The relative localization patterns of class B penicillin-binding proteins Pbp2x and Pbp2b were used as positional indicators of septal and peripheral (side-wall-like) peptidoglycan (PG) synthesis, respectively, in the midcell regions of Streptococcus pneumoniae cells at different stages of division. We confirm that Pbp2x and Pbp2b are essential in the strain D39 genetic background, which differs from that of laboratory strains. We show that Pbp2b, like Pbp2x and class A Pbp1a, follows a different localization pattern than FtsZ and remains at division septa after FtsZ reappears at the equators of daughter cells. Pulse-experiments with fluorescent D-amino acids (FDAAs) were performed in wild-type cells and in cells in which Pbp2x activity was preferentially inhibited by methicillin or Pbp2x amount was depleted. These experiments show that Pbp2x activity separates from that of other PBPs to the centers of constricting septa in mid-to-late divisional cells resolved by high-resolution 3D-SIM microscopy. Dual-protein and protein-fluorescent vancomycin 2D and 3D-SIM immunofluorescence microscopy (IFM) of cells at different division stages corroborate that Pbp2x separates to the centers of septa surrounded by an adjacent constricting ring containing Pbp2b, Pbp1a, and regulators, StkP and MreC. The separate localization of Pbp2x suggests distinctive roles in completing septal PG synthesis and remodeling.

Summary Bacterial cell shapes are manifestations of programs carried out by multi-protein machines that synthesize and remodel the peptidoglycan (PG) mesh and other polymers surrounding cells. GpsB protein is conserved in low-GC Gram-positive bacteria and is not essential in rod-shaped Bacillus subtilis, where it plays a role in shuttling penicillin binding proteins (PBPs) between septal side-wall sites of PG synthesis. In contrast, we report here that GpsB is essential in ellipsoid-shaped, ovococcal Streptococcus pneumoniae (pneumococcus), and depletion of GpsB leads to formation of elongated, enlarged cells containing unsegregated nucleoids and multiple, unconstricted rings of fluorescent-vancomycin staining, and eventual lysis. These phenotypes are similar to those caused by selective inhibition of Pbp2x by methicillin that prevents septal PG synthesis. Dual-protein 2D and 3D-SIM (structured illumination) immunofluorescence microscopy (IFM) showed that GpsB and FtsZ have overlapping, but not identical, patterns of localization during cell division and that multiple, unconstricted rings of division proteins FtsZ, Pbp2x, Pbp1a, and MreC are in elongated cells depleted of GpsB. These patterns suggest that GpsB, like Pbp2x, mediates septal ring closure. This first dual-protein 3D-SIM IFM analysis also revealed separate positioning of Pbp2x and Pbp1a in constricting septa, consistent with two separable PG synthesis machines.

The MutL, MutS, and MutH proteins mediate methyl-directed mismatch (MDM) repair and help to maintain chromosome stability in Escherichia coli. We determined the amounts of the MDM repair proteins in exponentially growing, stationary-phase, and nutrient-starved bacteria by quantitative Western immunoblotting. Extracts of null mutants containing various amounts of purified MDM repair proteins were used as quantitation standards. In bacteria growing exponentially in enriched minimal salts-glucose medium, about 113 MutL dimers, 186 MutS dimers, and 135 MutH monomers were present per cell. Calculations with the in vitro dissociation constants of MutS binding to different mismatches suggested that MutS is not present in excess, and may be nearly limiting in some cases, for MDM repair in exponentially growing cells. Remarkably, when bacteria entered late stationary phase or were deprived of a utilizable carbon source for several days, the cellular amount of MutS dropped at least 10-fold and became barely detectable by the methods used. In contrast, the amount of MutH dropped only about threefold and the amount of MutL remained essentially constant in late-stationary-phase and carbon-starved cells compared with those in exponentially growing bacteria. RNase T2 protection assays showed that the amounts of mutS, mutH, and mutL, but not miaA, transcripts decreased to undetectable levels in late-stationary-phase cells. These results suggested that depletion of MutS in nutritionally stressed cells was possibly caused by the relative instability of MutS compared with MutL and MutH. Our findings suggest that the MDM repair capacity is repressed in nutritionally stressed bacteria and correlate with conclusions from recent studies of adaptive mutagenesis. On the other hand, we did not detect induction of MutS or MutL in cells containing stable mismatches in multicopy single-stranded DNA encoded by bacterial retrons.The MutS, MutL, and MutH proteins play crucial roles in methyl-directed mismatch (MDM) repair in Escherichia coli ( Fig. 1) (35)(36)(37)45). MDM repair corrects mismatched base pairs and small bulge loops that arise as replication errors and thereby helps to set the spontaneous mutation rate (39,57,58). The MutL and MutS proteins also prevent homeologous recombination between related bacterial species (32,46,64,65), suppress chromosomal rearrangements (41), and play an ancillary role in very-short-patch repair, which corrects mismatches that arise by deamination of 5-methyl-cytosine residues in certain contexts (29,66). Together, these diverse functions indicate that the MutS, MutL, and MutH MDM repair proteins are part of a major system that maintains the genetic integrity and stability of bacterial chromosomes (36, 37, 45). The strand-break-directed mismatch repair exemplified by the E. coli MDM repair system is ancient and ubiquitous, and homologs of E. coli MutS and MutL have been found in yeasts, humans, and other organisms (7, 14, 23, 26a, 38, 42, 47). The recent discovery that human colon and sporadic cancers a...

