Genomic Comparison of Plant Pathogenic and Nonpathogenic Serratia marcescens Strains by Suppressive Subtractive Hybridization

Cucurbit yellow vine disease (CYVD) is caused by disease-associated Serratia marcescens strains that have phenotypes significantly different from those of nonphytopathogenic strains. To identify the genetic differences responsible for pathogenicity-related phenotypes, we used a suppressive subtractive hybridization (SSH) strategy. S. marcescens strain Z01-A, isolated from CYVD-affected zucchini, was used as the tester, whereas rice endophytic S. marcescens strain R02-A (IRBG 502) was used as the driver. SSH revealed 48 sequences, ranging from 200 to 700 bp, that were present in Z01-A but absent in R02-A. Sequence analysis showed that a large proportion of these sequences resembled genes involved in synthesis of surface structures. By construction of a fosmid library, followed by colony hybridization, selection, and DNA sequencing, a phage gene cluster and a genome island containing a fimbrial-gene cluster were identified. Arrayed dot hybridization showed that the conservation of subtracted sequences among CYVD pathogenic and nonpathogenic S. marcescens strains varied. Thirty-four sequences were present only in pathogenic strains. Primers were designed based on one Z01-A-specific sequence, A79, and used in a multiplex PCR to discriminate between S. marcescens strains causing CYVD and those from other ecological niches.     

抑制差减杂交筛选禽致病性大肠杆菌基因组差异片段及其分析

采用抑制差减杂交技术(Suppression subtractive hybridization,SSH)对禽致病性大肠杆菌E037株(血清型O78)与非致病菌株K-12MG1655以及同一O2血清型高致病菌株E058与低致病菌株E526进行基因组差异片段克隆与分析。从E037株中共检出17个特异性差异片段,E058株中共检出32个特异性差异片段。经同源分析,这些序列可分为4类:质粒相关序列、噬菌体相关序列、已知功能序列、未知功能序列。这些差异片段包含许多重要的大肠杆菌毒力相关基因,如大肠杆菌素、气杆菌素受体、铁基因簇等。49个片段中,14个片段与其它微生物基因组同源性较高。结果表明,大肠杆菌高致病株与低致病菌株或非致病菌株基因组间存在较多差异基因,其中包括毒力、毒力相关基因、代谢以及噬菌体等基因成分。     

Novel endophytes of rice form a taxonomically distinct subgroup of Serratia marcescens

Six endophytic strains isolated from surface-sterilized rice roots and stems of different rice varieties grown in the Philippines were characterized. They were analyzed by physiological and biochemical tests, SDS-PAGE of whole-cell protein patterns, DNA-DNA hybridization and 16S rDNA sequencing. SDS-PAGE of whole-cell patterns showed that the six isolates fell into two subgroups which were similar but not identical in protein patterns to S. marcescens. The phylogenetic analysis of 16S rDNA sequences of two representative strains IRBG 500 and IRBG 501 indicated that they were closely related to S. marcescens (more than 99% identity). Physiological and biochemical tests corroborated that the isolates were highly related to each other and to S. marcescens. In cluster analysis, all six isolates were clustered together at 93% similarity level and grouped closely with Serratia marcescens at 86% similarity level. DNA-DNA hybridization studies revealed that the isolates shared high similarity levels with S. marcescens (> or =86% DNA-DNA binding), indicating they belong to the same species. However, the isolates differed in several biochemical characteristics from the type strain. They produce urease and utilize urea and L(+) sorbose as a substrate, which is different from all known Serratia reference strains. These results suggest that the six endophytic isolates represent a novel, non-pigmented subgroup of S. marcescens.     

