Regulation of peptidoglycan synthesis by outer-membrane proteins.

Growth of the mesh-like peptidoglycan (PG) sacculus located between the bacterial inner and outer membranes (OM) is tightly regulated to ensure cellular integrity, maintain cell shape, and orchestrate division. Cytoskeletal elements direct placement and activity of PG synthases from inside the cell, but precise spatiotemporal control over this process is poorly understood. We demonstrate that PG synthases are also controlled from outside of the sacculus. Two OM lipoproteins, LpoA and LpoB, are essential for the function, respectively, of PBP1A and PBP1B, the major E. coli bifunctional PG synthases. Each Lpo protein binds specifically to its cognate PBP and stimulates its transpeptidase activity, thereby facilitating attachment of new PG to the sacculus. LpoB shows partial septal localization, and our data suggest that the LpoB-PBP1B complex contributes to OM constriction during cell division. LpoA/LpoB and their PBP-docking regions are restricted to γ-proteobacteria, providing models for niche-specific regulation of sacculus growth.

Decreased expression of cell proliferation-related genes in clonally derived skin fibroblasts from children with Silver-Russell syndrome is independent of the degree of 11p15 ICR1 hypomethylation.

BACKGROUND: The in vitro analysis of the hypomethylation of imprinting control region 1 (ICR1) within the IGF2/H19 locus is challenged by the mosaic distribution of the epimutation in tissues from children with Silver-Russell syndrome (SRS). To exclude mosaicism, clonal cultures of skin fibroblasts from four children with SRS and three controls were analyzed. Cell proliferation, IGF-II secretion, and IGF2 and H19 expression were measured, and a microarray expression analysis was performed. RESULTS: Single-cell expansion established severely ICR1 hypomethylated clones (SRShypo) and normomethylated clones (SRSnormo) from the patients and controls (Cnormo). IGF2 expression was below the detection limit of the quantitative real-time PCR (qRT-PCR) assay, whereas H19 expression was detectable, without differences between fibroblast clones. Cell count-related IGF-II release was comparable in SRShypo and Cnormo supernatants. Cell proliferation was diminished in SRShypo compared to Cnormo (p = 0.035). The microarray analysis revealed gene expression changes in SRS clones, predicting a decrease in cell proliferation and a delay in mitosis. CONCLUSIONS: The analysis of severely ICR1 hypomethylated clonal fibroblasts did not reveal functional differences compared to normomethylated clones with respect to IGF2 and H19 expression. A difference compared to the clones from healthy individuals was present in the form of a lower proliferation rate, presumably due to impaired cell cycle progression.

IGF2/H19 hypomethylation is tissue, cell, and CpG site dependent and not correlated with body asymmetry in adolescents with Silver-Russell syndrome.

BACKGROUND: Silver-Russell syndrome (SRS) is characterized by severe intrauterine and postnatal growth failure and frequent body asymmetry. Half of the patients with SRS carry a DNA hypomethylation of the imprinting center region 1 (ICR1) of the insulin-like growth factor 2 (IGF2)/H19 locus, and the clinical phenotype is most severe in these patients. We aimed to elucidate the epigenetic basis of asymmetry in SRS and the cellular consequences of the ICR1 hypomethylation. RESULTS: The ICR1 methylation status was analyzed in blood and in addition in buccal smear probes and cultured fibroblasts obtained from punch biopsies taken from the two body halves of 5 SRS patients and 3 controls. We found that the ICR1 hypomethylation in SRS patients was stronger in blood leukocytes and oral mucosa cells than in fibroblasts. ICR1 CpG sites were affected differently. The severity of hypomethylation was not correlated to body asymmetry. IGF2 expression and IGF-II secretion of fibroblasts were not correlated to the degree of ICR1 hypomethylation. SRS fibroblasts responded well to stimulation by recombinant human IGF-I or IGF-II, with proliferation rates comparable with controls. Clonal expansion of primary fibroblasts confirmed the complexity of the cellular mosaicism. CONCLUSIONS: We conclude that the ICR1 hypomethylation SRS is tissue, cell, and CpG site specific. The correlation of the ICR1 hypomethylation to IGF2 and H19 expression is not strict, may depend on the investigated tissue, and may become evident only in case of more severe methylation defects. The body asymmetry in juvenile SRS patients is not related to a corresponding ICR1 hypomethylation gradient, rendering more likely an intrauterine origin of asymmetry. Overall, it may be instrumental to consider not only the ICR1 methylation status as decisive for IGF2/H19 expression regulation.

Silver-Russell syndrome.

The Silver-Russell syndrome (SRS) is a sporadic clinically and genetically heterogeneous disorder. Diagnosis is based on the variable combination of the following characteristics: intrauterine growth retardation, short stature because of lack of catch-up growth, underweight, relative macrocephaly, typical triangular face, body asymmetry and several minor anomalies including clinodactyly V. Different diagnostic scores have been proposed. The main genetic defects detected are at the epigenetic level: hypomethylation of the imprinting control region 1 (ICR1) on 11p15 in around 44% of cases and maternal uniparental disomy of chromosome 7 (UPD(7)mat) in 5-10% of cases. Severe phenotype is frequently associated with hypomethylation of ICR1 while mild phenotype is more often seen in combination with UPD(7)mat. Origins and biological consequences of these epimutations are still obscure. For genetic testing, we recommend a methylation-specific PCR-approach for both 7p and 7q loci (confirmed by microsatellite typing) for the detection of UPD(7)mat, and the methylation-specific multiplex ligation dependent probe amplification (MS-MLPA) approach for methylation analysis of the 11p15 loci. Short stature in SRS can be treated by use of pharmacological doses of recombinant GH resulting in good short-term catch-up; sufficient information on the therapeutic effect in terms of final height is still missing.

Interaction between two murein (peptidoglycan) synthases, PBP3 and PBP1B, in Escherichia coli.

The murein (peptidoglycan) sacculus is an essential polymer embedded in the bacterial envelope. The Escherichia coli class B penicillin-binding protein (PBP) 3 is a murein transpeptidase and essential for cell division. In an affinity chromatography experiment, the bifunctional transglycosylase-transpeptidase murein synthase PBP1B was retained by PBP3-sepharose when a membrane fraction of E. coli was applied. The direct protein-protein interaction between purified PBP3 and PBP1B was characterized in vitro by surface plasmon resonance. The interaction was confirmed in vivo employing two different methods: by a bacterial two-hybrid system, and by cross-linking/co-immunoprecipitation. In the bacterial two-hybrid system, a truncated PBP3 comprising the N-terminal 56 amino acids interacted with PBP1B. Both synthases could be cross-linked in vivo in wild-type cells and in cells lacking FtsW or FtsN. PBP1B localized diffusely and in foci at the septation site and also at the side wall. Statistical analysis of the immunofluorescence signals revealed that the localization of PBP1B at the septation site depended on the physical presence of PBP3, but not on the activity of PBP3. These studies have demonstrated, for the first time, a direct interaction between a class B PBP (PBP3) and a class A PBP (PBP1B) in vitro and in vivo, indicating that different murein synthases might act in concert to enlarge the murein sacculus during cell division.