Theta replication of the lactococcal plasmid pWVO2

pWVO2 is a 3.8 kb narrow-host-range plasmid from Lactococcus lactis ssp. cremoris Wg2, which does not replicate in Bacillus subtilis or Escherichia coli. Single-stranded pWVO2 DNA was not observed in lactococcal cells, indicating that this plasmid does not replicate via a rolling-circle mechanism. The sequence of pWVO2 neither showed the structural organization typical for rolling-circle plasmids, nor were sequence similarities with known rolling-circle plasmids present. By 2-D agarose gel electrophoresis of replication intermediates, it was shown that pWVO2 replicates via a theta mechanism. This is the first proof for the existence of theta-replicating plasmids in lactococci. The pWVO2 minimal replicon is strongly related to that of several other lactococcal plasmid replicons. It contains one open reading frame encoding the replication protein, which is preceded by a 22 bp sequence tandemly repeated three and a half times. Further upstream is another 10bp direct repeat present in an A/T-rich sequence. This structural organization resembles that of several iteroncontaining theta-type plasmids from E. coli. Derivatives of pWVO2 were stably maintained in L. lactis and are good candidates for the development of stable food-grade cloning vectors for this organism.     

The specific growth rate of Pseudomonas putida PAW1 influences the conjugal transfer rate of the TOL plasmid.

The kinetics of the conjugal transfer of a TOL plasmid were investigated by using Pseudomonas putida PAW1 as the donor strain and P. aeruginosa PAO 1162 as the recipient strain. Short-term batch mating experiments were performed in a nonselective medium, while the evolution of the different cell types was determined by selective plating techniques. The experimental data were analyzed by using a mass action model that describes plasmid transfer kinetics. This method allowed analysis of the mating experiments by a single intrinsic kinetic parameter for conjugal plasmid transfer. Further results indicated that the specific growth rate of the donor strain antecedent to the mating experiment had a strong impact on the measured intrinsic plasmid transfer rate coefficient, which ranged from 1 x 10(-14) to 5 x 10(-13) ml per cell per min. Preliminary analysis suggested that the transfer rates of the TOL plasmid are large enough to maintain the TOL plasmid in a dense microbial community without selective pressures.     

Genetic analysis of a lactococcal plasmid replicon

Summary The sequence and genetic organization was determined of the 2508 by lactococcal portion of pFX2, which was derived from a crypticLactococcus lactis subsp.lactis plasmid and used as the basis for construction of a series of lactococcal vectors. A lactococcal plasmid plus origin and two replication protein-coding regions (repA andrepB) were located. RepA has a helix-turn-helix motif, a geometry typical of DNA-binding proteins. RepB shows a high degree of homology to the plasmid replication initiation proteins from other gram-positive bacteria andMycoplasma. The transcribed inverted repeat sequence betweenrepA andrepB could form an attenuator to regulate pFX2 replication. Upstream of theori site, and in a region which was non-essential for replication, a 215 by sequence identical to the staphylococcal plasmid pE194 and carrying the RS_A site was identified. The genetic organization of this lactococcal plasmid replicon shares significant similarity with pE194 group plasmids.     

Nucleotide sequence analysis of the lactococcal EPS plasmid pNZ4000

The complete 42180-bp nucleotide sequence of the mobilization plasmid pNZ4000, coding for exopolysaccharide (EPS) production in Lactococcus lactis, was determined. This plasmid contains a region involved in EPS biosynthesis, four functional replicons, a region containing mobilization genes, and three origin of transfer (oriT) sequences. Sequences identical to these oriT sequences were also found on two other lactococcal plasmids and a plasmid from Lactobacillus helveticus. Several complete and partial IS elements were identified on pNZ4000, including iso-ISS1, iso-IS946, and iso-IS982 sequences. Furthermore, pNZ4000 contains a gene cluster that may encode a cobalt transport system and a gene that encodes a CorA homologue which may function as a magnesium transporter.     

Clonal heterogeneity in specific growth rate of Succhuromyces cerevisiue cells

The cell volume increase in individual clones of cells of the yeast Saccharomyces cerevisiae has been measured using time lapse cinematography in populations showing steady state balanced exponential growth. There were significant differences in clonal specific growth rates within the population in each of 10 experiments using different strains on different media supporting different growth rates. The results suggest that specific growth rates of cells which are either genetically identical or very closely related can be different and this difference can be propagated over at least three generations. Since the proliferation rate in yeast is determined by growth rate, these observed differences provide an additional source of cell cycle variability for yeast cells that has not been considered before. The implications for the theoretical analysis of cell cycle kinetics are examined.     

