Transposon mediated transgenesis in a marine invertebrate chordate: Ciona intestinalis

Achievement of transposon mediated germline transgenesis in a basal chordate, Ciona intestinalis, is discussed. A Tc1/mariner superfamily transposon, Minos, has excision and transposition activities in Ciona. Minos enables the creation of stable transgenic lines, enhancer detection, and insertional mutagenesis

GlmS and NagB Regulate Amino Sugar Metabolism in Opposing Directions and Affect Streptococcus mutans Virulence

Streptococcus mutans is a cariogenic pathogen that produces an extracellular polysaccharide (glucan) from dietary sugars, which allows it to establish a reproductive niche and secrete acids that degrade tooth enamel. While two enzymes (GlmS and NagB) are known to be key factors affecting the entrance of amino sugars into glycolysis and cell wall synthesis in several other bacteria, their roles in S. mutans remain unclear. Therefore, we investigated the roles of GlmS and NagB in S. mutans sugar metabolism and determined whether they have an effect on virulence. NagB expression increased in the presence of GlcNAc while GlmS expression decreased, suggesting that the regulation of these enzymes, which functionally oppose one another, is dependent on the concentration of environmental GlcNAc. A glmS-inactivated mutant could not grow in the absence of GlcNAc, while nagB-inactivated mutant growth was decreased in the presence of GlcNAc. Also, nagB inactivation was found to decrease the expression of virulence factors, including cell-surface protein antigen and glucosyltransferase, and to decrease biofilm formation and saliva-induced S. mutans aggregation, while glmS inactivation had the opposite effects on virulence factor expression and bacterial aggregation. Our results suggest that GlmS and NagB function in sugar metabolism in opposing directions, increasing and decreasing S. mutans virulence, respectively

Sugar Allocation to Metabolic Pathways is Tightly Regulated and Affects the Virulence of Streptococcus mutans

Bacteria take up and metabolize sugar as a carbohydrate source for survival. Most bacteria can utilize many sugars, including glucose, sucrose, and galactose, as well as amino sugars, such as glucosamine and N-acetylglucosamine. After entering the cytoplasm, the sugars are mainly allocated to the glycolysis pathway (energy production) and to various bacterial component biosynthesis pathways, including the cell wall, nucleic acids and amino acids. Sugars are also utilized to produce several virulence factors, such as capsule and lipoteichoic acid. Glutamine-fructose-6-phosphate aminotransferase (GlmS) and glucosamine-6-phosphate deaminase (NagB) have crucial roles in sugar distribution to the glycolysis pathway and to cell wall biosynthesis. In Streptococcus mutans, a cariogenic pathogen, the expression levels of glmS and nagB are coordinately regulated in response to the presence or absence of amino sugars. In addition, the disruption of this regulation affects the virulence of S. mutans. The expression of nagB and glmS is regulated by NagR in S. mutans, but the precise mechanism underlying glmS regulation is not clear. In Staphylococcus aureus and Bacillus subtilis, the mRNA of glmS has ribozyme activity and undergoes self-degradation at the mRNA level. However, there is no ribozyme activity region on glmS mRNA in S. mutans. In this review article, we summarize the sugar distribution, particularly the coordinated regulation of GlmS and NagB expression, and its relationship with the virulence of S. mutans

Strains and plasmids used in this study.

<p>Strains and plasmids used in this study.</p

Expression of genes involved in the resistance to oxidative stress and <i>hmp</i>.

<p>The expression of <i>katA</i>, <i>dps</i>, <i>ahpC</i> and <i>hmp</i> in <i>S</i>. <i>aureus</i> MW2 WT, <i>srrA</i>-inactivated mutant and the complemented strain incubated with or without 0.4 mM H₂O₂ was determined by quantitative PCR as described in the Materials and Methods section. The data are the mean ± SD of five biological independent experiments. **, <i>P</i> < 0.01; ***, <i>P</i> < 0.001; N.S., not significant by Tukey's honestly significant difference test.</p

Primers used in this study.

<p>Primers used in this study.</p

Co-culture of the <i>srrA</i>-inactivated mutant with <i>S</i>. <i>sanguinis</i>.

<p>(A) The population percentages of <i>S</i>. <i>aureus</i> MW2 WT harbouring an empty pCL8 vector (MW2::pCL8), the <i>srrA</i>-inactivated mutant and the complemented strain when co-cultured with <i>S</i>. <i>sanguinis</i> were measured by co-culture assay as described in the Materials and Methods section. (B) The population percentage of MW2 strains co-cultured with pre-cultured (37°C under 5% CO₂ for 1 h) <i>S</i>. <i>sanguinis</i>. The data are the mean ± SD of three biological independent experiments. Significant differences compared with WT were determined by Dunnett’s test (*, P < 0.05; **, P < 0.001).</p

Susceptibility to H₂O₂ and expression of <i>katA</i>, <i>dps and ahpC</i> in the <i>perR</i>-inactive mutant.

<p>(A) The susceptibilities of <i>S</i>. <i>aureus</i> MW2, the <i>srrA</i>-inactivated mutant and the <i>perR</i>-inactivated mutant to the H₂O₂ produced by <i>S</i>. <i>sanguinis</i> were determined by direct assay under aerobic conditions (5% CO₂). (B) The expression of <i>katA</i>, <i>dps</i> and <i>ahpC</i> in <i>S</i>. <i>aureus</i> MW2 WT and in the <i>perR</i>-inactivated mutant grown in TSB to mid-log phase was determined by quantitative PCR as described in the Materials and Methods section. The data shown represent the mean ± SD of three biological independent experiments. Significant differences compared with WT were determined by Student’s <i>t</i>-test (***, P < 0.001).</p