Keeping it together: absence of genetic variation and DNA incorporation by the predatory bacteria Micavibrio aeruginosavorus and Bdellovibrio bacteriovorus during predation

The use of predatory bacteria as a potential live therapeutic to control human infection is gaining increased attention. Earlier work with Micavibrio spp. and Bdellovibrio spp. has demonstrated the ability of these predators to control drug-resistant Gram-negative pathogens, Tier-1 select agents and biofilms. Additional studies also confirmed that introducing high doses of the predators into animals does not negatively impact animal well-being and might assist in reducing bacterial burden in vivo. The survival of predators requires extreme proximity to the prey cell, which might bring about horizontal transfer of genetic material, such as genes encoding for pathogenic genetic islands that would indirectly facilitate the spread of genetic material to other organisms. In this study, we examined the genetic makeup of several lab isolates of the predators Bdellovibriobacteriovorus and Micavibrioaeruginosavorus that were cultured repeatedly and stored over a course of 13 years. We also conducted controlled experiments in which the predators were sequentially co-cultured on Klebsiella pneumoniae followed by genetic analysis of the predator. In both cases, we saw little genetic variation and no evidence of horizontally transferred chromosomal DNA from the prey during predator–prey interaction. Culturing the predators repeatedly did not cause any change in predation efficacy.     

ST239-spa t037 MRSA与ST239-spa t030 MRSA的比较基因组分析

目的 寻找金黄色葡萄球菌基因组进化相关的关键基因.方法 利用含有2457个金黄色葡萄球菌基因的比较基因组芯片对23株不同时间、不同地点分离的ST239-spa t037和ST239-spa t030的甲氧西林耐药的金黄色葡萄球菌(methicillin-resistant S.aureus,MRSA)进行比较基因组分析,鉴定差异区段,并利用PCR验证差异基因.结果 通过对北京地区MRSA早期克隆(ST239-spat037,1994-1998年)和晚期克隆(ST239-spa t030,2000-2006年)的比较基因组分析,发现4个基因簇仅存在于晚期克隆,主要定位于3个已知的基因组岛(vSa4,前噬菌体ΦSa1和ΦSa3).不同地区的ST239-spa t030 MRSA的比较基因组分析发现:在不同地区的菌株中有8个基因存在差异.结论 北京地区MRSA从t037进化到t030的过程中获得3个基因组岛(vSa4、前噬菌体ΦSa1和ΦSa3),从而增强了ST239-spa t030 MRSA的毒力和适应性,对其取代ST239-spa t037 MRSA发挥关键作用.     

The complete genomes of Staphylococcus aureus bacteriophages 80 and 80α—Implications for the specificity of SaPI mobilization

Staphylococcus aureus pathogenicity islands (SaPIs) are mobile elements that are induced by a helper bacteriophage to excise and replicate and to be encapsidated in phage-like particles smaller than those of the helper, leading to high-frequency transfer. SaPI mobilization is helper phage specific; only certain SaPIs can be mobilized by a particular helper phage. Staphylococcal phage 80α can mobilize every SaPI tested thus far, including SaPI1, SaPI2 and SaPIbov1. Phage 80, on the other hand, cannot mobilize SaPI1, and ?11 mobilizes only SaPIbov1. In order to better understand the relationship between SaPIs and their helper phages, the genomes of phages 80 and 80α were sequenced, compared with other staphylococcal phage genomes, and analyzed for unique features that may be involved in SaPI mobilization.     

Dichloromethane-degrading bacteria in the genomic age

Dichloromethane (DCM) is a volatile toxic halogenated solvent mainly produced and used industrially. DCM-degrading bacteria have long been models of choice for studying bacterial dehalogenation metabolism at the physiological, biochemical and genetic levels, and have also been used in bioremediation processes. DCM-degrading strains isolated in recent years will be discussed in the context of enzymes known to catalyze dehalogenation of DCM. Insights into the modes of adaptation of bacteria to DCM gained by comparative genomic analysis, highlight the importance of horizontal gene transfer in the dissemination of genes for DCM metabolism in the environment.     

