The novel regulatory ncRNA, NfiS, optimizes nitrogen fixation via base pairing with the nitrogenase gene
            <i>nifK</i>
            mRNA in
            <i>Pseudomonas stutzeri</i>
            A1501

Zhan, Yuhua; Yan, Yongliang; Deng, Zhiping; Chen, Ming; Lu, Wei; Lu, Chao; Shang, Lei; Yang, Ziyin; Zhang, Wei; Wang, Wei; Li, Yun; Qin, Ke; Lu, Jiasi; Xu, Yuquan; Zhang, Liwen; Xie, Zhihong; Cheng, Qi; Elmerich, Claudine; Lin, Min

doi:10.1073/pnas.1604514113

Cited by 54 publications

(80 citation statements)

References 62 publications

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“…3B). The same stress-response phenotype has been detected with other Pseudomonas species in regard to the deletion of rpoS [39].…”

Section: Mutant Phenotype and Transcriptional Analysessupporting

confidence: 74%

High-Frequency Targeted Mutagenesis in Pseudomonas stutzeri Using a Vector-Free Allele-Exchange Protocol

Gomaa¹,

Deng²,

Yang³

et al. 2017

Journal of Microbiology and Biotechnology

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The complexity of the bacterial recombination system is a barrier for the construction of bacterial mutants for the further functional investigation of specific genes. Several protocols have been developed to inactivate genes from the genus . Those protocols are complicated and time-consuming and mostly do not enable easy construction of multiple knock-ins/outs. The current study describes a single and double crossover-recombination system using an optimized vector-free allele-exchange protocol for gene disruption and gene replacement in a single species of the family Pseudomonadaceae. The protocol is based on self-ligation (circularization) for the DNA cassette which has been obtained by overlapping polymerase chain reaction (Fusion-PCR), and carries an antibiotic resistance cassette flanked by homologous internal regions of the target locus. To establish the reproducibility of the approach, three different chromosomal genes () were knocked-out from the root-associative bacterium A1501. The results showed that the A1501 mutants, which are free of any plasmid backbone, could be obtained via a single or double crossover recombination. In order to optimize this protocol, three key factors that were found to have great effect on the efficiency of the homologous recombination were further investigated. Moreover, the modified protocol does not require further cloning steps, and it enables the construction of multiple gene knock-in/out mutants sequentially. This work provides a simple and rapid mutagenesis strategy for genome editing in , which may also be applicable for other gram-negative bacteria.

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“…3B). The same stress-response phenotype has been detected with other Pseudomonas species in regard to the deletion of rpoS [39].…”

Section: Mutant Phenotype and Transcriptional Analysessupporting

confidence: 74%

High-Frequency Targeted Mutagenesis in Pseudomonas stutzeri Using a Vector-Free Allele-Exchange Protocol

Gomaa¹,

Deng²,

Yang³

et al. 2017

Journal of Microbiology and Biotechnology

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“…Examples of ncRNAs controlling and enhancing key functions can be found elsewhere (Fröhlich and Vogel, 2009), such as in Pseudomonas stutzeri A1501 with NfiS, a positive regulator of the Nitrogenase (Zhan et al, 2016). In this system, a compact ncRNA structure acts on the mRNA of nifK , encoding the β -subunit of the MoFe protein of the nitrogenase enzymatic complex, enhancing the translation.…”

Section: Discussionmentioning

confidence: 99%

Pulling the strings of cell cycle: a non-coding RNA, CcnA, modulates the master regulators CtrA and GcrA in Caulobacter crescentus

