A new mathematical approach predicts individual cell growth behavior using bacterial population information

A theoretical methodology has been developed for studying the growth kinetics of bacterial cells. It utilizes the steady-state cell length distribution in a bacterial population to predict the dependency of growth and division rates on cell length and age. The mathematical model has been applied to the analysis of two bacterial populations, a wild-type strain of Bacillus subtilis, and a minicell-producing strain that carries the divIVB1 mutation. The results show that our model describes the wild-type population very well and that the assumptions typically used in traditional methods are unrealistic. In the case of the minicell-producing mutant we find evidence that the rate of cell division must be a function not only of cell size but also of cell age.     

Cell division suppression in the Bacillus subtilis div IC-A1 minicell-producing mutant.

Growth and division patterns of Bacillus subtilis wild-type (div IV-A1+) and minicell-producing mutant (div IV-A1) clones were studied after spore germination during microcolony development in chambers that facilitate continuous observation with a phase contrast microscope. Data obtained from 13 div IV-A1+ clones were used to derive the equation DE equals [(mum minus 17.6)/8.8], which expresses the relationship of cell divisions present in clones of various lengths. This equation was used to determine the number of divisions expected in div IV-A1 clones if the mutant clones were able to divide as often as wild-type clones. The observed number of divisions present in mutant clones was found to be only 25.27% of the number expected on the basis of this equation. Although individual div IV-A1 clones varied in the percentage of division equivalents expressed, there appeared to be no correlation between the overall clone growth rate and the number of divisions expressed. Culturing div IV-A1+ and div IV-A1 clones together in the same growth chamber revealed that there were no diffusible interactions influencing the division phenotypes of either mutant or wild-type cells. At later stages of growth, mixed microcolonies containing cells of both genotypes were formed. A length analysis of individual cells in these populations indicated that the relative division suppression of mutant compared with wild-type cells characteristic of the initial stages of clone development was maintained. It is likely, therefore, that the excessive length of minicell-producing cells (div IV-A1) is a reflection primarily of division suppression in the mutant and not simply of mislocation of division along cell length.     

Quantal Behavior of a Diffusible Factor Which Initiates Septum Formation at Potential Division Sites in Escherichia coli

Analysis of nucleated cell size in a minicell-producing strain of Escherichia coli and in its parental strain shows that the two distributions are considerably different. A model is proposed to account for this difference. The model states that: (i) in the mutant population, the cell poles are available as potential division sites in addition to the normally located division sites; (ii) the probability of a division occurring at any of the potential division sites is equal; and (iii) only enough "division factor" arises at each unit cell doubling to permit a single division. This factor is utilized entirely in the formation of a single septum. Thus, the occurrence of a polar division with the production of an anucleate minicell (which occurs only in the mutant strain) prevents the occurrence of a non-polar division, with the result that the average nucleated cell length is increased in minicell-producing strains. The model has been used to construct a theoretical population, and a number of parameters of the real and theoretical populations have been compared. The two populations are very similar in all of the parameters measured.     

Clonal Analysis of Cell Division in the Bacillus subtilis div IV-B1 Minicell-Producing Mutant

Spores of the Bacillus subtilis minicell-producing mutant div IV-B1 were germinated and grown to microcolonies in chambers which facilitate continuous observation of the developing clones with a phase-contrast microscope. Time lapse photographs were taken of 46 clones, covering the period from the beginning of outgrowth until at least two rounds of cell division had been completed. Cell lineages were constructed from contour length measurements of the photographs. These data include cell lengths, division site locations, and cell numbers in clones of various ages. From these data we have determined that the probability of a minicell being produced at any division by the div IV-B1 mutant is 0.31. The location of the abnormal division site which generates the first minicell produced in the outgrowing clone appears to be random with respect to the existing cell poles. In contrast, the location of the second abnormal division site, and hence the second minicell, is not random but rather occurs preferentially in proximity to the first minicell. This clustering of abnormal events suggests that division site location is related to pole age (generations), although other influences on minicell clustering cannot be ruled out at present.     

Minicell-forming mutants of Escherichia coli: production of minicells and anucleate rods.

The Escherichia coli minB mutant originally isolated is known to septate at cell poles to form spherical anucleate minicells. Three new minicell-producing mutants were isolated during a screening by autoradiography for chromosome partition mutants giving rise spontaneously to normal-sized anucleate cells. These min mutants were affected close to or in the minB locus. Autoradiography analysis as well as fluorescent staining of DNA showed that in addition to minicells, these strains and the original minB mutant also spontaneously produced anucleate rods of normal size and had an abnormal DNA distribution in filaments. These aberrations were not associated with spontaneous induction of the SOS response. Inhibition of DNA synthesis in these mutants gave rise to anucleate cells whose size was longer than unit cell length, suggesting that the min defect allows septation to take place at normally forbidden sites not only at cell poles but also far from poles. Abnormal DNA distribution and production of anucleate rods suggest that the Min product(s) could be involved in DNA distribution.     

