Publications

3843

“Candidatus Berkiella cookevillensis” (strain CC99) and “Candidatus Berkiella aquae” (strain HT99), belonging to the Coxiellaceae family, are gram-negative bacteria isolated from amoebae in biofilms present in human-constructed water systems. Both bacteria are obligately intracellular, requiring host cells for growth and replication. The intracellular bacteria-containing vacuoles of both bacteria closely associate with or enter the nuclei of their host cells. In this study, we analyzed the genome sequences of CC99 and HT99 to better understand their biology and intracellular lifestyles. The CC99 genome has a size of 2.9Mb (37.9% GC) and contains 2,651 protein-encoding genes (PEGs) while the HT99 genome has a size of 3.6Mb (39.4% GC) and contains 3,238 PEGs. Both bacteria encode high proportions of hypothetical proteins (CC99: 46.5%; HT99: 51.3%). The central metabolic pathways of both bacteria appear largely intact. Genes for enzymes involved in the glycolytic pathway, the non-oxidative branch of the phosphate pathway, the tricarboxylic acid pathway, and the respiratory chain were present. Both bacteria, however, are missing genes for the synthesis of several amino acids, suggesting reliance on their host for amino acids and intermediates. Genes for type I and type IV (dot/icm) secretion systems as well as type IV pili were identified in both bacteria. Moreover, both bacteria contain genes encoding large numbers of putative effector proteins, including several with eukaryotic-like domains such as, ankyrin repeats, tetratricopeptide repeats, and leucine-rich repeats, characteristic of other intracellular bacteria.

IntroductionHuanglonbing (HLB) is the most serious disease of citrus in the world, associated with three non-cultivable phloem-restricted bacteria Candidatus Liberibacter asiaticus (CLas), Ca L. africanus (CLaf) and Ca L. americanus (CLam). CLas is transmitted by the Asian citrus psyllid Diaphorina citri, and has spread to several countries. The African psyllid Trioza erytreae, the vector of CLaf occurs in Africa and neighbouring islands. Only two major citrus-growing regions - Australia/New Zealand and the Mediterranean Basin - are still HLB-free in the world. However, T. erytreae has recently been introduced into continental Europe (Portugal and Spain) and has become a potential threat to citrus production. The transmission of CLas by T. erytreae had been postulated but never tested. To evaluate the risk of T. erytreae transmitting CLas, comparative transmissions of CLas by T. erytreae and D. citri were assessed.MethodsTransmission tests were performed on excised leaves and seedlings of Citrus volkameriana with different inoculation access periods (in series) for both insect species. Quantifications of bacterial titers were made in excised leaves, seedlings three and six months after inoculation and on individual insects.ResultsOur results showed that T. erytreae was able to efficiently acquire CLas. Furthermore, T. erytreae carried significantly higher bacterial titers than D. citri, and was able to efficiently transmit the bacteria to seedlings at a similar rate that D. citri highlighting the high risk of spread of the most aggressive variant of HLB (CLas) by T. erytreae in Europe.DiscussionThus, extreme precautions to prevent any entry of CLas into Europe should be adopted.

The hypervariable genomic regions derived from 35 published C Las genomes were used to decipher the genetic diversity of C Las strains and identify 10 new strains with high variations in prophage regions. Characterizing these variations in the C Las bacteria might provide insight into their evolution and adaptation to host plants and insects in China.