The impact of resource dependence of the mechanisms of life on the spatial population dynamics of an in silico microbial community

Biodiversity has a critical impact on ecosystem functionality and stability, and thus the current biodiversity crisis has motivated many studies of the mechanisms that sustain biodiversity, a notable example being non-transitive or cyclic competition. We therefore extend existing microscopic models of communities with cyclic competition by incorporating resource dependence in demographic processes, characteristics of natural systems often oversimplified or overlooked by modellers. The spatially explicit nature of our individual-based model of three interacting species results in the formation of stable spatial structures, which have significant effects on community functioning, in agreement with experimental observations of pattern formation in microbial communities. Published by AIP Publishing

In-Frame and Unmarked Gene Deletions in Burkholderia cenocepacia via an Allelic Exchange System Compatible with Gateway Technology

Burkholderia cenocepacia is an emerging opportunistic pathogen causing life-threatening infections in immunocompromised individuals and in patients with cystic fibrosis, which are often difficult, if not impossible, to treat. Understanding the genetic basis of virulence in this emerging pathogen is important for the development of novel treatment regimes. Generating deletion mutations in genes predicted to encode virulence determinants is fundamental to investigating the mechanisms of pathogenesis. However, there is a lack of appropriate selectable and counter-selectable markers for use in B. cenocepacia, making its genetic manipulation problematic. Here we describe a Gateway-compatible allelic exchange system based on the counter-selectable pheS gene and I-SceI homing endonuclease. The system provides efficiency in cloning homology regions of target genes, and allows the generation of precise and unmarked gene deletions in B. cenocepacia. As a proof of concept, we demonstrate its utility by deleting the Bcam1349 gene, encoding a c-di-GMP responsive regulator protein important for biofilm formation

Carbon starvation of Pseudomonas aeruginosa biofilms selects for dispersal insensitive mutants.

BACKGROUND: Biofilms disperse in response to specific environmental cues, such as reduced oxygen concentration, changes in nutrient concentration and exposure to nitric oxide. Interestingly, biofilms do not completely disperse under these conditions, which is generally attributed to physiological heterogeneity of the biofilm. However, our results suggest that genetic heterogeneity also plays an important role in the non-dispersing population of P. aeruginosa in biofilms after nutrient starvation. RESULTS: In this study, 12.2% of the biofilm failed to disperse after 4 d of continuous starvation-induced dispersal. Cells were recovered from the dispersal phase as well as the remaining biofilm. For 96 h starved biofilms, rugose small colony variants (RSCV) were found to be present in the biofilm, but were not observed in the dispersal effluent. In contrast, wild type and small colony variants (SCV) were found in high numbers in the dispersal phase. Genome sequencing of these variants showed that most had single nucleotide mutations in genes associated with biofilm formation, e.g. in wspF, pilT, fha1 and aguR. Complementation of those mutations restored starvation-induced dispersal from the biofilms. Because c-di-GMP is linked to biofilm formation and dispersal, we introduced a c-di-GMP reporter into the wild-type P. aeruginosa and monitored green fluorescent protein (GFP) expression before and after starvation-induced dispersal. Post dispersal, the microcolonies were smaller and significantly brighter in GFP intensity, suggesting the relative concentration of c-di-GMP per cell within the microcolonies was also increased. Furthermore, only the RSCV showed increased c-di-GMP, while wild type and SCV were no different from the parental strain. CONCLUSIONS: This suggests that while starvation can induce dispersal from the biofilm, it also results in strong selection for mutants that overproduce c-di-GMP and that fail to disperse in response to the dispersal cue, starvation

A broad range quorum sensing inhibitor working through sRNA inhibition

Abstract For the last decade, chemical control of bacterial virulence has received considerable attention. Ajoene, a sulfur-rich molecule from garlic has been shown to reduce expression of key quorum sensing regulated virulence factors in the opportunistic pathogen Pseudomonas aeruginosa. Here we show that the repressing effect of ajoene on quorum sensing occurs by inhibition of small regulatory RNAs (sRNA) in P. aeruginosa as well as in Staphylococcus aureus, another important human pathogen that employs quorum sensing to control virulence gene expression. Using various reporter constructs, we found that ajoene lowered expression of the sRNAs RsmY and RsmZ in P. aeruginosa and the small dual-function regulatory RNA, RNAIII in S. aureus, that controls expression of key virulence factors. We confirmed the modulation of RNAIII by RNA sequencing and found that the expression of many QS regulated genes encoding virulence factors such as hemolysins and proteases were lowered in the presence of ajoene in S. aureus. Importantly, our findings show that sRNAs across bacterial species potentially may qualify as targets of anti-virulence therapy and that ajoene could be a lead structure in search of broad-spectrum compounds transcending the Gram negative-positive borderline

