Molecular characterization of a carbapenem-hydrolyzing beta-lactamase from Chryseobacterium (Flavobacterium) indologenes

Chryseobacterium (Flavobacterium) indologenes 001 clinical strain was resistant to several beta-lactam classes including carbapenems. Shotgun cloning experiments of Sau3AI restricted genomic DNA of C. indologenes 001 into pBKCMV cloning vector followed by transformation into Escherichia coli DH10B gave one recombinant plasmid possessing a 4.2-kb DNA insert. It encoded a pI 7.2 beta-lactamase of 239 amino acids (IND-1) which is a metallo-enzyme with a broad spectrum beta-lactam hydrolysis profile. This class B carbapenem-hydrolyzing beta-lactamase shares the highest identity (43%) with BlaB from C. meningosepticum, thus showing heterogeneity of carbapenem-hydrolyzing beta-lactamases in Chryseobacterium spp.     

Pectolytic Bacteria Associated with Soft Rot of Calla Lily (Zantedeschia spp.) Tubers

Soft rot is the most important disease on calla lily in Poland. The isolation of the presumptive pathogen from symptomatic tubers on nutrient agar yielded bacteria with different colony morphology. Of 41 isolates collected, 10 showed pectolytic activity on crystal violet pectate medium and caused soft rot on potato slices. All pectolytic bacteria appeared to be Gram‐negative rods producing typical soft rot on inoculated leaf petioles of calla lily. Bacteria with colonies which morphologically resembled those used for inoculation were re‐isolated from diseased petioles. Their identification was based on phenotypic characters and sequence of the gene fragment coding 16S rRNA. It was found that, in addition to Pectobacterium carotovorum subsp. carotovorum, soft rot of calla lily can be caused by Pectobacterium carotovorum subsp. atrosepticum, Pseudomonas marginalis, Pseudomonas veronii and Chryseobacterium indologenes. The latter two are described for the first time as plant pathogens. The pectolytic activity of all identified bacteria, except that of P. carotovorum subsp. atrosepticum, was lower than that of P. carotovorum subsp. carotovorum, but strains of P. veronii showed a higher activity than P. marginalisand C. indologenes species.     

Production and characterization of an intracellular bioflocculant by Chryseobacterium daeguense W6 cultured in low nutrition medium

A novel intracellular bioflocculant (named MBF-W6) produced by Chryseobacterium daeguense W6 cultured in low nutrition medium was investigated in this study. The effects of carbon source, nitrogen source, C/N ratio, initial pH, inoculum size, culture temperature and shaking speed on MBF-W6 production were studied. Chemical analysis showed that the purified MBF-W6 was mainly composed of 32.4% protein, 13.1% polysaccharide and 6.8% nucleic acid. Fourier-transform infrared (FTIR) spectroscopy indicated the presence of carboxyl, hydroxyl, and methoxyl groups. The elemental analysis of purified MBF-W6 revealed that the mass proportion of C, H, O, N and S was 40.92:6.53:44.01:8.53:1.01 (w/w) correspondingly. MBF-W6 had good flocculating rate in Kaolin suspension without any cation addition. The highest flocculating rate of 96.9% was achieved under the optimal conditions (bioflocculant dosage 1.2 mg l⁻¹, pH 5.6 and temperature 15 °C).     

Impact of incubation period on biodegradation of petroleum hydrocarbons from refinery wastewater in Kuantan,Malaysia by indigenous bacteria

