pheS * , an effective host-genotype-independent counter-selectable marker for marker-free chromosome deletion in Bacillus amyloliquefaciens

Zhou, Changyu; Shi, Lingling; Ye, Bin; Feng, Haichao; Zhang, Ji; Zhang, Ruifu; Yan, Xin

doi:10.1007/s00253-016-7906-9

Cited by 46 publications

(30 citation statements)

References 44 publications

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“…The double‐complementary strain expressing mcpA and mcpC was constructed by integrating the two genes into the amyE locus of SQR9Δ 8mcp , following the experimental protocol reported by Zhou et al . (). The primers used are listed in Supporting Information Table S2.…”

Section: Methodsmentioning

confidence: 97%

Recognition of dominant attractants by key chemoreceptors mediates recruitment of plant growth‐promoting rhizobacteria

Feng

Zhang

et al. 2019

Environmental Microbiology

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Summary Chemotaxis to plant root exudates is supposed to be a prerequisite for efficient root colonization by rhizobacteria. This is a highly multifactorial process since root exudates are complex compound mixtures of which components are recognized by different chemoreceptors. Little information is available as to the key components in root exudates and their receptors that drive colonization related chemotaxis. We present here the first global assessment of this issue using the plant growth‐promoting rhizobacterium (PGPR) Bacillus velezensis SQR9 (formerly B. amyloliquefaciens). This strain efficiently colonizes cucumber roots, and here, we show that chemotaxis to cucumber root exudates was essential in this process. We conducted chemotaxis assays using cucumber root exudates at different concentrations, individual exudate components as well as recomposed exudates, taking into account their concentrations detected in root exudates. Results indicated that two key chemoreceptors, McpA and McpC, were essential for root exudate chemotaxis and root colonization. Both receptors possess a broad ligand range and recognize most of the exudate key components identified (malic, fumaric, gluconic and glyceric acids, Lys, Ser, Ala and mannose). The remaining six chemoreceptors did not contribute to exudate chemotaxis. This study provides novel insight into the evolution of the chemotaxis system in rhizobacteria.

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Section: Methodsmentioning

confidence: 97%

Recognition of dominant attractants by key chemoreceptors mediates recruitment of plant growth‐promoting rhizobacteria

Feng

Zhang

et al. 2019

Environmental Microbiology

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“…Construction of a pheS* marker for counterselection in M. capsulatus (Bath). Instead of the sacB marker, we attempted to employ a pheS* marker, which has been established as a counterselectable marker in various bacteria (18,(22)(23)(24)(25), for counterselection in M. capsulatus (Bath). The amino acid sequence of PheS from M. capsulatus (Bath) was aligned with those from E. coli and other representative methanotrophs ( Fig.…”

Section: Resultsmentioning

confidence: 99%

“…Furthermore, Miyazaki recently reported that substitution of T251 in PheS from E. coli with alanine or serine increased the efficiency of counterselection (21). Although individual pheS* genes have been constructed and used for counterselection in various bacteria (18,(22)(23)(24)(25), counterselection using pheS* has not been carried out in methanotrophs.…”

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confidence: 99%

Efficient Counterselection for Methylococcus capsulatus (Bath) by Using a Mutated pheS Gene

