Restriction endonuclease EcoO109 from Escherichia coli H709c with heptanucleotide recognition site 5'-PuG/GNCCPy

A new restriction endonuclease, EcoO109, has been isolated from Escherichia coli H709c by polyethyleneimine (PEI) precipitation, DEAE-cellulose chromatography and heparin agarose chromatography. The yield was high, more than 3000 units/g of wet cells. The EcoO109 endonuclease recognizes and cleaves a nucleotide sequence of (formula: see text), in the presence of 10 mM Mg2+. The enzyme will be useful for structural analysis and molecular cloning of DNA because of the stability, high yield and easy handling of the producer strain.     

On the serology of Plesiomonas shigelloides

The serology of 87 strains of Plesiomonas shigelloides was studied. Thirty O antigenic groups and 11 H antigens were defined within the 87 strains, and an antigenic schema consisting of 40 serovars was established. Some O antigens of P. shigelloides were identical or closely related to those of some Shigella serovars.     

RsrII--a novel restriction endonuclease with a heptanucleotide recognition site.

A sequence-specific endonuclease present in extracts of Rhodopseudomonas sphaeroides 630 has been purified and characterized. The enzyme, Rsr II, recognises and cleaves the palindromic heptanucleotide sequence: (sequence; see test) By virtue of its unusual specificity, RsrII cuts most DNA molecules very infrequently which should facilitate the physical mapping of large genomes.     

Structural studies of the O-specific polysaccharide from Plesiomonas shigelloides strain CNCTC 113/92.

The structure of the O-specific side chain of the lipopolysaccharide (LPS) of Plesiomonas shigelloides, strain CNCTC 113/92 has been investigated by NMR spectroscopy, matrix-assisted laser desorption/ionization time of flight mass spectrometry and sugar and methylation analysis. It was concluded that the polysaccharide is composed of a hexasaccharide repeating unit with the following structure: in which D-beta-D-Hepp is Dglycero-beta-Dmanno-heptopyranose and 6d-beta-D-Hep is 6-deoxy-beta-Dmanno-heptopyranose. This structure represents a novel hexasaccharide repeating unit of bacterial O-antigen that is characteristic and unique to the Plesiomonas shigelloides strain. Using the high-resolution magic angle spinning technique, 1H-NMR spectra were also obtained for the O-polysaccharide components of isolated LPS and in their original form directly on the surface of bacterial cells.     

Experiences with serology of Plesiomonas shigelloides. 1. O-antigenic structure

With a set of 30 O-antisera, O-antigens were identified in 80% of 158 Plesiomonas shigelloides strains. Only strains of one serovar (018) regularly contained capsular antigen that caused their inagglutinability in the live state. Two groups of serovars displayed some O-antigenic relationship: 03 and 029; 08, 011 and 012. Each serovar in either group possessed a specific O-antigen and "group-common" minor antigens, which were designated I, II and III. Serovar 017 possessed O-antigen identical with that of Shigella sonnei phase I; this serovar was the most frequent one. Some serovars seemed to be ubiquitous; this was indicated by their wide geographic distribution and findings in man, domestic and feral animals, and water.     

Experiences with serology of Plesiomonas shigelloides. 2. H-antigenic structure

A total of 140 out of 144 motile Plesiomonas shigelloides strains were typable with the aid of a set of 15 H-antisera designated by letter symbols a through o. Commonly, strains of different O-serovars turned out to share the identical H-antigen and for some O-serovars it was typical to possess the certain type of H-antigen, as e.g. strains of serovar 017 which all have been to date characterized by the presence of H-antigens c, d or f. This set of 15 H-sera together with the previously described O-antigenic scheme of Plesiomonas may help to determine the frequency rates of OH serovars.     

