Characterization of mleR, a positive regulator of malolactic fermentation and part of the acid tolerance response in Streptococcus mutans

BACKGROUND: One of the key virulence determinants of Streptococcus mutans, the primary etiological agent of human dental caries, is its strong acid tolerance. The acid tolerance response (ATR) of S. mutans comprises several mechanisms that are induced at low pH and allow the cells to quickly adapt to a lethal pH environment. Malolactic fermentation (MLF) converts L-malate to L-lactate and carbon dioxide and furthermore regenerates ATP, which is used to translocate protons across the membrane. Thus, MLF may contribute to the aciduricity of S. mutans but has not been associated with the ATR so far. RESULTS: Here we show that the malolactic fermentation (mle) genes are under the control of acid inducible promoters which are induced within the first 30 minutes upon acid shock in the absence of malate. Thus, MLF is part of the early acid tolerance response of S. mutans. However, acidic conditions, the presence of the regulator MleR and L-malate were required to achieve maximal expression of all genes, including mleR itself. Deletion of mleR resulted in a decreased capacity to carry out MLF and impaired survival at lethal pH in the presence of L-malate. Gel retardation assays indicated the presence of multiple binding sites for MleR. Differences in the retardation patterns occurred in the presence of L-malate, thus demonstrating its role as co-inducer for transcriptional regulation. CONCLUSION: This study shows that the MLF gene cluster is part of the early acid tolerance response in S. mutans and is induced by both low pH and L-malate

Uptake of radiolabelled modified fragment of human alfa-fetoptrotein by experimental mammary adenocarcinoma: in vitro and in vivo studies

BACKGROUND: The aim of the study was to examine in vitro and in vivo binding of radiolabelled analogues of P149 peptide by experimental mammary adenocarcinoma with the intention of potential application for diagnosis and internal radiotherapy of tumours. MATERIAL AND METHODS: The 36-amino acid peptide (P149-QY) of 90% homology to 447–480 peptide fragment of hAFP was synthesised and radiolabelled with iodine-125. The biodistribution of P149-Q[125I]-Y was studied in experimental mammary tumours. For in vitro experiments, extract from mouse mammary tumours were prepared and incubated with radioiodinated P149-QY peptide in the presence of a cross-linking reagent. RESULTS: The gel electrophoresis analysis (SDS-PAGE) showed that radioiodinated P149-QY peptide formed a complex with adenocarcinoma proteins of about 30 kDa. The biodistribution of P149-Q[125I]-Y studied in experimental mammary tumours revealed a higher pharmacokinetic rate in comparison with the whole radioiodinated AFP molecule. A moderate uptake of P149-Q[125I]-Y in the tumour tissue was observed (3.2% ID/g at 30-min p.i.v). However, a faster radioactivity clearance from blood and normal tissues resulted in an increase in the tumour/muscle (T/M) ratio, i.e. from 2.3 to 3.4 after 30 mins and 24 h p.i.v, respectively. CONCLUSIONS: The present study shows that radioiodinated P149-QY peptide reveals some positive features as the AFP receptor radioligand, however, some additional structural modifications of the initial peptide molecule are necessary for full retention of the ligand-receptor interaction of its radiolabelled forms

Strategi Pembangunan Pariwisata melalui Sinergitas Dinas Pariwisata dengan Desa Adat ( Studi Kasus pada Pengelolaan Obyek Wisata Pantai Labuan Sait dalam Meningkatkan Retribusi Daerah di Kabupaten Badung)

A gradual Tourism development is very important to improve the quality of tourism each year to compete with other tourist attraction. The synergy between the Central Government with local government plays an important role to the development of tourism. The background to this research is the development of tourism which is still insufficient in Labuan Sait both in terms of means and infrastructure, promotion, as well as structuring tourism. This study measures how does tourism development strategy through the synergy with the customary village tourism office on the management of Beach Tourism Labuan Sait in increasing the levy County in Badung Regency with the theory of development that uses the concept of planning development by Sjahrizal in the regional development planning in the era of autonomy. The indicator consists of planning, implementation, monitoring and evaluation. In addition also use the concept of synergy from Najiyati and Rahmat which consists of indicators communication and coordination as well as indicators of the SWOT by Freddy Rangkuti. Method used in this study is a qualitative method with descriptive approach with data collection techniques in the form of in-depth interviews to several informants associated with this research. The results of the research showed that the development strategy of tourism through the synergy with the customary village Tourism Office on the management of Beach Tourism Labuan Sait in improving regional levies in Badung Regency are still insufficient. That is because the is still lacking from the indicator monitoring and implementation and evaluation of the impact against the decline of levy of admission attractions Labuan Sait in the 2017.     Keywords: Development, Tourism, Synergy, and Strateg

