Exploring the human hair follicle microbiome.

Human hair follicles (HFs) carry complex microbial communities that differ from the skin surface microbiota. This likely reflects that the HF epithelium differs from the epidermal barrier in that it provides a moist, less acidic, and relatively ultraviolet light-protected environment, part of which is immune-privileged, thus facilitating microbial survival. Here we review the current understanding of the human HF microbiome and its potential physiological and pathological functions, including in folliculitis, acne vulgaris, hidradenitis suppurativa, alopecia areata and cicatricial alopecias. While reviewing the main human HF bacteria (such as Propionibacteria, Corynebacteria, Staphylococci and Streptococci), viruses, fungi and parasites as human HF microbiome constituents, we advocate a broad view of the HF as an integral part of the human holobiont. Specifically, we explore how the human HF may manage its microbiome via the regulated production of antimicrobial peptides (such as cathelicidin, psoriasin, RNAse7 and dermcidin) by HF keratinocytes, how the microbiome may impact on cytokine and chemokine release from the HF, and examine hair growth-modulatory effects of antibiotics, and ask whether the microbiome affects hair growth in turn. We highlight major open questions and potential novel approaches to the management of hair diseases by targeting the HF microbiome.

Regulation of Streptococcus mutans PTS Bio by the transcriptional repressor NigR.

Streptococcus mutans is implicated in human dental caries, and the carbohydrate metabolism of this organism plays an important role in the formation of this disease. Carbohydrate transport and metabolism are essential for the survival of S. mutans in the oral cavity. It is known that a unique phosphoenolpyruvate-sugar phosphotransferase system PTS(B) (io) of S. mutans UA159 is expressed in sucrose-grown biofilms (Mol Oral Microbiol 28: 2013; 114). In this study we analyzed the transcriptional regulation of the operon (O(B) (io) ) encoding the PTS(B) (io) and showed that it was repressed by NigR, a LacI-like transcriptional regulator. Using electro-mobility shift assay, we described two operators to which NigR bound with different affinities. We also identified the transcriptional start site and showed that one of the operators overlaps with the promoter and presumably represses initiation of transcription. Mutational analyses revealed the key nucleotides in the operators required for high-affinity binding of NigR. PTS(B) (io) is expressed in S. mutans biofilms so understanding its regulation may provide improved strategies for caries treatment and prevention.

A novel phosphotransferase system of Streptococcus mutans is responsible for transport of carbohydrates with α-1,3 linkage.

The most common type of carbohydrate-transport system in Streptococcus mutans is the phosphoenolpyruvate-sugar phosphotransferase system (PTS). Fourteen PTS exist in S. mutans UA159. Several studies have shown that microorganisms growing in biofilms express different genes compared with their planktonic counterparts. In this study, we showed that one PTS of S. mutans was expressed in sucrose-grown biofilms. Furthermore, the same PTS was also responsible for the transport and metabolism of disaccharide nigerose (3-O-α-d-glucopyranosyl-d-glucose). Additionally, the results indicate that the studied PTS might be involved in the transport and metabolism of carbohydrates synthesized by glucosyltransferase B and glucosyltransferase C of S. mutans. To our knowledge, this is the first report that shows PTS transport of a disaccharide (and possibly extracellular oligosaccharides) with α-1,3 linkage.

Manganese affects Streptococcus mutans virulence gene expression.

