Identification of two new genes,mukE andmukF,involved in chromosome partitioning inEscherichia coli

We have previously reported that the MukB protein is essential for chromosome partitioning inEscherichia coli and thatmukB mutants produce anucleate cells and are temperature-sensitive for colony formation. ThemukB gene maps at 21 min on theE. coli chromosome andsmtA-mukF-mukE-mukB genes might comprise an operon, which is transcribed in a clockwise direction. Here, we report thatmukF andmukE null mutants are both temperature-sensitive for colony formation and produce anucleate cells even at the permissive temperature. These phenotypes are the same as those observed in themukB null mutant. The primary sequence of MukF includes a leucine zipper structure and an acidic domain. Mutational analysis revealed that both are required for MukF function. When the MukF protein was overproduced in the wild-type strain, anucleate cells were produced. In contrast, overproduction of either MukE or MukB did not cause the defect. In null mutants for themukF, mukE, andmukB genes, the synchronous initiation of chromosome replication was not affected. The mini-F plasmid was as stably maintained in these mutants as in the wild-type strain. These results indicate that the MukF, MukE, and MukB proteins are involved in the chromosome partitioning steps, but are not required for mini-F plasmid partitioning.     

New genes with old modus operandi. The connection between supercoiling and partitioning of DNA in Escherichia coli

The process of partitioning bacterial sister chromosomes into daughter cells seems to be distinct from chromatid segregation during eukaryotic mitosis. In Escherichia coli, partitioning starts soon after initiation of replication, when the two newly replicated oriCs move from the cell centre to quarter positions within the cell. As replication proceeds, domains of the compact, supercoiled chromosome are locally decondensed ahead of the replication fork. The nascent daughter chromosomes are recondensed and moved apart through the concerted activities of topoisomerases and the SeqA (sequestration) and MukB (chromosome condensation) proteins, all of which modulate nucleoid superhelicity. Thus, genes involved in chromosome topology, once set aside as ‘red herrings’ in the search for ‘true’ partition functions, are again recognized as being important for chromosome partitioning in E. coli.     

New killing system controlled by two genes located immediately upstream of the mukB gene in Escherichia coli

The nucleotide sequence was determined of the region upstream of the mukB gene of Escherichia coli. Two new genes were found, designated kicA and kicB (killing of cell); the gene order is kicB-kicA-mukB. Promoter activities were detected in the regions immediately upstream of kicB and kicA, but not in front of mukB. Gene disruption experiments revealed that the kicA disruptant was nonviable, but the kicB-disrupted mutant and the mutant lacking both the kicB and kicA genes were able to grow. When kicA disruptant cells bearing a temperature-sensitive replication plasmid carrying the kicA ⁺ gene were grown at 30° C and then transferred to 42° C, the mutant cells gradually lost colony-forming ability, even in the presence of a mukB ⁺ plasmid. Rates of protein synthesis, but not of RNA or DNA synthesis, fell dramatically during incubation at 42° C. These results suggested that the kicB gene encodes a killing factor and the kicA gene codes for a protein that suppresses the killing function of the kicB gene product. It was also demonstrated that KicA and KicB can function as a post-segregational killing system, when the genes are transferred from the E. coli chromosome onto a plasmid.     

Identification of two new genes,mukE andmukF, involved in chromosome partitioning inEscherichia coli

We have previously reported that the MukB protein is essential for chromosome partitioning inEscherichia coli and thatmukB mutants produce anucleate cells and are temperature-sensitive for colony formation. ThemukB gene maps at 21 min on theE. coli chromosome andsmtA-mukF-mukE-mukB genes might comprise an operon, which is transcribed in a clockwise direction. Here, we report thatmukF andmukE null mutants are both temperature-sensitive for colony formation and produce anucleate cells even at the permissive temperature. These phenotypes are the same as those observed in themukB null mutant. The primary sequence of MukF includes a leucine zipper structure and an acidic domain. Mutational analysis revealed that both are required for MukF function. When the MukF protein was overproduced in the wild-type strain, anucleate cells were produced. In contrast, overproduction of either MukE or MukB did not cause the defect. In null mutants for themukF, mukE, andmukB genes, the synchronous initiation of chromosome replication was not affected. The mini-F plasmid was as stably maintained in these mutants as in the wild-type strain. These results indicate that the MukF, MukE, and MukB proteins are involved in the chromosome partitioning steps, but are not required for mini-F plasmid partitioning.     