Ishikawa

Yokoe

Kato

et al. 2018

Appl Environ Microbiol

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Methylococcus capsulatus (Bath) is a representative gammaproteobacterial methanotroph that has been studied extensively in diverse research fields. The sacB gene, which encodes levansucrase, causing cell death in the presence of sucrose, is widely used as a counterselectable marker for disruption of a target gene in Gram-negative bacteria. However, sacB is not applicable to all Gram-negative bacteria, and its efficiency for the counterselection of M. capsulatus (Bath) is low. Here, we report the construction of an alternative counterselectable marker, pheS*, by introduction of two point mutations (A306G and T252A) into the pheS gene from M. capsulatus (Bath), which encodes the α-subunit of phenylalanyl-tRNA synthetase. The transformant harboring pheS* on an expression plasmid showed sensitivity to 10 mM p-chloro-phenylalanine, whereas the transformant harboring an empty plasmid showed no sensitivity, indicating the availability of pheS* as a counterselectable marker in M. capsulatus (Bath). To validate the utility of the pheS* marker in counterselection, we attempted to obtain an unmarked mutant of xoxF, a gene encoding the major subunit of Xox methanol dehydrogenase, which we failed to obtain by counterselection using the sacB marker. PCR, immunodetection using an anti-XoxF antiserum, and a cell growth assay in the absence of calcium demonstrated successful disruption of the xoxF gene in M. capsulatus (Bath). The difference in counterselection efficiencies of the markers indicated that pheS* is more suitable than sacB for counterselection in M. capsulatus (Bath). This study provides a new genetic tool enabling efficient counterselection in M. capsulatus (Bath). IMPORTANCE Methanotrophs have long been considered promising strains for biologically reducing methane from the environment and converting it into valuable products, because they can oxidize methane at ambient temperatures and pressures. Although several methodologies and tools for the genetic manipulation of methanotrophs have been developed, their mutagenic efficiency remains lower than that of tractable strains such as Escherichia coli. Therefore, further improvements are still desired. The significance of our study is that we increased the efficiency of counterselection in M. capsulatus (Bath) by employing pheS*, which was newly constructed as a counterselectable marker. This will allow for the efficient production of gene-disrupted and gene-integrated mutants of M. capsulatus (Bath). We anticipate that this counterselection system will be utilized widely by the methanotroph research community, leading to improved productivity of methane-based bioproduction and new insights into methanotrophy.

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“…Unmarked gene knockout in B. subtilis was performed according to a previously reported method (Zhou et al, ). Taking the example of aprE deletion, the upstream homologous arm (UHA), the direct repeat sequence (DR), and the downstream homologous arm (DHA) were amplified from B. subtilis 168 genomic DNA using the primer pairs P1/P2, P3/P4 and P7/P8, respectively.…”

Section: Methodsmentioning

confidence: 99%

Construction of second generation protease‐deficient hosts of Bacillus subtilis for secretion of foreign proteins

Zhao

Zhang

et al. 2019

Biotech & Bioengineering

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Although one of the major factors limiting the application of Bacillus subtilis as an expression host has been its production of at least eight extracellular proteases, researchers have also noticed that some proteases benefited the secretion of foreign proteins at times. Therefore, to maximize the yield of a foreign protein, the proteases should be selectively inactivated. This raises a new question that how to identify the favorable and unfavorable proteases for a target protein. Here, an evaluation system containing nine mutant strains of B. subtilis 168 was developed to address this question. The mutant strain PD8 has all the eight proteases inactivated whereas each of the other eight mutant strains expresses only one kind of these eight proteases.The target protein is secreted in these nine mutant strains; if the production of target protein in a mutant strain is higher than that in strain PD8, the corresponding protease is regarded as favorable. Accordingly, the optimal protease-deficient host is constructed through inactivating the unfavorable proteases. The effectiveness of this system was confirmed by expressing three foreign proteins. This study provides a strategy for improving the secretion of a foreign protein in B. subtilis through tailoring a personalized protease-deficient host.

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pheS * , an effective host-genotype-independent counter-selectable marker for marker-free chromosome deletion in Bacillus amyloliquefaciens

Cited by 46 publications

References 44 publications

Recognition of dominant attractants by key chemoreceptors mediates recruitment of plant growth‐promoting rhizobacteria

Recognition of dominant attractants by key chemoreceptors mediates recruitment of plant growth‐promoting rhizobacteria

Efficient Counterselection for Methylococcus capsulatus (Bath) by Using a Mutated pheS Gene

Construction of second generation protease‐deficient hosts of Bacillus subtilis for secretion of foreign proteins

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