Characterization of Escherichia coli donor-specific RNA-containing bacteriophages

The biological properties of 9 clones of Ri bacteriophages isolated from sewage water in 1981 were studied. On the basis of the activity of Ri phages with respect to E. coli donor-specific strains K12, the type of negative colonies, the ultrastructure of the virion and its sizes, adsorption on the pili of host cells, the latent period, the amount of harvest obtained from one infected cell, the clones under study were classified with small spherical RNA-bacteriophages. The neutralization of Ri phages with antiphage sera to standard phages f2 and fr made it possible to classify them with the first serological group and to divide them into 3 subgroups.     

Transfer-deficient mutants of the narrow-host-range plasmid R91-5 of Pseudomonas aeruginosa.

Three methods have been successful in the isolation of transfer-deficient mutants of the narrow-host-range R plasmid R91-5 of Pseudomonas aeruginosa: (i) selection for donor-specific phage resistance; (ii) direct screening after mutagenic treatment with either ethyl methane sulfonate or N-methyl-N'-nitro-N-nitrosoguanidine; (iii) in vitro mutagenesis of plasmid DNA by hydroxylamine followed by transformation and direct screening. The majority of transfer-deficient mutants were donor-specific phage resistant, supporting the view that sex pili and other surface components are essential for conjugal transfer (since the phages PRD1 and PR4 adsorb to these sites). Some of the transfer-deficient mutants were also unable to inhibit the replication of phage G101 or lost entry exclusion or both phenotypes. The ability to revert these pleiotropic mutants to wild type implicates the latter two functions in R91-5 transfer. Suppressor mutations in P. aeruginosa enabled the detection of suppressor-sensitive, transfer-deficient mutants. Such mutants should prove useful in conjugational complementation tests for the identification of the transfer cistrons of R91-5.     

Identification and characterization of RP1 Tra1 cistrons involved in pilus function and plasmid mobilization.

Transfer-defective mutants of the Tra1 region of RP1 were isolated. Complementation studies involving stable heterozygotes combined with the mapping of Tn5 insertion mutations revealed two pilus cistrons, pilA and pilB, at positions 46.9 to 48.2 kb and 46.0 to 46.4 kb, respectively. All pilB mutants were Dps- (i.e., resistant to donor-specific phages PR4 and PRR1), whereas pilA mutants were Dps- (promoter-proximal mutations), Dps+/- (sensitive only to PR4 [more centrally located mutations]), or Dps+ (sensitive to both phages [promoter-distal mutations]). The correlation between the site mutated and the Dps phenotype, together with the finding that certain Dps+ pilA mutants continued to mobilize nonconjugative plasmids, suggested that pilA is bifunctional, contributing both to pilus function (at the promoter-proximal end) and to RP1 mobilization. It was also shown that the 43.5- to 49.5-kb region that includes pilA and pilB encodes all of the Tra1 pilus functions required for propagation of donor-specific phages and hence, probably, for pili that are active in conjugation. Finally, three cistrons that specifically affect RP1 mobilization were identified. Two of these, mobA and mobB, occur immediately anticlockwise to oriT and probably correspond to the traJ and traI genes characterized by other workers. The third cistron, mobC, occurs clockwise to oriT and may be a new mobilization gene, since its function can be substituted by IncP beta plasmids, a feature different from that of the traK mobilization gene which occurs in the same region but is RP1 specific. None of the mob cistrons was required for mobilization of nonconjugative plasmids, except for mobB, which was required by pVS99.     

Effect of plasmids from various incompatibility groups on the development of bacteriophages specific to Pseudomonas aeruginosa and Pseudomonas putida

The aim of this work was to study the effect of plasmids belonging to different incompatibility groups on the growth of bacteriophages in Pseudomonas aeruginosa and Pseudomonas putida strains. The growth of bacteriophages was shown to be limited most often due to the presence in cells of plasmids belonging to the P-2 incompatibility group. Plasmids of the Inc P-2 group differed from one another in the spectrum of bacteriophages whose growth they limited. Phages whose growth was suppressed in strains containing plasmids of the P-5, P-9 or P-10 incompatibility groups were found. Some plasmids showed no specific interaction with bacteriophages. The plasmids investigated differed in the studied trait in P. aeruginosa and P. putida cells. In contrast to P. aeruginosa PAO, P. putida PpGI plasmid containing cells did not maintain the growth of donor-specific bacteriophages and, to a lesser degree, limited the growth of phages specific for P. putida PpGI.     

