Skin-associated Corynebacterium amycolatum shares cobamides.

The underlying interactions that occur to maintain skin microbiome composition, function, and overall skin health are largely unknown. Often, these types of interactions are mediated by microbial metabolites. Cobamides, the vitamin B12 family of cofactors, are essential for metabolism in many bacteria, but are only synthesized by a small fraction of prokaryotes, including certain skin-associated species. Therefore, we hypothesize that cobamide sharing mediates skin community dynamics. Preliminary work predicts that several skin-associated Corynebacterium species encode de novo cobamide biosynthesis and that their abundance is associated with skin microbiome diversity. Here, we show that commensal Corynebacterium amycolatum produces cobamides and that this synthesis can be tuned by cobalt limitation. To demonstrate cobamide sharing by C. amycolatum, we employed a co-culture assay using an E. coli cobamide auxotroph and show that C. amycolatum produces sufficient cobamides to support E. coli growth, both in liquid co-culture and when separated spatially on solid medium. We also generated a C. amycolatum non-cobamide-producing strain (cob-) using UV mutagenesis that contains mutated cobamide biosynthesis genes cobK and cobO and confirm that disruption of cobamide biosynthesis abolishes support of E. coli growth through cobamide sharing. Our study provides a unique model to study metabolite sharing by microorganisms, which will be critical for understanding the fundamental interactions that occur within complex microbiomes and for developing approaches to target the human microbiota for health advances.

Symbioses: a key driver of insect physiological processes, ecological interactions, evolutionary diversification, and impacts on humans.

Symbiosis is receiving increased attention among all aspects of biology because of the unifying themes it helps construct across ecological, evolutionary, developmental, semiochemical, and pest management theory. Insects show a vast array of symbiotic relationships with a wide diversity of microorganisms. These relationships may confer a variety of benefits to the host (macrosymbiont), such as direct or indirect nutrition, ability to counter the defenses of plant or animal hosts, protection from natural enemies, improved development and reproduction, and communication. Benefits to the microsymbiont (including a broad range of fungi, bacteria, mites, nematodes, etc.) often include transport, protection from antagonists, and protection from environmental extremes. Symbiotic relationships may be mutualistic, commensal, competitive, or parasitic. In many cases, individual relationships may include both beneficial and detrimental effects to each partner during various phases of their life histories or as environmental conditions change. The outcomes of insect-microbial interactions are often strongly mediated by other symbionts and by features of the external and internal environment. These outcomes can also have important effects on human well being and environmental quality, by affecting agriculture, human health, natural resources, and the impacts of invasive species. We argue that, for many systems, our understanding of symbiotic relationships will advance most rapidly where context dependency and multipartite membership are integrated into existing conceptual frameworks. Furthermore, the contribution of entomological studies to overall symbiosis theory will be greatest where preoccupation with strict definitions and artificial boundaries is minimized, and integration of emerging molecular and quantitative techniques is maximized. We highlight symbiotic relations involving bark beetles to illustrate examples of the above trends.

Molecular biological access to the chemistry of unknown soil microbes: a new frontier for natural products.

Cultured soil microorganisms have provided a rich source of natural-product chemistry. Because only a tiny fraction of soil microbes from soil are readily cultured, soil might be the greatest untapped resource for novel chemistry. The concept of cloning the metagenome to access the collective genomes and the biosynthetic machinery of soil microflora is explored here.

Archaeal and bacterial communities across a chronosequence of drained lake basins in Arctic Alaska.

We examined patterns in soil microbial community composition across a successional gradient of drained lake basins in the Arctic Coastal Plain. Analysis of 16S rRNA gene sequences revealed that methanogens closely related to Candidatus 'Methanoflorens stordalenmirensis' were the dominant archaea, comprising >50% of the total archaea at most sites, with particularly high levels in the oldest basins and in the top 57 cm of soil (active and transition layers). Bacterial community composition was more diverse, with lineages from OP11, Actinobacteria, Bacteroidetes, and Proteobacteria found in high relative abundance across all sites. Notably, microbial composition appeared to converge in the active layer, but transition and permafrost layer communities across the sites were significantly different to one another. Microbial biomass using fatty acid-based analysis indicated that the youngest basins had increased abundances of gram-positive bacteria and saprotrophic fungi at higher soil organic carbon levels, while the oldest basins displayed an increase in only the gram-positive bacteria. While this study showed differences in microbial populations across the sites relevant to basin age, the dominance of Candidatus 'M. stordalenmirensis' across the chronosequence indicates the potential for changes in local carbon cycling, depending on how these methanogens and associated microbial communities respond to warming temperatures.

The Earth's bounty: assessing and accessing soil microbial diversity.

