Jesús Mingorance

Concentration and assembly of the division ring proteins FtsZ, FtsA, and ZipA during the Escherichia coli cell cycle.

The concentration of the cell division proteins FtsZ, FtsA, and ZipA and their assembly into a division ring during the Escherichia coli B/r K cell cycle have been measured in synchronous cultures obtained by the membrane elution technique. Immunostaining of the three proteins revealed no organized structure in newly born cells. In a culture with a doubling time of 49 min, assembly of the Z ring started around minute 25 and was detected first as a two-dot structure that became a sharp band before cell constriction. FtsA and ZipA localized into a division ring following the same pattern and time course as FtsZ. The concentration (amount relative to total mass) of the three proteins remained constant during one complete cell cycle, showing that assembly of a division ring is not driven by changes in the concentration of these proteins. Maintenance of the Z ring during the process of septation is a dynamic energy-dependent event, as evidenced by its disappearance in cells treated with sodium azide.

Septum Enlightenment: Assembly of Bacterial Division Proteins

When observed under the microscope, cell division is just another dark event in the secret life of a bacterium: the cell grows up to a certain size, and then a constriction appears in its center that finally separates the cell into two daughters without any other perceptible changes. Upon a more

Essential cell division protein FtsZ assembles into one monomer-thick ribbons under conditions resembling the crowded intracellular environment

Experimental conditions that simulate the crowded bacterial cytoplasmic environment have been used to study the assembly of the essential cell division protein FtsZ from Escherichia coli. In solutions containing a suitable concentration of physiological osmolytes, macromolecular crowding promotes the GTP-dependent assembly of FtsZ into dynamic two-dimensional polymers that disassemble upon GTP depletion. Atomic force microscopy reveals that these FtsZ polymers adopt the shape of ribbons that are one subunit thick. When compared with the FtsZ filaments observed in vitro in the absence of crowding, the ribbons show a lag in the GTPase activity and a decrease in the GTPase rate and in the rate of GTP exchange within the polymer. We propose that, in the crowded bacterial cytoplasm under assembly-promoting conditions, the FtsZ filaments tend to align forming dynamic ribbon polymers. In vivo these ribbons would fit into the Z-ring even in the absence of other interactions. Therefore, the presence of mechanisms to prevent the spontaneous assembly of the Z-ring in non-dividing cells must be invoked.

Cell division in cocci: localization and properties of the Streptococcus pneumoniae FtsA protein

We studied the cytological and biochemical properties of the FtsA protein of Streptococcus pneumoniae. FtsA is a widespread bacterial cell division protein that belongs to the actin superfamily. In Escherichia coli and Bacillus subtilis, FtsA localizes to the septal ring after FtsZ, but its exact role in septation is not known. In S. pneumoniae, we found that, during exponential growth, the protein localizes to the nascent septa, at the equatorial zones of the dividing cells, where an average of 2200 FtsA molecules per cell are present. Likewise, FtsZ was found to localize with the same pattern and to be present at an average of 3000 molecules per cell. Consistent with the colocalization, FtsA was found to interact with FtsZ and with itself. Purified FtsA is able to bind several nucleotides, the affinity being highest for adenosine triphosphate (ATP), and lower for other triphosphates and diphosphates. The protein polymerizes in vitro, in a nucleotide‐dependent manner, forming long corkscrew‐like helixes, composed of 2 + 2 paired protofilaments. No nucleotide hydrolytic activity was detected. Consistent with the absence of an ATPase activity, the polymers are highly stable and not dynamic. These results suggest that the FtsA protein could also polymerize in vivo and the polymers participate in septation.

Strong FtsZ is with the force: mechanisms to constrict bacteria

FtsZ, the best-known prokaryotic division protein, assembles at midcell with other proteins forming a ring during septation. Widely conserved in bacteria, FtsZ represents the ancestor of tubulin. In the presence of GTP it forms polymers able to associate into multi-stranded flexible structures. FtsZ research is aimed at determining the role of the Z-ring in division, describing the polymerization and potential force-generating mechanisms and evaluating the roles of nucleotide exchange and hydrolysis. Systems to reconstruct the FtsZ ring in vitro have been described and some of its mechanical properties have been reproduced using in silico modeling. We discuss current research in FtsZ, some of the controversies, and finally propose further research needed to complete a model of FtsZ action that reconciles its in vitro properties with its role in division.

Bringing gene order into bacterial shape.

A different arrangement of a cluster of genes involved in division and cell-wall synthesis separates bacilli from other bacteria in a phylogenetic analysis. We conclude that the relationships between these genes are not random and might reflect significant events in the evolution of the coupling between growth and division in bacteria.

Escherichia coli FtsZ polymers contain mostly GTP and have a high nucleotide turnover

The cell division protein FtsZ is a GTPase structurally related to tubulin and, like tubulin, it assembles in vitro into filaments, sheets and other structures. To study the roles that GTP binding and hydrolysis play in the dynamics of FtsZ polymerization, the nucleotide contents of FtsZ were measured under different polymerizing conditions using a nitrocellulose filter‐binding assay, whereas polymerization of the protein was followed in parallel by light scattering. Unpolymerized FtsZ bound 1 mol of GTP mol−1 protein monomer. At pH 7.5 and in the presence of Mg2+ and K+, there was a strong GTPase activity; most of the bound nucleotide was GTP during the first few minutes but, later, the amount of GTP decreased in parallel with depolymerization, whereas the total nucleotide contents remained invariant. These results show that the long FtsZ polymers formed in solution contain mostly GTP. Incorporation of nucleotides into the protein was very fast either when the label was introduced at the onset of the reaction or subsequently during polymerization. Molecular modelling of an FtsZ dimer showed the presence of a cleft between the two subunits maintaining the nucleotide binding site open to the medium. These results show that the FtsZ polymers are highly dynamic structures that quickly exchange the bound nucleotide, and this exchange can occur in all the subunits.

