The FtsEX ABC transporter directs cellular differentiation in Bacillus subtilis

A fundamental challenge in developmental biology is to elucidate the regulatory events that trigger cellular differentiation. Sporulation in the Gram-positive bacterium Bacillus subtilis serves as a simple experimental model system to address this challenge. The hallmark of sporulation is the formation of an asymmetrically positioned septum that divides the cell into unequally sized progeny. Here we describe the role of an ABC transporter, comprising the FtsE and FtsX proteins, in the initiation of spore formation. We discovered that in the absence of this transporter, entry into sporulation is delayed and an atypical symmetric septum is formed instead of a polar one. We show that this phenotype can be suppressed by artificially activating the master regulator of sporulation, Spo0A, or by activating the histidine kinases that function upstream of Spo0A. Our data indicate that the FtsEX transporter is one of the top components in the hierarchy of factors required to initiate sporulation, and thus it is essential for establishing proper temporal activation of the process.     

The metD D-methionine transporter locus of Escherichia coli is an ABC transporter gene cluster

The metD D-methionine transporter locus of Escherichia coli was identified as the abc-yaeE-yaeC cluster (now renamed metNIQ genes). The abc open reading frame is preceded by tandem MET boxes bracketed by the -10 and -35 boxes of a promoter. The expression driven by this promoter is controlled by the MetJ repressor and the level of methionine.     

Drug-lipid A interactions on the Escherichia coli ABC transporter MsbA

MsbA is an essential ATP-binding cassette half-transporter in the cytoplasmic membrane of the gram-negative Escherichia coli and is required for the export of lipopolysaccharides (LPS) to the outer membrane, most likely by transporting the lipid A core moiety. Consistent with the homology of MsbA to the multidrug transporter LmrA in the gram-positive Lactococcus lactis, our recent work in E. coli suggested that MsbA might interact with multiple drugs. To enable a more detailed analysis of multidrug transport by MsbA in an environment deficient in LPS, we functionally expressed MsbA in L. lactis. MsbA expression conferred an 86-fold increase in resistance to the macrolide erythromycin. A kinetic characterization of MsbA-mediated ethidium and Hoechst 33342 transport revealed apparent single-site kinetics and competitive inhibition of these transport reactions by vinblastine with K(i) values of 16 and 11 microM, respectively. We also detected a simple noncompetitive inhibition of Hoechst 33342 transport by free lipid A with a K(i) of 57 microM, in a similar range as the K(i) for vinblastine, underscoring the relevance of our LPS-less lactococcal model for studies on MsbA-mediated drug transport. These observations demonstrate the ability of heterologously expressed MsbA to interact with free lipid A and multiple drugs in the absence of auxiliary E. coli proteins. Our transport data provide further functional support for direct LPS-MsbA interactions as observed in a recent crystal structure for MsbA from Salmonella enterica serovar Typhimurium (C. L. Reyes and G. Chang, Science 308:1028-1031, 2005).     

Role of FtsEX in cell division of Escherichia coli: viability of ftsEX mutants is dependent on functional SufI or high osmotic strength

In Escherichia coli, at least 12 proteins, FtsZ, ZipA, FtsA, FtsE/X, FtsK, FtsQ, FtsL, FtsB, FtsW, FtsI, FtsN, and AmiC, are known to localize to the septal ring in an interdependent and sequential pathway to coordinate the septum formation at the midcell. The FtsEX complex is the latest recruit of this pathway, and unlike other division proteins, it is shown to be essential only on low-salt media. In this study, it is shown that ftsEX null mutations are not only salt remedial but also osmoremedial, which suggests that FtsEX may not be involved in salt transport as previously thought. Increased coexpression of cell division proteins FtsQ-FtsA-FtsZ or FtsN alone restored the growth defects of ftsEX mutants. ftsEX deletion exacerbated the defects of most of the mutants affected in Z ring localization and septal assembly; however, the ftsZ84 allele was a weak suppressor of ftsEX. The viability of ftsEX mutants in high-osmolarity conditions was shown to be dependent on the presence of a periplasmic protein, SufI, a substrate of twin-arginine translocase. In addition, SufI in multiple copies could substitute for the functions of FtsEX. Taken together, these results suggest that FtsE and FtsX are absolutely required for the process of cell division in conditions of low osmotic strength for the stability of the septal ring assembly and that, during high-osmolarity conditions, the FtsEX and SufI functions are redundant for this essential process.     

