Antibiotic-resistance cassettes for Bacillus subtilis

Guérout‐Fleury, Anne‐Marie; Shazand, Kamran; Frandsen, Niels; Stragier, Patrick

doi:10.1016/0378-1119(95)00652-4

Cited by 557 publications

(377 citation statements)

References 6 publications

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“…Mutant strains were constructed using an overlap-extension PCR technique (Kobayashi, 2007). Antibiotic-resistance cassettes were amplified by PCR using the primers pUC-F and pUC-R from pCBB31(cat), pAE41(erm), pDG780(kan) and pDG1726(spc) (Guerout-Fleury et al, 1995;Kobayashi et al, 1995). As ATCC 6051 has low competence, the mutant alleles were first introduced into strain 168 and then transferred into ATCC 6051 by transformation with chromosomal DNA from the newly generated 168 mutant strains.…”

Section: Methodsmentioning

confidence: 99%

kinA mRNA is missing a stop codon in the undomesticated Bacillus subtilis strain ATCC 6051

2008

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Several features distinguish laboratory and undomesticated strains of Bacillus subtilis. For example, unlike the laboratory strain 168, the undomesticated strain ATCC 6051 is deficient in sporulation in a rich sporulation medium, 2¾ SG. ATCC 6051 cannot induce transcription of the spoIIG operon, suggesting that this strain has a defect in initiation of sporulation. To determine the genetic difference between 168 and ATCC 6051, the DNA region responsible for the Spo " phenotype was transferred to strain 168. Genetic mapping and DNA sequencing analysis revealed that a stop codon (TAA) for kinA in 168 is replaced with Lys (TAT) in ATCC 6051, making the kinA open reading frame 201 bp longer in the undomesticated strain ATCC 6051. Introduction of kinA from strain 168 restored sporulation in ATCC 6051, indicating that the difference in kinA is responsible for the Spo " phenotype of ATCC 6051. A potential rindependent terminator is located upstream of a stop codon for the extended kinA open reading frame in ATCC 6051. Northern blot analysis showed that transcription of kinA terminated at this terminator, and kinA mRNA is missing a stop codon in ATCC 6051. Moreover, deletion of tmRNA suppresses the sporulation defect in ATCC 6051. These observations indicate that in ATCC 6051 the absence of a stop codon in kinA mRNA affects sporulation, probably by leading to rapid degradation of KinA via the trans-translation process. In ATCC 6051, the kinA mutation affects sporulation but not other Spo0A-dependent phenomena such as biofilm formation, which can be activated by a low level of Spo0A~P. This is due to the fact that KinA activity is kept low during the exponential phase via transcriptional and post-translational regulation. Thus, the stop-codon-less kinA probably affects only sporulation. DNA sequencing of 30 B. subtilis strains revealed that another strain also produces stop-codon-less kinA mRNA. This observation suggests that the lack of a stop codon for kinA mRNA may give rise to a selective advantage under certain conditions. INTRODUCTIONThe Gram-positive soil bacterium Bacillus subtilis makes spores in response to nutrient depletion. Initiation of sporulation is controlled by the multi-component phosphotransfer system, the phosphorelay (Hoch, 1991(Hoch, , 1993. In response to nutrient depletion, a histidine kinase phosphorylates itself, and then this phosphate residue is sequentially transferred to Spo0F, Spo0B and finally Spo0A. Spo0A is a member of a response-regulator family of transcriptional regulators, and phosphorylated Spo0A (Spo0A~P) activates transcription of early sporulation genes.Spo0A~P directly regulates about 120 genes required for various stationary-phase phenomena including sporulation, competence development, production of degradative enzymes, cannibalism and biofilm formation (Molle et al., 2003). Spo0A-regulated genes can be classified into two groups based on Spo0A dose dependency: low-threshold genes require a low level of Spo0A~P for activation or repression of transcription, whereas high-thres...