The study of microbial diversity represents a major opportunity for advances in biology and biotechnology. Recent progress in molecular microbial ecology shows that the extent of microbial diversity in nature is far greater than previously thought. Here, we discuss methods to analyse microorganisms from natural environments without culturing them and new approaches for gaining access to the genetic and chemical resources of these microorganisms.

chvA locus may be involved in export of neutral cyclic beta-1,2-linked D-glucan from Agrobacterium tumefaciens.

Extracellular and intracellular neutral beta-1,2-linked D-glucan content was determined in a virulent, attachment-deficient mutants of Agrobacterium tumefaciens that map in the chvA locus. chvA mutants contained approximately the same amount of intracellular glucan as cells of the virulent control strain A759, but released into the culture medium only 2% of the glucan released by strain A759. Introduction of a cosmid carrying the wild-type chv region restored attachment and virulence and restored extracellular glucan production to chvA mutant A2505. Exogenous glucan did not enhance or inhibit attachment or tumorigenesis of the virulent control strain or the chvA or chvB mutants. Our results suggest that the chvA locus is involved in the export of glucan from the cell and that export may be required for tumorigenesis.

rosR, a determinant of nodulation competitiveness in Rhizobium etli.

We previously described a Tn5 mutant of Rhizobium etli strain CE3, designated CE3003, that is decreased in nodulation competitiveness, reduced in competitive growth in the rhizosphere, and has a hydrophobic cell surface (R. S. Araujo, E. A. Robleto, and J. Handelsman, Appl. Environ. Microbiol., 60:1430-1436, 1994). To determine the molecular basis for the mutant phenotypes, we identified a 1.2-kb fragment of DNA derived from the parent that restored the wild-type phenotypes to the mutant. DNA sequence analysis indicated that this 1.2-kb fragment contained a single open reading frame that we designated rosR. The Tn5 insertion in CE3003 was within rosR. We constructed a derivative of CE3 that contained a deletion in rosR, and this mutant was phenotypically indistinguishable from CE3003 in cell surface and competitive characteristics. Based on the nucleotide sequence, the deduced RosR amino acid sequence is 80% identical to that of the Ros protein from Agrobacterium tumefaciens and the MucR protein from Rhizobium meliloti. Both Ros and MucR are transcriptional repressors that contain a putative zinc-finger DNA-binding domain. This study defines a gene, rosR, that is homologous to a family of transcriptional regulators and contributes to nodulation competitiveness of R. etli. Moreover, we established that a single gene affects nodulation competitiveness, competitive growth in the rhizosphere, and cell surface hydrophobicity.

Rhizobium etli CE3 carries vir gene homologs on a self-transmissible plasmid.

RosR is a transcriptional regulator important for determining cell-surface characteristics and nodulation competitiveness in Rhizobium etli CE3. We identified a 15-kb region that contains genes with similarity to members of the virB, virC, virG, and virE operons of Agrobacterium tumefaciens and demonstrated that RosR directly regulates one operon in this region. These genes were located on plasmid pa of R. etli CE3, which is self-transmissible between R. etli and A. tumefaciens.

A vector for promoter trapping in Bacillus cereus.

We constructed a promoter-trap plasmid, pAD123, for Bacillus cereus. This plasmid contains a promoterless gene that encodes a mutant version of the green fluorescent protein, GFPmut3a, that is optimized for fluorescence-activated cell sorting [Cormack, B.P., Valdivia, R.H., Falkow, S., 1996. FACS-optimized mutants of the green fluorescent protein (GFP). Gene 173, 33-38.]. The plasmid replicates and confers drug resistance in both Escherichia coli and B. cereus. We constructed a library in pAD123, which consists of 29000 clones containing chromosomal DNA from B. cereus strain UW85. A portion of the library (988 clones) was screened for GFP expression in B. cereus UW85 using a 96-well microtiter dish assay. GFP expression was detected by visual inspection with a fluorimager. We identified 21 clones as fluorescing in the initial screen, and further characterized these clones by restriction analysis, sequencing, and quantification of fluorescence intensity. Flow cytometry and cell sorting efficiently separated B. cereus cells expressing GFP from a 10000-fold excess of non-expressing cells. Selected clones provided useful markers to follow B. cereus populations on plant surfaces. Our results indicate that GFP and pAD123 are useful tools for identifying regulatory sequences in Bacillus cereus, and that flow cytometry and cell sorting is a useful method for screening large libraries constructed in this vector.

Zwittermicin A biosynthetic cluster.

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.

