Lars Fieseler

Discovery of the novel candidate phylum "Poribacteria" in marine sponges.

ABSTRACT Marine sponges (Porifera) harbor large amounts of commensal microbial communities within the sponge mesohyl. We employed 16S rRNA gene library construction using specific PCR primers to provide insights into the phylogenetic identity of an abundant sponge-associated bacterium that is morphologically characterized by the presence of a membrane-bound nucleoid. In this study, we report the presence of a previously unrecognized evolutionary lineage branching deeply in the domain Bacteria that is moderately related to the Planctomycetes, Verrucomicrobia, and Chlamydia lines of decent. Because members of this lineage showed <75% 16S rRNA gene sequence similarity to known bacterial phyla, we suggest the status of a new candidate phylum, named “Poribacteria”, to acknowledge the affiliation of the new bacterium with sponges. The affiliation of the morphologically conspicuous sponge bacterium with the novel phylogenetic lineage was confirmed by fluorescence in situ hybridization with newly designed probes targeting different sites of the poribacterial 16S rRNA. Consistent with electron microscopic observations of cell compartmentalization, the fluorescence signals appeared in a ring-shaped manner. PCR screening with “Poribacteria”-specific primers gave positive results for several other sponge species, while samples taken from the environment (seawater, sediments, and a filter-feeding tunicate) were PCR negative. In addition to a report for Planctomycetes, this is the second report of cell compartmentalization, a feature that was considered exclusive to the eukaryotic domain, in prokaryotes.

Biocontrol of Salmonella Typhimurium in RTE foods with the virulent bacteriophage FO1-E2.

Foodborne Salmonella infections are a major public health concern worldwide. Bacteriophages offer highly specific and effective biocontrol of such pathogens. We evaluated the broad host range, virulent phage FO1-E2 for reduction of Salmonella Typhimurium in different RTE foods. Samples were spiked with 1×10³ Salmonella cells and treated with 3×10⁸ pfu/g phage, and incubated for 6 days at 8 °C or 15 °C. At 8 °C, no viable cells remained following FO1-E2 application, corresponding to a more than 3 log₁₀ unit reduction. At 15 °C, application of phage lowered S. Typhimurium counts by 5 log units on turkey deli meat and in chocolate milk, and by 3 logs on hot dogs and in seafood. In egg yolk, an effect was observed only after 2 days, but not after 6 days. Phage particles retained their infectivity, although they were readily immobilized by the food matrix, resulting in loss of their ability to diffuse and infect target cells. At the end of the incubation period, phage-resistant Salmonella strains appeared which, however, were not able to compensate for the initial killing effect. Altogether, our data show that virulent phages such as FO1-E2 offer an effective biocontrol measure for Salmonella in foods.

Widespread occurrence and genomic context of unusually small polyketide synthase genes in microbial consortia associated with marine sponges.

ABSTRACT Numerous marine sponges harbor enormous amounts of as-yet-uncultivated bacteria in their tissues. There is increasing evidence that these symbionts play an important role in the synthesis of protective metabolites, many of which are of great pharmacological interest. In this study, genes for the biosynthesis of polyketides, one of the most important classes of bioactive natural products, were systematically investigated in 20 demosponge species from different oceans. Unexpectedly, the sponge metagenomes were dominated by a ubiquitously present, evolutionarily distinct, and highly sponge-specific group of polyketide synthases (PKSs). Open reading frames resembling animal fatty acid genes were found on three corresponding DNA regions isolated from the metagenomes of Theonella swinhoei and Aplysina aerophoba. Their architecture suggests that methyl-branched fatty acids are the metabolic product. According to a phylogenetic analysis of housekeeping genes, at least one of the PKSs belongs to a bacterium of the Deinococcus-Thermus phylum. The results provide new insights into the chemistry of sponge symbionts and allow inference of a detailed phylogeny of the diverse functional PKS types present in sponge metagenomes. Based on these qualitative and quantitative data, we propose a significantly simplified strategy for the targeted isolation of biomedically relevant PKS genes from complex sponge-symbiont associations.

