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

Is autoinducer-2 a universal signal for interspecies communication: a comparative genomic and phylogenetic analysis of the synthesis and signal transduction pathways

BACKGROUND: Quorum sensing is a process of bacterial cell-to-cell communication involving the production and detection of extracellular signaling molecules called autoinducers. Recently, it has been proposed that autoinducer-2 (AI-2), a furanosyl borate diester derived from the recycling of S-adenosyl-homocysteine (SAH) to homocysteine, serves as a universal signal for interspecies communication. RESULTS: In this study, 138 completed genomes were examined for the genes involved in the synthesis and detection of AI-2. Except for some symbionts and parasites, all organisms have a pathway to recycle SAH, either using a two-step enzymatic conversion by the Pfs and LuxS enzymes or a one-step conversion using SAH-hydrolase (SahH). 51 organisms including most Gamma-, Beta-, and Epsilonproteobacteria, and Firmicutes possess the Pfs-LuxS pathway, while Archaea, Eukarya, Alphaproteobacteria, Actinobacteria and Cyanobacteria prefer the SahH pathway. In all 138 organisms, only the three Vibrio strains had strong, bidirectional matches to the periplasmic AI-2 binding protein LuxP and the central signal relay protein LuxU. The initial two-component sensor kinase protein LuxQ, and the terminal response regulator luxO are found in most Proteobacteria, as well as in some Firmicutes, often in several copies. CONCLUSIONS: The genomic analysis indicates that the LuxS enzyme required for AI-2 synthesis is widespread in bacteria, while the periplasmic binding protein LuxP is only present in Vibrio strains. Thus, other organisms may either use components different from the AI-2 signal transduction system of Vibrio strains to sense the signal of AI-2, or they do not have such a quorum sensing system at all

Metabolic fluxes in the central carbon metabolism of Dinoroseobacter shibae and Phaeobacter gallaeciensis, two members of the marine Roseobacter clade

<p>Abstract</p> <p>Background</p> <p>In the present work the central carbon metabolism of <it>Dinoroseobacter shibae </it>and <it>Phaeobacter gallaeciensis </it>was studied at the level of metabolic fluxes. These two strains belong to the marine <it>Roseobacter </it>clade, a dominant bacterial group in various marine habitats, and represent surface-associated, biofilm-forming growth (<it>P. gallaeciensis</it>) and symbiotic growth with eukaryotic algae (<it>D. shibae</it>). Based on information from recently sequenced genomes, a rich repertoire of pathways has been identified in the carbon core metabolism of these organisms, but little is known about the actual contribution of the various reactions <it>in vivo</it>.</p> <p>Results</p> <p>Using ¹³C labelling techniques in specifically designed experiments, it could be shown that glucose-grown cells of <it>D. shibae </it>catabolise the carbon source exclusively via the Entner-Doudoroff pathway, whereas alternative routes of glycolysis and the pentose phosphate pathway are obviously utilised for anabolic purposes only. Enzyme assays confirmed this flux pattern and link the lack of glycolytic flux to the absence of phosphofructokinase activity. The previously suggested formation of phosphoenolpyruvate from pyruvate during mixotrophic CO₂assimilation was found to be inactive under the conditions studied. Moreover, it could be shown that pyruvate carboxylase is involved in CO₂assimilation and that the <it>cyclic </it>respiratory mode of the TCA cycle is utilised. Interestingly, the use of intracellular pathways was highly similar for <it>P. gallaeciensis</it>.</p> <p>Conclusion</p> <p>The present study reveals the first insight into pathway utilisation within the <it>Roseobacter </it>group. Fluxes through major intracellular pathways of the central carbon metabolism, which are closely linked to the various important traits found for the <it>Roseobacter </it>clade, could be determined. The close similarity of fluxes between the two physiologically rather different species might provide the first indication of more general key properties among members of the <it>Roseobacter </it>clade which may explain their enormous success in the marine realm.</p

Functional profiling of mercuric reductase (mer A) genes in biofilm communities of a technical scale biocatalyzer

BACKGROUND: Bacterial mercury resistance is based on enzymatic reduction of ionic mercury to elemental mercury and has recently been demonstrated to be applicable for industrial wastewater clean-up. The long-term monitoring of such biocatalyser systems requires a cultivation independent functional community profiling method targeting the key enzyme of the process, the merA gene coding for the mercuric reductase. We report on the development of a profiling method for merA and its application to monitor changes in the functional diversity of the biofilm community of a technical scale biocatalyzer over 8 months of on-site operation. RESULTS: Based on an alignment of 30 merA sequences from Gram negative bacteria, conserved primers were designed for amplification of merA fragments with an optimized PCR protocol. The resulting amplicons of approximately 280 bp were separated by thermogradient gelelectrophoresis (TGGE), resulting in strain specific fingerprints for mercury resistant Gram negative isolates with different merA sequences. The merA profiling of the biofilm community from a technical biocatalyzer showed persistence of some and loss of other inoculum strains as well as the appearance of new bands, resulting in an overall increase of the functional diversity of the biofilm community. One predominant new band of the merA community profile was also detected in a biocatalyzer effluent isolate, which was identified as Pseudomonas aeruginosa. The isolated strain showed lower mercury reduction rates in liquid culture than the inoculum strains but was apparently highly competitive in the biofilm environment of the biocatalyzer where moderate mercury levels were prevailing. CONCLUSIONS: The merA profiling technique allowed to monitor the ongoing selection for better adapted strains during the operation of a biocatalyzer and to direct their subsequent isolation. In such a way, a predominant mercury reducing Ps. aeruginosa strain was identified by its unique mercuric reductase gene

