to allow rapid germination and outgrowth.

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“…(c) Synthesis of the I-SceI restriction enzyme directed by pBKJ223 resulted in the cleavage of the integrated pBKJ236 vector, creating a potent substrate for bacterial-host recombinational repair systems. Integrant colonies arising in the previous step were inoculated into LB medium plus 0.1% glucose and 5 g/ml erythromycin, grown with shaking overnight at 37°C, subcultured into fresh medium of the same composition, and used to prepare electrocompetent cells by the method of Quinn and Dancer (18). Plasmid DNA was prepared from SCS110(pBKJ223) using a spin mini-prep kit (QIAGEN, Inc., Valencia, CA).…”

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confidence: 99%

Routine Markerless Gene Replacement in Bacillus anthracis

2006

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An improved genetic tool suitable for routine markerless allelic exchange in Bacillus anthracis has been constructed. Its utility was demonstrated by the introduction of insertions, deletions, and missense mutations on the chromosome and plasmid pXO1 of the Sterne strain of B. anthracis.Bacillus anthracis, a gram-positive, spore-forming bacterium, is the causative agent of anthrax. Full virulence requires the production of a toxin and the formation of a protective capsule. The primary genes involved in the production of these two virulence factors are contained within two large nonessential plasmids, pXO1 and pXO2 (13). Distributed elsewhere on the two plasmids and on the chromosome are genes involved in regulating the expression of anthrax toxin, the capsule, and a number of other genes potentially involved in virulence (2, 6, 11). The entire genomic sequence of the Ames strain of B. anthracis has recently been determined, enabling the identification of interesting, potentially virulence-related genes by reverse genetics (19).The ideal mutation to initially introduce in such "genomemining" studies is an in-frame deletion of the candidate gene. Such a mutation avoids problems with polarity and other effects on the expression of surrounding genes, which accompany either insertion or less-precise deletion mutations and which can complicate interpretation. In addition, the ability to readily introduce missense mutations, which enables a determination of the effects of single-amino-acid substitutions, is necessary for more-sophisticated genetic analyses of structure-function relationships. Both of these types of desirable mutations are "markerless" in that they are not necessarily associated with a phenotype that can be selected or screened for during genetic manipulation (e.g., antibiotic resistance in the case of an insertion mutation). For B. anthracis, markerless gene replacements for some loci have been reported (3), but the methods by which these mutations have been isolated can be time and labor-intensive. This is due to the lack of a counterselection scheme, in which, by selecting for the loss of a plasmid vector, one can select for the second of two successive crossovers between such a vector and the chromosome in order to achieve gene replacement.An alternative to counterselection schemes involves the use of the intron-encoded homing restriction enzyme I-SceI. The ability of this enzyme, which recognizes an 18-bp sequence, to cleave an introduced site that is essentially unique in a genome has been exploited in the promotion of homologous recombination in organisms as diverse as bacteria, Drosophila, and other higher eukaryotes (4,20,22). In one case, the use of I-SceI in promoting allelic exchange in Escherichia coli has been reported (17). In such a scheme, the integration of a suicide plasmid by a cloned region of homology containing the desired genetic change results in one of the two crossovers required to effect allelic exchange. To promote the second, the synthesis of the I-SceI enzyme results in cl...

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Transformation of vegetative cells of Bacillus anthracis with plasmid DNA

Cited by 32 publications

References 28 publications

Roles of Germination-Specific Lytic Enzymes CwlJ and SleB in Bacillus anthracis

Roles of Germination-Specific Lytic Enzymes CwlJ and SleB in Bacillus anthracis

The Bacillus anthracis SleL (YaaH) Protein Is an N -Acetylglucosaminidase Involved in Spore Cortex Depolymerization

Routine Markerless Gene Replacement in Bacillus anthracis

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