Evolution of the long range structure of satellite DNAs in the genus Apodemus.

The temperate bacteriophage Mu causes mutations by inserting its DNA randomly into the genes of its host bacterium Escherichia coli. It is shown here that Mu DNA can be precisely excised from the different integration sites and that as a result wild-type function of the gene into which Mu was inserted is restored. The excision of Mu DNA is observable only if the Mu prophage carries mutations at the X locus. Thus, lac⁺ revertants from six strains, containing heat-inducible prophage Mu cts62 at different locations in the Z gene of the lac operon, were readily obtained by first introducing the X mutation into Mu cts62. The lac⁺ revertants produced wild-type β-galactosidase, and no trace of Mu DNA could be detected in them; this indicates that the junction of Mu DNA and host DNA can be specifically recognized. However, the excision of Mu DNA is generally not perfect, because in most cases it does not lead to the wild-type genotype. The function of gene A of Mu appears to be required for excision. Since the lethal functions of Mu are completely blocked in the Mu cts62 X prophage, the X locus probably has a regulatory function. At least one X mutation is caused by an insertion of about 900 base-pairs in Mu DNA. The discovery of the X mutants opens the way for studying the reversible interaction of the host and Mu chromosomes, and for using Mu to manipulate the host genome in various ways.     

A spontaneous Escherichia coli K12 mutant which inhibits the excision-reintegration process of Mu gem2ts

Escherichia coli K12 strains lysogenic for Mu gem2ts with the prophage inserted in a target gene (i.e., lacZ::Mu gem2ts lysogenic strains) revert to Lac⁺ by prophage precise excision with a relatively high frequency (about 1×10⁻⁶). The revertants obtained are still lysogens with the prophage inserted elsewhere in the bacterial chromosome. We have observed that, with the time of storage in stabs, bacterial cultures lysogenic for Mu gem2ts lose the ability to excise the prophage. The mutation responsible for this effect was co-transducible with the gyrB gene. After the removal of the prophage by P1 vir transduction from these strains, one randomly chosen clone, R3538, was further analyzed. It shows an increment of DNA supercoiling of plasmid pAT153, used as a reporter, and a reduced β-galactosidase activity. On the other hand, R3538 is totally permissive to both lytic and lysogenic cycles of bacteriophage Mu.     

Crystal structure of the C-terminal domain of Mu phage central spike and functions of bound calcium ion

Bacteriophage Mu, which has a contractile tail, is one of the most famous genus of Myoviridae. It has a wide host range and is thought to contribute to horizontal gene transfer. The Myoviridae infection process is initiated by adhesion to the host surface. The phage then penetrates the host cell membrane using its tail to inject its genetic material into the host. In this penetration process, Myoviridae phages are proposed to puncture the membrane of the host cell using a central spike located beneath its baseplate. The central spike of the Mu phage is thought to be composed of gene 45 product (gp45), which has a significant sequence homology with the central spike of P2 phage (gpV). We determined the crystal structure of shortened Mu gp45Δ1-91 (Arg92–Gln197) at 1.5 Å resolution and showed that Mu gp45 is a needlelike structure that punctures the membrane. The apex of Mu gp45 and that of P2 gpV contained iron, chloride, and calcium ions. Although the C-terminal domain of Mu gp45 was sufficient for binding to the E. coli membrane, a mutant D188A, in which the Asp amino acid residue that coordinates the calcium ion was replaced by Ala, did not exhibit a propensity to bind to the membrane. Therefore, we concluded that calcium ion played an important role in interaction with the host cell membrane.     

Bacteriophage Mu-1: A tool to transpose and to localize bacterial genes

In a previous publication (Faelen et al., 1975), it was predicted that the temperate phage Mu-1 would mediate transposition of bacterial genes. Here we show that this is indeed the case. By mating either induced F′ strains (which carry a thermoinducible Mu prophage in the bacterial chromosome), or sensitive F′ infected with Mu, with appropriate recipients, we were able to isolate new F′ episomes which carry various lengths of bacterial DNA. The frequency of transposition of a given marker can be as high as 10^?4. The episomes which carry the transposed DNA always carry Mu as well. When this is coupled with the fact that induction or infection with Mu is necessary for transposition to occur, it is probable that both Mu enzymes and Mu DNA are required by the transposition process. Episomes selected for the presence of a given marker were analyzed for the presence of unselected markers. It was found that: (1) only markers linked to the selected marker can be cotransposed with it; (2) when two markers are simultaneously transposed, all markers lying between them on the chromosome are also transposed; (3) the frequency at which an unselected marker is cotransposed is in some way related to the distance between that marker and the selected marker; (4) the transposition process occurs in both Rec⁺ and Rec^? strains. Mu-mediated transposition offers a new way to isolate F′ episomes and to localize and order bacterial genes as far apart as three minutes.     

