The chitinase C gene PsChiC from Pseudomonas sp. and its synergistic effects on larvicidal activity

Pseudomonas sp. strain TXG6-1, a chitinolytic gram-negative bacterium, was isolated from a vegetable field in Taixing city, Jiangsu Province, China. In this study, a Pseudomonas chitinase C gene (PsChiC) was isolated from the chromosomal DNA of this bacterium using a pair of specific primers. The PsChiC gene consisted of an open reading frame of 1443 nucleotides and encoded 480 amino acid residues with a calculated molecular mass of 51.66 kDa. The deduced PsChiC amino acid sequence lacked a signal sequence and consisted of a glycoside hydrolase family 18 catalytic domain responsible for chitinase activity, a fibronectin type III-like domain (FLD) and a C-terminal chitin-binding domain (ChBD). The amino acid sequence of PsChiCshowed high sequence homology (> 95%) with chitinase C from Serratia marcescens. SDS-PAGE showed that the molecular mass of chitinase PsChiC was 52 kDa. Chitinase assays revealed that the chitobiosidase and endochitinase activities of PsChiCwere 51.6- and 84.1-fold higher than those of pET30a, respectively. Although PsChiC showed little insecticidal activity towards Spodoptera litura larvae, an insecticidal assay indicated that PsChiC increased the insecticidal toxicity of SpltNPV by 1.78-fold at 192 h and hastened death. These results suggest that PsChiC from Pseudomonas sp. could be useful in improving the pathogenicity of baculoviruses.     

Molecular characterization of a cryptic plasmid from the psychrotrophic antarctic bacterium Pseudoalteromonas sp. 643A

We report the identification and nucleotide sequence analysis of pKW1, a plasmid of the psychrotrophic bacterium Pseudoalteromonas sp. 643A isolated from the stomach of Antarctic krill Euphasia superba. pKW1 consists of 4583 bp, has a G+C content of 43% and seven putative open reading frames (ORFs). The deduced amino acid sequence from ORF-1 shared significant similarity with the plasmid replicase protein of Psychrobacter cryohalolentis, strain K5. The DNA region immediately downstream of the ORF-1 showed some homology with the Rep-binding sequence of the theta-replicating ColE2-type plasmids. The ORF-3 amino acid sequence revealed amino acid sequence homology with the mobilization protein of Psychrobacter sp. PRwf-1 and Moraxella catarrhalis, with identities of 28% and 25%, respectively. The ORF-4 showed 46% amino acid sequence homology with the putative relaxase/mobilization nuclease MobA of Hafnia alvei and 44% homology with the putative mobilization protein A of Pasterulla multocida. The copy number of pKW1 in Pseudoalteromonas sp. 643A was estimated of 15 copies per chromosome.     

Nucleotide sequence of the gene encoding NADH dehydrogenase from an alkalophile, Bacillus sp. strain YN-1

The gene encoding NADH dehydrogenase from an alkalophile, Bacillus sp., was cloned and sequenced. The cloned DNA fragment contained an open reading frame of 1,557 nucleotides which encodes a polypeptide composed of 519 amino acid residues (Mr 55,830). The predicted amino acid sequence was consistent with the partial amino acid sequences including the N-terminal and C-terminal sequences determined in a previous study. Sequence comparison with other flavoenzymes revealed high homology between the present dehydrogenase and Escherichia coli thioredoxin reductase.     

节杆菌A．pascens DMDC12中SOD基因的克隆和序列分析

根据基因库中细菌SOD的基因保守序列和Arthrobacter pascens DMDC12的N-末端氨基酸序列设计引物,采用PCR技术,以A.pascens DMDC12基因组为模板,克隆了A.pascens DMDC12中Mn-SOD的567 bp的基因片段;再结合该基因片段和A.pascens DMDC12中SOD的N-末端氨基酸序列的有关信息,设计包含该sod完整ORF区域的引物,成功扩增了A.pascens DMDC12中Mn-SOD的基因序列,新sod序列(sodAP)已提交Gen-Bank(收录号DQ779150)。利用生物学软件对该序列进行分析表明,该sodAP序列的ORF区域全长651 bp,编码216个氨基酸;该序列和Arthrobacter sp.FB24的SOD基因序列同源性为86%。     