Influence of the growth rate calculation on the relationship between growth rate and temperature

Three calculations of the growth rate (e.g. slope of a plot of the log10 of cfu ml-1 vs time, mum of the Gompertz equation and the reciprocal of time to obtain 108 cfu ml-1) were compared for Escherichia coli TG1 growing in tryptone soy broth medium at temperatures ranging from 14 to 39 degrees C. Up to now, the influence of using such different definitions on the relationship between microbial growth rate and temperature has never been investigated. In order to compare these calculation procedures, a dimensionless analysis based on the following normalized variables, mudim = mu/muopt and Tdim = [T-Tmin]/[Topt-Tmin], was used (Dantigny 1998). The influence of suboptimal temperatures on the growth rate was represented by means of a Belehràdek-type model based on a power function law: [mudim] = [Tdim]alpha. The influence of the different growth rate calculations on the model constants was assessed. Despite the great dependence of the raw growth rate values on the calculation procedure, the dimensionless analysis demonstrated that the alpha-value is independent of the growth rate definition. This result suggests that any definition for the growth rate can be utilized in studies aimed at determining the influence of temperature on microbial growth and highlights the interest of using dimensionless variables to overcome differences in the order of magnitude of the growth rate data and to avoid confusion between definitions.     

Osmotic effects on radial growth rate and specific growth rate of three soil fungi

The radial growth rate on osmotically adjusted agar medium and the relative specific growth rate in osmotically adjusted liquid medium were determined for Rhizoctonia solani, Pythium ultimum, and Verticillium dahliae. On basal medium, an isolate of P. ultimum and R. solani had similar radial growth rates of 0.52 and 0.47 mm/h, respectively, whereas V. dahliae grew at a rate of 0.08 mm/h. Radial growth rate was reduced 50% at osmotic potentials of -16, -27, and -32 bars for P. ultimum, R. solani, and V. dahliae, respectively. No growth occurred at -32 bars for P. ultimum, -56.2 bars for R. solani, and -100 bars for V. dahlia. Specific growth rates in liquid culture were 0.011 h-1 for P. ultimum, 0.008 h-1 for V. dahliae, and 0.026 h-1 for R. solani. Ratios of radial growth rate (Kr) to specific growth rate (alphas) were computed for each fungus growing at different osmotic potentials. There was not a constant relationship between Kr on agar and alphas in liquid medium, e.g., Kr/alphas ratios varied from 8-41% from a mean ratio for a particular species. The results indicated that radial growth rate on osmotic agar was not useful as a measure of relative specific growth rate of a fungus in osmotically adjusted liquid medium.     

Nucleotide sequence and characterization of the broad-host-range lactococcal plasmid pWVO1.

The nucleotide sequence of the Lactococcus lactis broad-host-range plasmid pWVO1, replicating in both gram-positive and gram-negative bacteria, was determined. This analysis revealed four open reading frames (ORFs). ORF A appeared to encode a trans-acting 26.8-kDa protein (RepA), necessary for replication. The ORF C product was assumed to play a regulatory role in replication. Both RepA and the ORF C product showed substantial sequence similarity with the Rep proteins of the streptococcal plasmid pLS1. In addition, the plus origin of replication was identified on the basis of strong similarity with the plus origin of pLS1. Derivatives of pWVO1 produced single-stranded (ss) DNA in Bacillus subtilis and L. lactis, suggesting that this plasmid uses the rolling-circle mode of replication. In B. subtilis, but not in L. lactis, the addition of rifampicin resulted in increased levels of ssDNA, indicating that in the former organism the host-encoded RNA polymerase is involved in the conversion of the ssDNA to double-stranded plasmid DNA (dsDNA). Apparently, in L. lactis the conversion of ss to ds pWVO1 DNA occurs by a mechanism which does not require the host RNA polymerase.     

Cloning of two bacteriocin genes from a lactococcal bacteriocin plasmid

Lactococcus lactis subsp. cremoris 9B4 plasmid p9B4-6 (60 kilobases [kb]), which specifies bacteriocin production and immunity, was analyzed with restriction endonucleases, and fragments of this plasmid were cloned into shuttle vectors based on the broad-host-range plasmid pWVO1. Two regions on p9B4-6 were identified which specify inhibitory activity on L. lactis indicator strains: one that could be confined to a 1.8-kb ScaI-ClaI fragment with low antagonistic activity and a 15-kb XbaI-SalI fragment specifying high antagonistic activity. The inhibitory substances produced by these two clones were sensitive to proteolysis. A 4-kb HindIII fragment derived from the 15-kb fragment strongly hybridized with the 1.8-kb fragment. The antagonistic activity specified by the 4-kb fragment was somewhat reduced as compared with that of the 15-kb fragment. A 1.3-kb ScaI-HindIII subfragment of the 4-kb fragment contained both the immunity and bacteriocin genes. Inhibition studies showed that the two bacteriocins had different specificities.     

Effect of growth rate on the maintenance of a recombinant plasmid inBacillus subtilis

Summary The rate of fall in the proportion of plasmid-containing cells in a population ofBacillus subtilis 1A297[pVC102] grown in continuous culture was independent of growth rate. Plasmid loss could not be ascribed to faulty partitioning during cell division. At a low dilution rate, the specific rate of plasmid loss exceeded the specific growth rate of the plasmid-containing cells.     

Cloning of two bacteriocin genes from a lactococcal bacteriocin plasmid.

