Microbiology

Summary Previously uncultured nitrite‐oxidizing bacteria affiliated to the genus Nitrospira have for the first time been successfully enriched from activated sludge from a municipal wastewater treatment plant. During the enrichment procedure, the abundance of the Nitrospira ‐like bacteria increased to approximately 86% of the total bacterial population. This high degree of purification was achieved by a novel enrichment protocol, which exploits physiological features of Nitrospira ‐like bacteria and includes the selective repression of coexisting Nitrobacter cells and heterotrophic contaminants by application of ampicillin in a final concentration of 50 µg ml −1 . The enrichment process was monitored by electron microscopy, fluorescence in situ hybridization (FISH) with rRNA‐targeted probes and fatty acid profiling. Phylogenetic analysis of 16S rRNA gene sequences revealed that the enriched bacteria represent a novel Nitrospira species closely related to uncultured Nitrospira ‐like bacteria previously found in wastewater treatment plants and nitrifying bioreactors. The enriched strain is provisionally classified as ‘ Candidatus Nitrospira defluvii’.

A moderately thermophilic, anaerobic bacterium (strain SB91T) was isolated from a freshwater hot spring at Barguzin Valley, Buryatiya, Russia. Cells of strain SB91Twere straight to slightly curved rods, 0·5–0·6 μm in diameter and 3·0–7·0 μm in length. Formation of endospores was not observed. The temperature range for growth was 26–62 °C, with an optimum at 50 °C. The pH range for growth was 5·5–9·5, with an optimum at pH 7·5–8·0. The substrates utilized by strain SB91Tin the presence of 9,10-anthraquinone 2,6-disulfonate included peptone, tryptone, Casamino acids, yeast extract, beef extract, casein hydrolysate, alanine plus glycine, alanine plus proline,l-valine and n-propanol. Carbohydrates were not utilized. Strain SB91Treduced amorphous Fe(III) oxide, Fe(III) citrate, Fe(III) EDTA or Fe(III) nitrilotriacetate with peptone,l-valine or n-propanol as an electron donor. Strain SB91Treduced 9,10-anthraquinone 2,6-disulfonate, thiosulfate, elemental sulfur, fumarate and selenite. Strain SB91Tsurvived after exposure to gamma-radiation at a dose of 5·4 kGy. The G+C content of the DNA of strain SB91Twas 33 mol%. Analysis of the 16S rRNA gene sequence revealed that the isolated organism belonged to cluster XII of the clostridia. On the basis of its physiological properties and the results of phylogenetic analyses, it is proposed that strain SB91Trepresents the sole species of a novel genus,Tepidimicrobium; the nameTepidimicrobium ferriphilumgen. nov., sp. nov. is proposed, with strain SB91T(=DSM 16624T=VKM B-2348T) as the type strain.

During 2003, surveys of sugarcane yellow leaf disease and papaya bunchy top-like disease were carried out on plantations in Havana province, Cuba, to determine the roles of weeds and Auchenorrhyncha insects in the epidemiology of these diseases. More than 250 plant and insect samples were collected and indexed by using a nested PCR for phytoplasma 16S rDNA with the generic primer pairs P1/P7 and R16F2n/R16R2. The PCR products were further characterized by restriction fragment length polymorphism using HaeIII, AluI, Sau3AI, Tru9I, HhaI, HpaII and TaqI endonucleases, giving patterns that distinguished them from those of the other reference phytoplasmas analysed. Phylogenetic analysis of 16S rRNA gene sequences identified the phytoplasmas present in sugarcane (Saccharum officinarum L.), Cynodon dactylon L., Conyza canadensis L. Cronq., Sorghum halepense L. Pers., Macroptilium lathyroides L. Urb., Saccharosydne saccharivora (Westwood) and Cedusa spp., and those in papaya (Carica papaya L.) and Empoasca papayae, as two novel provisional phytoplasma species. We propose that these phytoplasmas should be given Candidatus status, as ‘Candidatus Phytoplasma graminis' and ‘Candidatus Phytoplasma caricae’, respectively.