Altered biological properties of cell membranes in Escherichia coli dnaA and seqA mutants

We present evidence that biological properties of cell membranes are altered in dnaA and seqA mutants of Escherichia coli relative to wild-type bacteria. We found that bacteriophage λ forms extremely large plaques on the dnaA seqA double mutants. On the single mutants, dnaA and seqA, the plaques are also bigger than those formed on the wild-type host. However, no significant differences in intracellular phage λ development were observed between wild-type and mutant hosts, indicating that differences in burst size do not account for the observed differences in plaque size. On the other hand, more efficient release of the phage lytic proteins and/or higher sensitivity of the cell membranes to these proteins may result in more efficient cell lysis. We found that the efficiency of adsorption of bacteriophage λ to the dnaA seqA mutant cells is decreased at 0°?C , but not at 30°?C, relative to the wild-type strain. A considerable increase in the permeability of membranes of the mutant cells for β-galactosidase is demonstrated. The dnaA and seqA mutants are more sensitive to ethanol (an organic solvent) than wild-type bacteria, and the seqA strain and the double mutant dnaA seqA are very sensitive to deoxycholate (a detergent). We conclude that lesions in the genes dnaA and seqA result in alterations in cell membranes, such that the permeability and possibly also other properties of the membranes are significantly altered relative to wild-type bacteria.     

Role of the C Terminus of FtsK in Escherichia coli Chromosome Segregation

FtsK is essential for Escherichia coli cell division. We report that cells lacking the C terminus of FtsK are defective in chromosome segregation as well as septation, often exhibiting asymmetrically positioned nucleoids and large anucleate regions. Combining the corresponding truncated ftsK gene with a mukB null mutation resulted in a synthetic lethal phenotype. When the truncated ftsK was combined with a minCDE deletion, chains of minicells were generated, many of which contained DNA. These results suggest that the C terminus of FtsK has an important role in chromosome partitioning.     

New killing system controlled by two genes located immediately upstream of the mukB gene in Escherichia coli

The nucleotide sequence was determined of the region upstream of the mukB gene of Escherichia coli. Two new genes were found, designated kicA and kicB (killing of cell); the gene order is kicB-kicA-mukB. Promoter activities were detected in the regions immediately upstream of kicB and kicA, but not in front of mukB. Gene disruption experiments revealed that the kicA disruptant was nonviable, but the kicB-disrupted mutant and the mutant lacking both the kicB and kicA genes were able to grow. When kicA disruptant cells bearing a temperature-sensitive replication plasmid carrying the kicA ⁺ gene were grown at 30° C and then transferred to 42° C, the mutant cells gradually lost colony-forming ability, even in the presence of a mukB ⁺ plasmid. Rates of protein synthesis, but not of RNA or DNA synthesis, fell dramatically during incubation at 42° C. These results suggested that the kicB gene encodes a killing factor and the kicA gene codes for a protein that suppresses the killing function of the kicB gene product. It was also demonstrated that KicA and KicB can function as a post-segregational killing system, when the genes are transferred from the E. coli chromosome onto a plasmid.     

Excess SeqA leads to replication arrest and a cell division defect in Vibrio cholerae

Although most bacteria contain a single circular chromosome, some have complex genomes, and all Vibrio species studied so far contain both a large and a small chromosome. In recent years, the divided genome of Vibrio cholerae has proven to be an interesting model system with both parallels to and novel features compared with the genome of Escherichia coli. While factors influencing the replication and segregation of both chromosomes have begun to be elucidated, much remains to be learned about the maintenance of this genome and of complex bacterial genomes generally. An important aspect of replicating any genome is the correct timing of initiation, without which organisms risk aneuploidy. During DNA replication in E. coli, newly replicated origins cannot immediately reinitiate because they undergo sequestration by the SeqA protein, which binds hemimethylated origin DNA. This DNA is already methylated by Dam on the template strand and later becomes fully methylated; aberrant amounts of Dam or the deletion of seqA leads to asynchronous replication. In our study, hemimethylated DNA was detected at both origins of V. cholerae, suggesting that these origins are also subject to sequestration. The overproduction of SeqA led to a loss of viability, the condensation of DNA, and a filamentous morphology. Cells with abnormal DNA content arose in the population, and replication was inhibited as determined by a reduced ratio of origin to terminus DNA in SeqA-overexpressing cells. Thus, excessive SeqA negatively affects replication in V. cholerae and prevents correct progression to downstream cell cycle events such as segregation and cell division.     