Characterization and phylogenetic analysis of Varicella-zoster virus strains isolated from Korean patients

Varicella-zoster virus (VZV) is a causative agent of chickenpox in primary infection and shingles after its reactivation from latency. Complete or almost-complete genomic DNA sequences for various VZV strains have been reported. Recently, clinical VZV strains were isolated from Korean patients whose genome was sequenced using high-throughput sequencing technology. In this study, we analyzed single nucleotide polymorphism (SNP) of VZV strains to genetically characterize Korean clinical isolates. Phylogenetic analyses revealed that three Korean strains, YC01, YC02, and YC03, were linked to clade 2. Comprehensive SNP analysis identified 86 sites specific for the 5 VZV clades. VZV strains isolated from Korea did not form a phylogenetic cluster. Rather, YC02 and YC03 clustered strongly with Chinese strain 84-7 within clade 2, more specifically cluster 2a. Signature sequences for the cluster 2a were identified and found to play an important role in the separation of cluster 2a strains from other clade 2 strains, as shown in substitution studies. Further genetic analysis with additional strains isolated from Japan, China, and other Asian countries would provide a novel insight into the significance of two distinct subclades within clade 2.     

Bacterial diversity and function of aerobic granules engineered in a sequencing batch reactor for phenol degradation

Aerobic granules are self-immobilized aggregates of microorganisms and represent a relatively new form of cell immobilization developed for biological wastewater treatment. In this study, both culture-based and culture-independent techniques were used to investigate the bacterial diversity and function in aerobic phenol- degrading granules cultivated in a sequencing batch reactor. Denaturing gradient gel electrophoresis (DGGE) analysis of PCR-amplified 16S rRNA genes demonstrated a major shift in the microbial community as the seed sludge developed into granules. Culture isolation and DGGE assays confirmed the dominance of beta-Proteobacteria and high-G+C gram-positive bacteria in the phenol-degrading aerobic granules. Of the 10 phenol-degrading bacterial strains isolated from the granules, strains PG-01, PG-02, and PG-08 possessed 16S rRNA gene sequences that matched the partial sequences of dominant bands in the DGGE fingerprint belonging to the aerobic granules. The numerical dominance of strain PG-01 was confirmed by isolation, DGGE, and in situ hybridization with a strain-specific probe, and key physiological traits possessed by PG-01 that allowed it to outcompete and dominate other microorganisms within the granules were then identified. This strain could be regarded as a functionally dominant strain and may have contributed significantly to phenol degradation in the granules. On the other hand, strain PG-08 had low specific growth rate and low phenol degradation ability but showed a high propensity to autoaggregate. By analyzing the roles played by these two isolates within the aerobic granules, a functional model of the microbial community within the aerobic granules was proposed. This model has important implications for rationalizing the engineering of ecological systems.     

Bacterial Diversity and Function of Aerobic Granules Engineered in a Sequencing Batch Reactor for Phenol Degradation

Aerobic granules are self-immobilized aggregates of microorganisms and represent a relatively new form of cell immobilization developed for biological wastewater treatment. In this study, both culture-based and culture-independent techniques were used to investigate the bacterial diversity and function in aerobic phenol- degrading granules cultivated in a sequencing batch reactor. Denaturing gradient gel electrophoresis (DGGE) analysis of PCR-amplified 16S rRNA genes demonstrated a major shift in the microbial community as the seed sludge developed into granules. Culture isolation and DGGE assays confirmed the dominance of β-Proteobacteria and high-G+C gram-positive bacteria in the phenol-degrading aerobic granules. Of the 10 phenol-degrading bacterial strains isolated from the granules, strains PG-01, PG-02, and PG-08 possessed 16S rRNA gene sequences that matched the partial sequences of dominant bands in the DGGE fingerprint belonging to the aerobic granules. The numerical dominance of strain PG-01 was confirmed by isolation, DGGE, and in situ hybridization with a strain-specific probe, and key physiological traits possessed by PG-01 that allowed it to outcompete and dominate other microorganisms within the granules were then identified. This strain could be regarded as a functionally dominant strain and may have contributed significantly to phenol degradation in the granules. On the other hand, strain PG-08 had low specific growth rate and low phenol degradation ability but showed a high propensity to autoaggregate. By analyzing the roles played by these two isolates within the aerobic granules, a functional model of the microbial community within the aerobic granules was proposed. This model has important implications for rationalizing the engineering of ecological systems.     