Lack of the Delta Subunit of RNA Polymerase Increases Virulence Related Traits of Streptococcus mutans

The delta subunit of the RNA polymerase, RpoE, maintains the transcriptional specificity in Gram-positive bacteria. Lack of RpoE results in massive changes in the transcriptome of the human dental caries pathogen Streptococcus mutans. In this study, we analyzed traits of the ΔrpoE mutant which are important for biofilm formation and interaction with oral microorganisms and human cells and performed a global phenotypic analysis of its physiological functions. The ΔrpoE mutant showed higher self-aggregation compared to the wild type and coaggregated with other oral bacteria and Candida albicans. It formed a biofilm with a different matrix structure and an altered surface attachment. The amount of the cell surface antigens I/II SpaP and the glucosyltransferase GtfB was reduced. The ΔrpoE mutant displayed significantly stronger adhesion to human extracellular matrix components, especially to fibronectin, than the wild type. Its adhesion to human epithelial cells HEp-2 was reduced, probably due to the highly aggregated cell mass. The analysis of 1248 physiological traits using phenotype microarrays showed that the ΔrpoE mutant metabolized a wider spectrum of carbon sources than the wild type and had acquired resistance to antibiotics and inhibitory compounds with various modes of action. The reduced antigenicity, increased aggregation, adherence to fibronection, broader substrate spectrum and increased resistance to antibiotics of the ΔrpoE mutant reveal the physiological potential of S. mutans and show that some of its virulence related traits are increased

Characterization of mleR, a positive regulator of malolactic fermentation and part of the acid tolerance response in Streptococcus mutans

Abstract Background One of the key virulence determinants of Streptococcus mutans, the primary etiological agent of human dental caries, is its strong acid tolerance. The acid tolerance response (ATR) of S. mutans comprises several mechanisms that are induced at low pH and allow the cells to quickly adapt to a lethal pH environment. Malolactic fermentation (MLF) converts L-malate to L-lactate and carbon dioxide and furthermore regenerates ATP, which is used to translocate protons across the membrane. Thus, MLF may contribute to the aciduricity of S. mutans but has not been associated with the ATR so far. Results Here we show that the malolactic fermentation (mle) genes are under the control of acid inducible promoters which are induced within the first 30 minutes upon acid shock in the absence of malate. Thus, MLF is part of the early acid tolerance response of S. mutans. However, acidic conditions, the presence of the regulator MleR and L-malate were required to achieve maximal expression of all genes, including mleR itself. Deletion of mleR resulted in a decreased capacity to carry out MLF and impaired survival at lethal pH in the presence of L-malate. Gel retardation assays indicated the presence of multiple binding sites for MleR. Differences in the retardation patterns occurred in the presence of L-malate, thus demonstrating its role as co-inducer for transcriptional regulation. Conclusion This study shows that the MLF gene cluster is part of the early acid tolerance response in S. mutans and is induced by both low pH and L-malate

The Delta Subunit of RNA Polymerase, RpoE, Is a Global Modulator of Streptococcus mutans Environmental Adaptation▿ †

The delta subunit of RNA polymerase, RpoE, is widespread in low-G+C Gram-positive bacteria and is thought to play a role in enhancing transcriptional specificity by blocking RNA polymerase binding at weak promoter sites and stimulating RNA synthesis by accelerating core enzyme recycling. Despite the well-studied biochemical properties of RpoE, a role for this protein in vivo has not been defined in depth. In this study, we show that inactivation of rpoE in the human dental caries pathogen Streptococcus mutans causes impaired growth and loss of important virulence traits, including biofilm formation, resistance to antibiotics, and tolerance to environmental stresses. Complementation of the mutant with rpoE expressed in trans restored its phenotype to wild type. The luciferase fusion reporter showed that rpoE was highly transcribed throughout growth and that acid and hydrogen peroxide stresses repressed rpoE expression. Transcriptome profiling of wild-type and ΔrpoE cells in the exponential and early stationary phase of growth, under acid and hydrogen peroxide stress and under both stresses combined, revealed that genes involved in histidine synthesis, malolactic fermentation, biofilm formation, and antibiotic resistance were downregulated in the ΔrpoE mutant under all conditions. Moreover, the loss of RpoE resulted in dramatic changes in transport and metabolism of carbohydrates and amino acids. Interestingly, differential expression, mostly upregulation, of 330 noncoding regions was found. In conclusion, this study demonstrates that RpoE is an important global modulator of gene expression in S. mutans which is required for optimal growth and environmental adaptation

