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The product of the human C21orf57 (huYBEY) gene is predicted to be a homologue of the highly conserved YbeY proteins found in nearly all bacteria. We show that, like its bacterial and chloroplast counterparts, the HuYbeY protein is an RNase and that it retains sufficient function in common with bacterial YbeY proteins to partially suppress numerous aspects of the complex phenotype of an Escherichia coli ΔybeY mutant. Expression of HuYbeY in Saccharomyces cerevisiae, which lacks a YbeY homologue, results in a severe growth phenotype. This observation suggests that the function of HuYbeY in human cells is likely regulated through specific interactions with partner proteins similarly to the way YbeY is regulated in bacteria., National Institutes of Health (U.S.) (Grant GM31010), National Institutes of Health (U.S.) (Grant GM17151)

Acinetobacter baumannii rapidly acquires antibiotic resistance, and its genome encodes mechanisms to tolerate biocides and desiccation, enhancing its persistence in hospital settings. Tools to rapidly dissect the A. baumannii genome are needed to understand cellular factors that contribute to its resiliency at a genetic and mechanistic level. While a substantial amount of clinical data has documented the global rise of A. baumannii as an antibiotic-resistant pathogen, genetic tools to dissect its molecular details have been limited. This procedure describes a recombination-mediated genetic engineering (recombineering) system for targeted genome editing of A. baumannii. This system can perform directed mutagenesis on wide-ranging genes and operons and has broad application in various strains of A. baumannii.

With the increasing occurrence of antibiotic resistance among Acinetobacter sp., the race is on for researchers to not only isolate resistant isolates but also utilize basic and applied microbiological techniques to study mechanisms of resistance. For many antibiotics, the limit of efficacy against Gram-negative bacteria is dependent on its ability to permeate the outer membrane and access its target. As such, it is critical that researchers be able to isolate and analyze the lipid components of the cell envelope from any number of Acinetobacter sp. that are either resistant or sensitive to antibiotics of interest. The following chapter provides in-depth protocols to confirm the presence or absence of lipooligosaccharide (LOS) in Acinetobacter sp., isolate lipid A, and glycerophospholipids and analyze them using qualitative (mass spectrometry) and semiquantitative (thin-layer chromatography) methods.

Acinetobacter baumannii is a hospital-associated pathogen of growing importance and is a paradigm for endemic hospital contamination. Desiccation tolerance has been implicated as an important characteristic that potentiates the spread of A. baumannii in clinical settings through contaminated healthcare equipment and personnel. Desiccation is a multifaceted stress, and many physiological and environmental factors can influence its impact on bacterial survival. This chapter provides a protocol for assessing desiccation survival that facilitates comparisons among A. baumannii strains under various environmental conditions.

Three new coinage metal carbene complexes of silver and gold were synthesized from a thiamine inspired proligand. The compounds were characterized by HRMS, NMR spectroscopy ((1)H, (19)F, (31)P and (13)C), FT-IR and elemental analysis. The coordination environment around the metal centers was correlated to the diffusion coefficients obtained from DOSY-NMR experiments and was in agreement with the nuclearity observed in the solid-state by single crystal X-ray crystallography. The silver and gold carbene compounds were subjected to MIC studies against a panel of pathogenic bacteria, including multidrug resistant strains, with the gold carbene derivative showing the most potent antimicrobial activity against Gram-positive methicillin resistant Staphylococcus aureus (MRSA).

Enterotoxigenic Escherichia coli (ETEC) is a global diarrheal pathogen that utilizes adhesins and secreted enterotoxins to cause disease in mammalian hosts. Decades of research on virulence factor regulation in ETEC has revealed a variety of environmental factors that influence gene expression, including bile, pH, bicarbonate, osmolarity, and glucose. However, other hallmarks of the intestinal tract, such as low oxygen availability, have not been examined. Further, determining how ETEC integrates these signals in the complex host environment is challenging. To address this, we characterized ETEC’s response to the human host using samples from a controlled human infection model. We found ETEC senses environmental oxygen to globally influence virulence factor expression via the oxygen-sensitive transcriptional regulator fumarate and nitrate reduction (FNR) regulator. In vitro anaerobic growth replicates the in vivo virulence factor expression profile, and deletion of fnr in ETEC strain H10407 results in a significant increase in expression of all classical virulence factors, including the colonization factor antigen I (CFA/I) adhesin operon and both heat-stable and heat-labile enterotoxins. These data depict a model of ETEC infection where FNR activity can globally influence virulence gene expression, and therefore proximity to the oxygenated zone bordering intestinal epithelial cells likely influences ETEC virulence gene expression in vivo. Outside of the host, ETEC biofilms are associated with seasonal ETEC epidemics, and we find FNR is a regulator of biofilm production. Together these data suggest FNR-dependent oxygen sensing in ETEC has implications for human infection inside and outside of the host.