Streptomyces poriferorum sp. nov., a novel marine sponge-derived Actinobacteria species expressing anti-MRSA activity

Marine sponges represent a rich source of uncharacterized microbial diversity, and many are host to microorganisms that produce biologically active specialized metabolites. Here, a polyphasic approach was used to characterize two Actinobacteria strains, P01-B04^T and P01-F02, that were isolated from the marine sponges Geodia barretti (Bowerbank, 1858) and Antho dichotoma (Esper, 1794), respectively. Phylogenetic analysis based on 16S rRNA gene sequences indicated that strains P01-B04^T and P01-F02 are closely related to Streptomyces beijiangensis DSM 41794^T, Streptomyces laculatispora NRRL B-24909^T, and Streptomyces brevispora NRRL B-24910^T. The two strains showed nearly identical 16S rRNA gene sequences (99.93%), and the average nucleotide identity (ANI) and digital DNA-DNA hybridization (dDDH) relatedness values were 99.96% and 99.6%, respectively, suggesting that these strains are affiliated with the same species. Chemotaxonomic and culture characteristics of both strains were also consistent with the genus Streptomyces, while phenotypic properties, genome-based comparisons, and phylogenomic analyses distinguished strains P01-B04^T and P01-F02 from their closest phylogenetic relatives. In silico analysis predicted that the 8.9 Mb genome of P01-B04^T contains at least 41 biosynthetic gene clusters (BGCs) encoding secondary metabolites, indicating that this strain could express diverse bioactive metabolites; in support of this prediction, this strain expressed antibacterial activity against Gram-positive bacteria including a clinical isolate of methicillin-resistant Staphylococcus aureus (MRSA) EAMC30. Based on these results, the marine sponge-associated isolates represent a novel species of the genus Streptomyces, for which the name Streptomyces poriferorum sp. nov. is proposed, with P01-B04^T (=DSM 111306^T = CCM 9048^T) as the type strain.     

Nucleotide sequence changes between Streptococcus pneumoniae R6 and D39 strains determined by an oligonucleotide hybridization DNA sequencing technology

The Gram-positive pathogen Streptococcus pneumoniae, which can be responsible for serious cases of pneumonia and meningitis, has been intensely studied for almost 100 years. Many of the key experiments have been performed in two strains; the non-pathogenic S. pneumoniae R6 and its pathogenic progenitor, S. pneumoniae D39. Whereas the genomic sequence of the R6 strain has been published, there is relatively little genomic information available on D39. Since R6 was derived from D39, we wished to explore the utility of a new technology, Comparative Genome Sequencing, which uses a set of custom oligonucleotide arrays to compare DNA sequences between similar strains. We report here the nucleotide polymorphisms identified between the R6 strain and D39 based on an R6 sequencing array. During the process, we were also able to confirm all of the high confidence changes reported by the oligonucleotide array chip by sequencing the region in the genome around the changes identified with the genome hybridization chip. We also discuss the potential impact of some of the amino acid changes found between these two widely used strains of pneumococci.     

产甾体皂甙华重楼内生菌的筛选与鉴定

从华重楼(Paris polyphyllavar.chinensisFranch)的地下块茎中分离筛选得到2株可能产生甾体皂甙的内生菌(SS01和SS02),薄层层析检测菌株SS01、SS02的发酵产物分别有3条和2条层析带与重楼总皂甙的层析带迁移率相当。形态和生理生化特征初步表明SS01和SS02分属于肠杆菌科(Enterobacteriaceae)和芽孢杆菌属(Bacillussp.)细菌。扩增、测序得到SS01和SS02的部分16S rDNA序列,GenBank接收号分别为AY842143和AY842144。用Blastn调出与菌株16SrDNA同源的序列,用Clustal W进行多重序列对比,用软件Phylip按Neighbor-Joining方法构建16SrDNA系统发育树。菌株SS01和SS02分别与Cedecea davisae DSM4568、Paenibacillus daejeonensis处于同一分支,相似性分别为98.9%和97.7%,将它们鉴定为Cedecea davisae SS01和Paenibacillus daejeonensis SS02。     

