Alfonso Jiménez-Sánchez

Functional Taxonomy of Bacterial Hyperstructures

SUMMARY The levels of organization that exist in bacteria extend from macromolecules to populations. Evidence that there is also a level of organization intermediate between the macromolecule and the bacterial cell is accumulating. This is the level of hyperstructures. Here, we review a variety of spatially extended structures, complexes, and assemblies that might be termed hyperstructures. These include ribosomal or “nucleolar” hyperstructures; transertion hyperstructures; putative phosphotransferase system and glycolytic hyperstructures; chemosignaling and flagellar hyperstructures; DNA repair hyperstructures; cytoskeletal hyperstructures based on EF-Tu, FtsZ, and MreB; and cell cycle hyperstructures responsible for DNA replication, sequestration of newly replicated origins, segregation, compaction, and division. We propose principles for classifying these hyperstructures and finally illustrate how thinking in terms of hyperstructures may lead to a different vision of the bacterial cell.

On the origin and evolution of the genetic code

Two ideas have essentially been used to explain the origin of the genetic code: Cricks frozen accident and Woeses amino acid-codon specific chemical interaction. Whatever the origin and codon-amino acid correlation, it is difficult to imagine the sudden appearance of the genetic code in its present form of 64 codons coding for 20 amino acids without appealing to some evolutionary process. On the contrary, it is more reasonable to assume that it evolved from a much simpler initial state in which a few triplets were coding for each of a small number of amino acids. Analysis of genetic code through information theory and the metabolism of pyrimidine biosynthesis provide evidence that suggests that the genetic code could have begun in an RNA world with the two letters A and U grouped in eight triplets coding for seven amino acids and one stop signal. This code could have progressively evolved by making gradual use of letters G and C to end with 64 triplets coding for 20 amino acids and three stop signals. According to proposed evidence, DNA could have appeared after the four-letter structure was already achieved. In the newborn DNA world, T substituted U to get higher physicochemical and genetic stability.

Ribonucleoside diphosphate reductase is a component of the replication hyperstructure in Escherichia coli

Although the nrdA101 allele codes for a ribonucleoside diphosphate (rNDP) reductase that is essentially destroyed in less than 2 min at 42°C, and chemical inhibition of the enzyme by hydroxyurea stops DNA synthesis at once, we found that incubation at 42°C of an Escherichia coli strain containing this allele allows DNA replication for about 40 min. This suggests that mutant rNDP reductase is protected from thermal inactivation by some hyperstructure. If, to‐gether with the temperature upshift, RNA or protein synthesis is inhibited, the thermostability time of the mutant rNDP reductase becomes at least as long as the replication time and residual DNA synthesis becomes a run‐out replication producing fully replicated chromosomes. This suggests that cessation of replication in the nrdA101 mutant strain is not the result of inactivation of its gene product but of the activity of a protein reflecting the presence of a partially altered enzyme. The absence of Tus protein, which specifically stops the replication complex by inhibiting replicative helicase activity, allows forks to replicate for a longer time at the restrictive temperature in the nrdA101 mutant strain. We therefore propose that rNDP reductase is a component of the replication complex, and that this association with other proteins protects the protein coded by allele nrdA101 from thermal inactivation.

Differential inhibition of the initiation of DNA replication in stringent and relaxed strains of Escherichia coli

Starvation for isoleucine inhibits chromosome, minichromosome and pBR322 DNA replication in a stringent strain of E. coli , but does not do so in a relaxed mutant. Starvation for other amino acids inhibits either chromosome and minichromosome replication in both strains. From these results we conclude that oriC and pBR322 replication are stringently regulated and that isoleucine seems not to be essential for the protein synthesis required at the initiation of oriC replication. Deprivation of isoleucine in a Rel − strain gives rise to amplification of minichromosome and pBR322 with a better yield of the latter plasmid than that following treatment with chloramphenicol.

Production of biosurfactants by Cladosporium resinae

SummaryCladosporium resinae produces extracellular biosurfactants when growing in a hydrocarbon source such as the jet fuel JP8. This production of biosurfactants was observed by the reduction of the surface tension of the aqueous phase of growing medium, and by the increase in emulsion and foaming properties. A partial purification by collapsed foam gave better physical properties by decreasing surface tension and increasing foaming power and stabilization of emulsions. Surface active substances were purified by reversed phase chromatography. Six compounds representing over 75% of fraction containing surface activity were present. This fraction gave an improvement of all surface properties.

