Publications

4475

A man with a well-controlled HIV infection, previously diagnosed with lymphogranuloma venereum and treated for Hodgkin’s lymphoma, was suffering from chronic diarrhea. He travelled to Indonesia in the month prior to the start of complaints. Over a 15-month period, sequences related to Campylobactertroglodytis/upsaliensis, C. pinnepediorum/mucosalis/concisus and C. hominis were detected by 16S rRNA qPCR-based assays in various stool samples and in a colon biopsy. Culture revealed the first isolation of “candidatus Campylobacter infans”, a species identified recently by molecular methods only. The patient was treated with azithromycin, ciprofloxacin and tetracycline. To identify potential continuous exposure of the patient to Campylobacter, stool samples of the partner and the cat of the patient were analyzed and C. pinnepediorum/mucosalis/concisus and C. helveticus, respectively, were detected. The diversity in detected species in this immunocompromised patient with a lack of repeatedly consistent findings resulted in the conclusion that not any of the Campylobacter species was the primary cause of the clinical condition. This study shows the challenges in detection and interpretation of diagnostic results regarding Campylobacter.

Citrus Greening, also known as HuangLongbing (HLB), is considered one of the most dangerous citrus diseases, and limiting the production of citrus trees all over the world. Production of antibodies against Ompa protein of Candidatus Liberibacter asiaticus (CLas) for detection of citrus greening disease is considered as promising research direction. In this study, for the purpose of producting antibodies against Ompa of CLas, we firstly used the camel VHH antibody library for screening VHH antibodies against Ompa using phage-display technique. Next, phages which had strong interaction with Ompa as shown in ELISA were selected for phagemid isolation and the DNA fragments encoding VHH antibodies were sequenced. The DNA fragment encoding the best VHH antibody was then selected and inserted into the expression vector pET-21a (+), then cloned in Ecoli DH5α strain and expressed in BL21 (DE3) strain. The expression of VHH antibodies against Ompa was optimized at different temperatures with an inductive concentration of 0.1 M IPTG. Anti-Ompa VHH antibodies were purified under denatured conditions then re-folded. The biological activity of the VHH antibody with Ompa antigen was assessed by indirect-ELISA reaction. Results indicated that the VHH antibody had a very strong interaction with the Ompa antigen. This opens up the prospect of applying VHH antibody in the detection of citrus greening disease.

Here, we demonstrate how microbial storage metabolism can adjust to a wide range of environmental conditions. Such flexibility generates a selective advantage under fluctuating environmental conditions. It can also explain the different observations reported in PAO literature, including the capacity of “ Ca . Accumulibacter phosphatis” to act like glycogen-accumulating organisms (GAOs). These observations stem from slightly different experimental conditions, and controversy arises only when one assumes that metabolism can operate only in a single mode. Furthermore, we also show how the study of metabolic strategies is possible when combining omics data with functional cofactor assays and modeling. Genomic information can only provide the potential of a microorganism. The environmental context and other complementary approaches are still needed to study and predict the functional expression of such metabolic potential.

Grapevine Bois noir (BN) is associated with infection by “Candidatus Phytoplasma solani” (CaPsol). In this study, an array of CaPsol strains was identified from 142 symptomatic grapevines in vineyards of northern, central, and southern Italy and North Macedonia. Molecular typing of the CaPsol strains was carried out by analysis of genes encoding 16S rRNA and translation elongation factor EF-Tu, as well as eight other previously uncharacterized genomic fragments. Strains of tuf-type a and b were found to be differentially distributed in the examined geographic regions in correlation with the prevalence of nettle and bindweed. Two sequence variants were identified in each of the four genomic segments harboring hlyC, cbiQ-glyA, trxA-truB-rsuA, and rplS-tyrS-csdB, respectively. Fifteen CaPsol lineages were identified based on distinct combinations of sequence variations within these genetic loci. Each CaPsol lineage exhibited a unique collective restriction fragment length polymorphism (RFLP) pattern and differed from each other in geographic distribution, probably in relation to the diverse ecological complexity of vineyards and their surroundings. This RFLP-based typing method could be a useful tool for investigating the ecology of CaPsol and the epidemiology of its associated diseases. Phylogenetic analyses highlighted that the sequence variants of the gene hlyC, which encodes a hemolysin III-like protein, separated into two clusters consistent with the separation of two distinct lineages on the basis of tufB gene sequences. Alignments of deduced full protein sequences of elongation factor-Tu (tufB gene) and hemolysin III-like protein (hlyC gene) revealed the presence of critical amino acid substitutions distinguishing CaPsol strains of tuf-type a and b. Findings from the present study provide new insights into the genetic diversity and ecology of CaPsol populations in vineyards.