Role of two essential domains of Escherichia coli FtsA in localization and progression of the division ring

The FtsA protein is a member of the actin superfamily that localizes to the bacterial septal ring during cell division. Deletions of domain 1C or the S12 and S13 β‐strands in domain 2B of the Escherichia coli FtsA, previously postulated to be involved in dimerization, result in partially active proteins that do not allow the normal progression of septation. The truncated FtsA protein lacking domain 1C (FtsAΔ1C) localizes in correctly placed division rings, together with FtsZ and ZipA, but does not interact with other FtsA molecules in the yeast two‐hybrid assay, and fails to recruit FtsQ and FtsN into the division ring. The rings containing FtsAΔ1C are therefore incomplete and do not support division. The production of high levels of FtsAΔ1C causes filamentation, an effect that has been reported to result as well from the imbalance between FtsA+ and FtsZ+ molecules. These data indicate that the domain 1C of FtsA participates in the interaction of the protein with other FtsA molecules and with the other proteins that are incorporated at later stages of ring assembly, and is not involved in the interaction with FtsZ and the localization of FtsA to the septal ring. The deletion of the S12–S13 strands of domain 2B generates a protein (FtsAΔS12–13) that retains the ability to interact with FtsA+. When the mutated protein is expressed at wild‐type levels, it localizes into division rings and recruits FtsQ and FtsN, but it fails to sustain septation at normal levels resulting in filamentation. A fivefold overexpression of FtsAΔS12–13 produces short cells that have normal division rings, but also cells with polar localization of the mutated protein, and cells with rings at abnormal positions that result in the production of a fraction (15%) of small nucleoid‐free cells. The S12–S13 strands of domain 2B are not essential for septation, but affect the localization of the division ring.

Infections caused by OXA-48-producing Klebsiella pneumoniae in a tertiary hospital in Spain in the setting of a prolonged, hospital-wide outbreak

OBJECTIVES We describe clinical and microbiological features of infections caused by OXA-48-producing Klebsiella pneumoniae (O48KP) in the setting of a prolonged, hospital-wide outbreak detected in January 2011. METHODS Clinical, demographic and microbiological data of patients with growth of O48KP in clinical specimens were collected until December 2011. PCR was used to detect carbapenemase and β-lactamase genes. The genetic relationships were determined by automated repetitive-sequence-based PCR. RESULTS Seventy-one patients with clinically guided cultures showing growth of O48KP were identified. Nine were considered to be colonizing rather than causing infection. The most frequent source of infection was the urinary tract (22/62), followed by surgical site infections (17/62). Blood cultures were positive in 23/62 patients. Many patients had significant comorbidity and prolonged hospital stays. In-hospital mortality among patients with O48KP infections was 43.5%. The MIC(90)s of ertapenem, imipenem and meropenem were >32, 16 and 16 mg/L, respectively. No single antimicrobial was active against all the isolates. The antibiotics most active against O48KP were amikacin (97.2% susceptible), colistin (90.1%), tigecycline (73%) and fosfomycin (66.2%). Although eight clones were identified, a predominant clone caused 73.2% of the infections. Multilocus sequence typing (MLST) of the predominant clone gave sequence type (ST) 405 and bla(TEM-1), bla(SHV-76), bla(CTX-M-15) and bla(OXA-1) genes and the insertion sequence IS1999 of the Tn1999 transposon were associated with bla(OXA-48) in this clone. CONCLUSIONS To our knowledge, this is the largest reported series of infections caused by O48KP in the setting of a single-centre outbreak and provides further input on the clinical relevance of infections caused by O48KP and the difficulties associated with its detection and control.

Role of the carboxy terminus of Escherichia coli FtsA in self-interaction and cell division.

The role of the carboxy terminus of the Escherichia coli cell division protein FtsA in bacterial division has been studied by making a series of short sequential deletions spanning from residue 394 to 420. Deletions as short as 5 residues destroy the biological function of the protein. Residue W415 is essential for the localization of the protein into septal rings. Overexpression of the ftsA alleles harboring these deletions caused a coiled cell phenotype previously described for another carboxy-terminal mutation (Gayda et al., J. Bacteriol. 174:5362-5370, 1992), suggesting that an interaction of FtsA with itself might play a role in its function. The existence of such an interaction was demonstrated using the yeast two-hybrid system and a protein overlay assay. Even these short deletions are sufficient for impairing the interaction of the truncated FtsA forms with the wild-type protein in the yeast two-hybrid system. The existence of additional interactions between FtsA molecules, involving other domains, can be postulated from the interaction properties shown by the FtsA deletion mutant forms, because although unable to interact with the wild-type and with FtsADelta1, they can interact with themselves and cross-interact with each other. The secondary structures of an extensive deletion, FtsADelta27, and the wild-type protein are indistinguishable when analyzed by Fourier transform infrared spectroscopy, and moreover, FtsADelta27 retains the ability to bind ATP. These results indicate that deletion of the carboxy-terminal 27 residues does not alter substantially the structure of the protein and suggest that the loss of biological function of the carboxy-terminal deletion mutants might be related to the modification of their interacting properties.