Publications

4526

Abstract The understanding and manipulation of microbial communities toward the conversion of lignocellulose and plastics are topics of interest in microbial ecology and biotechnology. In this study, the polymer-degrading capability of a minimal lignocellulolytic microbial consortium (MELMC) was explored by genome-resolved metagenomics. The MELMC was mostly composed (>90%) of three bacterial members (Pseudomonas protegens; Pristimantibacillus lignocellulolyticus gen. nov., sp. nov; and Ochrobactrum gambitense sp. nov) recognized by their high-quality metagenome-assembled genomes (MAGs). Functional annotation of these MAGs revealed that Pr. lignocellulolyticus could be involved in cellulose and xylan deconstruction, whereas Ps. protegens could catabolize lignin-derived chemical compounds. The capacity of the MELMC to transform synthetic plastics was assessed by two strategies: (i) annotation of MAGs against databases containing plastic-transforming enzymes; and (ii) predicting enzymatic activity based on chemical structural similarities between lignin- and plastics-derived chemical compounds, using Simplified Molecular-Input Line-Entry System and Tanimoto coefficients. Enzymes involved in the depolymerization of polyurethane and polybutylene adipate terephthalate were found to be encoded by Ps. protegens, which could catabolize phthalates and terephthalic acid. The axenic culture of Ps. protegens grew on polyhydroxyalkanoate (PHA) nanoparticles and might be a suitable species for the industrial production of PHAs in the context of lignin and plastic upcycling.

Abstract Objective ‘Candidatus Liberibacter asiaticus’ (CLas) is associated with the devastating citrus ‘greening’ disease. All attempts to achieve axenic growth and complete Koch’s postulates with CLas have failed to date, at best yielding complex cocultures with very low CLas titers detectable only by PCR. Reductive genome evolution has rendered all pathogenic ‘Ca. Liberibacter’ spp. deficient in multiple key biosynthetic, metabolic and structural pathways that are highly unlikely to be rescued in vitro by media supplementation alone. By contrast, Liberibacter crescens (Lcr) is axenically cultured and its genome is both syntenic and highly similar to CLas. Our objective is to achieve replicative axenic growth of CLas via addition of missing culturability-related Lcr genes. Results Bioinformatic analyses identified 405 unique ORFs in Lcr but missing (or truncated) in all 24 sequenced CLas strains. Site-directed mutagenesis confirmed and extended published EZ-Tn5 mutagenesis data, allowing elimination of 310 of these 405 genes as nonessential, leaving 95 experimentally validated Lcr genes as essential for CLas growth in axenic culture. Experimental conditions for conjugation of large GFP-expressing plasmids from Escherichia coli to Lcr were successfully established for the first time, providing a practical method for transfer of large groups of ‘essential’ Lcr genes to CLas.

AbstractThe endosymbiotic Wolbachia is one of the most common intracellular bacteria known in arthropods and nematodes. Its ability for reproductive manipulation can cause unequal inheritance to male and female offspring, allowing the manipulator to spread, but potentially also impact the evolutionary dynamics of infected hosts. Estimated to be present in up to 66% of insect species, little is known about the phenotypic impact of Wolbachia within the order Coleoptera. Here, we describe the reproductive manipulation by the Wolbachia strain wSur harboured by the sawtoothed grain beetle Oryzaephilus surinamensis (Coleoptera, Silvanidae), through a combination of genomics approaches and bioassays. The Wolbachia strain wSur belongs to supergroup B that contains well‐described reproductive manipulators of insects and encodes a pair of cytoplasmic incompatibility factor (cif) genes, as well as multiple homologues of the WO‐mediated killing (wmk) gene. A phylogenetic comparison with wmk homologues of wMel of Drosophila melanogaster identified 18 wmk copies in wSur, including one that is closely related to the wMel male‐killing homologue. However, further analysis of this particular wmk gene revealed an eight‐nucleotide deletion leading to a stop‐codon and subsequent reading frame shift midsequence, probably rendering it nonfunctional. Concordantly, utilizing a Wolbachia‐deprived O. surinamensis population and controlled mating pairs of wSur‐infected and noninfected partners, we found no experimental evidence for male‐killing. However, a significant ~50% reduction of hatching rates in hybrid crosses of uninfected females with infected males indicates that wSur is causing cytoplasmic incompatibility. Thus, Wolbachia also represents an important determinant of host fitness in Coleoptera.