The expression of the DNA ligase gene of Escherichia coli is stimulated by relaxation of chromosomal supercoiling

Many genes of Escherichia coli have been shown to be sensitive to DNA superhelicity. The superhelicity of the chromosome is itself also supercoiling-dependent. We have developed a general strategy for investigating how a particular gene responds to changes in DNA topology. This approach is used to study the E. coli ligase gene. The thermosensitivity of the E. coli ligts251 mutation can be phenotypically suppressed by mutations which map close to, or in, the gyrB gene and which affect the degree of DNA supercoiling. The level of suppression correlates with the degree of DNA relaxation observed, suggesting that the gene encoding the E. coli DNA ligase is activated by relaxation of the chromosomal DNA.     

FRUIT,a Scar-Free System for Targeted Chromosomal Mutagenesis,Epitope Tagging,and Promoter Replacement in Escherichia coli and Salmonella enterica

Recombineering is a widely-used approach to delete genes, introduce insertions and point mutations, and introduce epitope tags into bacterial chromosomes. Many recombineering methods have been described, for a wide range of bacterial species. These methods are often limited by (i) low efficiency, and/or (ii) introduction of “scar” DNA into the chromosome. Here, we describe a rapid, efficient, PCR-based recombineering method, FRUIT, that can be used to introduce scar-free point mutations, deletions, epitope tags, and promoters into the genomes of enteric bacteria. The efficiency of FRUIT is far higher than that of the most widely-used recombineering method for Escherichia coli. We have used FRUIT to introduce point mutations and epitope tags into the chromosomes of E. coli K-12, Enterotoxigenic E. coli, and Salmonella enterica. We have also used FRUIT to introduce constitutive and inducible promoters into the chromosome of E. coli K-12. Thus, FRUIT is a versatile, efficient recombineering approach that can be applied in multiple species of enteric bacteria.     

CRISPRi engineering E. coli for morphology diversification

Microbial morphology engineering has recently become interesting for biotechnology. Genes ftsZ and mreB encoding proteins of bacterial fission ring and skeletons, respectively, are essential for cell growth, they both are the most important genes keeping the bacterial shapes including the cell length and width, respectively. Clustered regularly interspaced short palindromic repeats interference, abbreviated as CRISPRi, was for the first time used in this study to regulate expression intensities of ftsZ or/and mreB in E. coli. Five sgRNAs associated with CRISPRi were designed and synthesized, respectively, to target five various locations on genes ftsZ or mreB encoded in the E. coli chromosome, resulting in various reduced expression levels of ftsZ or/and mreB, respectively, forming elongated or/and fatter cells. Repressions on gene expressions of ftsZ or/and mreB could be further intensified by combining various sgRNAs together. It was found that the stronger the repression on genes ftsZ or/and mreB, the longer the E. coli fibers, and the larger the E. coli cells. Combined repressions on expressions of ftsZ and mreB generated long and larger E. coli with diverse morphologies including various sizes of gourds, bars, coccus, spindles, multi-angles and ellipsoids. In all cases, accumulations of intracellular biopolyester polyhydroxybutyrate (PHB) were in direct proportional to the intracellular volumes, ranging from 40% to 80% PHB in bacterial cell dry weights, depending on the cell volumes increases by the above CRISPRi applications.     

