Genome sequence of Rickettsia bellii illuminates the role of amoebae in gene exchanges between intracellular pathogens

The recently sequenced Rickettsia felis genome revealed an unexpected plasmid carrying several genes usually associated with DNA transfer, suggesting that ancestral rickettsiae might have been endowed with a conjugation apparatus. Here we present the genome sequence of Rickettsia bellii, the earliest diverging species of known rickettsiae. The 1,552,076 base pair–long chromosome does not exhibit the colinearity observed between other rickettsia genomes, and encodes a complete set of putative conjugal DNA transfer genes most similar to homologues found in Protochlamydia amoebophila UWE25, an obligate symbiont of amoebae. The genome exhibits many other genes highly similar to homologues in intracellular bacteria of amoebae. We sought and observed sex pili-like cell surface appendages for R. bellii. We also found that R. bellii very efficiently multiplies in the nucleus of eukaryotic cells and survives in the phagocytic amoeba, Acanthamoeba polyphaga. These results suggest that amoeba-like ancestral protozoa could have served as a genetic “melting pot” where the ancestors of rickettsiae and other bacteria promiscuously exchanged genes, eventually leading to their adaptation to the intracellular lifestyle within eukaryotic cells.     

Isolation of Rickettsia rhipicephali and Rickettsia bellii from Haemaphysalis juxtakochi Ticks in the State of São Paulo, Brazil

In the present study, attempts to isolate Rickettsia in cell culture were performed individually in seven specimens of Haemaphysalis juxtakochi ticks collected in the state of São Paulo (southeastern Brazil). Rickettsia was successfully isolated by the shell vial technique and established in Vero cell culture from six ticks (six isolates). DNA extracted from infected cells of these isolates was tested by PCR and DNA sequencing, using genus-specific Rickettsia primers targeting the genes gltA, htrA, ompA, and ompB. After the generated sequences were compared with available sequences in GenBank, five out of the six isolates were identified as Rickettsia bellii (isolates HJ#1, HJ#2, HJ#3, HJ#4, and HJ#7). The sixth isolate (HJ#5) was closest to Rickettsia sp. strain R300, previously detected in H. juxtakochi in northern Brazil, and to Rickettsia rhipicephali, isolated from ticks in the United States. Following recent gene sequence-based criteria proposed for the identification of Rickettsia isolates, both isolate HJ#5 and strain R300 were identified as South American strains of R. rhipicephali, which was confirmed in this continent for the first time. Isolation of R. bellii from H. juxtakochi ticks, added to eight other tick species that have been reported to be infected with this bacterium in Brazil, indicates that R. bellii is indeed the most frequent Rickettsia species infecting ticks in Brazil. Currently, the role of both R. rhipicephali and R. bellii as human pathogens is regarded as unknown.     

Transformation and Conjugal Transfer of Foreign Genes into the Filamentous Multicellular Cyanobacteria (Subsection V) Fischerella and Chlorogloeopsis

Cyanobacteria of subsection V grow as filaments with asymmetrical cell divisions that can generate a true-branching phenotype. Members of the genera Fischerella and Chlorogloeopsis furthermore differentiate akinetes (spore-like resting stages), heterocysts (specialized in nitrogen fixation) and hormogonia (cell aggregates with gliding motility for colonization and dispersal). Genetic approaches to studying the complex morphology and differentiations of these prokaryotes require transformation techniques. For Fischerella and Chlorogloeopsis reliable protocols for introducing foreign genes are lacking. Here, we explored conjugation, electroporation, and biolistic DNA transfer methods in Fischerella and Chlorogloeopsis, using the cyanobacterial replicon pRL25C as a marker. We successfully transformed Fischerella muscicola PCC 7414 and Chlorogloeopsis fritschii PCC 6912 and were able to express the GFP reporter protein under two different promoters: the nitrogen regulated _p glnA and the strong E. coli hybrid _p trc. For Fischerella all methods worked, for Chlorogloeopsis electroporation was unsuccessful. For both strains conjugation delivered the most reproducible results, whereby partial removal of the exopolysaccharide sheath by salt washing was a critical step.     