Beroual

Prévost

Lalaouna

et al. 2019

Preprint

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20Bacterial cells are powerful models for understanding how cells divide and accomplish global 21 regulatory programs. In Caulobacter crescentus a cascade of essential master regulators regulate the 22 correct and sequential activation of DNA replication, cell division and development of different cell 23 types. Among them CtrA plays a crucial role coordinating all those functions. Despite decades of 24 investigation, no control by non-coding RNAs (ncRNAs) has been linked to Caulobacter cell cycle. 25Here, for the first time we describe the role of a novel essential factor named CcnA, a ncRNA located 26 at the origin of replication, activated by CtrA and responsible for the rapid and strong accumulation of 27 CtrA itself. In addition CcnA is also responsible for the inhibition of GcrA translation by direct 28 interaction with its UTR region. By a combination of probing experiments and mutagenesis, we 29 propose a new mechanism by liberation (CtrA) or sequestration (GcrA) of the Ribosome Binding Site 30 (RBS). CcnA role is conserved in other alphaproteobacterial species, such as Sinorhizobium meliloti, 31 representing indeed a conserved and fundamental process regulating cell cycle in Rhizobiales and32 Caulobacterales.73 responsible for the specific and highly regulated proteolysis of CtrA. 74In Caulobacter, the regulation of gene expression by ncRNAs has revealed few examples. Initially 75 only 27 ncRNAs were described in this organism (Landt et al., 2008). CrfA is an sRNA involved in 76 adaptation to carbon starvation (Landt et al., 2010). GsrN is involved in the response to σ T -dependent 77 multiple stresses (Tien et al., 2017). Finally ChvR has been recently characterized as a ncRNA that is 78 expressed in response to DNA damage, low pH, and growth in minimal medium (Fröhlich et al.,79 2018). However as new recent approaches using RNAseq and post-genomic techniques expanded the 80 plethora of ncRNA candidates to more than 100 (Zhou et al., 2015), predictions of their integration 81 into the cell cycle circuit (Beroual et al., 2018) has suggested that several new candidate ncRNAs 82 should be deeply studied. 83Here we investigated the role of a ncRNA, named CcnA, that belongs to the origin of replication of 84 Caulobacter chromosome. We studied its role by the construction of deletion mutants, silencing by 85 expression of its antisense and over expression. Results presented in this work identified the mRNAs 86 of CtrA and GcrA, two master regulators of cell cycle, as main targets of this ncRNA. Data were 87 88 closely related organism such as Sinorhizobium meliloti suggested its potential conservation across 89 bacteria. 91 Results 92CcnA expression is regulated by CtrA 94Based on previous results (Zhou et al., 2015) we observed that CCNA_R0094, here named Cell Cycle 95 non-coding RNA A (CcnA), expression peaks after few minutes from the accumulation of ctrA 96 transcript and protein, in the second half of the S-phase, when P2, the second ctrA promoter, is 97 activated (Fig 1A). 98We designed primers to dete...

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“…Microscale thermophoresis (MST) experiments were performed as described previously (Zhan et al ., ). The purified proteins (DrHD, CehD, YlHD and BnHD) were labelled with the Monolith NTTM Protein Labeling Kit RED‐NHS according to the supplied labelling protocol (Cat# L011; NanoTemper Technologies Co.,Ltd., Chaoyang, Beijing, China).…”

Section: Methodsmentioning

confidence: 97%

Functional assessment of hydrophilic domains of late embryogenesis abundant proteins from distant organisms

Liu

Zhang

Han

et al. 2019

Microbial Biotechnology

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Summary Late embryogenesis abundant (LEA) proteins play a protective role during desiccation and oxidation stresses. LEA3 proteins are a major group characterized by a hydrophilic domain (HD) with a highly conserved repeating 11‐amino acid motif. We compared four different HD orthologs from distant organisms: (i) DrHD from the extremophilic bacterium Deinococcus radiodurans; (ii) CeHD from the nematode Caenorhabditis elegans; (iii) YlHD from the yeast Yarrowia lipolytica; and (iv) BnHD from the plant Brassica napus. Circular dichroism spectroscopy showed that all four HDs were intrinsically disordered in phosphate buffer and then folded into α‐helical structures with the addition of glycerol or trifluoroethanol. Heterologous HD expression conferred enhanced desiccation and oxidation tolerance to Escherichia coli. These four HDs protected the enzymatic activities of lactate dehydrogenase (LDH) by preventing its aggregation under desiccation stress. The HDs also interacted with LDH, which was intensified by the addition of hydrogen peroxide (H2O2), suggesting a protective role in a chaperone‐like manner. Based on these results, the HDs of LEA3 proteins show promise as protectants for desiccation and oxidation stresses, especially DrHD, which is a potential ideal stress‐response element that can be applied in synthetic biology due to its extraordinary protection and stress resistance ability.

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The novel regulatory ncRNA, NfiS, optimizes nitrogen fixation via base pairing with the nitrogenase gene nifK mRNA in Pseudomonas stutzeri A1501

Cited by 54 publications

References 62 publications

High-Frequency Targeted Mutagenesis in Pseudomonas stutzeri Using a Vector-Free Allele-Exchange Protocol

High-Frequency Targeted Mutagenesis in Pseudomonas stutzeri Using a Vector-Free Allele-Exchange Protocol

Pulling the strings of cell cycle: a non-coding RNA, CcnA, modulates the master regulators CtrA and GcrA in Caulobacter crescentus

Functional assessment of hydrophilic domains of late embryogenesis abundant proteins from distant organisms

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