Properties of Serratia marcescens strains isolated in septic diseases of newborn infants

As the result of the study of blood and liquor samples from 120 newborns, Serratia marcescens was isolated in 21 cases (17.5 %). 8 strains were isolated from the environment of these patients. Almost all strains isolated from both the patients and the environment (with the exception of one environmental strain) belonged to serotype 04. The isolated S. marcescens strains were resistant to penicillin, ampicillin, streptomycin, kanamycin, oxacillin, methicillin, ceporin and moderately sensitive to polymixin. 2 strains from the environment and 9 strains from the patients were mildly sensitive to gentamicin. In one hospital all isolated strains were found to have 2 transmissive R plasmids with the molecular weight 40 and 60 megadaltons. The presence of R plasmids with the same molecular weight in all S. marcescens strains isolated in this hospital, as well as their serological identity, suggest that in all patients infection originated from a common source.     

Novel Endophytes of Rice form a Taxonomically Distinct Subgroup of Serratia marcescens

Six endophytic strains isolated from surface-sterilized rice roots and stems of different rice varieties grown in the Philippines were characterized. They were analyzed by physiological and biochemical tests, SDS-PAGE of whole-cell protein patterns, DNA-DNA hybridization and 16S rDNA sequencing. SDS-PAGE of whole-cell patterns showed that the six isolates fell into two subgroups which were similar but not identical in protein patterns to S. marcescens. The phylogenetic analysis of 16S rDNA sequences of two representative strains IRBG 500 and IRBG 501 indicated that they were closely related to S. marcescens(more than 99% identity). Physiological and biochemical tests corroborated that the isolates were highly related to each other and to S. marcescens. In cluster analysis, all six isolates were clustered together at 93% similarity level and grouped closely with Serratia marcescens at 86% similarity level. DNA-DNA hybridization studies revealed that the isolates shared high similarity levels with S. marcescens(≥86% DNA-DNA binding), indicating they belong to the same species. However, the isolates differed in several biochemical characteristics from the type strain. They produce urease and utilize urea and L(+) sorbose as a substrate, which is different from all known Serratia reference strains. These results suggest that the six endophytic isolates represent a novel, non-pigmented subgroup of S. marcescens.     

一株红球菌(Rhodococcus sp.)二噁英降解质粒的稳定性与接合转移特性

【目的】考察一株红球菌Rhodococcus sp.strain p52中的二噁英降解质粒pDF01(170 kb)和pDF02(242 kb)的稳定性和接合转移特性。【方法】在无选择压力的条件下对菌株p52进行连续传代培养,考察质粒pDF01、pDF02的丢失;以菌株p52为供体菌,以不同种属的菌株作受体菌,通过平板接合实验探讨质粒pDF01、pDF02接合转移的受体菌范围以及接合转移频率,利用菌落杂交、Southern杂交对质粒转移结果进行确认,利用降解实验测试转移质粒降解基因的表达。【结果】质粒pDF01和pDF02在红球菌p52中均具有较高的稳定性,在LB培养基上连续传代少于47次时pDF02可保持,连续传代少于65次时pDF01可保持。质粒pDF01和pDF02具备在同属和属间接合转移的能力,可向受体菌——紫红红球菌(Rhodococcus rhodochrous)、红串红球菌(Rhodococcus erythropolis)、大地两面神菌(Terrabacter tumescens)和节杆菌(Arthrobacter sp.)转移,其中以节杆菌作受体菌时质粒pDF01和pDF02接合转移频率最高,达到3.5×10~(-6)(接合子/受体菌);对节杆菌接合子质粒进行Southern杂交进一步确认了质粒pDF01、pDF02的存在。另外获得质粒pDF01、pDF02后的节杆菌接合子可以对二苯并呋喃高效利用,且降解能力与红球菌供体菌株p52相当。【结论】红球菌菌株p52可通过降解质粒转移强化生物修复过程,在去除环境中二噁英污染中具有良好的应用前景。     