Effect of canavanine from alfalfa seeds on the population biology of bacillus cereus

Bacillus cereus UW85 suppresses diseases of alfalfa seedlings, although alfalfa seed exudate inhibits the growth of UW85 in culture (J. L. Milner, S. J. Raffel, B. J. Lethbridge, and J. Handelsman, Appl. Microbiol. Biotechnol. 43:685-691, 1995). In this study, we determined the chemical basis for and biological role of the inhibitory activity. All of the alfalfa germ plasm tested included seeds that released inhibitory material. We purified the inhibitory material from one alfalfa cultivar and identified it as canavanine, which was present in the cultivar Iroquois seed exudate at a concentration of 2 mg/g of seeds. Multiple lines of evidence suggested that canavanine activity accounted for all of the inhibitory activity. Both canavanine and seed exudate inhibited the growth of UW85 on minimal medium; growth inhibition by either canavanine or seed exudate was prevented by arginine, histidine, or lysine; and canavanine and crude seed exudate had the same spectrum of activity against B. cereus, Bacillus thuringiensis, and Vibrio cholerae. The B. cereus UW85 populations surrounding canavanine-exuding seeds were up to 100-fold smaller than the populations surrounding non-canavanine-exuding seeds, but canavanine did not affect the growth of UW85 on seed surfaces. The spermosphere populations of canavanine-resistant mutants of UW85 were larger than the spermosphere populations of UW85, but the mutants and UW85 were similar in spermoplane colonization. These results indicate that canavanine exuded from alfalfa seeds affects the population biology of B. cereus.

Cloning and heterologous expression of a natural product biosynthetic gene cluster from eDNA.

[reaction: see text] To study the natural products produced by uncultured microorganisms, an environmental DNA (eDNA) cosmid library was constructed and screened for the heterologous production of small molecules. A blue clone, CSL51, found in the eDNA library produces deoxyviolacein and the broad spectrum antibiotic violacein. The full sequence of the 6.7 kb eDNA violacein gene cluster and the characterization of violacein and deoxyviolacein from an eDNA clone are reported here.

Stabilization of ileostomy position with fascia.

An ileostomy that maintains its protrusion in a stable manner can be fashioned by the technique of Brooke. However, in a significant number of patients, recession or prolapse of the ileostomy occurs, tending to displace an appliance. A safe and effective technique is described whereby the ileostomy is stabilized without danger. A ribbon of fascia, obtained from the abdominal wall, is passed through the mesentery adjacent to the bowel between vessels, at its point of exit from the peritoneum. It is neither wrapped around the bowel nor sutured to it. The ends of the fascia are securely sutured to the peritoneum and transversalis fascia. This secures the position of the ileostomy without the danger of fistula from suturing the bowel wall. It may be used for recession or prolapse. It may be supplemented by passing additional fascia or suture elsewhere in the mesentery or into other available tissue.

Endorectal pull-through operation in adults after colectomy and excision of rectal mucosa.

Ileal endorectal pull-through can be performed in adults after abdominal colectomy and excision of rectal mucosa. The procedure requires a painstaking dissection of the mucous membrane of the rectum to ensure cure of the polyposis or ulcerative colitis for which it is carried out. Some cases of ulcerative colitis are inappropriate for this dissection, and Crohn's disease prohibits the use of this operation. Fifteen operations in adults are described herein. Certain technical maneuvers have been of help to us and these are described. We have favored a diverting ileostomy maintained for 3 months. A pouch, positioned in the rectum, has hastened the return toward normal control. Sphincter control and responses have been uniformly good. Dilatations and sphincter exercises are routinely employed. Strictures are not rare but are amenable to correction by digital stretching.

Salvage after anastomotic leak in Crohn disease. Utilization of a new, simple diverting ileostomy.

It may be possible to manage an anastomotic leak following resection for Crohn disease by drainage, suture, and proximal loop ileostomy. This report describes success with a simple diverting ileostomy.

Experience with ambulatory preoperative bowel preparation at the Johns Hopkins Hospital.

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.

Biocontrol of plant disease: a (gram-) positive perspective.

Biological control offers an environmentally friendly alternative to the use of pesticides for controlling plant diseases. Unfortunately, growers continue to use chemical control over biological agents, and lack of knowledge often contributes to the downfall of a biocontrol agent. Knowledge of the biological environment in which the agent will be used and of how to produce a stable formulation are both critical to successful biocontrol. Certain Gram-positive bacteria have a natural formulation advantage over their Gram-negative counterparts: the spore. Although the Gram-positive bacteria have not been as well represented in the biocontrol literature, their spore-forming abilities and historical industrial uses bode well for biocontrol success. Here we describe several systems utilizing Gram-positive biocontrol agents that have been researched in depth and provide models for the future of biocontrol.

