Publications

3843

An intracellular bacterium with the unique ability to enter mitochondria exists in the European vector of Lyme disease, the hard tick Ixodes ricinus. Previous phylogenetic analyses based on 16S rRNA gene sequences suggested that the bacterium formed a divergent lineage within the Rickettsiales (Alphaproteobacteria). Here, we present additional phylogenetic evidence, based on the gyrB gene sequence, that confirms the phylogenetic position of the bacterium. Based on these data, as well as electron microscopy (EM), in situ hybridization and other observations, we propose the name ‘Candidatus Midichloria mitochondrii’ for this bacterium. The symbiont appears to be ubiquitous in females of I. ricinus across the tick's distribution, while lower prevalence is observed in males (44 %). Based on EM and in situ hybridization studies, the presence of ‘Candidatus M. mitochondrii’ in females appears to be restricted to ovarian cells. The bacterium was found to be localized both in the cytoplasm and in the intermembrane space of the mitochondria of ovarian cells. ‘Candidatus M. mitochondrii’ is the first bacterium to be identified that resides within animal mitochondria.

ABSTRACT To survive in host cells, intracellular pathogens or symbiotic bacteria require protective mechanisms to overcome the oxidative stress generated by phagocytic activities of the host. By genomic library tagging, we cloned a dps (stands for D NA-binding p rotein from s tarved cells) gene of the symbiotic “ Candidatus Legionella jeonii” organism (called the X bacterium) ( dps X ) that grows in Amoeba proteus . The gene encodes a 17-kDa protein (pI 5.19) with 91% homology to Dps and DNA-binding ferritin-like proteins of other organisms. The cloned gene complemented the dps mutant of Escherichia coli and conferred resistance to hydrogen peroxide. Dps X proteins purified from E. coli transformed with the dps X gene were in oligomeric form, formed a complex with pBlueskript SKII DNA, and protected the DNA from DNase I digestion and H 2 O 2 -mediated damage. The expression of the dps X gene in “ Candidatus Legionella jeonii” was enhanced when the host amoeba was treated with 2 mM H 2 O 2 and by phagocytic activities of the host cell. These results suggested that the Dps protein has a function protective of the bacterial DNA and that its gene expression responds to oxidative stress generated by phagocytic activities of the host cell. With regard to the fact that invasion of Legionella sp. into respiratory phagocytic cells causes pneumonia in mammals, further characterization of dps X expression in the Legionella sp. that multiplies in a protozoan host in the natural environment may provide valuable information toward understanding the protective mechanisms of intracellular pathogens.

Two Gram-positive bacilli, designated as strains SMC 4352-1T and SMC 4352-2T, were isolated sequentially from the blood of a newborn child with sepsis. They could not be identified by using conventional clinical microbiological methods. 16S rRNA gene sequencing and phylogenetic analysis revealed that both strains belonged to the genus Bacillus but clearly diverged from known Bacillus species. Strain SMC 4352-1T and strain SMC 4352-2T were found to be closely related to Bacillus firmus NCIMB 9366T (98.2 % sequence similarity) and Bacillus cibi JG-30T (97.1 % sequence similarity), respectively. They also displayed low DNA–DNA reassociation values (less than 40 %) with respect to the most closely related Bacillus species. On the basis of their polyphasic characteristics, strain SMC 4352-1T and strain SMC 4352-2T represent two novel species of the genus Bacillus, for which the names Bacillus infantis sp. nov. (type strain SMC 4352-1T=KCCM 90025T=JCM 13438T) and Bacillus idriensis sp. nov. (type strain SMC 4352-2T=KCCM 90024T=JCM 13437T) are proposed.

Blood transfusions are commonly administered to cats; associated risks include the transmission of various infectious diseases including Mycoplasma haemofelis (Mhf) and ‘ Candidatus Mycoplasma haemominutum’ (Mhm). Blood transfusions in citrate-phosphate-dextrose-adenine (CPDA-1) solution are commonly administered immediately or stored for up to 1 month prior to administration. It is unknown whether Mhf or Mhm survive in this solution or temperature. The purpose of this study was to determine if Mhf or Mhm remain viable after storage in CPDA-1 for varying periods of time. The results provide evidence that transmission of hemoplasmas to naïve cats occurs after administration of infected feline blood that has been stored in CPDA-1 solution for 1 h (Mhf) and 1 week (Mhm). These findings support the recommendation that cats used as blood donors be screened for Mhf and Mhm infections by polymerase chain reaction (PCR) assay prior to use.

