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Microbial interactions dictate the structure and function of microbiomes, but the complexity of natural communities can obscure the individual interactions. Model microbial communities constructed with genetically tractable strains known to interact in natural settings can untangle these networks and reveal underpinning mechanisms. Our model system, T he H itchhikers o f the R hizosphere (THOR), is composed of three species-Bacillus cereus, Flavobacterium johnsoniae, and Pseudomonas koreensis-that co-isolate from field-grown soybean roots. Comparative metatranscriptomics on THOR revealed global patterns of interspecies transcriptional regulation. When grown in pairs, each member of THOR exhibits unique signaling behavior. In the community setting, gene expression is dominated by pairwise interactions with Pseudomonas koreensis mediated either directly or indirectly by its production of the antibiotic koreenceine-the apparent "hammer" of THOR. In pairwise interactions, the koreenceine biosynthetic cluster is responsible for 85 and 22% of differentially regulated genes in F. johnsoniae and B. cereus, respectively. Although both deletion of the koreenceine locus and reduction of P. koreensis inoculum size increase F. johnsoniae populations, the transcriptional response of P. koreensis is only activated when it is a relative minority member at the beginning of coculture. The largest group of upregulated P. koreensis genes in response to F. johnsoniae are those without functional annotation, indicating that focusing on genes important for community interactions may offer a path toward functional assignments for unannotated genes. This study illustrates the power of comparative metatranscriptomics of microorganisms encountering increasing microbial complexity for understanding community signal integration, antibiotic responses, and interspecies communication. IMPORTANCE The diversity, ubiquity, and significance of microbial communities is clear. However, the predictable and reliable manipulation of microbiomes to impact human, environmental, and agricultural health remains a challenge. Effective remodeling of microbiomes will be enabled by understanding the interspecies interactions that govern community processes. The extreme complexity of most microbiomes has impeded characterization of the relevant interactions. Investigating the genetics and biochemistry of simplified, model microbiomes could unearth specific interactions and generate predictions about community-governing principles. Here, we use one such model community to quantify changes in gene expression of individual species as they encounter stimuli from one or more species, directly mapping combinatorial interspecies interactions. A surprising amount of gene expression is regulated by a single molecule, the antibiotic koreenceine, which appears to impact gene regulation across community networks.

The world faces two seemingly unrelated challenges-a shortfall in the STEM workforce and increasing antibiotic resistance among bacterial pathogens. We address these two challenges with Tiny Earth, an undergraduate research course that excites students about science and creates a pipeline for antibiotic discovery., (Copyright © 2021 Hurley et al.)

Antibiotics produced by bacteria play important roles in microbial interactions and competition Antibiosis can induce resistance mechanisms in target organisms, and at sublethal doses, antibiotics have been shown to globally alter gene expression patterns. Here, we show that hygromycin A from Streptomyces sp. strain 2AW. induces Chromobacterium violaceum ATCC 31532 to produce the purple antibiotic violacein. Sublethal doses of other antibiotics that similarly target the polypeptide elongation step of translation likewise induced violacein production, unlike antibiotics with different targets. C. violaceum biofilm formation and virulence against Drosophila melanogaster were also induced by translation-inhibiting antibiotics, and we identified an a ntibiotic- i nduced r esponse ( air ) two-component regulatory system that is required for these responses. Genetic analyses indicated a connection between the Air system, quorum-dependent signaling, and the negative regulator VioS, leading us to propose a model for induction of violacein production. This work suggests a novel mechanism of interspecies interaction in which a bacterium produces an antibiotic in response to inhibition by another bacterium and supports the role of antibiotics as signal molecules. IMPORTANCE Secondary metabolites play important roles in microbial communities, but their natural functions are often unknown and may be more complex than appreciated. While compounds with antibiotic activity are often assumed to underlie microbial competition, they may alternatively act as signal molecules. In either scenario, microorganisms might evolve responses to sublethal concentrations of these metabolites, either to protect themselves from inhibition or to change certain behaviors in response to the local abundance of another species. Here, we report that violacein production by C. violaceum ATCC 31532 is induced in response to hygromycin A from Streptomyces sp. 2AW, and we show that this response is dependent on inhibition of translational polypeptide elongation and a previously uncharacterized two-component regulatory system. The breadth of the transcriptional response beyond violacein induction suggests a surprisingly complex metabolite-mediated microbe-microbe interaction and supports the hypothesis that antibiotics evolved as signal molecules. These novel insights will inform predictive models of soil community dynamics and the unintended effects of clinical antibiotic administration., (Copyright © 2020 Lozano et al.)

