76 research outputs found
Nucleotide sequence analysis of the inversion termini located within IS3 elements α3β3 and β5α5 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 α3β3 and β5α5 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 α3β3 and β5α5 elements, but they do not reveal new sequences within recomhined IS3 elements that could influence the expression of nearby genes
Structure and activity of the Streptococcus pyogenes family GH1 6-phospho β-glycosidase, Spy1599
The group A streptococcus Streptococcus pyogenes is the causative agent of a wide spectrum of invasive infections, including necrotizing fasciitis, scarlet fever and toxic shock syndrome. In the context of its carbohydrate chemistry, it is interesting that S. pyogenes (in this work strain M1 GAS SF370) displays a spectrum of oligosaccharide-processing enzymes that are located in close proximity on the genome but that the in vivo function of these proteins remains unknown. These proteins include different sugar transporters (SPy1593 and SPy1595), both GH125 -1,6- and GH38 -1,3-mannosidases (SPy1603 and SPy1604), a GH84 -hexosaminidase (SPy1600) and a putative GH2 -galactosidase (SPy1586), as well as SPy1599, a family GH1 `putative -glucosidase'. Here, the solution of the three-dimensional structure of SPy1599 in a number of crystal forms complicated by unusual crystallographic twinning is reported. The structure is a classical (/)8-barrel, consistent with CAZy family GH1 and other members of the GH-A clan. SPy1599 has been annotated in sequence depositions as a -glucosidase (EC 3.2.1.21), but no such activity could be found; instead, three-dimensional structural overlaps with other enzymes of known function suggested that SPy1599 contains a phosphate-binding pocket in the active site and has possible 6-phospho--glycosidase activity. Subsequent kinetic analysis indeed showed that SPy1599 has 6-phospho--glucosidase (EC 3.2.1.86) activity. These data suggest that SPy1599 is involved in the intracellular degradation of 6-phosphoglycosides, which are likely to originate from import through one of the organism's many phosphoenolpyruvate phosphotransfer systems (PEP-PTSs)
Detection of putative new mutacins by bioinformatic analysis using available web tools
In order to characterise new bacteriocins produced by Streptococcus mutans we perform a complete bioinformatic analyses by scanning the genome sequence of strains UA159 and NN2025. By searching in the adjacent genomic context of the two-component signal transduction system we predicted the existence of many putative new bacteriocins' maturation pathways and some of them were only exclusive to a group of Streptococcus. Computational genomic and proteomic analysis combined to predictive functionnal analysis represent an alternative way for rapid identification of new putative bacteriocins as well as new potential antimicrobial drugs compared to the more traditional methods of drugs discovery using antagonism tests
Gene expression of bacterial collagenolytic proteases in root caries
Objective: It is unknown whether bacteria play a role in the collagen matrix degradation that occurs during caries progression. Our aim was to characterize the expression level of genes involved in bacterial collagenolytic proteases in root biofilms with and without caries. Method: we collected samples from active cavitated root caries lesions (RC, n = 30) and from sound root surfaces (SRS, n = 10). Total microbial RNA was isolated and cDNA sequenced on the Illumina Hi-Seq2500. Reads were mapped to 162 oral bacterial reference genomes. Genes encoding putative bacterial collagenolytic proteases were identified. Normalization and differential expression analysis was performed on all metatranscriptomes (FDR8) but none in SRS were Pseudoramibacter alactolyticus [HMPREF0721_RS02020; HMPREF0721_RS04640], Scardovia inopinata [SCIP_RS02440] and Olsenella uli DSM7084 [OLSU_RS02990]. Conclusion: Our findings suggest that the U32 proteases may be related to carious dentine. The contribution of a small number of species to dentine degradation should be further investigated. These proteases may have potential in future biotechnological and medical applications, serving as targets for the development of therapeutic agents
Differential proteome and cellular adhesion analyses of the probiotic bacterium <i>Lactobacillus acidophilus</i> NCFM grown on raffinose - an emerging prebiotic
Global Transcriptional Analysis of Streptococcus mutans Sugar Transporters Using Microarrays▿ †
The transport of carbohydrates by Streptococcus mutans is accomplished by the phosphoenolpyruvate-phosphotransferase system (PTS) and ATP-binding cassette (ABC) transporters. To undertake a global transcriptional analysis of all S. mutans sugar transporters simultaneously, we used a whole-genome expression microarray. Global transcription profiles of S. mutans UA159 were determined for several monosaccharides (glucose, fructose, galactose, and mannose), disaccharides (sucrose, lactose, maltose, and trehalose), a β-glucoside (cellobiose), oligosaccharides (raffinose, stachyose, and maltotriose), and a sugar alcohol (mannitol). The results revealed that PTSs were responsible for transport of monosaccharides, disaccharides, β-glucosides, and sugar alcohol. Six PTSs were transcribed only if a specific sugar was present in the growth medium; thus, they were regulated at the transcriptional level. These included transporters for fructose, lactose, cellobiose, and trehalose and two transporters for mannitol. Three PTSs were repressed under all conditions tested. Interestingly, five PTSs were always highly expressed regardless of the sugar source used, presumably suggesting their availability for immediate uptake of most common dietary sugars (glucose, fructose, maltose, and sucrose). The ABC transporters were found to be specific for oligosaccharides, raffinose, stachyose, and isomaltosaccharides. Compared to the PTSs, the ABC transporters showed higher transcription under several tested conditions, suggesting that they might be transporting multiple substrates
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
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
GlaR (YugA)—a novel RpiR‐family transcription activator of the Leloir pathway of galactose utilization in Lactococcus lactis IL1403
Abstract Bacteria can utilize diverse sugars as carbon and energy source, but the regulatory mechanisms directing the choice of the preferred substrate are often poorly understood. Here, we analyzed the role of the YugA protein (now designated GlaR—Galactose–lactose operon Regulatory protein) of the RpiR family as a transcriptional activator of galactose (gal genes) and lactose (lac genes) utilization genes in Lactococcus lactis IL1403. In this bacterium, gal genes forming the Leloir operon are combined with lac genes in a single so‐called gal–lac operon. The first gene of this operon is the lacS gene encoding galactose permease. The glaR gene encoding GlaR lies directly upstream of the gal–lac gene cluster and is transcribed in the same direction. This genetic layout and the presence of glaR homologues in the closest neighborhood to the Leloir or gal–lac operons are highly conserved only among Lactococcus species. Deletion of glaR disabled galactose utilization and abrogated or decreased expression of the gal–lac genes. The GlaR‐dependent regulation of the gal–lac operon depends on its specific binding to a DNA region upstream of the lacS gene activating lacS expression and increasing the expression of the operon genes localized downstream. Notably, expression of lacS‐downstream genes, namely galMKTE, thgA and lacZ, is partially independent of the GlaR‐driven activation likely due to the presence of additional promoters. The glaR transcription itself is not subject to catabolite control protein A (CcpA) carbon catabolite repression (CRR) and is induced by galactose. Up to date, no similar mechanism has been reported in other lactic acid bacteria species. These results reveal a novel regulatory protein and shed new light on the regulation of carbohydrate catabolism in L. lactis IL1403, and by similarity, probably also in other lactococci
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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 α-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
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