Despite differences in their morphologies, comparative analyses of 16S rRNA gene sequences revealed high levels of similarity (>94 %) between strains of the filamentous bacterium ‘Candidatus Nostocoida limicola’ and the cocci Tetrasphaera australiensis and Tetrasphaera japonica and the rod Tetrasphaera elongata, all isolated from activated sludge. These sequence data and their chemotaxonomic characters, including cell wall, menaquinone and lipid compositions and fingerprints of their 16S–23S rRNA intergenic regions, support the proposition that these isolates should be combined into a single genus containing six species, in the family Intrasporangiaceae in the Actinobacteria. This suggestion receives additional support from DNA–DNA hybridization data and when partial sequences of the rpoC1 gene are compared between these strains. Even though few phenotypic characterization data were obtained for these slowly growing isolates, it is proposed, on the basis of the extensive chemotaxonomic and molecular evidence presented here, that ‘Candidatus N. limicola’ strains Ben 17, Ben 18, Ben 67, Ben 68 and Ben 74 all be placed into the species Tetrasphaera jenkinsii sp. nov. (type strain Ben 74T=DSM 17519T=NCIMB 14128T), ‘Candidatus N. limicola’ strain Ben 70 into Tetrasphaera vanveenii sp. nov. (type strain Ben 70T=DSM 17518T=NCIMB 14127T) and ‘Candidatus N. limicola’ strains Ver 1 and Ver 2 into Tetrasphaera veronensis sp. nov. (type strain Ver 1T=DSM 17520T=NCIMB 14129T).

Huanglongbing (greening) disease caused by a nonculturable, phloem-limited bacterium is a severe disease of citrus. On the basis of the influence of temperature on host symptoms and the causal agent, this disease can be categorized as Asian caused by “Candidatus Liberibacter asiaticus”, African caused by “Ca. L. africanus”, and American caused by “Ca. L. americanus”. Kumquat (Fortunella margarita (Lour.) Swingle), a member of the Rutaceae is an economically important crop for export and local consumption in Taiwan. Recently, a Huanglongbing-like disease was found on kumquat in Yilan County, the largest kumquat-producing area in northeastern Taiwan. Even though the disease has been reported on Citrus spp. from Taiwan, it has never been reported on kumquat. Symptoms of infected kumquat were mottling, yellowing, hardening, and curling of leaves followed by premature defoliation, twig dieback, decay of feeder rootlets and lateral roots, and ultimately the death of the entire plant. Typical sieve-tube-restricted bacteria were observed in kumquat cells by electron microscopy (1). In addition, psyllid-transmission tests demonstrated that the Asian psyllid (Diaphorina citri) could transmit this bacterium to healthy kumquats. Positive bud graft transmissions were obtained to F. margarita, F. japonica (Thunb.) Swingle, F. obovata Hort. ex Tanaka, Luchen sweet orange (Citrus sinensis (L.) Osb.), and Wentan pummelo (C. maxima f. sp. butan Hay.). These inoculated plants showed symptoms in 3 to 8 months, and bacteria could be detected by polymerase chain reaction (PCR) using a common primer pair that amplified a 226-bp specific DNA fragment (2). For further molecular identification, the bacterial DNA was extracted from the inoculated plants and PCR was performed by using two sets of primers selected from the 16S rRNA region (GenBank Accession No. L22532) and 16S/23S intergenic spacer region (GenBank Accession No. AB019793). The expected DNA fragments of 1,389 bp and 862 bp were, respectively, amplified from symptomatic plants but not from healthy plants. The PCR products were cloned and sequenced (GenBank Accession Nos. DQ302750 and DQ207841). The 16S rRNA has 98 to 99% identity and 16S/23S intergenic spacer region has 99% identity to the corresponding region of “Ca. L. asiaticus” in GenBank. These molecular analyses confirm the presence of “Ca. L. asiaticus” in kumquat. Since Huanglongbing has been rarely reported naturally on kumquat, further analysis of this bacterium as a special strain of “Ca. L. asiaticus” is needed. References: (1) M. Garnier et al. Ann. Microbiol. 135A:169, 1984. (2) T. H. Hung et al. J. Phytopathol. 147:599, 1999.