This work studied the biodegradation of petroleum hydrocarbons (PHCs) extracted from refinery wastewater to produce industrially important by-products at different incubation periods. Two out of 13 bacterial isolates, KRD2 and KRA4 were isolated. Dichloromethane was used to extract the PHC, and gas chromatography-mass spectrometry (GC-MS) analysis revealed that the refinery wastewater PHC was successfully biodegraded using the selected bacterial isolates within 15 days of incubation. Both KRD2 and KRA4 isolates degraded all 13 initially extracted PHC compounds within 5 days, except C13BD and C9BD, which produced 6 and 4 compounds as secondary metabolites with peak area percentages of 1.58, 1.38, 0.85, 29.94, 7.59, and 11.16% and 3.55, 2.88, 52.31, and 6.14%, respectively. These metabolites have been reported in industrial and medical applications. After 10 days, only 6 and 8 compounds were degraded by both isolates, respectively, and C11PAD compound was produced, as well as C5PAD, C7PAD, and C13PAD. After 15 days, it was clear that all the initial PHC compounds have been completely degraded by both isolates. Metabolites C5PAD, C6PAD, C8PAD, and C13PAD were produced by KRD2, and metabolites C5PAD, C6PAD, C8PAD, and C9PAD were produced by KRA4 at different peak areas. The alignment revealed that the KRA4 isolate was included in the genus Chryseobacterium gambrini, while KRD2 isolate was successfully identified as Mycobacterium confluentis using the Biolog microbial identification system. The incubation period evidently affected biodegradation process by indigenous degraders. These effective bacteria were shown to be of great potential for further application in biodegradation technology of PHC contaminated refinery wastewater to produce industrially important by-products.     

DDT降解菌株Chryseobacterium sp. PYR2对小麦的促生作用及其机理

【背景】前期结果表明,DDT降解菌株Chryseobacterium sp. PYR2可高效去除土壤中的DDT等污染物,具有潜在的应用价值,但该菌对植物的影响尚不清楚。【目的】探讨菌株Chryseobacterium sp. PYR2对植物的促生作用及其机理,为后续开发DDT降解及植物促生双效功能菌剂提供理论依据。【方法】配制该菌株的不同梯度稀释菌悬液,用纸卷发芽法和盆栽法研究菌悬液对小麦种子萌发和植株生长的影响;Salkowski法测定PYR2合成吲哚-3-乙酸(Indole-3-acetic acid,IAA)量;单因素实验研究不同培养条件对菌株生长及IAA合成的影响;液相色谱-串联质谱-多反应监测(LC-MS/MS-MRM)方法分析IAA在PYR2菌体内的生物合成途径。【结果】PYR2菌悬液可明显提高小麦种子萌发率并促进小麦植株的生长,小麦的侧根数、株高、鲜重、干重等指标均明显提高。该作用是由于菌株PYR2可以合成植物生长激素IAA。最适IAA合成条件:温度30°C,pH 7.0-8.0,盐浓度0.5%,L-色氨酸50mg/L。代谢液中检测到色醇、色胺和吲哚-3-乙酰胺3种中间代谢产物,推测PYR2体内存在3条IAA合成途径,分别为吲哚-3-丙酮酸(IPy A)、TAM和IAM途径。【结论】菌株PYR2对小麦具有明显的促生效果,是由于其具有多条高效合成IAA的代谢途径,表明其在农药污染土壤的生物修复及作物种植中具有潜在的应用前景。     

一株牛蛙源金黄杆菌的分离鉴定及其致病特性

【背景】美国牛蛙养殖过程中病害问题非常突出,尤其是细菌性病害,其病原种类多、病原菌复杂多样、蔓延速度快、发病死亡率高,一直是牛蛙养殖过程中防控的难点。【目的】确定从患病牛蛙体内分离到的一株细菌NW1203的分类地位和致病性。【方法】无菌操作从牛蛙体内取样划线分离细菌,通过形态观察、生理生化试验、16S rRNA基因序列比对进行种属鉴定,通过人工感染、溶血性试验和病理切片观察分析其致病特性。【结果】经形态和生理生化鉴定及16S rRNA基因序列比对,菌株NW1203为金黄杆菌属细菌,与Chryseobacterium sp. F30的相似性达100%,进化树也显示该菌与金黄杆菌属细菌聚类;溶血性试验表明,菌株NW1203对绵羊、小鼠和牛蛙的血细胞都呈完全溶血;人工感染试验及感染病蛙的组织切片观察显示,菌株NW1203对牛蛙具有较强致病性,可引起牛蛙肝、肾、脾等主要组织严重病变,LD50为4.753×103 CFU/g。【结论】明确了菌株NW1203为牛蛙新病原,为牛蛙疾病的防控提供了理论依据。     

