A family of shuttle vectors for lactobacilli and other gram-positive bacteria based on the plasmid pLF1311 replicon

A set of broad-host-range single-replicon shuttle vectors for cloning nucleotide sequences in gram-positive bacteria (lactobacilli, enterococci, lactococci, bacilli, etc.) was created. The vectors are based on the cryptic plasmid pLF1311 from Lactobacillus fermentum VKM 1311 belonging to a family of the sigma-type pE194-like plasmids. The vectors can replicate in gram-positive bacteria and Escherichia coli. They are stable in many gram-positive bacteria, have small sizes, and allow the selection of recombinants on media with X-Gal. The vectors that contain the region of initiation of the conjugal transfer of plasmid RP4 belonging to the incompatibility group IncP alpha can be mobilized in a great number of bacteria using a helper plasmid from E. coli but not from gram-positive bacteria.     

A family of shuttle vectors for lactic acid bacteria and other gram-positive bacteria based on the plasmid pLF1311 replicon

A set of broad-host-range single-replicon shuttle vectors for cloning nucleotide sequences in gram-positive bacteria (lactobacilli, enterococci, lactococci, bacilli, etc.) was created. The vectors are based on the cryptic plasmid pLF 1311 fromLactobacillus fermentum VKM 1311, belonging to the family of the σ-type pE194-like plasmids. The vectors can replicate in gram-positive bacteria andEscherichia coli. They are stable in many gram-positive bacteria, have small sizes, and allow the selection of recombinants on media with X-Gal. The vectors that contain the region of initiation of the conjugal transfer of plasmid RP4 belonging to the incompatibility group IncPα can be mobilized in a great number of bacteria using a helper plasmid fromE. coli but not from gram-positive bacteria     

乳酸菌食品级表达载体的研究与应用

乳酸菌是能够发酵糖类产生大量有机酸的革兰氏阳性菌的通称,在发酵食品中有着悠久的应用历史。乳酸菌通常被认为是安全菌株,这些微生物的基因工程操作在食品、医学等方面具有广阔的应用前景。表达载体是基因工程中常用的工具之一,大多数乳酸菌的表达载体通常以抗生素抗性基因作为选择标记,然而抗性基因具有潜在的转移性,因此需要开发食品级表达载体。食品级表达载体不含有抗生素的抗性基因,仅包含来自同源宿主或通常被认为是安全生物的DNA。本文介绍了乳酸菌食品级表达载体的构成及其常用宿主,同时对乳酸菌食品级表达载体的应用进行了归纳总结。     

Genome organization of the plasmid of IncQ/P4 group and its vector derivatives

The genome organization and functioning of IncQ/P4 plasmids are reviewed. Based on these plasmids, cloning vectors have been constructed for broad host range of gram-negative bacteria. Together with one- and two-replicon vectors for cloning via insertion inactivation of markers, specialized plasmid vectors are described: cosmids, promoter-probe vectors, vectors for direct selection of recombinant molecules. Examples of using broad host range vectors for gene cloning and expression in non-enteric gram-negative bacteria are presented.     

Using bacteria to express and display anti-parasite molecules in mosquitoes: current and future strategies

Vector-borne diseases impose enormous health and economical burdens throughout the world. Unfortunately, as insecticide and drug resistance spread, these burdens will increase unless new control measures are developed. Genetically modifying vectors to be incapable of transmitting parasites is one possible control strategy and much progress has been made towards this goal. Numerous effector molecules have been identified that interfere with parasite development in its insect vectors, and techniques for transforming the vectors with genes encoding these molecules have been established. While the ability to generate refractory vectors is close at hand, a mechanism for replacing a wild vector population with a refractory one remains elusive. This review examines the feasibility of using bacteria to deliver the anti-parasitic effector molecules to wild vector populations. The first half briefly examines paratransgenic approaches currently being tested in both the triatomine bug and tsetse fly. The second half explores the possibility of using midgut bacteria to control malaria transmission by Anopheles mosquitoes.     

Use of green fluorescent protein color variants expressed on stable broad-host-range vectors to visualize rhizobia interacting with plants

We developed two sets of broad-host-range vectors that drive expression of the green fluorescent protein (GFP) or color variants thereof (henceforth collectively called autofluorescent proteins [AFPs]) from the lac promoter. These two sets are based on different replicons that are maintained in a stable fashion in Escherichia coli and rhizobia. Using specific filter sets or a dedicated confocal laser scanning microscope setup in which emitted light is split into its color components through a prism, we were able to unambiguously identify bacteria expressing enhanced cyan fluorescent protein (ECFP) or enhanced yellow fluorescent protein (EYFP) in mixtures of the two. Clearly, these vectors will be valuable tools for competition, cohabitation, and rescue studies and will also allow the visualization of interactions between genetically marked bacteria in vivo. Here, we used these vectors to visualize the interaction between rhizobia and plants. Specifically, we found that progeny from different rhizobia can be found in the same nodule or even in the same infection thread. We also visualized movements of bacteroids within plant nodule cells.     

