Horizontal Transfer of Bacteriocin Biosynthesis Genes Requires Metabolic Adaptation To Improve Compound Production and Cellular Fitness.

Biosynthetic gene clusters (BGCs) encoding the production of bacteriocins are widespread among bacterial isolates and are important genetic determinants of competitive fitness within a given habitat. Staphylococci produce a tremendous diversity of compounds, and the corresponding BGCs are frequently associated with mobile genetic elements, suggesting gain and loss of biosynthetic capacity. Pharmaceutical biology has shown that compound production in heterologous hosts is often challenging, and many BGC recipients initially produce small amounts of compound or show reduced growth rates. To assess whether transfer of BGCs between closely related Staphylococcus aureus strains can be instantly effective or requires elaborate metabolic adaptation, we investigated the intraspecies transfer of a BGC encoding the ribosomally synthesized and posttranslationally modified peptide (RiPP) micrococcin P1 (MP1). We found that acquisition of the BGC by S. aureus RN4220 enabled immediate MP1 production but also imposed a metabolic burden, which was relieved after prolonged cultivation by adaptive mutation. We used a multiomics approach to study this phenomenon and found adaptive evolution to select for strains with increased activity of the tricarboxylic acid cycle (TCA), which enhanced metabolic fitness and levels of compound production. Metabolome analysis revealed increases of central metabolites, including citrate and α-ketoglutarate in the adapted strain, suggesting metabolic adaptation to overcome the BGC-associated growth defects. Our results indicate that BGC acquisition requires genetic and metabolic predispositions, allowing the integration of bacteriocin production into the cellular metabolism. Inappropriate metabolic characteristics of recipients can entail physiological burdens, negatively impacting the competitive fitness of recipients within natural bacterial communities. IMPORTANCE Human microbiomes are critically associated with human health and disease. Importantly, pathogenic bacteria can hide in human-associated communities and can cause disease when the composition of the community becomes unbalanced. Bacteriocin-producing commensals are able to displace pathogens from microbial communities, suggesting that their targeted introduction into human microbiomes might prevent pathogen colonization and infection. However, to develop probiotic approaches, strains are needed that produce high levels of bioactive compounds and retain cellular fitness within mixed bacterial communities. Our work offers insights into the metabolic burdens associated with the production of the bacteriocin micrococcin P1 and highlights evolutionary strategies that increase cellular fitness in the context of production. Metabolic adaptations are most likely broadly relevant for bacteriocin producers and need to be considered for the future development of effective microbiome editing strategies.

Rapid Microscopic Detection of Bacillus anthracis by Fluorescent Receptor Binding Proteins of Bacteriophages.

Bacillus anthracis, the etiological agent of anthrax disease, is typically diagnosed by immunological and molecular methods such as polymerase chain reaction (PCR). Alternatively, mass spectrometry techniques may aid in confirming the presence of the pathogen or its toxins. However, because of the close genetic relationship between B. anthracis and other members of the Bacillus cereus sensu lato group (such as Bacillus cereus or Bacillus thuringiensis) mis- or questionable identification occurs frequently. Also, bacteriophages such as phage gamma (which is highly specific for B. anthracis) have been in use for anthrax diagnostics for many decades. Here we employed host cell-specific receptor binding proteins (RBP) of (pro)-phages, also known as tail or head fibers, to develop a microscopy-based approach for the facile, rapid and unambiguous detection of B. anthracis cells. For this, the genes of (putative) RBP from Bacillus phages gamma, Wip1, AP50c and from lambdoid prophage 03 located on the chromosome of B. anthracis were selected. Respective phage genes were heterologously expressed in Escherichia coli and purified as fusions with fluorescent proteins. B. anthracis cells incubated with either of the reporter fusion proteins were successfully surface-labeled. Binding specificity was confirmed as RBP fusion proteins did not bind to most isolates of a panel of other B. cereus s.l. species or to more distantly related bacteria. Remarkably, RBP fusions detected encapsulated B. anthracis cells, thus RBP were able to penetrate the poly-γ-d-glutamate capsule of B. anthracis. From these results we anticipate this RBP-reporter assay may be useful for rapid confirmative identification of B. anthracis.

Quantitative Oxygen Consumption and Respiratory Activity of Meat Spoiling Bacteria Upon High Oxygen Modified Atmosphere.

High oxygen modified atmosphere packaging is a commonly applied method to prolong the minimum shelf life of fresh (red) meats. Upon spoilage, changes of the initial oxygen concentration and microbiome composition can be observed. Thus, we classified the typical representative meat spoiling bacteria Brochothrix (B.) thermosphacta TMW2.2101 and the four lactic acid bacteria (LAB) Carnobacterium (C.) divergens TMW2.1577, C. maltaromaticum TMW2.1581, Leuconostoc (L.) gelidum subsp. gelidum TMW2.1618, and L. gelidum subsp. gasicomitatum TMW2.1619 along their oxygen consuming capacity, which can indicate the timeline of microbiome and sensorial changes. All bacteria were grown in a model system employing gas tight glass bottles containing meat simulation media and under modified atmosphere (70% O2 and 30% CO2). Oxygen concentrations of media and headspaces were monitored over time and the oxygen uptake rate (OUR) was calculated for all species. All bacteria were able to consume dissolved oxygen, with B. thermosphacta TMW2.2101 exhibiting a 31-times higher OUR per single cell in 60 h. Furthermore, all strains showed significant growth enhancement in the presence of heme indicating respiratory activity. Comparative genomic and physiological analyses predict the activity of a respiratory chain for all species upon high oxygen atmosphere. An additional cytochrome aa3 oxidase is suggested to be responsible for the increased OUR of B. thermosphacta TMW2.2101. Furthermore, B. thermosphacta TMW2.2101 revealed highest oxidative stress tolerance compared to the other bacteria, facilitating a higher respiratory activity. Coupling of respiration and fermentation via regeneration of NADH can be a competitive advantage for meat spoiling bacteria resulting in a higher cell count and possibly accelerated spoilage. The exhibited highest capacity for oxygen consumption of B. thermosphacta compared to LAB in vitro also suggests a higher contribution of this bacterium to the change in the atmosphere upon spoilage of MAP meats in situ.