Lactococcus lactis subsp. cremoris 9B4 plasmid p9B4-6 (60 kilobases [kb]), which specifies bacteriocin production and immunity, was analyzed with restriction endonucleases, and fragments of this plasmid were cloned into shuttle vectors based on the broad-host-range plasmid pWVO1. Two regions on p9B4-6 were identified which specify inhibitory activity on L. lactis indicator strains: one that could be confined to a 1.8-kb ScaI-ClaI fragment with low antagonistic activity and a 15-kb XbaI-SalI fragment specifying high antagonistic activity. The inhibitory substances produced by these two clones were sensitive to proteolysis. A 4-kb HindIII fragment derived from the 15-kb fragment strongly hybridized with the 1.8-kb fragment. The antagonistic activity specified by the 4-kb fragment was somewhat reduced as compared with that of the 15-kb fragment. A 1.3-kb ScaI-HindIII subfragment of the 4-kb fragment contained both the immunity and bacteriocin genes. Inhibition studies showed that the two bacteriocins had different specificities.     

In vivo definition of the functional origin of leading strand replication on the lactococcal plasmid pFX2

The lactococcal plasmid pFX2 belongs to a family of plasmids, whose prototype is the streptococcal plasmid pMV158, that replicates by the rolling circle mechanism. Determination of the nucleotide sequence of the repX gene of pFX2 allowed us to make some minor corrections in the published sequence, and to show that the repX gene is identical to the rep gene of plasmid pWV01. We have established pFX2 in Escherichia coli and in Streptococcus pneumoniae. In the latter host, we have defined in vivo the nick site introduced by the RepX protein. Plasmid pFX2 and the pMV158 derivative pLS1 exhibit a moderate degree of incompatibility in S. pneumoniae. Cloning of the double strand origin (dso) of pFX2 into a high-copy-number plasmid that is compatible with the pMV158 replicon led to an increase in incompatibility toward pLS1. Plasmids pFX2 and pLS1 exhibit homologies in their Rep proteins and in their dso sequences, but not in their negative control elements. Thus, the observed incompatibility indicates that cross-recognition of Rep proteins and dso takes place. Received: 25 May 1998 / Accepted: 8 July 1998     

The effects of adding lactococcal proteinase on the growth rate of Lactococcus lactis in milk depend on the type of enzyme.

Increasing the proteolytic activity of Lactococcus lactis cultures in milk by adding the corresponding proteinase resulted in a stimulation of the growth rate regardless of the strain and the type of proteinase, demonstrating that the rate of casein degradation was responsible for the growth rate limitation of L. lactis in milk. However, the stimulation was only transient, and the reduction in growth rate in the poststimulation phase depended on the type of cell envelope proteinase. When a PI-type proteinase was added, three causes were involved in the subsequent reduction in growth rate: degradation of the added proteinase, repression of the proteolytic activity expressed by the cells, and competition for peptide uptake. When a PIII-type proteinase was added, the cessation of stimulation was due to the autoproteolysis of the added enzyme only.     

The stability of the yeast plasmid pJDB248 depends on growth rate of the culture

Summary The stability of the plasmid pJDB 248 has been measured in theS. cerevisiae strain S150-2B growing in a chemostat under conditions of glucose limitation. It was found that reducing the growth rate of the culture led to a more rapid loss of the plasmid from the cells.     

Dependence of the specific growth rate of methanogenic mutualistic cocultures on the methanogen

Methanogenesis from ethanol was studied in batch cocultures of a proton-reducing acetogenic Desulfovibrio sp. together with one of eight methanogenic bacteria representing five genera. A mathematical model of mutualistic cocultures predicts an equalisation in the specific growth rates of the two species which defines a specific growth rate for the coculture. At non-limiting ethanol concentrations the model predicts that the specific growth rate of the coculture is dependent upon the K _s (H₂) of the methanogen and its maximum specific growth rate in axenic culture when utilising H₂ as the energy source. We demonstrate experimentally that with methanogens known to have similar K _s (H₂) values, the specific growth rates of methanogenic mutualistic cocultures are dependent upon the maximum specific growth rates of the methanogens.     

Influence of specific growth rate on specific productivity and glycosylation of a recombinant avidin produced by a Pichia pastoris Mut+ strain

A recombinant avidin-producing Mut+ Pichia pastoris strain was used as a model organism to study the influence of the methanol feeding strategy on the specific product productivity (q(p)) and protein glycosylation. Fed-batch cultivations performed at various specific growth rates (micro) and residual methanol concentrations showed that the specific avidin productivity is growth-dependent. The specific productivity increases strongly with the specific growth rate for micro ranging from 0 to 0.02 h(-1), and increases only slightly with the specific growth rate above this limit. N-terminal glycosylation was also found to be influenced by the specific growth rate, since 9-mannose glycans were the most abundant form at low growth rates, whereas 10-mannose carbohydrate chains were favored at higher micro. These results show that culture parameters, such as the specific growth rate, may significantly affect the activity of glycoproteins produced in Pichia pastoris. In terms of process optimization, this suggests that a compromise on the specific growth rate may have to be found, in certain cases, to work with an acceptable productivity while avoiding the addition of many mannoses.