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; decreased in stationary phase), Spd-sr37 (80 nt; strongly expressed in all growth phases), and CcnA (93 nt; induced by competence stimulatory peptide). Spd-sr17 and CcnA likely fold into structures containing single-stranded regions between hairpin structures, whereas Spd-sr37 forms a base-paired structure. Primer extension mapping and ectopic expression in deletion/insertion mutants confirmed the independent expression of the three sRNAs. Microarray analyses indicated that insertion/deletion mutants in spd-sr37 and ccnA exerted strong cis-acting effects on the transcription of adjacent genes, indicating that these sRNA regions are also cotranscribed in operons. Deletion or overexpression of the three sRNAs did not cause changes in growth, certain stress responses, global transcription, or virulence. Constitutive ectopic expression of CcnA reversed some phenotypes of D39 ⌬ciaR mutants, but attempts to link CcnA to -E to comC as a target were inconclusive in ciaR ؉ strains. These results show that S. pneumoniae, which lacks known RNA chaperones, expresses numerous sRNAs, but three of these sRNAs do not strongly affect common phenotypes or transcription patterns.A large number of noncoding small RNAs (sRNAs) 50 to 400 nucleotides (nt) in length have been detected and characterized recently in numerous bacterial species (reviewed in references 3, 18, and 78). Some abundant, stable sRNAs, such as RNase P (14), tmRNA (34), and scRNA (4.5S RNA) (23, 33), are highly conserved and play important housekeeping and stress-related functions in RNA metabolism, protein degradation, and secretion. But most regulatory sRNAs are conserved only among closely related species (42). Many sRNAs play key roles in responses to stress conditions, such as iron limitation, osmotic shock, temperature shift, stationary phase, and metabolic imbalance, in different bacterial species (3,15,17,25,26,46,76,78,79). Other sRNAs are expressed during growth or developmental phases that are specific for particular bacterial species (38,64,68,75). In addition, sRNAs have been postulated to mediate virulence gene expression in several pathogenic bacteria and their survival in hosts (3,6,37,55,68,73).Little is known about RNA metabolism in Streptococcus pneumoniae (pneumococcus), which is a major human respiratory pathogen that causes several serious invasive diseases, including pneumonia, otitis media (ear infection), sinusitis, meningitis, and septicemia (49). Pneumococcus exists as a commensal bacterium that inhabits and colonizes the nasopharynx of up to 20 and 50% of healthy adults and children, respectively, at any time (10). The transition from commensal bacterium to opportunistic pathogen often occurs after a respiratory tract infection, and invasive pneumococcal diseases result in over 1.6 million deaths annually worldwide, especially among young, elderly, debilitated, and immunosuppressed individuals (reviewed in references 13 and 30). Clearly, S. pneumoniae has the ability to inhabit numerous niches in the human body (31,32), and responses ...