AbstractTaxonomy is the science of defining and naming groups of biological organisms based on shared characteristics and, more recently, on evolutionary relationships. With the birth of novel genomics/bioinformatics techniques and the increasing interest in microbiome studies, a further advance of taxonomic discipline appears not only possible but highly desirable. The present work proposes a new approach to modern taxonomy, consisting in the inclusion of novel descriptors in the organism characterization: (1) the presence of associated microorganisms (e.g.: symbionts, microbiome), (2) the mitochondrial genome of the host, (3) the symbiont genome. This approach aims to provide a deeper comprehension of the evolutionary/ecological dimensions of organisms since their very first description. Particularly interesting, are those complexes formed by the host plus associated microorganisms, that in the present study we refer to as “holobionts”. We illustrate this approach through the description of the ciliateEuplotes vanleeuwenhoekisp. nov. and its bacterial endosymbiont “CandidatusPinguicoccus supinus” gen. nov., sp. nov. The endosymbiont possesses an extremely reduced genome (~ 163 kbp); intriguingly, this suggests a high integration between host and symbiont.

AbstractCandidatus Izimaplasma, an intermediate in the reductive evolution from Firmicutes to Mollicutes, was proposed to represent a novel class of free-living wall-less bacteria within the phylum Tenericutes found in deep-sea methane seeps. Unfortunately, the paucity of marine isolates currently available has limited further insights into their physiological and metabolic features as well as ecological roles. Here, we present a detailed description of the phenotypic traits, genomic data and central metabolisms tested in both laboratorial and deep-sea environments of the novel strain zrk13, which allows for the first time the reconstruction of the metabolic potential and lifestyle of a member of the tentatively defined Candidatus Izimaplasma. On the basis of the description of strain zrk13, the novel species and genus Xianfuyuplasma coldseepsis is proposed. Notably, DNA degradation driven by X. coldseepsis zrk13 was detected in both laboratorial and in situ conditions, strongly indicating it is indeed a key DNA degrader. Moreover, the putative genes determining degradation broadly distribute in the genomes of other Izimaplasma members. Given extracellular DNA is a particularly crucial phosphorus as well as nitrogen and carbon source for microorganisms in the seafloor, Izimaplasma bacteria are thought to be important contributors to the biogeochemical cycling in the deep ocean.

AbstractA major bottleneck in identifying therapies to control citrus greening and other devastating plant diseases caused by fastidious pathogens is our inability to culture the pathogens in defined media or axenic cultures. As such, conventional approaches for antimicrobial evaluation (genetic or chemical) rely on time-consuming, low-throughput and inherently variable whole-plant assays. Here, we report that plant hairy roots support the growth of fastidious pathogens likeCandidatusLiberibacter spp., the presumptive causal agents of citrus greening, potato zebra chip and tomato vein greening diseases. Importantly, we leverage the microbial hairy roots for rapid, reproducible efficacy screening of multiple therapies. We identify six antimicrobial peptides, two plant immune regulators and eight chemicals which inhibitCandidatusLiberibacter spp. in plant tissues. The antimicrobials, either singly or in combination, can be used as near- and long-term therapies to control citrus greening, potato zebra chip and tomato vein greening diseases.

The recently described superphylum DPANN includes several phyla of uncultivated archaea with small cell sizes, reduced genomes, and limited metabolic capabilities. One of these phyla, “ Ca . Micrarchaeota,” comprises an enigmatic group of archaea found in acid mine drainage environments, the archaeal Richmond Mine acidophilic nanoorganisms (ARMAN) group. Analysis of their reduced genomes revealed the absence of key metabolic pathways consistent with their partner-associated lifestyle, and physical associations of ARMAN cells with their hosts were documented. However, “ Ca . Micrarchaeota” include several lineages besides the ARMAN group found in nonacidic environments, and none of them have been characterized. Here, we report a complete genome of “ Ca . Micrarchaeota” from a non-ARMAN lineage. Analysis of this genome revealed the presence of metabolic capacities lost in ARMAN genomes that could enable a free-living lifestyle. These results expand our understanding of genetic diversity, lifestyle, and evolution of “ Ca . Micrarchaeota.”