A new cell division operon in Escherichia coli

Summary At 76 min on theE. coli genetic map there is a cluster of genes affecting essential cellular functions, including the heat shock response and cell division. A combination ofin-vivo andin-vitro genetic analysis of cell division mutants suggests that the cell division genefts E is the second gene in a 3 gene operon. A cold-sensitive mutant, defective in the third gene, is also unable to divide at the restrictive temperature, and we designate this new cell division genefts X. Another cell division gene,fts S, is very close to, but distinct from, the 3 genes of the operon. Thefts E product is a 24.5 Kd polypeptide which shows strong homology with a small group of proteins involved in transport. Both thefts E product and the protein coded by the first gene (fts Y) in the operon have a sequence motif found in a wide range of heterogeneous proteins, including the Ras proteins of yeast. This common domain is indicative of a nucleotide-binding site.     

A new Escherichia coli cell division gene, ftsK.

A mutation in a newly discovered Escherichia coli cell division gene, ftsK, causes a temperature-sensitive late-stage block in division but does not affect chromosome replication or segregation. This defect is specifically suppressed by deletion of dacA, coding for the peptidoglycan DD-carboxypeptidase, PBP 5. FtsK is a large polypeptide (147 kDa) consisting of an N-terminal domain with several predicted membrane-spanning regions, a proline-glutamine-rich domain, and a C-terminal domain with a nucleotide-binding consensus sequence. FtsK has extensive sequence identity with a family of proteins from a wide variety of prokaryotes and plasmids. The plasmid proteins are required for intercellular DNA transfer, and one of the bacterial proteins (the SpoIIIE protein of Bacillus subtilis) has also been implicated in intracellular chromosomal DNA transfer.     

Escherichia coli XerC recombinase is required for chromosomal segregation at cell division

XerC is a site-specific recombinase of the bacteriophage lambda integrase family that is encoded by xerC at 3700 kbp on the genetic map of Escherichia coli. The protein was originally identified through its role in converting multimers of plasmid ColE1 to monomers; only monomers are stably inherited. Here we demonstrate that XerC also has a role in the segregation of replicated chromosomes at cell division. xerC mutants form filaments with aberrant nucleotides that appear unable to partition correctly. A DNA segment (dif) from the replication terminus region of the E. coli chromosome binds XerC and acts as a substrate for XerC-mediated site-specific recombination when inserted into multicopy plasmids. This dif segment contains a region of 28 bp with sequence similarity to the crossover region of ColE1 cer. The cell division phenotype of xerC mutants is suppressed in strains deficient in homologous recombination, suggesting that the role of XerC/dif in chromosomal metabolism is to convert any chromosomal multimers (arising through homologous recombination) to monomers.     

ftsZ is an essential cell division gene in Escherichia coli.

The ftsZ gene is thought to be an essential cell division gene in Escherichia coli. We constructed a null allele of ftsZ in a strain carrying additional copies of ftsZ on a plasmid with a temperature-sensitive replication defect. This strain was temperature sensitive for cell division and viability, confirming that ftsZ is an essential cell division gene. Further analysis revealed that after a shift to the nonpermissive temperature, cell division ceased when the level of FtsZ started to decrease, indicating that septation is very sensitive to the level of FtsZ. Subsequent studies showed that nucleoid segregation was normal while FtsZ was decreasing and that ftsZ expression was not autoregulated. The null allele could not be complemented by lambda 16-2, even though this bacteriophage can complement the thermosensitive ftsZ84 mutation and carries 6 kb of DNA upstream of the ftsZ gene.     

