Evolution of Rhodopseudomonas palustris to degrade halogenated aromatic compounds involves changes in pathway regulation and enzyme specificity.

Halogenated aromatic compounds are used in a variety of industrial applications but can be harmful to humans and animals when released into the environment. Microorganisms that degrade halogenated aromatic compounds anaerobically have been isolated but the evolutionary path that they may have taken to acquire this ability is not well understood. A strain of the purple nonsulfur bacterium, Rhodopseudomonas palustris, RCB100, can use 3-chlorobenzoate (3-CBA) as a carbon source whereas a closely related strain, CGA009, cannot. To reconstruct the evolutionary events that enabled RCB100 to degrade 3-CBA, we isolated an evolved strain derived from CGA009 capable of growing on 3-CBA. Comparative whole-genome sequencing of the evolved strain and RCB100 revealed both strains contained large deletions encompassing badM, a transcriptional repressor of genes for anaerobic benzoate degradation. It was previously shown that in strain RCB100, a single nucleotide change in an alicyclic acid coenzyme A ligase gene, named aliA, gives rise to a variant AliA enzyme that has high activity with 3-CBA. When the RCB100 aliA allele and a deletion in badM were introduced into R. palustris CGA009, the resulting strain grew on 3-CBA at a similar rate as RCB100. This work provides an example of pathway evolution in which regulatory constraints were overcome to enable the selection of a variant of a promiscuous enzyme with enhanced substrate specificity.IMPORTANCEBiodegradation of man-made compounds often involves the activity of promiscuous enzymes whose native substrate is structurally similar to the man-made compound. Based on the enzymes involved, it is possible to predict what microorganisms are likely involved in biodegradation of anthropogenic compounds. However, there are examples of organisms that contain the required enzyme(s) and yet cannot metabolize these compounds. We found that even when the purple nonsulfur bacterium, Rhodopseudomonas palustris, encodes all the enzymes required for degradation of a halogenated aromatic compound, it is unable to metabolize that compound. Using adaptive evolution, we found that a regulatory mutation and a variant of promiscuous enzyme with increased substrate specificity were required. This work provides insight into how an environmental isolate evolved to use a halogenated aromatic compound.

Bacterial communities associated with wood rot fungi that use distinct decomposition mechanisms.

Wood decomposer fungi are grouped by how they extract sugars from lignocellulose. Brown rot fungi selectively degrade cellulose and hemicellulose, leaving lignin intact, and white rot fungi degrade all components. Many trees are susceptible to both rot types, giving carbon in Earth's woody biomass, specifically lignin, a flexible fate that is affected not only by the fungal decomposition mechanism but also the associated microbial community. However, little is understood about how rot type may influence the microbial community in decaying wood. In this study, we quantified bacterial communities associated with Fomes fomentarius (white rot) and Fomitopsis betulina (brown rot) found on a shared tree host species, birch (Betula papyrifera). We collected 25 wood samples beneath sporocarps  of F. fomentarius (n = 13) and F. betulina (n = 12) on standing dead trees, and coupled microbial DNA sequencing with chemical signatures of rot type (pH and lignin removal). We found that bacterial communities for both fungi were dominated by Proteobacteria, a commonly reported association. However, rot type exerted significant influence on less abundant taxa in ways that align logically with fungal traits. Amplicon sequence variants (ASVs) were enriched in Firmicutes in white-rotted wood, and were enriched in Alphaproteobacteria, Actinobacteria and Acidobacteria in lower pH brown rot. Our results suggest that wood decomposer strategies may exert significant selection effects on bacteria, or vice versa, among less-abundant taxa that have been overlooked when using abundance as the only measure of influence.

The Response of Paraburkholderia terrae Strains to Two Soil Fungi and the Potential Role of Oxalate.

