Bistability and hysteresis in epigenetic regulation of the lactose operon. Since Delbrück, a long series of ignored models.

Bistability is the capacity of a system to switch in an "all-or-none" manner between alternative steady states. This powerful concept originates from the analysis of non-linear equations driving open systems. It is one of the various patterns of regulation associated with a particular class of dynamic structures that Glansdorff and Prigogine baptised "dissipative structures". The idea of discontinuous transitions between alternative states was first formulated much earlier, by Delbrück, in 1949. Cohn and Horibata and Novick and Weiner confirmed that such transitions occur in experiments on the lactose operon carried out ten years later. Modelling with non-linear differential equations made it possible to simulate the dynamic behaviour of the lac operon, and modelling by asynchronous logical analysis elucidated the determinant role played by positive feedback circuits in the emergence of multistationarity. Nevertheless, these studies were largely ignored until the recent demonstration of the hysteretic nature of the bistable transition between alternative states of the lac operon. As originally suggested by Delbrück, the pattern of lactose consumption adopted by the bacterium is controlled epigenetically rather than genetically: the true key determinant is the direction of change of an environmental variable with respect to the structural components of the operon.

A novel gene from Myxococcus xanthus that facilitates membrane translocation of an extracellular endoglucanase in Escherichia coli?

Expression in Escherichia coli of the Myxococcus xanthus gene celA, which encodes an extracellular endoglucanase, resulted in CelA being distributed between cytoplasm, periplasm and membrane. The presence of an adjacent open reading frame downstream from the full celA gene, or the absence of a putative lipoprotein signal sequence, confined CelA distribution to the periplasm and membrane, or to the cytoplasm and periplasm, respectively.

Positive feedback circuits and adaptive regulations in bacteria.

The mechanisms by which bacteria adapt to changes in their environment involve transcriptional regulation in which a transcriptional regulator responds to signal(s) from the environment and regulates (positively or negatively) the expression of several genes or operons. Some of these regulators exert a positive feedback on their own expression. This is a necessary (although not sufficient) condition for the occurrence of multistationarity. One biological consequence of multistationarity may be epigenetic modifications, a hypothesis unusual to microbiologists, in spite of some well-known epigenetic modifications in bacteria. We propose here that the occurrence of mucoidy in the opportunistic pathogen Pseudomonas aeruginosa, which is currently attributed to mutations only, may also be an epigenetic modification. A theoretical approach using a generalised logical analysis lends credit to this hypothesis and suggests experiments to ascertain it.

Characterization of an OprF-deficient mutant suggests that OprF is an essential protein for Pseudomonas fluorescens strain MF0.

A stable OprF-deficient mutant for Pseudomonas fluorescens strain MF0 was constructed using reverse genetics. This mutant, called MF372, showed a rounded morphology and grew more slowly in minimal medium, but not in rich medium. Contrary to other Pseudomonas strains, the loss of OprF for strain MF0 was accompanied by an altered outer membrane composition. At least three outer membrane proteins were overexpressed, apparently as a consequence of adaptive mutations. The N-terminal sequence of two of them revealed strong similarities with porins of the OprD family from P. aeruginosa. The data presented here shows that OprF may be an essential protein for this P. fluorescens strain.

Production of pectate lyases and cellulases by Chryseomonas luteola strain MFCL0 depends on the growth temperature and the nature of the culture medium: evidence for two critical temperatures.

Several extracellular enzymes that are responsible for plant tissue maceration were detected in culture supernatant of the psychrotrophic bacterium Chryseomonas luteola MFCL0. Isoelectrofocusing experiments showed that pectate lyase (PL) activity resulted from the cumulative action of three major isoenzymes, designated PLI, PLII, and PLIII. Cellulolytic activity was also detected in culture supernatants. These enzymes exhibited different behaviors with respect to growth temperature. PLII was not regulated by temperature, whereas PLI and PLIII were regulated similarly by growth temperature. Maximal levels of PLI and PLIII were produced at 14 degrees C when cells were grown in polygalacturonate-containing synthetic medium and at around 20 to 24 degrees C in nutrient broth. In contrast, thermoregulation of cellulolytic activity production differed from thermoregulation of PL. The level of cellulolytic activity was low in all media at temperatures up to 20 degrees C, and then it increased dramatically until the temperature was 28 degrees C, which is the optimal temperature for growth of C. luteola. Previously, we defined the critical temperature by using the modified Arrhenius equation to characterize bacterial behavior. This approach consists of monitoring changes in the maximal specific growth rate as a function of temperature. Our most striking result was the finding that the temperature at which maximum levels of PLI and PLIII were produced in two different media was the same as the critical temperature for growth observed in these two media.

