Linear plasmids and chromosomes of the bacterial genus Streptomyces have proteins of unknown characteristics and function linked covalently to their 5 DNA termini. We purified protein attached to the end of the pSLA2 linear plasmid of Streptomyces rochei, determined the N-terminal amino acid sequence, and used this information to clone corresponding genes from a S. rochei cosmid library. Three separate terminal protein genes (here designated as tpgR1, tpgR2, and tpgR3), which map to the S. rochei chromosome and to 100-kb and 206-kb linear plasmids contained in S. rochei, were isolated and found to encode a family of similar but distinct 21-kD proteins. Using tpgR1 to probe a genomic DNA library of Streptomyces lividans ZX7, whose linear chromosome can undergo transition to a circular form, we isolated a S. lividans chromosomal gene (tpgL) that we found specifies a protein closely related to, and functionally interchangeable with, TpgR proteins for pSLA2 maintenance in S. lividans. Mutation of tpgL precluded propagation of the pSLA2 plasmid in a linear form and also prevented propagation of S. lividans cells that contain linear, but not circular, chromosomes, indicating a specific and essential role for tpg genes in linear DNA replication. Surprisingly, Tpg proteins were observed to contain a reverse transcriptase-like domain rather than sequences in common with proteins that attach covalently to the termini of linear DNA replicons.

Periodontal diseases are among the most prevalent worldwide, but largely silent, chronic diseases. They affect the tooth-supporting tissues with multiple ramifications on life quality. Their early diagnosis is still challenging, due to lack of appropriate molecular diagnostic methods. Saliva offers a non-invasively collectable reservoir of clinically relevant biomarkers, which, if utilized efficiently, could facilitate early diagnosis and monitoring of ongoing disease. Despite several novel protein markers being recently enlisted by discovery proteomics, their routine diagnostic application is hampered by the lack of validation platforms that allow for rapid, accurate and simultaneous quantification of multiple proteins in large cohorts. Here we carried out a pipeline of two proteomic platforms; firstly, we applied open ended label-free quantitative (LFQ) proteomics for discovery in saliva ( = 67, including individuals with health, gingivitis, and periodontitis), followed by selected-reaction monitoring (SRM)-targeted proteomics for validation in an independent cohort ( = 82). The LFQ platform led to the discovery of 119 proteins with at least 2-fold significant difference between health and disease. The 65 proteins chosen for the subsequent SRM platform included 50 functionally related proteins derived from the significantly enriched processes of the LFQ data, 11 from literature-mining, and four house-keeping ones. Among those, 60 were reproducibly quantifiable proteins (92% success rate), represented by a total of 143 peptides. Machine-learning modeling led to a narrowed-down panel of five proteins of high predictive value for periodontal diseases with maximum area under the receiver operating curve >0.97 (higher in disease: Matrix metalloproteinase-9, Ras-related protein-1, Actin-related protein 2/3 complex subunit 5; lower in disease: Clusterin, Deleted in Malignant Brain Tumors 1). This panel enriches the pool of credible clinical biomarker candidates for diagnostic assay development. Yet, the quantum leap brought into the field of periodontal diagnostics by this study is the application of the biomarker discovery-through-verification pipeline, which can be used for validation in further cohorts.