Chemical characterization of enterobacterial common antigen isolated from Plesiomonas shigelloides ATCC 14029

Serologically characterized samples of enterobacterial common antigen (ECA) from Plesiomonas shigelloides, Salmonella montevideo and Shigella sonnei were investigated by chemical methods including methylation and NMR techniques. All showed the same sugar composition and contained a lipid moiety with palmitic acid as main fatty acid and with a phosphodiester group. Additional enzymatic studies, reported in the preceding paper, provided evidence that the lipid moiety is an L-glycerophosphatidyl residue attached via a phosphodiester linkage to C-1 of GlcNAc as the reducing end of the ECA sugar chain. ECA of P. shigelloides showed the best-resolved 13C-NMR spectra, especially after the removal of non-stoichiometric O-acetyl groups at C-6 of GlcNAc of the ECA repeating unit and of the lipid moiety by mild acid hydrolysis (0.01 M HCl, 100 degrees C, 10 min). Subsequent 13C-NMR studies were therefore carried out with the mild-acid-treated ECA of P. shigelloides which allowed a tentative assignment of all resonances of the ECA repeating unit. 13C-NMR spectra of Salmonella and Shigella ECA were essentially the same as those obtained with Plesiomonas ECA. The same trisaccharide repeating unit was encountered as demonstrated previously in the cyclic form of ECA isolated from S. sonnei by Dell et al. [Carbohydr. Res. 133, 95-104 (1984)]. Methylation analysis, however, afforded small amounts of terminal GlcNAc thus proving, in combination with the demonstration of the attached lipid moiety, an acyclic nature of ECA from P. shigelloides and from the two enterobacterial species. The question of whether the cyclic form co-exists in S. sonnei phase I and possibly in other enterobacterial species or, whether it had been formed during extraction as an artifact, has not yet been answered. The way in which ECA was isolated in our studies would preclude the presence of a non-amphiphilic (cyclic) polysaccharide. The finding that the sugar chain of ECA is attached to an L-glycerophosphatidyl residue is in full corroboration with serological, enzymatic and gel electrophoretic studies shown in the preceding paper and with the character of ECA as a surface antigen being anchored by hydrophobic interactions in the outer membrane of Enterobacteriaceae and P. shigelloides.     

草鱼致病性类志贺邻单胞菌的分离与鉴定

摘要:【目的】分离鉴定引起草鱼(Ctenopharyngodon idellus)肌肉糜烂的类志贺邻单胞菌(Plesiomonas shigeloide)菌株JX-09。【方法】从患病草鱼分离致病菌,经形态学观察、人工感染试验、生理生化特性测定和16S rRNA基因序列分析对其进行分类鉴定和致病性检验;同时对分离到的菌株做药物敏感性试验。【结果】从回归感染后的草鱼体内再次分离到菌株JX-09,表明该菌株为致病菌;菌株JX-09对草鱼的半致死量为6.4×104cfu/g。通过生化特征和分子系统学分析,将菌株JX-09鉴定为类志贺邻单胞菌。药敏试验表明该菌株对氨曲南、头孢唑林、头孢噻吩、头孢曲松等头孢类药物敏感,对卡那霉素、麦迪霉素、万古霉素和哌拉西林等耐药。【结论】类志贺邻单胞菌是草鱼肌肉糜烂病的致病菌,这是首次报道了该菌对草鱼有致病性。     

鲟致病性类志贺邻单胞菌的鉴定及药物敏感性

【目的】2012年夏季北京地区多地养殖的鲟鱼发病,主要临床症状为肛门红肿、伴有黄色分泌物,腹腔内有大量腹水,腹腔内壁有出血点,肝脏点状出血,脾脏肿大等,累计死亡率达60%。本文目的为研究其病原。【方法】从具有临床症状的濒死鱼中分离病原菌,分析病原菌的形态特征、理化特性、分类地位及药物敏感性等特性,经过人工感染及引起的组织病理确认致病性。【结果】结果显示病原菌的16S rDNA序列构建的进化树,与类志贺邻单胞菌同源性最高,在99%以上;结合其生理生化特征和API细菌鉴定系统的结果,确认为类志贺邻单胞菌。该菌对鲟鱼的半致死量LD50为1.0×105.8CFU/mL,引起肝、肾和脾组织病变。胞外产物不具有淀粉酶、蛋白酶、脂肪酶和明胶酶活性,也无溶血性,推测其毒性可能来源于内毒素。该菌对恩诺沙星、盐酸多西环素、氟苯尼考和甲枫霉素敏感,药物敏感浓度均小于2μg/mL;而对试验的其它抗菌药物不敏感。【结论】确认类志贺邻单胞菌是引起北京地区鲟鱼发生上述临床症状疾病的主要致病菌,可优选盐酸多西环素、氟苯尼考和恩诺沙星进行防治。     

Restriction endonuclease BsoFI is sensitive to the 5'-methylation of deoxycytidines in its recognition sequence.

