Local adaptation of bacteriophages to their bacterial hosts in soil.

Microbes are incredibly abundant and diverse and are key to ecosystem functioning, yet relatively little is known about the ecological and evolutionary mechanisms that shape their distributions. Bacteriophages, viral parasites that lyse their bacterial hosts, exert intense and spatially varying selection pressures on bacteria and vice versa. We measured local adaptation of bacteria and their associated phages in a centimeter-scale soil population. We first demonstrate that a large proportion of bacteria is sensitive to locally occurring phages. We then show that sympatric phages (isolated from the same 2-gram soil samples as the bacteria) are more infective than are phages from samples some distance away. This study demonstrates the importance of biotic interactions for the small-scale spatial structuring of microbial genetic diversity in soil.

Isolation of new Stenotrophomonas bacteriophages and genomic characterization of temperate phage S1.

Twenty-two phages that infect Stenotrophomonas species were isolated through sewage enrichment and prophage induction. Of them, S1, S3, and S4 were selected due to their wide host ranges compared to those of the other phages. S1 and S4 are temperate siphoviruses, while S3 is a virulent myovirus. The genomes of S3 and S4, about 33 and 200 kb, were resistant to restriction digestion. The lytic cycles lasted 30 min for S3 and about 75 min for S1 and S4. The burst size for S3 was 100 virions/cell, while S1 and S4 produced about 75 virus particles/cell. The frequency of bacteriophage-insensitive host mutants, calculated by dividing the number of surviving colonies by the bacterial titer of a parallel, uninfected culture, ranged between 10(-5) and 10(-6) for S3 and 10(-3) and 10(-4) for S1 and S4. The 40,287-bp genome of S1 contains 48 open reading frames (ORFs) and 12-bp 5' protruding cohesive ends. By using a combination of bioinformatics and experimental evidence, functions were ascribed to 21 ORFs. The morphogenetic and lysis modules are well-conserved, but no lysis-lysogeny switch or DNA replication gene clusters were recognized. Two major clusters of genes with respect to transcriptional orientation were observed. Interspersed among them were lysogenic conversion genes encoding phosphoadenosine phosphosulfate reductase and GspM, a protein involved in the general secretion system II. The attP site of S1 may be located within a gene that presents over 75% homology to a Stenotrophomonas chromosomal determinant.

Fluorescence correlation spectroscopy to study diffusion and reaction of bacteriophages inside biofilms.

In the natural environment, most of the phages that target bacteria are thought to exist in biofilm ecosystems. The purpose of this study was to gain a clearer understanding of the reactivity of these viral particles when they come into contact with bacteria embedded in biofilms. Experimentally, we quantified lactococcal c2 phage diffusion and reaction through model biofilms using in situ fluorescence correlation spectroscopy with two-photon excitation. Correlation curves for fluorescently labeled c2 phage in nonreacting Stenotrophomonas maltophilia biofilms indicated that extracellular polymeric substances did not provide significant resistance to phage penetration and diffusion, even though penetration and diffusion were sometimes restricted because of the noncontractile tail of the viral particle. Fluctuations in the fluorescence intensity of the labeled phage were detected throughout the thickness of biofilms formed by c2-sensitive and c2-resistant strains of Lactococcus lactis but could never be correlated with time, revealing that the phage was immobile. This finding confirmed that recognition binding receptors for the viral particles were present on the resistant bacterial cell wall. Taken together, our results suggest that biofilms may act as "active" phage reservoirs that can entrap and amplify viral particles and protect them from harsh environments.

In situ measurements of viral particles diffusion inside mucoid biofilms.

Fluorescence correlation spectroscopy (FCS) under two-photon excitation was used successfully to characterize the diffusion properties of model virus particles (bacteriophages) in bacterial biofilm of Stenotrophonas maltophilia. The results are compared to those obtained with fluorescent latex beads used as a reference. The FCS data clearly demonstrated the possibility for viral particles to penetrate inside the exopolymeric matrix of mucoid biofilms, and hence to benefit from its protective effect toward antimicrobials (antibiotics and biocides). Microbial biofilms should hence be considered as potential reservoirs of pathogenic viruses, and are probably responsible for numerous persistent viral infections.