Viral communities in a pH>10 serpentinite-like environment: insight into diversity and potential roles in modulating the microbiomes by bioactive vitamin B9 synthesis.

Viral communities exist in a variety of ecosystems and play significant roles in mediating biogeochemical processes, whereas viruses inhabiting strongly alkaline geochemical systems remain underexplored. In this study, the viral diversity, potential functionalities, and virus-host interactions in a strongly alkaline environment (pH = 10.4-12.4) exposed to the leachates derived from the serpentinization-like reactions of smelting slags were investigated. The viral populations (e.g., Herelleviridae, Queuovirinae, and Inoviridae) were closely associated with the dominating prokaryotic hosts (e.g., Meiothermus, Trueperaceae, and Serpentinomonas) in this ultrabasic environment. Auxiliary metabolic genes (AMGs) suggested that viruses may enhance hosts' fitness by facilitating cofactor biosynthesis, hydrogen metabolism, and carbon cycling. To evaluate the activity of synthesis of essential cofactor vitamin B9 by the viruses, a viral folA (vfolA) gene encoding dihydrofolate reductase (DHFR) was introduced into a thymidine-auxotrophic strain Escherichia coli MG1655 ΔfolA mutant, which restored the growth of the latter in the absence of thymidine. Notably, the homologs of the validated vDHFR were globally distributed in the viromes across various ecosystems. The present study sheds new light on the unique viral communities in hyperalkaline ecosystems and their potential beneficial impacts on the coexisting microbial consortia by supplying essential cofactors. IMPORTANCE: This study presents a comprehensive investigation into the diversity, potential functionalities, and virus-microbe interactions in an artificially induced strongly alkaline environment. Functional validation of the detected viral folA genes encoding dihydrofolate reductase substantiated the synthesis of essential cofactors by viruses, which may be ubiquitous, considering the broad distribution of the viral genes associated with folate cycling.

The robustness of porin-cytochrome gene clusters from Geobacter metallireducens in extracellular electron transfer.

To investigate their roles in extracellular electron transfer (EET), the porin-cytochrome (pcc) gene clusters Gmet0825-0828, Gmet0908-0910, and Gmet0911-0913 of the Gram-negative bacterium Geobacter metallireducens were deleted. Failure to delete all pcc gene clusters at the same time suggested their essential roles in extracellular reduction of Fe(III)-citrate by G. metallireducens. Deletion of Gmet0825-0828 had no impact on bacterial reduction of Fe(III)-citrate but diminished bacterial reduction of ferrihydrite and abolished anode reduction and direct interspecies electron transfer (DIET) to Methanosarcina barkeri and Geobacter sulfurreducens. Although it had no impact on the bacterial reduction of Fe(III)-citrate, deletion of Gmet0908-0910 delayed ferrihydrite reduction, abolished anode reduction, and diminished DIET. Deletion of Gmet0911-0913 had little impact on DIET but diminished bacterial reductions of Fe(III)-citrate, ferrihydrite, and anodes. Most importantly, deletions of both Gmet0825-0828 and Gmet0908-0910 restored bacterial reduction of ferrihydrite and anodes and DIET. Enhanced expression of Gmet0911-0913 in this double mutant when grown in coculture with G. sulfurreducens ΔhybLΔfdnG suggested that this cluster might compensate for impaired EET functions of deleting Gmet0825-0828 and Gmet0908-0910. Thus, these pcc gene clusters played essential, distinct, overlapping, and compensatory roles in EET of G. metallireducens that are difficult to characterize as deletion of some clusters affected expression of others. The robustness of these pcc gene clusters enabled G. metallireducens to mediate EET to different acceptors for anaerobic growth even when two of its three pcc gene clusters were inactivated by mutation. The results from this investigation provide new insights into the roles of pcc gene clusters in bacterial EET. IMPORTANCE: The Gram-negative bacterium Geobacter metallireducens is of environmental and biotechnological significance. Crucial to the unique physiology of G. metallireducens is its extracellular electron transfer (EET) capability. This investigation sheds new light on the robust roles of the three porin-cytochrome (pcc) gene clusters, which are directly involved in EET across the bacterial outer membrane, in the EET of G. metallireducens. In addition to their essential roles, these gene clusters also play distinct, overlapping, and compensatory roles in the EET of G. metallireducens. The distinct roles of the pcc gene clusters enable G. metallireducens to mediate EET to a diverse group of electron acceptors for anaerobic respirations. The overlapping and compensatory roles of the pcc gene clusters enable G. metallireducens to maintain and restore its EET capability for anaerobic growth when one or two of its three pcc gene clusters are deleted from the genome.

Flagella act as Geobacter biofilm scaffolds to stabilize biofilm and facilitate extracellular electron transfer.

Flagella are widely expressed in electroactive biofilms; however, their actual role is unknown. To understand the role of flagella, two Geobacter sulfurreducens strains (KN400 and PCA, with and without flagella, respectively) were selected. We restored flagellum expression in trans in strain PCA and prevented flagellum expression in strain KN400. Electrochemical results showed that flagellum restoration in strain PCA promoted current generation, while flagellum deletion in strain KN400 impaired current production. However, the expression of conductive pili and outer surface c-type cytochromes was not affected. Further microscopic analyses demonstrated that flagella promoted the formation of thicker biofilms and served as biofilm matrix scaffolds to accommodate more extracellular cytochromes with an orderly arrangement, which increased the electron diffusion rate within the biofilm. Our findings reveal an unprecedented structural role for flagella in stabilizing electroactive biofilms and highlight the importance of cytochromes in electron transfer across biofilms, which will deepen our understanding of biofilm conductivity.

Syntrophic growth with direct interspecies electron transfer between pili-free Geobacter species.

Direct interspecies electron transfer (DIET) may prevail in microbial communities that show methanogenesis and anaerobic methane oxidation and can be an electron source to support anaerobic photosynthesis. Previous mutagenic studies on cocultures of defined Geobacter species indicate that both conductive pili and extracellular cytochromes are essential for DIET. However, the actual functional role of the pili in DIET is uncertain, as the pilus mutation strategy used in these studies affected the extracellular cytochrome profile. Here we repressed the function of pili by deleting the pilus polymerization motor PilB in both Geobacter species. The PilB mutation inhibited the pilus assembly but did not alter the pattern of extracellular cytochromes. We report that the two pilus-free Geobacter species can form aggregates and grow syntrophically with DIET. The results demonstrate that the Gmet_2896 cytochrome of Geobacter metallireducens plays a key role in DIET and that conductive pili are not necessary to facilitate DIET in cocultures of Geobacter species, and they suggest cytochromes by themselves can meditate DIET, deepening the understanding of DIET.