An unusual lipid a from a marine bacterium Chryseobacterium scophtalmum CIP 104199T

The hydrolysis of defatted cells of the marine bacterium Chryseobacterium scophtalmum CIP 104199^T with 10% acetic acid (3 h, 100°C) led to an unusual lipid A (LA) (yield 0.6%), obtained for the first time. Using chemical analysis, FAB MS, and NMR spectroscopy, it was shown to be D-glucosamine 1-phosphate acylated with (R)-3-hydroxy-15-methylhexadecanoic and (R)-3-hydroxy-13-methyltetroadecanoic acids at the C2 and C3 atoms, respectively. It is similar to the monosaccharide biosynthetic precursor of lipopolysaccharide (LPS), so-called lipid X (LX). Unlike LX, LA can be isolated by the treatment of bacteria with organic solvents only after the preliminary acidic hydrolysis of the cells, which suggests that LA might be strongly, probably chemically, linked to other components of the outer membrane. However, LPS cannot be such a component, because extraction with phenol-water or phenol-chloroform-petroleum ether mixtures in high yields (5.34% and 0.5%, respectively) leads to preparations that do not contain 3-deoxy-D-manno-oct-2-ulopyranosonic acid, 3-hydroxyalkanoic acids, or LA.     

Purification and characterization of a metalloprotease from Chryseobacterium indologenes Ix9a and determination of the amino acid specificity with electrospray mass spectrometry

The heat-stable protease from Chryseobacterium indologenes Ix9a was purified to homogeneity using immobilized metal affinity chromatography. The enzyme was characterized as a metalloprotease with an approximate relative molecular mass of 24,000, a pH optimum of 6.5, and a high temperature optimum (50 degrees C). The metal chelator EDTA and the Zn2+-specific chelator 1,10-phenanthroline were identified as inhibitors and atomic absorption analysis showed that the enzyme contained Ca2+ and Zn2+. The activity of the apoenzyme could be restored with Ca2+, Zn2+, Mg2+, and Co2+. Phosphoramidon and Gly-d-Phe did not inhibit Chryseobacterium indologenes Ix9a protease. Heat inactivation did not follow first order kinetics, but showed biphasic inactivation curves. The protease has a Km of 0.813 microg. ml-1 for casein as substrate. Amino acid analysis showed that the protease contains a high amount of small amino acids like glycine, alanine, and serine, but a low concentration of methionine and no cysteine at all. Electrospray mass spectrometry of proteolysis fragments formed when insulin B chain was hydrolyzed showed cleavage at the amino terminal of leucine, tyrosine, and phenylalanine. A hydrophobic amino acid at the carboxyl donating side seems to increase the rate of reaction.     

Purification and characterization of three novel keratinolytic metalloproteases produced by Chryseobacterium indologenes TKU014 in a shrimp shell powder medium

A protease-producing bacterium was isolated and identified as Chryseobacterium indologenes TKU014. The optimized condition for protease production was found when the culture was shaken at 30 °C for one day in 50 mL of medium containing 0.5% shrimp shell powder (w/v), 0.1% K₂HPO₄, and 0.05% MgSO₄ · 7H₂O. Three extracellular proteases (P1, P2, and P3) were purified from culture by DEAE-Sepharose and Phenyl Sepharose chromatography. Three enzymes all showed activities of keratinase and elastase with molecular weights of 56, 40, 40 kDa, respectively. The inhibitory effect of metal chelator EDTA and Zn-specific chelator 1,10-phenanthroline characterized three enzymes as Zn-metalloproteases. Peptide mass fingerprints of P1, P2, and P3 were determined by using liquid chromatography–tandem mass spectrometry (LC–MS/MS). Similarity search in the NCBI non-redundant protein sequence database revealed that three enzymes exhibited no significant homology to any other reported microbial peptides. Therefore, P1, P2, and P3 are most likely novel proteins.