Many insects are associated with obligate symbiotic bacteria, which are localized in specialized cells called bacteriocytes, vertically transmitted through host generations via ovarial passage, and essential for growth and reproduction of their hosts. Although vertical transmission is pivotal for maintenance of such intimate host–symbiont associations, molecular and cellular mechanisms underlying the process are largely unknown. Here we report a cellular mechanism for vertical transmission of the obligate symbiont Buchnera in the pea aphid Acyrthosiphon pisum . In the aphid body, Buchnera cells are transmitted from maternal bacteriocytes to adjacent blastulae at the ovariole tips in a highly coordinated manner. By making use of symbiont-manipulated strains of A. pisum , we demonstrated that the facultative symbiont Serratia is, unlike Buchnera , not transmitted from maternal bacteriocytes to blastulae, suggesting a specific mechanism for Buchnera transmission. EM observations revealed a series of exo-/endocytotic processes operating at the bacteriocyte–blastula interface: Buchnera cells are exocytosed from the maternal bacteriocyte, temporarily released to the extracellular space, and endocytosed by the posterior syncytial cytoplasm of the blastula. These results suggest that the selective Buchnera transmission is likely attributable to Buchnera -specific exocytosis by the maternal bacteriocyte, whereas both Buchnera and Serratia are nonselectively incorporated by the endocytotic activity of the posterior region of the blastula. The sophisticated cellular mechanism for vertical transmission of Buchnera must have evolved to ensure the obligate host–symbiont association, whereas facultative symbionts like Serratia may coopt the endocytotic component of the mechanism for their entry into the host embryos.

In natural populations of the pea aphid Acyrthosiphon pisum, a facultative bacterial symbiont of the genus Rickettsia has been detected at considerable infection frequencies worldwide. We investigated the effects of the Rickettsia symbiont on the host aphid and also on the coexisting essential symbiont Buchnera. In situ hybridization revealed that the Rickettsia symbiont was specifically localized in two types of host cells specialized for endosymbiosis: secondary mycetocytes and sheath cells. Electron microscopy identified bacterial rods, about 2 m long and 0.5 m thick, in sheath cells of Rickettsia-infected aphids. Virus-like particles were sometimes observed in association with the bacterial cells. By an antibiotic treatment, we generated Rickettsia-infected and Rickettsia-eliminated aphid strains with an identical genetic background. Comparison of these strains revealed that Rickettsia infection negatively affected some components of the host fitness. Quantitative PCR analysis of the bacterial population dynamics identified a remarkable interaction between the coexisting symbionts: Buchnera population was significantly suppressed in the presence of Rickettsia, particularly at the young adult stage, when the aphid most actively reproduces. On the basis of these results, we discussed the possible mechanisms that enable the prevalence of Rickettsia infection in natural host populations in spite of the negative fitness effects observed in the laboratory.

The Japanese common broad-headed bugs Riptortus clavatus and Leptocorisa chinensis possess a number of crypts in the posterior region of the midgut, whose lumen contains a copious amount of bacterial cells. We characterized the gut symbiotic bacteria by using molecular phylogenetic analysis, light and electron microscopy, in situ hybridization, and PCR-based detection techniques. Restriction fragment length polymorphism analysis of 16S rRNA gene clones suggested that a single bacterium dominated the microbiota in the crypts of the both bug species. The predominant 16S rRNA gene sequences obtained from different individuals and species of the bugs were not identical but were very similar to each other. Homology searches in the DNA databases revealed that the sequences showed the highest levels of similarity (96% to 99%) to the sequences of Burkholderia spp. belonging to the ␤ subdivision of the class Proteobacteria. In situ hybridization with specific oligonucleotide probes confirmed the localization of the Burkholderia symbiont in the lumen of the midgut crypts. Electron microscopy showed that the lumen of the crypts was filled with rod-shaped bacteria of a single morphotype. Molecular phylogenetic analysis demonstrated that the Burkholderia symbionts of the bugs formed a well-defined monophyletic group, although the group also contained several environmental Burkholderia strains. The phylogenetic relationship of the Burkholderia symbionts did not reflect the relationship of the host bug species at all. The sequences from R. clavatus and the sequences from L. chinensis did not form clades but were intermingled in the phylogeny, suggesting that horizontal transmission of the symbiont might have occasionally occurred between populations and species of the bugs.

