Keyun Zhang

Anti-infection Peptidomics of Amphibian Skin

Peptidomics and genomics analyses were used to study an anti-infection array of peptides of amphibian skin. 372 cDNA sequences of antimicrobial peptides were characterized from a single individual skin of the frog Odorrana grahami that encode 107 novel antimicrobial peptides. This contribution almost triples the number of currently reported amphibian antimicrobial peptides. The peptides could be organized into 30 divergent groups, including 24 novel groups. The diversity in peptide coding cDNA sequences is, to our knowledge, the most extreme yet described for any animal. The patterns of diversification suggest that point mutations as well as insertion, deletion, and “shuffling” of oligonucleotide sequences were responsible for the diversity. The diversity of antimicrobial peptides may have resulted from the diversity of microorganisms. These diverse peptides exhibited both diverse secondary structure and “host defense” properties. Such extreme antimicrobial peptide diversity in a single amphibian species is amazing. This has led us to reconsider the strong capability of innate immunity and molecular genetics of amphibian ecological diversification and doubt the general opinion that 20–30 different antimicrobial peptides can protect an animal because of the relatively wide specificity of the peptide antibiotics. The antimicrobial mechanisms of O. grahami peptides were investigated. They exerted their antimicrobial functions by various means, including forming lamellar mesosome-like structures, peeling off the cell walls, forming pores, and inducing DNA condensation. With respect to the development of antibiotics, these peptides provide potential new templates to explore further.

Heterorhabditidoides chongmingensis gen. nov., sp. nov. (Rhabditida: Rhabditidae), a novel member of the entomopathogenic nematodes.

During a recent soil sample survey in Eastern China, a new entomopathogenic nematode species, collected from the Chongming Islands in the southern-eastern area of Shanghai, was discovered. Morphological characteristics of different developmental stages of the nematode combined with molecular data showed that this nematode is a new genus of Rhabditidae, and described as Heterorhabditidoides chongmingensis gen. nov., sp. nov., for that it shares more morphological characteristics with heterorhabditids than with steinernematids. For males, the papillae formula of bursa is 1, 2, 3, 3, with constant papillae number in the terminal group, stoma tubular-shaped and about 1.5 head width; cheilorhabdions cuticularized, esophageal collar present and long, median bulb present. For infective juveniles, EP=90 (80-105)microm, ES=104 (92-120)microm, tail length=111 (89-159)microm, and a=19.1 (15-21). The percentages of the nucleotides A, T, C and G in the ITS1 regions of the new species are significantly different from those of heterorhabditids and other rhabditids. Molecular phylogenetic trees based on 18S rDNA and the internal transcribed spacer (ITS) sequences data revealed that the new entomopathogenic nematode species forms a monophyletic group, which is a sister group of the clade comprised of some genera of Rhabditidae.

Flavobacterium anhuiense sp. nov., isolated from field soil.

A novel strain, D3T, isolated from a field-soil sample obtained from Anhui Province, PR China, was characterized taxonomically by using a polyphasic approach. The cells were Gram-negative, yellow-pigmented rods devoid of flagella, but showing gliding motility. The organism was able to grow at 5-37 degrees C and at pH 4.0-10.0. A comparative 16S rRNA gene sequence analysis indicated that strain D3T is a member of the genus Flavobacterium, sharing highest sequence similarity with the type strain of Flavobacterium defluvii (96.7 %). The major isoprenoid quinone was MK-6 and the predominant fatty acids were iso-C15 : 0, summed feature 3 (C16 : 1 omega 7c and/or iso-C15 : 0 2-OH) and C16 : 0. The DNA G+C content was 31.4 mol%. On the basis of phylogenetic and phenotypic data, strain D3T represents a novel species within the genus Flavobacterium, for which the name Flavobacterium anhuiense sp. nov. is proposed. The type strain is D3T (=KCTC 22128T = CGMCC 1.6859T).

