Escherichia coli division inhibitor MinCD blocks septation by preventing Z-ring formation.

The min system spatially regulates division through the topological regulation of MinCD, an inhibitor of cell division. MinCD was previously shown to inhibit division by preventing assembly of the Z ring (E. Bi and J. Lutkenhaus, J. Bacteriol. 175:1118-1125, 1993); however, this was questioned in a recent report (S. S. Justice, J. Garcia-Lara, and L. I. Rothfield, Mol. Microbiol. 37:410-423, 2000) which indicated that MinCD acted after Z-ring formation and prevented the recruitment of FtsA to the Z ring. This discrepancy was due in part to alternative fixation conditions. We have therefore reinvestigated the action of MinCD and avoided fixation by using green fluorescent protein (GFP) fusions to division proteins. MinCD prevented the localization of both FtsZ-GFP and ZipA-GFP, consistent with it preventing Z-ring assembly. Consistent with a direct interaction between FtsZ and the MinCD inhibitor, we find that increased FtsZ, but not FtsA, suppresses MinCD-induced lethality. Furthermore, strains carrying various alleles of ftsZ, selected on the basis of resistance to the inhibitor SulA, displayed variable resistance to MinCD. These results are consistent with FtsZ as the target of MinCD and confirm that this inhibitor prevents Z-ring assembly.

The MinC component of the division site selection system in Escherichia coli interacts with FtsZ to prevent polymerization.

Positioning of the Z ring at the midcell site in Escherichia coli is assured by the min system, which masks polar sites through topological regulation of MinC, an inhibitor of division. To study how MinC inhibits division, we have generated a MalE-MinC fusion that retains full biological activity. We find that MalE-MinC interacts with FtsZ and prevents polymerization without inhibiting FtsZ's GTPase activity. MalE-MinC19 has reduced ability to inhibit division, reduced affinity for FtsZ, and reduced ability to inhibit FtsZ polymerization. These results, along with MinC localization, suggest that MinC rapidly oscillates between the poles of the cell to destabilize FtsZ filaments that have formed before they mature into polar Z rings.

Escherichia coli gene ydeA encodes a major facilitator pump which exports L-arabinose and isopropyl-beta-D-thiogalactopyranoside.

Inactivation of the Escherichia coli gene ydeA, which encodes a member of the major facilitator superfamily, decreased the efflux of L-arabinose, thereby affecting the expression of AraC-regulated genes. In addition, overexpression of ydeA decreased the expression of genes regulated by isopropyl-beta-D-thiogalactopyranoside.

An Escherichia coli gene (yaeO) suppresses temperature-sensitive mutations in essential genes by modulating Rho-dependent transcription termination.

An extragenic multicopy suppressor of the cell division inhibition caused by a MalE-MinE fusion protein in Escherichia coli has been mapped and identified as yaeO, one of the two short open reading frames (ORFs) of an operon located at 4.6 min. Overexpressed yaeO also suppressed some temperature-sensitive mutations in division genes ftsA and ftsQ, in chaperone gene groEL and in co-chaperone gene grpE. Gene yaeO, whose expression is regulated by growth rate, codes for a 9 kDa acidic protein with no obvious resemblance to other proteins. Transcription termination protein Rho co-purified with a histidine-tagged derivative of YaeO protein on Ni2+-NTA agarose columns in a manner that suggested direct YaeO-Rho interaction. In vivo, yaeO expression reduced termination at rho-dependent bacteriophage terminator tL1 and at the terminator of autogenously regulated gene rho. The suppression of temperature-sensitive phenotypes was a consequence of anti-termination, as it could be mimicked by a Prho::Tn10 mutation that reduces the expression and activity of gene rho. Our data indicate that the suppression is not caused by overexpression of the mutated genes, but presumably by indirect stabilization of the mutated proteins.

On the birth and fate of bacterial division sites.