Termination of DNA replication is required for cell division in Escherichia coli.

The correlation between termination of DNA replication and cell division in Escherichia coli was studied under conditions in which DNA replication was slowed down without inducing SOS functions. The experimental system used involved amino acid starvation of synchronized cells in the presence of methionine. The results further support the essential correlation between termination of DNA replication and initiation of division processes.     

The Escherichia coli cell division mutation ftsM1 is in serU.

The ftsM1 mutation is believed to be in a gene implicated in the regulation of cell division in Escherichia coli because it displayed the lon mutation phenotypes. In this study, we show that this mutation is located in serU, a gene which codes for tRNA(Ser)2, and has the phenotypes of the serU allele supH. Both ftsM1 and supH suppressed the leuB6 and ilvD145 missense mutations, and both conferred temperature and UV light irradiation sensitivity to the harboring cells. Cells which carried the ftsM1 mutation or the supH suppressor had very low colony-forming abilities on salt-free L agar, and this phenotype was almost completely abolished by the presence of plasmids bearing the ftsZ+ gene. Furthermore, sensitivity of the mutant cells to UV irradiation was also markedly diminished when they carried a ftsZ+-bearing plasmid. These results suggest that supH-containing cells have reduced FtsZ activities, in accordance with their displaying the phenotypes of the lon mutant cells. The possibility that ftsM1 (supH) is functionally involved in the biosynthesis of a specific protein which affects cell division is discussed.     

Export of recombinant proteins in Escherichia coli using ABC transporter with an attached lipase ABC transporter recognition domain (LARD)

Background  

ATP binding cassette (ABC) transporter secretes the protein through inner and outer membranes simultaneously in gram negative bacteria. Thermostable lipase (TliA) of Pseudomonas fluorescens SIK W1 is secreted through the ABC transporter. TliA has four glycine-rich repeats (GGXGXD) in its C-terminus, which appear in many ABC transporter-secreted proteins. From a homology model of TliA derived from the structure of P. aeruginosa alkaline protease (AprA), lipase ABC transporter domains (LARDs) were designed for the secretion of fusion proteins.     

In vitro functional characterization of BtuCD-F, the Escherichia coli ABC transporter for vitamin B12 uptake

BtuCD is an ATP binding cassette (ABC) transporter that facilitates uptake of vitamin B(12) into the cytoplasm of Escherichia coli. The crystal structures of BtuCD and its cognate periplasmic binding protein BtuF have been recently determined. We have now explored BtuCD-F function in vitro, both in proteoliposomes and in various detergents. BtuCD reconstituted into proteoliposomes has a significant basal ATP hydrolysis rate that is stimulated by addition of BtuF and inhibited by sodium ortho-vanadate. When using different detergents to solubilize BtuCD, the basal ATP hydrolysis rate, the ability of BtuF to stimulate hydrolysis, and the extent to which sodium ortho-vanadate inhibits ATP hydrolysis all vary significantly. Reconstituted BtuCD can mediate transport of vitamin B(12) against a concentration gradient when coupled to ATP hydrolysis by BtuD in the liposome lumen and BtuF outside the liposomes. These in vitro studies establish the functional competence of the BtuCD and BtuF preparations used in the crystallographic analyses for both ATPase and transport activities. Furthermore, the tight binding of BtuF to BtuCD under the conditions studied suggests that the binding protein may not dissociate from the transporter during the catalytic cycle, which may be relevant to the mechanisms of other ABC transporter systems.     

Replication of a specific terminal chromosome segment in Escherichia coli which is required for cell division

In the absence of protein synthesis, DNA replication does not proceed to the very end of the chromosome. A terminal segment remains unreplicated. This segment can be selectively labelled. Its size is about 0·5% of the chromosome and its replication is required for subsequent cell division.     

Apparent minimal size required for cell division in Escherichia coli.