Transfer of an autonomously replicating vector between vegetatively incompatible biotypes of Colletotrichum gloeosporioides

Previous research has indicated that biotypes A and B of Colletotrichum gloeosporioides that infect Stylosanthes spp. in Australia are asexual and vegetatively incompatible. Selectable marker genes conferring resistance either to hygromycin or phleomycin were introduced into isolates of these biotypes. Vectors conferring resistance to hygromycin and carrying telomeric sequences from Fusarium oxysporum replicated autonomously in C. gloeosporioides and gave frequencies of transformation 100-times higher than vectors that integrated into the genome. Monoconidial colonies resistant to both antibiotics were recovered when hygromycin-resistant biotype-A transformants carrying an autonomously replicating vector were paired in culture with a phleomycin-resistant biotype-B transformant carrying integrative vector sequences. Molecular analysis of double antibiotic-resistant progeny indicated that they contained the autonomous vector in a biotype-B genetic background. Results indicate that transfer of the autonomous vector had occurred from biotype A to biotype B, demonstrating the potential for transfer of genetic information between these biotypes. Received: 13 January / 24 March 1997     

Poxvirus protein evolution: Family wide assessment of possible horizontal gene transfer events

To investigate the evolutionary origins of proteins encoded by the Poxviridae family of viruses, we examined all poxvirus protein coding genes using a method of characterizing and visualizing the similarity between these proteins and taxonomic subsets of proteins in GenBank. Our analysis divides poxvirus proteins into categories based on their relative degree of similarity to two different taxonomic subsets of proteins such as all eukaryote vs. all virus (except poxvirus) proteins. As an example, this allows us to identify, based on high similarity to only eukaryote proteins, poxvirus proteins that may have been obtained by horizontal transfer from their hosts. Although this method alone does not definitively prove horizontal gene transfer, it allows us to provide an assessment of the possibility of horizontal gene transfer for every poxvirus protein. Potential candidates can then be individually studied in more detail during subsequent investigation.Results of our analysis demonstrate that in general, proteins encoded by members of the subfamily Chordopoxvirinae exhibit greater similarity to eukaryote proteins than to proteins of other virus families. In addition, our results reiterate the important role played by host gene capture in poxvirus evolution; highlight the functions of many genes poxviruses share with their hosts; and illustrate which host-like genes are present uniquely in poxviruses and which are also present in other virus families.     

Mobile genetic elements of Pseudomonas aeruginosa isolates from hydrotherapy facility and respiratory infections

The content of mobile genetic elements in Pseudomonas aeruginosa isolates of a pristine natural mineral water system associated with healthcare was compared with clinical isolates from respiratory infections. One isolate, from the therapy pool circuit, presented a class 1 integron, with 100% similarity to a class 1 integron contained in plasmid p4800 of the Klebsiella pneumoniae Kp4800 strain, which is the first time it has been reported in P. aeruginosa. Class 1 integrons were found in 25.6% of the clinical isolates. PAGI1 orf3 was more prevalent in environmental isolates, while PAGI2 c105 and PAGI3 sg100 were more prevalent in clinical isolates. Plasmids were not observed in either population.     

O139霍乱弧菌CTAKΦ的基因水平转移的研究

目的 探讨CTAKΦ介导的霍乱弧菌毒素基因水平转移。方法 用抗生素敏感试验筛选出几组基因水平转移试验组合的供、受体菌，以便于用抗性筛选来确定基因转移的情况。在这几组试验组合中分别用接合转移、上清水平转移、培养液水平转移的方法，检查供体菌将CTAKΦ的A^RK^R抗性转移给受体菌的能力。结果 供体菌FJ97129、DX29能通过多种转移方式，以很高的频率将CTAKΦ的A^RK^R抗性转移给受体菌HK42、XJ93172、GD3188、7763、569B和94001；而受体菌IEM101对CTAKΦ有一定的抗感染能力。结论 CTAKΦ能够介导霍乱弧菌中霍乱毒素基因的水平转移。     

FRET的理论基础及应用

2001年,《自然》和《科学》杂志先后公布了人类基因组的工作草图,人类从此进入了功能基因组时代。目前,研究生物大分子,特别是蛋白质的结构、功能、特点及其相互作用已经成为生命科学的工作重点,随着蛋白质研究的进展,一项古老的技术-荧光共振能量转移(fluorescenceresonanceen     

Site-specific in situ growth of a cyclized protein-polymer conjugate with improved stability and tumor retention

A major disadvantage of therapeutic proteins is their instability to external stressors during storage, transport and use. Here, we report site-specific in situ growth of a cyclized protein-polymer conjugate with improved in vitro and in vivo stability. Green fluorescence protein (GFP) was genetically fused at its N- and C-termini with two sortase recognition sequences pentaglycine and LPETG, respectively to yield a linear GFP (l-GFP). A cyclized GFP (c-GFP) was generated from the l-GFP by sortase-catalyzed cyclization. A maleimide-functionalized atom transfer radical polymerization (ATRP) initiator was selectively attached to a free cysteine residue genetically engineered at the C-terminus of GFP to form a macroinitiator (c-GFP-Br). Subsequent in situ ATRP of oligo(ethylene glycol) methyl ether methacrylate (OEGMA) from the c-GFP-Br generated a site-specific (C-terminal) and stoichiometric (1:1) c-GFP-POEGMA conjugate with almost quantitative conversion and highly retained activity. Notably, the c-GFP-POEGMA conjugate showed 9- and 310-fold increases in thermal stability as compared to the l-GFP and its counterpart l-GFP-POEGMA, respectively. Additionally, the conjugate displayed significantly improved tumor retention relative to the l-GFP and l-GFP-POEGMA. The method developed may be applicable to a variety of therapeutic proteins to improve their in vitro and in vivo stability.     

