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Troubleshooting is an important part of experimental research, but graduate students rarely receive formal training in this skill. In this article, we describe an initiative called Pipettes and Problem Solving that we developed to teach troubleshooting skills to graduate students at the University of Texas at Austin. An experienced researcher presents details of a hypothetical experiment that has produced unexpected results, and students have to propose new experiments that will help identify the source of the problem. We also provide slides and other resources that can be used to facilitate problem solving and teach troubleshooting skills at other institutions.

ABSTRACTExtracellular cytochrome filaments are proposed to serve as conduits for long-range extracellular electron transfer. The primary functional physiological evidence has been the reported inhibition of Geobacter sulfurreducens Fe(III) oxide reduction when the gene for the filament-forming cytochrome OmcS is deleted. Here we report that the OmcS-deficient strain from that original report reduces Fe(III) oxide as well as the wild-type, as does a triple mutant in which the genes for the other known filament-forming cytochromes were also deleted. The triple cytochrome mutant displayed filaments with the same 3 nm diameter morphology and conductance as those produced by Escherichia coli heterologously expressing the G. sulfurreducens PilA pilin gene. Fe(III) oxide reduction was inhibited when the pilin gene in cytochrome-deficient mutants was modified to yield poorly conductive 3 nm diameter filaments. The results are consistent with the concept that 3 nm diameter electrically conductive pili (e-pili) are required for G. sulfurreducens long-range extracellular electron transfer. In contrast, rigorous physiological functional evidence is lacking for cytochrome filaments serving as conduits for long-range electron transport.IMPORTANCEUnraveling microbial extracellular electron transfer mechanisms has profound implications for environmental processes and advancing biological applications. This study on Geobacter sulfurreducens challenges prevailing beliefs on cytochrome filaments as crucial components thought to facilitate long-range electron transport. The discovery of an OmcS-deficient strain’s unexpected effectiveness in Fe(III) oxide reduction prompted a reevaluation of the key conduits for extracellular electron transfer. By exploring the impact of genetic modifications on G. sulfurreducens’ performance, this research sheds light on the importance of 3-nm diameter electrically conductive pili in Fe(III) oxide reduction. Reassessing these mechanisms is essential for uncovering the true drivers of extracellular electron transfer in microbial systems, offering insights that could revolutionize applications across diverse fields.

Biocides are widely used for the mitigation of microbial contamination, especially in the field of the aviation fuel industry. However, the long-term use of biocide has raised the concerns regarding the environmental contamination and microbial drug resistance. In this study, the effect of a mixture of D-amino acids (D-tyrosine and D-methionine) on the enhancement of the bactericidal effect of 5-Chloro-2-Methyl-4-isothiazolin-3-one/2-Methyl-2H-isothiazole-3-one (CMIT/MIT) against corrosive Vibrio harveyi biofilm was evaluated. The results revealed that D-Tyr and D-Met alone can enhance the biocidal efficacy of CMIT/MIT, while the treatment of 5 ppm CMIT/MIT, 1 ppm D-Tyr and 100 ppm D-Met showed the best efficacy comparable to that of 25 ppm CMIT/MIT alone. The triple combination treatment successfully prevented the establishment of the corrosive V. harveyi biofilm and effectively removed the mature V. harveyi biofilm. These conclusions were confirmed by the results of sessile cell counts, images obtained by scanning electron microscope and confocal laser scanning microscope, and the ATP test kit.

Pectobacterium brasiliense (Pbr) is considered as one of the most virulent species among the Pectobacteriaceae. This species has a broad host range within horticulture crops and is well distributed elsewhere. It has been found to be pathogenic not only in the field causing blackleg and soft rot of potato, but it is also transmitted via storage causing soft rot of other vegetables. Genomic analysis and other cost-effective molecular detection methods such as a quantitative polymerase chain reaction (qPCR) are essential to investigate the ecology and pathogenesis of the Pbr. The lack of fast, field deployable point-of-care testing (POCT) methods, specific control strategies and current limited genomic knowledge make management of this species difficult. Thus far, no comprehensive review exists about Pbr, however there is an intense need to research the biology, detection, pathogenicity and management of Pbr, not only because of its fast distribution across Europe and other countries but also due to its increased survival to various climatic conditions. This review outlines the information available in peer-reviewed literature regarding host range, detection methods, genomics, geographical distribution, nomenclature and taxonomical evolution along with some of the possible management and control strategies. In summary, the conclusions and a further directions highlight the management of this species.