The isoschizomeric restriction endonucleases Fnu4HI and BsoFI cleave DNA at 5'-GCdecreasesNGC-3' sequences. Fnu4HI has been shown to be inhibited by 5'-CG-3'methylation in the sequences 5'-GmCGGC-3' or 5'-GCGGmCG-3'. We have now investigated the methylation sensitivity of BsoFI by testing its activity on plasmid DNA 5'-CG-3' methylated with the M.SssI DNA methyltransferase or on synthetic (CGG)n repetitive oligodeoxyribonucleotides which have been partly or completely C methylated. The data demonstrate that BsoFI cannot cleave at its recognition sequence when it is completely 5'-CG-3' methylated. These enzymes have proven to be useful in analyses of the methylation status in (CGG)n repeats of the human genome.     

Restriction generated oligonucleotides utilizing the two base recognition endonuclease CviJI*.

The conversion of an anonymous DNA sample into numerous oligonucleotides is enzymatically feasible using an unusual restriction endonuclease, CviJI. Depending on reaction conditions, CviJI is capable of digesting DNA at a two or three base recognition sequence. CviJI normally cleaves RGCY sites between the G and C to leave blunt ends. Under 'relaxed' conditions CviJI* cleaves RGCY, and RGCR/YGCY, but not YGCR sites. In theory, CviJI* restriction of pUC19 (2686 bp) should produce 157 fragments, 75% of which are smaller than 20 bp. Instead, 96% of the CviJI* fragments were 18-56 bp long and none of the fragments were smaller than 18 bp. Thermal denaturation of these fragments generates sequence specific oligonucleotides homologous for the cognate template. The enzymatic conversion of anonymous DNA into sequence specific oligomers has implications for several conventional and novel molecular biology procedures.     

Intracellular vacuolation induced by culture filtrates of Plesiomonas shigelloides isolated from environmental sources

AIMS: Potential virulence factors produced by culture filtrates of Plesiomonas shigelloides isolated from water were investigated. METHODS AND RESULTS: Culture filtrates of P. shigelloides strains were assayed for cytotoxic activity in CHO (Chinese hamster ovary), Vero (African green monkey kidney), HeLa (human cervix), HT29 (human epithelial intestinal) and SK6 (swine epithelial kidney) cells. Microscopic analyses revealed intensive cytoplasmic vacuolation including cell rounding and swelling, with gradual destruction of the monolayer in filtrate-treated cells. Neutral red assays showed that CHO, HeLa and Vero cells were the most sensitive to the vacuolating activity, which was evident within 30 min of culture filtrate exposure. This activity was inactived by heating at 56 degrees C for 15 min and partially neutralized by antiserum to the cytotoxin of Aeromonas hydrophila. All P. shigelloides strains had a cell-associated haemolysin in the agar plate assay. Three isolates were found to produce a cell-free haemolytic activity at 37 degrees C. In the suckling mouse test, two P. shigelloides culture supernatants were positive for enterotoxic activity. CONCLUSIONS: P. shigelloides culture filtrates isolated from aquatic environment cause intracellular vacuolation on mammalian cells, and produce haemolytic and enterotoxic activities. SIGNIFICANCE AND IMPACT OF THE STUDY: This work revealed the presence of putative virulence factors that could be associated with human infections involving Plesiomonas strains.     

Restriction endonuclease EcaI from Enterobacter cloacae

Restriction endonuclease EcaI obtained from Enterobacter cloacae DSM30056 recognizes the group of heptanucleotide palindromes 5′-G[unk]G-T-N-A-C-C-3′, and on cleavage (arrow) produces fragments with 5′-terminal pentanucleotide extensions. It is identical in specificity with restriction endonuclease BstEII from Bacillus stearothermophilus ET.     