BACKGROUND/AIMS: Studies of trace metals in drinking water and tooth enamel have suggested a caries-promoting potential for manganese (Mn). Additionally, Mn has been shown to be essential for the expression of mutans streptococci virulence factors such as the glucan-binding lectin (GBL) of Streptococcus sobrinus. The Streptococcus mutans glucan-binding protein (Gbp) GbpC is the functional analogue of the S. sobrinus GBL. S. mutans Gbps have been shown to contribute to biofilm architecture and virulence. This study was undertaken to examine the effects of Mn on the transcription of genes encoding S. mutans Gbps, including gbpC, along with other critical S. mutans virulence genes. METHODS: Microarray analyses suggested the potential for an Mn effect on Gbp genes. Further investigation of the Mn effects on selected genes was undertaken by performing Northern blots, Western blots, and RT-PCR under conditions of planktonic and biofilm growth in Mn-depleted media or in media containing 50 mircoM Mn. RESULTS: Mn resulted in increased expression of gbpC and gtfB, and decreased expression of wapA, in both planktonic and biofilm cultures. The expression levels of gbpA and gbpD were also decreased in the presence of Mn, but only in biofilms. The expression of gtfC was increased in the presence of Mn only in planktonic cultures. The spaP gene was expressed more highly in Mn-supplemented planktonic cultures but less in Mn-supplemented biofilms. CONCLUSION: Mn availability affects the expression of multiple S. mutans genes involved in adhesion and biofilm formation. Furthermore, these effects depend on the growth state of the organism.

Complete genome sequence of an M1 strain of Streptococcus pyogenes.

The 1,852,442-bp sequence of an M1 strain of Streptococcus pyogenes, a Gram-positive pathogen, has been determined and contains 1,752 predicted protein-encoding genes. Approximately one-third of these genes have no identifiable function, with the remainder falling into previously characterized categories of known microbial function. Consistent with the observation that S. pyogenes is responsible for a wider variety of human disease than any other bacterial species, more than 40 putative virulence-associated genes have been identified. Additional genes have been identified that encode proteins likely associated with microbial "molecular mimicry" of host characteristics and involved in rheumatic fever or acute glomerulonephritis. The complete or partial sequence of four different bacteriophage genomes is also present, with each containing genes for one or more previously undiscovered superantigen-like proteins. These prophage-associated genes encode at least six potential virulence factors, emphasizing the importance of bacteriophages in horizontal gene transfer and a possible mechanism for generating new strains with increased pathogenic potential.

The NAD-glycohydrolase (nga) gene of Streptococcus pyogenes.

The gene for NAD-glycohydrolase (nga) of group A streptococci (Streptococcus pyogenes) was identified and shown to be located immediately adjacent to the gene for streptolysin O (slo). The nga gene contains 1341 base pairs and encodes a protein of 447 amino acids, including an N-terminal signal peptide. Results from analysis with the polymerase chain reaction indicated that the nga gene is present in all of the strains tested. Functional extracellular NAD-glycohydrolase, also known as NADase, was detected among a wide variety of clinical isolates and known laboratory strains and shown to be present in 72% of 100 strains examined. In contrast, 92% of strains isolated from patients with invasive streptococcal infections were positive for NADase production.

The rgg gene of Streptococcus pyogenes NZ131 positively influences extracellular SPE B production.

Streptococcus pyogenes produces several extracellular proteins, including streptococcal erythrogenic toxin B (SPE B), also known as streptococcal pyrogenic exotoxin B and streptococcal proteinase. Several reports suggest that SPE B contributes to the virulence associated with S. pyogenes; however, little is known about its regulation. Nucleotide sequence data revealed the presence, upstream of the speB gene, of a gene, designated rgg, that was predicted to encode a polypeptide similar to previously described positive regulatory factors. The putative Rgg polypeptide of S. pyogenes NZ131 consisted of 280 amino acids and had a predicted molecular weight of 33,246. To assess the potential role of Rgg in the production of SPE B, the rgg gene was insertionally inactivated in S. pyogenes NZ131, which resulted in markedly decreased SPE B production, as determined both by immunoblotting and caseinolytic activity on agar plates. However, the production of other extracellular products, including streptolysin O, streptokinase, and DNase, was not affected. Complementation of the rgg mutant with an intact rgg gene copy in S. pyogenes NZ131 could restore SPE B production and confirmed that the rgg gene product is involved in the production of SPE B.

Transcriptional regulation of the Streptococcus mutans gal operon by the GalR repressor.

