Six patients from northern Minnesota and Wisconsin with a febrile illness accompanied by granulocytic cytoplasmic morulae suggestive of ehrlichial infection were identified. Two patients died, and splenic granulocytes of one patient contained cytoplasmic vacuoles with organisms ultrastructurally characteristic of ehrlichiae. From one patient, a 1.5-kb DNA product was amplified by PCR with universal eubacterial primers of 16S rDNA. Analysis of the nucleotide sequence of the amplified product revealed 99.9 and 99.8% similarities with E. phagocytophila and E. equi, respectively, neither of which has previously been known to infect humans. From the variable regions of the determined sequence, a forward primer specific for three organisms (human granulocytic ehrlichia, E. phagocytophila, and E. equi) and a reverse primer for these ehrlichiae and E. platys were designed. By nested PCR with amplification by the universal primers and then reamplification with the specific primers described above, the expected 919-bp product was generated from the blood of the index patient and three additional patients. Blood from these four patients and two more patients with granulocytic morulae contained DNA which was amplified by nested PCR involving a combination of a universal primer and the human granulocytic ehrlichia-E. phagocytophila-E. equi-E. platys group-specific primer. This apparently vector-borne human granulocytic ehrlichia has only 92.5% 16S rDNA homology with E. chaffeensis. Nested PCR with group-specific primers did not amplify E. chaffeensis DNA, and E. chalfeensis-specific primers did not amplify DNAs of the human granulocytic ehrlichia. Thus, six patients were shown to be infected by an Ehrlichia species never previously reported to infect humans.

The study of electrically conductive protein nanowires in Geobacter sulfurreducens has led to new concepts for long-range extracellular electron transport, as well as for the development of sustainable conductive materials and electronic devices with novel functions. Until recently, electrically conductive pili (e-pili), assembled from the PilA pilin monomer, were the only known Geobacter protein nanowires. However, filaments comprised of the multi-heme c-type cytochrome, OmcS, are present in some preparations of G. sulfurreducens outer-surface proteins. The purpose of this review is to evaluate the available evidence on the in vivo expression of e-pili and OmcS filaments and their biological function. Abundant literature demonstrates that G. sulfurreducens expresses e-pili, which are required for long-range electron transport to Fe (III) oxides and through conductive biofilms. In contrast, there is no definitive evidence yet that wild-type G. sulfurreducens express long filaments of OmcS extending from the cells, and deleting the gene for OmcS actually increases biofilm conductivity. The literature does not support the concern that many previous studies on e-pili were mistakenly studying OmcS filaments. For example, heterologous expression of the aromatic-rich pilin monomer of Geobacter metallireducens in G. sulfurreducens increases the conductivity of individual nanowires more than 5,000-fold, whereas expression of an aromatic-poor pilin reduced conductivity more than 1,000-fold. This more than million-fold range in nanowire conductivity was achieved while maintaining the 3-nm diameter characteristic of e-pili. Purification methods that eliminate all traces of OmcS yield highly conductive e-pili, as does heterologous expression of the e-pilin monomer in microbes that do not produce OmcS or any other outer-surface cytochromes. Future studies of G. sulfurreducens expression of protein nanowires need to be cognizant of the importance of maintaining environmentally relevant growth conditions because artificial laboratory culture conditions can rapidly select against e-pili expression. Principles derived from the study of e-pili have enabled identification of non-cytochrome protein nanowires in diverse bacteria and archaea. A similar search for cytochrome appendages is warranted. Both e-pili and OmcS filaments offer design options for the synthesis of protein-based “green” electronics, which may be the primary driving force for the study of these structures in the near future.