急性腹泻患者类志贺邻单胞菌流行及药物敏感性分析

目的了解类志贺邻单胞菌在急性腹泻患者中的分布及对常用抗菌药物的敏感性,为疾病的预防和临床治疗提供依据。方法收集2012年6月至2013年12月期间浙江省4家医院急性腹泻患者病例,采用常规微生物检验程序对常见肠道致病菌进行分离培养及检测。分离到的类志贺邻单胞菌采用K-B法进行药物敏感性试验。结果收集1 640例病例,共分离到类志贺邻单胞菌共41株,检出率为2.5%,占所有分离菌株的7.5%,居细菌性病原第四。41例病例中,男女检出率分别为2.6%和2.4%,差异无统计学意义;患者半数以上分布在18~44岁;每月均能够分离到类志贺邻单胞菌,其中夏季(6-8月)分离率最高,达3.9%。类志贺邻单胞菌对氨苄西林耐药率高达89.3%,对阿米卡星、复方新诺明、庆大霉素耐药率分别在30%~50%,未出现对头孢哌酮-舒巴坦、哌拉西林-他唑巴坦、亚胺培南及美罗培南耐药株。结论类志贺邻单胞菌是沿海地区夏秋季腹泻较为常见的病原菌之一,是临床肠道感染中不可忽视的病原。     

Characterization of the Plesiomonas shigelloides genes encoding the heme iron utilization system

Plesiomonas shigelloides is a gram-negative pathogen which can utilize heme as an iron source. In previous work, P. shigelloides genes which permitted heme iron utilization in a laboratory strain of Escherichia coli were isolated. In the present study, the cloned P. shigelloides sequences were found to encode ten potential heme utilization proteins: HugA, the putative heme receptor; TonB and ExbBD; HugB, the putative periplasmic binding protein; HugCD, the putative inner membrane permease; and the proteins HugW, HugX, and HugZ. Three of the genes, hugA, hugZ, and tonB, contain a Fur box in their putative promoters, indicating that the genes may be iron regulated. When the P. shigelloides genes were tested in E. coli K-12 or in a heme iron utilization mutant of P. shigelloides, hugA, the TonB system genes, and hugW, hugX, or hugZ were required for heme iron utilization. When the genes were tested in a hemA entB mutant of E. coli, hugWXZ were not required for utilization of heme as a porphyrin source, but their absence resulted in heme toxicity when the strains were grown in media containing heme as an iron source. hugA could replace the Vibrio cholerae hutA in a heme iron utilization assay, and V. cholerae hutA could complement a P. shigelloides heme utilization mutant, suggesting that HugA is the heme receptor. Our analyses of the TonB system of P. shigelloides indicated that it could function in tonB mutants of both E. coli and V. cholerae and that it was similar to the V. cholerae TonB1 system in the amino acid sequence of the proteins and in the ability of the system to function in high-salt medium.     

Restriction endonuclease analysis of ribosomal DNA from Saccharomyces cerevisiae

Summary Temperature-sensitive mutants of Escherichia coli that are unable to grow at high temperature can be obtained among those selected for resistance to streptovaricin or rifampicin at low temperature (Yura et al., 1970). One of these mutants (KY5323) that was supposed to carry a single mutation affecting both rifampicin resistance and temperature sensitivity was further investigated. Using purified RNA polymerase preparations obtained from the mutant and the wild type, it was found that the activity for DNA chain elongation is more sensitive to heat treatment than that for RNA chain initiation or DNA binding, and that the mutant enzyme is significantly more labile than the wild-type enzyme with respect to RNA chain elongation, when heat treatment is carried out at high salt concentration. These results, taken together with those of the enzyme reconstitution experiments, strongly suggest that the subunit of the polymerase is directly involved in both RNA chain initiation and elongation reactions. Enzyme reconstitution experiments using isolated subunits derived from the mutant and the wild-type polymerases demonstrate that the alteration of subunit is primarily responsible for both rifampicin resistance and thermolability of the mutant enzyme. In addition, the results suggested the apparent alteration of both and subunits in this mutant. Extensive transduction experiments provided genetic evidence that are consistent with the view that the strain KY5323 carries a second mutation affecting the subunit, beside the primary mutation affecting the subunit. The hypothetical subunit mutation seems to modify quantitatively the rifampicin resistance caused by the subunit mutation.