A bioluminescence assay for screening thermoregulated genes in a psychrotrophic bacterium Pseudomonas fluorescens.

Random transcription fusion delivery, with bacterial luciferase genes as reporter, was performed in the psychrotrophic bacterium Pseudomonas fluorescens. Direct screening on plates of the insertions allowed the isolation of fusions into thermoregulated genes with good accuracy, either in a library of insertion fusions, or after genetic transfer of a putative regulatory mutation. Using this method, it was shown that in Ps. fluorescens, nearly 40% of the genes are thermoregulated and belong to at least three classes according to the maximal temperature of expression of the fused genes. This is more than had been estimated by a previous method, and demonstrates the importance of thermoregulation in psychrotrophic bacteria. As this reporter is the first to be used for direct screening for genes regulated by temperature, it should be of great value in the study of mechanisms involved in adaptation to this environmental factor.

Genetic studies of a thermoregulated gene in the psychrotrophic bacterium Pseudomonas fluorescens.

In the psychrotrophic bacterium Pseudomonas fluorescens, some genes are thermoregulated: they are maximally expressed at a particular temperature within the broad range of temperatures that allow growth of this bacterium. To study this regulation, random transcriptional insertion fusions were obtained by means of mini-Tn5lacZ1 or mini-Tn5luxAB transposition. One fusion was studied in which beta-galactosidase production was maximal at a low-growth temperature. The mutated gene (that we call xsf) was highly homologous to xseA from Escherichia coli (and from other bacteria) which encodes the large subunit of exonuclease VII. Genetic tools were constructed in order to analyse and manipulate this fusion: a plasmid derived from R68.45 was used for chromosome transfer and a replacement vector was constructed to allow in situ marker exchange of the mini-Tn5lacZ1 by an Hg(r) interposon. This vector was used to make double mutants and hence to study the effect of the insertion in xsf on the expression of other fusions. Six genes were thereby identified with a decreased expression in an xsf- background and with different characteristics of thermoregulation.

Nucleic acid extraction from polluted estuarine water for detection of viruses and bacteria by PCR and RT-PCR analysis.

We describe an extraction protocol for genomic DNA and RNA of both viruses and bacteria from polluted estuary water. This procedure was adapted to the molecular study of microflora of estuarine water where bacteria and viruses are found free, forming low-density biofilms, or intimately associated with organo-mineral particles. The sensitivity of the method was determined with seeded samples for RT-PCR and PCR analysis of viruses (10 virions/mL), and bacteria (1 colony-forming unit mL). We report an example of molecular detection of both poliovirus and Salmonella in the Seine estuary (France) and an approach to studying their association with organo-mineral particles.

Regulation of the expression of a gene encoding beta-endoglucanase secreted by Myxococcus xanthus during growth: role of genes involved in developmental regulation.

An endoglucanase, CelA, is secreted by Myxococcus xanthus only during exponential growth. The production of this enzyme is decreased by mutations in 5 different genes (Exc +/- phenotype), three of which correspond to asg genes which regulate the production of an early cell-to-cell signal in development. Transcription of celA is decreased in two of these Exc +/- mutants, whereas a post-transcriptional step is affected in two other Exc- mutants. Thus, asg genes, in addition to regulating the onset of development, also regulate a gene (celA) that is expressed during exponential growth and that is not involved in development.

Cloning and sequencing of two genes, prtA and prtB, from Myxococcus xanthus, encoding PrtA and PrtB proteases, both of which are required for the protease activity.