依纽小单孢菌接合转移体系的构建

以依纽小单孢菌HP变种基因组DNA为模板,扩增位于西索米星3′,4′-双脱羟基酶基因sisI上下游序列的两端同源交换臂,在两臂之间添加抗性筛选标记ermE基因,并在该基因上游加入组成型强启动子ermE*,以强化筛选标记.将该外源DNA序列插入到质粒pKC1139,构建重组质粒pFD57.转化大肠杆菌ET12567后,经接合转移导入依纽小单孢菌中,经抗性筛选得到两株阳性菌株,命名为HP-I-1和HP-I-2.经PCR验证和测序,结果表明,重组质粒已整合到染色体DNA上.依纽小单孢菌接合转移体系的构建达到了预期目的,并实现了对该体系的优化.     

Conjugal Transfer of Genetic Information in Group N Streptococci

Streptococcus lactis strains ML3 and C₂O and S. lactis subsp. diacetylactis strains DRC3, 11007, and WM₄ were found to transfer lactose-fermenting ability to LM0230, an S. lactis C2 lactose-negative (Lac⁻) derivative which is devoid of plasmid deoxyribonucleic acid (DNA). Lactose-positive streptomycin-resistant (Lac⁺ Str^r) recombinants were found when the Lac⁺ Str^s donor was mixed with Lac⁻ Str^r LM0230 in solid-surface matings. Transduction and transformation were ruled out as the mechanism of genetic exchange in strains ML3, DRC3, 11007, and WM₄, nor was reversion responsible for the high number of Lac⁺ Str^r recombinants. Furthermore, chloroform treatment of the donor prevented the appearance of recombinants, indicating that transfer of lactose-fermenting ability required viable cell-to-cell contact. Strain C₂O demonstrated transduction as well as conjugation. Transfer of plasmid DNA during conjugation for all strains was confirmed by demonstrating the presence of plasmid DNA in the transconjugants by using agarose gel electrophoresis. In some instances, a cryptic plasmid was transferred in conjunction with the lactose plasmid by using strains DRC3, 11007, and WM₄. In S. lactis C2 × LM0230 matings, the Str^r marker was transferred from LM0230 to C2, suggesting conjugal transfer of chromosomal DNA. The results confirm conjugation as another mechanism of genetic exchange occurring in dairy starter cultures.     

The Infidelity of Conjugal DNA Transfer in ESCHERICHIA COLI

The accuracy of replication and transfer of a lacI gene on an F' plasmid was measured. Following conjugal transfer of the F', a small but reproducible increase (1.8-fold) in the frequency of lacI- mutations was detected. Among these, however, the frequency of nonsense mutations was 15-fold higher than in the absence of transfer. This corresponds to a 300-fold increase in the rate of base substitutions per round of replication compared with normal vegetative DNA replication. The amber mutational spectra revealed that, following conjugal transfer, mutation frequencies were increased markedly at all sites detected. In addition, an increase in G:C leads to A:T transitions was noted and was due almost entirely to an enhanced proportion of mutants recovered at the spontaneous hotspots (amber sites 6, 15 and 34). recA-dependent processes were not responsible for the increase in mutation, since similar results were observed with various recA- donor and recipient combinations. These results demonstrate that the fidelity of conjugal DNA replication is considerably lower than that of vegetative DNA replication.     