The obligately intracellular coccoid bacterium UWE25, a symbiont of Acanthamoeba spp., was previously identified as being related to chlamydiae based upon the presence of a chlamydia-like developmental cycle and its 16S rRNA gene sequence. Analysis of its complete genome sequence demonstrated that UWE25 shows many characteristic features of chlamydiae, including dependency on host-derived metabolites, composition of the cell envelope and the ability to thrive as an energy parasite within the cells of its eukaryotic host. Phylogenetic analysis of 44 ribosomal proteins further confirmed the affiliation of UWE25 to the ‘Chlamydiae’. Within this phylum, UWE25 could be assigned to the family Parachlamydiaceae based on comparative analyses of the 16S rRNA, 23S rRNA and endoribonuclease P RNA genes. The distinct dissimilarities from its closest relative, Parachlamydia acanthamoebae Bn9 T (7·1, 9·7 and 28·8 %, respectively), observed in this analysis justify its classification in a new genus. Therefore, the name ‘Candidatus Protochlamydia amoebophila’ is proposed for the designation of the Acanthamoeba sp. symbiont UWE25 (=ATCC PRA-7).

Symptoms of huanglongbing (HLB) were reported in São Paulo State (SPS), Brazil, in March 2004. In Asia, HLB is caused by ‘Candidatus Liberibacter asiaticus' and in Africa by ‘Candidatus Liberibacter africanus’. Detection of the liberibacters is based on PCR amplification of their 16S rRNA gene with specific primers. Leaves with blotchy mottle symptoms characteristic of HLB were sampled in several farms of SPS and tested for the presence of liberibacters. ‘Ca. L. asiaticus' was detected in a small number of samples but most samples gave negative PCR results. Therefore, a new HLB pathogen was suspected. Evidence for an SPS-HLB bacterium in symptomatic leaves was obtained by PCR amplification with universal primers for prokaryotic 16S rRNA gene sequences. The amplified 16S rRNA gene was cloned and sequenced. Sequence analysis and phylogeny studies showed that the 16S rRNA gene possessed the oligonucleotide signatures and the secondary loop structure characteristic of the α-Proteobacteria, including the liberibacters. The 16S rRNA gene sequence phylogenetic tree showed that the SPS-HLB bacterium clustered within the α-Proteobacteria, the liberibacters being its closest relatives. For these reasons, the SPS-HLB bacterium is considered a member of the genus ‘Ca. Liberibacter’. However, while the 16S rRNA gene sequences of ‘Ca. L. asiaticus' and ‘Ca. L. africanus' had 98·4 % similarity, the 16S rRNA gene sequence of the SPS-HLB liberibacter had only 96·0 % similarity with the 16S rRNA gene sequences of ‘Ca. L. asiaticus' or ‘Ca. L. africanus’. This lower similarity was reflected in the phylogenetic tree, where the SPS-HLB liberibacter did not cluster within the ‘Ca. L asiaticus’/‘Ca. L. africanus group’, but as a separate branch. Within the genus ‘Candidatus Liberibacter’ and for a given species, the 16S/23S intergenic region does not vary greatly. The intergenic regions of three strains of ‘Ca. L. asiaticus’, from India, the People's Republic of China and Japan, were found to have identical or almost identical sequences. In contrast, the intergenic regions of the SPS-HLB liberibacter, ‘Ca. L. asiaticus' and ‘Ca. L. africanus' had quite different sequences, with similarity between 66·0 and 79·5 %. These results confirm that the SPS-HLB liberibacter is a novel species for which the name ‘Candidatus Liberibacter americanus' is proposed. Like the African and the Asian liberibacters, the ‘American’ liberibacter is restricted to the sieve tubes of the citrus host. The liberibacter could also be detected by PCR amplification of the 16S rRNA gene in Diaphorina citri, the psyllid vector of ‘Ca. L. asiaticus’, suggesting that this psyllid is also a vector of ‘Ca. L. americanus' in SPS. ‘Ca. L. americanus' was detected in 216 of 218 symptomatic leaf samples from 47 farms in 35 municipalities, while ‘Ca. L. asiaticus' was detected in only 4 of the 218 samples, indicating that ‘Ca. L. americanus' is the major cause of HLB in SPS.