Novel and uncommon antimicrobial resistance genes in livestock-associated methicillin-resistant Staphylococcus aureus

Livestock-associated methicillin-resistant Staphylococcus aureus (LA-MRSA) isolates have been the subject of numerous studies during recent years. The characterization of such isolates has usually also included the determination of their resistance phenotypes and associated resistance genotypes. Analysis of the resistance genes present in LA-MRSA isolates has revealed a number of genes commonly found in S. aureus and coagulase-negative staphylococci of humans and animals. In addition, novel resistance genes and/or resistance genes that have been rarely detected in staphylococci so far have been encountered. These include the phenicol exporter gene fexA, the multiresistance gene cfr, the tetracycline resistance gene tet(L), the trimethoprim resistance gene dfrK, the macrolide–lincosamide–streptogramin B resistance gene erm(T), the lincosamide–streptogramin A–pleuromutilin resistance genes vga(C) and vga(E), and the apramycin resistance gene apmA. Most of these genes were located on multiresistance plasmids in LA-MRSA. The co-localization of these resistance genes with other resistance genes enables their co-selection and persistence. LA-MRSA can therefore act as a donor and a recipient of antimicrobial resistance genes within the Gram-positive gene pool.     

Evolution and phylogeny of the corticotropin-releasing factor (CRF) family of peptides: Expansion and specialization in the vertebrates

New sequence data on CRF family members from a number of genomes has led to the modification of our understanding of CRF evolution in the Metazoa. The corticotropin-releasing factor (CRF) family of peptides include four paralogous lineages in jawed vertebrates; CRF, urotensin-I/urocortin/sauvagine, urocortin 2 (Ucn2) and urocortin 3 (Ucn3). CRF and the urotensin-I/urocortin/sauvagine group represent a gene duplication from one lineage, whereas Ucns 2 and 3 are the result of a gene duplication in the other paralogous lineage. Both paralogous lineages are the result of a gene duplication from a single ancestral peptide that occurred after the divergence of the tunicates from the ancestor that led to the evolution of chordates and vertebrates. The presence of a single CRF-like peptide in tunicates and insects suggests that a single CRF-like ancestor was present before the separation of deuterostomes and protostomes. Currently there is no strong evidence that indicates that CRF-like peptides were present in metazoan taxa that evolved before this time although the structural similarity between some CRF peptides in insects, tunicates and vertebrates with the calcitonin family of peptides hints that prior to the formation of deuterostomes and protostomes the ancestral peptide possessed both CRF and calcitonin-like structural attributes. Here, we show evidences of conservation of CRF-like function dating back to early prokaryotes. This ancestral CRF–calcitonin-like peptide may have initially resulted from a horizontal gene transfer event from prokaryotes to a protistan species that later gave rise to the metazoans.     

Distribution of the pilS gene in Escherichia coli pathovars,its transfer ability and influence in the typical enteropathogenic E. coli adherence phenotype

Typical enteropathogenic Escherichia coli strains (tEPEC) cause attaching/effacing lesions in eukaryotic cells and produce the bundle-forming pilus (BFP), which interweaves and aggregates bacteria, resulting in the localized adherence (LA) pattern on eukaryotic cells. Previously, we identified tEPEC strains (serotype O119:H6) that exhibited LA simultaneously with an aggregative adherence (AA)-like pattern (LA/AA-like+). Remarkably, AA is characteristically produced by strains of enteroaggregative E. coli (EAEC), another diarrheagenic E. coli pathovar. In one LA/AA-like?+?strain (Ec404/03), we identified a conjugative plasmid containing the pil operon, which encodes the Pil fimbriae. Moreover, a pil operon associated with an AA pattern and plasmid transfer had been previously described in the EAEC C1096 strain. In this study, we investigated the occurrence of the two pilS alleles (pilS_Ec404 and pilS_C1096) in tEPEC strains of different serotypes, origins and years of isolation. We also examined the potential relationship of pilS with the AA-like phenotype, its ability to be transferred by conjugation, and occurrence among strains of the other E. coli pathovars. The pilS alleles were found in 90 (55.2%) of 163 tEPEC strains, with pilS_Ec404 occurring more often (30.7%) than pilS_C1096 (25.1%). About 21 tEPEC serotypes carried pilS. The pilS alleles were found in tEPEC strains from Chile, Peru and different Brazilian cities, with the oldest strain being isolated in 1966. No absolute correlation was found between the presence of pilS and the AA-like pattern. Conjugative pilS transfer was detected in 26.2% of pilS_Ec404+ strains and in 65.1% of pilS_C1096+ strains, but only pilS_Ec404+ transconjugants were AA-like+, thus suggesting that the latter allele might need a different genetic background to express this phenotype. pilS was found in all other E. coli pathovars, where it was most prevalent in enterotoxigenic E. coli. More studies are needed to understand the mechanisms involved in the regulation of Pil expression and production.     

