Molecular Dissection of Mycobacterium tuberculosis Integration Host Factor Reveals Novel Insights into the Mode of DNA Binding and Nucleoid Compaction

The annotated whole-genome sequence of Mycobacterium tuberculosis revealed that Rv1388 (Mtihf) is likely to encode for a putative 20-kDa integration host factor (mIHF). However, very little is known about the functional properties of mIHF or the organization of the mycobacterial nucleoid. Molecular modeling of the mIHF three-dimensional structure, based on the cocrystal structure of Streptomyces coelicolor IHF duplex DNA, a bona fide relative of mIHF, revealed the presence of Arg-170, Arg-171, and Arg-173, which might be involved in DNA binding, and a conserved proline (Pro-150) in the tight turn. The phenotypic sensitivity of Escherichia coli ΔihfA and ΔihfB strains to UV and methyl methanesulfonate could be complemented with the wild-type Mtihf but not its alleles bearing mutations in the DNA-binding residues. Protein-DNA interaction assays revealed that wild-type mIHF, but not its DNA-binding variants, binds with high affinity to fragments containing attB and attP sites and curved DNA. Strikingly, the functionally important amino acid residues of mIHF and the mechanism(s) underlying its binding to DNA, DNA bending, and site-specific recombination are fundamentally different from that of E. coli IHFαβ. Furthermore, we reveal novel insights into IHF-mediated DNA compaction depending on the placement of its preferred binding sites; mIHF promotes DNA compaction into nucleoid-like or higher order filamentous structures. We therefore propose that mIHF is a distinct member of a subfamily of proteins that serve as essential cofactors in site-specific recombination and nucleoid organization and that these findings represent a significant advance in our understanding of the role(s) of nucleoid-associated proteins.     

Protein-DNA Complexes in Mycobacteriophage L5 Integrative Recombination

The temperate mycobacteriophage L5 integrates site specifically into the genomes of Mycobacterium smegmatis, Mycobacterium tuberculosis, and Mycobacterium bovis bacillus Calmette-Guérin. This integrative recombination event occurs between the phage L5 attP site and the mycobacterial attB site and requires the phage-encoded integrase and mycobacterial-encoded integration host factor mIHF. Here we show that attP, Int-L5, and mIHF assemble into a recombinationally active complex, the intasome, which is capable of attB capture and formation of products. The arm-type integrase binding sites within attP play specialized roles in the formation of specific protein-DNA architectures; the intasome is constructed by the formation of intramolecular integrase bridges between one pair of sites, P4-P5, and the attP core, while an additional pair of sites, P1-P2, is required for interaction with attB.     

Integration Host Factor of Mycobacterium tuberculosis,mIHF, Compacts DNA by a Bending Mechanism

The bacterial chromosomal DNA is folded into a compact structure called as ‘nucleoid’ so that the bacterial genome can be accommodated inside the cell. The shape and size of the nucleoid are determined by several factors including DNA supercoiling, macromolecular crowding and nucleoid associated proteins (NAPs). NAPs bind to different sites of the genome in sequence specific or non-sequence specific manner and play an important role in DNA compaction as well as regulation. Until recently, few NAPs have been discovered in mycobacteria owing to poor sequence similarities with other histone-like proteins of eubacteria. Several putative NAPs have now been identified in Mycobacteria on the basis of enriched basic residues or histone-like “PAKK” motifs. Here, we investigate mycobacterial Integration Host Factor (mIHF) for its architectural roles as a NAP using atomic force microscopy and DNA compaction experiments. We demonstrate that mIHF binds DNA in a non-sequence specific manner and compacts it by a DNA bending mechanism. AFM experiments also indicate a dual architectural role for mIHF in DNA compaction as well as relaxation. These results suggest a convergent evolution in the mechanism of E. coli and mycobacterial IHF in DNA compaction.     