Fungal-associated Paraburkholderia terrae strains in soil have been extensively studied, but their sensing strategies to locate fungi in soil have remained largely elusive. In this study, we investigated the behavior of five mycosphere-isolated P. terrae strains [including the type-3 secretion system negative mutant BS001-ΔsctD and the type strain DSM 17804T] with respect to their fungal-sensing strategies. The putative role of oxalic acid as a signaling molecule in the chemotaxis toward soil fungi, as well as a potential carbon source, was assessed. First, all P. terrae strains, including the type strain, were found to sense, and show a chemotactic response toward, the different levels of oxalic acid (0.1, 0.5, and 0.8%) applied at a distance. The chemotactic responses were faster and stronger at lower concentrations (0.1%) than at higher ones. We then tested the chemotactic responses of all strains toward exudates of the soil fungi Lyophyllum sp. strain Karsten and Trichoderma asperellum 302 used in different dilutions (undiluted, 1:10, 1:100 diluted) versus the control. All P. terrae strains showed significant directed chemotactic behavior toward the exudate source, with full-strength exudates inciting the strongest responses. In a separate experiment, strain BS001 was shown to be able to grow on oxalate-amended (0.1 and 0.5%) mineral medium M9. Chemical analyses of the fungal secretomes using proton nuclear magnetic resonance (1H NMR), next to high-performance liquid chromatography (HPLC), indeed revealed the presence of oxalic acid (next to glycerol, acetic acid, formic acid, and fumaric acid) in the supernatants of both fungi. In addition, citric acid was found in the Lyophyllum sp. strain Karsten exudates. Given the fact that, next to oxalic acid, the other compounds can also serve as C and energy sources for P. terrae, the two fungi clearly offer ecological benefits to this bacterium. The oxalic acid released by the two fungi may have primarily acted as a signaling molecule, and, as a "second option," a carbon source for P. terrae strains like BS001.

Draft genome sequences of three fungal-interactive Paraburkholderia terrae strains, BS007, BS110 and BS437.

Here, we report the draft genome sequences of three fungal-interactive 10.1601/nm.27008 strains, denoted BS110, BS007 and BS437. Phylogenetic analyses showed that the three strains belong to clade II of the genus 10.1601/nm.1619, which was recently renamed 10.1601/nm.26956. This novel genus primarily contains environmental species, encompassing non-pathogenic plant- as well as fungal-interactive species. The genome of strain BS007 consists of 11,025,273 bp, whereas those of strains BS110 and BS437 have 11,178,081 and 11,303,071 bp, respectively. Analyses of the three annotated genomes revealed the presence of (1) a large suite of substrate capture systems, and (2) a suite of genetic systems required for adaptation to microenvironments in soil and the mycosphere. Thus, genes encoding traits that potentially  confer fungal interactivity were found, such as type 4 pili, type 1, 2, 3, 4 and 6 secretion systems, and biofilm formation (PGA, alginate and pel) and glycerol uptake systems. Furthermore, the three genomes also revealed the presence of a highly conserved five-gene cluster that had previously been shown to be upregulated upon contact with fungal hyphae. Moreover, a considerable number of prophage-like and CRISPR spacer sequences was found, next to genetic systems responsible for secondary metabolite production. Overall, the three 10.1601/nm.27008 strains possess the genetic repertoire necessary for adaptation to diverse soil niches, including those influenced by soil fungi.

Transcriptional Responses of the Bacterium Burkholderia terrae BS001 to the Fungal Host Lyophyllum sp. Strain Karsten under Soil-Mimicking Conditions.

