f Streptococcus mutans, a major etiological agent of human dental caries, lives primarily on the tooth surface in biofilms. Limited information is available concerning the extracellular DNA (eDNA) as a scaffolding matrix in S. mutans biofilms. This study demonstrates that S. mutans produces eDNA by multiple avenues, including lysis-independent membrane vesicles. Unlike eDNAs from cell lysis that were abundant and mainly concentrated around broken cells or cell debris with floating open ends, eDNAs produced via the lysis-independent pathway appeared scattered but in a structured network under scanning electron microscopy. Compared to eDNA production of planktonic cultures, eDNA production in 5-and 24-h biofilms was increased by >3-and >1.6-fold, respectively. The addition of DNase I to growth medium significantly reduced biofilm formation. In an in vitro adherence assay, added chromosomal DNA alone had a limited effect on S. mutans adherence to saliva-coated hydroxylapatite beads, but in conjunction with glucans synthesized using purified glucosyltransferase B, the adherence was significantly enhanced. Deletion of sortase A, the transpeptidase that covalently couples multiple surface-associated proteins to the cell wall peptidoglycan, significantly reduced eDNA in both planktonic and biofilm cultures. Sortase A deficiency did not have a significant effect on membrane vesicle production; however, the protein profile of the mutant membrane vesicles was significantly altered, including reduction of adhesin P1 and glucan-binding proteins B and C. Relative to the wild type, deficiency of protein secretion and membrane protein insertion machinery components, including Ffh, YidC1, and YidC2, also caused significant reductions in eDNA.

The transcriptional repressor Rex has been implicated in regulation of energy metabolism and fermentative growth in response to redox potential. Streptococcus mutans, the primary causative agent of human dental caries, possesses a gene that encodes a protein with high similarity to members of the Rex family of proteins. In this study, we showed that Rex-deficiency compromised the ability of S. mutans to cope with oxidative stress and to form biofilms. The Rex-deficient mutant also accumulated less biofilm after 3-days than the wild-type strain, especially when grown in sucrose-containing medium, but produced more extracellular glucans than the parental strain. Rex-deficiency caused substantial alterations in gene transcription, including those involved in heterofermentative metabolism, NAD+ regeneration and oxidative stress. Among the up-regulated genes was gtfC, which encodes glucosyltransferase C, an enzyme primarily responsible for synthesis of water-insoluble glucans. These results reveal that Rex plays an important role in oxidative stress responses and biofilm formation by S. mutans.

Increasing evidence suggests that sulfur in ubiquitous ironsulfur clusters is derived from L-cysteine via cysteine desulfurases. In Escherichia coli, the major cysteine desulfurase activity for biogenesis of iron-sulfur clusters has been attributed to IscS. The gene that encodes IscS is a member of an operon isc-SUA, which also encodes two highly conserved proteins: IscU and IscA. Previous studies suggested that both IscU and IscA may act as the iron-sulfur cluster assembly scaffold proteins. However, recent evidence indicated that IscA is an iron-binding protein that can provide iron for the iron-sulfur cluster assembly in IscU (Ding, H., Harrison, K., and Lu, J. Iron-sulfur clusters are one of the most ancient and ubiquitous redox centers in almost all living organisms (1-3). Throughout evolution, iron-sulfur clusters have become integral parts of diverse biological processes including energy conversion and the regulation of gene expression. It is now clear that biogenesis of iron-sulfur clusters is not a spontaneous process. The pioneering work by Dean's group (4) revealed that sulfur in iron-sulfur clusters is derived from L-cysteine via cysteine desulfurases, a group of pyridoxal 5-phosphate-dependent enzymes that are conserved from bacteria to humans (5-7). In Escherichia coli, there are at least three cysteine desulfurases: IscS 2 (5), SufS (8), and CSD (cysteine sulfinate desulfinase) (9, 10). Deletion of gene iscS greatly diminishes the specific activities of iron-sulfur proteins in E. coli (11,12), suggesting that IscS is the major cysteine desulfurase for biogenesis of ironsulfur clusters. Gene iscS is a member of an operon iscSUA, which also encodes two highly conserved proteins: IscU and IscA (13,14). Biochemical studies indicated that IscS catalyzes desulfurization of L-cysteine and transfers sulfane sulfur for the iron-sulfur cluster assembly in IscU via specific protein-protein interactions (15-18). Accordingly, IscU was characterized as an iron-sulfur cluster assembly scaffold protein (15-21).The function of IscA, however, still remains elusive. Previous studies suggested that IscA is an alternative iron-sulfur cluster assembly scaffold protein (22-29), because IscA, like IscU, can bind iron-sulfur clusters in the presence of ferrous iron and sulfide in vitro. On the other hand, recent studies indicated that IscA is a novel iron binding protein with an iron association constant of 2.0 -3.0 ϫ 10 19 M Ϫ1 in the presence of the thioredoxin reductase system (30) or dithiothreitol (31) and that the iron-loaded IscA can provide iron for the iron-sulfur cluster assembly in IscU (32, 33). To reconcile the two models proposed for the function of IscA, here we re-evaluated the ironsulfur cluster binding activity of IscA and IscU under physiologically relevant conditions and found that in the presence of ferrous iron, L-cysteine, and cysteine desulfurase IscS, IscU is a preferred iron-sulfur cluster assembly scaffold protein. On the other hand, when L-cysteine is not present in the incubation solution, Isc...

