Publications

4366

The anaerobic oxidation of fatty acids and alcohols occurs near the thermodynamic limit of life. This process is driven by syntrophic bacteria that oxidize fatty acids and/or alcohols, their syntrophic partners that consume the products of this oxidation, and the pathways for interspecies electron exchange via these products or direct interspecies electron transfer (DIET). Due to the interdependence of syntrophic microorganisms on each other’s metabolic activity, their isolation in pure cultures is almost impossible. Thus, little is known about their physiology, and the only available way to fill in the knowledge gap on these organisms is genomic and metabolic analysis of syntrophic cultures. Here we report the results of genome sequencing and analysis of an obligately syntrophic alkaliphilic bacterium ‘Candidatus Contubernalis alkaliaceticus’. The genomic data suggest that acetate oxidation is carried out by the Wood–Ljungdahl pathway, while a bimodular respiratory system involving an Rnf complex and a Na+-dependent ATP synthase is used for energy conservation. The predicted genomic ability of ‘Ca. C. alkaliaceticus’ to outperform interspecies electron transfer both indirectly, via H2 or formate, and directly, via pili-like appendages of its syntrophic partner or conductive mineral particles, was experimentally demonstrated. This is the first indication of DIET in the class Dethiobacteria.

Candidatus Accumulibacter belongs to phosphate-accumulating organisms (PAOs) which exhibit a cyclic metabolism and are capable of intracellular polyphosphate accumulation and their hydrolysis under feast-famine anaerobic-aerobic cycling. In consortia of activated sludge microorganisms, these bacteria are responsible for enhanced biological phosphorus removal (EBPR). The spectrum of the substrates used by Ca. Accumulibacter remains insufficiently studied. It was investigated by measuring the oxygen uptake rates (OUR) of Ca. Accumulibacter-enriched culture supplemented with 17 different organic substrates. The highest oxygen uptake rate values were observed in the presence of tryptone, volatile fatty acids (acetate, propionate, and butyrate), succinate, pyruvate, and amino acids (aspartate and glutamate). Phosphate dynamics in the medium under shifts from anaerobic to aerobic cultivation in batch experiments were studied for these compounds (except for tryptone). All tested substrates were shown to cause phosphate cycling (release in the anaerobic phase and uptake in the aerobic one), with OURs for the substrates correlating with the number of phosphates consumed during the aerobic phase. It was concluded that OUR may be used as an indicator of the monosubstrates used by Ca. Accumulibacter in the anaerobic/aerobic cycle. The possible pathways for substrate transport and metabolism by Ca. Accumulibacter are discussed using stoichiometric data and the results of metagenomic analysis.

ABSTRACT Autophagy plays an important role against pathogen infection in many organisms; however, little has been done with regard to vector-borne plant and animal pathogens, that sometimes replicate and cause deleterious effects in their vectors. Candidatus Liberibacter solanacearum (CLso) is a fastidious gram-negative phloem-restricted plant pathogen and vectored by the carrot psyllid, Bactericera trigonica . The plant disease caused by this bacterium is called carrot yellows and has recently gained much importance due to worldwide excessive economical losses. Here, we demonstrate that calcium ATPase, cytosolic calcium, and most importantly Beclin-1 have a role in regulating autophagy and its association with Liberibacter inside the psyllid. The presence of CLso generates reactive oxygen species and induces the expression of detoxification enzymes in the psyllid midguts, a main site for bacteria transmission. CLso also induces the expression of both sarco/endoplasmic reticulum Ca2+pump (SERCA) and 1,4,5-trisphosphate receptors (ITPR) in midguts, resulting in high levels of calcium in the cellular cytosol. Silencing these genes individually disrupted the calcium levels in the cytosol and resulted in direct effects on autophagy and subsequently on Liberibacter persistence and transmission. Inhibiting Beclin1-phosphorylation through different calcium-induced kinases altered the expression of autophagy and CLso titers and persistence. Based on our results obtained from the midgut, we suggest the existence of a direct correlation between cytosolic calcium levels, autophagy, and CLso persistence and transmission by the carrot psyllid. IMPORTANCE Plant diseases caused by vector-borne Liberibacter species are responsible for the most important economic losses in many agricultural sectors. Preventing these diseases relies mostly on chemical sprays against the insect vectors. Knowledge-based interference with the bacteria-vector interaction remains a promising approach as a sustainable solution. For unravelling how Liberibacter exploits molecular pathways in its insect vector for transmission, here, we show that the bacterium manipulates calcium levels on both sides of the endoplasmic reticulum membrane, resulting in manipulating autophagy. Silencing genes associated with these pathways disrupted the calcium levels in the cytosol and resulted in direct effects on autophagy and Liberibacter transmission. These results demonstrate major pathways that could be exploited for manipulating and controlling the disease transmission.

