Conjugation-Mediated Plasmid Transfer Enables Genetic Modification of Diverse Bacillus Species.

Performing genetic manipulations in Bacillus strains is often hindered by difficulty in identifying conditions appropriate for DNA uptake. This shortcoming limits our understanding of the functional diversity within this genus and the practical application of new strains. We have developed a simple method for increasing the genetic tractability of Bacillus spp. through conjugation-mediated plasmid transfer via a diaminopimelic acid (DAP) auxotrophic Escherichia coli donor strain. We observe transfer into representatives of the Bacillus clades subtilis, cereus, galactosidilyticus, and Priestia megaterium and successfully applied this protocol to 9 out of 12 strains attempted. We utilized the BioBrick 2.0 plasmids pECE743 and pECE750, as well as the CRISPR plasmid pJOE9734.1, to generate a xylose-inducible green-fluorescent protein (GFP)-expressing conjugal vector, pEP011. The use of xylose-inducible GFP ensures ease of confirming transconjugants, which enables users to quickly rule out false positives. Additionally, our plasmid backbone offers the flexibility to be used in other contexts, including transcriptional fusions and overexpression, with only a few modifications. IMPORTANCE Bacillus species are widely used to produce proteins and to understand microbial differentiation. Unfortunately, outside a few lab strains, genetic manipulation is difficult and can prevent thorough dissection of useful phenotypes. We developed a protocol that utilizes conjugation (plasmids that initiate their own transfer) to introduce plasmids into a diverse range of Bacillus spp. This will facilitate a deeper study of wild isolates for both industrial and pure research uses.

Improved chemical and isotopic labeling of biomembranes in Bacillus subtilis by leveraging CRISPRi inhibition of beta-ketoacyl-ACP synthase (fabF).

Assessing the structure of living microbial cell membranes is a challenging analytical goal. The cell membrane is defined by its transverse structure, an approximately 5 nm-thick selectively permeable bilayer that serves many important cellular functions. Compositionally complex, dynamic, and organized in both the transverse and lateral dimensions, understanding the cell membrane structure-and the role that structure plays in cellular function, communication, and environmental sensing is an active scientific effort. Previously, we have devised a novel isotopic labeling approach for membrane lipids to enable direct in vivo structural studies of the cell membrane in the Gram-positive bacterium, Bacillus subtilis, using small-angle neutron scattering. This was accomplished through a genetic inhibition of fatty acid (FA) degradation (ΔfadN) and a chemical inhibition of FA biosynthesis using cerulenin, an irreversible inhibitor of type II fatty acid synthases. Here, we improve upon the previous system by introducing a dCas9/sgRNA-fabF complex that blocks transcription of the essential fabF gene when under xylose induction. This leads to greater sensitivity to cerulenin in the mutant strain (JEBS102) and more robust cell growth when supplementary FAs are introduced to the culture medium. A subtle change in FA uptake is noted when compared to the prior labeling strategy. This is seen in the gas chromatography/mass spectrometry (GC/MS) data as a higher ratio of n16:0 to a15:0, and manifests in an apparent increase in the membrane thickness determined via neutron scattering. This represents an improved method of isotopic labeling for the cell membrane of Bacillus subtilis; enabling improved investigations of cellular uptake and utilization of FAs, cell membrane structure and organization as a phenotypic response to metabolic and environmental changes.

Bacillus pumilus B12 Degrades Polylactic Acid and Degradation Is Affected by Changing Nutrient Conditions.

Poly-lactic acid (PLA) is increasingly used as a biodegradable alternative to traditional petroleum-based plastics. In this study, we identify a novel agricultural soil isolate of Bacillus pumilus (B12) that is capable of degrading high molecular weight PLA films. This degradation can be detected on a short timescale, with significant degradation detected within 48-h by the release of L-lactate monomers, allowing for a rapid identification ideal for experimental variation. The validity of using L-lactate as a proxy for degradation of PLA films is corroborated by loss of rigidity and appearance of fractures in PLA films, as measured by atomic force microscopy and scanning electron microscopy (SEM), respectively. Furthermore, we have observed a dose-dependent decrease in PLA degradation in response to an amino acid/nucleotide supplement mix that is driven mainly by the nucleotide base adenine. In addition, amendments of the media with specific carbon sources increase the rate of PLA degradation, while phosphate and potassium additions decrease the rate of PLA degradation by B. pumilus B12. These results suggest B. pumilus B12 is adapting its enzymatic expression based on environmental conditions and that these conditions can be used to study the regulation of this process. Together, this work lays a foundation for studying the bacterial degradation of biodegradable plastics.

A Student's Guide to Giant Viruses Infecting Small Eukaryotes: From Acanthamoeba to Zooxanthellae.

The discovery of infectious particles that challenge conventional thoughts concerning "what is a virus" has led to the evolution a new field of study in the past decade. Here, we review knowledge and information concerning "giant viruses", with a focus not only on some of the best studied systems, but also provide an effort to illuminate systems yet to be better resolved. We conclude by demonstrating that there is an abundance of new host-virus systems that fall into this "giant" category, demonstrating that this field of inquiry presents great opportunities for future research.

Colloidal and antibacterial properties of novel triple-headed, double-tailed amphiphiles: exploring structure-activity relationships and synergistic mixtures.

Two novel series of tris-cationic, tripled-headed, double-tailed amphiphiles were synthesized and the effects of tail length and head group composition on the critical aggregation concentration (CAC), thermodynamic parameters, and minimum inhibitory concentration (MIC) against six bacterial strains were investigated. Synergistic antibacterial combinations of these amphiphiles were also identified. Amphiphiles in this study are composed of a benzene core with three benzylic ammonium bromide groups, two of which have alkyl chains, each 8-16 carbons in length. The third head group is a trimethylammonium or pyridinium. Log of critical aggregation concentration (log[CAC]) and heat of aggregation (ΔHagg) were both inversely proportional to the length of the linear hydrocarbon chains. Antibacterial activity increases with tail length until an optimal tail length of 12 carbons per chain, above which, activity decreased. The derivatives with two 12 carbon chains had the best antibacterial activity, killing all tested strains at concentrations of 1-2µM for Gram-positive and 4-16µM for Gram-negative bacteria. The identity of the third head group (trimethylammonium or pyridinium) had minimal effect on colloidal and antibacterial activity. The antibacterial activity of several binary combinations of amphiphiles from this study was higher than activity of individual amphiphiles, indicating that these combinations are synergistic. These amphiphiles show promise as novel antibacterial agents that could be used in a variety of applications.