Deficiency of BrpB causes major defects in cell division, stress responses and biofilm formation by Streptococcus mutans

Abstract

Streptococcus mutans, the primary aetiological agent of dental caries, possesses an YjeE-like protein that is encoded by locus SMU.409, herein designated brpB. In this study, a BrpBdeficient mutant, JB409, and a double mutant deficient of BrpB and BrpA (a paralogue of the LytR-CpsA-Psr family of cell wall-associated proteins), JB819, were constructed and characterized using function assays and microscopy analysis. Both JB409 and JB819 displayed extended lag phases and drastically slowed growth rates during growth in brain heart infusion medium as compared to the wild-type, UA159. Relative to UA159, JB409 and JB819 were more than 60-and 10-fold more susceptible to acid killing at pH 2.8, and more than 1 and 2 logs more susceptible to hydrogen peroxide, respectively. Complementation of the deficient mutants with a wild-type copy of the respective gene(s) partly restored the acid and oxidative stress responses to a level similar to the wild-type. As compared to UA159, biofilm formation by JB409 and JB819 was drastically reduced (P,0.001), especially during growth in medium containing sucrose. Under a scanning electron microscope, JB409 had significantly more giant cells with an elongated, rod-like morphology, and JB819 formed marble-like super cells with apparent defects in cell division. As revealed by transmission electron microscopy analysis, BrpB deficiency in both JB409 and JB819 resulted in the development of low electron density patches and formation of a loose nucleoid structure. Taken together, these results suggest that BrpB likely functions together with BrpA in regulating cell envelope biogenesis/homeostasis in Strep. mutans. Further studies are under way to elucidate the mechanism that underlies the BrpA-and BrpB-mediated regulation.

Development and functional characterization of extrahepatic cholangiocyte lines from normal rats

“…For ultrastructural analysis [30, 31], EBDs were fixed in 2% paraformaldehyde/2.5% glutaraldehyde in 100 mM phosphate buffer, pH 7.2 for 1 hr at room temperature. Samples were washed in phosphate buffer and post-fixed in 1% osmium tetroxide (Polysciences Inc.) for 1 hr.…”

Essentiality of threonylcarbamoyladenosine (t6A), a universal tRNA modification, in bacteria

“…Single gene knockout (Bitoun et al, 2014) All genes are essential except those highlighted in bold. Essentiality of gene names underlined has not been determined.…”

“…Finally, phenotypes deriving from the absence or reduction of t 6 A enzymes in prokaryotes are very diverse, and include defects in cell division or in stress responses, increased glycation of proteins, or cyanophycin accumulation (Zuther et al ., ; Allali‐Hassani et al ., ; Handford et al ., ; Oberto et al ., ; Katz et al ., ; Bergmiller et al ., ; Bitoun et al ., ). However, the molecular basis for these pleiotropic phenotypes has not been established, and it is unknown whether these phenotypes are all due to translation defects caused by the absence of the modification, whether t 6 A has roles in the cell not linked to translation, or whether the genes for t 6 A biosynthesis have roles in the cell unrelated to t 6 A synthesis.…”

“…Interestingly, the V. cholerae genomes contain both the tsaC and sua5 (tsaC2) forms, and either gene alone is sufficient for growth (Chao et al, 2013;Kamp et al, 2013). Finally, t 6 A synthesis genes can be deleted in some bacteria, for example, tsaD and tsaB in Deinococcus radiodurans (Onodera et al, 2013); tsaD in Synechocystis PCC 6803 (Zuther et al, 1998); and, tsaE in Streptococcus mutans (Bitoun et al, 2014). Some discrepancies were found in Bacillus subtilis, where all four t 6 A genes were found to be essential in a global transposon study (Table 2), but two of them, ywlC (sua5 homolog) and ydiB (tsaE homolog), were found to be dispensable in follow-up targeted studies (Hunt et al, 2006).…”

Modification of the CpsA Protein Reveals a Role in Alteration of the Streptococcus agalactiae Cell Envelope

“…In Streptococcus mutans, mutation of the LCP family member brpA led to abnormal cell division events and atypical cell wall morphology (43,46). S. mutans lytR mutants also have dysregulated cell division, leading to the formation of long chains of cocci (47).…”