Interdomain Conjugal Transfer of DNA from Bacteria to Archaea

Escherichia coli transforms the methanogenic archaeon Methanococcus maripaludis at frequencies ranging from 0.2 × 10⁻⁶ to 2 × 10⁻⁶ per recipient cell. Transformation requires cell-to-cell contact, oriT, and tra functions, is insensitive to DNase I, and otherwise displays hallmarks of conjugation.Conjugal transfer of DNA involves a specific set of transfer (tra) functions that mediate the mobilization of DNA containing an origin of transfer (oriT) from a donor to a recipient in a process requiring cell-to-cell contact (9). While conjugation is often very efficient between members of a given species or genus, it can also occur at a lower efficiency between phylogenetically distant microorganisms with structurally distinct cell surfaces. Escherichia coli, for example, mediates conjugal transfer of DNA to such diverse bacterial recipients as cyanobacteria (23), spirochetes (14), and a variety of Gram-positive bacteria (17, 22); E. coli even mediates conjugal DNA transfer to members of the domain Eukarya, such as to Saccharomyces cerevisiae (6) and mammalian (20) cells. Because of its broad range of potential recipients, conjugation has proven to be a valuable genetic tool (11) and may be an important mechanism of horizontal gene transfer and a driver of genome evolution (7). Conjugation-like DNA transfer has also been demonstrated in members of the domain Archaea (5, 15). However, conjugation between Bacteria and Archaea has not been demonstrated, despite the observation that many whole-genome sequences of Archaea harbor DNA that appears to be of bacterial origin (7).To investigate whether conjugation can occur between Bacteria and Archaea, the RP4 (IncPα group) conjugal-transfer system was used to attempt to mobilize DNA from E. coli to the anaerobic, methanogenic archaeon Methanococcus maripaludis strain S2 (21). The RP4 system was selected because previous work demonstrated that this plasmid supports the transfer of DNA from E. coli to phylogenetically distant recipients, including yeast (3) and mammalian (20) cells. Additionally, E. coli has been shown to successfully conjugate with strictly anaerobic bacterial strains (22). M. maripaludis was chosen as a recipient because it has growth parameters similar to those of E. coli and has readily available selectable markers (1). For all the experiments described, M. maripaludis was grown in liquid or solid (excluding cysteine) McCas medium (12), supplemented with 2.5 μg/ml puromycin (Pur) where appropriate, using standard anaerobic techniques (2). All plating for conjugation experiments, except for determination of viable-E. coli cell counts, was performed in an anaerobic chamber (Coy, Grass Lake, MI) with an atmosphere of 5:5:90 H₂-CO₂-N₂. E. coli was grown in Difco LB medium (Becton-Dickinson, Sparks, MD) supplemented where appropriate with 50 μg/ml kanamycin sulfate (Kan) and ampicillin (Amp).To interrogate conjugal DNA transfer between E. coli and M. maripaludis, a set of vectors that either contained or lacked cis-acting sites required for mobilization by RP4 transfer functions were constructed (Table ​(Table1).1). Each of these vectors contained a Pur resistance (Pur^r) gene cassette (pac) (4) flanked by ∼0.5 kb DNA homologous to regions 5′ and 3′ of the M. maripaludis nrpR gene (nrpR::pac), which allows for selection by Pur in M. maripaludis and provides sites for homologous recombination into the nrpR locus of the M. maripaludis chromosome. This construct was selected because it has previously been used to transform M. maripaludis to Pur resistance by recombination into the nrpR locus using a polyethylene glycol (PEG)-mediated transformation protocol (10, 18). After it was demonstrated that plasmids of the appropriate genotypes support conjugation from donor strain E. coli S17-1, which contains the RP4 trans-acting transfer (tra) functions on the chromosome via an integrated RP4-2-Tc::Mu-Km::Tn7 cassette (16), to E. coli recipient cells (Table ​(Table1;1; see also the supplemental material), we investigated whether these same donor strains could support DNA transfer to M. maripaludis.

TABLE 1.

Transformation of M. maripaludis by E. coli

Inter- and Intraspecies Conjugal Transfer of Different Plasmids in Bacilli

Conjugal transfer of plasmid pUB110 between different strains of bacilli was studied. The plasmid transfer was possible not only between various strains of B. subtilis, but also when many other species of bacilli served as recipients. Conjugation of a donor strain B. subtilis 19 (p19 pUB110) was accompanied by a transfer of plasmid p19 along with plasmid pUB110 to the B. subtilis recipient strains lacking a large plasmid p19. If, like the donor cells, the recipient B. subtilis strain carried plasmid p19, the frequency of conjugation decreased. The small plasmid pBC16 was also capable of conjugative transfer. However, if this plasmid carried the mob gene with an inverted region, the frequency of its transmission dramatically decreased. If the donor strain contained another small plasmid, pV, which also carried the mob gene, the efficiency of transmission was partially restored.     