Assembly of Both the Head and Tail of Bacteriophage Mu Is Blocked in Escherichia coli groEL and groES Mutants

Like several other Escherichia coli bacteriophages, transposable phage Mu does not develop normally in groE hosts (M. Pato, M. Banerjee, L. Desmet, and A. Toussaint, J. Bacteriol. 169:5504–5509, 1987). We show here that lysates obtained upon induction of groE Mu lysogens contain free inactive tails and empty heads. GroEL and GroES are thus essential for the correct assembly of both Mu heads and Mu tails. Evidence is presented that groE mutations inhibit processing of the phage head protein gpH as well as the formation of a 25S complex suspected to be an early Mu head assembly intermediate.     

Use of Mu phages to isolate transposon insertions juxtaposed to given genes ofEscherichia coli

The small sizes of the DNA fragments transduced by lysates of phage Mu and of mixed lysates of Mu and mini-Mu18A-1 (an internally deleted Mu phage) provide a method for the selection of insertions of transposon Tn10 located very close to givenEscherichia coli genes. Generalized transduction with Mu lysates selected for those insertions located within 38 kilobase pairs of the gene of interest whereas insertions located within about half that distance are directly selected by use of mini-Mu phages. Use of these transduction systems avoids screening of individual colonies by phage P1 transduction for those transposon insertions closely linked to a given gene. Such insertions are most useful for localized mutagenesis and for in vitro molecular cloning.     

Analysis of the killing effect of Mu ligts mutants on host cells

Infection of Escherichia coli with the mutant lig ts2 of bacteriophage Mu at a temperature nonpermissive for this mutant is lethal for the host cells. This effect is insensitive to phage immunity of the host cells, to inhibitors of protein synthesis and is not suppressed in trans in bacterial strains producing the Lig⁺ active protein. These data suggest that the killing effect of this mutant is different from the other kil functions identified in Mu [1].     

Congruence of in vivo and in vitro insertion patterns in hot E. coli gene targets of transposable element Mu: opposing roles of MuB in target capture and integration

Phage Mu transposes promiscuously, employing protein MuB for target capture. MuB forms stable filaments on A/T-rich DNA, and a correlation between preferred MuB binding and Mu integration has been observed. We have investigated the relationship between MuB-binding and Mu insertion into hot and cold Mu targets within the Escherichia coli genome. Although higher binding of MuB to select hot versus cold genes was seen in vivo, the hot genes had an average A/T content and were less preferred targets in vitro, whereas cold genes had higher A/T values and were more efficient targets in vitro. These data suggest that A/T-rich regions are unavailable for MuB binding, and that A/T content is not a good predictor of Mu behavior in vivo. Insertion patterns within two hot genes in vivo could be superimposed on those obtained in vitro in reactions employing purified MuA transposase and MuB, ruling out the contribution of a special DNA structure or additional host factors to the hot behavior of these genes. While A/T-rich DNA is a preferred target in vitro, a fragment made up exclusively of A/T was an extremely poor target. A continuous MuB filament assembled along the A/T region likely protects it against the action of MuA. Our results suggest that MuB binds E. coli DNA in an interspersed manner utilizing local A/T richness, and facilitates capture of these bound regions by the transpososome. Actual integration events are then directed to sites that are in proximity to MuB filaments but are themselves free of MuB.     

Bidesmoside triterpenoid glycosides from Stauntonia chinensis and relationship to anti-inflammation

Ten triterpenoid glycosides, yemuoside YM_26-35 (1-9 and 12), were isolated from a traditional Chinese medicine known as “Ye Mu Gua” (Stauntonia chinensis DC.) along with two known ones, kalopanax saponin C (10) and sieboldianoside A (11). Their structures, as elucidated by spectroscopic analyses and chemical methods, were either penta-saccharidic or hexa-saccharidic bidesmoside triterpenoid glycosides. To help explain the clinical applications of “Ye Mu Gua” for its anti-inflammatory effects, the inhibitory activity on the release of inflammatory mediators (nitric oxide, TNF-α and IL-6) of 1-12 and the related aglycone, hederagenin (13), was evaluated in vitro. It was found that compound 13, but not 1-12, exhibited significant inhibitory activity. The abundant triterpenoid glycosides in “Ye Mu Gua” might therefore be transformed into their respective aglycones, and thus inhibit the release of inflammatory factors in vivo. This could then account for the clinical value of “Ye Mu Gua” as regards anti-inflammatory effects. This proposed explanation of how “Ye Mu Gua” may have an effect is similar to the concept of prodrugs for chemical drugs which could be extended to some traditional medicines. That is, the major components might be biologically active not directly, but via biochemical transformation in vivo. Hence, we propose a “traditional medicine’s prodrug characteristic” concept.     