Widespread Distribution of Poribacteria in Demospongiae

ABSTRACT Poribacteria were found in nine sponge species belonging to six orders of Porifera from three oceans. Phylogenetic analysis revealed four distinct poribacterial clades, which contained organisms obtained from several different geographic regions, indicating that the distribution of poribacteria is cosmopolitan. Members of divergent poribacterial clades were also found in the same sponge species in three different sponge genera.

Novel virulent and broad-host-range Erwinia amylovora bacteriophages reveal a high degree of mosaicism and a relationship to Enterobacteriaceae phages.

ABSTRACT A diverse set of 24 novel phages infecting the fire blight pathogen Erwinia amylovora was isolated from fruit production environments in Switzerland. Based on initial screening, four phages (L1, M7, S6, and Y2) with broad host ranges were selected for detailed characterization and genome sequencing. Phage L1 is a member of the Podoviridae, with a 39.3-kbp genome featuring invariable genome ends with direct terminal repeats. Phage S6, another podovirus, was also found to possess direct terminal repeats but has a larger genome (74.7 kbp), and the virus particle exhibits a complex tail fiber structure. Phages M7 and Y2 both belong to the Myoviridae family and feature long, contractile tails and genomes of 84.7 kbp (M7) and 56.6 kbp (Y2), respectively, with direct terminal repeats. The architecture of all four phage genomes is typical for tailed phages, i.e., organized into function-specific gene clusters. All four phages completely lack genes or functions associated with lysogeny control, which correlates well with their broad host ranges and indicates strictly lytic (virulent) lifestyles without the possibility for host lysogenization. Comparative genomics revealed that M7 is similar to E. amylovora virus ΦEa21-4, whereas L1, S6, and Y2 are unrelated to any other E. amylovora phage. Instead, they feature similarities to enterobacterial viruses T7, N4, and ΦEcoM-GJ1. In a series of laboratory experiments, we provide proof of concept that specific two-phage cocktails offer the potential for biocontrol of the pathogen.

Genomic Mining for Novel FADH2-Dependent Halogenases in Marine Sponge-Associated Microbial Consortia

Many marine sponges (Porifera) are known to contain large amounts of phylogenetically diverse microorganisms. Sponges are also known for their large arsenal of natural products, many of which are halogenated. In this study, 36 different FADH2-dependent halogenase gene fragments were amplified from various Caribbean and Mediterranean sponges using newly designed degenerate PCR primers. Four unique halogenase-positive fosmid clones, all containing the highly conserved amino acid motif “GxGxxG”, were identified in the microbial metagenome of Aplysina aerophoba. Sequence analysis of one halogenase-bearing fosmid revealed notably two open reading frames with high homologies to efflux and multidrug resistance proteins. Single cell genomic analysis allowed for a taxonomic assignment of the halogenase genes to specific symbiotic lineages. Specifically, the halogenase cluster S1 is predicted to be produced by a deltaproteobacterial symbiont and halogenase cluster S2 by a poribacterial sponge symbiont. An additional halogenase gene is possibly produced by an actinobacterial symbiont of marine sponges. The identification of three novel, phylogenetically, and possibly also functionally distinct halogenase gene clusters indicates that the microbial consortia of sponges are a valuable resource for novel enzymes involved in halogenation reactions.

The tail-associated depolymerase of Erwinia amylovora phage L1 mediates host cell adsorption and enzymatic capsule removal, which can enhance infection by other phage