We report the screening of 16,000 synthetic compounds for induction and inhibition of quorum sensing in a Pseudomonas putida N-acylated l-homoserine lactone (AHL) sensor strain engineered with the LasR transcriptional activator. LasR controls virulence gene expression in the opportunistic pathogen Pseudomonas aeruginosa and is of significant interest as a therapeutic target. Nine compounds that inhibit and 14 compounds that induce LasR activity were identified in our high-throughput screen.

Functional metagenomics was used to search for florfenicol resistance genes in libraries of cloned DNA isolated from Alaskan soil. A gene that mediated reduced susceptibility to florfenicol was identified and designated pexA. The predicted PexA protein showed a structure similar to that of efflux pumps of the major facilitator superfamily.

The use of combination antimicrobial therapy for bacteraemia caused by Gram-negative bacilli is controversial. We did a meta-analysis of published studies to determine whether a combination of two or more antimicrobials reduces mortality in patients with Gram-negative bacteraemia. Criteria for inclusion were: analytic studies of patients with documented Gram-negative bacteraemia that included patients receiving a single antibiotic (monotherapy) and patients receiving two or more antibiotics (combination therapy). Data on mortality (outcome) had to be provided. A pooled odds ratio was calculated with the random effects model of DerSimonian and Laird. Assessment of heterogeneity was done with the Breslow-Day test and reasons for heterogeneity were explored. 17 studies met the inclusion criteria, five prospective cohort studies, two prospective randomised trials, and ten retrospective cohort studies. Most studies used beta-lactams or aminoglycosides alone and in combination. The summary odds ratio was 0.96 (95% CI 0.70-1.32), indicating no mortality benefit with combination therapy. Subgroup analyses adjusting for year of publication, study design, and severity of illness did not change the results. Considerable heterogeneity was present in the main analyses. Analysis of only Pseudomonas aeruginosa bacteraemias showed a significant mortality benefit (OR 0.50, 95% CI 0.30-0.79). Our analysis does not support the routine use of combination antimicrobial therapy for Gram-negative bacteraemia, beyond settings where infection by P aeruginosa is strongly suspected or more than one drug would be desirable to assure in-vitro efficacy.

To access the genetic and biochemical potential of soil microorganisms by culture-independent methods, a 24,546-member library in Escherichia coli with DNA extracted directly from soil had previously been constructed (M. R. Rondon, P. R. August, A. D. Bettermann, S. F. Brady, T. H. Grossman, M. R. Liles, K. A. Loiacono, B. A. Lynch, I. A. MacNeil, M. S. Osburne, J. Clardy, J. Handelsman, and R. M. Goodman, Appl. Environ. Microbiol. 66:2541-2547, 2000). Three clones, P57G4, P89C8, and P214D2, produced colonies with a dark brown melanin-like color. We fractionated the culture supernatant of P57G4 to identify the pigmented compound or compounds. Methanol extracts of the acid precipitate from the culture supernatant contained a red and an orange pigment. Structural analysis revealed that these were triaryl cations, designated turbomycin A and turbomycin B, respectively; both exhibited broad-spectrum antibiotic activity against gram-negative and gram-positive organisms. Mutagenesis, subcloning, and sequence analysis of the 25-kb insert in P57G4 demonstrated that a single open reading frame was necessary and sufficient to confer production of the brown, orange, and red pigments on E. coli; the predicted product of this sequence shares extensive sequence similarity with members of the 4-hydroxyphenylpyruvate dioxygenase (4HPPD) family of enzymes. Another member of the same family of genes, lly, which is required for production of the hemolytic pigment in Legionella pneumophila, also conferred production of turbomycin A and B on E. coli. We further demonstrated that turbomycin A and turbomycin B are produced from the interaction of indole, normally secreted by E. coli, with homogentisic acid synthesized by the 4HPPD gene products. The results demonstrate successful heterologous expression of DNA extracted directly from soil as a means to access previously uncharacterized small organic compounds, serving as an example of a chimeric pathway for the generation of novel chemical structures.

The goal of this study was to identify the biosynthetic cluster for zwittermicin A, a novel, broad spectrum, aminopolyol antibiotic produced by Bacillus cereus. The nucleotide sequence of 2.7kb of DNA flanking the zwittermicin A self-resistance gene, zmaR, from B. cereus UW85 revealed three open reading frames (ORFs). Of these ORFs, two had sequence similarity to acyl-CoA dehydrogenases and polyketide synthases, respectively. Insertional inactivation demonstrated that orf2 is necessary for zwittermicin A production and that zmaR is necessary for high-level resistance to zwittermicin A but is not required for zwittermicin A production. Expression of ZmaR was temporally associated with zwittermicin A production. The results suggest that zmaR is part of a cluster of genes that is involved in zwittermicin A biosynthesis, representing the first biosynthetic pathway for an aminopolyol antibiotic.

