We have identified a gene involved in bacterial cell division, located immediately upstream of the ftsI gene in the min 2 region of the Escherichia coli chromosome. This gene, which we named ftsL , was detected through characterization of Tn phoA insertions in a plasmid containing this chromosomal region. Tn phoA topological analysis and fractionation of alkaline phosphatase fusion proteins indicated that the ftsL gene product is a 13.6-kDa cytoplasmic membrane protein with a cytoplasmic amino terminus, a single membrane-spanning segment, and a periplasmic carboxy terminus. The ftsL gene is essential for cell growth and division. A null mutation in ftsL resulted in inhibition of cell division, formation of long, nonseptate filaments, ultimate cessation of growth, and lysis. Under certain growth conditions, depletion of FtsL or expression of the largest ftsL-phoA fusion produced a variety of cell morphologies, including Y-shaped bacteria, indicating a possible general weakening of the cell wall. The FtsL protein is estimated to be present at about 20 to 40 copies per cell. The periplasmic domain of the protein displays a sequence with features characteristic of leucine zippers, which are involved in protein dimerization.

SummaryFtsI, also known as penicillin-binding protein 3, is a transpeptidase required for the synthesis of peptidoglycan in the division septum of the bacterium, Escherichia coli. FtsI has been estimated to be present at about 100 molecules per cell, well below the detection limit of immunoelectron microscopy. Here, we confirm the low abundance of FtsI and use immunofluorescence microscopy, a highly sensitive technique, to show that FtsI is localized to the division site during the later stages of cell growth. FtsI was also sometimes observed at the cell pole; polar localization was not anticipated and its significance is not known. We conclude (i) that immunofluorescence microscopy can be used to localize proteins whose abundance is as low as approximately 100 molecules per cell; and (ii) that spatial and temporal regulation of FtsI activity in septum formation is achieved, at least in part, by timed localization of the protein to the division site.

In vivo genetic footprinting was developed in the yeast Saccharomyces cerevisiae to simultaneously assess the importance of thousands of genes for the fitness of the cell under any growth condition. We have developed in vivo genetic footprinting for Escherichia coli, a model bacterium and pathogen. We further demonstrate the utility of this technology for rapidly discovering genes that affect the fitness of E. coli under a variety of growth conditions. The definitive features of this system include a conditionally regulated Tn10 transposase with relaxed sequence specificity and a conditionally regulated replicon for the vector containing the transposase and mini-Tn10 transposon with an outwardly oriented promoter. This system results in a high frequency of randomly distributed transposon insertions, eliminating the need for the selection of a population containing transposon insertions, stringent suppression of transposon mutagenesis, and few polar effects. Successful footprints have been achieved for most genes longer than 400 bp, including genes located in operons. In addition, the ability of recombinant proteins to complement mutagenized hosts has been evaluated by genetic footprinting using a bacteriophage transposon delivery system.Large-scale sequencing of the genomes of many different microorganisms has yielded information for thousands of genes. However, the functions of a significant number of these genes remain unknown. Conventional knockout techniques have been used to examine the effect of deleting or disrupting a gene, but these techniques are difficult to apply on a genomic scale. Genetic footprinting allows the rapid, simultaneous determination of the importance of a large number of genes required for growth under a chosen condition (19,20).Genetic footprinting is a three-step process involving transposon mutagenesis, outgrowth of the mutagenized cell population, and analysis of the fate of cells carrying mutations in specific genes. The first step involves insertional mutagenesis using a transposon that inserts randomly throughout the genome. Following this period of mutagenesis where transposase expression is induced, a sample of the initial population, designated T0 (time zero), is taken. The second step is growth of the mutagenized T0 culture over many population doublings under conditions that repress transposase expression and plasmid replication. The third step is the comparative evaluation of transposon insertions present within specific genes based on analysis of PCR products generated from DNA isolated from the T0 mutagenized culture versus samples from the outgrowth culture (e.g., T15, T30, and T45 [15, 30, 45 population doublings, respectively]). Bacteria containing transposon insertions in genes that are important for the fitness or viability of the organism under a specific growth condition will not be represented in the outgrowth population, and therefore loss or diminution of PCR products corresponding to insertions within that gene will be observed. By measuring the loss of gene-s...

We have identified a gene involved in bacterial cell division, located immediately upstream of theftsI gene in the min 2 region of the Escherichia coli chromosome. This gene, which we namedftsL, was detected through characterization of TnphoA insertions in a plasmid containing this chromosomal region. TnphoA topological analysis and fractionation of alkaline phosphatase fusion proteins indicated that the jtsL gene product is a 13.6-kDa cytoplasmic membrane protein with a cytoplasmic amino terminus, a single membrane-spanning segmient, and a periplasmic carboxy terminus. TheftsL gene is essential for cell growth and division. A null mutation in ftsL resulted in inhibition of cell division, formation of long, nonseptate filaments, ultimate cessation of growth, and lysis. Under certain growth conditions, depletion of FtsL or expression of the largest ftsL-phoA fusion produced a variety of cell morphologies, including Y-shaped bacteria, indicating a possible general weakening of the cell wall. The FtsL protein is estimated to be present at about 20 to 40 copies per cell. The periplasmic domain of the protein displays a sequence with features characteristic of leucine zippers, which are involved in protein dimerization.