鹅副粘病毒SF02 F基因的序列分析及SF02的多重RT—PCR鉴别

对新近分离的鹅副粘病毒SF0 2采用RT PCR方法 ,扩增F基因后测序 ,得到全长的F基因。该基因的ORF总长为 16 6 2nt,编码 5 5 3个氨基酸 ,其裂解位点的序列为112 R R Q K R F117,与新城疫病毒强毒株的特征相符。其核苷酸和氨基酸同源性分析 ,并与国内新城疫病毒标准强毒株F4 8E9相比较 ,表明该毒株在F基因上已发生了较大的变异 ,而与近年来在我国台湾和部分西欧国家流行的禽副粘病毒有很高的亲缘关系。在分析F基因序列的基础上 ,设计 3条引物 ,建立了一种新的多重RT PCR方法 ,能区分鸡新城疫病毒与鹅副粘病毒。     

Genome comparison of Bifidobacterium longum strains NCC2705 and CRC-002 using suppression subtractive hybridization

Because probiotic effects are strain dependent, genomic explanations of these differences will contribute to understanding their mechanisms of action. The genomic sequence of the Bifidobacterium longum probiotic strain NCC2705 was determined, but little is known about the genetic diversity between strains of this species. Suppression subtractive hybridization (SSH) is a powerful method for generating a set of DNA fragments differing between two closely related bacterial strains. The purpose of this study was to identify genetic differences between genomes of B. longum strains NCC2705 and CRC-002 using PCR-based SSH. Strain CRC-002 produces exopolysaccharides whereas NCC2705 is not known for reliable exopolysaccharide production. Thirty-five and 30 different sequences were obtained from the SSH libraries of strains CRC-002 and NCC2705, respectively. Specific CRC-002 genes found were predicted to be involved in the biosynthesis of exopolysaccharides and metabolism of other carbohydrates, and these genes were not present in the genome of strain NCC2705. The identification of an endo-1,4-beta-xylanase gene in the CRC-002 SSH library is an important difference because xylanase genes have previously been proposed as a defining characteristic of the NCC2705 strain. The results demonstrate that the SSH technique was useful to highlight potential genes involved in complex sugar metabolism that differ between the two probiotic strains.     

食用菌病毒脱毒方法的比较

应用dsRNA技术从平菇天达300(Pleurotus ostreatus TD300)菌丝中提取到4条dsRNA条带,大小分别是8.2、2.5、2.1和1.1kb。采用菌丝尖端脱毒法、原生质体再生法、冷冻真空干燥法和单孢杂交法进行脱毒,各得到一株脱毒菌株,分别是HTC8、PR15、FL01和SSH11。脱毒菌株HTC8仅残留有低含量的8.2kbdsRNA,PR15和SSH11完全脱去dsRNA,而FL01仍残留有低浓度的8.2kb和2.5kbdsRNA。该4株脱毒菌株的菌丝生长速度和漆酶活力均比出发菌种有不同程度的提高,尤其是HTC8和PR15,菌丝生长速度比出发菌种分别提高22.73%和18.18%,漆酶活力比出发菌种分别提高145.83%和134.38%。综合比较此4种脱毒方法,菌丝尖端脱毒法和原生质体再生脱毒法比冷冻真空干燥法和单孢杂交法脱毒效果好,制备脱毒菌株用时短、效率高。     

Short, interspersed, and repetitive DNA sequences in Spiroplasma species

Small fragments of DNA from an 8-kbp plasmid, pRA1, from a plant pathogenic strain of Spiroplasma citri were shown previously to be present in the chromosomal DNA of at least two species of Spiroplasma. We describe here the shot-gun cloning of chromosomal DNA from S. citri Maroc and the identification of two distinct sequences exhibiting homology to pRA1. Further subcloning experiments provided specific molecular probes for the identification of these two sequences in chromosomal DNA from three distinct plant pathogenic species of Spiroplasma. The results of Southern blot hybridization indicated that each of the pRA1-associated sequences is present as multiple copies in short, dispersed, and repetitive sequences in the chromosomes of these three strains. None of the sequences was detectable in chromosomal DNA from an additional nine Spiroplasma strains examined.     