In this study, the mycosphere isolate Burkholderia terrae BS001 was confronted with the soil fungus Lyophyllum sp. strain Karsten on soil extract agar plates in order to examine its transcriptional responses over time. At the initial stages of the experiment (T1-day 3; T2-day 5), contact between both partner organisms was absent, whereas in the final stage (T3-day 8), the two populations made intimate physical contact. Overall, a strong modulation of the strain BS001 gene expression patterns was found. First, the stationary-phase sigma factor RpoS, and numerous genes under its control, were strongly expressed as a response to the soil extract agar, and this extended over the whole temporal regime. In the system, B. terrae BS001 apparently perceived the presence of the fungal hyphae already at the early experimental stages (T1, T2), by strongly upregulating a suite of chemotaxis and flagellar motility genes. With respect to specific metabolism and energy generation, a picture of differential involvement in different metabolic routes was obtained. Initial (T1, T2) up- or downregulation of ethanolamine and mandelate uptake and utilization pathways was substituted by a strong investment, in the presence of the fungus, in the expression of putative metabolic gene clusters (T3). Specifically at T3, five clustered genes that are potentially involved in energy generation coupled to an oxidative stress response, and two genes encoding short-chain dehydrogenases/oxidoreductases (SDR), were highly upregulated. In contrast, the dnaE2 gene (related to general stress response; encoding error-prone DNA polymerase) was transcriptionally downregulated at this stage. This study revealed that B. terrae BS001, from a stress-induced state, resulting from the soil extract agar milieu, responds positively to fungal hyphae that encroach upon it, in a temporally dynamic manner. The response is characterized by phases in which the modulation of (1) chemotaxis, (2) metabolic activity, and (3) oxidative stress responses are key mechanisms.

Chemotaxis and adherence to fungal surfaces are key components of the behavioral response of Burkholderia terrae BS001 to two selected soil fungi.

Burkholderia terrae BS001 has previously been proposed to be a 'generalist' associate of soil fungi, but its strategies of interaction have been largely ignored. Here, we studied the chemotactic behavior of B. terrae BS001 towards Lyophyllum sp. strain Karsten and Trichoderma asperellum 302 and the role of fungal surface molecules in their physical interaction with the bacteria. To assess the involvement of the type 3 secretion system (T3SS), wild-type strain BS001 and T3SS mutant strain BS001-ΔsctD were used in the experiments. First, the two fungi showed divergent behavior when confronted with B. terrae BS001 on soil extract agar medium. Lyophyllum sp. strain Karsten revealed slow growth towards the bacterium, whereas T. asperellum 302 grew avidly over it. Both on soil extract and M9 agar, B. terrae BS001 and BS001-ΔsctD moved chemotactically towards the hyphae of both fungi, with a stronger response to Lyophyllum sp. strain Karsten than to T. asperellum 302. The presence of a progressively increasing glycerol level in the M9 agar enhanced the level of movement. Different oxalic acid concentrations exerted varied effects, with a significantly raised chemotactic response at lower, and a subdued response at higher concentrations. Testing of the adherence of B. terrae BS001 and BS001-ΔsctD to Lyophyllum sp. strain Karsten and to cell envelope-extracted ceramide monohexosides (CMHs) revealed that CMHs in both conidia and hyphae could bind strain BS001 cells. As BS001-ΔsctD adhered significantly less to the CMHs than BS001, the T3SS was presumed to have a role in the interaction. In contrast, such avid adherence was not detected with T. asperellum 302. Thus, B. terrae BS001 shows a behavior characterized by swimming towards Lyophyllum sp. strain Karsten and T. asperellum 302 and attachment to the CMH moiety in the cell envelope, in particular of the former.

The interactions of bacteria with fungi in soil: emerging concepts.

In this chapter, we review the existing literature on bacterial-fungal interactions in soil, exploring the role fungi may play for soil bacteria as providers of hospitable niches. A focus is placed on the mycosphere, i.e., the narrow zone of influence of fungal hyphae on the external soil milieu, in which hypha-associated bacterial cells dwell. Evidence is brought forward for the contention that the hyphae of both mycorrhizal and saprotrophic fungi serve as providers of ecological opportunities in a grossly carbon-limited soil, as a result of their release of carbonaceous compounds next to the provision of a colonizable surface. Soil bacteria of particular nature are postulated to have adapted to such selection pressures, evolving to the extent that they acquired capabilities that allow them to thrive in the novel habitat created by the emerging fungal hyphae. The mechanisms involved in the interactions and the modes of genetic adaptation of the mycosphere dwellers are discussed, with an emphasis on one key mycosphere-adapted bacterium, Burkholderia terrae BS001. In this discussion, we interrogate the positive interactions between soil fungi and bacteria, and refrain from considering negative interactions.

The genome of the fungal-interactive soil bacterium Burkholderia terrae BS001-a plethora of outstanding interactive capabilities unveiled.