Construction of Plasmid Vectors for Screening Replicons from Gram-Positive Bacteria and Their Use as Shuttle Cloning Vectors

Plasmids play a central role in engineering recombinant bacteria because they are the primary vehicles used to manipulate targeted sequences. In some cases, bacteria of interest are poorly provided with suitable tools for these molecular or genetic manipulations. In this context, we constructed from two shuttle cloning vectors, pUCB2871 and pUCB2872, the basic vectors pUCB30 and pUCB31, which could represent suitable tools to isolate replicons from Gram-positive bacteria. These plasmid vectors are characterized by the following after-features: (a) the pUC origin of replication is unable to replicate in Gram-positive bacteria; (b) an erythromycin-resistance encoding gene that is functional in both Gram-negative and -positive bacteria; (c) the pUC19 multiple cloning site (MCS) within the lacZα reporter gene; and (4) an additional multiple cloning site (MCS). Cloning replicons from Gram-positive bacteria in this additional MCS would allow the derivative vectors to function directly as shuttle cloning vectors.     

Obtaining of fluorescent-labeled nodule bacteria strains of wild legumes for their detection in vive and in vitro

A series of expression vectors containing genes of fluorescent proteins TurboGFP and TurboRFP under the phage T5 constitutive promoter regulation, intended for lifetime marking of nodule bacteria is created: a series of vectors based on a broad-host-range replicon BBRI, for marking strains with an expression of reporter gene from a transformed plasmid and a series of vectors based on a plasmid pRL765gfp for marking strains by introduction genes of fluorescent proteins in a bacterial chromosome. It was shown that transformation is the most preferable method of constructions transfer in nodule bacteria cells, as in the presence of mob locus in the vectors necessary for conjugation, exists the possibility of occasional plasmid mobilization and its transition from marked strain cells in other soil bacteria. With application of the created vector constructions we obtained fluorescent tagged strains of Rhizobium sp., Mesorhizobium sp., Ensifer (Sinorhizobium) sp., Bradyrhizobium sp., Phyllobacterium sp., Agrobacterium sp. Also their suitability for experiments in vivo and in vitro is shown.     

在大肠杆菌中表达人防御素-1

构建了一组HNP-1原核表达载体,其中有几个含SD或序列的载体,在表达产物中检测到了HNP-1的存在,说明宿主菌是否在任何情况下都降解外源小分子多肽是一个值得考虑的问题．另外从表达载体在大肠杆菌JM109和JM109（DE3）中蛋白表达量的差异也可推测HNP-1发生了低水平的表达,HNP-1对细菌的作用与HNP-1浓度和细菌本身的生理状况密切相关.     

Selection and characterization of a promoter for expression of single-copy recombinant genes in Gram-positive bacteria

Background  

In the past ten years there has been a growing interest in engineering Gram-positive bacteria for biotechnological applications, including vaccine delivery and production of recombinant proteins. Usually, bacteria are manipulated using plasmid expression vectors. The major limitation of this approach is due to the fact that recombinant plasmids are often lost from the bacterial culture upon removal of antibiotic selection. We have developed a genetic system based on suicide vectors on conjugative transposons allowing stable integration of recombinant DNA into the chromosome of transformable and non-transformable Gram-positive bacteria.     

Cloning and stable maintenance of DNA fragments over 300 kb in Escherichia coli with conventional plasmid-based vectors.

