Janine Guespin-Michel

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.

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.

The gene encoding the β-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.

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.

Transfer of incP plasmids into Stigmatella aurantiaca leading to insertional mutants affected in spore development

SummaryDerivatives of the broad-host-range plasmid RP4, containing the wild-type or modified transposon Tn5 were transferred by conjugation to various Stigmatella aurantiaca isolates. The transposons and in some cases fragments of the plasmid as well were integrated into the chromosome. Thus, insertional mutants have been obtained affected in spore formation in liquid culture.

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 δ 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.

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.

Utilization of IncP-1 plasmids as vectors for transposon mutagenesis in Myxobacteria

No free plasmid has ever been found in the myxobacterium Myxococcus xanthus, but IncP-1 plasmids are able to integrate into the chromosome of this bacterium. The frequency of integration depends greatly upon the structure of the IncP-1 plasmid used. This property has been used to devise new delivery systems for transposon mutagenesis in this species. Plasmids with low integration efficiencies have proved to be efficient donors of Tn5, while plasmids with very high frequencies of integration could be used directly to generate mutations. These vectors have also proved efficient for Tn5 transfer into other species of myxobacteria, which have not so far been susceptible to genetic analysis.

Regulation of the expression of a gene encoding β-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.