A new family of antimicrobial peptides from skin secretions of Rana pleuraden

While conducting experiments to investigate antimicrobial peptides of amphibians living in the Yunnan-Guizhou region of southwest China, a new family of antimicrobial peptides was identified from skin secretions of the Yunnan frog, Rana pleuraden. Members of the new peptide family named pleurain-As are composed of 26 amino acids with a unique N-terminal sequence (SIIT) and a disulfide-bridged heptapeptide sequence (CRLYNTC). By BLAST search, pleurain-As had no significant similarity to any known peptides. Native and synthetic peptides showed antimicrobial activities against tested microorganisms including Gram-negative and Gram-positive bacteria and fungi. Twenty different cDNAs encoding pleurain-As were cloned from the skin cDNA library of R. pleuraden. The precursors of pleurain-As are composed of 69 amino acid residues including predicted signal peptides, acidic propieces, and cationic mature antimicrobial peptides. The preproregion of pleurain-A precursor comprises a hydrophobic signal peptide of 22 residues followed by an 18 residue acidic propiece which terminates by a typical prohormone processing signal Lys-Arg. The preproregions of precursors are very similar to other amphibian antimicrobial peptide precursors but the mature pleurain-As are different from other antimicrobial peptide families. The remarkable similarity of preproregions of precursors that give rise to very different antimicrobial peptides in distantly related frog species suggests that the corresponding genes form a multigene family originating from a common ancestor. Furthermore, pleurain-As could exert antimicrobial capability against Helicobacter pylori. This is the first report of naturally occurring peptides with anti-H. pylori activity from Rana amphibians.

Sphingobacterium siyangense sp nov., isolated from farm soil

The taxonomic position of a novel Gram-negative strain, designated SY1(T), isolated from a farm-soil sample obtained from Jiangsu Province, PR China, was characterized by using a polyphasic approach. The cells were non-motile, non-spore-forming rods. The organism grew optimally at 30-37 degrees C and at pH 6.0-8.0. Based on 16S rRNA gene sequence analysis, strain SY1(T) is a member of the genus Sphingobacterium; Sphingobacterium multivorum JCM 21156(T) was the nearest relative (98.5 % sequence similarity). The predominant fatty acids of strain SY1(T) were iso-C(15 : 0) (32.9 %), C(16 : 0) (10.9 %) and summed feature 3 (iso-C(15 : 0) 2-OH and/or C(16 : 1)omega7c; 24.1 %). The DNA G+C content was 38.5 mol%. The low level of DNA-DNA relatedness (2.2 %) to S. multivorum JCM 21156(T) in combination with differential morphological and biochemical properties demonstrated that strain SY1(T) (=KCTC 22131(T)=CGMCC 1.6855(T)) should be classified as representing a novel species of the genus Sphingobacterium for which the name Sphingobacterium siyangense sp. nov. is proposed.

Purification, characterization and cloning of two novel tigerinin-like peptides from skin secretions of Fejervarya cancrivora

While investigating the innate defense of brackish water-living amphibian and its comparison with freshwater-living amphibians, two novel 12-residue antimicrobial peptides were purified from the skin secretions of the crab-eating frog, Fejervarya cancrivora which typically inhabits brackish water of mangrove forests of Southeast Asia. These two antimicrobial peptides, tigerinin-RC1 and -RC2 share significant structural similarity with tigerinins found in the skin of Indian frog, Hoplobatrachus tigerinus. cDNAs encoding tigerinin-RC1 and -RC2 were also cloned from the skin cDNA library of F. cancrivora. Tigerinin-RC precursors are composed of 71 amino acid residues including a signal peptide, acidic spacer peptide, which are very similar to other amphibian antimicrobial peptide precursors and mature tigerinin-RC. The current results confirmed that both amphibians inhabiting freshwater and brackish water share the same antimicrobial peptide family to exert innate defense. Furthermore, the current work was also the first report of precursor and cDNA cloning of the tigerinin antimicrobial peptide family.