Bacterial artificial chromosome (BAC) and P1-derived artificial chromosome (PAC) systems were previously developed for cloning of very large eukaryotic DNA fragments in bacteria. We report the feasibility of cloning very large fragments of eukaryotic DNA in bacteria using conventional plasmid-based vectors. One conventional plasmid vector (pGEM11), one conventional binary plasmid vector (pSLJ1711) and one conventional binary cosmid vector (pCLD04541) were investigated using the widely used BAC (pBeloBAC11 and pECBAC1) and BIBAC (BIBAC2) vectors as controls. The plasmid vector pGEM11 yielded clones ranging in insert sizes from 40 to 100 kb, whereas the two binary vectors pCLD04541 and pSLJ1711 yielded clones ranging in insert sizes from 40 to 310 kb. Analysis of the pCLD04541 and pSLJ1711 clones indicated that they had insert sizes and stabilities similar to the BACs and BIBACs. Our findings indicate that conventional plasmid-based vectors are capable of cloning and stably maintaining DNA fragments as large as BACs and PACs in bacteria. These results suggest that many existing plasmid-based vectors, including plant and animal transformation and expression binary vectors, could be directly used for cloning of very large eukaryotic DNA fragments. The pCLD04541 and pSLJ1711 clones were shown to be present at at least 4-5 copies/cell. The high stability of these clones indicates that stability of clones does not seem contingent on single-copy status. The insert sizes and the copy numbers of the pCLD04541 and pSLJ1711 clones indicate that Escherichia coli can stably maintain at least 1200 kb of foreign DNA per cell. These results provide a new conceptual and theoretical basis for development of improved and new vectors for large DNA fragment cloning and transformation. According to this discovery, we have established a system for large DNA fragment cloning in bacteria using the two binary vectors, with which several very large-insert DNA libraries have been developed.     

ColD-derived cloning vectors that autoamplify in the stationary phase of bacterial growth

The construction of cloning vectors based on the replicon of plasmid ColD-CA23 is reported. These vectors, like ColD itself, autoamplify when cultures of host bacteria enter the stationary phase of growth, thereby resulting in a substantial increase in the expression of cloned genes as a consequence of the increase in gene dosage. The principal advantage of these vectors is that, unlike the situation pertaining to other expression vectors, the increase in expression of genes cloned in ColD vectors does not require any experimental intervention (i.e., occurs naturally), and takes place at high cell densities. The vectors show high stability in Escherichia coli strains and are compatible with ColE1-type cloning vectors.     

Construction and characterization of shuttle vectors for succinic acid-producing rumen bacteria

Shuttle vectors carrying the origins of replication that function in Escherichia coli and two capnophilic rumen bacteria, Mannheimia succiniciproducens and Actinobacillus succinogenes, were constructed. These vectors were found to be present at ca. 10 copies per cell. They were found to be stably maintained in rumen bacteria during the serial subcultures in the absence of antibiotic pressure for 216 generations. By optimizing the electroporation condition, the transformation efficiencies of 3.0 x 10(6) and 7.1 x 10(6) transformants/mug DNA were obtained with M. succiniciproducens and A. succinogenes, respectively. A 1.7-kb minimal replicon was identified that consists of the rep gene, four iterons, A+T-rich regions, and a dnaA box. It was found that the shuttle vector replicates via the theta mode, which was confirmed by sequence analysis and Southern hybridization. These shuttle vectors were found to be suitable as expression vectors as the homologous fumC gene encoding fumarase and the heterologous genes encoding green fluorescence protein and red fluorescence protein could be expressed successfully. Thus, the shuttle vectors developed in this study should be useful for genetic and metabolic engineering of succinic acid-producing rumen bacteria.     

细菌抵抗消毒剂及其对抗生素共耐药

消毒剂是一种可杀灭物体表面、器材设备、皮肤、空气和水源等传播媒介上携带的病原微生物的有机分子。它在体外能杀灭病原微生物,切断其传播途径,进而达到控制污染的目的,在生命安全防控中起着重要的作用。但是不合理地使用消毒剂导致细菌对消毒剂产生耐药。消毒剂耐药基因在不同种属间的水平转移加剧其传播风险,使消毒剂耐药情况进一步恶化。更令人担忧的是,细菌对消毒剂的耐药可能会导致对抗生素产生共耐药,给公共安全带来巨大的威胁。但目前为止,对消毒剂耐药以及共耐药的认识还不够全面。本文总结了关于细菌对消毒剂耐药的研究报道,对消毒剂的作用机制、细菌对消毒剂的耐药机制进行了论述,另外针对消毒剂耐药基因的传播以及细菌对消毒剂和抗生素的共耐药进行了综述,为减少消毒剂耐药性的产生和制定合理的消毒剂使用规范奠定基础。     

Ecological characteristics of flea species relate to their suitability as plague vectors