SynopsisIscA/SufA paralogs are the members of the iron-sulfur cluster assembly machinery in Escherichia coli. While deletion of either IscA or SufA has only a mild effect on cell growth, deletion of both IscA and SufA results in a null-growth phenotype in minimal medium under aerobic growth conditions. Here we report that cell growth of the iscA/sufA double mutant (E. coli strain in which both iscA and sufA had been in-frame-deleted) can be partially restored by supplementing with BCAAs (branched-chain amino acids) and thiamin. We further demonstrate that deletion of IscA/ SufA paralogs blocks the [4Fe-4S] cluster assembly in IlvD (dihydroxyacid dehydratase) of the BCAA biosynthesis pathway in E. coli cells under aerobic conditions and that addition of the ironbound IscA/SufA efficiently promotes the [4Fe-4S] cluster assembly in IlvD and restores the enzyme activity in vitro, suggesting that IscA/SufA may act as an iron donor for the [4Fe-4S] cluster assembly under aerobic conditions. Additional studies reveal that IscA/SufA are also required for the cluster assembly in protein ThiC of the thiamin biosynthesis pathway, aconitase B of the citrate acid cycle, and endonuclease III of the DNA base excision repair pathway in E. coli under aerobic conditions. Nevertheless, deletion of IscA/SufA does not significantly affect the [2Fe-2S] cluster assembly in the redox transcription factor SoxR, ferredoxin, and the siderophore-iron reductase FhuF. The results suggest that the biogenesis of the [4Fe-4S] clusters and the [2Fe-2S] clusters may have distinct pathways and that IscA/SufA paralogs are essential for the [4Fe-4S] cluster assembly, but are dispensable for the [2Fe-2S] cluster assembly in E. coli under aerobic conditions. Keywords aconitase; branched-chain amino acids; dihydroxyacid dehydratase; iron-sulfur clusters; IscA/SufA paralogs; thiamin IntroductionIron-sulfur clusters are one of the most ancient and ubiquitous redox centers in biology. They are involved in diverse physiological processes including respiratory electron transfer, nitrogen fixation, photosynthesis, biosynthesis of amino acids, thiamin, heme, biotin, and lipoic acid, DNA synthesis and repair, RNA modification, and the regulation of gene expression [1,2]. However, the mechanism underlying the iron-sulfur cluster assembly is still not fully understood [3]. The discovery of cysteine desulfurase (NifS) Experimental Mutant strains and cell growthThe E. coli deletion mutants in which iscA and sufA were in-frame deleted were previously constructed [28]. Each deletion in E. coli cells was confirmed by PCR as described in [28]. For cell growth analysis, overnight cell cultures grown in Luria-Bertani (LB) medium were washed twice with minimal medium containing glucose (0.2%) before inoculated on minimal medium plates or in liquid minimal medium at 37°C with aeration (250 rpm). Cell growth was recorded by measuring the optical density of cell culture at 600 nm. When indicated, minimal medium was supplemented with the three branched-chain amino acids ...

ABSTRACTPrevious studies have shown that BrpA plays a major role in acid and oxidative stress tolerance and biofilm formation byStreptococcus mutans. Mutant strains lacking BrpA also display increased autolysis and decreased viability, suggesting a role for BrpA in cell envelope integrity. In this study, we examined the impact of BrpA deficiency on cell envelope stresses induced by envelope-active antimicrobials. Compared to the wild-type strain UA159, the BrpA-deficient mutant (TW14D) was significantly more susceptible to antimicrobial agents, especially lipid II inhibitors. Several genes involved in peptidoglycan synthesis were identified by DNA microarray analysis as downregulated in TW14D. Luciferase reporter gene fusion assays also revealed that expression ofbrpAis regulated in response to environmental conditions and stresses induced by exposure to subinhibitory concentrations of cell envelope antimicrobials. In aGalleria mellonella(wax worm) model, BrpA deficiency was shown to diminish the virulence ofS. mutansOMZ175, which, unlikeS. mutansUA159, efficiently kills the worms. Collectively, these results suggest that BrpA plays a role in the regulation of cell envelope integrity and that deficiency of BrpA adversely affects the fitness and diminishes the virulence of OMZ175, a highly invasive strain ofS. mutans.