Most RNA molecules require Mg(2+) for their structure and enzymatic properties. Here we report the first example of an RNA serving as sensor for cytoplasmic Mg(2+). We establish that expression of the Mg(2+) transporter MgtA of Salmonella enterica serovar Typhimurium is controlled by its 5' untranslated region (5'UTR). We show that the 5'UTR of the mgtA gene can adopt different stem-loop structures depending on the Mg(2+) levels, which determine whether transcription reads through into the mgtA coding region or stops within the 5'UTR. We could recapitulate the Mg(2+)-regulated transcription using a defined in vitro transcription system with RNA polymerase as the only protein component. The initiation of mgtA transcription responds to extracytoplasmic Mg(2+) and its elongation into the coding region to cytoplasmic Mg(2+), providing a singular example in which the same ligand is sensed in different cellular compartments to regulate disparate steps in gene transcription.

Porphyromonas gingivalis produces arginine-specific cysteine proteinase (Arg-gingipain, RGP) and lysinespecific cysteine proteinase (Lys-gingipain, KGP) in the extracellular and cell-associated forms. Two separate genes (rgpA and rgpB) and a single gene (kgp) have been found to encode RGP and KGP, respectively. We constructed rgpA rgpB kgp triple mutants by homologous recombination with cloned rgp and kgp DNA interrupted by drug resistance gene markers. The triple mutants showed no RGP or KGP activity in either cell extracts or culture supernatants. The culture supernatants of the triple mutants grown in a rich medium had no proteolytic activity toward bovine serum albumin or gelatin derived from human type I collagen. Moreover, the mutants did not grow in a defined medium containing bovine serum albumin as the sole carbon/energy source. These results indicate that the proteolytic activity of P. gingivalis toward bovine serum albumin and gelatin derived from human type I collagen appears to be attributable to RGP and KGP. The hemagglutinin gene hagA of P. gingivalis possesses the adhesin domain regions responsible for hemagglutination and hemoglobin binding that are also located in the Cterminal regions of rgpA and kgp. A rgpA kgp hagA triple mutant constructed in this study exhibited no hemagglutination using sheep erythrocytes or hemoglobin binding activity, as determined by a solid-phase binding assay with horseradish peroxidase-conjugated human hemoglobin, indicating that the adhesin domains seem to be particularly important for P. gingivalis cells to agglutinate erythrocytes and bind hemoglobin, leading to heme acquisition.

Background Previous reports suggest that peripheral airways are associated with asthma control. Patient history, although subjective is used largely to assess asthma control in children because spirometry is many times normal. Impulse oscillometry (IOS) is an objective non-invasive measurement of lung function, which has the potential to examine independently both small and large airway obstruction. Objective To determine the utility of IOS in assessing asthma control in children. Methods Asthmatic and healthy children (6–17 yrs) were enrolled in the study. Spirometry and IOS (resistance at 5 and 20 Hz, R5 and R20, respectively, reactance at 5 Hz, X5, resonant frequency, Fres, and area under the reactance curve between 5 Hz and Fres, AX) were collected in triplicate before and after a bronchodilator was administered. The physicians were blinded to the IOS measurements and assessed asthma control using ATS guidelines. Results Small airway IOS measurements, including R5-20, X5, Fres and AX, of children with uncontrolled asthma (n=44) were significantly different from those of controlled asthmatic (n=57) and healthy (n=14) children, especially prior to the administration of a bronchodilator. However, there was no difference in large airway IOS (R20). No differences were found between controlled asthmatic and healthy children in any of the endpoints. ROC analysis showed cut-points for baseline R5-20 (1.5 cmH2O·L−1·s) and AX (9.5 cmH2O·L−1) that effectively discriminated controlled versus uncontrolled asthma (AUC=0.86 and 0.84), and correctly classified more than 80% of the population. Conclusion Uncontrolled asthma is associated with small airways dysfunction, and IOS may be a reliable non-invasive method to assess asthma control in children.

The PhoP/PhoQ two-component system is a master regulator that governs the ability of Salmonella to cause a lethal infection in mice, the adaptation to low Mg 2؉ environments, and resistance to a variety of antimicrobial peptides. We have recently established that the PhoP-activated ugtL gene is required for resistance to the antimicrobial peptides magainin 2 and polymyxin B. Here we report that ugtL transcription requires not only the PhoP protein but also the virulence regulatory protein SlyA. The PhoP protein footprinted two regions of the ugtL promoter, mutation of either one of which was sufficient to abolish ugtL transcription. Although the SlyA protein is a transcriptional activator of the ugtL gene, it footprinted the ugtL promoter at a region located downstream of the transcription start site. The PhoP protein footprinted the slyA promoter, indicating that it controls slyA transcription directly. The slyA mutant was hypersensitive to magainin 2 and polymyxin B, suggesting that the virulence attenuation exhibited by slyA mutants may be caused by hypersensitivity to antimicrobial peptides. We propose that the PhoP and SlyA proteins control ugtL transcription using a feed-forward loop design.

A nonpigmented mutant of Porphyromonas gingivalis was constructed by using transposon mutagenesis. The mutant possessed the transposon DNA at the novel gene porR. Gene targeted mutagenesis revealed that porR was responsible for pigmentation. The porR gene shared similarities with genes of the degT family, the products of which are now considered to be transaminases involved in biosynthesis of sugar portions of cell-surface polysaccharides and aminoglycosides. The porR mutant showed a pleiotropic phenotype : delayed maturation of fimbrillin, preferential presence of Rgp and Kgp proteinases in culture supernatants, and no haemagglutination. The porR mutant had altered phenol extractable polysaccharide compared to the porR M sibling strain. A mAb, 1B5, that reacts with sugar portions of P. gingivalis cell surface polysaccharide and membrane-type Rgp proteinase showed no reaction with the cell lysates of the porR mutant. These results indicate that porR is involved in biosynthesis of cell surface polysaccharide that may function as an anchorage for Rgp, Kgp, haemagglutinins and the haemoglobin receptor protein.