Characterization of Pseudomonas aeruginosa derepressed R-plasmids.

A genetic study of conjugal transmissibility of two R-plasmids was undertaken in Pseudomonas aeruginosa. Conjugally derepressed mutants of the R-plasmids were isolated, and examination of 11 independent mutants revealed that 10 were recessive to the wild-type transfer repressor, whereas 1 mutant was cis dominant. Cross-repression was observed between the two R-plasmids, suggesting that they have functionally equivalent systems for regulating the expression of tra loci. The derepressed R-plasmid mutants exhibited several characteristics, in addition to derepressed transfer, that were not expressed by the parental plasmids. These included sensitivity to certain donor-specific phages, inhibition of multiplication of a transducing phage, and, in the one case examined, a high degree of entry exclusion. The coexpression of these different functions suggests that their respective genetic loci are controlled by the same regulatory system as that of tra, or else that they are part of the tra complex.     

Physical and genetic analysis of deletion mutants of plasmid R91-5 and the cloning of transfer genes in Pseudomonas aeruginosa.

We isolated deletion mutants of Pseudomonas aeruginosa plasmid R91-5 by both in vitro and in vivo means. Many of the deletion mutants selected on the basis of resistance to donor-specific phages fell into a few groups of apparently identical mutants, although the mutants were nonsibs. By analyzing plasmids with large deletions, we found that the essential replication genes of R91-5 were within a 3.85-kilobase region between coordinates 45.5 and 48.9. The origin of plasmid transfer (oriT) was mapped to a 4.5-kilobase region between coordinates 1.7 and 6.2. We indirectly determined the direction of plasmid transfer from oriT. By combining the data from our analysis of the deletions with data from complementation tests between cloned R91-5 fragments and known reference mutants, we ordered and mapped the 10 known transfer (tra) cistrons of R91-5. All of the tra cistrons mapped within the Tra2 region, and their order was as follows: traX, -Y, -T, -Q, -(V, R), -U, -(S, Z), -W (the cistrons in parentheses could not be ordered with respect to each other).     

Morphology of the Bacteriophages of Lactic Streptococci

Electron microscope studies have been made of a number of phages of lactic streptococci, seven of which were phages of Streptococcus lactis C10. Two of the phages are thought to be identical; five have been classified by the method of Tikhonenko as belonging to group IV (phages with noncontractile tails) with type III tail plates; one belongs to group V (phages with tails possessing a contractile sheath). Both prolate polyhedral heads and isometric polyhedral heads are represented among the group IV phages. The phage drc3 of S. diacetilactis DRC3 has been shown to have similar structure to the group IV phages of S. lactis C10 with prolate polyhedral heads. The phages ml1, hp, c11, and z8 of the S. cremoris strains ML1, HP, C11, and Z8, respectively, were shown to belong to the group IV phages with type III tail plates by the method of Tikhonenko. All had octahedral heads and tended to be larger than most of the other phages studied.     

Phage specificity of the freshwater fish pathogen Flavobacterium columnare

Flavobacteria and their phages were isolated from Finnish freshwaters and fish farms. Emphasis was placed on finding phages infecting the fish pathogen Flavobacterium columnare for use as phage therapy agents. The host ranges of the flavobacterial phages varied, phages infecting F. columnare being more host specific than the other phages.     

Two loci in the genome of the transposable phage B39 of Pseudomonas aeruginosa affecting the process of integration. I. Study of the properties of phages with the Pde- phenotype and mapping of the rms site

Mutants and recombinants of transposable Pseudomonas aeruginosa bacteriophage B39 with a specific phenotype Pde- (pleiotropic developmental effect) were studied. Pde- phages produce clear minute plaques on lawns of P. aeruginosa PAO1 and fail to grow in cells of PAO1 harbouring Rms 163 (Inc P5) plasmid. Pde+ character is under control of the two loci in phage genome which were designated pdeX and pdeY. In hybrid phages the pdeX and pdeY loci originating from different transposable phages (pdeX from B39 and pdeY from PH132) do not accomplish their function and, as a result, the hybrid phages have the Pde- phenotype. The frequency of integration (f.o.i.) of Pde- phages into bacterial chromosome is lower than f.o.i. for Pde+ phages, as well as the frequency of stable lysogenization of infected bacteria; lytic development of the Pde- phages is also limited. The great difference among the transposable phages in their reaction to the presence of Rms163 plasmid is caused by some differences in the specific rms site in the phage genome. The site is located inside the interval 1.1-3.9 kb of the physical genome map, being closely linked to cI gene of phage B39. The growth of Pde- phages in cells with Rms163 can be restored, due to additional mutations in phage genes affecting lysogenization.     

