Role of the Single-Stranded DNA–Binding Protein SsbB in Pneumococcal Transformation: Maintenance of a Reservoir for Genetic Plasticity

Bacteria encode a single-stranded DNA (ssDNA) binding protein (SSB) crucial for genome maintenance. In Bacillus subtilis and Streptococcus pneumoniae, an alternative SSB, SsbB, is expressed uniquely during competence for genetic transformation, but its precise role has been disappointingly obscure. Here, we report our investigations involving comparison of a null mutant (ssbB−) and a C-ter truncation (ssbBΔ7) of SsbB of S. pneumoniae, the latter constructed because SSBs' acidic tail has emerged as a key site for interactions with partner proteins. We provide evidence that SsbB directly protects internalized ssDNA. We show that SsbB is highly abundant, potentially allowing the binding of ∼1.15 Mb ssDNA (half a genome equivalent); that it participates in the processing of ssDNA into recombinants; and that, at high DNA concentration, it is of crucial importance for chromosomal transformation whilst antagonizing plasmid transformation. While the latter observation explains a long-standing observation that plasmid transformation is very inefficient in S. pneumoniae (compared to chromosomal transformation), the former supports our previous suggestion that SsbB creates a reservoir of ssDNA, allowing successive recombination cycles. SsbBΔ7 fulfils the reservoir function, suggesting that SsbB C-ter is not necessary for processing protein(s) to access stored ssDNA. We propose that the evolutionary raison d'être of SsbB and its abundance is maintenance of this reservoir, which contributes to the genetic plasticity of S. pneumoniae by increasing the likelihood of multiple transformation events in the same cell

A point mutation in cpsE renders Streptococcus pneumoniae nonencapsulated and enhances its growth, adherence and competence.

BACKGROUND: The polysaccharide capsule is a major virulence factor of the important human pathogen Streptococcus pneumoniae. However, S. pneumoniae strains lacking capsule do occur. RESULTS: Here, we report a nasopharyngeal isolate of Streptococcus pneumoniae composed of a mixture of two phenotypes; one encapsulated (serotype 18C) and the other nonencapsulated, determined by serotyping, electron microscopy and fluorescence isothiocyanate dextran exclusion assay.By whole genome sequencing, we demonstrated that the phenotypes differ by a single nucleotide base pair in capsular gene cpsE (C to G change at gene position 1135) predicted to result in amino acid change from arginine to glycine at position 379, located in the cytoplasmic, enzymatically active, region of this transmembrane protein. This SNP is responsible for loss of capsule production as the phenotype is transferred with the capsule operon. The nonencapsulated variant is superior in growth in vitro and is also 117-fold more adherent to and more invasive into Detroit 562 human epithelial cells than the encapsulated variant.Expression of six competence pathway genes and one competence-associated gene was 11 to 34-fold higher in the nonencapsulated variant than the encapsulated and transformation frequency was 3.7-fold greater. CONCLUSIONS: We identified a new single point mutation in capsule gene cpsE of a clinical S. pneumoniae serotype 18C isolate sufficient to cause loss of capsule expression resulting in the co-existence of the encapsulated and nonencapsulated phenotype. The mutation caused phenotypic changes in growth, adherence to epithelial cells and transformability. Mutation in capsule gene cpsE may be a way for S. pneumoniae to lose its capsule and increase its colonization potential

A Novel Manganese Efflux System, YebN, Is Required for Virulence by Xanthomonas oryzae pv. oryzae

Manganese ions (Mn2+) play a crucial role in virulence and protection against oxidative stress in bacterial pathogens. Such pathogens appear to have evolved complex mechanisms for regulating Mn2+ uptake and efflux. Despite numerous studies on Mn2+ uptake, however, only one efflux system has been identified to date. Here, we report on a novel Mn2+ export system, YebN, in Xanthomonas oryzae pv. oryzae (Xoo), the causative agent of bacterial leaf blight. Compared with wild-type PXO99, the yebN mutant was highly sensitive to Mn2+ and accumulated high concentrations of intracellular manganese. In addition, we found that expression of yebN was positively regulated by Mn2+ and the Mn2+-dependent transcription regulator, MntR. Interestingly, the yebN mutant was more tolerant to methyl viologen and H2O2 in low Mn2+ medium than PXO99, but more sensitive in high Mn2+ medium, implying that YebN plays an important role in Mn2+ homoeostasis and detoxification of reactive oxygen species (ROS). Notably, deletion of yebN rendered Xoo sensitive to hypo-osmotic shock, suggesting that YebN may protect against such stress. That mutation of yebN substantially reduced the Xoo growth rate and lesion formation in rice implies that YebN could be involved in Xoo fitness in host. Although YebN has two DUF204 domains, it lacks homology to any known metal transporter. Hence, this is the first report of a novel metal export system that plays essential roles in hypo-osmotic and oxidative stress, and virulence. Our results lay the foundations for elucidating the complex and fascinating relationship between metal homeostasis and host-pathogen interactions