A genome-wide study of two-component signal transduction systems in eight newly sequenced mutans streptococci strains

<p>Abstract</p> <p>Background</p> <p>Mutans streptococci are a group of gram-positive bacteria including the primary cariogenic dental pathogen <it>Streptococcus mutans </it>and closely related species. Two component systems (TCSs) composed of a signal sensing histidine kinase (HK) and a response regulator (RR) play key roles in pathogenicity, but have not been comparatively studied for these oral bacterial pathogens.</p> <p>Results</p> <p>HKs and RRs of 8 newly sequenced mutans streptococci strains, including <it>S. sobrinus </it>DSM20742, <it>S. ratti </it>DSM20564 and six <it>S. mutans </it>strains, were identified and compared to the TCSs of <it>S. mutans </it>UA159 and NN2025, two previously genome sequenced <it>S. mutans </it>strains. Ortholog analysis revealed 18 TCS clusters (HK-RR pairs), 2 orphan HKs and 2 orphan RRs, of which 8 TCS clusters were common to all 10 strains, 6 were absent in one or more strains, and the other 4 were exclusive to individual strains. Further classification of the predicted HKs and RRs revealed interesting aspects of their putative functions. While TCS complements were comparable within the six <it>S. mutans </it>strains, <it>S. sobrinus </it>DSM20742 lacked TCSs possibly involved in acid tolerance and fructan catabolism, and <it>S. ratti </it>DSM20564 possessed 3 unique TCSs but lacked the quorum-sensing related TCS (ComDE). Selected computational predictions were verified by PCR experiments.</p> <p>Conclusions</p> <p>Differences in the TCS repertoires of mutans streptococci strains, especially those of <it>S. sobrinus </it>and <it>S. ratti </it>in comparison to <it>S. mutans</it>, imply differences in their response mechanisms for survival in the dynamic oral environment. This genomic level study of TCSs should help in understanding the pathogenicity of these mutans streptococci strains.</p

Integrated Transcriptional Regulatory Network of Quorum Sensing, Replication Control, and SOS Response in Dinoroseobacter shibae

Quorum sensing (QS) coordinates population wide gene expression of bacterial species. Highly adaptive traits like gene transfer agents (GTA), morphological heterogeneity, type 4 secretion systems (T4SS), and flagella are QS controlled in Dinoroseobacter shibae, a Roseobacter model organism. Its QS regulatory network is integrated with the CtrA phosphorelay that controls cell division in alphaproteobacteria. To elucidate the network topology, we analyzed the transcriptional response of the QS-negative D. shibae strain ΔluxI1 toward externally added autoinducer (AI) over a time period of 3 h. The signaling cascade is initiated by the CtrA phosphorelay, followed by the QS genes and other target genes, including the second messenger c-di-GMP, competence, flagella and pili. Identification of transcription factor binding sites in promoters of QS induced genes revealed the integration of QS, CtrA phosphorelay and the SOS stress response mediated by LexA. The concentration of regulatory genes located close to the origin or terminus of replication suggests that gene regulation and replication are tightly coupled. Indeed, addition of AI first stimulates and then represses replication. The restart of replication comes along with increased c-di-GMP levels. We propose a model in which QS induces replication followed by differentiation into GTA producing and non-producing cells. CtrA-activity is controlled by the c-di-GMP level, allowing some of the daughter cells to replicate again. The size of the GTA producing subpopulation is tightly controlled by QS via the AI Synthase LuxI2. Finally, induction of the SOS response allows for integration of GTA DNA into the host chromosome

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. &nbsp; &nbsp; Keywords: Development, Tourism, Synergy, and Strateg

The luxS mutation causes loosely-bound biofilms in Shewanella oneidensis

<p>Abstract</p> <p>Background</p> <p>The <it>luxS </it>gene in <it>Shewanella oneidensis </it>was shown to encode an autoinducer-2 (AI-2)-like molecule, the postulated universal bacterial signal, but the impaired biofilm growth of a <it>luxS </it>deficient mutant could not be restored by AI-2, indicating it might not have a signalling role in this organism.</p> <p>Findings</p> <p>Here, we provide further evidence regarding the metabolic role of a <it>luxS </it>mutation in <it>S. oneidensis</it>. We constructed a <it>luxS </it>mutant and compared its phenotype to a wild type control with respect to its ability to remove AI-2 from the medium, expression of secreted proteins and biofilm formation. We show that <it>S. oneidensis </it>has a cell-dependent mechanism by which AI-2 is depleted from the medium by uptake or degradation at the end of the exponential growth phase. As AI-2 depletion is equally active in the <it>luxS </it>mutant and thus does not require AI-2 as an inducer, it appears to be an unspecific mechanism suggesting that AI-2 for <it>S. oneidensis </it>is a metabolite which is imported under nutrient limitation. Secreted proteins were studied by iTraq labelling and liquid chromatography mass spectrometry (LC-MS) detection. Differences between wild type and mutant were small. Proteins related to flagellar and twitching motility were slightly up-regulated in the <it>luxS </it>mutant, in accordance with its loose biofilm structure. An enzyme related to cysteine metabolism was also up-regulated, probably compensating for the lack of the LuxS enzyme. The <it>luxS </it>mutant developed an undifferentiated, loosely-connected biofilm which covered the glass surface more homogenously than the wild type control, which formed compact aggregates with large voids in between.</p> <p>Conclusions</p> <p>The data confirm the role of the LuxS enzyme for biofilm growth in <it>S. oneidensis </it>and make it unlikely that AI-2 has a signalling role in this organism.</p

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