Differences in the allele state of genes controlling the specificity of adsorption in transposable phages of Pseudomonas aeruginosa

To reveal possible differences in adsorptional specificities of transposable phages of Pseudomonas aeruginosa and to study the genetical control of this character, we isolated a group of phage-resistant P. aeruginosa mutants using some temperate and virulent phages. The study of resistance of the mutants to all the phages permitted us to find some types of mutants and to build a formal scheme of distribution of adsorptional receptors on the surface of P. aeurginosa cell. According to the results obtained, there are two main "receptor chains", where the receptors for all phages under study are grouped. For the majority of phages, just a single adsorptional receptor is obligatory, and at least two essential receptors are needed for adsorption of virulent phage E79. Two receptors were found also for another virulent phage, phi 11, one of them only being essential. Transposable phages can be grouped into three types, according to their adsorptional specificities. No correlations of adsorptional specificity types and all other characteristics of transposable phages studied (including the sub-groups of transposable phages belonging to different DNA homology types) were found. Genes of natural transposable phages controlling the differences in adsorptional specificities revealed can recombine in phage crosses.     

Virulence factors and resistance mechanisms of Serratia marcescens. A short review

Serratia marcescens, a Gram-negative bacillus that belongs to the family Enterobacteriaceae, is a human opportunistic pathogen bacterium that causes many diseases, such as urinary tract infections, respiratory tract infections, bacteremia, conjunctivitis, endocarditis, meningitis and wound infections. Many plasmides that confers multi-drug resistance were discovered, such as virulence factors, like cytotoxins that damage epithelial cells. The main topic of this paper presents a review about the molecular traits evolved in the pathogenic processes mediated by Serratia and its mechanism of resistance to drugs.     

Continuous survey of the distribution of RNA coliphages in Japan

In order to demonstrate the stability and continuity of RNA coliphages (phages) in their natural habitats, we investigated the amount and group types of RNA phages in sewage samples collected continuously from domestic drainage in Japan proper and islands in the seas adjacent to Japan (abbreviated simply as islands, hereafter) over a 5-yr period from 1973 to 1977. It was found that the frequencies of isolation of RNA phages were fairly high and constant. The group types of RNA phages isolated were also stable in the three cities. Choshi, Niigata, and Toyama in Japan proper. The average for the three cities was group II:III = 3:1. The investigation in islands revealed that the frequencies of isolation of RNA phages were fairly high as in the case of the above three cities in Japan proper and the group types of RNA phages isolated were also stable. That is to say, group II phages were predominant on Rishiri Island, Rebun I., Iki I., and Tsushima I., which are located relatively near to mainland Japan, while group III phages were predominant on Amamiohshima I., mainland Okinawa, Ishigakijima I., and Iriomotejima I., which are located south of Kyushu. It can thus be said that the RNA phages in the domestic drainage of Japan proper and islands remained more or less stable over at least the 5-yr period, and an apparent difference in the geographical distribution of RNA phages in Japan exists between Kyushu and Amamiohshima I.     

Rapamycin in combination with donor-specific CD4CD25Treg cells amplified in vitro might be realize the immune tolerance in clinical organ transplantation