人管家基因启动子序列中的组合转录调控元件分析

研究表明,第一内含子可能参与基因转录调控.利用统计方法提取人管家基因上游至第一内含子序列中潜在的组合转录调控模体,分析模体间的距离、区域分布等特征,探讨内含子参与基因转录调控的可能性及其参与方式.在管家基因中共获得960对潜在转录调控模体对,其中57%与实验已知的具有转录相互作用的因子对吻合,共涉及12组因子对.分析发现,绝大多数模体对(80%)偏向于上游区域及"上游-内含子"区域,进一步支持了内含子参与基因转录调控的假设,并据此推测内含子与上游序列之间具有转录协同作用,模体在基因转录起始位点(TSS)附近较为集中,模体对的两个模体之间距离较近,60%左右距离在200 bp以内,特别地,65%的模体对特征距离在100 bp以内,短距离间隔有利于转录因子间的协同作用.这些结果将有助于对人基因转录调控机制及内含子功能的深入认识.     

抑癌基因的负转录调控

抑癌基因在正常细胞中适度表达,抑制细胞永生及转化,其转录下调见于某些肿瘤,而在衰老细胞中常见表达上调或活性增强。抑癌基因INK4a／ARF、p53和p21^Cipl的表达及其负调控与肿瘤及细胞衰老的关系十分密切。     

真核细胞中基因的转录调控

叙述了真核细胞三种RNA聚合酶合成的基因的转录调控.由于真核细胞DNA含量非常大,其基因的转录调控具有以下特点：参与的转录因子多;与顺式DNA序列元件结合呈一定顺序.这反映了真核细胞中基因的转录调控是由多个转录因子间的相互作用来实现的．     

Conjugal Transfer of a Virulence Plasmid in the Opportunistic Intracellular Actinomycete Rhodococcus equi

Rhodococcus equi is a facultative intracellular, Gram-positive, soilborne actinomycete which can cause severe pyogranulomatous pneumonia with abscessation in young horses (foals) and in immunocompromised people, such as persons with AIDS. All strains of R. equi isolated from foals and approximately a third isolated from humans contain a large, ∼81-kb plasmid which is essential for the intramacrophage growth of the organism and for virulence in foals and murine in vivo model systems. We found that the entire virulence plasmid could be transferred from plasmid-containing strains of R. equi (donor) to plasmid-free R. equi strains (recipient) at a high frequency and that plasmid transmission reestablished the capacity for intracellular growth in macrophages. Plasmid transfer required living cells and cell-to-cell contact and was unaffected by the presence of DNase, factors pointing to conjugation as the major means of genetic transfer. Deletion of a putative relaxase-encoding gene, traA, located in the proposed conjugative region of the plasmid, abolished plasmid transfer. Reversion of the traA mutation restored plasmid transmissibility. Finally, plasmid transmission to other Rhodococcus species and some additional related organisms was demonstrated. This is the first study showing a virulence plasmid transfer in R. equi, and it establishes a mechanism by which the virulence plasmid can move among bacteria in the soil.     

心脏基因转录潜能的区域化

心脏基因在心肌中的区域化表达是一个非常普遍的现象,通过转基因动物研究心肌中的转录潜能的区域化已取得了重大的进展.房室转录区域化最早是在线状心管形成时开始出现的.之后,在胚胎心脏的各个部分均可以发现转基因区域化表达.对于转基因在心肌中区域化表达的分子机理的研究,现在主要集中在对转基因调控区域的顺式调控元件和反式调控元件的解析,以及在心肌细胞中对于转录很重要的基因或是已被证明会介导位置信息的基因的表达模式和功能的研究上.