ZmaR is a resistance determinant of unusual abundance in the environment and confers on gram-positive and gram-negative bacteria resistance to zwittermicin A, a novel broad-spectrum antibiotic produced by species of Bacillus. The ZmaR protein has no sequence similarity to proteins of known function; thus, the purpose of the present study was to determine the function of ZmaR in vitro. Cell extracts of E. coli containing zmaR inactivated zwittermicin A by covalent modification. Chemical analysis of inactivated zwittermicin A by 1H NMR, 13C NMR, and high- and low-resolution mass spectrometry demonstrated that the inactivated zwittermicin A was acetylated. Purified ZmaR protein inactivated zwittermicin A, and biochemical assays for acetyltransferase activity with [14C]acetyl coenzyme A demonstrated that ZmaR catalyzes the acetylation of zwittermicin A with acetyl coenzyme A as a donor group, suggesting that ZmaR may constitute a new class of acetyltransferases. Our results allow us to assign a biochemical function to a resistance protein that has no sequence similarity to proteins of known function, contributing fundamental knowledge to the fields of antibiotic resistance and protein function.

Zwittermicin A is a novel antibiotic produced by Bacillus cereus UW85, which suppresses certain plant diseases in the laboratory and in the field. We developed a rapid method for large-scale purification of zwittermicin A and then studied the in vitro activity of zwittermicin A against bacteria, fungi, and protists. Zwittermicin A was highly active against the Oomycetes and their relatives, the algal protists, and had moderate activity against diverse Gram-negative bacteria and certain Gram-positive bacteria as well as against a wide range of plant pathogenic fungi. Zwittermicin A was more active against bacteria and fungi at pH 7-8 than at pH 5-6. When zwittermicin A was combined with kanosamine, another antibiotic produced by B. cereus, the two acted synergistically against Escherichia coli and additively against Phytophthora medicaginis, an Oomycete. The results indicate that there are diverse potential applications of this new class of antibiotic.

Zwittermicin A is a novel aminopolyol antibiotic that represents a new structural class of antibiotic and has diverse biological activities, including the suppression of plant disease and the ability to inhibit prokaryotic and eukaryotic cells. To enhance our fundamental understanding and applications of zwittermicin A, we elucidated mechanisms of zwittermicin A resistance in Escherichia coli. Two classes of zwittermicin A-resistant mutants of E. coli were selected and characterized. One class included mutants altered in hemA, hemB, hemL, ubi, cydAB or atp, which were defective in generating a proton motive force (PMF) and resistant to aminoglycosides. The mutant analysis, coupled with physiological data, indicated an association between the electrical membrane potential (deltapsi) component of PMF and zwittermicin A sensitivity. A second class of zwittermicin A-resistant mutants was aminoglycoside sensitive and was affected in rpoB and rpoC, genes that encode subunits of RNA polymerase. The rpoB and rpoC mutants suggested that zwittermicin A might inhibit transcription, DNA replication, DNA gyrase or topoisomerase I; however, we found no further evidence to support any of these as the target for zwittermicin A. This study elucidated the genetic mechanisms of zwittermicin A resistance in E. coli. The results suggest that deltapsi drives zwittermicin A uptake, and that, unlike other antibiotics for which resistance maps in rpoB or rpoC, zwittermicin A does not cause the rapid cessation of DNA or RNA synthesis, suggesting a unique mechanism of antibiosis.

Many strains of Bacillus cereus produce zwittermicin A, a novel antibiotic that contributes to the ability of B. cereus to suppress certain plant diseases. The purpose of this study was to identify molecular indicators of zwittermicin A production in B, cereus strains, contribute to an understanding of the ecology and evolution of this group of bacteria, and identify potential agents for control of plant disease. The fatty acid composition of 20 strains known to be zwittermicin A producers and 20 strains known to be non-producers was determined. Cluster analysis of the fatty acid methyl ester (FAME) profiles revealed that zwittermicin A producers grouped together in two clusters, apart from most non-producers. Discriminant analysis of the FAME profiles generated models that correctly predicted the zwittermicin A-production phenotype in 17 of 20 zwittermicin A producers and 17 of 20 non-producers. Sixteen random oligonucleotide primers were tested in PCR, and one primer was identified that generated a fragment of 0.48 kb or 0.49 kb from total DNA from 26 of 28 strains known to produce zwittermicin A, whereas PCR with this primer did not generate bands of that size from 16 of 20 non-producing strains. PCR with primers designed to amplify zmaR, a gene from B. cereus that confers resistance to zwittermicin A, generated DNA fragments of 1.1 kb and 1.0 kb in all 29 zwittermicin A-producing strains tested, amplified a fragment of 0.3 kb in some of the zwittermicin A-producing strains, and amplified no fragments in 20 of 23 non-producing strains in a stock collection of B. cereus strains. The zmaR primers were tested for their ability to identify new zwittermicin A-producing isolates of B. cereus from two soils. All 12 of the isolates that produced the banding pattern characteristic of this primer pair produced zwittermicin A, and none of the 12 isolates that did not have the banding pattern produced detectable zwittermicin A. Seven of the 12 isolates initially identified as zwittermicin A producers with the zmaR primers significantly suppressed damping-off of alfalfa, whereas only one of the non-producers suppressed this disease. The results show that FAME and PCR analyses distinguish B. cereus strains that produce zwittermicin A from other B. cereus strains, that PCR with the primers designed to amplify zmaR is the most reliable method of those tested for identification of zwittermicin A producers, and that this method can be used to identify new strains with disease-suppressive activity.

