Characterization of a Novel, Antifungal, Chitin-Binding Protein from Streptomyces tendae Tü901 That Interferes with Growth Polarity

ABSTRACT The afp1 gene, which encodes the antifungal protein AFP1, was cloned from nikkomycin-producing Streptomyces tendae Tü901, using a nikkomycin-negative mutant as a host and screening transformants for antifungal activity againstPaecilomyces variotii in agar diffusion assays. The 384-bpafp1 gene has a low G+C content (63%) and a transcription termination structure with a poly(T) region, unusual attributes forStreptomyces genes. AFP1 was purified from culture filtrate of S. tendae carrying the afp1 gene on the multicopy plasmid pIJ699. The purified protein had a molecular mass of 9,862 Da and lacked a 42-residue N-terminal peptide deduced from the nucleotide sequence. AFP1 was stable at extreme pH values and high temperatures and toward commercial proteinases. AFP1 had limited similarity to cellulose-binding domains of microbial plant cell wall hydrolases and bound to crab shell chitin, chitosan, and cell walls ofP. variotii but showed no enzyme activity. The biological activity of AFP1, which represents the first chitin-binding protein from bacteria exhibiting antifungal activity, was directed against specific ascomycetes, and synergistic interaction with the chitin synthetase inhibitor nikkomycin inhibited growth ofAspergillus species. Microscopy studies revealed that fluorescein-labeled AFP1 strongly bound to the surface of germinated conidia and to tips of growing hyphae, causing severe alterations in cell morphogenesis that gave rise to large spherical conidia and/or swollen hyphae and to atypical branching.

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Characteristics and Function of the Chitin Binding Protein from Xenorhabdus nematophila

Protein and Peptide Letters ◽

10.2174/0929866526666190327143335 ◽

2019 ◽

Vol 26 (6) ◽

pp. 414-422

Author(s):

Jia Liu ◽

Ping Song ◽

Jie Zhang ◽

Ziyan Nangong ◽

Xiaobei Liu ◽

...

Keyword(s):

Antifungal Activity ◽

Growth Inhibition ◽

Insecticidal Activity ◽

Spore Germination ◽

Binding Protein ◽

Colloidal Chitin ◽

Inhibition Rate ◽

Xenorhabdus Nematophila ◽

Chitin Binding Protein ◽

And Function

Background: Genome sequence analysis (GenBank access No.: FN667742.1) shows that Xenorhabdus nematophila ATCC19061 contains one gene (Xn-cbp) encoding chitin binding protein (Xn-CBP). Objective: The present work aims to clarify the characteristics and function of Xn-CBP from X. nematophila HB310. Methods: In this study, the Xn-cbp gene was cloned and expressed in Escherichia coli BL21 (DE3). Substrate binding assays were performed to explain the ability of Xn-CBP combined with the polysaccharide. The insecticidal toxicity of Xn-CBP against the second-instar larvae of Helicoverpa armigera was determined by feeding method. Besides, the antifungal activity of Xn-CBP against Coniothyrium diplodiella, Verticillium dahlia, and Fusarium oxysporum was tested by spore germination assay and hyphal extension assay. Results: Xn-CBP encoded 199 amino acids with a calculated mass of 28 kDa, which contained a signal peptide and a chitin binding domain. The Bmax and Kd values of Xn-CBP to colloidal chitin were 2.46 and 4.08, respectively. Xn-CBP had insecticidal activity against the H. armigera with a growth inhibition rate of 84.08%. Xn-CBP had the highest spore germination inhibitory effect on C. diplodiella with the inhibition rate of 83.11%. The hyphal growth inhibition rate of Xn-CBP to F. oxysporum, 41.52%, was higher than the other two fungi. Conclusion: The Xn-CBP had the highest binding ability to colloidal chitin and it showed insecticidal activity and antifungal activity. The present study laid a foundation for further exploitation and utilization of X. nematophila.

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The type II and X cellulose-binding domains of Pseudomonas xylanase A potentiate catalytic activity against complex substrates by a common mechanism

Biochemical Journal ◽

10.1042/bj3420473 ◽

1999 ◽

Vol 342 (2) ◽

pp. 473-480 ◽

Cited By ~ 27

Author(s):

Jaitinder GILL ◽

Jane E. RIXON ◽

David N. BOLAM ◽

Simon MCQUEEN-MASON ◽

Peter J. SIMPSON ◽

...

Keyword(s):

Microcrystalline Cellulose ◽

Plant Cell Wall ◽

Catalytic Domain ◽

Wall Material ◽

Common Mechanism ◽

Type Ii ◽

Binding Domains ◽

Covalently Linked ◽

Cellulose Binding ◽

Internal Domain

Xylanase A (Pf Xyn10A), in common with several other Pseudomonas fluorescens subsp. cellulosa polysaccharidases, consists of a Type II cellulose-binding domain (CBD), a catalytic domain (Pf Xyn10ACD) and an internal domain that exhibits homology to Type X CBDs. The Type X CBD of Pf Xyn10A, expressed as a discrete entity (CBDX) or fused to the catalytic domain (Pf Xyn10A′), bound to amorphous and bacterial microcrystalline cellulose with a Ka of 2.5×105 M-1. CBDX exhibited no affinity for soluble forms of cellulose or cello-oligosaccharides, suggesting that the domain interacts with multiple cellulose chains in the insoluble forms of the polysaccharide. Pf Xyn10A′ was 2-3 times more active against cellulose-hemicellulose complexes than Pf Xyn10ACD; however, Pf Xyn10A′ and Pf Xyn10ACD exhibited the same activity against soluble substrates. CBDX did not disrupt the structure of plant-cell-wall material or bacterial microcrystalline cellulose, and did not potentiate Pf Xyn10ACD when not covalently linked to the enzyme. There was no substantial difference in the affinity of full-length Pf Xyn10A and the enzyme's Type II CBD for cellulose. The activity of Pf Xyn10A against cellulose-hemicellulose complexes was similar to that of Pf Xyn10A′, and a derivative of Pf Xyn10A in which the Type II CBD is linked to the Pf Xyn10ACD via a serine-rich linker sequence [Bolam, Cireula, McQueen-Mason, Simpson, Williamson, Rixon, Boraston, Hazlewood and Gilbert (1998) Biochem J. 331, 775-781]. These data indicate that CBDX is functional in Pf Xyn10A and that no synergy, either in ligand binding or in the potentiation of catalysis, is evident between the Type II and X CBDs of the xylanase.