Abstract Background The appearance of the novel porcine haemotrophic mycoplasma (HM) species ‘Candidatus Mycoplasma haemosuis’ was reported in apparently healthy but also in clinically sick animals in China, Korea and in a case report from Germany. Outside of Asia, however, nothing further is known about the frequency of ‘Ca. M. haemosuis’ in pigs to date. To investigate the distribution of this novel HM species in Germany, fattening pigs, sows and pre-suckling piglets were examined using a herein developed quantitative real-time PCR assay (qPCR). Because the piglets were sampled before the first colostrum uptake, additional information on a possible vertical transmission from dams to their offspring was obtained. Results Our novel qPCR assay successfully detected ‘Ca. M. haemosuis’ in all blood samples from the ‘Ca. M. haemosuis’-infected pigs. No cross-reactivity was detected when DNA from non-target Mycoplasma spp. and other bacterial species representing 105 bacteria/reaction were used as a template. The lower limit of detection of the qPCR was thus 10 gap gene copies per reaction and 2.5 x 103 genome equivalents (GE) per mL blood. ‘Candidatus M. haemosuis’ was detected by this qPCR in blood samples from a total out of 6.25% sows (13/208), 4.50% pre-suckling piglets (28/622) and 17.50% fattening pigs (35/200). On farm level, 3 out of 21 piglet producing farms (14.28%) and 9 out of 20 fattening farms (45.00%) were positive for ‘Ca. M. haemosuis’. Co-infections with M. suis were evident in all age groups. Conclusion ‘Candidatus M. haemosuis’ infection is present in German pig farms and the detection of the novel porcine HM species in piglets immediately after birth before colostrum intake indicates vertical transmission. The novel qPCR assay specific for ‘Ca. M. haemosuis’ described herein will be a prerequisite for future studies on the prevalence, epidemiology as well as the clinical and economic impact of ‘Ca. M. haemosuis’ infections.

The Anaplasmataceae family encompasses obligate intracellular α-proteobacteria of human and veterinary medicine importance. This study performed multi-locus sequencing to characterize Ehrlichia and Anaplasma in coati’s blood samples in Midwestern Brazil. Twenty-five samples (25/165—15.1%) were positive in the screening PCR based on the dsb gene of Ehrlichia spp. and were characterized using 16S rRNA, sodB, groEL, and gltA genes and the 23S-5S intergenic space region (ITS). Phylogenetic analyses based on all six molecular markers positioned the sequences into a new clade, with a common origin of Ehrlichia ruminantium. Haplotype analyses of 16S RNA sequences revealed the presence of two distinct Ehrlichia genotypes. Six samples (6/165, 3.6%) were positive in the screening nPCR for the 16S rRNA gene of Anaplasma spp. and were submitted to an additional PCR targeting the ITS for molecular characterization. Phylogenetic analyses based on both 16S rRNA gene and ITS positioned the Anaplasma sp. detected in the present study in a large clade with other Anaplasma sp. previously detected in ticks and wild animals and in a clade with ‘Candidatus Anaplasma brasiliensis’, respectively. Based on distinct molecular markers, the present work described a putative novel Anaplasmataceae agent, namely ‘Candidatus Ehrlichia dumleri’, and Anaplasma sp. closely related to the previously described ‘Candidatus Anaplasma brasiliensis’.

AbstractEleven haplotypes of the bacterium, ‘Candidatus Liberibacter solanacearum’, have been identified worldwide, several of which infect important agricultural crops. In the United States, haplotypes A and B are associated with yield and quality losses in potato, tomato, and other crops of the Solanaceae. Both haplotypes are vectored by potato psyllid, Bactericera cockerelli. Recently, a third haplotype, designated F, was identified in southern Oregon potato fields. To identify the vector of this haplotype, psyllids of multiple species were collected from yellow sticky cards placed near potato fields during two growing seasons. Over 2700 specimens were tested for ‘Ca. L. solanacearum’ by polymerase chain reaction. Forty-seven psyllids harbored the bacterium. The infected specimens comprised four psyllid species in two families, Aphalaridae and Triozidae (Hemiptera: Psylloidea). Nucleic acid and/or amino acid sequence analysis of the ‘Ca. L. solanacearum’ 16S ribosomal RNA, 50S ribosomal proteins L10/L12, and outer membrane protein identified three new haplotypes of the bacterium, designated as Aph1, Aph2 and Aph3, including two variants of Aph2 (Aph2a and Aph2b). The impact of these new haplotypes on solanaceous or other crops is not known. The vector of ‘Ca. L. solanacearum’ haplotype F was not detected in this study.