Coevolution of Bacteriophage PP01 and Escherichia coli O157:H7 in Continuous Culture

The interaction between Escherichia coli O157:H7 and its specific bacteriophage PP01 was investigated in chemostat continuous culture. Following the addition of bacteriophage PP01, E. coli O157:H7 cell lysis was observed by over 4 orders of magnitude at a dilution rate of 0.876 h⁻¹ and by 3 orders of magnitude at a lower dilution rate (0.327 h⁻¹). However, the appearance of a series of phage-resistant E. coli isolates, which showed a low efficiency of plating against bacteriophage PP01, led to an increase in the cell concentration in the culture. The colony shape, outer membrane protein expression, and lipopolysaccharide production of each escape mutant were compared. Cessation of major outer membrane protein OmpC production and alteration of lipopolysaccharide composition enabled E. coli O157:H7 to escape PP01 infection. One of the escape mutants of E. coli O157:H7 which formed a mucoid colony (Mu) on Luria-Bertani agar appeared 56 h postincubation at a dilution rate of 0.867 h⁻¹ and persisted until the end of the experiment (~200 h). Mu mutant cells could coexist with bacteriophage PP01 in batch culture. Concentrations of the Mu cells and bacteriophage PP01 increased together. The appearance of mutant phage, which showed a different host range among the O157:H7 escape mutants than wild-type PP01, was also detected in the chemostat culture. Thus, coevolution of phage and E. coli O157:H7 proceeded as a mutual arms race in chemostat continuous culture.     

BCG-mediated protection against Mycobacterium ulcerans infection in the mouse

Background

Vaccination with Mycobacterium bovis bacille Calmette-Guérin (BCG) is widely used to reduce the risk of childhood tuberculosis and has been reported to have efficacy against two other mycobacterial diseases, leprosy and Buruli ulcer caused by M. ulcerans (Mu). Studies in experimental models have also shown some efficacy against infection caused by Mu. In mice, most studies use the C57BL/6 strain that is known to develop good cell-mediated protective immunity. We hypothesized that there may be differences in vaccination efficacy between C57BL/6 and the less resistant BALB/c strain.

Methods

We evaluated BCG vaccine efficacy against challenge with ∼3×10⁵ M. ulcerans in the right hind footpad using three strains: initially, the Australian type strain, designated Mu1617, then, a Malaysian strain, Mu1615, and a recent Ghanaian isolate, Mu1059. The latter two strains both produce mycolactone while the Australian strain has lost that capacity. CFU of both BCG and Mu and splenocyte cytokine production were determined at intervals after infection. Time to footpad swelling was assessed weekly.

Principal Findings

BCG injection induced visible scars in 95.5% of BALB/c mice but only 43.4% of C57BL/6 mice. BCG persisted at higher levels in spleens of BALB/c than C57BL/6 mice. Vaccination delayed swelling and reduced Mu CFU in BALB/c mice, regardless of challenge strain. However, vaccination was only protective against Mu1615 and Mu1617 in C57BL/6 mice. Possible correlates of the better protection of BALB/c mice included 1) the near universal development of BCG scars in these mice compared to less frequent and smaller scars observed in C57BL/6 mice and 2) the induction of sustained cytokine, e.g., IL17, production as detected in the spleens of BALB/c mice whereas cytokine production was significantly reduced, e.g., IL17, or transient, e.g., Ifnγ, in the spleens of C57BL/6 mice.

Conclusions

The efficacy of BCG against M. ulcerans, in particular, and possibly mycobacteria in general, may vary due to differences in both host and pathogen.     

Detection of Mycolactone A/B in Mycobacterium ulcerans–Infected Human Tissue

Background

Mycobacterium ulcerans disease (Buruli ulcer) is a neglected tropical disease common amongst children in rural West Africa. Animal experiments have shown that tissue destruction is caused by a toxin called mycolactone.

Methodology/Principal Findings

A molecule was identified among acetone-soluble lipid extracts from M. ulcerans (Mu)-infected human lesions with chemical and biological properties of mycolactone A/B. On thin layer chromatography this molecule had a retention factor value of 0.23, MS analyses showed it had an m/z of 765.6 [M+Na⁺] and on MS:MS fragmented to produce the core lactone ring with m/z of 429.4 and the polyketide side chain of mycolactone A/B with m/z of 359.2. Acetone-soluble lipids from lesions demonstrated significant cytotoxic, pro-apoptotic and anti-inflammatory activities on cultured fibroblast and macrophage cell lines. Mycolactone A/B was detected in all of 10 tissue samples from patients with ulcerative and pre-ulcerative Mu disease.

