Hundreds of antimicrobial peptides create a selective barrier for insect gut symbionts.

The spatial organization of gut microbiota is crucial for the functioning of the gut ecosystem, although the mechanisms that organize gut bacterial communities in microhabitats are only partially understood. The gut of the insect Riptortus pedestris has a characteristic microbiota biogeography with a multispecies community in the anterior midgut and a monospecific bacterial population in the posterior midgut. We show that the posterior midgut region produces massively hundreds of specific antimicrobial peptides (AMPs), the Crypt-specific Cysteine-Rich peptides (CCRs) that have membrane-damaging antimicrobial activity against diverse bacteria but posterior midgut symbionts have elevated resistance. We determined by transposon-sequencing the genetic repertoire in the symbiont Caballeronia insecticola to manage CCR stress, identifying different independent pathways, including AMP-resistance pathways unrelated to known membrane homeostasis functions as well as cell envelope functions. Mutants in the corresponding genes have reduced capacity to colonize the posterior midgut, demonstrating that CCRs create a selective barrier and resistance is crucial in gut symbionts. Moreover, once established in the gut, the bacteria differentiate into a CCR-sensitive state, suggesting a second function of the CCR peptide arsenal in protecting the gut epithelia or mediating metabolic exchanges between the host and the gut symbionts. Our study highlights the evolution of an extreme diverse AMP family that likely contributes to establish and control the gut microbiota.

Transposon sequencing reveals the essential gene set and genes enabling gut symbiosis in the insect symbiont Caballeronia insecticola.

Caballeronia insecticola is a bacterium belonging to the Burkholderia genus sensu lato, which is able to colonize multiple environments like soils and the gut of the bean bug Riptortus pedestris. We constructed a saturated Himar1 mariner transposon library and revealed by transposon-sequencing that 498 protein-coding genes constitute the essential genome of Caballeronia insecticola for growth in free-living conditions. By comparing essential gene sets of Caballeronia insecticola and seven related Burkholderia s.l. strains, only 120 common genes were identified, indicating that a large part of the essential genome is strain-specific. In order to reproduce specific nutritional conditions that are present in the gut of Riptortus pedestris, we grew the mutant library in minimal media supplemented with candidate gut nutrients and identified several condition-dependent fitness-defect genes by transposon-sequencing. To validate the robustness of the approach, insertion mutants in six fitness genes were constructed and their growth deficiency in media supplemented with the corresponding nutrient was confirmed. The mutants were further tested for their efficiency in Riptortus pedestris gut colonization, confirming that gluconeogenic carbon sources, taurine and inositol, are nutrients consumed by the symbiont in the gut. Thus, our study provides insights about specific contributions provided by the insect host to the bacterial symbiont.

Intercontinental Diversity of Caballeronia Gut Symbionts in the Conifer Pest Bug Leptoglossus occidentalis.

Many stinkbugs in the superfamily Coreoidea (Hemiptera: Heteroptera) develop crypts in the posterior midgut, harboring Caballeronia (Burkholderia) symbionts. These symbionts form a monophyletic group in Burkholderia sensu lato, called the "stinkbug-associated beneficial and environmental (SBE)" group, recently reclassified as the new genus Caballeronia. SBE symbionts are separated into the subclades SBE-α and SBE-ß. Previous studies suggested a regional effect on the symbiont infection pattern; Japanese and American bug species are more likely to be associated with SBE-α, while European bug species are almost exclusively associated with SBE-ß. However, since only a few insect species have been investigated, it remains unclear whether region-specific infection is general. We herein investigated Caballeronia gut symbionts in diverse Japanese, European, and North American populations of a cosmopolitan species, the Western conifer seed bug Leptoglossus occidentalis (Coreoidea: Coreidae). A mole-cular phylogenetic ana-lysis of the 16S rRNA gene demonstrated that SBE-ß was the most dominant in all populations. Notably, SBE-α was rarely detected in any region, while a third clade, the "Coreoidea clade" occupied one fourth of the tested populations. Although aposymbiotic bugs showed high mortality, SBE-α- and SBE-ß-inoculated insects both showed high survival rates; however, a competition assay demonstrated that SBE-ß outcompeted SBE-α in the midgut crypts of L. occidentalis. These results strongly suggest that symbiont specificity in the Leptoglossus-Caballeronia symbiotic association is influenced by the host rather than geography, while the geographic distribution of symbionts may be more important in other bugs.

Caenorhabditis elegans, a pluricellular model organism to screen new genes involved in mitochondrial genome maintenance.