Control of directionality in L5 integrase-mediated site-specific recombination

Mycobacteriophage L5 is a temperate phage that forms lysogens in Mycobacterium smegmatis. These lysogens carry an integrated L5 prophage inserted at a specific chromosomal location and undergo subsequent excision during induction of lytic growth. Both the integrative and excisive site-specific recombination events are catalyzed by the phage-encoded tyrosine integrase (Int-L5) and require the host-encoded protein, mIHF. The directionality of these recombination events is determined by a second phage-encoded protein, Excise, the product of gene 36 (Xis-L5); integration occurs efficiently in the absence of Xis-L5 while excision is dependent upon it. We show here that Xis-L5 binds to attR DNA, introduces a DNA bend, and facilitates the formation of an intasome-R complex. This complex, which requires mIHF, Xis-L5 and Int-L5, readily recombines with a second intasome formed by Int-L5, mIHF and attL DNA (intasome-L) to generate the attP and attB products of excision. Xis-L5 also strongly inhibits Int-L5-mediated integrative recombination but does not prevent either the protein-DNA interactions that form the attP intasome (intasome-P) or the capture of attB, but acts later in the reaction presumably by preventing the formation of a recombinagenic synaptic intermediate. The mechanism of action of Xis-L5 appears to be purely architectural, influencing the assembly of protein-DNA structures solely through its DNA-binding and DNA-bending properties.     

Revision of the paraphyletic genus Koerneria Meyl, 1960 and resurrection of two other genera of Diplogastridae (Nematoda)

Recent inferences of phylogeny from molecular characters, as well as a reexamination of morphological and biological characters, reject the monophyly of the nematode genus Koerneria Meyl, 1960 (Diplogastridae). Here, Koerneria sensu lato is revised. The genus, which previously consisted of 40 species, is separated into three genera. Almost all of the transferred species are moved to the resurrected genus Allodiplogaster Paramonov & Sobolev in Skrjabin et al. (1954). Koerneria and Allodiplogaster are distinguished from each other by a weakly vs. clearly striated body surface, an undivided vs. divided stomatal cheilostom, and arrangement of the terminal ventral triplet of male genital papillae, namely in that v5 and v6 are paired and separated from v7 vs. v5–v7 being close to each other. Allodiplogaster is further divided into two groups of species, herein called the henrichae and striata groups, based on both morphological and life-history traits. The henrichae group is characterized by papilliform labial sensilla and male genital papillae, a conical tail in both males and females, and an association with terrestrial habitats and insects, whereas the striata group is characterized by setiform labial sensilla and male genital papillae, an elongated conical tail in both sexes, and an association with aquatic habitats. A second genus, Anchidiplogaster Paramonov, 1952, is resurrected to include a single species that is characterized by its miniscule stoma and teeth, unreflexed testis, and a distinct lack of male genital papillae or stomatal apodemes. Lastly, one further species that was previously included in Koerneria sensu lato is transferred to the genus Pristionchus Kreis, 1932. The revision of Koerneria sensu lato is necessitated by the great variability in its subordinate taxa, which occupy a variety of habitats, in addition to the increased attention to Diplogastridae as a model system for comparative mechanistic biology.     

Revision of the subgenus Orthoscymnus Canepari of Scymnus Kugelann (Coleoptera,Coccinellidae), with descriptions of four new species

The subgenus Orthoscymnus Canepari, 1997 of Scymnus Kugelann, 1794 is herein revised. Seven species of the Orthoscymnus fauna are recognized, of which four species, Scymnus (Orthoscymnus) jilongicus sp. n., Scymnus (Orthoscymnus) paradoxus sp. n., Scymnus (Orthoscymnus) crispatus sp. n. and Scymnus (Orthoscymnus) duomaculatus sp. n., are described as new to science. Scymnus (Orthoscymnus) rhododendri Canepari is recorded from China for the first time. Scymnus (Pullus) robustibasalis Yu is transferred to the subgenus Orthoscymnus (comb. n.). All species are diagnosed, described and illustrated, and distributions are provided for each species. A key to the species is included.     

On the validity of Epeorella Ulmer, 1939 (Ephemeroptera,Heptageniidae) with general considerations on the Heptageniidae of the Sunda Islands

The type material of Epeorella borneonia Ulmer, 1939, the sole species of the genus Epeorella Ulmer, 1939 is reinvestigated and a lectotype (male imago) is designated. Based on several morphological structures, the synonymy with Epeorus Eaton, 1881 (Rhithrogeninae) is rejected. Epeorella stat. prop., known only at the winged stages, belongs to the subfamily Ecdyonurinae, and is a probable endemic of the island of Borneo. The newly erected genus Darthus Webb & McCafferty, 2007, also endemic to Borneo and only known by one species at the nymphal stage, is shown to be a junior subjective synonym of Epeorella. The new combination Epeorella vadora (Webb & McCafferty, 2007) is proposed for the species. The distribution of known heptageniid species from the Sunda Islands is discussed.     