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Characterization of hybrid proteins consisting of the catalytic domains of Clostridium and Ruminococcus endoglucanases, fused to Pseudomonas non-catalytic cellulose-binding domains

Biochemical Journal ◽

10.1042/bj2790787 ◽

1991 ◽

Vol 279 (3) ◽

pp. 787-792 ◽

Cited By ~ 22

Author(s):

D M Poole ◽

A J Durrant ◽

G P Hazlewood ◽

H J Gilbert

Keyword(s):

Catalytic Domain ◽

Binding Capacity ◽

Binding Domains ◽

Hybrid Proteins ◽

C Terminus ◽

N Terminus ◽

Fusion Enzymes ◽

Cellulose Binding

The N-terminal 160 or 267 residues of xylanase A from Pseudomonas fluorescens subsp. cellulosa, containing a non-catalytic cellulose-binding domain (CBD), were fused to the N-terminus of the catalytic domain of endoglucanase E (EGE') from Clostridium thermocellum. A further hybrid enzyme was constructed consisting of the 347 N-terminal residues of xylanase C (XYLC) from P. fluorescens subsp. cellulosa, which also constitutes a CBD, fused to the N-terminus of endoglucanase A (EGA) from Ruminococcus albus. The three hybrid enzymes bound to insoluble cellulose, and could be eluted such that cellulose-binding capacity and catalytic activity were retained. The catalytic properties of the fusion enzymes were similar to EGE' and EGA respectively. Residues 37-347 and 34-347 of XYLC were fused to the C-terminus of EGE' and the 10 amino acids encoded by the multiple cloning sequence of pMTL22p respectively. The two hybrid proteins did not bind cellulose, although residues 39-139 of XYLC were shown previously to constitute a functional CBD. The putative role of the P. fluorescens subsp. cellulosa CBD in cellulase action is discussed.

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Modification of polysaccharides and plant cell wall by endo-1,4-β-glucanase and cellulose-binding domains

Biomolecular Engineering ◽

10.1016/s1389-0344(02)00007-2 ◽

2002 ◽

Vol 19 (1) ◽

pp. 17-30 ◽

Cited By ~ 63

Author(s):

Ilan Levy ◽

Ziv Shani ◽

Oded Shoseyov

Keyword(s):

Cell Wall ◽

Plant Cell ◽

Plant Cell Wall ◽

Binding Domains ◽

Cellulose Binding

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Characterization of a chitin-binding protein GP37 of Spodoptera litura multicapsid nucleopolyhedrovirus

Virus Research ◽

10.1016/s0168-1702(03)00179-5 ◽

2003 ◽

Vol 96 (1-2) ◽

pp. 113-122 ◽

Cited By ~ 40

Author(s):

Zhaofei Li ◽

Chongbi Li ◽

Kai Yang ◽

Lihua Wang ◽

Chong Yin ◽

...

Keyword(s):

Spodoptera Litura ◽

Binding Protein ◽

Chitin Binding Protein ◽

Spodoptera Litura Multicapsid Nucleopolyhedrovirus

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Characterization of Microtubule Binding Domains in the Arabidopsis Kinesin-like Calmodulin Binding Protein

The Plant Cell ◽

10.1105/tpc.10.6.957 ◽

1998 ◽

Vol 10 (6) ◽

pp. 957-965 ◽

Cited By ~ 60

Author(s):

Soma B. Narasimhulu ◽

Anireddy S. N. Reddy

Keyword(s):

Binding Protein ◽

Microtubule Binding ◽

Calmodulin Binding ◽

Binding Domains

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Substrate-binding domains of glycanases from Streptomyces lividans: characterization of a new family of xylan-binding domains

Biochemical Journal ◽

10.1042/bj3300041 ◽

1998 ◽

Vol 330 (1) ◽

pp. 41-45 ◽

Cited By ~ 36

Author(s):

Claude DUPONT ◽

Martin ROBERGE ◽

François SHARECK ◽

Rolf MOROSOLI ◽

Dieter KLUEPFEL

Keyword(s):

Amino Acid ◽

Amino Acid Sequence ◽

Substrate Binding ◽

Streptomyces Lividans ◽

Cellulose Binding Domain ◽

Binding Domains ◽

New Family ◽

Cellulose Binding ◽

Sequence Similarities

The substrate-binding domains of six glycanases from Streptomyces lividans were investigated to determine their specificity towards cellulose and xylan. Based upon amino acid sequence similarities, four of the six domains could be assigned to existing cellulose-binding domain families. However, the binding domains of xylanase A and arabinofuranosidase B could not be classified in any of the known families and should therefore be classified as members of a new family. Evidence is also presented that this new family is one of true xylan-binding domains.

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