In today’s networks, IEEE 802.11 Wi-Fi is commonplace. While providing great ease of use and mobility, IEEE 802.11 also has many flaws. One of these is contention. Contention is the means that wireless stations compete to determine who gets to transmit. During the contention times, the medium is sitting idle. Idle time equates to wasted bandwidth. This research presents a novel technique to eliminate contention in a wireless network while actually increasing sustained throughput by the use of Direct Sequence Spread-Spectrum Code Division Multiple Access (DS-CDMA).

Engineering the bacteria present in animal microbiomes promises to lead to breakthroughs in medicine and agriculture, but progress is hampered by a dearth of tools for genetically modifying the diverse species that comprise these communities. Here we present a toolkit of genetic parts for the modular construction of broad-host-range plasmids built around the RSF1010 replicon. Golden Gate assembly of parts in this toolkit can be used to rapidly test various antibiotic resistance markers, promoters, fluorescent reporters, and other coding sequences in newly isolated bacteria. We demonstrate the utility of this toolkit in multiple species of Proteobacteria that are native to the gut microbiomes of honey bees ( Apis mellifera) and bumble bees (B ombus sp.). Expressing fluorescent proteins in Snodgrassella alvi, Gilliamella apicola, Bartonella apis, and Serratia strains enables us to visualize how these bacteria colonize the bee gut. We also demonstrate CRISPRi repression in B. apis and use Cas9-facilitated knockout of an S. alvi adhesion gene to show that it is important for colonization of the gut. Beyond characterizing how the gut microbiome influences the health of these prominent pollinators, this bee microbiome toolkit (BTK) will be useful for engineering bacteria found in other natural microbial communities.

Quaternary amine compounds (QAC) are potent antimicrobials used to prevent the spread of pathogenic bacteria. While they are known for their membrane-damaging properties, QAC action has been suggested to extend beyond the surface to intracellular targets. Here we characterize the range of action of the QAC biocide benzalkonium chloride (BZK) against the bacterial pathogen Acinetobacter baumannii. At high concentrations, BZK acts through membrane disruption, but at low concentrations we show that wide-spread protein aggregation is associated with BZK-induced cell death. Resistance to BZK is found to develop through ribosomal protein mutations that protect A. baumannii against BZK-induced protein aggregation. The multifunctional impact of BZK led us to discover that alternative QAC structures, with low human toxicity, retain potent action against multidrug-resistant A. baumannii, Staphylococcus aureus, and Clostridium difficile and present opportunities for their development as antibiotics., IMPORTANCE Quaternary amine compounds (QACs) are widely used to prevent the spread of bacterial pathogens, but our understanding of their mode of action is incomplete. Here we describe disruption of bacterial proteostasis as an unrecognized action of QAC antimicrobial action and uncover the potential of diverse QAC structures to act as multitarget antibiotics.

Peptide oligomers offer versatile scaffolds for the formation of potent antimicrobial agents due to their high sequence versatility, inherent biocompatibility, and chemical tunability. Though many methods exist for the formation of peptide-based macrocycles (MCs), increasingly pervasive in commercial antimicrobial therapeutics, the introduction of multiple looped structures into a single peptide oligomer remains a significant challenge. Herein, we report the utilization of dynamic hydrazone condensation for the versatile formation of single-, double-, and triple-loop peptide MCs using simple dialdehyde or dihydrazide small-molecule cross-linkers, as confirmed by MALDI-TOF MS, HPLC, and SDS-PAGE. Furthermore, incorporation of aldehyde-containing side chains onto peptides synthesized from hydrazide C-terminal resins resulted in tunable peptide MC assemblies formed directly upon resin cleavage post solid-phase peptide synthesis. Both of these types of dynamic covalent assemblies produced significant enhancements to overall antimicrobial properties when introduced into a known antimicrobial peptide, buforin II, when compared to the original unassembled sequence.