PRODUCTION AND SOME PROPERTIES OF PARTIALLY PURIFIED LIPASE FROM PENICILLIUM SIMPLICISSIMUM

Lipases (triacylglycerol acylhydrolases, EC 3.1.1.3) are widely distributed throughout fungi. Two lipolytic fungi commonly used in the manufacture of Camembert and Brie cheeses are Penicillium camembertii and P. caseicolum. Lipolytic activity was also discovered in a P. chrysogenum strain [1]. Lipase from P. cyclopium M1 specific to p-nitrophenyl laurate has been purified and crystallized [2]. Three lipases were discovered in the culture broth of P. crutosum [3], two of them were purified and crystallized [3]. We describe here a lipolytic activity of P. simplicissimum, the purification and some properties of the lipase

The global impact of the delta subunit RpoE of the RNA polymerase on the proteome of Streptococcus mutans.

Transcriptional specificity in low-G+C Gram-positive bacteria is maintained by RpoE, the delta subunit of the RNA polymerase. Here, we studied the effect of RpoE at the proteome level in the human dental pathogen Streptococcus mutans by comparing the ΔrpoE mutant with the wild-type under five conditions: (0) exponential growth, (1) early stationary phase, (2) acid stress, (3) oxidative stress, and (4) combined acid and oxidative stress. A total of 280 cellular protein spots were reproducibly detected, of which 97 differentially expressed protein spots were identified by MALDI-TOF MS. Lack of RpoE caused downregulation of proteins for carbohydrate metabolism and energy production, including phosphoglucomutase (PGM), the phosphopentomutase DeoB and the pyruvate formate-lyase Pfl. The ΔrpoE mutant had extensive changes in the abundance of proteins involved in acid and oxidative tolerance and protein turnover, and of chaperones, at exponential phase in the absence of stress, suggesting a potential internal stress. In addition, the mutant had reduced amounts of proteins for adaptation responses, e.g. the multiple sugar transport and metabolism enzymes required for entering early stationary phase, and the proteins for stress-defence mechanisms and glycolysis under oxidative stress. Comparison of the proteome data with the corresponding transcriptome data suggested that the effects were the result of altered transcriptional and post-transcriptional regulation. The data are consistent with the reduced transcriptional specificity of the RNA polymerase in the ΔrpoE mutant, and suggest a general impact, but not a specific regulatory role, of RpoE in stress adaptation

Worlds Apart – Transcriptome Profiles of Key Oral Microbes in the Periodontal Pocket Compared to Single Laboratory Culture Reflect Synergistic Interactions

Periodontitis is a worldwide prevalent oral disease which results from dysbiosis of the periodontal microbiome. Some of the most active microbial players, e.g., Porphyromonas gingivalis, Treponema denticola, and Fusobacterium nucleatum, have extensively been studied in the laboratory, but it is unclear to which extend these findings can be transferred to in vivo conditions. Here we show that the transcriptional profiles of P. gingivalis, T. denticola, and F. nucleatum in the periodontal niche are distinct from those in single laboratory culture and exhibit functional similarities. GO (gene ontology) term enrichment analysis showed up-regulation of transporters, pathogenicity related traits and hemin/heme uptake mechanisms for all three species in vivo. Differential gene expression analysis revealed that cysteine proteases, transporters and hemin/heme-binding proteins were highly up-regulated in the periodontal niche, while genes involved in DNA modification were down-regulated. The data suggest strong interactions between those three species regarding protein degradation, iron up-take, and mobility in vivo, explaining their enhanced synergistic pathogenicity. We discovered a strikingly high frequency of Single Nucleotide Polymorphisms (SNPs) in vivo. For F. nucleatum we discovered a total of 127,729 SNPs in periodontal niche transcripts, which were found in similar frequency in health and disease and covered the entire genome, suggesting continuous evolution in the host. We conclude that metabolic interactions shape gene expression in vivo. Great caution is required when inferring pathogenicity of microbes from laboratory data, and microdiversity is an important adaptive trait of natural communities