Application of the bacteriophage Mu-driven system for the integration/amplification of target genes in the chromosomes of engineered Gram-negative bacteria—mini review

The advantages of phage Mu transposition-based systems for the chromosomal editing of plasmid-less strains are reviewed. The cis and trans requirements for Mu phage-mediated transposition, which include the L/R ends of the Mu DNA, the transposition factors MuA and MuB, and the cis/trans functioning of the E element as an enhancer, are presented. Mini-Mu(LR)/(LER) units are Mu derivatives that lack most of the Mu genes but contain the L/R ends or a properly arranged E element in cis to the L/R ends. The dual-component system, which consists of an integrative plasmid with a mini-Mu and an easily eliminated helper plasmid encoding inducible transposition factors, is described in detail as a tool for the integration/amplification of recombinant DNAs. This chromosomal editing method is based on replicative transposition through the formation of a cointegrate that can be resolved in a recombination-dependent manner. (E-plus)- or (E-minus)-helpers that differ in the presence of the trans-acting E element are used to achieve the proper mini-Mu transposition intensity. The systems that have been developed for the construction of stably maintained mini-Mu multi-integrant strains of Escherichia coli and Methylophilus methylotrophus are described. A novel integration/amplification/fixation strategy is proposed for consecutive independent replicative transpositions of different mini-Mu(LER) units with “excisable” E elements in methylotrophic cells

Gain and Loss of Phototrophic Genes Revealed by Comparison of Two Citromicrobium Bacterial Genomes

Proteobacteria are thought to have diverged from a phototrophic ancestor, according to the scattered distribution of phototrophy throughout the proteobacterial clade, and so the occurrence of numerous closely related phototrophic and chemotrophic microorganisms may be the result of the loss of genes for phototrophy. A widespread form of bacterial phototrophy is based on the photochemical reaction center, encoded by puf and puh operons that typically are in a ‘photosynthesis gene cluster’ (abbreviated as the PGC) with pigment biosynthesis genes. Comparison of two closely related Citromicrobial genomes (98.1% sequence identity of complete 16S rRNA genes), Citromicrobium sp. JL354, which contains two copies of reaction center genes, and Citromicrobium strain JLT1363, which is chemotrophic, revealed evidence for the loss of phototrophic genes. However, evidence of horizontal gene transfer was found in these two bacterial genomes. An incomplete PGC (pufLMC-puhCBA) in strain JL354 was located within an integrating conjugative element, which indicates a potential mechanism for the horizontal transfer of genes for phototrophy

Can Insects Develop Resistance to Insect Pathogenic Fungi?

This paper presents new, important information on the microevolution of insect resistance to the insect pathogenic fungus Beauveria bassiana which will have far-reaching implications for the development of insect pathogenic fungi as biological control agents. We placed successive generations of a melanic population of the Greater wax moth, Galleria mellonella, under constant selective pressure from the insect pathogenic fungus, Beauveria bassiana. Enhanced fungal resistance was observed and larvae from the 25th generation were studied in detail to uncover mechanisms underpinning resistance, and the possible cost of those survival strategies. There are 3 novel, core findings from the study:1.Antifungal resistance in these insects is pathogen species-specific, and probably arises through trans-generational immune priming. The resistance was less obvious in earlier generations, suggesting subtle cumulative changes that are only fully apparent in the 25th generation. 2.The insect’s fecundity is already pushed close to minimum by its melanic phenotype. Therefore, the additional drain on resources required to boost antifungal defence still more, comes not from further compromising life history traits but via a re-allocation of the insect’s immune defences. Specifically during B. bassiana infection, systemic (fat body and hemocoel) responses, particularly the expression of antimicrobial peptides, are damped down in favour of a tailored repertoire of enhanced responses in the integument (cuticle and epidermis) – the foremost and most important barrier to natural fungal infection. 3.A previously-overlooked range of putative stress-management factors are activated during the specific response of selected insects to B. bassiana. This too occurs primarily in the integument, and contributes to antifungal defense and/or helps ameliorate the damage inflicted by the fungus or the host’s own immune responses during the battle between host and pathogen.No other study to date has examined so many genes in this context. Indeed, we show that the epidermis has a great capacity to express defense and stress-management genes as well as the fat body (which is the main tissue producing antimicrobial peptides and has been the traditional focus of attention). We therefore propose a “be specific / fight locally / de-stress” model to explain resource allocation and defence priorities for insects selected for superior resistance to insect-pathogenic fungi. However, we also show that these insects are less fecund and probably at no evolutionary advantage in the wild, implying that the risk is small of biological control agents failing in the field

The invertible segment of bacteriophage Mu DNA determines the adsorption properties of Mu particles

Interest in bacteriophage Mu stems from the highly promiscuous insertion of its DNA into the genome of its host bacterium Escherichia coli (see ref. 1 for review). There are two characterstic features of Mu DNA: first, mature Mu DNA contains heterogeneous host sequences at both ends2,3, and second, near the right end, the S end, of Mu DNA there is a 3,000 base pair sequence that can undergo inversion. The structure of Mu DNA is diagrammed in Fig. 1. The invertible sequence, called the G segment, is remarkable in that it is also found in bacteriophage P1 (ref. 4). The inversion of the G segment in Mu occurs in the prophage state and is independent of the recA function of the host5. According to Hsu and Davidson6, the inversion occurs by recombination between identical but inverted sequences of about 50 base pairs flanking the G segment. Allet and Bukhari7 have presented evidence that a Mu function, located within or close to the G segment, is required for the inversion reaction. When Mu particles are grown by induction of a lysogen, about half of the particles contain DNA with one orientation (referred to as the + or flip orientation) and the rest of the particles have the G segment in the reverse orientation (the − or flop orientation). However, when Mu particles are grown by infection, almost all of the particles contain DNA with the flip orientation. The predominance of the flip orientation is obtained even if the phage lysate used for infection contained equal numbers of the flip and the flop orientations. We show here that the predominance of the flip orientation after infection results from the inability of the flop containing particles to adsorb properly to the bacterial cells

Involvement of phage Mu-1 early functions in Mu-mediated chromosomal rearrangements

SCOPUS: ar.jinfo:eu-repo/semantics/publishe

Connecting two unrelated DNA sequences with a Mu Dimer

SCOPUS: ar.jinfo:eu-repo/semantics/publishe

Nucleotide sequences of the attachment sites of bacteriophage Mu DNA

THE temperate bacteriophage Mu has the remarkable ability to insert its DNA in apparently random sites of the Escherichia coli chromosome (1–3). All Mu prophages have the same gene order4, and the finding that the prophage and the phage maps are identical suggests that specific sites (attachment sites) at the ends of the Mu genome are used for the integration of Mu5. Phage particles do not contain the excised, free form of the Mu DNA; instead, the Mu genome is flanked by what seems to be heterogeneous bacterial DNA6,7. The variable DNA at both ends of Mu is thought to be generated when Mu DNA is transposed to many new chromosomal sites during replication and is subsequently packaged into phage particles together with adjacent host DNA. The nucleotide sequence analysis of the ends of Mu DNA reported here substantiates this hypothesis and identifies the attachment sites of Mu. Structural features of the attachment sites are presented