Density dynamics of diverse Spiroplasma strains naturally infecting different species of Drosophila

Facultative heritable bacterial endosymbionts can have dramatic effects on their hosts, ranging from mutualistic to parasitic. Within-host bacterial endosymbiont density plays a critical role in maintenance of a symbiotic relationship, as it can affect levels of vertical transmission and expression of phenotypic effects, both of which influence the infection prevalence in host populations. Species of genus Drosophila are infected with Spiroplasma, whose characterized phenotypic effects range from that of a male-killing reproductive parasite to beneficial defensive endosymbiont. For many strains of Spiroplasma infecting at least 17 species of Drosophila, however, the phenotypic effects are obscure. The infection prevalence of these Spiroplasma vary within and among Drosophila species, and little is known about the within-host density dynamics of these diverse strains. To characterize the patterns of Spiroplasma density variation among Drosophila we used quantitative PCR to assess bacterial titer at various life stages of three species of Drosophila naturally-infected with two different types of Spiroplasma. For naturally infected Drosophila species we found that non-male-killing infections had consistently lower densities than the male-killing infection. The patterns of Spiroplasma titer change during aging varied among Drosophila species infected with different Spiroplasma strains. Bacterial density varied within and among populations of Drosophila, with individuals from the population with the highest prevalence of infection having the highest density. This density variation underscores the complex interaction of Spiroplasma strain and host genetic background in determining endosymbiont density.     

Predicted ATP-binding cassette systems in the phytopathogenic mollicute <Emphasis Type="Italic">Spiroplasma kunkelii</Emphasis>

Spiroplasma kunkelii is a cell wall-free, helical, and motile mycoplasma-like organism that causes corn stunt disease in maize. The bacterium has a compact genome with a gene set approaching the minimal complement necessary for cellular life and pathogenesis. A set of 21 ATP-binding cassette (ABC) domains was identified during the annotation of a draft S. kunkelii genome sequence. These 21 ABC domains are present in 18 predicted proteins, and are components of 16 functional systems, which account for 5% of the protein coding capacity of the S. kunkelii genome. Of the 16 systems, 11 are membrane-bound transporters, and two are cytosolic systems involved in DNA repair and the oxidative stress response; the genes for the remaining three hypothetical systems harbor nonsense and/or frameshift mutations, so their functional status is doubtful. Assembly of the 11 multicomponent transporters, and comparisons with other known systems permitted functional predictions for the S. kunkelii ABC transporter systems. These transporters convey a wide variety of substrates, and are critical for nutrient uptake, multidrug resistance, and perhaps virulence. Our findings provide a framework for functional characterization of the ABC systems in S. kunkelii.Communicated by W. Goebel     

Gene content and organization of an 85-kb DNA segment from the genome of the phytopathogenic mollicute Spiroplasma kunkelii

Spiroplasma kunkelii, the causative agent of corn stunt disease in maize ( Zea mays L.), is a helical, cell wall-less prokaryote assigned to the class Mollicutes. As part of a project to sequence the entire S. kunkelii genome, we analyzed an 85-kb DNA segment from the pathogenic strain CR2-3x. This genome segment contains 101 ORFs and two tRNA genes. The majority of the ORFs code for predicted proteins that can be assigned to respective clusters of orthologous groups (COGs). These COGs cover diverse functional categories including genetic information storage and processing, cellular processes, and metabolism. The most notable gene cluster in this genome segment is a super-operon capable of encoding 24 ribosomal proteins. The organization of genes in this operon reflects the unique evolutionary position of the spiroplasma. Gene duplications, domain rearrangements, and frameshift mutations in the segment are interpreted as indicators of phase variation in the spiroplasma. To our knowledge, this is the first analysis of a large genome segment from a plant pathogenic spiroplasma.Communicated by W. Goebel     

Spiroplasma species in the Costa Rican highlands: implications for biogeography and biodiversity

More than 1,000 Spiroplasma isolates have been obtained from horse flies and deer flies (Diptera:Tabanidae) in the United States and Canada. However, the spiroplasma biota of Central America is poorly known. In August of 1995 and 1998, 13 isolates were obtained in 14 attempts from horse flies of a single species, Poeciloderas quadripunctatus, taken in the Costa Rican highlands (1,100–2,000 m). The majority of the “isolates” proved to be mixtures of two or more Spiroplasma species, but after filter cloning, single strains emerged that were designated as representatives of the 13 accessions. Six distinct spiroplasma serogroups were identified from these isolations. Three of the strains are putative new species with no serological relationship to any other Spiroplasma species. A fourth strain is a putative new species that may be distantly related to S. helicoides, a southeastern U.S. species. These four strains are accorded herein status as representatives of new serogroups: strain BARC 4886 (group XXXV); strain BARC 4900 (group XXXVI); strain BARC 4908 (group XXXVII); and GSU5450 (group XXXVIII). A fifth Spiroplasma species was very closely related to S. lineolae, known previously only from the Georgia (U.S.) coast. The sixth was most closely related to subgroup VIII-3, known from Texas and the southeastern U.S. Discovery of six spiroplasma species in only 13 attempted isolations reflects diversity seldom equaled in southeast Georgia, and never elsewhere in the U.S. These results are consistent with a hypothesis that spiroplasma diversity increases from north (Nova Scotia) to south (Georgia and Costa Rica). The discovery of significant affinity between some spiroplasmas of the southeastern U.S. and the Costa Rican highlands was unexpected, but may reflect a climatically complex Pleistocene history.