The sequence of a 1955-bp TaqI DNA fragment from Myxococcus xanthus was determined. This fragment contains two complete genes, designated prtA and prtB. The prtA and prtB ORFs extend over 828 and 798 bp, respectively. They are separated only by 3 nt and appear to be present in a polycistronic transcriptional unit. A typical lipoprotein signal sequence is present at the N terminus of the two deduced polypeptides. The aa sequence of PrtA shows a high degree of identity to the region adjacent to the Ser residue belonging to the catalytic triad of serine proteases from Staphylococcus aureus and Enterococcus faecalis. It also exhibits features characteristic of trypsin-like serine proteases in that it contains the same pattern of variable and conserved regions. The deduced aa sequence of PrtB reveals a signature zinc-binding consensus motif (HEXXHXXGXXH/Met-turn) characteristic of the class of metalloproteases called metzincins. Plasmids containing prtA, prtB, or both were constructed. Protease activity studies of Escherichia coli clones containing these plasmids showed that both genes are necessary for this activity, whatever their cis or trans position. As prtB produces a putative membrane-bound lipoprotein of 266 aa, the protease activation must occur at the membrane level.

A gene involved in both protein secretion during growth and starvation-induced development encodes a subunit of the NADH:ubiquinone oxidoreductase in Myxococcus xanthus.

The secretion of numerous proteins during vegetative growth of Myxococcus xanthus, and the multicellular development cycle induced upon starvation of these bacteria, are partially interrelated in so far as mutants impaired in extracellular protein production are unable to undergo development. We have cloned and sequenced a gene in which a Tn5 insertion leads to a decrease in the production of most, if not all, extracellular proteins, and prevents development and sporulation. The deduced protein is homologous to the putative ubiquinone-binding subunit of bacterial and mitochondrial NADH:ubiquinone oxidoreductases (complex I). This is the first example of the presence of this complex in a bacterium from subclass delta of the proteobacteria. This gene is expressed during growth and during early development. As its disruption by Tn5 does not impair growth of the mutant strain, we assume the presence of a second alternative NADH oxidoreductase, and suggest that the phenotypic alterations caused by the mutation are due to a decrease in the proton-motive force.

Isolation of a soil psychrotrophic toluene-degrading Pseudomonas strain: influence of temperature on the growth characteristics on different substrates.

Two psychrotrophic toluene-degrading Pseudomonas putida strains were isolated at low temperature from a toluene-polluted soil, thereby demonstrating that toluene degradation at low temperature occurred in nature, a finding of possible interest for soil bioremediation procedures. In one of these strains, two aromatic compounds (toluene and benzoate) were degraded, most likely through different pathways. To study the effect of the growth temperature on the metabolism of these substrates, we studied the evolution of the maximal growth rates with respect to both temperature and substrate. It was shown that not only cardinal temperatures but also temperature characteristics deduced from the Arrhenius plot of maximal growth rates differed when the different substrates were used as sole carbon and energy source.

Evidence for two domains of growth temperature for the psychrotrophic bacterium Pseudomonas fluorescens MF0.

The variations in the maximal specific growth rate of the psychrotrophic bacterium Pseudomonas fluorescens MF0 with respect to temperature were studied between 0 and 30 degrees C (optimal for growth). The Arrhenius plot showed a drastic change in slope at the intermediate temperature of 17 degrees C. Over the cold domain from 0 to 17 degrees C, the temperature characteristic was twofold higher than over the suboptimal domain from 17 to 30 degrees C. The macromolecular composition of exponentially growing cells was invariant over the entire range from 0 to 30 degrees C. Variations of temperature and growth rate were independently investigated through chemostat experiments in order to characterize their respective effects on cell macromolecular composition and size. The effect of growth rate in this psychrotrophic strain is identical to that of all other bacteria assayed so far. In contrast, an original biphasic variation of total protein concentration was demonstrated in strain MF0 with respect to temperature, with a maximum at 17 to 20 degrees C. Indeed, increasing the temperature in the chemostat resulted in a biphasic decrease in the net protein production rate: a very slight decrease below 17 degrees C and a much larger decrease from 17 to 28 degrees C. These results could signify an increase in the cellular protein degradation rate with increasing temperature, especially above 17 degrees C.

Starvation yields a drastic decrease in outer-membrane permeability to a periplasmic foreign protein in Myxococcus xanthus.

A recombinant Myxococcus xanthus strain was constructed that constitutively produces two proteins from Escherichia coli, the cytoplasmic beta-galactosidase and the periplasmic pH 2.5 acid phosphatase (AppA protein). We have previously shown that during vegetative growth, AppA protein is partly accumulated in the periplasm of M. xanthus and partly released into the medium. We demonstrate here that during starvation-induced development, release of periplasmic AppA protein to the medium did not occur over a period of 20 h. This was coincident with, but not caused by, the arrest of the synthesis of the foreign proteins. We have shown that this lack of secretion could be attributed to starvation per se and did not depend on the ability of the cells to undergo development. Our findings suggest that protein secretion which occurs during the first hours of starvation-induced development might therefore take place via a different route from that which occurs in vegetative cells.