Change in State following Transposition of a Regulatory Element of the Enhancer System in Maize

From an original A2 allele (colored aleurone), a mutable allele, a2-m-4 1629, that changes from a2 to A2 is described. Mutability is expressed as a very distinct pattern limited to the last cell division.—The mutability of a2-m-4 1629 is autonomously controlled by an En at the a2 locus. This En, inactive on standard a testers for En, is partially active on a2-m-1, an a2 tester for En, and expresses varied levels of activity from limited to nearly full suppression of the a2-m-1 color phenotype.—When the En of the a2-m-4 1629 allele transposes from the a2 locus, it behaves, at the new position, like a standard En in triggering a2-m-1, a-m-1 and a-m(r), which express colored spots on a colorless background. The activity of En is therefore different following the change in chromosome location. This finding supports the "position" hypothesis that has been proposed to explain diverse patterns observed among controlling elements. In this case mutation is related to the terminal cell state and not to tissue differences as shown with some phase-variation regulatory elements.     

Rediscovery of Eremobittacus spinulatus Byers (Mecoptera,Bittacidae) in Mexico,with description of the female and comments on sexual dimorphism and potential mimicry

The female of Eremobittacus spinulatus Byers, 1997 is described for the first time. A key to the two species known of this genus endemic to Mexico is provided, and species distribution is illustrated. A case is made for adults of Eremobittacus to be sexually dimorphic, which appears to be an exceptional occurrence in Bittacidae. It is claimed that Eremobittacus spinulatus habitus has a wasp-like appearance, which may potentially depict a case of mimicry.     

An unusual new species of paguroid (Crustacea,Anomura, Paguridae) from deep waters of the Gulf of Mexico

A new hermit crab species of the family Paguridae, Tomopaguropsis ahkinpechensis sp. n., is described from deep waters (780–827 m) of the Gulf of Mexico. This is the second species of Tomopaguropsis known from the western Atlantic, and the fifth worldwide. The new species is morphologically most similar to a species from Indonesia, Tomopaguropsis crinita McLaughlin, 1997, the two having ocular peduncles that diminish in width distally, reduced corneas, dense cheliped setation, and males lacking paired pleopods 1. The calcified figs on the branchiostegite and anterodorsally on the posterior carapace, and the calcified first pleonal somite that is not fused to the last thoracic somite, are unusual paguroid characters. A discussion of the affinities and characters that define this new species is included, along with a key to all five species of Tomopaguropsis.     

Different UmuC requirements for generation of different kinds of UV-induced mutations in Escherichia coli

An Escherichia coli strain bearing the dnaQ49 mutation, which results in a defective s subunit of DNA polymerase III, and carrying the lexA71 mutation, which causes derepression of the SOS regulon, is totally unable to maintain high-copy-number plasmids containing the umuDC operon. The strain is also unable to maintain the pAN4 plasmid containing a partial deletion of the umuD gene but retaining the wild-type umuC gene. These results suggest that a high cellular level of UmuC is exceptionally harmful to the defective DNA polymerase III of the dnaQ49 mutant. We have used this finding as a basis for selection of new plasmid umuC mutants. The properties of two such mutants, bearing the umuC61 or umuC95 mutation, are described in detail. In the umuC122:: Tn 5 strain harbouring the mutant plasmids, UV-induced mutagenesis is severely decreased compared to that observed with the parental umuDC ⁺ plasmid. Interestingly, while the frequency of UV-induced GC → AT transitions is greatly reduced, the frequency of AT → TA transversions is not affected. Both mutant plasmids bear frameshift mutations within the same run of seven A residues present in umuC ⁺; in umuC61 the run is shortened to six A whereas in umuC95 is lengthened to eight A. We have found in both umuC61 and umuC95 that translation is partially restored to the proper reading frame. We propose that under conditions of limiting amounts of UmuC, the protein preferentially facilitates processing of only some kinds of UV-induced lesions.     