Bidirectional replication ofStreptomyces species have multiple biological properties that have made them important subjects for the study of mechanisms that regulate morphological and biochemical development in prokaryotes. Their complex life cycle, the high degree of cellular organization and morphological differentiation that exists within their colonies, and the genetic control mechanisms that regulate these events and processes have been of great biological interest (for review, see Champness and Chater 1994;Hopwood et al. 1995). Streptomyces synthesize a multitude of antimicrobial compounds and other agents used widely in medicine and agriculture (Chater 1992;Hopwood et al. 1995). Additionally, the presence in these organisms of plasmids and chromosomes that are linear, but which can circularize readily, has provided, and is continuing to provide, an attractive experimental system for fundamental investigations of telomere function and replicon evolution (Shiffman and Cohen 1992;Chang and Cohen 1994;Chang et al. 1996;Chen 1996;Lin and Chen 1997;Volff et al. 1997;Huang et al. 1998;Qin andCohen 1998, 2000; Altenbuchner 1998, 2000;Wang et al. 1999;Bao and Cohen 2001;Yang and Losick 2001;Chen et al. 2002;Yang et al. 2002).The replication of Streptomyces linear plasmids has been shown to proceed divergently from a site located near the center of the molecule and to generate 3Ј-leading-strand overhangs at the telomeres (Chang and Cohen 1994). The recessed 5Ј ends of the lagging strands produced by the joining together of Okazaki fragments (Kurosawa et al. 1975) are then extended (i.e., "patched") to produce full-length duplex DNA molecules (Chang and Cohen 1994). As Streptomyces linear chromosomes and linear plasmids have similar termini (Huang et al. 1998;Qin and Cohen 1998), and the linear chromosomes also replicate bidirectionally from an internal origin of repli-

BackgroundPeriodontal diseases are polymicrobial diseases that cause the inflammatory destruction of the tooth-supporting (periodontal) tissues. Their initiation is attributed to the formation of subgingival biofilms that stimulate a cascade of chronic inflammatory reactions by the affected tissue. The Gram-negative anaerobes Porphyromonas gingivalis, Tannerella forsythia and Treponema denticola are commonly found as part of the microbiota of subgingival biofilms, and they are associated with the occurrence and severity of the disease. P. gingivalis expresses several virulence factors that may support its survival, regulate its communication with other species in the biofilm, or modulate the inflammatory response of the colonized host tissue. The most prominent of these virulence factors are the gingipains, which are a set of cysteine proteinases (either Arg-specific or Lys-specific). The role of gingipains in the biofilm-forming capacity of P. gingivalis is barely investigated. Hence, this in vitro study employed a biofilm model consisting of 10 “subgingival” bacterial species, incorporating either a wild-type P. gingivalis strain or its derivative Lys-gingipain and Arg-gingipan isogenic mutants, in order to evaluate quantitative and qualitative changes in biofilm composition.ResultsFollowing 64 h of biofilm growth, the levels of all 10 species were quantified by fluorescence in situ hybridization or immunofluorescence. The wild-type and the two gingipain-deficient P. gingivalis strains exhibited similar growth in their corresponding biofilms. Among the remaining nine species, only the numbers of T. forsythia were significantly reduced, and only when the Lys-gingipain mutant was present in the biofilm. When evaluating the structure of the biofilm by confocal laser scanning microscopy, the most prominent observation was a shift in the spatial arrangement of T. denticola, in the presence of P. gingivalis Arg-gingipain mutant.ConclusionsThe gingipains of P. gingivalis may qualitatively and quantitatively affect composition of polymicrobial biofilms. The present experimental model reveals interdependency between the gingipains of P. gingivalis and T. forsythia or T. denticola.

Several independent gene clusters containing varying lengths of type I polyketide synthase genes were isolated from ' Streptomyces nanchangensis ' NS3226, a producer of nanchangmycin and meilingmycin. The former is a polyether compound similar to dianemycin and the latter is a macrolide compound similar to milbemycin, which shares the same macrolide ring as avermectin but has different side groups. Clusters A-H spanned about 133, 132, 104, 174, 122, 54, 37 and 59 kb, respectively. Two systems were developed for functional analysis of the gene clusters by gene disruption or replacement. (1) Streptomyces phage φC31 and its derived vectors can infect and lysogenize this strain. (2) pSET152, an Escherichia coli plasmid with φC31 attP site, and pHZ1358, a Streptomyces-Escherichia coli shuttle cosmid vector, both carrying oriT from RP4, can be mobilized from E. coli into NS3226 by conjugation. pHZ1358 was shown to be generally useful for generating mutant strains by gene disruption and replacement in NS3226 as well as in several other Streptomyces strains. A region in cluster A (" 133 kb) seemed to be involved in nanchangmycin production because replacement of several DNA fragments in this region by an apramycin resistance gene [aac3(IV)] gave rise to nanchangmycin non-producing mutants.