Bacillus cereus UW85 produces two antibiotics that contribute to its ability to suppress certain plant diseases (L. Silo-Suh, B. Lethbridge, S. J. Raffel, H. He, J. Clardy, and J. Handelsman, Appl. Environ. Microbiol. 60:2023-2030, 1994). To enhance the understanding of disease suppression by UW85, we determined the chemical structure, regulation, and the target range of one of the antibiotics. The antibiotic was identified as 3-amino-3-deoxy-D-glucose, also known as kanosamine. Kanosamine was highly inhibitory to growth of plant-pathogenic oomycetes and moderately inhibitory to certain fungi and inhibited few bacterial species tested. Maximum accumulation of kanosamine in B. cereus UW85 culture supernatants coincided with sporulation. Kanosamine accumulation was enhanced by the addition of ferric iron and suppressed by addition of phosphate to rich medium. Kanosamine accumulation was also enhanced more than 300% by the addition of alfalfa seedling exudate to minimal medium.

Zwittermicin A is a novel aminopolyol antibiotic produced by Bacillus cereus that is active against diverse bacteria and lower eukaryotes (L.A. Silo-Suh, B.J. Lethbridge, S.J. Raffel, H. He, J. Clardy, and J. Handelsman, Appl. Environ. Microbiol. 60:2023-2030, 1994). To identify a determinant for resistance to zwittermicin A, we constructed a genomic library from B. cereus UW85, which produces zwittermicin A, and screened transformants of Escherichia coli DH5alpha, which is sensitive to zwittermicin A, for resistance to zwittermicin A. Subcloning and mutagenesis defined a genetic locus, designated zmaR, on a 1.2-kb fragment of DNA that conferred zwittermicin A resistance on E. coli. A DNA fragment containing zmaR hybridized to a corresponding fragment of genomic DNA from B. cereus UW85. Corresponding fragments were not detected in mutants of B. cereus UW85 that were sensitive to zwittermicin A, and the plasmids carrying zmaR restored resistance to the zwittermicin A-sensitive mutants, indicating that zmaR was deleted in the zwittermicin A-sensitive mutants and that zmaR is functional in B. cereus. Sequencing of the 1.2-kb fragment of DNA defined an open reading frame, designated ZmaR. Neither the nucleotide sequence nor the predicted protein sequence had significant similarity to sequences in existing databases. Cell extracts from an E. coli strain carrying zmaR contained a 43.5-kDa protein whose molecular mass and N-terminal sequence matched those of the protein predicted by the zmaR sequence. The results demonstrate that we have isolated a gene, zmaR, that encodes a zwIttermicin A resistance determinant that is functional in both B. cereus and E. coli.

Bacillus cereus strain UW85 produces an antibiotic, designated zwittermicin A, that is associated with the ability of UW85 to suppress damping-off disease of alfalfa (Medicago sativa) caused by the oomycete pathogen, Phytophthora medicaginis, in a laboratory bioassay. We have identified certain culture conditions that promote or suppress zwittermicin A accumulation by UW85. Maximum accumulation was detected in supernatants of trypticase soy broth cultures after sporulation, which is when cultures of UW85 provide the greatest suppression of damping-off on alfalfa. Inorganic amendments to trypticase soy broth cultures had the following effects on zwittermicin A accumulation and disease suppression: phosphate (50 mM or more) reduced zwittermicin A accumulation and disease suppression; ferric iron (0.25-1.0 mM) enhanced zwittermicin A accumulation and disease suppression; micronutrients (manganese, boron, copper, molybdenum, zinc) had no effect on zwittermicin A accumulation or disease suppression. Cultures of UW85 grown in chemically defined minimal medium supplemented with casein hydrolysate or grown in defined medium containing the minimal requirements for growth supplemented with five amino acids (Gln, Arg, Met, Phe, Ile) accumulated zwittermicin A.(ABSTRACT TRUNCATED AT 250 WORDS)

