Characterization and genome comparisons of three Achromobacter phages of the family Siphoviridae.

In this study, we present the characterization and genomic data of three Achromobacter phages belonging to the family Siphoviridae. Phages 83-24, JWX and JWF were isolated from sewage samples in Paris and Braunschweig, respectively, and infect Achromobacter xylosoxidans, an emerging nosocomial pathogen in cystic fibrosis patients. Analysis of morphology and growth parameters revealed that phages 83-24 and JWX have similar properties, both have nearly the same head and tail measurements, and both have a burst size between 85 and 100 pfu/cell. In regard to morphological properties, JWF had a much longer and more flexible tail compared to other phages. The linear double-stranded DNAs of all three phages are terminally redundant and not circularly permutated. The complete nucleotide sequences consist of 81,541 bp for JWF, 49,714 bp for JWX and 48,216 bp for 83-24. Analysis of the genome sequences showed again that phages JWX and 83-24 are quite similar. Comparison to the GenBank database via BLASTN revealed partial similarities to Roseobacter phage RDJL phi1 and Burkholderia phage BcepGomr. In contrast, BLASTN analysis of the genome sequence of phage JWF revealed only few similarities to non-annotated prophage regions in different strains of Burkholderia and Mesorhizobium.

Isolation and characterization of numerous novel phages targeting diverse strains of the ubiquitous and opportunistic pathogen Achromobacter xylosoxidans.

The clinical relevance of nosocomially acquired infections caused by multi-resistant Achromobacter strains is rapidly increasing. Here, a diverse set of 61 Achromobacter xylosoxidans strains was characterized by MultiLocus Sequence Typing and Phenotype MicroArray technology. The strains were further analyzed in regard to their susceptibility to 35 antibiotics and to 34 different and newly isolated bacteriophages from the environment. A large proportion of strains were resistant against numerous antibiotics such as cephalosporines, aminoglycosides and quinolones, whereas piperacillin-tazobactam, ticarcillin, mezlocillin and imipenem were still inhibitory. We also present the first expanded study on bacteriophages of the genus Achromobacter that has been so far a blank slate with respect to phage research. The phages were isolated mainly from several waste water treatment plants in Germany. Morphological analysis of all of these phages by electron microscopy revealed a broad diversity with different members of the order Caudovirales, including the families Siphoviridae, Myoviridae, and Podoviridae. A broad spectrum of different host ranges could be determined for several phages that lysed up to 24 different and in part highly antibiotic resistant strains. Molecular characterisation by DNA restriction analysis revealed that all phages contain linear double-stranded DNA. Their restriction patterns display distinct differences underlining their broad diversity.

First genome sequences of Achromobacter phages reveal new members of the N4 family.

BACKGROUND: Multi-resistant Achromobacter xylosoxidans has been recognized as an emerging pathogen causing nosocomially acquired infections during the last years. Phages as natural opponents could be an alternative to fight such infections. Bacteriophages against this opportunistic pathogen were isolated in a recent study. This study shows a molecular analysis of two podoviruses and reveals first insights into the genomic structure of Achromobacter phages so far. METHODS: Growth curve experiments and adsorption kinetics were performed for both phages. Adsorption and propagation in cells were visualized by electron microscopy. Both phage genomes were sequenced with the PacBio RS II system based on single molecule, real-time (SMRT) technology and annotated with several bioinformatic tools. To further elucidate the evolutionary relationships between the phage genomes, a phylogenomic analysis was conducted using the genome Blast Distance Phylogeny approach (GBDP). RESULTS: In this study, we present the first detailed analysis of genome sequences of two Achromobacter phages so far. Phages JWAlpha and JWDelta were isolated from two different waste water treatment plants in Germany. Both phages belong to the Podoviridae and contain linear, double-stranded DNA with a length of 72329 bp and 73659 bp, respectively. 92 and 89 putative open reading frames were identified for JWAlpha and JWDelta, respectively, by bioinformatic analysis with several tools. The genomes have nearly the same organization and could be divided into different clusters for transcription, replication, host interaction, head and tail structure and lysis. Detailed annotation via protein comparisons with BLASTP revealed strong similarities to N4-like phages. CONCLUSIONS: Analysis of the genomes of Achromobacter phages JWAlpha and JWDelta and comparisons of different gene clusters with other phages revealed that they might be strongly related to other N4-like phages, especially of the Escherichia group. Although all these phages show a highly conserved genomic structure and partially strong similarities at the amino acid level, some differences could be identified. Those differences, e.g. the existence of specific genes for replication or host interaction in some N4-like phages, seem to be interesting targets for further examination of function and specific mechanisms, which might enlighten the mechanism of phage establishment in the host cell after infection.

Genomic and molecular analysis of phage CMP1 from Clavibacter michiganensis subspecies michiganensis.