In silico analyses of metagenomes from human atherosclerotic plaque samples

Background Through several observational and mechanistic studies, microbial infection is known to promote cardiovascular disease. Direct infection of the vessel wall, along with the cardiovascular risk factors, is hypothesized to play a key role in the atherogenesis by promoting an inflammatory response leading to endothelial dysfunction and generating a proatherogenic and prothrombotic environment ultimately leading to clinical manifestations of cardiovascular disease, e.g., acute myocardial infarction or stroke. There are many reports of microbial DNA isolation and even a few studies of viable microbes isolated from human atherosclerotic vessels. However, high-resolution investigation of microbial infectious agents from human vessels that may contribute to atherosclerosis is very limited. In spite of the progress in recent sequencing technologies, analyzing host-associated metagenomes remain a challenge. Results To investigate microbiome diversity within human atherosclerotic tissue samples, we employed high-throughput metagenomic analysis on: (1) atherosclerotic plaques obtained from a group of patients who underwent endarterectomy due to recent transient cerebral ischemia or stroke. (2) Presumed stabile atherosclerotic plaques obtained from autopsy from a control group of patients who all died from causes not related to cardiovascular disease. Our data provides evidence that suggest a wide range of microbial agents in atherosclerotic plaques, and an intriguing new observation that shows these microbiota displayed differences between symptomatic and asymptomatic plaques as judged from the taxonomic profiles in these two groups of patients. Additionally, functional annotations reveal significant differences in basic metabolic and disease pathway signatures between these groups. Conclusions We demonstrate the feasibility of novel high-resolution techniques aimed at identification and characterization of microbial genomes in human atherosclerotic tissue samples. Our analysis suggests that distinct groups of microbial agents might play different roles during the development of atherosclerotic plaques. These findings may serve as a reference point for future studies in this area of research

Utilization and control of ecological interactions in polymicrobial infections and community-based microbial cell factories

Microbial activities are most often shaped by interactions between co-existing microbes within mixed-species communities. Dissection of the molecular mechanisms of species interactions within communities is a central issue in microbial ecology, and our ability to engineer and control microbial communities depends, to a large extent, on our knowledge of these interactions. This review highlights the recent advances regarding molecular characterization of microbe-microbe interactions that modulate community structure, activity, and stability, and aims to illustrate how these findings have helped us reach an engineering-level understanding of microbial communities in relation to both human health and industrial biotechnology

Burkholderia Type VI Secretion Systems Have Distinct Roles in Eukaryotic and Bacterial Cell Interactions

Bacteria that live in the environment have evolved pathways specialized to defend against eukaryotic organisms or other bacteria. In this manuscript, we systematically examined the role of the five type VI secretion systems (T6SSs) of Burkholderia thailandensis (B. thai) in eukaryotic and bacterial cell interactions. Consistent with phylogenetic analyses comparing the distribution of the B. thai T6SSs with well-characterized bacterial and eukaryotic cell-targeting T6SSs, we found that T6SS-5 plays a critical role in the virulence of the organism in a murine melioidosis model, while a strain lacking the other four T6SSs remained as virulent as the wild-type. The function of T6SS-5 appeared to be specialized to the host and not related to an in vivo growth defect, as ΔT6SS-5 was fully virulent in mice lacking MyD88. Next we probed the role of the five systems in interbacterial interactions. From a group of 31 diverse bacteria, we identified several organisms that competed less effectively against wild-type B. thai than a strain lacking T6SS-1 function. Inactivation of T6SS-1 renders B. thai greatly more susceptible to cell contact-induced stasis by Pseudomonas putida, Pseudomonas fluorescens and Serratia proteamaculans—leaving it 100- to 1000-fold less fit than the wild-type in competition experiments with these organisms. Flow cell biofilm assays showed that T6S-dependent interbacterial interactions are likely relevant in the environment. B. thai cells lacking T6SS-1 were rapidly displaced in mixed biofilms with P. putida, whereas wild-type cells persisted and overran the competitor. Our data show that T6SSs within a single organism can have distinct functions in eukaryotic versus bacterial cell interactions. These systems are likely to be a decisive factor in the survival of bacterial cells of one species in intimate association with those of another, such as in polymicrobial communities present both in the environment and in many infections

Phenotypic Variation and Bistable Switching in Bacteria

Microbial research generally focuses on clonal populations. However, bacterial cells with identical genotypes frequently display different phenotypes under identical conditions. This microbial cell individuality is receiving increasing attention in the literature because of its impact on cellular differentiation, survival under selective conditions, and the interaction of pathogens with their hosts. It is becoming clear that stochasticity in gene expression in conjunction with the architecture of the gene network that underlies the cellular processes can generate phenotypic variation. An important regulatory mechanism is the so-called positive feedback, in which a system reinforces its own response, for instance by stimulating the production of an activator. Bistability is an interesting and relevant phenomenon, in which two distinct subpopulations of cells showing discrete levels of gene expression coexist in a single culture. In this chapter, we address techniques and approaches used to establish phenotypic variation, and relate three well-characterized examples of bistability to the molecular mechanisms that govern these processes, with a focus on positive feedback.