The inheritance of functional mitochondria depends on faithful replication and transmission of mitochondrial DNA (mtDNA). A large and heterogeneous group of human disorders is associated with mitochondrial genome quantitative and qualitative anomalies. Several nuclear genes have been shown to account for these severe OXPHOS disorders. However, in several cases, the disease-causing mutations still remain unknown. Caenorhabditis elegans has been largely used for studying various biological functions because this multicellular organism has short life cycle and is easy to grow in the laboratory. Mitochondrial functions are relatively well conserved between human and C.elegans, and heteroplasmy exists in this organism as in human. C. elegans therefore represents a useful tool for studying mtDNA maintenance. Suppression by RNA interference of genes involved in mtDNA replication such as polg-1, encoding the mitochondrial DNA polymerase, results in reduced mtDNA copy number but in a normal phenotype of the F1 worms. By combining RNAi of genes involved in mtDNA maintenance and EtBr exposure, we were able to reveal a strong and specific phenotype (developmental larval arrest) associated to a severe decrease of mtDNA copy number. Moreover, we tested and validated the screen efficiency for human orthologous genes encoding mitochondrial nucleoid proteins. This allowed us to identify several genes that seem to be closely related to mtDNA maintenance in C. elegans. This work reports a first step in the further development of a large-scale screening in C. elegans that should allow to identify new genes of mtDNA maintenance whose human orthologs will obviously constitute new candidate genes for patients with quantitative or qualitative mtDNA anomalies.

A nonproteolytic proteasome activity controls organelle fission in yeast.

To understand the processes underlying organelle function, dynamics and inheritance, it is necessary to identify and characterize the regulatory components involved. Recently in yeast and mammals, proteins of the membrane fission machinery (Dnm1-Mdv1-Caf4-Fis1 in yeast and DLP1-FIS1 in human) have been shown to have a dual localization on mitochondria and peroxisomes, where they control mitochondrial fission and peroxisome division. Here, we show that whereas vacuole fusion is regulated by the proteasome degradation function, mitochondrial fission and peroxisomal division are not controlled by the proteasome activity but rather depend on a new function of the proteasomal lid subunit Rpn11. Rpn11 was found to regulate the Fis1-dependent fission machinery of both organelles. These findings indicate a unique role of the Rpn11 protein in mitochondrial fission and peroxisomal proliferation that is independent of its role in proteasome-associated deubiquitylation.

Proteolytic cleavage of the hyperthermophilic topoisomerase I from Thermotoga maritima does not impair its enzymatic properties.

Using limited proteolysis, we show that the hyperthermophilic topoisomerase I from Thermotoga maritima exhibits a unique hot spot susceptible to proteolytic attack with a variety of proteases. The remaining of the protein is resistant to further proteolysis, which suggests a compact folding of the thermophilic topoisomerase, when compared to its mesophilic Escherichia coli homologue. We further show that a truncated version of the T. maritima enzyme, lacking the last C-terminal 93 amino acids is more susceptible to proteolysis, which suggests that the C-terminal region of the topoisomerase may be important to maintain the compact folding of the enzyme. The hot spot of cleavage is located around amino acids 326-330 and probably corresponds to an exposed loop of the protein, near the active site tyrosine in charge of DNA cleavage and religation. Location of this protease sensitive region in the vicinity of bound DNA is consistent with the partial protection observed in the presence of different DNA substrates. Unexpectedly, although proteolysis splits the enzyme in two halves, each containing part of the motifs involved in catalysis, trypsin-digested topoisomerase I retains full DNA binding, cleavage, and relaxation activities, full thermostability and also the same hydrodynamic and spectral properties as undigested samples. This supports the idea that the two fragments which are generated by proteolysis remain correctly folded and tightly associated after proteolytic cleavage.

Caenorhabditis elegans expressing the Saccharomyces cerevisiae NADH alternative dehydrogenase Ndi1p, as a tool to identify new genes involved in complex I related diseases.

Isolated complex I deficiencies are one of the most commonly observed biochemical features in patients suffering from mitochondrial disorders. In the majority of these clinical cases the molecular bases of the diseases remain unknown suggesting the involvement of unidentified factors that are critical for complex I function. The Saccharomyces cerevisiae NDI1 gene, encoding the mitochondrial internal NADH dehydrogenase was previously shown to complement a complex I deficient strain in Caenorhabditis elegans with notable improvements in reproduction and whole organism respiration. These features indicate that Ndi1p can functionally integrate the respiratory chain, allowing complex I deficiency complementation. Taking into account the Ndi1p ability to bypass complex I, we evaluate the possibility to extend the range of defects/mutations causing complex I deficiencies that can be alleviated by NDI1 expression. We report here that NDI1 expressing animals unexpectedly exhibit a slightly shortened lifespan, a reduction in the progeny, and a depletion of the mitochondrial genome. However, Ndi1p is expressed and targeted to the mitochondria as a functional protein that confers rotenone resistance to those animals without affecting their respiration rate and ATP content. We show that the severe embryonic lethality level caused by the RNAi knockdowns of complex I structural subunit encoding genes (e.g., NDUFV1, NDUFS1, NDUFS6, NDUFS8, or GRIM-19 human orthologs) in wild type animals is significantly reduced in the Ndi1p expressing worm. All together these results open up the perspective to identify new genes involved in complex I function, assembly, or regulation by screening an RNAi library of genes leading to embryonic lethality that should be rescued by NDI1 expression.

Dissection of the carboxyl-terminal domain of the proteasomal subunit Rpn11 in maintenance of mitochondrial structure and function.