Characterization of Chitinase C from a Marine Bacterium, Alteromonas sp. Strain O-7, and Its Corresponding Gene and Domain Structure

One of the chitinase genes of Alteromonas sp. strain O-7, the chitinase C-encoding gene (chiC), was cloned, and the nucleotide sequence was determined. An open reading frame coded for a protein of 430 amino acids with a predicted molecular mass of 46,680 Da. Alignment of the deduced amino acid sequence demonstrated that ChiC contained three functional domains, the N-terminal domain, a fibronectin type III-like domain, and a catalytic domain. The N-terminal domain (59 amino acids) was similar to that found in the C-terminal extension of ChiA (50 amino acids) of this strain and furthermore showed significant sequence homology to the regions found in several chitinases and cellulases. Thus, to evaluate the role of the domain, we constructed the hybrid gene that directs the synthesis of the fusion protein with glutathione S-transferase activity. Both the fusion protein and the N-terminal domain itself bound to chitin, indicating that the N-terminal domain of ChiC constitutes an independent chitin-binding domain.     

Purification,cDNA cloning,and characterization of LysM-containing plant chitinase from horsetail (Equisetum arvense)

Chitinase-A (EaChiA), molecular mass 36 kDa, was purified from the vegetative stems of a horsetail (Equisetum arvense) using a series of column chromatography. The N-terminal amino acid sequence of EaChiA was similar to the lysin motif (LysM). A cDNA encoding EaChiA was cloned by rapid amplification of cDNA ends and polymerase chain reaction. It consisted of 1320 nucleotides and encoded an open reading frame of 361 amino acid residues. The deduced amino acid sequence indicated that EaChiA is composed of a N-terminal LysM domain and a C-terminal plant class IIIb chitinase catalytic domain, belonging to the glycoside hydrolase family 18, linked by proline-rich regions. EaChiA has strong chitin-binding activity, however, no antifungal activity. This is the first report of a chitinase from Equisetopsida, a class of fern plants, and the second report of a LysM-containing chitinase from a plant.     

Amino acid sequence of the N-terminal domain of yam (Dioscorea japonica) aerial tuber acidic chitinase. Evidence for the presence of a wheat germ agglutinin domain in matured acidic chitinase from unstressed tuber

The amino acid sequence of the N-terminal domain of acidic chitinase from unstressed aerial tuber was determined and proved the presence of an N-terminal domain in acidic chitinase. The amino acid sequence was determined on a pyroglutamylaminopeptidase-treated N-terminal fragment of V8 protease and on chymotryptic peptides of this fragment. The sequence determined revealed 8 residues deletion and 2 residues insertion as compared with the N-terminal domain of tobacco basic chitinase. The N-terminal domain determined showed a homology of 40% and 52% with the N-terminal domain of tobacco basic chitinase and wheat germ agglutinin, respectively.Abbreviations DABITC,4-N,N dimethylaminoazobenzene 4-isothiocyanate - PITC phenylisothiocyanate - Cm carboxymethyl - WGA wheat germ agglutinin - TFA trifluoroacetic acid - PGAP pyroglutamylaminopeptidase     

Cloning and expression of chitinase A gene from Streptomyces cyaneus SP-27: the enzyme participates in protoplast formation of Schizophyllum commune

Chitinase A of Streptomyces cyaneus SP-27 or chitinase I of Bacillus circulans KA-304 showed the protoplast-forming activity when combined with alpha-1,3-glucanase of B. circulans KA-304. The gene of chitinase A was cloned. It consisted of 903 nucleotides encoding 301 amino acid residues, including a putative signal peptide (35 amino acid residues). The deduced N-terminal moiety of chitinase A showed sequence homology with the chitin-binding domain of chitinase F from Streptomyces coelicolor and chitinase 30 from Streptomyces olivaceoviridisis. The C-terminal moiety also showed high sequence similarity to the catalytic domain of several Streptomyces family 19 chitinases as well as that of chitinase I of B. circulans KA-304. Recombinant chitinase A was expressed in Escherichia coli Rosetta-gami B (DE 3). The properties of the recombinant enzyme were almost the same as those of chitinase A purified from a culture filtrate of S. cyaneus SP-27. The recombinant enzyme was superior to B. circulans KA-304 chitinase I not only in respect to protoplast forming activity in a mixture containing alpha-1,3-glucanase, but also to antifungal activity and powder chitin-hydrolyzing activity.     