Bacillus solisalsi sp. nov., a halotolerant, alkaliphilic bacterium isolated from soil around a salt lake

A novel Gram-positive, motile, rod-shaped bacterium isolated from a saline soil in China was characterized by a polyphasic taxonomic approach. The strain, designated YC1(T), was halotolerant [tolerating up to 15 % (w/v) NaCl] and alkaliphilic (growing at a broad pH range of 5-13). 16S rRNA gene sequence analysis revealed that the isolate belonged to the genus Bacillus, showing highest similarity to Bacillus macauensis JCM 13285(T) (98.0 %). However, DNA-DNA hybridization indicated low levels of genomic relatedness with B. macauensis JCM 13285(T) (8.5 %). The major isoprenoid quinone was MK-7 and the cellular fatty acid profile consisted of significant amounts of iso-C(15 : 0) (38.6 %) and anteiso-C(15 : 0) (35.9 %). The predominant polar lipids were diphosphatidylglycerol, phosphatidylglycerol and phosphatidylethanolamine. The G+C content of the genomic DNA was 41.8 mol%. On the basis of the polyphasic evidence from this study, strain YC1(T) (=KCTC 13181(T)=CGMCC 1.6854(T)) should be classified as the type strain of a novel species of the genus Bacillus, for which the name Bacillus solisalsi sp. nov. is proposed.

Synthesis and antimicrobial activities of 4-aryl-3,4-dihydrocoumarins and 4-arylcoumarins

A new series of 4-aryl-3,4-dihydrocoumarins and 4-arylcoumarins were synthesized by the reaction of substituted cinnamic acids and 3-arylpropiolic acid with the corresponding phenols. These compounds were evaluated for antibacterial activity in vitro. The synthesized compounds displayed different degrees of antimicrobial activity against Staphylococcus aureus, Escherichia coli, and Bacillus dysenteriae, and Candida albicans (a fungus). Compounds with catechol moieties and 7,8-substituted dihydroxyls in the A ring were the most active antimicrobial agents.

Saccharibacillus kuerlensis sp. nov., isolated from a desert soil.

A taxonomic study was performed on strain HR1(T), which was isolated from a desert soil sample collected from Xinjiang Province (China). Cells were aerobic, Gram-positive-staining, pink-pigmented, sporulating rods with a single lateral flagellum. The organism can grow at 15-42 degrees C and pH 5.0-10.0, optimally at 30-37 degrees C and pH 6.0-8.0. Growth is inhibited by 6 % NaCl. Analysis of almost-complete 16S rRNA gene sequence revealed that the isolate represents a distinct taxon within the genus Saccharibacillus; Saccharibacillus sacchari LMG 24085(T) was the nearest relative (97.9 % sequence similarity). DNA-DNA hybridization showed 29.6 % genetic relatedness between strain HR1(T) and S. sacchari LMG 24085(T). The major isoprenoid quinone was MK-7 and the predominant fatty acid was anteiso-C(15 : 0) (50.3 %). The G+C content of the DNA was 50.5 mol%. Therefore, based on phenotypic criteria and the phylogenetic position, strain HR1(T) belongs to a previously unidentified species of the genus Saccharibacillus, for which the name Saccharibacillus kuerlensis sp. nov. is proposed. The type strain is HR1(T) (=KCTC 13182(T) =JCM 14865(T) =CGMCC 1.6964(T)).

Pseudomonas duriflava sp. nov., isolated from a desert soil

A Gram-negative, rod-shaped, non-motile, non-spore-forming bacterium, designated strain HR2(T), was isolated from a soil sample from the Taklimaken Desert in Xinjiang Province, China. Strain HR2(T) grew optimally at pH 7.0-8.0 and 30-37 degrees C in the presence of 0-1 % (w/v) NaCl. An analysis of 16S rRNA gene sequences revealed that strain HR2(T) fell within the radiation of the genus Pseudomonas, the highest level of similarity being found with respect to Pseudomonas luteola IAM 13000(T) (97.5 %); the levels of sequence similarity with respect to other recognized Pseudomonas species were <96.4 %. DNA-DNA hybridization showed that the genetic relatedness between strain HR2(T) and P. luteola IAM 13000(T) was 53.2 %. The G+C content of the genomic DNA of strain HR2(T) was 55.2 mol%. The major fatty acids were 18 : 1, summed feature 3 and 16 : 0. The hydroxylated fatty acids 10 : 0 3-OH, 12 : 0 3-OH and 12 : 0 2-OH were also present. The data obtained in this polyphasic study indicated that this isolate represents a novel species of the genus Pseudomonas, for which the name Pseudomonas duriflava sp. nov. is proposed. The type strain is HR2(T) (=KCTC 22129(T)=CGMCC 1.6858(T)).