FLP-FRT-Based Method To Obtain Unmarked Deletions of
 CHU
 _
 3237
 (
 porU
 ) and Large Genomic Fragments of Cytophaga hutchinsonii

Wang, Ying; Wang, Zhiquan; Cao, Jing; Guan, Zhonghong; Lu, Xuemei

doi:10.1128/aem.01785-14

Cited by 35 publications

(51 citation statements)

References 53 publications

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“…Inverse PCR showed that the transposon was inserted into gene locus chu_3220. To confirm this, chu_3220 was disrupted by linear DNA double-crossover deletion (27). The transformant was confirmed by PCR tests with primers 3220F/3220R and 3220F/3220E and named Δ3220.…”

Section: Resultsmentioning

confidence: 99%

“…According to the potential start codon, the protein would contain 2,040 instead of 1,980 amino acids. To verify the actual start codon of chu_3220, we used the active promoter P 1284 that we had discovered previously (27) to replace the annotated and potential promoter of chu_3220 and obtained the Δ3220-A and Δ3220-P mutants, respectively (Fig. 1a).…”

Section: Resultsmentioning

confidence: 99%

“…A 1.5-kbp fragment spanning part of chu_3220 (bp 322 to 1858), referred to as 3220H2, was amplified from C. hutchinsonii genomic DNA with primers 3220H2F and 3220H2R. 3220H2 was digested with BamHI and KpnI and cloned into plasmid pTKS (27). A 1.5-kbp fragment spanning part of chu_3220 (bp 4126 to 5650), referred to as 3220H1, was amplified with primers 3220H1F and 3220H1R and digested with SalI and SacI.…”

Section: Methodsmentioning

confidence: 99%

“…1 filter paper overlaid on Stainer agar. The plates were incubated at 30°C to observe cellulose degradation (27).…”

Section: Methodsmentioning

confidence: 99%

“…C. hutchinsonii ATCC 33406 was grown in PY6 medium (6 g/liter peptone, 0.5 g/liter yeast extract, 4 g/liter glucose, pH 7.3) (27) (21) plus 0.2% (wt/vol) glucose at 30°C with shaking at 160 rpm, and Escherichia coli strains were grown in Luria-Bertani medium at 37°C with shaking at 170 rpm. Antibiotics were used at the following concentrations: ampicillin, 100 g/ml; erythromycin, 30 g/ml.…”

Section: Methodsmentioning

confidence: 99%

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Identification and Characterization of a Large Protein Essential for Degradation of the Crystalline Region of Cellulose by Cytophaga hutchinsonii

Wang

Zhao

Bai

et al. 2017

Appl Environ Microbiol

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Cytophaga hutchinsonii is a Gram-negative bacterium that can efficiently degrade crystalline cellulose by a unique mechanism different from the free cellulase or cellulosome strategy. In this study, chu_3220, encoding the hypothetical protein CHU_3220 (205 kDa), was identified by insertional mutation and gene deletion as the first gene essential for degradation of the crystalline region but not the amorphous region of cellulose by C. hutchinsonii. A chu_3220 deletion mutant was defective in the degradation of crystalline cellulose and increased the degree of crystallinity of Avicel PH101 but could still degrade amorphous cellulose completely. CHU_3220 was found to be located on the outer surface of the outer membrane and could bind to cellulose. It contains 15 PbH1 domains and a C-terminal domain (CHU_C) that was proved to be critical for the localization of CHU_3220 on the cell surface and the function of CHU_3220 in crystalline cellulose degradation. Moreover, the degradation of crystalline cellulose was intact-cell dependent and inhibited by NaN 3 . Further study showed that chu_3220 was induced by cellulose and that the endoglucanase activity on the cell surface was significantly reduced without chu_3220. Real-time PCR revealed that the transcription of most genes encoding endoglucanases located on the cell surface was decreased in the chu_3220 deletion mutant, indicating that chu_3220 might also play a role in the regulation of the expression of some endoglucanases.IMPORTANCE Cytophaga hutchinsonii could efficiently degrade crystalline cellulose with a unique mechanism without cellulosomes and free cellulases. It lacks proteins that are thought to play important roles in disruption of the crystalline region of cellulose, including exoglucanases, lytic polysaccharide monooxygenases, expansins, expansin-like proteins, or swollenins, and most of its endoglucanases lack carbohydrate binding modules. The mechanism of the degradation of crystalline cellulose is still unknown. In this study, chu_3220 was identified as the first gene essential for the degradation of the crystalline region but not the amorphous region of cellulose. CHU_3220 is a high-molecular-weight protein located on the outer surface of the outer membrane and could bind to cellulose. We proposed that CHU_3220 might be an essential component of a protein complex on the cell surface in charge of the decrystallization of crystalline cellulose. The degradation of crystalline cellulose by C. hutchinsonii was not only dependent on intact cells but also required the energy supplied by the cells. This was obviously different from other known cellulose depolymerization system. Our study has shed more light on the novel strategy of crystalline cellulose degradation by C. hutchinsonii.