Correlation between ribonucleoside-diphosphate reductase and three replication proteins in Escherichia coli

BackgroundThere has long been evidence supporting the idea that RNR and the dNTP-synthesizing complex must be closely linked to the replication complex or replisome. We contributed to this body of evidence in proposing the hypothesis of the replication hyperstructure. A recently published work called this postulate into question, reporting that NrdB is evenly distributed throughout the cytoplasm. Consequently we were interested in the localization of RNR protein and its relationship with other replication proteins.ResultsWe tagged NrdB protein with 3×FLAG epitope and detected its subcellular location by immunofluorescence microscopy. We found that this protein is located in nucleoid-associated clusters, that the number of foci correlates with the number of replication forks at any cell age, and that after the replication process ends the number of cells containing NrdB foci decreases.We show that the number of NrdB foci is very similar to the number of SeqA, DnaB, and DnaX foci, both in the whole culture and in different cell cycle periods. In addition, interfoci distances between NrdB and three replication proteins are similar to the distances between two replication protein foci.ConclusionsNrdB is present in nucleoid-associated clusters during the replication period. These clusters disappear after replication ends. The number of these clusters is closely related to the number of replication forks and the number of three replication protein clusters in any cell cycle period. Therefore we conclude that NrdB protein, and most likely RNR protein, is closely linked to the replication proteins or replisome at the replication fork. These results clearly support the replication hyperstructure model.

DIFFERENCES IN THE DEGREE OF INHIBITION OF NDP REDUCTASE BY CHEMICAL INACTIVATION AND BY THE THERMOSENSITIVE MUTATION nrdA101 IN Escherichia coli SUGGEST AN EFFECT ON CHROMOSOME SEGREGATION

NDP reductase activity can be inhibited either by treatment with hydroxyurea or by incubation of an nrdAts mutant strain at the non-permissive temperature. Both methods inhibit replication, but experiments on these two types of inhibition yielded very different results. The chemical treatment immediately inhibited DNA synthesis but did not affect the cell and nucleoid appearance, while the incubation of an nrdA101 mutant strain at the non-permissive temperature inhibited DNA synthesis after more than 50 min, and resulted in aberrant chromosome segregation, long filaments, and a high frequency of anucleate cells. These phenotypes are not induced by SOS. In view of these results, we suggest there is an indirect relationship between NDP reductase and the chromosome segregation machinery through the maintenance of the proposed replication hyperstructure.

A temperature upshift induces initiation of replication at oriC on the Escherichia coli chromosome

A temperature upshift of 10 or more degrees in the growth temperature of a bacterial culture causes induction of extra rounds of chromosome replication. This heat‐induced replication (HIR) initiates at oriC, is transitory, requires RNase H1 and RecA proteins and requires neither RNA polymerase activity nor de novo protein synthesis. The number of origins activated by heat is growth rate and temperature differential dependent. An origin activation higher than 20% increases the DNA:mass ratio around twofold, and this value is kept constant for the subsequent generations of growth at 41°C. We have also shown that HIR is neither related to SDR nor induced by the heat shock response. We suggest that a thermodynamic alteration of oriC structure or of membrane fluidity could explain the observed HIR.

A calcium-binding protein that may be required for the initiation of chromosome replication in Escherichia coli.

Starvation for isoleucine but not for other amino acids in an ilv- strain or the addition of valine in an ilv+ strain inhibits initiation of chromosome and minichromosome replication in stringent (Rel+) Escherichia coli, but it does not inhibit replication in relaxed (relA) mutants (Guzman et al, 1988). From these results, we concluded that, (1) oriC initiation of replication is inhibited by ppGpp, and (2) isoleucine is not needed for the protein synthesis required at initiation. These results led us to find an isoleucine-free protein whose de novo synthesis is the sole protein synthesis requirement for oriC initiation. We also present evidence that this protein may be a calcium-binding protein located at 73 min in the genetic map.

Initiation of Heat-Induced Replication Requires DnaA and the L-13-mer of oriC

An upshift of 10 degrees C or more in the growth temperature of an Escherichia coli culture causes induction of extra rounds of chromosome replication. This stress replication initiates at oriC but has functional requirements different from those of cyclic replication. We named this phenomenon heat-induced replication (HIR). Analysis of HIR in bacterial strains that had complete or partial oriC deletions and were suppressed by F integration showed that no sequence outside oriC is used for HIR. Analysis of a number of oriC mutants showed that deletion of the L-13-mer, which makes oriC inactive for cyclic replication, was the only mutation studied that inactivated HIR. The requirement for this sequence was strictly correlated with Benhams theoretical stress-induced DNA duplex destabilization. oriC mutations at DnaA, FIS, or IHF binding sites showed normal HIR activation, but DnaA was required for HIR. We suggest that strand opening for HIR initiation occurs due to heat-induced destabilization of the L-13-mer, and the stable oligomeric DnaA-single-stranded oriC complex might be required only to load the replicative helicase DnaB.