The experiments described in this report were designed to find out whether there is a minimal size threshold for cell division or for DNA replication in Escherichia coli. Cells with decreasing size (or mass) were obtained by successive amino acid starvations. Following two starvations, the cells were at least 30% smaller than unstarved newborn cells. The results suggest that this size is below a minimal size threshold for cell division but not for initiation of DNA replication.     

Cell division defects in Escherichia coli deficient in the multidrug efflux transporter AcrEF-TolC

The Escherichia coli chromosome contains several operons encoding confirmed and predicted multidrug transporters. Among these transporters only the inactivation of components of the AcrAB-TolC complex leads to substantial changes in susceptibility to multiple drugs. This observation prompted a conclusion that other transporters are silent or expressed at levels insufficient to contribute to multidrug resistance phenotype. We found that increased expression of AcrA, the periplasmic membrane fusion protein, is toxic only in cells lacking the multidrug efflux transporter AcrEF. AcrEF-deficient cells with increased expression of AcrA have a severe cell division defect that results in cell filamentation (>50 microm). Similar defects were obtained in cells lacking the outer membrane channel TolC, which acts with AcrEF, suggesting that cell filamentation is caused by the loss of AcrEF function. Green fluorescent protein-AcrA fusion studies showed that in normal and filamentous cells AcrA is associated with membranes in a confined manner and that this localization is not affected by the lack of AcrEF. Similarly, the structure and composition of membranes were normal in filamentous cells. Fluorescence microscopy showed that the filamentous AcrEF-deficient E. coli cells are defective in chromosome condensation and segregation. Our results suggest that the E. coli AcrEF transporter is expressed under standard laboratory conditions and plays an important role in the normal maintenance of cell division.     

Nucleoid-independent identification of cell division sites in Escherichia coli

The mechanism used by Escherichia coli to determine the correct site for cell division is unknown. In this report, we have attempted to distinguish between a model in which septal position is determined by the position of the nucleoids and a model in which septal position is predetermined by a mechanism that does not involve nucleoid position. To do this, filaments with extended nucleoid-free regions adjacent to the cell poles were produced by simultaneous inactivation of cell division and DNA replication. The positions of septa that formed within the nucleoid-free zones after division was allowed to resume were then analyzed. The results showed that septa were formed at a uniform distance from cell poles when division was restored, with no relation to the distance from the nearest nucleoid. In some cells, septa were formed directly over nucleoids. These results are inconsistent with models that invoke nucleoid positioning as the mechanism for determining the site of division site formation.     

Cyclic AMP and cell division in Escherichia coli.

We examined several aspects of cell division regulation in Escherichia coli which have been thought to be controlled by cyclic AMP (cAMP) and its receptor protein (CAP). Mutants lacking adenyl cyclase (cya) or CAP (crp) were rod shaped, not spherical, during exponential growth in LB broth or glucose-Casamino Acids medium, and lateral wall elongation was normal; in broth, stationary-phase cells became ovoid. Cell mass was smaller for the mutants than for the wild type, but it remained appropriate for their slower growth rate and thus probably does not reflect early (uncontrolled) septation. The slow growth did not seem to reflect a gross metabolic disorder, since the mutants gave a normal yield on limiting glucose; surprisingly, however, the cya mutant (unlike crp) was unable to grow anaerobically on glucose, suggesting a role for cAMP (but not for CAP) in the expression of some fermentation enzyme. Both cya and crp mutants are known to be resistant to mecillinam, an antibiotic which inhibits penicillin-binding protein 2 (involved in lateral wall elongation) and also affects septation. This resistance does not reflect a lack of PBP2. Furthermore, it was not simply the result of slow growth and small cell mass, since small wild-type cells growing in acetate remained sensitive. The cAMP-CAP complex may regulate the synthesis of some link between PBP2 and the septation apparatus. The ftsZ gene, coding for a cell division protein, was expressed at a higher level in the absence of cAMP, as measured with an ftsZ::lacZ fusion, but the amount of protein per cell, shown by others to be invariable over a 10-fold range of cell mass, was independent of cAMP, suggesting that ftsZ expression is not regulated by the cAMP-CAP complex.