Use of Cluster and Discriminant Analyses to Compare Rhizosphere Bacterial Communities Following Biological Perturbation

We present an approach to comparing the diversity and composition of bacterial communities from different habitats and for identifying which members of a community are most affected by an introduced bacterium. We use this method to explore both previously published and new data from field and growth chamber experiments in which we isolated heterotrophic bacteria from samples of root-free soil, roots of nontreated soybean seedlings, and from the roots of soybean seedlings grown from Bacillus cereus UW85n1-treated seeds. We characterize bacterial isolates for 40 physiological attributes, and grouped the isolates hierarchically using two-stage density-linkage cluster analysis. Multivariate analysis of variance and discriminant analysis of the relative frequencies of the clusters in the soil and rhizosphere habitats were then used to determine whether there were differences among the bacterial communities from the various habitats, and which of the clusters were most useful in discriminating among the communities. We used rarefied estimates of richness as a measure of community diversity in the various habitats. Introduction of UW85n1 affected the composition and/or diversity of rhizosphere communities in three of four experiments.

Modeling dose-response relationships in biological control: partitioning host responses to the pathogen and biocontrol agent.

ABSTRACT Breeding plants to improve the effectiveness of biocontrol agents is a promising approach to enhance disease suppression by microorganisms. Differences in biocontrol efficacy among cultivars suggest there is genetic variation for this trait within crop germplasm. The ability to quantify host differences in support of biological control is influenced by variation in host response to the pathogen and the dose of pathogen and biocontrol agent applied to the host. To assess the contribution of each of these factors to successful biocontrol interactions, we measured disease over a range of pathogen (Pythium) and biocontrol agent (Bacillus cereus UW85) inoculum doses. We fit dose-response models to these data and used model parameter estimates to quantify host differences in response to the pathogen and biocontrol agent. We first inoculated eight plant species separately with three species of Pythium and evaluated three dose-response models for their ability to describe the disease response to pathogen inoculum level. All three models fit well to at least some of the host-pathogen combinations; the hyperbolic saturation model provided the best overall fit. To quantify the host contribution to biological control, we next evaluated these models with data from a tomato assay, using six inbred tomato lines, P. torulosum, and UW85. The lowest dose of pathogen applied revealed the greatest differences in seedling mortality among the inbred lines, ranging from 40 to 80%. The negative exponential (NE) pathogen model gave the best fit to these pathogen data, and these differences corresponded to model parameter values, which quantify pathogen efficiency, of 0.023 and 0.091. At a high pathogen dose, we detected the greatest differences in biocontrol efficacy among the inbred lines, ranging from no effect to a 68% reduction in mortality. The NE pathogen model with a NE biocontrol component, the NE/NE biocontrol model, gave the best fit to these biocontrol data, and these reductions corresponded to model parameter values, which quantify biocontrol efficiency, of 0.00 and 0.038, respectively. There was no correlation between the host response to the pathogen and biocontrol agent for these inbred lines. This work demonstrates the utility of epidemiological modeling approaches for the study of biological control and lays the groundwork to employ manipulation of host genetics to improve biocontrol efficacy.

Regression Analyses for Evaluating the Influence of Bacillus cereus on Alfalfa Yield Under Variable Disease Intensity.

ABSTRACT We developed and tested regression methods to exploit the variability in disease inherent in field experiments, and applied the methods to evaluate strains of Bacillus cereus for biocontrol efficacy. Four B. cereus strains were tested for their effect on alfalfa (Medicago sativa) performance in 16 field trials planted during 1993 to 1996 at multiple sites in Wisconsin. To evaluate performance of the strains, we used the ratio of (metalaxyl response)/(untreated control response) as a measure of disease intensity within the experiments. The ratio of (Bacillus response)/(untreated control response) was then regressed as a function of disease intensity. The slope of the resulting line provides a statistical test to compare performance of the Bacillus strain with that of the untreated seed (H(o): slope = 0) and metalaxyl controls (H(o): slope = 1). Under conditions in which disease occurred, forage yield of plots planted with seed treated with B. cereus strain AS4-12 exceeded yield from the untreated control plots (P = 0.002) and was similar to yield of plots planted with metalaxyl-treated seed (P = 0.14). Yield gain associated with AS4-12 and metalaxyl seed treatment averaged 6.1 +/- 2.8% (+/-standard error) and 3.0 +/- 2.8%, respectively. In contrast to the regression approach, means analysis by analysis of variance did not detect differences among treatments. Three other B. cereus strains either did not increase alfalfa yield or increased yield less than did AS4-12. Metalaxyl and three of the Bacillus strains increased seedling emergence, but the improved stands were not predictive of increased forage yield. In six additional studies conducted for one season in 1997, AS4-12 enhanced yield of two cultivars at diverse locations in Wisconsin, but there was an apparent cultivar-location interaction. A strong correlation between response to AS4-12 and metalaxyl treatment suggests that these treatments controlled similar pathogens, most likely the oomycete pathogens Phytophthora medicaginis and Pythium spp.