Physiologic effects of forced down-regulation of dnaK and groEL expression in Streptococcus mutans.

Department of Oral Biology, University of Florida College of Dentistry, Gainesville, FL 32610-0424, USA.
Journal of Bacteriology (Impact Factor: 3.19). 04/2007; 189(5):1582-8. DOI: 10.1128/JB.01655-06
Source: PubMed

ABSTRACT Strains of Streptococcus mutans lacking DnaK or GroEL appear not to be isolable. To better distinguish the roles played by these chaperones/chaperonins in the physiology of S. mutans, we created a knockdown strategy to lower the levels of DnaK by over 95% in strain SM12 and the level of GroEL about 80% in strain SM13. Interestingly, GroEL levels were approximately twofold higher in SM12 than in the parent strain, but the levels of DnaK were not altered in the GroEL knockdown strain. Both SM12 and SM13 grew slower than the parent strain, had a strong tendency to aggregate in broth culture, and showed major changes in their proteomes. Compared with the wild-type strain, SM12 and SM13 had impaired biofilm-forming capacities when grown in the presence of glucose. The SM12 strain was impaired in its capacity to grow at 44 degrees C or at pH 5.0 and was more susceptible to H(2)O(2), whereas SM13 behaved like the wild-type strain under these conditions. Phenotypical reversions were noted for both mutants when cells were grown in continuous culture at a low pH, suggesting the occurrence of compensatory mutations. These results demonstrate that DnaK and GroEL differentially affect the expression of key virulence traits, including biofilm formation and acid tolerance, and support that these chaperones have evolved to accommodate unique roles in the context of this organism and its niche.

  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Protein chaperones are essential in all domains of life to prevent and resolve protein misfolding during translation and proteotoxic stress. HSP70 family chaperones, including E. coli DnaK, function in stress induced protein refolding and degradation, but are dispensable for cellular viability due to redundant chaperone systems that prevent global nascent peptide insolubility. However, the function of HSP70 chaperones in mycobacteria, a genus that includes multiple human pathogens, has not been examined. We find that mycobacterial DnaK is essential for cell growth and required for native protein folding in Mycobacterium smegmatis. Loss of DnaK is accompanied by proteotoxic collapse characterized by the accumulation of insoluble newly synthesized proteins. DnaK is required for solubility of large multimodular lipid synthases, including the essential lipid synthase FASI, and DnaK loss is accompanied by disruption of membrane structure and increased cell permeability. Trigger Factor is nonessential and has a minor role in native protein folding that is only evident in the absence of DnaK. In unstressed cells, DnaK localizes to multiple, dynamic foci, but relocalizes to focal protein aggregates during stationary phase or upon expression of aggregating peptides. Mycobacterial cells restart cell growth after proteotoxic stress by isolating persistent DnaK containing protein aggregates away from daughter cells. These results reveal unanticipated essential nonredunant roles for mycobacterial DnaK in mycobacteria and indicate that DnaK defines a unique susceptibility point in the mycobacterial proteostasis network.
    PLoS Genetics 07/2014; 10(7):e1004516. · 8.52 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Xanthomonas citri subsp. citri (X. citri) is the causative agent of the citrus canker, a disease that affects several citrus plants in Brazil and across the world. Although many studies have demonstrated the importance of genes for infection and pathogenesis in this bacterium, there are no data related to phosphate uptake and assimilation pathways. To identify the proteins that are involved in the phosphate response, we performed a proteomic analysis of X. citri extracts after growth in three culture media with different phosphate concentrations. Using mass spectrometry and bioinformatics analysis, we showed that X. citri conserved orthologous genes from Pho regulon in Escherichia coli, including the two-component system PhoR/PhoB, ATP binding cassette (ABC transporter) Pst for phosphate uptake, and the alkaline phosphatase PhoA. Analysis performed under phosphate starvation provided evidence of the relevance of the Pst system for phosphate uptake, as well as both periplasmic binding proteins, PhoX and PstS, were formed in high abundance. The results from this study are the first evidence of the Pho regulon activation in X. citri and bring new insights for studies related to the bacterial metabolism and physiology. Biological Significance Using proteomics and bioinformatics analysis we showed for the first time that the phytopathogenic bacterium Xathomonas citri conserves a set of proteins that belong to the Pho regulon, which are induced during phosphate starvation. The most relevant in terms of conservation and up-regulation were the periplasmic-binding proteins PstS and PhoX from the ABC transporter PstSBAC for phosphate, the two-component system composed by PhoR/PhoB and the alkaline phosphatase PhoA.
    Journal of Proteomics 05/2014; · 4.09 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: In this study, we are interested in comparing the protein profiles of acid-shocked and control cells of S. mutans isolated from Korean children with caries. The results of 2D gel electrophoresis showed that twelve proteins are up-regulated when the cells were grown under 20 mM lactic acid stress in the exponential phase. Up-proteins under acid stress were estimated a major key of the survival and proliferation of S. mutans in low pH environments. These proteins are estimated generally associated with three biochemical pathways: glycolysis, alternative acid production and branched-chain amino acid biosynthesis.
    Journal of the Korea Academia-Industrial cooperation Society. 01/2009; 10(7).


1 Download
Available from