Dataset Information

A Linear Plasmid-Like Prophage of Actinomyces odontolyticus Promotes Biofilm Assembly.

ABSTRACT: The human oral cavity is home to a large number of bacteria and bacteriophages (phages). However, the biology of oral phages as members of the human microbiome is not well understood. Recently, we isolated Actinomyces odontolyticus subsp. actinosynbacter strain XH001 from the human oral cavity, and genomic analysis revealed the presence of an intact prophage named xhp1. Here, we demonstrated that xhp1 is a linear plasmid-like prophage, which is a newly identified phage of A. odontolyticus The prophage xhp1 genome is a 35-kb linear double-stranded DNA with 10-bp single-stranded, 3' cohesive ends. xhp1 exists extrachromosomally, with an estimated copy number of 5. Annotation of xhp1 revealed 54 open reading frames, while phylogenetic analysis suggests that it has limited similarity with other phages. xhp1 phage particles can be induced by mitomycin C and belong to the Siphoviridae family, according to transmission electron microscopic examination. The released xhp1 particles can reinfect the xhp1-cured XH001 strain and result in tiny blurry plaques. Moreover, xhp1 promotes XH001 biofilm formation through spontaneous induction and the release of host extracellular DNA (eDNA). In conclusion, we identified a linear plasmid-like prophage of A. odontolyticus, which enhances bacterial host biofilm assembly and could be beneficial to the host for its persistence in the oral cavity.IMPORTANCE The biology of phages as members of the human oral microbiome is understudied. Here, we report the characterization of xhp1, a novel linear plasmid-like prophage identified from a human oral isolate, Actinomyces odontolyticus subsp. actinosynbacter strain XH001. xhp1 can be induced and reinfect xhp1-cured XH001. The spontaneous induction of xhp1 leads to the lysis of a subpopulation of bacterial hosts and the release of eDNA that promotes biofilm assembly, thus potentially contributing to the persistence of A. odontolyticus within the oral cavity.

SUBMITTER: Shen M

PROVIDER: S-EPMC6102993 | biostudies-literature | 2018 Sep

REPOSITORIES: biostudies-literature

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A Linear Plasmid-Like Prophage of Actinomyces odontolyticus Promotes Biofilm Assembly.

Shen Mengyu M Yang Yuhui Y Shen Wei W Cen Lujia L McLean Jeffrey S JS Shi Wenyuan W Le Shuai S He Xuesong X

Applied and environmental microbiology 20180817 17

The human oral cavity is home to a large number of bacteria and bacteriophages (phages). However, the biology of oral phages as members of the human microbiome is not well understood. Recently, we isolated Actinomyces odontolyticus subsp. actinosynbacter strain XH001 from the human oral cavity, and genomic analysis revealed the presence of an intact prophage named xhp1. Here, we demonstrated that xhp1 is a linear plasmid-like prophage, which is a newly identified phage of A. odo ...[more]

PMID: 29915115

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Project description:BackgroundActinomyces naeslundii genospecies 1 and 2 express type-2 fimbriae (FimA subunit polymers) with variant Galbeta binding specificities and Actinomyces odontolyticus a sialic acid specificity to colonize different oral surfaces. However, the fimbrial nature of the sialic acid binding property and sequence information about FimA proteins from multiple strains are lacking.ResultsHere we have sequenced fimA genes from strains of A.naeslundii genospecies 1 (n = 4) and genospecies 2 (n = 4), both of which harboured variant Galbeta-dependent hemagglutination (HA) types, and from A.odontolyticus PK984 with a sialic acid-dependent HA pattern. Three unique subtypes of FimA proteins with 63.8-66.4% sequence identity were present in strains of A. naeslundii genospecies 1 and 2 and A. odontolyticus. The generally high FimA sequence identity (> 97.2%) within a genospecies revealed species specific sequences or segments that coincided with binding specificity. All three FimA protein variants contained a signal peptide, pilin motif, E box, proline-rich segment and an LPXTG sorting motif among other conserved segments for secretion, assembly and sorting of fimbrial proteins. The highly conserved pilin, E box and LPXTG motifs are present in fimbriae proteins from other Gram-positive bacteria. Moreover, only strains of genospecies 1 were agglutinated with type-2 fimbriae antisera derived from A. naeslundii genospecies 1 strain 12104, emphasizing that the overall folding of FimA may generate different functionalities. Western blot analyses with FimA antisera revealed monomers and oligomers of FimA in whole cell protein extracts and a purified recombinant FimA preparation, indicating a sortase-independent oligomerization of FimA.ConclusionThe genus Actinomyces involves a diversity of unique FimA proteins with conserved pilin, E box and LPXTG motifs, depending on subspecies and associated binding specificity. In addition, a sortase independent oligomerization of FimA subunit proteins in solution was indicated.

Project description:The Gram-positive bacterium Actinomyces oris, a key colonizer in the development of oral biofilms, contains 18 LPXTG motif-containing proteins, including fimbrillins that constitute two fimbrial types critical for adherence, biofilm formation, and polymicrobial interactions. Export of these protein precursors, which harbor a signal peptide, is thought to be mediated by the Sec machine and require cleavage of the signal peptide by type I signal peptidases (SPases). Like many Gram-positive bacteria, A. oris expresses two SPases, named LepB1 and LepB2. The latter has been linked to suppression of lethal "glyco-stress," caused by membrane accumulation of the LPXTG motif-containing glycoprotein GspA when the housekeeping sortase srtA is genetically disrupted. Consistent with this finding, we show here that a mutant lacking lepB2 and srtA was unable to produce high levels of glycosylated GspA and hence was viable. However, deletion of neither lepB1 nor lepB2 abrogated the signal peptide cleavage and glycosylation of GspA, indicating redundancy of SPases for GspA. In contrast, the lepB2 deletion mutant failed to assemble the wild-type levels of type 1 and 2 fimbriae, which are built by the shaft fimbrillins FimP and FimA, respectively; this phenotype was attributed to aberrant cleavage of the fimbrillin signal peptides. Furthermore, the lepB2 mutants, including the catalytically inactive S101A and K169A variants, exhibited significant defects in polymicrobial interactions and biofilm formation. Conversely, lepB1 was dispensable for the aforementioned processes. These results support the idea that LepB2 is specifically utilized for processing of fimbrial proteins, thus providing an experimental model with which to study the basis of type I SPase specificity.Sec-mediated translocation of bacterial protein precursors across the cytoplasmic membrane involves cleavage of their signal peptide by a signal peptidase (SPase). Like many Gram-positive bacteria, A. oris expresses two SPases, LepB1 and LepB2. The latter is a genetic suppressor of lethal "glyco-stress" caused by membrane accumulation of glycosylated GspA when the housekeeping sortase srtA is genetically disrupted. We show here that LepB1 and LepB2 are capable of processing GspA, whereas only LepB2 is required for cleavage of fimbrial signal peptides. This is the first example of a type I SPase dedicated to LPXTG motif-containing fimbrial proteins. Thus, A. oris provides an experimental model with which to investigate the specificity mechanism of type I SPases.

Dataset Information

A Linear Plasmid-Like Prophage of Actinomyces odontolyticus Promotes Biofilm Assembly.

Publications

A Linear Plasmid-Like Prophage of Actinomyces odontolyticus Promotes Biofilm Assembly.

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