The rate of allelism of lethal genes in a geographically structured population

The expected rate of allelism, E[I(x)], of lethal genes between two colonies with distance x in a structured population is studied by using one- and two-dimensional stepping-stone models. It is shown that E[I(x)] depends on the magnitude of selection in heterozygous condition (h), the rate of migration among adjacent colonies (m), the number of loci which produce lethal mutations (n) and the effective population size of each colony (N).——E[I(x)] always decreases with distance x. The rate of decrease is affected strongly by the magnitude of m. The rate of decrease is faster when m is small. E[I(x)] also decreases with increasing N and n. The effect of h on E[I(x)] is somewhat complicated. However, E[I(0)] is always smaller when h is small than when it is large.——For large x, the following approximate formulae may be obtained: (see PDF) where q and Var (q) are the mean and the variance of gene frequencies in each colony, t is approximated as t=h, (see PDF), -h for the partially recessive, completely recessive, and overdominant lethals, respectively, and C₀ is a function of m and t. It is clear that E[I(x)] declines exponentially with x in a one-dimensional habitat. The decrease E[I(x)] is faster in a two-dimensional habitat than in a one-dimensional habitat. The present result is applied to some of the existing data and the estimation of population parameters is also discussed.     

Phylogeny of the Augochlora clade with the description of four new species (Hymenoptera,Apoidea)

The Augochlora clade includes four genera: Augochlora Smith, Augochlorella Sandhouse, Ceratalictus Moure, and Pereirapis Moure. This is one of the richest and most widespread groups of Augochlorini bees. There are about 150 species, which occur from Argentina to Canada. The species of Augochlora clade are considered solitary to facultatively social, except Ceratalictus for which nothing is known. Wood nesting behavior arose once in the clade, in Augochlora sensu strictu. The objective of this study is to describe four new species and to present a revised phylogenetic analysis of the Augochlora clade for the placement of these species. The morphological matrix comprised 77 characters and 42 terminals, and resulted in two most parsimonious trees. The monophyly of the Augochlora clade is corroborated. Ceratalictus and Pereirapis are considered as sister groups and Ceratalictus inflexus sp. nov. came as sister to other species of Ceratalictus. Augochlora and Augochlorella are monophyletic and sister groups. Both extant subgenera of Augochlora were corroborated as monophyletic. Augochlorella comis is considered as sister group to the rest of Augochlorella species. All Augochlorella new species described belong to the Augochlorella ephyra group. Augochlorella kelliae sp. nov. is phylogenetically related to Augochlorella una. Augochlorella procliva sp. nov. and Augochlorella mavricera sp. nov. constitute a clade with Augochlorella acarinata. Including the new species, Augochlorella has 19 species and Ceratalictus 11 species. A revised key for species of Augochlorella and Ceratalictus is also presented in the Supplementary Information.     

A new species of Isoperla (Insecta,Plecoptera) from the Karawanken,with considerations on the Southern Limestone Alps as centers of endemism

A new species of the genus Isoperla (Plecoptera, Perlodidae), belonging to the oxylepis species-group is described, and the male mating call is characterized. Its range falls within a small region of the Southern Limestone Alps which is well known to be one endemism-centre of aquatic insects.     

Bathycamptus eckmani gen. et spec. nov. (Copepoda,Harpacticoida) with a review of the taxonomic status of certain other deepwater harpacticoids

Bathycamptus eckmani gen. et spec. nov., which is associated with mudballs produced by the. cirratulid Tharyx luticastellus, is described from bathyal muds in San Diego Trough, off California. ?Heteropsyllus minutus Wells from the Haden Ground, Scotland is considered to be its closest relative and is placed in the same genus. The genera Bathycamptus and Psammocamptus Mielke are regarded as sister groups on the basis of the shared sexual dimorphism shown by P3-P4. Relationships with other marine Canthocamptidae are discussed, and a re-evaluation of the genus Hemimesochra Sars is made. It is concluded that this genus should encompass only the type species H. clavularis Sars. ?Leimia dubia Wells and H. nympha Por are transferred to the new genus Boreolimella, which is closely related to Bathycamptus but not to Leimia Willey. The genus Perucamptus gen. nov. is established to include H. rapiens Becker and shows no clear relationship with the other genera. H. trisetosa Coull is assigned to Caroliiaicola gen. nov., which is regarded as being an advanced member of the Paranannopidae. H. secunda Wells is recognised as belonging to Mesopsyllus Por; whilst H. nixe Por is considered the type species of a new genus Pusillargillus.