Comparative genomics identifies genes shared by distantly related insect-transmitted plant pathogenic mollicutes

Phytoplasmas and spiroplasmas are distantly related insect-transmitted plant pathogens within the class Mollicutes. Genome sequencing projects of phytoplasma strain Aster Yellows-Witches' Broom (AY-WB) and Spiroplasma kunkelii are near completion. Complete genome sequences of seven obligate animal and human pathogenic mollicutes (Mycoplasma and Ureaplasma spp.), and OY phytoplasma have been reported. Putative ORFs predicted from the genome sequences of AY-WB and S. kunkelii were compared to those of the completed genomes. This resulted in identification of at least three ORFs present in AY-WB, OY and S. kunkelii but not in the obligate animal and human pathogenic mollicutes. Moreover, we identified ORFs that seemed more closely related between AY-WB and S. kunkelii than to their mycoplasma counterparts. Phylogenetic analyses using parsimony were employed to study the origin of these genes, resulting in identification of one gene that may have undergone horizontal gene transfer. The possible involvement of these genes in plant pathogenicity is discussed.     

Extensive chromosome aberrations inSpiroplasma citri strain BR3

Genetic variations in the plant pathogen,Spiroplasma citri strain BR3, were characterized through physical genome mapping of the original isolate, BR3-3X, and two derivatives, BR3-T and BR3-G, obtained after several years of different maintenance conditions. BR3-T was transmitted from plant to plant via its natural insect vector, the leafhopperCirculifer tenellus, while BR3-G was maintained only in plants by periodic grafting and has lost its ability to be insect transmitted. By pulsed field gel electrophoresis (PFGE) analysis and DNA hybridization, extensive changes in chromosomal DNA restriction patterns relative to the parent, BR3-3X, were observed in both BR3-T and BR3-G, each of which also had a larger genome size than the parent line. Genetic organization was relatively conserved between BR3-T and BR3-3X. In contrast, a large chromosomal inversion and deletions of approximately 10 kb near each of the inversion borders were observed in BR3-G. One of the deletions, which included several possibly functional genes, was closely linked to a SpV1-related transposase gene. The locations of the deletion borders were also determined. The results of this study demonstrated remarkable genome instability of spiroplasmas.     

Interspecific transmission of endosymbiotic Spiroplasma by mites

The occurrence of closely related strains of maternally transmitted endosymbionts in distantly related insect species indicates that these infections can colonize new host species by lateral transfer, although the mechanisms by which this occurs are unknown. We investigated whether ectoparasitic mites, which feed on insect haemolymph, can serve as interspecific vectors of Spiroplasma poulsonii, a male-killing endosymbiont of Drosophila. Using Spiroplasma-specific primers for PCR, we found that mites can pick up Spiroplasma from infected Drosophila nebulosa females and subsequently transfer the infection to Drosophila willistoni. Some of the progeny of the recipient D. willistoni were infected, indicating successful maternal transmission of the Spiroplasma within the new host species. However, the transmission rate of the infection from recipient flies to their offspring was low, perhaps due to low Spiroplasma density in the recipient flies.     

Wolbachia and other endosymbiont infections in spiders

Maternally inherited endosymbiotic bacteria, such as Wolbachia, Rickettsia and Spiroplasma, have been shown to have wide-ranging effects on the reproduction of their hosts. We present data on the presence of each of these sorts of bacteria in spiders, a group for which there are currently few data, but where such infections could explain many observed reproductive characteristics, such as sex ratio skew. The Wolbachia and Spiroplasma variants that we find in spiders belong to the same clades previously found to infect other arthropods, but many of the rickettsias belong to two, novel, hitherto spider-specific bacterial lineages. We find evidence for coexistence of different bacterial types within species, and in some cases, within individuals. We suggest that spiders present a useful opportunity for studying the effect of these sorts of bacteria on the evolution of host traits, such as those that are under sexual selection.