The possibility that (formerly) and species cooperate via direct interspecies electron transfer (DIET) in terrestrial methanogenic environments was investigated in rice paddy soils. Genes with high sequence similarity to the gene for the PilA pilin monomer of the electrically conductive pili (e-pili) of accounted for over half of the PilA gene sequences in metagenomic libraries and 42% of the mRNA transcripts in RNA sequencing (RNA-seq) libraries. This abundance of e-pilin genes and transcripts is significant because e-pili can serve as conduits for DIET. Most of the e-pilin genes and transcripts were affiliated with species, but sequences most closely related to putative e-pilin genes from genera such as, ,, and , were also detected. Approximately 17% of all metagenomic and metatranscriptomic bacterial sequences clustered with species, and the finding that spp. were actively transcribing growth-related genes indicated that they were metabolically active in the soils. Genes coding for e-pilin were among the most highly transcribed genes. In addition, homologs of genes encoding OmcS, a -type cytochrome associated with the e-pili of and required for DIET, were also highly expressed in the soils. species in the soils highly expressed genes for enzymes involved in the reduction of carbon dioxide to methane. DIET is the only electron donor known to support CO reduction in Thus, these results are consistent with a model in which species were providing electrons to species for methane production through electrical connections of e-pili. species are some of the most important microbial contributors to global methane production, but surprisingly little is known about their physiology and ecology. The possibility that DIET is a source of electrons for in methanogenic rice paddy soils is important because it demonstrates that the contribution that makes to methane production in terrestrial environments may extend beyond the conversion of acetate to methane. Furthermore, defined coculture studies have suggested that when species receive some of their energy from DIET, they grow faster than when acetate is their sole energy source. Thus, growth and metabolism in methanogenic soils may be faster and more robust than generally considered. The results also suggest that the reason that species are repeatedly found to be among the most metabolically active microorganisms in methanogenic soils is that they grow syntrophically in cooperation with spp., and possibly other methanogens, via DIET.

The electrically conductive pili (e-pili) of Geobactersulfurreducens have environmental and practical significance because they can facilitate electron transfer to insoluble Fe(III) oxides; to other microbial species; and through electrically conductive biofilms. E-pili conductivity has been attributed to the truncated PilA monomer, which permits tight packing of aromatic amino acids to form a conductive path along the length of e-pili. In order to better understand the evolution and distribution of e-pili in the microbial world, type IVa PilA proteins from various Gram-negative and Gram-positive bacteria were examined with a particular emphasis on Fe(III)-respiring bacteria. E-pilin genes are primarily restricted to a tight phylogenetic group in the order Desulfuromonadales. The downstream gene in all but one of the Desulfuromonadales that possess an e-pilin gene is a gene previously annotated as 'pilA-C' that has characteristics suggesting that it may encode an outermembrane protein. Other genes associated with pilin function are clustered with e-pilin and 'pilA-C' genes in the Desulfuromonadales. In contrast, in the few bacteria outside the Desulfuromonadales that contain e-pilin genes, the other genes required for pilin function may have been acquired through horizontal gene transfer. Of the 95 known Fe(III)-reducing micro-organisms for which genomes are available, 80 % lack e-pilin genes, suggesting that e-pili are just one of several mechanisms involved in extracellular electron transport. These studies provide insight into where and when e-pili are likely to contribute to extracellular electron transport processes that are biogeochemically important and involved in bioenergy conversions.

Syntrophic interspecies electron exchange is essential for the stable functioning of diverse anaerobic microbial communities. Hydrogen/formate interspecies electron transfer (HFIT), in which H 2 and/or formate function as diffusible electron carriers, has been considered to be the primary mechanism for electron sharing because most common syntrophs were thought to lack biochemical components, such as electrically conductive pili (e-pili), necessary for direct interspecies electron transfer (DIET). Here we report that Syntrophus aciditrophicus, one of the most intensively studied microbial models for HFIT, produces e-pili and can grow via DIET. Pilin genes likely to yield e-pili were found in other genera of hydrogen/formate-producing syntrophs. The finding that DIET is a likely option for diverse syntrophs that are abundant in many anaerobic environments necessitates a reexamination of the paradigm that HFIT is the predominant mechanism for syntrophic electron exchange within anaerobic microbial communities of biogeochemical and practical significance.