Conclusions/Significance

Mycolactone can be detected in human tissue infected with Mu. This could have important implications for successful management of Mu infection by antibiotic treatment but further studies are needed to measure its concentration.     

Fusions of the lac operon to the transfer RNA gene tyrT of Escherichia coli.

The tyrT gene codes for one of the tyrosirie tRNA species. Using the Casadabatn (1976a) technique, strains of Escherichia coli were isolated in which the lac structural genes are fused to the promoter of the tyrT gene. This procedure involved obtaining a number of insertions of phage Mu DNA in the tyrT gene, lysogenizing the Mu insertion strains with a λplac-Mu hybrid phage, and selecting Lac⁺ derivatives of such lysogens. In a number of Lac⁺ strains thus obtained, the synthesis of β-galactosidase, the product of the lacZ gene, is regulated in a similar fashion to the synthesis of stable RNA. The fusion strains were shown directly to be tyrT-lac fusions by demonstrating that a Mu insertion in the tyrT gene when genetically recombined into the presumed fusion, inactivates the expression of the lac genes. This result shows that tyrT gene sequences are fused to and control the expression of the lac genes in these strains. This is the first report in which genes which code for proteins have been fused to a stable RNA gene in vivo.     

Efficient Insertion Mutagenesis Strategy for Bacterial Genomes Involving Electroporation of In Vitro-Assembled DNA Transposition Complexes of Bacteriophage Mu

An efficient insertion mutagenesis strategy for bacterial genomes based on the phage Mu DNA transposition reaction was developed. Incubation of MuA transposase protein with artificial mini-Mu transposon DNA in the absence of divalent cations in vitro resulted in stable but inactive Mu DNA transposition complexes, or transpososomes. Following delivery into bacterial cells by electroporation, the complexes were activated for DNA transposition chemistry after encountering divalent metal ions within the cells. Mini-Mu transposons were integrated into bacterial chromosomes with efficiencies ranging from 10⁴ to 10⁶ CFU/μg of input transposon DNA in the four species tested, i.e., Escherichia coli, Salmonella enterica serovar Typhimurium, Erwinia carotovora, and Yersinia enterocolitica. Efficiency of integration was influenced mostly by the competence status of a given strain or batch of bacteria. An accurate 5-bp target site duplication flanking the transposon, a hallmark of Mu transposition, was generated upon mini-Mu integration into the genome, indicating that a genuine DNA transposition reaction was reproduced within the cells of the bacteria studied. This insertion mutagenesis strategy for microbial genomes may be applicable to a variety of organisms provided that a means to introduce DNA into their cells is available.     

Strongyloides stercoralis and Trypanosoma cruzi coinfections in a highly endemic area in Argentina

BackgroundStrongyloidiasis and Chagas disease are endemic in northern Argentina. In this study we evaluate the association between S. stercoralis and T. cruzi infections in villages with diverse prevalence levels for these parasites. Further understanding in the relationship between these Neglected Tropical Diseases of South America is relevant for the design of integrated control measures as well as exploring potential biologic interactions.MethodologyCommunity based cross-sectional studies were carried in different villages of the Chaco and Yungas regions in Argentina. Individuals were diagnosed by serology for S. stercoralis and T. cruzi. The association between S. stercoralis and T. cruzi, and between anemia and the two parasites was evaluated using two approaches: marginal (Ma) and multilevel regression (Mu).ResultsA total of 706 individuals from six villages of northern Argentina were included. A total of 37% were positive for S. stercoralis, 14% were positive for T. cruzi and 5% were positive for both. No association was found between infection with S. stercoralis and T. cruzi in any of the models, but we found a negative correlation between the prevalence of these species in the different villages (r = -0.91). Adults (> 15 years) presented association with S. stercoralis (Ma OR = 2.72; Mu OR = 2.84) and T. cruzi (Ma OR = 5.12; Mu OR = 5.48). Also, 12% and 2% of the variance of infection with S. stercoralis and T. cruzi, respectively, could be explained by differences among villages. On the other hand, anemia was associated with infection with S. stercoralis (Ma OR = 1.73; Mu OR = 1.78) and was more prevalent in adults (Ma OR = 2.59; Mu OR = 2.69).ConclusionWe found that coinfection between S. stercoralis and T. cruzi is not more frequent than chance in endemic areas. However, the high prevalence for both parasites, raises the need for an integrated strategy for the control of STH and Chagas disease.