Cloning and Expression of a Chitinase Gene from Sanguibacter sp. C4

The chitinase Chi58 is an extracellular chitinase produced by Sanguibacter sp.strain C4. The gene-specific PCR primers were used to detect the presence of the chiA gene in strain C4. A chiA fragment (chiA-F) was amplified from the C4 genomic DNA and was used to blast-search the related sequences from the GenBank dadabase. By alignment and selection of the highly conserved regions of the homologous sequences, two pairs of primers were designed to amplify the open reading frame (ORF) of the chitinase from strain C4 by nested PCR. The results revealed that the Chi58 ORF consisted of 1 692 nucleotides encoding a protein of 563 amino acid residues. The molecular weight of the mature protein was predicted to be 58.544 kDa. The Chi58 ORF was a modular enzyme composed of a signal peptide sequence, a polycystic kidney disease I domain, and a glycosyl hydrolase family 18 domain. The chitinase of C4 exhibited a high level of similarity to the chitinase A of Serratia (88.9%-99.6%) at the amino acid sequence level. The Chi58 gene was cloned into the expression vector pET32a to construct the recombinant plasmid pChi58 and was expressed in E. coli BL-21 (DE3) cells with IPTG induction. The molecular weight of the Trx-Chi58 fusion protein was estimated to be 81.1 kDa by SDS-PAGE.     

斑茅δ-OAT基因克隆及其序列分析

利用RT-PCR和RACE技术从斑茅（Erianthus arundinaceus）中分离出编码鸟氨酸-δ-氨基转氨酶基因的全长cDNA序列,序列全长1 680 bp,编码454个氨基酸。通过对哺乳动物、高等植物、微生物的δ-OAT基因编码的氨基酸序列进行同源比对,发现斑茅δ-OAT基因同其近缘属植物甘蔗的同源性最高（87%）,同其他高等植物的同源性次之（约为70%）,而同动物的同源性最低（约为60%）。在斑茅δ-OAT基因编码的氨基酸序列的5′端未发现线粒体定位序列,同甘蔗δ-OAT基因一样。斑茅δ-OAT基因具有完整的鸟氨酸转氨酶功能区rocD。利用定量RCR（real-time PCR）对30%PEG胁迫下的斑茅δ-OAT基因表达量进行研究,结果表明δ-OAT基因在胁迫12 h表达量达到最高,约为对照的4.1倍;胁迫2 h δ-OAT基因表达量反而有所降低。     

Cloning, sequence, and expression of a chitinase gene from a marine bacterium, Altermonas sp. strain O-7.

The gene encoding an extracellular chitinase from marine Alteromonas sp. strain O-7 was cloned in Escherichia coli JM109 by using pUC18. The chitinase produced was not secreted into the growth medium but accumulated in the periplasmic space. A chitinase-positive clone of E. coli produced two chitinases with different molecular weights from a single chitinase gene. These proteins showed almost the same enzymatic properties as the native chitinase of Alteromonas sp. strain O-7. The N-terminal sequences of the two enzymes were identical. The nucleotide sequence of the 3,394-bp SphI-HindIII fragment that included the chitinase gene was determined. A single open reading frame was found to encode a protein consisting of 820 amino acids with a molecular weight of 87,341. A putative ribosome-binding site, promoter, and signal sequence were identified. The deduced amino acid sequence of the cloned chitinase showed sequence homology with chitinases A (33.4%) and B (15.3%) from Serratia marcescens. Regardless of origin, the enzymes of the two bacteria isolated from marine and terrestrial environments had high homology, suggesting that these organisms evolved from a common ancestor.     