ABSTRACT Bacterial cells can vary greatly in size, from a few hundred nanometers to hundreds of micrometers in diameter. Filamentous cable bacteria also display substantial size differences, with filament diameters ranging from 0.4 to 8 µm. We analyzed the genomes of cable bacterium filaments from 11 coastal environments of which the resulting 23 new genomes represent 10 novel species-level clades of Candidatus Electrothrix and two clades that putatively represent novel genus-level diversity. Fluorescence in situ hybridization with a species-level probe showed that large-sized cable bacteria belong to a novel species with the proposed name Ca . Electrothrix gigas. Comparative genome analysis suggests genes that play a role in the construction or functioning of large cable bacteria cells: the genomes of Ca . Electrothrix gigas encode a novel actin-like protein as well as a species-specific gene cluster encoding four putative pilin proteins and a putative type II secretion platform protein, which are not present in other cable bacteria. The novel actin-like protein was also found in a number of other giant bacteria, suggesting there could be a genetic basis for large cell size. This actin-like protein (denoted big bacteria protein, Bbp) may have a function analogous to other actin proteins in cell structure or intracellular transport. We contend that Bbp may help overcome the challenges of diffusion limitation and/or morphological complexity presented by the large cells of Ca . Electrothrix gigas and other giant bacteria. IMPORTANCE In this study, we substantially expand the known diversity of marine cable bacteria and describe cable bacteria with a large diameter as a novel species with the proposed name Candidatus Electrothrix gigas. In the genomes of this species, we identified a gene that encodes a novel actin-like protein [denoted big bacteria protein (Bbp)]. The bbp gene was also found in a number of other giant bacteria, predominantly affiliated to Desulfobacterota and Gammaproteobacteria, indicating that there may be a genetic basis for large cell size. Thus far, mostly structural adaptations of giant bacteria, vacuoles, and other inclusions or organelles have been observed, which are employed to overcome nutrient diffusion limitation in their environment. In analogy to other actin proteins, Bbp could fulfill a structural role in the cell or potentially facilitate intracellular transport.

AbstractPhotosynthesis is a fundamental biogeochemical process, thought to be restricted to a few bacterial and eukaryotic phyla. However, understanding the origin and evolution of phototrophic organisms can be impeded and biased by the difficulties of cultivation. Here, we analyzed metagenomic datasets and found potential photosynthetic abilities encoded in the genomes of uncultivated bacteria within the phylum Myxococcota. A putative photosynthesis gene cluster encoding a type-II reaction center appears in at least six Myxococcota families from three classes, suggesting vertical inheritance of these genes from an early common ancestor, with multiple independent losses in other lineages. Analysis of metatranscriptomic datasets indicate that the putative myxococcotal photosynthesis genes are actively expressed in various natural environments. Furthermore, heterologous expression of myxococcotal pigment biosynthesis genes in a purple bacterium supports that the genes can drive photosynthetic processes. Given that predatory abilities are thought to be widespread across Myxococcota, our results suggest the intriguing possibility of a chimeric lifestyle (combining predatory and photosynthetic abilities) in members of this phylum.

Highly purified cultures of alkaliphilic aceticlastic methanogens were collected for the first time using methanogenic enrichments with acetate from a soda lake and a terrestrial mud volcano. The cells of two strains were non-motile rods forming filaments. The mud volcano strain M04Ac was alkalitolerant, with the pH range for growth from 7.5 to 10.0 (optimum at 9.0), while the soda lake strain Mx was an obligate alkaliphile growing in the pH range 7.7–10.2 (optimum 9.3–9.5) in the presence of optimally 0.2–0.3 M total Na+. Genomes of both strains encoded all enzymes required for aceticlastic methanogenesis and different mechanisms of (halo)alkaline adaptations, including ectoine biosynthesis, which is the first evidence for the formation of this osmoprotectant in archaea. According to 16S rRNA gene phylogeny, the strains possessed 98.3–98.9% sequence identity and belonged to the obligately aceticlastic genus Methanothrix with M. harundinaceae as the most closely related species. However, a more advanced phylogenomic reconstruction based on 122 conserved single-copy archaeal protein-coding marker genes clearly indicated a polyphyletic origin of the species included in the genus Methanothrix. We propose to reclassify Methanothrix harrundinacea (type strain 8AcT) into a new genus, Methanocrinis gen. nov., with the type species Methanocrinis harrundinaceus comb. nov. We also propose under SeqCode the complete genome sequences of strain MxTs (GCA_029167045.1) and strain M04AcTs (GCA_029167205.1) as nomenclatural types of Methanocrinis natronophilus sp. nov. and Methanocrinis alkalitolerans sp. nov., respectively, which represent other species of the novel genus. This work demonstrates that the low energy aceticlastic methanogenesis may function at extreme conditions present in (halo)alkaline habitats.

AbstractHuanglongbing (HLB, or citrus greening disease) affects all citrus varieties world-wide. In the USA, Asia, and South America the causal agent is “CandidatusLiberibacter asiaticus” (CLas), a phloem-limited, uncultured, alphaproteobacterium. The hemipteran insect vector,Diaphorina citri(Asian citrus psyllid) acquires and transmitsCLas in a circulative, propagative manner. In addition toCLas,D. citrihosts multiple symbiotic bacterial species includingWolbachia(wDi). InD. citri, wDi has been sequenced and studied but specific roles inD. citribiology are unknown. Using well established quantitative PCR methods we measuredCLas titer inD. citricollected from four groves in central Florida with distinct HLB management strategies and tested whetherCLas and wDi titer were correlated in a sub-set of these insects. Grove site had the largest effect onCLas titer. Sex had no effect onCLas titer, while higher wDi titer was correlated with non-infected insects. Our results suggest that more directed follow-up research is necessary and important to clarify whether field management tactics influenceCLas titer inD. citriand to better understand gene-by-environment interactions amongD. citri, wDi andCLas. Now that millions of trees in Florida have been treated with injectable formulations of oxytetracycline, which is likely to decrease bacterial populations inD. citri, this study may represent the last biologically meaningful snapshot of grove-level vector-pathogen ecology in the state during the HLB epidemic.