The depolymerase enzyme (DpoL1) encoded by the T7-like phage L1 efficiently degrades amylovoran, an important virulence factor and major component of the extracellular polysaccharide (EPS) of its host, the plant pathogen Erwinia amylovora. Mass spectrometry analysis of hydrolysed EPS revealed that DpoL1 cleaves the galactose-containing backbone of amylovoran. The enzyme is most active at pH 6 and 50°C, and features a modular architecture. Removal of 180 N-terminal amino acids was shown not to affect enzyme activity. The C-terminus harbours the hydrolase activity, while the N-terminal domain links the enzyme to the phage particle. Electron microscopy demonstrated that DpoL1-specific antibodies cross-link phage particles at their tails, either lateral or frontal, and immunogold staining confirmed that DpoL1 is located at the tail spikes. Exposure of high-level EPS-producing Er. amylovora strain CFBP1430 to recombinant DpoL1 dramatically increased sensitivity to the Dpo-negative phage Y2, which was not the case for EPS-negative mutants or low-level EPS-producing Er. amylovora. Our findings indicate that enhanced phage susceptibility is based on enzymatic removal of the EPS capsule, normally a physical barrier to Y2 infection, and that use of DpoL1 together with the broad host range, virulent phage Y2 represents an attractive combination for biocontrol of fire blight.

Acanthamoeba feature a unique backpacking strategy to trap and feed on Listeria monocytogenes and other motile bacteria

Despite its prominent role as an intracellular human pathogen, Listeria monocytogenes normally features a saprophytic lifestyle, and shares many environmental habitats with predatory protozoa. Earlier studies claimed that Acanthamoeba may act as environmental reservoirs for L. monocytogenes, whereas others failed to confirm this hypothesis. Our findings support the latter and provide clear evidence that L. monocytogenes is unable to persist in Acanthamoeba castellanii and A. polyphaga. Instead, external Listeria cells are rapidly immobilized on the surface of Acanthamoeba trophozoites, forming large aggregates of densely packed bacteria that we termed backpacks. While the assembly of backpacks is dependent on bacterial motility, flagellation alone is not sufficient. Electron micrographs showed that the aggregates are held together by filaments of likely amoebal origin. Time-lapse microscopy revealed that shortly after the bacteria are collected, the amoeba can change direction of movement, phagocytose the backpack and continue to repeat the process. The phenomenon was also observed with avirulent L. monocytogenes mutants, non-pathogenic Listeria, and other motile bacteria, indicating that formation of backpacks is not specific for L. monocytogenes, and independent of bacterial pathogenicity or virulence. Hence, backpacking appears to represent a unique and highly effective strategy of Acanthamoeba to trap and feed on motile bacteria.

Acanthamoeba release compounds which promote growth of Listeria monocytogenes and other bacteria

Listeria monocytogenes can grow as a saphrophyte in diverse habitats, e.g., soil, rivers, lakes, and on decaying plant material. In these environments, the bacteria are frequently exposed to predatory protozoa such as Acanthamoeba. Although L. monocytogenes is a facultative intracellular pathogen it does not infect or survive intracellular in Acanthamoeba castellanii, unlike several other facultative intracellular bacteria. Instead, motile L. monocytogenes can form large aggregates on amoebal cells and are effectively phagocytosed and eventually digested by Acanthamoeba. Here, we demonstrate that non-motile L. monocytogenes represent a less preferred prey in co-cultures with A. castellanii. Moreover, we found that the presence of Acanthamoeba strongly promotes growth of the bacteria in non-nutrient saline, by releasing nutrients or other growth promoters. Thus, the lack of motility and ability to utilize amoebal metabolites may aid to avoid eradication by amoebal predation in low-nutrient environments.

Rhamnose-Inducible Gene Expression in Listeria monocytogenes

Acid production from rhamnose is a characteristic phenotype of Listeria monocytogenes. We report the identification of the rhamnose transport and utilization operon located at lmo2846 to lmo2851, including the rhamnose-dependent promoter Prha. Expression of reporter genes under control of Prha on a single copy integration vector demonstrated its suitability for inducible gene expression in L. monocytogenes. Transcription initiation from Prha is dose dependent, and a concentration as low as 100 µM rhamnose was found sufficient for induction. Moreover, Prha is subject to glucose catabolite repression, which provides additional options for strict control of expression. Infection of human THP1 macrophages revealed that Prha is repressed in intracellular L. monocytogenes, which is explained by the absence of rhamnose in the cytosol and possible interference by catabolite repression. The Prha promoter provides a novel and useful tool for triggering gene expression in extracellular L. monocytogenes, whereas intracellular conditions prevent transcription from this promoter.