It is an urgent need to induce and keep the donor-specific immune tolerance without affecting the function of normal immune defense and immune surveillance in clinical organ transplantation. Large number of studies showed that both the establishment of donor-recipient chimerism and the application of antibodies or drugs could obtain the donor-specific immune tolerance in animal transplantation model. However, the former as treatment of clinical practice has a poor feasibility, the latter has a very low success rate in clinical organ transplantation. There is a group of naturally occurring CD4⁺CD25⁺ regulatory T cells (Tregs) that mediate immune tolerance by suppressing alloreactive T cells in vivo. These cells are unable to curb the occurrence of allograft rejection owing their low content. And donor-specific Tregs amplified in vitro alone can not induce donor-specific immune tolerance for recipient. Rapamycin (RPM) as a proliferation signal inhibitor, studies have shown it can effectively inhibit allograft rejection and maybe contribute to induction of immune tolerance. But there exist still many dose-dependent adverse reactions which could prevent the establishment of immune tolerance and reduce the life quality of recipients in the clinical application of RPM. Therefore, we speculate a small amount of RPM combined with donor-specific Tregs amplified in vitro may be not only induce the achievement of donor-specific tolerance, but also reduce or eliminate the side effects of RPM in clinical organ transplantation.     

Optimization of the detection of bacteriophages induced from Listeria sp.

It is necessary to isolate new phages in order to improve the rate of typeability of Listeria monocytogenes strains. We propose a method which increases the detection of induced phages in the presence of inhibitory substances synthesized or liberated by the cells during phage production. Of the 29 phages isolated, 11 (38%) were detected by the spot-on-the-lawn technique and 18 (62%) were revealed by the soft-agar technique. To increase the rate of phage detection, both techniques appear useful. Listeria cultures were subjected to phage typing procedures utilizing these newly isolated phages and the French International set of phages. It appears that the newly isolated phages are good tools for the differentiation of Listeria strains. Among them, one phage seems to be complementary to the French International set.     

Effect of the detergent Metaupon on replication of various phages]

As several other surfactants do, the detergent Metaupon acts on the multiplication of bacteriophages. We investigated the influence of Metaupon on the phages phi and lambda, the cyanophage LPP-1, and the RNA-phages f 2, M 12, and Q beta by means of the agar diffusion test, pour plate test, adsorption test, and one-step growth test. The action of Metaupon on the free phages was also tested. Metaupon inhibits the formation of plaques by the phages with exception of lambda. With the phages f 2 and M 12 the substance increases the amount of plaques depending on concentration. The main mode of action of Metaupon was found to be the inhibition of the adsorption of the phages to the host cells. Only in the case of phi 105 free phages were inactivated.     

短尾噬菌体识别宿主机制的研究进展

噬菌体可以作为抗生素的替代物,用于致病菌的防控和治疗。有尾噬菌体是最常见的噬菌体类型,可以根据尾部形态的不同分为短尾噬菌体、肌尾噬菌体和长尾噬菌体3类。不同噬菌体间不仅具有明显的形态差异,其对宿主细菌的识别机制也不相同。短尾噬菌体由于其较小的基因组长度和相对简单的结构组成,成为研究宿主与噬菌体的共进化关系、以及通过基因工程改造噬菌体的良好模型。本文综述了短尾噬菌体的分类特征及不同短尾噬菌体识别宿主受体的分子机制。通过明确短尾噬菌体的识别宿主机制,有助于对相应噬菌体进行工程化改造,解决噬菌体应用中存在的关键问题,使噬菌体更广泛地应用于生物、医学与食品工业等领域中。     

Application of filamentous phages in environment: A tectonic shift in the science and practice of ecorestoration

Theories in soil biology, such as plant–microbe interactions and microbial cooperation and antagonism, have guided the practice of ecological restoration (ecorestoration). Below‐ground biodiversity (bacteria, fungi, invertebrates, etc.) influences the development of above‐ground biodiversity (vegetation structure). The role of rhizosphere bacteria in plant growth has been largely investigated but the role of phages (bacterial viruses) has received a little attention. Below the ground, phages govern the ecology and evolution of microbial communities by affecting genetic diversity, host fitness, population dynamics, community composition, and nutrient cycling. However, few restoration efforts take into account the interactions between bacteria and phages. Unlike other phages, filamentous phages are highly specific, nonlethal, and influence host fitness in several ways, which make them useful as target bacterial inocula. Also, the ease with which filamentous phages can be genetically manipulated to express a desired peptide to track and control pathogens and contaminants makes them useful in biosensing. Based on ecology and biology of filamentous phages, we developed a hypothesis on the application of phages in environment to derive benefits at different levels of biological organization ranging from individual bacteria to ecosystem for ecorestoration. We examined the potential applications of filamentous phages in improving bacterial inocula to restore vegetation and to monitor changes in habitat during ecorestoration and, based on our results, recommend a reorientation of the existing framework of using microbial inocula for such restoration and monitoring. Because bacterial inocula and biomonitoring tools based on filamentous phages are likely to prove useful in developing cost‐effective methods of restoring vegetation, we propose that filamentous phages be incorporated into nature‐based restoration efforts and that the tripartite relationship between phages, bacteria, and plants be explored further. Possible impacts of filamentous phages on native microflora are discussed and future areas of research are suggested to preclude any potential risks associated with such an approach.     