Molecular cloning of an alanine aminotransferase from NAD-malic enzyme type C4 plant Panicum miliaceum

We have determined the nucleotide sequence of a cDNA encoding AlaAT-2, which is believed to function in the C4-pathway of Panicum miliaceum. An open reading frame (1446 bp) encodes a protein of 482 amino acid residues. The deduced amino acid sequence of AlaAT-2 shows 44.2 and 44.8% homology with the amino acid sequences of AlaATs from rat and human livers, respectively. Northern blot analysis showed that the gene encoding AlaAT-2 in mesophyll and bundle sheath cells was the same and transcribed similarly in the cells. The level of translatable mRNA for AlaAT-2 increased dramatically during greening.     

Isolation and characterization of the 54-kDa and 22-kDa chitinase genes of Serratia marcescens KCTC2172

A DNA fragment (pCHI5422) containing two genes encoding a 54-kDa and a 22-kDa chitinase was isolated from a cosmid DNA library of Serratia marcescens KCTC2172. The complete nucleotide sequence of pCHI5422 consisting of 4581 bp was determined. The nucleotide sequence of the 22-kDa chitinase consists of 681 bp of open reading frame encoding 227 amino acids and is located 1422 bp downstream of the translation termination codon of the 54-kDa chitinase sequence. The 54-kDa chitinase gene consisted of 1497 bp in a single open reading frame encoding 499 amino acids. The genes encoding the 54-kDa and 22-kDa chitinase were separately subcloned in Escherichia coli and the individual chitinases were expressed and purified from the culture broth using chitin affinity chromatography. When chitohexaose was used as substrate, the major product of the enzymatic reaction of both the 54-kDa and 22-kDa chitinases was a (GlcNAc)₂ dimer with a minor amount of monomer. The specific activity of the 54-kDa and 22-kDa chitinases were 300 μM (min)⁻¹ mg⁻¹ and 17 μM (min)⁻¹ mg⁻¹ on the natural swollen chitin, respectively.     

Isolation and characterization of genes encoding two chitinase enzymes from Serratia marcescens

Analysis of clones isolated from a cosmid DNA library indicates that the Serratia marcescens chromosome contains at least two genes, chiA and chiB, which encode distinct secreted forms of the enzyme chitinase. These genes have been characterized by inspection of chitinase activity and secreted proteins in Escherichia coli strains containing subclones of these cosmids. The two chitinase genes show no detectable homology to each other. DNA sequence analysis of one of the genes predicts an amino acid sequence with an N-terminal signal peptide typical of genes encoding secreted bacterial proteins. This gene was mutagenized by cloning a neomycin phosphotransferase gene within its coding region, and the insertion mutation was recombined into the parental S. marcescens strain. The resulting chiA mutant transconjugant showed reduced chitinase production, reduced inhibition of fungal spore germination and reduced biological control of a fungal plant pathogen.     

Cloning of the 52-kDa chitinase gene from Serratia marcescens KCTC2172 and its proteolytic cleavage into an active 35-kDa enzyme

A chitinase gene (pCHI52) encoding the 52-kDa chitinase was isolated from a Serratia marcescens KCTC2172 cosmid library. This chitinase gene consists of 2526 bp with an open reading frame that encodes 485 amino acids. Escherichia coli harboring the pCHI52 gene secreted not only a 52-kDa but also a 35-kDa chitinase into the culture supernatant. We purified both 52-kDa and 35-kDa chitinases using a chitin affinity column and Sephacryl-S-300 gel filtration chromatography. We determined that the 17 N-terminal amino acid sequences of the 52-kDa and the 35-kDa chitinase are identical. Furthermore, a protease obtained from S. marcescens KCTC2172 cleaved the 52-kDa chitinase into the 35-kDa protein with chitinase activity. These results suggest that the 35-kDa chitinase derives from the 52-kDa chitinase by post-translational proteolytic modification. The optimal reaction temperature of 45°C and the optimal pH of 5.5 were identical for both enzymes. The specific activities of the 52-kDa and 35-kDa chitinases on natural swollen chitin were 67 μmol min⁻¹ mg⁻¹ and 60 μmol min⁻¹ mg⁻¹, respectively.     