Symbiosis has significantly contributed to organismal adaptation and diversification. For establishment and maintenance of such hostsymbiont associations, host organisms must have evolved mechanisms for selective incorporation, accommodation, and maintenance of their specific microbial partners. Here we report the discovery of a previously unrecognized type of animal organ for symbiont sorting. In the bean bug Riptortus pedestris, the posterior midgut is morphologically differentiated for harboring specific symbiotic bacteria of a beneficial nature. The sorting organ lies in the middle of the intestine as a constricted region, which partitions the midgut into an anterior nonsymbiotic region and a posterior symbiotic region. Oral administration of GFP-labeled Burkholderia symbionts to nymphal stinkbugs showed that the symbionts pass through the constricted region and colonize the posterior midgut. However, administration of food colorings revealed that food fluid enters neither the constricted region nor the posterior midgut, indicating selective symbiont passage at the constricted region and functional isolation of the posterior midgut for symbiosis. Coadministration of the GFP-labeled symbiont and red fluorescent protein-labeled Escherichia coli unveiled selective passage of the symbiont and blockage of E. coli at the constricted region, demonstrating the organ's ability to discriminate the specific bacterial symbiont from nonsymbiotic bacteria. Transposon mutagenesis and screening revealed that symbiont mutants in flagella-related genes fail to pass through the constricted region, highlighting that both host's control and symbiont's motility are involved in the sorting process. The blocking of food flow at the constricted region is conserved among diverse stinkbug groups, suggesting the evolutionary origin of the intestinal organ in their common ancestor.stinkbug | gut symbiosis | partner choice | Burkholderia | flagellar motility

Serpentinization, or the aqueous alteration of ultramafic rocks, results in challenging environments for life in continental sites due to the combination of extremely high pH, low salinity and lack of obvious electron acceptors and carbon sources. Nevertheless, certain Betaproteobacteria have been frequently observed in such environments. Here we describe physiological and genomic features of three related Betaproteobacterial strains isolated from highly alkaline (pH 11.6) serpentinizing springs at The Cedars, California. All three strains are obligate alkaliphiles with an optimum for growth at pH 11 and are capable of autotrophic growth with hydrogen, calcium carbonate and oxygen. The three strains exhibit differences, however, regarding the utilization of organic carbon and electron acceptors. Their global distribution and physiological, genomic and transcriptomic characteristics indicate that the strains are adapted to the alkaline and calcium-rich environments represented by the terrestrial serpentinizing ecosystems. We propose placing these strains in a new genus ‘Serpentinomonas’.

Despite the omnipresence of specific host–symbiont associations with acquisition of the microbial symbiont from the environment, little is known about how the specificity of the interaction evolved and is maintained. The bean bug Riptortus pedestris acquires a specific bacterial symbiont of the genus Burkholderia from environmental soil and harbors it in midgut crypts. The genus Burkholderia consists of over 100 species, showing ecologically diverse lifestyles, and including serious human pathogens, plant pathogens, and nodule-forming plant mutualists, as well as insect mutualists. Through infection tests of 34 Burkholderia species and 18 taxonomically diverse bacterial species, we demonstrate here that nonsymbiotic Burkholderia and even its outgroup Pandoraea could stably colonize the gut symbiotic organ and provide beneficial effects to the bean bug when inoculated on aposymbiotic hosts. However, coinoculation revealed that the native symbiont always outcompeted the nonnative bacteria inside the gut symbiotic organ, explaining the predominance of the native Burkholderia symbiont in natural bean bug populations. Hence, the abilities for colonization and cooperation, usually thought of as specific traits of mutualists, are not unique to the native Burkholderia symbiont but, to the contrary, competitiveness inside the gut is a derived trait of the native symbiont lineage only and was thus critical in the evolution of the insect gut symbiont.