Two groups of bacteriophages infecting Streptococcus thermophilus can be distinguished on the basis of mode of packaging and genetic determinants for major structural proteins.

A comparative study of 30 phages of Streptococcus thermophilus was performed based on DNA restriction profiles, DNA homology, structural proteins, packaging mechanisms, and host range data. All phages exhibited distinct DNA restriction profiles, with some phages displaying similarly sized restriction fragments. DNA homology was shown to be present among all 30 phages. The phages could be divided into two groups on the basis of their packaging mechanism as was derived from the appearance of submolar DNA fragments in restriction enzyme digests and the presence (cos-containing phages) or absence (pac-containing phages) of cohesive genomic extremities. Interestingly, the 19 identified cos-containing phages possessed two major structural proteins (32 and 26 kDa) in contrast to the remaining 11 pac-containing phages, which possessed three major structural proteins (41, 25, and 13 kDa). Southern hybridization demonstrated that all pac-containing phages tested contain homologs of the genes encoding the three major structural proteins of the pac-containing phage O1205, whereas all cos-containing phages tested exhibit homology to the gene specifying one of the structural components of the cos-containing phage phi 7201. Fifty-seven percent of the phages (both cos and pac containing) possessed the previously identified 2.2-kb EcoRI fragment of the temperate S. thermophilus phage Sfi18 (H. Brüssow, A. Probst, M. Frémont, and J. Sidoti, Virology 200:854-857, 1994). No obvious correlation was detected between grouping based on packaging mechanism and host range data obtained with 39 industrial S. thermophilus strains.     

Plaque morphology and antigenic relationship of the Salmonella weltevreden typing phages

The plaque morphology and antigenic relationship of the six typing phages of Salmonella weltevreden were studied. Under identical conditions of plating, the phages could be classified into three groups based on plaque morphology. Neutralization tests with anti-phage sera showed that typing phages phi I and phi II were antigenically similar. Phages phi III, phi IV and phi VI also showed antigenic similarity. Typing phage phi V was antigenically distinct from all other phages. Thus the phages could be classified into three groups on the basis of both plaque morphology on their respective indicator strains and velocities of neutralization by homologous/heterologous anti-sera.     

Identification of the receptor-binding protein in 936-species lactococcal bacteriophages

The aim of this work was to identify genes responsible for host recognition in the lactococcal phages sk1 and bIL170 belonging to species 936. These phages have a high level of DNA identity but different host ranges. Bioinformatic analysis indicated that homologous genes, orf18 in sk1 and orf20 in bIL170, could be the receptor-binding protein (RBP) genes, since the resulting proteins were unrelated in the C-terminal part and showed homology to different groups of proteins hypothetically involved in host recognition. Consequently, chimeric bIL170 phages carrying orf18 from sk1 were generated. The recombinant phages were able to form plaques on the sk1 host Lactococcus lactis MG1614, and recombination was verified by PCR analysis directly with the plaques. A polyclonal antiserum raised against the C-terminal part of phage sk1 ORF18 was used in immunogold electron microscopy to demonstrate that ORF18 is located at the tip of the tail. Sequence analysis of corresponding proteins from other lactococcal phages belonging to species 936 showed that the N-terminal parts of the RBPs were very similar, while the C-terminal parts varied, suggesting that the C-terminal part plays a role in receptor binding. The phages investigated could be grouped into sk1-like phages (p2, fd13, jj50, and phi 7) and bIL170-like phages (P008, P113G, P272, and bIL66) on the basis of the homology of their RBPs to the C-terminal part of ORF18 in sk1 and ORF20 in bIL170, respectively. Interestingly, sk1-like phages bind to and infect a defined group of L. lactis subsp. cremoris strains, while bIL170-like phages bind to and infect a defined group of L. lactis subsp. lactis strains.