The galactose operon of Streptococcus mutans is transcriptionally regulated by a repressor protein (GalR) encoded by the galR gene, which is divergently oriented from the structural genes of the gal operon. To study the regulatory function of GalR, we partially purified the protein and examined its DNA binding activity by gel mobility shift and DNase I footprinting experiments. The protein specifically bound to the galR-galK intergenic region at an operator sequence, the position of which would suggest that GalR plays a role in the regulation of the gal operon as well as autoregulation. To further examine this hypothesis, transcriptional start sites of the gal operon and the galR gene were determined. Primer extension analysis showed that both promoters overlap the operator, indicating that GalR most likely represses transcription initiation of both promoters. Finally, the results from in vitro binding experiments with potential effector molecules suggest that galactose is a true intracellular inducer of the galactose operon.

Organization and nucleotide sequence of the Streptococcus mutans galactose operon.

The galactose operon encoding a repressor and genes for the Leloir pathway for galactose metabolism (galactokinase, galactose-1-phosphate-uridyl transferase and UDP glucose-4-epimerase) was located adjacent to the multiple sugar metabolism (msm) operon on the chromosome of Streptococcus mutans Ingbritt (serotype c) and the complete nucleotide sequence of this 5-kilobase region was determined. The Leloir pathway was induced by the presence of galactose in the growth medium or following the release of intracellular galactose after uptake and cleavage of alpha-galactosides by the multiple sugar metabolism system. Analysis of the mechanism of galactose transport confirmed the absence of a galactose-specific phosphotransferase system and suggested the presence of an inducible galactose permease. Evidence is presented that galactose transport is independent of the proton motive force and may be ATP-dependent.

Nucleotide sequence, mutational analysis, transcriptional start site, and product analysis of nov, the gene which affects Escherichia coli K-12 resistance to the gyrase inhibitor novobiocin.

In a previous study, we demonstrated the existence of a gene locus, nov, which affects resistance of Escherichia coli K-12 to the gyrase inhibitor novobiocin and, to a lesser degree, coumermycin (J. Rakonjac, M. Milic, D. Ajdic, D. Santos, R. Ivanisevic, and D. J. Savic, Mol. Microbiol. 6:1547-1553, 1992). In the present study, sequencing of the nov gene locus revealed one open reading frame that encodes a protein of 54,574 Da, a value. found to be in correspondence with the size of the Nov protein identified in an in vitro translation system. We also located the 5' end of the nov transcript 8 bp downstream from a classical sigma70 promoter. Transcription of the gene is in the counterclockwise direction on the E. coli chromosome. Transposon mutagenesis of nov followed by complementation analyses and replacement of chromosomal alleles with mutated nov confirmed our previous assumption that the nov gene exists in two allelic forms and that the Novr gene is an active allele while the Novs gene is an inactive form. After comparing nucleotide sequences flanking the nov gene with existing data, we conclude that the gene order in this region of the E. coli K-12 map is att phi 80-open reading frame of unknown function-kch (potassium channel protein)-nov-opp. Finally, the possible identity of the nov gene with cls, the gene that codes for cardiolipin synthase, is also discussed.

Nucleotide sequence analysis of the inversion termini located within IS3 elements alpha 3 beta 3 and beta 5 alpha 5 of Escherichia coli K-12.

This paper presents the first detailed structural analysis of termini of an inversion mediated by recombination between Escherichia coli native IS elements. The complete nucleotide sequence of the inversion termini in the lactose region of Escherichia coli K-12 confirms our previous suggestion that the inversion occurred by homologous recombination between alpha 3 beta 3 and beta 5 alpha 5 IS3 elements (D. J. Savic, J. Bacteriol. 140:311-319, 1979; D. J. Savic, S. Romac, and S. D. Ehrlich, J. Bacteriol. 155:943-946, 1983). The data show a slight structural divergence of alpha 3 beta 3 and beta 5 alpha 5 elements, but they do not reveal new sequences within recombined IS3 elements that could influence the expression of nearby genes.

[Analysis of herpes simplex virus DNA using restriction endonucleases]. / Analiza DNK herpes simpleks virusa pomocu restrikcionih endonukleaza.

DNA of herpes simplex virus was analysed by restriction endonucleases EcoRI, BgII and HindIII. By this method it is possible to differentiate type 1 and type 2 of viruses. DNA of herpes simplex virus was prepared according to the method of Kinnunen and his collaborators which is described in detail.