The gene encoding the beta-1,4-endoglucanase (CelA) from Myxococcus xanthus: evidence for independent acquisition by horizontal transfer of binding and catalytic domains from actinomycetes.

The celA gene encoding a beta-1,4 endoglucanase (CelA) from Myxococcus xanthus has been cloned in Escherichia coli and sequenced. The C-terminal region of CelA displayed a high level of similarity with the catalytic domain of several Egl belonging to the glycosyl hydrolases family 6 (CenA from Cellulomonas fimi, CelA from Microbispora bispora, E2 from Thermonospora fusca, CasA from Streptomyces KSM9 and CelA1 from Streptomyces halstedii) and less similarity to the cellobiohydrolases of the fungi Trichoderma reesei and Agaricus bisporus. Using PCR amplification we found in another myxobacterium, Stigmatella aurantiaca, a part of a glycosyl hydrolase belonging to the same family. The N-terminal part of CelA displayed significant similarities with the cellulose-binding domain of other cellulases belonging to a rare subset of family II, such as the avicelase I from Streptomyces reticuli, both tandem repeats N1 and N2 of the cellulase CenC from Cellulomonas fimi, and the N-terminal part of the Egl E1 from Thermonospora fusca. Analyses of the multiple alignments and reconstruction of phylogenetic trees strongly suggest that both domains of CelA were acquired by independent horizontal transfers between Gram+ soil bacteria and scavenging myxobacteria followed by domain shuffling.

An endo-N-acetyl-beta-D-glucosaminidase, acting on the di-N-acetylchitobiosyl part of N-linked glycans, is secreted during sporulation of Myxococcus xanthus.

After the demonstration that Stigmatella aurantiaca DW4 secretes an endo-N-acetyl-beta-D-glucosaminidase (ENGase), acting on the di-N-acetylchitobiosyl part of N-linked glycans (S. Bourgerie, Y. Karamanos, T. Grard, and R. Julien, J. Bacteriol. 176:6170-6174, 1994), an ENGase activity having the same substrate specificity was also found to be secreted during vegetative growth of Myxococcus xanthus DK1622. The activity decreased in mutants known to secrete less protein than the wild type (Exc +/-). During submerged development, the activity was produced in two steps: the first increase occurred during the aggregation phase, and the second one occurred much later, during spore formation. This production was lower in developmental mutants impairing cell-cell signaling, the late mutants (csg and dsg) being the most deficient. Finally, when sporulation was obtained either by starvation in liquid shake flask culture or by glycerol induction, the activity was produced exclusively by the wild-type cells during the maturation of the coat.

Lipase and acidic phosphatase from the psychrotrophic bacterium Pseudomonas fluorescens: two enzymes whose synthesis is regulated by the growth temperature.

In the psychrotrophic bacterium Pseudomonas fluorescens, the production of several enzymes that otherwise differ in their cell location, growth phase production and inducibility appeared to be similarly regulated by the growth temperature. In order to determine the level of this regulation, the expression of the apo and lip genes encoding two of these enzymes, the acidic phosphatase and lipase, respectively, was studied at different temperatures. Both genes were optimally expressed at 17.5 degrees C, i.e., at the optimal temperature of production of the enzymes; however, the low level of activity at the highest temperature could be due to an additional post-transcriptional control.

Growth temperature controls the production of a single extracellular protease by Pseudomonas fluorescens MF0, in the presence of various inducers.

The psychotrophic strain Pseudomonas fluorescens MFO is known to express several enzymatic activities in milk, including extracellular proteolytic activity, optimally when cells are grown at 17.5 degrees C. In order to study the nature of the mechanisms controlling the production of the extracellular protease, we devised a defined medium in which this enzymatic activity was induced by an amino acid and small peptides. Regardless of the inducer, optimal proteolytic activity appeared at 17.5 degrees C. SDS-PAGE and isoelectrofocussing revealed a single protease produced by P. fluorescens MFO with all the inducers used and at all temperatures examined. The level of proteolytic activity correlated with the amount of enzyme in the supernatants.