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Section: Methodsmentioning

confidence: 99%

kinA mRNA is missing a stop codon in the undomesticated Bacillus subtilis strain ATCC 6051

2008

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“…DNA extending 1662 bp downstream of the fusion point was cloned after restricting chromosomal DNA of the strain harbouring the original lacZ fusion with StuI. The complete sequence was provided by P. Glaser and allowed us to precisely inactivate the various genes of the region by marker replacement with antibiotic-resistance cassettes (Gué rout-Fleury et al, 1995). Cells were grown for approx.…”

Section: Cloning Of Spoiiqmentioning

confidence: 99%

spoIIQ, a forespore‐expressed gene required for engulfment in Bacillus subtilis

Fréhel

Stragier

1997

Molecular Microbiology

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128

135

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SummaryA crucial step in converting an actively growing Bacillus subtilis cell into a dormant spore is the formation of a cell within a cell. This unusual structure is created by a phagocytosis-like process in which the larger mother cell progressively engulfs the adjacent smaller forespore. Only mutations blocking engulfment at an early stage and affecting genes expressed in the mother cell have been identified. Here we describe a new locus, spoIIQ, which is transcribed in the forespore and which encodes a membrane-bound protein required at a late stage of engulfment. Immunofluorescence microscopy analysis have shown that SpoIIQ is initially targeted to the septum at the boundary between the two cells and then spreads around the entire membrane of the forespore. Septum targeting requires only the first 52 residues of SpoIIQ as well as unidentified forespore-specific components. Electron-microscopy studies of cells engineered to activate the mother-cell program of gene expression independently of the forespore indicate that other as yet uncharacterized genes are involved in engulfment and that this morphological process is driven from both sides of the forespore envelope.

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“…Two hundred microliters of BAC at 750 ppm (disinfected biofilms; note that BAC concentration approximates the industrially used concentration) or 200 μL of NaCl at 150 mM (untreated biofilm) was added in the wells. After 5 min of contact at 20°C, 200 μL of a 30 g/L saponin) was added in wells to neutralize biocide activity. Biofilms were then mechanically detached from slides and dispersed in 5 mL of the quenching solution.…”

mentioning

confidence: 99%

Bacterial swimmers that infiltrate and take over the biofilm matrix

Houry

Gohar

Deschamps

et al. 2012

Proc. Natl. Acad. Sci. U.S.A.

177

149

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Bacteria grow in either planktonic form or as biofilms, which are attached to either inert or biological surfaces. Both growth forms are highly relevant states in nature and of paramount scientific focus. However, interchanges between bacteria in these two states have been little explored. We discovered that a subpopulation of planktonic bacilli is propelled by flagella to tunnel deep within a biofilm structure. Swimmers create transient pores that increase macromolecular transfer within the biofilm. Irrigation of the biofilm by swimmer bacteria may improve biofilm bacterial fitness by increasing nutrient flow in the matrix. However, we show that the opposite may also occur (i.e., swimmers can exacerbate killing of biofilm bacteria by facilitating penetration of toxic substances from the environment). We combined these observations with the fact that numerous bacteria produce antimicrobial substances in nature. We hypothesized and proved that motile bacilli expressing a bactericide can also kill a heterologous biofilm population, Staphylococcus aureus in this case, and then occupy the newly created space. These findings identify microbial motility as a determinant of the biofilm landscape and add motility to the complement of traits contributing to rapid alterations in biofilm populations.bacterial ecology | biofilm disruption | motile subpopulations | antimicrobials | time-lapse confocal imaging

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Antibiotic-resistance cassettes for Bacillus subtilis

Cited by 557 publications

References 6 publications

kinA mRNA is missing a stop codon in the undomesticated Bacillus subtilis strain ATCC 6051

kinA mRNA is missing a stop codon in the undomesticated Bacillus subtilis strain ATCC 6051

spoIIQ, a forespore‐expressed gene required for engulfment in Bacillus subtilis

Bacterial swimmers that infiltrate and take over the biofilm matrix

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