Researchers have described aquaporin water channels from diverse eubacterial and eukaryotic species but not from the third division of life, Archaea. Methanothermobacter marburgensis is a methanogenic archaeon that thrives under anaerobic conditions at 65°C. After transfer to hypertonic media, M. marburgensis sustained cytoplasmic shrinkage that could be prevented with HgCl 2 . We amplified aqpM by PCR from M. marburgensis DNA. Like known aquaporins, the open reading frame of aqpM encodes two tandem repeats each containing three membrane-spanning domains and a poreforming loop with the signature motif Asn-Pro-Ala (NPA). Unlike other known homologs, the putative Hg 2؉ -sensitive cysteine was found proximal to the first NPA motif in AqpM, rather than the second. Moreover, amino acids distinguishing water-selective homologs from glycerol-transporting homologs were not conserved in AqpM. A fusion protein, 10-His-AqpM, was expressed and purified from Escherichia coli. AqpM reconstituted into proteoliposomes was shown by stopped-flow light scattering assays to have elevated osmotic water permeability (P f ‫؍‬ 57 m⅐s ؊1 versus 12 m⅐s ؊1 of control liposomes) that was reversibly inhibited with HgCl 2 . Transient, initial glycerol permeability was also detected. AqpM remained functional after incubations at temperatures above 80°C and formed SDS-stable tetramers. Our studies of archaeal AqpM demonstrate the ubiquity of aquaporins in nature and provide new insight into protein structure and transport selectivity.To withstand environmental and physiological stresses, organisms must be able to rapidly absorb and release water. Facilitated transport of water across cell membranes must be highly selective to prevent uncontrolled movement of other solutes, protons, and ions. Discovery of the aquaporins provided a molecular explanation to these processes (2). More than 200 aquaporins have now been identified, and their presence has been established in most forms of life (3). No aquaporin from Archaea has yet been characterized, although functional roles for a water channel protein have been predicted in these organisms (4).Two major protein family subsets are presently recognized, water-selective channels (aquaporins) and glycerol-transporting homologs with varying water permeabilities (aquaglyceroporins). The permeation selectivity of new members of the protein family may be predicted by a small number of conserved residues (5, 6). Several prokaryotic aquaporins and aquaglyceroporins are known. The bacterial water channel, AqpZ, was first identified in Escherichia coli (7,8). Movement of water across the bacterial plasma membrane may be part of the osmoregulatory response by which microorganisms adjust cell turgor (9), although the regulation and physiological role of AqpZ are being reassessed (10). AqpZ is a highly stable tetramer with negligible permeability to glycerol. In contrast, the glycerol permeability of the glycerol facilitator (GlpF) from E. coli has long been recognized (11). GlpF has relatively limited water perm...

A microbial fuel cell (MFC) was inoculated with a random transposon insertion mutant library of Shewanella oneidensis MR-1 and operated with lactate as the sole fuel to select for mutants that preferentially grew in it. Agar plate cultivation of the resultant MFC enrichment culture detected an increased number of colonies exhibiting rough morphology. One such isolate, strain 4A, generated 50% more current in an MFC than wild-type MR-1. Determination of the transposon insertion site in strain 4A followed by deletion and complementation experiments revealed that the SO3177 gene, encoding a putative formyltransferase and situated in a cell surface polysaccharide biosynthesis gene cluster, was responsible for the increased current. Transmission electron microscopy showed that a layered structure at the cell surface, stainable with ruthenium red, was impaired in the SO3177 mutant (⌬SO3177), confirming that SO3177 is involved in the biosynthesis of cell surface polysaccharides. Compared to the wild type, ⌬SO3177 cells preferentially attached to graphite felt anodes in MFCs, while physicochemical analyses revealed that the cell surface of ⌬SO3177 was more hydrophobic. These results demonstrate that cell surface polysaccharides affect not only the cell adhesion to graphite anodes but also the current generation in MFCs.Dissimilatory metal-reducing bacteria (DMRB) conserve energy for growth by coupling the oxidation of organic compounds to the reduction of metal compounds (29). DMRB are of great interest not only for their importance in the biogeochemical cycling of metals (25) but also for their utility in biotechnological processes, such as microbial fuel cells (MFCs) (24,40). In recent years, the ability of many DMRB, including members of the genera Shewanella (5,12,20,31), Geobacter (2), Aeromonas (34), Desulfobulbus (19), and Phodoferax (9), to generate current in MFCs has been described.Among DMRB, Shewanella oneidensis MR-1 is one of the most extensively studied due to its metabolic versatility (28), annotated genome sequence (17), and genetic accessibility. In addition, since the first report in 1999 when this microorganism was shown to have the ability to transfer electrons to an anode without an exogenously added mediator (20), it has become a model organism for the study of microbial current generation in MFCs. Extensive studies have been performed to understand the mechanisms of extracellular electron transfer (EET) to solid materials, such as MFC anodes and metal oxides, in strain MR-1. Multiple mechanisms, including direct EET through the physical contact of bacterial cells via outer membrane (OM) cytochromes (42) and conductive nanowires (16) and mediated EET via self-produced electron shuttles such as quinones and flavins (27,30,39,41), have been identified.Although OM cytochromes and electron shuttles have been identified to play central roles in EET, it is reasonable to speculate that this complex catabolic process is also influenced by other (extra)cellular components. To date, only limited studies hav...