Minicell Yield and Cell Division Suppression in Bacillus subtilis Mutants

Minicell yield is determined by the probability of a minicell-producing division and the relationship of growth to division in Bacillus subtilis mutants.     

New mini-ColE1 as a molecular cloning vehicle.

A new mini-ColE1 plasmid, designated pAC105, was isolated. It has a molecular weight of 1.6 X 10(6) and carries information for its self-replication as well as information for conferring colicin E1 immunity upon its host. Furthermore, pAC105 undergoes replication in the presence of chloramphenicol even when a foreign deoxyribonucleic acid (pSC101) is inserted into its single EcoRI restriction site. Studies in minicell-producing strains demonstrate that pAC105 codes for only two or three polypeptides of low molecular weight. The advantages of using it as a molecular cloning vehicle are discussed.     

Genetic expression and gyrase dependence of methylated and undermethylated DNA in Escherichia coli

The genetic expression and dependence on gyrase of plasmid pBR322 were studied in dam-3, dcm-6, and dam-3 dcm-6 derivatives of a minicell-producing Escherichia coli strain. The results obtained with both methylated and undermethylated plasmid DNA were similar.     

Analysis of the genes and gene products of Xanthomonas transposable elements ISXc5 and ISXc4.

A series of deletion mutants have been constructed for the gene analyses of transposable elements, ISXc5 and ISXc4, derived from Xanthomonas. At least two element-encoded polypeptides of 48 kDa and 40 kDa have been identified in the minicell-producing Escherichia coli strain, TC410. A study of the element transposition and cointegrate resolution revealed that the 48-kDa and 40-kDa polypeptide are both involved in translocation of the elements, the 48-kDa product being involved in transposition of the elements and the 40-kDa product being involved in cointegrate resolution.     

Photoreactivation, Excision, and Strand-Rejoining Repair in R Factor-Containing Minicells of Escherchia coli K-12

Chromosomeless “minicells” are formed by misplaced cell fissions near the polar extremities of an Escherichia coli K-12 mutant strain. Resistance (R)-factor deoxyribonucleic acid (DNA) can be introduced into minicells by segregation from an R⁺ (R64-11) derivative of the original mutant. We have assessed the ability of R⁺ minicells to correct defects produced in their plasmid DNA by ultraviolet (UV) and gamma radiations. Minicells harboring plasmid DNA, in comparison with their repair-proficient minicell-producing parents, possess (i) an equal competence to rejoin single-strand breaks induced in DNA by gamma rays, (ii) a reduced capacity for the photoenzymatic repair of UV-induced pyrimidine dimers, and (iii) a total inability to excise dimers, apparently owing to a deficiency in UV-specific endonuclease activity responsible for mediating the initial incision step in excision repair. Assuming that the DNA repair properties of R⁺ minicells reflect the concentration of repair enzymes located in the plasmid-containing polar caps of entire cells, these findings suggest that: (i) the enzymes responsible for rejoining single-strand breaks are distributed throughout the cell; (ii) photoreactivating enzyme molecules tend to be concentrated near bacterial DNA and to a lesser extent near plasmid DNA; and (iii) UV-specific endonuclease molecules are primarily confined to the central region of the E. coli cell and, thus, seldom segregate with R-factor DNA into minicells.     

Relationship Between Prophage Induction and Transformation in Haemophilus influenzae

The interaction between transformation and prophages of HP1c1, S2, and a defective phage of Haemophilus influenzae has been investigated by measurement of (i) the effect of prophage on transformation frequency and (ii) the effect of transformation on phage induction. The presence of any of the prophages does not appreciably alter transformation frequencies in various Rec(+) and Rec(-) strains. However, exposure of competent lysogens to transforming deoxyribonucleic acid (DNA) may induce phage but only in Rec(+) strains, which are able to integrate transforming DNA into their genome. Transformation of Rec(+) lysogens with DNA irradiated with ultraviolet (UV) light causes the production of even more phage than results from unirradiated DNA, but this indirect UV induction is not as effective as direct induction by UV irradiation of lysogens. Both types of UV induction are influenced by the repair capacity of the host. Wild-type cells contain a prophage and can be induced by transformation to produce a defective phage, which kills a small fraction of the cells. Defective phage in wild-type cells are also induced by H. parainfluenzae DNA, and a much larger fraction of the cells is killed. Strain BC200, which is highly transformable but is not inducible for defective phage, is not killed by H. parainfluenzae DNA, suggesting that wild-type cells are killed by killed by this DNA because of phage induction. A minicell-producing mutant, LB11, has been isolated. Some phage induction occurs in this strain when the cells are made competent, unlike the wild type. A large majority of LB11 cells surviving the competence regime are killed by exposure to transforming DNA.     