Bacillus cereus UW85 produces a novel aminopolyol antibiotic, zwittermicin A, that contributes to the ability of UW85 to suppress damping-off of alfalfa caused by Phytophthora medicaginis. UW85 produces a second antibiotic, provisionally designated antibiotic B, which also contributes to suppression of damping-off but has not been structurally defined yet and is less potent than zwittermicin A. The purpose of this study was to isolate genetically diverse strains of B. cereus that produce zwittermicin A and suppress disease. We found that most isolates of B. cereus that were sensitive to phage P7 or inhibited the growth of Erwinia herbicola produced zwittermicin A; therefore, phage typing and E. herbicola inhibition provided indirect, but rapid screening tests for identification of zwittermicin A-producing isolates. We used these tests to screen a collection of 4,307 B. cereus and Bacillus thuringiensis isolates obtained from bacterial stock collections and from diverse soils collected in Honduras, Panama, Australia, The Netherlands, and the United States. A subset of the isolates screened by the P7 sensitivity and E. herbicola inhibition tests were assayed directly for production of zwittermicin A, leading to the identification of 57 isolates that produced zwittermicin A; 41 of these isolates also produced antibiotic B. Eight isolates produced antibiotic B but not zwittermicin A. The assay for phage P7 sensitivity was particularly useful because of its simplicity and rapidity and because 22 of the 23 P7-sensitive isolates tested produced zwittermicin A. However, not all zwittermicin A-producing isolates were sensitive to P7, and the more labor-intensive E. herbicola inhibition assay identified a larger proportion of the zwittermicin A producers.(ABSTRACT TRUNCATED AT 250 WORDS)

Cultures and culture filtrates of Bacillus cereus UW85 suppress damping-off of alfalfa caused by Phytophthora medicaginis. We studied the role in disease suppression of two antibiotics from culture filtrates of UW85 that reversibly inhibited growth of P. medicaginis. We purified the two antibiotics by cation-exchange chromatography and high-voltage paper electrophoresis and showed that one of them, designated zwittermicin A, was an aminopolyol of 396 Da that was cationic at pH 7.0; the second, designated antibiotic B, appeared to be an aminoglycoside containing a disaccharide. Both antibiotics prevented disease of alfalfa seedlings caused by P. medicaginis. Purified zwittermicin A reversibly reduced elongation of germ tubes derived from cysts of P. medicaginis, and antibiotic B caused swelling of the germ tubes. Mutants generated with Tn917 or mitomycin C treatment were screened either for antibiotic accumulation in an agar plate diffusion assay or for the ability to suppress damping-off disease of alfalfa. Of 2,682 mutants screened for antibiotic accumulation, 5 mutants were substantially reduced in antibiotic accumulation and disease-suppressive activity. Of the 1,700 mutants screened for disease-suppressive activity, 3 mutants had reduced activity and they accumulated less of both antibiotics than did the parent strain. The amount of antibiotic accumulated by the mutants was significantly correlated with the level of disease suppression. Addition of either zwittermicin A or antibiotic B to alfalfa plants inoculated with a culture of a nonsuppressive mutant resulted in disease suppression. These results demonstrate that B. cereus UW85 produces two fungistatic antibiotics that contribute to suppression of damping-off disease of alfalfa.

A transition to ambulatory preoperative antibiotic bowel preparation was carried out. The protocol included a liquid diet for 40 hours preceding surgery and coordination of purging with buffered oral saline laxative, 45 mL containing 8 g sodium phosphate and 22 g sodium biphosphate (Fleet Phospho-Soda, C.B. Fleet Co, Lynchburg, Va) and bisacodyl preparation with an oral erythromycin base-neomycin routine. Enemas were omitted. Personnel in the preoperative evaluation center had the responsibility of instructing patients, distributing directions and drugs, and reviewing for compliance and possible problems during the preoperative period. All patients scheduled for any of a variety of gastrointestinal procedures, as well as some other complex operations, were included in this study. Follow-up data were obtained. Surgeons' comments regarding efficacy were highly favorable. In only five cases was there comment regarding liquid stool, and this was no impediment to surgery. This incidence was comparable with that of the inpatient experience, as was the spectrum of postoperative complications. Transfer of responsibility to the department proceeded with ease. Results were entirely comparable with those of the former inpatient experience.