Structure and expression of the gene encoding ribosomal protein S1 from the cyanobacterium Synechococcus sp. strain PCC 6301: striking sequence similarity to the chloroplast ribosomal protein CS1

We isolated a 38 kDa ssDNA-binding protein from the unicellular cyanobacterium Synechococcus sp. strain PCC 6301 and determined its N-terminal amino acid sequence. A genomic clone encoding the 38 kDa protein was isolated by using a degenerate oligonucleotide probe based on the amino acid sequence. The nucleotide sequence and predicted amino acid sequence revealed that the 38 kDa protein is 306 amino acids long and homologous to the nuclear-encoded 370 amino acid chloroplast ribosomal protein CS1 of spinach (48% identity), therefore identifying it as ribosomal protein (r-protein) S1. Cyanobacterial and chloroplast S1 proteins differ in size from Escherichia coli r-protein S1 (557 amino acids). This provides an additional evidence that cyanobacteria are closely related to chloroplasts. The Synechococcus gene rps1 encoding S1 is located 1.1 kb downstream from psbB, which encodes the photosystem 11 P680 chlorophyll a apoprotein. An open reading frame encoding a potential protein of 168 amino acids is present between psbB and rps1 and its deduced amino acid sequence is similar to that of E. coli hypothetical 17.2 kDa protein. Northern blot analysis showed that rps1 is transcribed as a monocistronic mRNA.     

Cloning and analysis of a cDNA encoding a two-domain hemoglobin chain from the water flea Daphnia magna

A cDNA encoding a two-domain hemoglobin (Hb) chain of Daphnia magna was cloned and its nucleotide (nt) sequence of 1261 bp was determined. The nt sequence contained 74 bp of the leader sequence, 1047 bp of an open reading frame (ORF), and 119 bp of the 3′-untranslated region (UTR), excluding the polyadenylation tail. A sequence, AATACA, located 24 bp upstream from the polyA sequence was considered to be a polyadenylation signal. cDNA-derived amino acid (aa) sequence revealed that D. magna Hb chain is synthesized as a secretory precursor with a signal peptide of 18 aa. Mature D. magna Hb chain consists of 330-aa residues with a calculated molecular weight of 36 227, which is composed of two large repeated domains, domain 1 and 2. Several key aa that are invariant in all or most of other Hb and required for functional heme-binding are conserved in each of the two domains. The N-terminal extension (pre-A segment) of domain 1 was unusually long and contained an unusual threonine-rich sequence. The homology between the aa sequences of the two domains (24% identity) was much lower than that observed in other two-domain Hb chains from clams or nematode. Hb mRNA level in D. magna reared under low oxygen concentration was more than 12 times higher than that in D. magna reared with sufficient aeration, indicating that the expression of Hb gene is regulated by mRNA level.     

Highly thermostable and surfactant-activated chitinase from a subseafloor bacterium,Laceyella putida

A novel chitinase (LpChiA) was purified to homogeneity from a culture of Laceyella putida JAM FM3001. LpChiA hydrolyzed colloidal chitin optimally at a pH of 4 in an acetate buffer and temperature of 75?ºC. The enzyme was remarkably stable to incubation at 70?ºC up to 1 h at pH 5.2, and its activity half-life was 3 days. The molecular mass of the enzyme was around 38 kDa by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and around 75 kDa by gel filtration, suggesting it is a homodimer. The enzyme activity was enhanced about 60 % when pre-incubated with anionic, cationic, and nonionic surfactants. The gene for LpChiA was cloned by PCR and sequenced. The nucleotide sequence of the gene consisted of 1,683 bp encoding 560 amino acids. The N-terminal and internal amino acid sequences of the purified LpChiA from L. putida suggested that the mature enzyme was composed of 384 amino acids after cleaving its 176 N-terminal amino acids and dimerized to express its activity. The deduced amino acid sequence of the mature enzyme showed the highest similarity to chitinase of Laceyella sacchari with 79 % identity.