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Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

“…1 filter paper overlaid on Stainer agar. The plates were incubated at 30°C to observe cellulose degradation (27).…”

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

See 3 more Smart Citations

Identification and Characterization of a Large Protein Essential for Degradation of the Crystalline Region of Cellulose by Cytophaga hutchinsonii

Wang

Zhao

Bai

et al. 2017

Appl Environ Microbiol

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Techniques for Large-Scale Bacterial Genome Manipulation and Characterization of the Mutants with Respect to In Silico Metabolic Reconstructions

diCenzo

Finan

2017

Methods in Molecular Biology

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The rate at which all genes within a bacterial genome can be identified far exceeds the ability to characterize these genes. To assist in associating genes with cellular functions, a large-scale bacterial genome deletion approach can be employed to rapidly screen tens to thousands of genes for desired phenotypes. Here, we provide a detailed protocol for the generation of deletions of large segments of bacterial genomes that relies on the activity of a site-specific recombinase. In this procedure, two recombinase recognition target sequences are introduced into known positions of a bacterial genome through single cross-over plasmid integration. Subsequent expression of the site-specific recombinase mediates recombination between the two target sequences, resulting in the excision of the intervening region and its loss from the genome. We further illustrate how this deletion system can be readily adapted to function as a large-scale in vivo cloning procedure, in which the region excised from the genome is captured as a replicative plasmid. We next provide a procedure for the metabolic analysis of bacterial large-scale genome deletion mutants using the Biolog Phenotype MicroArray™ system. Finally, a pipeline is described, and a sample Matlab script is provided, for the integration of the obtained data with a draft metabolic reconstruction for the refinement of the reactions and gene-protein-reaction relationships in a metabolic reconstruction.

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Identification and characterization of a novel locus in Cytophaga hutchinsonii involved in colony spreading and cellulose digestion

Zhou

Wang

Yang

et al. 2015

Appl Microbiol Biotechnol

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Cytophaga hutchinsonii, an aerobic cellulolytic soil bacterium, is capable of degrading crystalline cellulose and gliding over surface rapidly. The involved mechanisms, however, are largely unknown. Here, we used the mariner-based transposon HimarEm1 to screen for C. hutchinsonii mutants deficient in utilizing filter paper as the sole carbon source. A novel gene locus, chu_1719, encoding a hypothetical protein was identified, whose inactivation resulted in a compromised growth of C. hutchinsonii on filter paper. Further analysis revealed that disruption of chu_1719 suppressed colony spreading but had no significant effect on Avicel degradation in liquid medium. Carboxymethylcellulase (CMCase) activity of the mutant membrane proteins was reduced by about 40% as compared with the wild-type strain. Moreover, profiles of cellulose-adsorbed outer membrane proteins were significantly different between the mutant and wild-type (WT) strains. These results suggest that chu_1719 plays an important role in controlling the spreading motility and cellulose utilization probably by affecting the appropriate production of membrane proteins in C. hutchinsonii.

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FLP-FRT-Based Method To Obtain Unmarked Deletions of CHU _ 3237 ( porU ) and Large Genomic Fragments of Cytophaga hutchinsonii

Cited by 35 publications

References 53 publications

Identification and Characterization of a Large Protein Essential for Degradation of the Crystalline Region of Cellulose by Cytophaga hutchinsonii

Identification and Characterization of a Large Protein Essential for Degradation of the Crystalline Region of Cellulose by Cytophaga hutchinsonii

Techniques for Large-Scale Bacterial Genome Manipulation and Characterization of the Mutants with Respect to In Silico Metabolic Reconstructions

Identification and characterization of a novel locus in Cytophaga hutchinsonii involved in colony spreading and cellulose digestion

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