We have previously demonstrated that the C-terminal part of Rpn11, a deubiquitinating enzyme in the lid of the proteasome, is essential for maintaining a correct cell cycle and normal mitochondrial morphology and function. The two roles are apparently unlinked as the mitochondrial role is mapped to the Carboxy-terminus, whereas the catalytic deubiquitinating activity is found within the N-terminal region. The mitochondrial defects are observed in rpn11-m1 (originally termed mpr1-1), a mutation that generates Rpn11 lacking the last 31 amino acids. No mitochondrial phenotypes are recorded for mutations in the MPN+/JAMM motif. In the present study, we investigated the participation of the last 31 amino acids of the Rpn11 protein by analysis of intragenic revertants and site-specific mutants. We identified a putative alpha-helix necessary for the maintenance of a correct cell cycle and determined that a very short region at the C-terminus of Rpn11 is essential for the maintenance of tubular mitochondrial morphology. Furthermore, we show that expression of the C-terminal part of Rpn11 is able to complement in trans all of the rpn11-m1 mitochondrial phenotypes. Finally, we investigate the mechanisms by which Rpn11 controls the mitochondrial shape and show that Rpn11 may regulate the mitochondrial fission and tubulation processes.

Mutational analysis of the helicase-like domain of Thermotoga maritima reverse gyrase.

Reverse gyrase is a unique type IA topoisomerase that is able to introduce positive supercoils into DNA in an ATP-dependent process. ATP is bound to the helicase-like domain of the enzyme that contains most of the conserved motifs found in helicases of the SF1 and SF2 superfamilies. In this paper, we have investigated the role of the conserved helicase motifs I, II, V, VI, and Q by generating mutants of the Thermotoga maritima reverse gyrase. We show that mutations in motifs I, II, V, and VI completely eliminate the supercoiling activity of reverse gyrase and that a mutation in the Q motif significantly reduces this activity. Further analysis revealed that for most mutants, the DNA binding and cleavage properties are not significantly changed compared with the wild type enzyme, whereas their ATPase activity is impaired. These results clearly show that the helicase motifs are tightly involved in the coupling of ATP hydrolysis to the topoisomerase activity. The zinc finger motif located at the N-terminal end of reverse gyrases was also mutated. Our results indicate that this motif plays an important role in DNA binding.

Interaction between apterous and early expression of vestigial in formation of the dorso-ventral compartments in the Drosophila wing disc.

BACKGROUND: Compartment formation is a developmental process that requires the existence of barriers against intermixing between cell groups. In the Drosophila wing disc, the dorso-ventral (D/V) compartment boundary is defined by the expression of the apterous (ap) selector gene in the dorsal compartment. AP activity is under control of dLMO which destabilizes the formation of the AP-CHIP complex. RESULTS: We report that D/V boundary formation in the wing disc also depends on early expression of vestigial (vg). Our data suggest that vg is already required for wing cell proliferation before D/V compartmentalization. In addition, we show that over-expression of vg can, to some extent, rescue the effect of the absence of ap on D/V boundary formation. Early VG product regulates AP activity by inducing dLMO and thus indirectly regulating ap target genes such as fringe and the PSalpha1 and PSalpha2 integrins. CONCLUSION: Normal cell proliferation is necessary for ap expression at the level of the D/V boundary. This would be mediated by vg, which interacts in a dose-dependent way with ap.

Thermotoga maritima-Escherichia coli chimeric topoisomerases. Answers about involvement of the carboxyl-terminal domain in DNA topoisomerase I-mediated catalysis.

Bacterial topoisomerases I are generally composed of two domains as follows: a core domain, which contains all the conserved motifs involved in the trans-esterification reactions, and a carboxyl-terminal domain, highly variable in size and sequence. In the present work, we have addressed the question of the respective roles of the two domains in the different steps of the topoisomerization cycle. For this purpose, we prepared various recombinant topoisomerases from two model enzymes: topoisomerase I from the hyperthermophilic bacterium Thermotoga maritima and topoisomerase I from Escherichia coli. We compared the properties of the two core domains to that of the topoisomerases formed by combining the core domain of one enzyme to the carboxyl-terminal domain of the other. We found that, contrary to E. coli (Lima, C. D., Wang, J. C., and Mondragon, A. (1993) J. Mol. Biol. 232, 1213-1216), the core domain from T. maritima (TmTop65) is able to sustain by itself a complete topoisomerization cycle, although with low efficiency. Fusion of TmTop65 to the entire carboxyl-terminal domain from E. coli considerably increases binding efficiency, thermal stability, and DNA relaxation activity. Moreover, the chimera predominantly acquires the cleavage specificity of E. coli full-length topoisomerase. For the chimera obtained by fusion of the T. maritima carboxyl-terminal domain to the core EcTop67, very low DNA relaxation activity and binding are recovered, but formation of a covalent DNA adduct is impaired. Taken together, our results show that the presence and the nature of the carboxyl-terminal domain of bacterial topoisomerases I strongly determine their DNA binding efficiency and cleavage specificity but is not strictly required for strand passage.