The ability of vector-borne diseases to persist and spread is closely linked to the ecological characteristics of the vector species they use. Yet there have been no investigations of how species used as vectors by pathogens such as the plague bacterium differ from closely related species that are not used as vectors. The plague bacterium uses mammals as reservoir hosts and fleas as vectors. The ability of different fleas to serve as vectors is assumed to depend on how likely they are to experience gut blockage following bacterial multiplication; the blockage causes fleas to regurgitate blood into a wound and thus inject bacteria into new hosts. Beyond these physiological differences, it is unclear whether there exist fundamental ecological differences between fleas that are effective vectors and those that are not. Here, using a comparative analysis, we identify clear associations between the ability of flea species to transmit plague and their ecological characteristics. First, there is a positive relationship between the abundance of flea species on their hosts and their potential as vectors. Second, although the number of host species exploited by a flea is not associated with its potential as a vector, there is a negative relationship between the ability of fleas to transmit plague and the taxonomic diversity of their host spectrum. This suggests a correlation between some ecological characteristics of fleas and their ability to develop the plague blockage. The plague pathogen thus uses mainly abundant fleas specialized on a narrow taxonomic range of mammals, features that should maximize the persistence of the disease in the face of high flea mortality, and its transmission to suitable hosts only. This previously unrecognized pattern of vector use is of importance for the persistence and transmission of the disease.Electronic Supplementary Material Supplementary material is available to authorised users in the online version of this article at .     

Versatile Escherichia coli-Bacillus shuttle vectors derived from runaway replication plasmids related to CloDF13

Summary Versatile cloning vectors were constructed employing a runaway replication mutant of the bacteriocinogenic plasmid CloDF13. These vectors can, under conditions where protein synthesis is not inhibited, be amplified in Escherichia coli to high levels by elevating the temperature and are therefore useful for the production of large quantities of DNA and protein. Since the constructed shuttle vectors, which harbour at least six unique restriction endonucleases sites, replicate in E. coli, Enterobacter cloacae, Staphylococcus aureus and a variety of Bacilli, they are applicable for the genetic engineering of both gramnegative and gram-positive bacteria.     

Selection of Dictyostelium discoideum transformants and analysis of vector maintenance using live bacteria resistant to G418.

A protocol that allows the rapid isolation and growth of large numbers of independent G418-resistant Dictyostelium discoideum transformant colonies on the surface of agar media with live bacteria was developed. Transformants grown under these conditions form normal fruiting bodies. Discovery that aggregation of nontransformants was inhibited at a nonselective level of G418 (25 to 35 micrograms/ml) led to the development of a vector maintenance assay. Using this assay we examined the stability of recombinant plasmids derived from the D. discoideum native plasmids Ddp1 and Ddp2. We conclude that the origin of replication of plasmid Ddp1 does not alone confer stable maintenance and thus, Ddp1 must bear additional sequences required for its own maintenance. Analysis of the maintenance of vectors derived from Ddp2 showed that autonomously replicating shuttle vectors that contained bacterial plasmid DNA and from which one element of the Ddp2 inverted repeat was removed were much less stable than vectors that contained a complete inverted repeat or that did not carry a bacterial plasmid. Sequences between the 3' end of the rep gene and the inverted repeat appear to play a role in plasmid maintenance. An intact rep gene and one copy of the inverted repeat element were required for extrachromosomal replication. Maintenance of extrachromosomal vectors was found to be strain dependent. Four traits distinguishing integrating vectors from those capable of autonomous replication were identified.     

A new distribution vector and its application in genome clustering

In this paper we report a novel mathematical method to transform the DNA sequences into the distribution vectors which correspond to points in the sixty dimensional space. Each component of the distribution vector represents the distribution of one kind of nucleotide in k segments of the DNA sequences. The mathematical and statistical properties of the distribution vectors are demonstrated and examined with huge datasets of human DNA sequences and random sequences. The determined expectation and standard deviation can make the mapping stable and practicable. Moreover, we apply the distribution vectors to the clustering of the Haemagglutinin (HA) gene of 60 H1N1 viruses from Human, Swine and Avian, the complete mitochondrial genomes from 80 placental mammals and the complete genomes from 50 bacteria. The 60 H1N1 viruses, 80 placental mammals and 50 bacteria are classified accurately and rapidly compared to the multiple sequence alignment methods. The results indicate that the distribution vectors can reveal the similarity and evolutionary relationship among homologous DNA sequences based on the distances between any two of these distribution vectors. The advantage of fast computation offers the distribution vectors the opportunity to deal with a huge amount of DNA sequences efficiently.     

Endosymbionts of arthropods and nematodes: allies to fight infectious diseases?

Arthropods and nematodes are important protagonists in human health because either they act as vectors of pathogens (bacteria, protozoa, viruses or fungus), or are themselves parasites. Fighting infectious diseases is based essentially on vaccination (prevention) or chemotherapeutic (curative) approaches in human, but one can envisage as an alternative to reduce the number of vectors or limit their ability to spread pathogens. Such strategies controlling dissemination will undoubtedly benefit from the knowledge accumulated by recent works on powerful mechanisms developed by symbiotic insect bacteria such as Wolbachia to popagate in arthropods and nematods. This review summarizes these recent data, and indicate how these mechanisms can be manipulated to reduce the dissemination of insect vectors propagating human diseases.