Cryptic Plasmids in a Minicell-Producing Strain of Salmonella typhimurium

A minicell-producing strain of Salmonella typhimurium contains two cryptic plasmids. One has a molecular weight of 2.6 x 10(6) to 2.8 x 10(6), is present in multiple copies per cell, and segregates into minicells. The other has a molecular weight of 130 x 10(6), is present in few copies per cell, and probably does not segregate into minicells.     

Expression of the replication region of phage lambda DNA cloned into pBR322 in E. coli minicells

Replication region of bacteriophage lambda DNA was cloned into pBR322 plasmid by the use of two restriction enzymes--PstI and HindIII. The restriction analysis of four obtained plasmids revealed that lambda DNA was cloned in both orientations. Recombinant plasmids were transferred to the minicell-producing strain of E. coli and synthesis of the plasmid-mediated proteins was analysed by polyacrylamide-gel electrophoresis. All four recombinant plasmids produced lambda DNA replication proteins pO and pP as well as some proteins specific for pBR322. The orientation of cloned fragment did not affect the synthesis of lambda DNA replication proteins.     

Localization of the structural gene for the ' subunit of RNA polymerase in Escherichia coli

A minicell-producing strain of $\text{E.}$$\text{coli}$ carrying an F′ factor, KLF10-1, forms minicells that contain plasmid but not chromosomal DNA. These minicells were found to synthesize two polypeptides corresponding precisely to the β and β′ subunits of RNA polymerase in SDS-polyacrylamide gel electrophoresis. In contrast, minicells obtained from an isogenic strain carrying F13-1 do not synthesize these proteins under similar conditions. These results indicate that the structural genes for the β′ as well as β subunits of the polymerase are located on the chromosomal segment (78 to 81 min on the standard genetic map of $\text{E.}$$\text{coli}$) carried by KLF10-1.     

Isolation of an ompC-like outer membrane protein gene from Salmonella typhi

We have isolated the structural gene for an outer membrane protein of Salmonella typhi, from a genomic library constructed in bacteriophage lambda 1059, using the Escherichia coli ompC gene as a heterologous probe. E. coli ompC codes for an outer membrane pore protein (porin) that is induced preferentially at high osmolarity and high temperature. The S. typhi ompC-like gene was subcloned in pBR322 and introduced into E. coli HB101 and into P678-54, a minicell-producing strain. In both strains it expressed a 38.5-kDa protein, which was incorporated into the outer membrane envelope and comigrated with an S. typhi outer membrane protein which was expressed both at low and high osmolarity in vivo.     

Intracellular location of catabolite activator protein of Escherichia coli.

The catabolite activator protein was assayed in extracts from the minicell-producing Escherichia coli strain P678-54. The level of catabolite activator protein was found to be the same in both parent cells and purified minicells, regardless of whether the bacteria were grown on glucose (which leads to low intracellular cyclic adenosine monophosphate levels) or on glycerol-yeast extract or LB broth (which lead to high cyclic adenosine monophosphate concentrations in the cell). Thus, at any given time most catabolite activator protein molecules are found in the cytoplasm. The implications of this for the mechanism of catabolite activator protein action at catabolite-sensitive operons are discussed.     

Enzymes synthesizing and hydrolyzing murein in Escherichia coli. Topographical distribution over the cell envelope.

Envelopes from regions of the cell which in vivo show very little, if any, murein synthesis were isolated using the minicell-producing strain P678-54. Envelopes from minicells, representing in fact cell ends, were able to synthesize murein and to carry out transpeptidation in vitro; also all four murein hydrolase activities tested, carboxypeptidase, endopeptidase, amidase and transglycosylase, were found to be present. The specific activities of the murein synthesizing and degrading enzymes in envelopes derived from cell poles and from actively growing cells were similar. The topological distribution of murein-synthesizing enzymes and of murein hydrolases over the cell envelope is discussed.     

Escherichia coli hemolysin is released extracellularly without cleavage of a signal peptide.

A 110-kilodalton polypeptide isolated from cell-free culture supernatants of hemolytic Escherichia coli was shown to be associated with hemolytic activity. The relative amount of the extracellular 110-kilodalton species detected directly reflects the extracellular hemolysin activity associated with Escherichia coli strains harboring different hemolysin recombinant plasmids. The predicted molecular mass of the hemolysin structural gene (hlyA) based on DNA sequence analysis was 109,858 daltons. Amino-terminal amino acid sequence analysis of the 110-kilodalton polypeptide provided direct evidence that it was encoded by hlyA. Based on this information, it was also demonstrated that the HlyA polypeptide was released extracellularly without signal peptidase-like cleavage. An examination of hemolysin-specific polypeptides detected by use of recombinant plasmids in a minicell-producing strain of Escherichia coli was performed. These studies demonstrated how hemolysin-associated 110- and 58-kilodalton polypeptides detected in the minicell background could be misinterpreted as a precursor-product relationship.