High Prevalence of Mucosa-Associated E. coli Producing Cyclomodulin and Genotoxin in Colon Cancer

Some Escherichia coli strains produce toxins designated cyclomodulins (CMs) which interfere with the eukaryotic cell cycle of host cells, suggesting a possible link between these bacteria and cancers. There are relatively few data available concerning the colonization of colon tumors by cyclomodulin- and genotoxic-producing E. coli. We did a qualitative and phylogenetic analysis of mucosa-associated E. coli harboring cyclomodulin-encoding genes from 38 patients with colorectal cancer (CRC) and 31 with diverticulosis. The functionality of these genes was investigated on cell cultures and the genotoxic activity of strains devoid of known CM-encoding gene was investigated. Results showed a higher prevalence of B2 phylogroup E. coli harboring the colibatin-producing genes in biopsies of patients with CRC (55.3%) than in those of patients with diverticulosis (19.3%), (p<0.01). Likewise, a higher prevalence of B2 E. coli harboring the CNF1-encoding genes in biopsies of patients with CRC (39.5%) than in those of patients with diverticulosis (12.9%), (p = 0.01). Functional analysis revealed that the majority of these genes were functional. Analysis of the ability of E. coli to adhere to intestinal epithelial cells Int-407 indicated that highly adherent E. coli strains mostly belonged to A and D phylogroups, whatever the origin of the strains (CRC or diverticulosis), and that most E. coli strains belonging to B2 phylogroup displayed very low levels of adhesion. In addition, 27.6% (n = 21/76) E. coli strains devoid of known cyclomodulin-encoding genes induced DNA damage in vitro, as assessed by the comet assay. In contrast to cyclomodulin-producing E. coli, these strains mainly belonged to A or D E. coli phylogroups, and exhibited a non significant difference in the distribution of CRC and diverticulosis specimens (22% versus 32.5%, p = 0.91). In conclusion, cyclomodulin-producing E. coli belonging mostly to B2 phylogroup colonize the colonic mucosa of patients with CRC.     

The use of selection in recovery of transgenic targets for mutation analysis

Transgenic animal mutagenesis assays using lambda shuttle vectors have recently been described for isolation and characterization of spontaneous and chemical induced DNA mutations. Extensive information on lambda and E. coli genetics provides a wealth of techniques to allow selection of mutant target genes. Here we describe the modification of an E. coli host which permits two methods for the direct selection of mutant genes. These methods reduce the number of plates needed to be screened for a comparable amount of frequency data by 20–100-fold and thus provide a significant savings of the materials and time required for the screening of mutations. In addition, mutants selected by these approaches described here may alter or broaden the spectrum of mutations that are recoverable. Ultimately, a combination of selective and nonselective techniques may prove valuable for the analysis of mutations produced in vivo in transgenic animals.     

A method for construction of E. coli strains with multiple DNA insertions in the chromosome

A system for construction of E. coli strains with multiple DNA insertions in the chromosome, based on elements of modules for site specific recombination of Tn1545 and phage λ, has been developed. Circular non-replicating DNA fragments containing the transposon attachment site (attTn), an excisable cassette with a selectable marker, and a gene of interest integrate randomly into the chromosome of a host E. coli strain when provided with transposon integrase, Int-Tn (the host strain was obtained by insertion of the fragment containing transposon int-Tn gene coding for Int-Tn into the chromosome). Integration of these fragments into the chromosome of int-Tn⁺ cells gives rise to a collection of antibiotic-resistant clones with single insertions at different locations in the chromosome. These insertions are transferred subsequently by P1 transduction into one strain and selected for antibiotic resistance provided by the cassette with the selectable marker. After transduction of each copy, a helper plasmid bearing phage λ xis and int genes is introduced into the cells to excise the drug resistance gene flanked with the λattL and λattR sites from the chromosome. Cells cured of the helper plasmid can undergo the next cycle of P1 transduction/drug resistance gene excision. Each cycle adds another chromosomal copy of the foreign gene. To show the utility of the system, we constructed an E. coli strain bearing several chromosomal copies of lacZ at different locations.     