Bacteriophage CMP1 is a member of the Siphoviridae family that infects specifically the plant-pathogen Clavibacter michiganensis subsp. michiganensis. The linear double- stranded DNA is terminally redundant and not circularly permuted. The complete nucleotide sequence of the bacteriophage CMP1 genome consists of 58,652 bp including the terminal redundant ends of 791 bp. The G+C content of the phage (57%) is significantly lower than that of its host (72.66%). 74 potential open reading frames were identified and annotated by different bioinformatic tools. Two large clusters which encode the early and the late functions could be identified which are divergently transcribed. There are only a few hypothetical gene products with conserved domains and significant similarity to sequences from the databases. Functional analyses confirmed the activity of four gene products, an endonuclease, an exonuclease, a single-stranded DNA binding protein and a thymidylate synthase. Partial genomic sequences of CN77, a phage of Clavibacter michiganensis subsp. nebraskensis, revealed a similar genome structure and significant similarities on the level of deduced amino acid sequences. An endolysin with peptidase activity has been identified for both phages, which may be good tools for disease control of tomato plants against Clavibacter infections.

The endolysins of bacteriophages CMP1 and CN77 are specific for the lysis of Clavibacter michiganensis strains.

Putative endolysin genes of bacteriophages CMP1 and CN77, which infect Clavibacter michiganensis subsp. michiganensis and C. michiganensis subsp. nebraskensis, respectively, were cloned and expressed in Escherichia coli. The His-tagged endolysin of CMP1 consists of 306 amino acids and has a calculated molecular mass of 34.8 kDa, while the His-tagged endolysin of CN77 has 290 amino acids with a molecular mass of 31.9 kDa. The proteins were purified and their bacteriolytic activity was demonstrated. The bacteriolytic activity of both enzymes showed a host range which was limited to the respective C. michiganensis subspecies and did not affect other bacteria, even those closely related to Clavibacter. Due to the high specificity of the CMP1 and CN77 endolysins they may be useful tools for biocontrol of plant-pathogenic C. michiganensis without affecting other bacteria in the soil.

Temperate bacteriophage PhiO18P from an Aeromonas media isolate: characterization and complete genome sequence.

A group of 74 Aeromonas isolates from surface water of three ponds in Bielefeld, Germany was screened for prophage induction after UV irradiation. The phage PhiO18P was induced from the Aeromonas media isolate O18. PhiO18P belongs to the Myoviridae phage family. The complete nucleotide sequence of the double stranded DNA genome of bacteriophage PhiO18P consists of 33,985 bp. The genome has 5' protruding cohesive ends of 16 bases. On the PhiO18P genome 46 open reading frames (orfs) were identified which are organized in the modules integration and regulation, replication, head, packaging, tail and lysis. Additionally the phage DNA includes a methylase gene. Comparison of the genome architecture with those of other bacteriophages revealed significant similarities to the P2 phage family and especially to the prophages of Aeromonas salmonicida and the Vibrio cholerae phage K139.

Plasmid incidence in the subgroups of two bacterial communities.

The plasmid incidence of two bacterial communities from soil and freshwater was determined by endogenous plasmid isolation. The overall plasmid incidence for the communities was about 10%, while the frequency of plasmid-containing members in different subgroups ranged from 0% to 100%. Both communities included a minor population where all members contained several plasmids.

Bacteriophages of freshwater Brevundimonas vesicularis isolates.

Nine strains of Brevundimonas vesicularis were isolated from surface water of three ponds in Bielefeld, Germany. With those strains as indicators seven bacteriophages with different host ranges were isolated. Molecular characterization showed that all phages contained linear double-stranded DNA with a similar genome size of about 37 kb. Restriction analysis and hybridization of phage DNAs revealed that three of these phages are closely related to each other. These phages had morphologies typical of the family Siphoviridae. Their genomes contained cohesive ends. Four phages were classified into the family of Podoviridae. Restriction analysis of the DNAs of these phages did not reveal any similarities. The DNA of these phages were terminally redundant. All phages were unable to transduce plasmids or marker genes.

A functional plasmid-borne rrn operon in soil isolates belonging to the genus Paracoccus.

Plasmid analysis of isolates from a small Paracoccus population revealed that all 15 representatives carried at least one endogenous plasmid of 23 or 15 kb in size, in addition to further plasmids of different sizes. It was shown by restriction analysis and hybridization that the 23 and 15 kb plasmids from the different isolates were identical or very similar to each other. By partial sequencing of pOL18/23, one of the 23 kb plasmids, a complete rrn operon with the structural genes for 16S, 23S and 5S rRNA, two genes for tRNA(Ile) and tRNA(Ala) within the spacer between the 16S and 23S rRNA genes, and a final tRNA(fMet) at the end of the operon were discovered. Expression of a green fluorescent protein gene (gfp) after insertion of a DNA fragment from the region upstream of the rRNA genes into a promoter-probe vector demonstrated that the rrn promoter region is functional. The rrn operon encoded by plasmid pOL18/23 is the first complete rrn operon sequenced from a strain of the genus Paracoccus, and only the second example of an rrn operon on a small plasmid.