抑制性差减杂交技术在蚊虫研究中的应用

抑制性差减杂交技术(suppression subtractive hybridization)是在差减杂交和抑制性PCR技术的基础上发展起来的鉴别差异表达基因的实验方法,用于分离2种具有相同或者相似遗传背景,但是表型上有差异的生物样品中差异表达的基因。这项技术在蚊虫许多研究领域,如抗药性、病原体感染、生理特性等得到广泛的应用。本文对抑制性差减杂交技术在蚊虫各研究领域的应用情况进行了综述。     

Overwintering squash bugs harbor and transmit the causal agent of cucurbit yellow vine disease

Since 1988, cucurbit crops, particularly watermelon, cantaloupe, and squash, grown in Oklahoma and Texas have experienced devastating losses from cucurbit yellow vine disease (CYVD), caused by the phloem-limited bacterium Serratia marcescens Bizio. Squash bug, Anasa tristis (De Geer), is a putative vector of the pathogen. In 2000-2001, overwintering populations of squash bug collected from DeLeon, TX, were tested for their ability to harbor and transmit the bacterium. Individual squash bugs (n = 73) were caged serially for periods of up to 7 d on at least four squash seedlings. Two studies were conducted, one with insects collected in November 2000 placed on first true leaf-stage seedlings and the second with insects from an April 2001 collection, placed on 3-5 true leaf-stage squash. Controls consisted of squash seedlings caged without insects. Squash bug transmission rates of the pathogen in studies I and II were 20 and 7.5%, respectively. Overall, 11.0% of the squash bugs harbored and successfully transmitted the bacterium to squash seedlings. All control plants tested negative for S. marcescens and did not exhibit CYVD. Female squash bugs killed a significantly greater proportion of young first leaf-stage seedlings than males. Feeding on 3-5 leaf-stage squash resulted in no plant mortality regardless of squash bug gender. This study demonstrated that the squash bug harbors S. marcescens in its overwintering state. The squash bug-S. marcescens overwintering relationship reported herein greatly elevates the pest status of squash bug and places more importance on development of integrated strategies for reducing potential overwintering and emerging squash bug populations.     

Conservation of symbiotic nitrogen fixation gene sequences in Rhizobium japonicum and Bradyrhizobium japonicum.

Southern hybridization with nif (nitrogen fixation) and nod (nodulation) DNA probes from Rhizobium meliloti against intact plasmid DNA of Rhizobium japonicum and Bradyrhizobium japonicum strains indicated that both nif and nod sequences are on plasmid DNA in most R. japonicum strains. An exception is found with R. japonicum strain USDA194 and all B. japonicum strains where nif and nod sequences are on the chromosome. In R. japonicum strains, with the exception of strain USDA205, both nif and nod sequences are on the same plasmid. In strain USDA205, the nif genes are on a 112-megadalton plasmid, and nod genes are on a 195-megadalton plasmid. Hybridization to EcoRI digests of total DNA to nif and nod probes from R. meliloti show that the nif and nod sequences are conserved in both R. japonicum and B. japonicum strains regardless of the plasmid or chromosomal location of these genes. In addition, nif DNA hybridization patterns were identical among all R. japonicum strains and with most of the B. japonicum strains examined. Similarly, many of the bands that hybridize to the nodulation probe isolated from R. meliloti were found to be common among R. japonicum strains. Under reduced hybridization stringency conditions, strong conservation of nodulation sequences was observed in strains of B. japonicum. We have also found that the plasmid pRjaUSDA193, which possess nif and nod sequences, does not possess sequence homology with any plasmid of USDA194, but is homologous to parts of the chromosome of USDA194. Strain USDA194 is unique, since nif and nod sequences are present on the chromosome instead of on a plasmid as observed with all other strains examined.