The origins of eukaryotic-like proteins in Legionella pneumophila

Legionella pneumophila, the causative agent of Legionnaires’ disease, is known to be an intracellular pathogen of multiple species of protozoa and is assumed to have co-evolved with these organisms for millions of years. Genome sequencing of L. pneumophila strains has revealed an abundance of eukaryotic-like proteins (ELPs). Here, we study the evolution of these ELPs, in order to investigate their origin. Thirty-four new ELPs were identified, based on a higher similarity to eukaryotic proteins than to bacterial ones. Phylogenetic analyses demonstrated that both lateral gene transfer from eukaryotic hosts and bacterial genes that became eukaryotic-like by gradual adaptation to the intracellular milieu or gene fragment acquisition, contributed to the existing repertoire of ELPs, which comprise over 3% of the putative proteome of L. pneumophila strains. A PCR survey of 72 L. pneumophila strains showed that most ELPs were conserved in nearly all of these strains, indicating that they are likely to play important roles in this species. Genes of different evolutionary origin have distinct patterns of selection, as reflected by their ratio of a synonymous vs. synonymous mutations. One ELP is common to several strains of Legionella, but outside this genus has homologs only in Acanthamoeba polyphaga mimivirus, indicating that gene exchange involving eukaryotic viruses and intracellular bacterial pathogens may also contribute to the evolution of virulence in either or both of these groups of organisms. Information on selection patterns and eukaryotic-like status was combined as a novel approach to predict type IV secretion system effectors of Legionella, which represent promising targets for future study.     

Some considerations on the nature of LUCA, and the nature of life

In addition to its scientific interest, research on the last universal common ancestor (LUCA) and the lower part of the tree of life raises important and difficult issues in biology, but also in the philosophy of science as well as in philosophy in general. The way inquiries are formulated has to be scrutinized to avoid unanswerable questions. Preconceived ideas, poorly defined notions and abuse of metaphors are obstacles which can induce bias in studies and in the interpretation of their results. In the background, the question of life has recently re-emerged.     

基因组重排的机制研究

基因组结构变异是人类基因组中常见的一种变异形式,往往涉及染色体或大片段染色体区域的改变,将原来两个分开的DNA序列重新连接在一起,从而形成染色体片段的缺失、重复复制、插入、倒位或者易位.在核苷酸水平上对大量重排断裂点接头序列特征的分析,为研究基因组重排的机制提供了宝贵的资源.本文综述了导致基因组重排的同源重组和非同源修复机制,并提出复制过程在基因组重排中发挥了重要作用,是细胞压力和结构变异的潜在桥梁,对于理解人类基因组进化和人类疾病的发生发展具有重要意义.     

Vegetative incompatibility in Neurospora: its effect on horizontal transfer of mitochondrial plasmids and senescence in natural populations

We have investigated the horizontal transfer of two mitochondrial plasmids and the Kalilo senescence phenotype in the fungus Neurospora without the use of heterokaryon-forcing markers. The Kalilo senescent state was only transferred between fully-compatible N. crassa strains, but not between strains differing at any of the loci het-c, het-d, het-e or mating-type. However, the linear plasmid kalDNA and the circular plasmid Han-2 were transferred following incompatible vegetative interactions. Our data suggest that vegetative incompatibility due to allelic differences at het-c is more effective in preventing transfer than that due to het-d, het-e or mating-type. Based on these observations we have developed a novel test for assessing vegetative incompatibility between Kalilo and non-Kalilo field isolates of N. intermedia. In this procedure combinations of Kalilo and non-Kalilo field isolates of N. intermedia were grown together and tested for senescence. Compatibility is inferred if the young non-Kalilo strain dies along with the senescent Kalilo strain, whereas incompatibility is inferred when the Kalilo strain dies without imposing its senescent state onto the non-Kalilo strain. Our results suggest that each of the nine Kalilo strains tested is incompatible with each of 20 non-Kalilo isolates from the same N. intermedia population of the Hawaiian island of Kauai. However, the observed incompatibility did not completely prevent cytoplasmic exchange, and in several cases plasmid transfer could be detected.