Thanks to genetics, to the study of protein-protein interactions and to direct viewing of subcellular structures by the use of immunofluorescence and green fluorescent protein (GFP) fusions, the organization of the constriction apparatus of walled bacteria is gradually coming to light. The tubulin-like protein FtsZ assembles as a ring around the site of constriction and operates as an organizer and activator of septum-shaping proteins. Much less is known about the factors specifying the location of FtsZ rings. Circumstantial evidence favours the presence at future ring positions of fixed elements, the potential division sites (PDS), before FtsZ assembles. FtsZ polymerization is initiated from a point on a PDS, the nucleation site, still to be identified, and proceeds bidirectionally around the cell. We hypothesize that new PDS are specified in a manner that depends on the functioning of an active chromosome partition apparatus. This view is supported by the fact that formation of mid-cell PDS requires initiation of DNA replication, and by recent studies supporting the existence of a specialized partition apparatus in a variety of microorganisms. Although PDS may be specified directly by the partition apparatus, indirect localization linked to compartmentalized gene expression during chromosome segregation is also possible. Once created, PDS are used in a regulated manner, and several mechanisms normally operate to direct constriction to selected PDS at the correct time. One, dedicated to the permanent suppression of polar PDS, rests on the minicell suppression system and involves a protein that is able to discriminate between polar and non-polar sites. Another is involved in asymmetric site selection at the early stages of sporulation in Bacillus subtilis. Finally, a mechanism observed only in certain multi-nucleated cells appears to favour division at non-polar PDS related to the most ancient replication/DNA segregation events.

MinCD-independent inhibition of cell division by a protein that fuses MalE to the topological specificity factor MinE.

We report that MinE, the topological specificity factor of cell division in Escherichia coli, inhibits septation when fused to the C terminus of the maltose-binding protein MalE. This contrasts with overexpression of MinE alone, which affects growth but has no effect on division. Inhibition by MalE-MinE was minCD independent and depended on MinE segments involved in dimerization and prevention of MinCD division inhibition. The SOS and the heat shock responses were not involved, suggesting that the inhibition comes from a direct interaction of MalE-MinE with the septation apparatus. MalE-MinE lethality was suppressed by overexpression of ftsZ, as well as by overexpression of ftsN, a suppressor of temperature-sensitive mutations in genes ftsQ, ftsA, and ftsI. We also report that high-level synthesis of MalE disturbs nucleoid partitioning.

Deletion analysis of gene minE which encodes the topological specificity factor of cell division in Escherichia coli.

Division inhibition caused by the minCD gene products of Escherichia coli is suppressed specifically at mid-cell by MinE protein expressed at physiological levels. Excess MinE allows division to take place also at the poles, leading to a minicell-forming (Min-) phenotype. In order to investigate the basis of this topological specificity, we have analysed the ability of truncated derivatives of MinE to suppress either minCD-dependent division inhibition in a chromosomal delta(minB) background, or the division inhibition exerted by MinCD at the cell poles in a minB+ strain. Our results indicate that these two effects are not mediated by identical interactions of MinE protein. In addition, gel filtration and the yeast two-hybrid system indicated that MinE interacts with itself by means of its central segment. Taken together, our results favour a model in which wild-type MinE dimer molecules direct the division inhibitor molecules to the cell poles, thus preventing polar divisions and allowing non-polar sites to divide. This model explains how excess MinE, or an excess of certain MinE derivatives which prevent the accumulation of the division inhibitor at the poles, can confer a Min- phenotype in a minB+ strain.

Novel Drosophila melanogaster genes encoding RRM-type RNA-binding proteins identified by a degenerate PCR strategy.

We are interested in identifying Drosophila melanogaster RNA-binding proteins involved in important developmental decisions made at the level of mRNA processing, stability, localization or translational control. A large subset of the proteins known to interact with specific RNA sequences shares an evolutionarily conserved 80-90-amino-acid (aa) domain referred to as an RNA-recognition motif (RRM), including two ribonucleoprotein identifier sequences known as RNP-1 and RNP-2. Hence, we have herein applied degenerate polymerase chain reaction (PCR) methodology to clone three additional members (termed rox2, rox8 and rox21) of the D. melanogaster RRM-protein gene superfamily encoding putative trans-acting regulatory factors. Representative cDNA clones were isolated, the conceptual aa sequences of the candidate Rox proteins were inferred from their nucleotide sequences, and database searches were conducted. Rox2 displays extensive aa sequence similarities to putative RNA-binding proteins encoded by the genomes of the plants Oryza sativa and Arabidopsis thaliana; Rox21 resembles essential metazoan pre-mRNA splicing factors; as described elsewhere, Rox8 is likely a fly homolog of the two human TIA-1-type nucleolysins [Brand and Bourbon, Nucleic Acids Res. 21 (1993) 3699-3704].