Segregation of Induced Frameshift Mutations and the Sequence of Gene Replication in ESCHERICHIA COLI K12

We have constructed a strain of E. coli K12 carrying six mutations induced by the acridine half-mustard ICR-191. The mutations are widely spaced on the E. coli linkage map and are all easily reverted by ICR-191. Mapping of ten independent revertants for each of five markers indicated that the reversions induced by ICR-191 occurred near the original mutations. Exponentially and nonsynchronously growing cultures of this strain were exposed to ICR-191 for 0.85 generation, quickly washed free of mutagen, and resuspended in the original medium minus mutagen. Total viable cell number maintained its exponential increase both during and immediately after exposure to mutagen, whereas the number of revertants of any particular type remained constant for a characteristic period after removal of mutagen before finally assuming an exponential increase. Theoretically, the length of such a segregation lag should depend on the position of the particular reverted gene in the sequence of gene replication: the earlier a gene is replicated in the chromosome replication cycle, the longer its segregation lag should be. Our results are consistent with this prediction and fit a unidirectional, clockwise replication scheme with an origin between 55 and 74 min on the E. coli linkage map. The results also fit a very asymmetric bidirectional replication scheme.     

A Hi–C data-integrated model elucidates E. coli chromosome’s multiscale organization at various replication stages

The chromosome of Escherichia coli is riddled with multi-faceted complexity. The emergence of chromosome conformation capture techniques are providing newer ways to explore chromosome organization. Here we combine a beads-on-a-spring polymer-based framework with recently reported Hi–C data for E. coli chromosome, in rich growth condition, to develop a comprehensive model of its chromosome at 5 kb resolution. The investigation focuses on a range of diverse chromosome architectures of E. coli at various replication states corresponding to a collection of cells, individually present in different stages of cell cycle. The Hi–C data-integrated model captures the self-organization of E. coli chromosome into multiple macrodomains within a ring-like architecture. The model demonstrates that the position of oriC is dependent on architecture and replication state of chromosomes. The distance profiles extracted from the model reconcile fluorescence microscopy and DNA-recombination assay experiments. Investigations into writhe of the chromosome model reveal that it adopts helix-like conformation with no net chirality, earlier hypothesized in experiments. A genome-wide radius of gyration map captures multiple chromosomal interaction domains and identifies the precise locations of rrn operons in the chromosome. We show that a model devoid of Hi–C encoded information would fail to recapitulate most genomic features unique to E. coli.     

Bacillus subtilis Cell Cycle as Studied by Fluorescence Microscopy: Constancy of Cell Length at Initiation of DNA Replication and Evidence for Active Nucleoid Partitioning

Fluorescence microscopic methods have been used to characterize the cell cycle of Bacillus subtilis at four different growth rates. The data obtained have been used to derive models for cell cycle progression. Like that of Escherichia coli, the period required by B. subtilis for chromosome replication at 37°C was found to be fairly constant (although a little longer, at about 55 min), as was the cell mass at initiation of DNA replication. The cell cycle of B. subtilis differed from that of E. coli in that changes in growth rate affected the average cell length but not the width and also in the relative variability of period between termination of DNA replication and septation. Overall movement of the nucleoid was found to occur smoothly, as in E. coli, but other aspects of nucleoid behavior were consistent with an underlying active partitioning machinery. The models for cell cycle progression in B. subtilis should facilitate the interpretation of data obtained from the recently introduced cytological methods for imaging the assembly and movement of proteins involved in cell cycle dynamics.     

Initiation of chromosome replication in Escherichia coli. I. Requirements for RNA and protein synthesis at different growth rates

The effects of inhibition of protein and RNA synthesis on initiation of chromosome replication in Escherichia coli$\text{B}\text{r}$ were determined by measuring rates of DNA synthesis during the division cycle before and after addition of chloramphenicol and rifampicin. The ability of cells to initiate a round of replication depended upon the pattern of chromosome replication during the division cycle. Initiation in the presence of chloramphenicol (200 μ/ml) and rifampicin (100 gmg/ml) was observed only in slowly growing cells which normally initiated a new round between the end of the previous round and the subsequent division (i.e. in the D period of the division cycle). The cells that initiated were in the D period at the time of addition of the drugs. Rapidly growing cells which normally initiated before the D period and slowly growing cells which normally initiated after the D period did not initiate in the presence of the drugs. The contrasting effects of the drugs in cells possessing different chromosome replication patterns, and the coupling between septum-crosswall formation (the D period) and initiation suggest that the timing of initiation of chromosome replication in E. coli is controlled by the cell envelope.