Burkholderia terrae strain BS001, obtained as an inhabitant of the mycosphere of Laccaria proxima (a close relative of Lyophyllum sp. strain Karsten), actively interacts with Lyophyllum sp. strain Karsten. We here summarize the remarkable ecological behavior of B. terrae BS001 in the mycosphere and add key data to this. Moreover, we extensively analyze the approximately 11.5-Mb five-replicon genome of B. terrae BS001 and highlight its remarkable features. Seventy-nine regions of genomic plasticity (RGP), that is, 16.48% of the total genome size, were found. One 70.42-kb RGP, RGP76, revealed a typical conjugal element structure, including a full type 4 secretion system. Comparative analyses across 24 related Burkholderia genomes revealed that 95.66% of the total BS001 genome belongs to the variable part, whereas the remaining 4.34% constitutes the core genome. Genes for biofilm formation and several secretion systems, under which a type 3 secretion system (T3SS), were found, which is consistent with the hypothesis that T3SSs play a role in the interaction with Lyophyllum sp. strain Karsten. The high number of predicted metabolic pathways and membrane transporters suggested that strain BS001 can take up and utilize a range of sugars, amino acids and organic acids. In particular, a unique glycerol uptake system was found. The BS001 genome further contains genetic systems for the degradation of complex organic compounds. Moreover, gene clusters encoding nonribosomal peptide synthetases (NRPS) and hybrid polyketide synthases/NRPS were found, highlighting the potential role of secondary metabolites in the ecology of strain BS001. The patchwork of genetic features observed in the genome is consistent with the notion that 1) horizontal gene transfer is a main driver of B. terrae BS001 adaptation and 2) the organism is very flexible in its ecological behavior in soil.

Characterization of a virulent bacteriophage LK1 specific for Citrobacter freundii isolated from sewage water.

Citrobacter freundii is a worldwide emerging nosocomial pathogen with escalating incidence of multidrug resistance. Citrobacter freundii exists in natural environment, especially in health care settings and is difficult to eradicate. Phage therapy is considered as an alternative way of controlling bacterial infections and contaminations. In this study, we have described isolation and characterization of a virulent bacteriophage LK1 capable of specifically infecting Citrobacter freundii. A virulent bacteriophage LK1, specific for Citrobacter freundii was isolated from sewage water sample. TEM showed that phage Lk1 has an icosahedral head 70 nm in diameter and short tail of 17 nm, and can be classified as a member of the Podoviridae family. Restriction analysis indicated that phage LK1 was a dsDNA virus with an approximate genome size of 20-23 kb. Proteomic pattern generated by SDS PAGE using purified LK1 phage particles, revealed three major and six minor protein bands with molecular weight ranging from 25 to 80 kDa. Adsorption rate of LK1 relative to the host bacterium was also determined which showed significant improvement in adsorption with the addition of CaCl2 . In a single step growth experiment, LK1 exhibited a latent period of 24 min and burst size of 801 particle/cell. Moreover, pH and thermal stability of phage LK1 demonstrated a pH range of 5.0-6.0 and phage viability decreased to 0% at 65 °C. When LK1 was used to infect six other clinically isolated pathogenic strains, it showed relatively narrow host range. LK1 was capable of eliciting efficient lysis of Citrobacter freundii, revealing its potential as a non-toxic sanitizer for controlling Citrobacter freundii infection and contamination in both hospital and other public environments.

Bacteriophages and their implications on future biotechnology: a review.

Recently it has been recognized that bacteriophages, the natural predators of bacteria can be used efficiently in modern biotechnology. They have been proposed as alternatives to antibiotics for many antibiotic resistant bacterial strains. Phages can be used as biocontrol agents in agriculture and petroleum industry. Moreover phages are used as vehicles for vaccines both DNA and protein, for the detection of pathogenic bacterial strain, as display system for many proteins and antibodies. Bacteriophages are diverse group of viruses which are easily manipulated and therefore they have potential uses in biotechnology, research, and therapeutics. The aim of this review article is to enable the wide range of researchers, scientists, and biotechnologist who are putting phages into practice, to accelerate the progress and development in the field of biotechnology.