Bet-hedging in stochastically switching environments.

We investigate the evolution of bet-hedging in a population that experiences a stochastically switching environment by means of adaptive dynamics. The aim is to extend known results to the situation at hand, and to deepen the understanding of the range of validity of these results. We find three different types of evolutionarily stable strategies (ESSs) depending on the frequency at which the environment changes: for a rapid change, a monomorphic phenotype adapted to the mean environment; for an intermediate range, a bimorphic bet-hedging phenotype; for slowly changing environments, a monomorphic phenotype adapted to the current environment. While the last result is only obtained by means of heuristic arguments and simulations, the first two results are based on the analysis of Lyapunov exponents for stochastically switching systems.

Characterization of a bordetella pertussis diaminopimelate (DAP) biosynthesis locus identifies dapC, a novel gene coding for an N-succinyl-L,L-DAP aminotransferase.

The functional complementation of two Escherichia coli strains defective in the succinylase pathway of meso-diaminopimelate (meso-DAP) biosynthesis with a Bordetella pertussis gene library resulted in the isolation of a putative dap operon containing three open reading frames (ORFs). In line with the successful complementation of the E. coli dapD and dapE mutants, the deduced amino acid sequences of two ORFs revealed significant sequence similarities with the DapD and DapE proteins of E. coli and many other bacteria which exhibit tetrahydrodipicolinate succinylase and N-succinyl-L,L-DAP desuccinylase activity, respectively. The first ORF within the operon showed significant sequence similarities with transaminases and contains the characteristic pyridoxal-5'-phosphate binding motif. Enzymatic studies revealed that this ORF encodes a protein with N-succinyl-L,L-DAP aminotransferase activity converting N-succinyl-2-amino-6-ketopimelate, the product of the succinylase DapD, to N-succinyl-L,L-DAP, the substrate of the desuccinylase DapE. Therefore, this gene appears to encode the DapC protein of B. pertussis. Apart from the pyridoxal-5'-phosphate binding motif, the DapC protein does not show further amino acid sequence similarities with the only other known enzyme with N-succinyl-L,L-DAP aminotransferase activity, ArgD of E. coli.

Molecular mechanisms of bacterial pathogenicity.

Cautious optimism has arisen over recent decades with respect to the long struggle against bacteria, viruses, and parasites. This has been offset, however, by a fatal complacency stemming from previous successes such as the development of antimicrobial drugs, the eradication of smallpox, and global immunization programs. Infectious diseases nevertheless remain the world's leading cause of death, killing at least 17 million persons annually [61]. Diarrheal diseases caused by Vibrio cholerae or Shigella dysenteriae kill about 3 million persons every year, most of them young children: Another 4 million die of tuberculosis or tetanus. Outbreaks of diphtheria in Eastern Europe threatens the population with a disease that had previously seemed to be overcome. Efforts to control infectious diseases more comprehensively are undermined not only by socioeconomic conditions but also by the nature of the pathogenic organisms itself; some isolates of Staphylococcus aureus and Enterobacter have become so resistant to drugs by horizontal gene transfer that they are almost untreatable. In addition, the mechanism of genetic variability helps pathogens to evade the human immune system, thus compromising the development of powerful vaccines. Therefore detailed knowledge of the molecular mechanisms of microbial pathogenicity is absolutely necessary to develop new strategies against infectious diseases and thus to lower their impact on human health and social development.

A new gene locus of Bordetella pertussis defines a novel family of prokaryotic transcriptional accessory proteins.

Recently, a novel type of regulatory mutation causing differential effects on the expression of virulence genes due to a slight overexpression of the RNA polymerase alpha subunit (RpoA) was found in Bordetella pertussis (N. H. Carbonetti, T. M. Fuchs, A. A. Patamawenu, T. J. Irish, H. Deppisch, and R. Gross, J. Bacteriol. 176:7267-7273, 1994). To gather information on the molecular events behind this phenomenon, we isolated suppressor mutants of the RpoA-overexpressing strains after random mutagenesis. Genetic characterization of these suppressor strains revealed the existence of at least three distinct groups of dominant alleles. Mutations occurred either in the rpoA locus itself, in the bvg locus, or in unknown gene loci. One mutant of the latter group was further characterized. By the introduction of a cosmid library containing genomic B. pertussis DNA into this suppressor strain, we isolated a cosmid which suppressed the phenotype of the suppressor strain, thus restoring the negative effect on transcription of the ptx and cya toxin genes. Mutagenesis of the cosmid with Tn5 led to the identification of the gene locus responsible for this phenomenon. Its DNA sequence revealed the presence of an open reading frame (ORF) consisting of 2,373 bp coding for a hypothetical 86-kDa protein with extensive sequence similarities to ORFs with not yet identified functions of Escherichia coli, Haemophilus influenzae, and Neisseria meningitidis. The new gene, termed tex, for toxin expression, seems to be an essential factor for B. pertussis, as it cannot be deleted from the bacterial chromosome. All members of this new protein family show significant sequence similarities with the mannitol repressor protein MtlR and with the presumptive RNA-binding domains of the Pnp and ribosomal S1 proteins of E. coli in their N- and C-terminal parts, respectively. These sequence similarities and the fact that the tex gene was isolated by virtue of its effects on gene expression in B. pertussis indicate that the members of this new protein family may play an important role in the transcription machinery of prokaryotic organisms.

Signal transduction and virulence regulation in Bordetella pertussis.

Bordetella pertussis, the causative agent of whooping cough, coordinately regulates the expression of its virulence factors in response to certain environmental stimuli. This coordinate regulation is accomplished by the bvg locus encoding the BvgS and the BvgA proteins, which are members of the two-component family of bacterial signal transducing proteins. The sensor protein BvgS shows an "unorthodox" domain structure, combining the characteristic communication modules both of the two component sensors and response regulators, each of which is indispensable for BvgS function. Although under global control of the BvgAS system, two subsets of virulence factors exemplified by the adhesin FHA and the toxins PTX and CYA exhibit, respectively, a differential mode of expression. This is reflected in a differential kinetics of transcriptional activation in vivo, and the different ability of the various virulence promoters to be expressed in the heterologous organism Escherichia coli. Evidence is accumulating that this differential regulation may be due to different affinities of the virulence promoters for the phosphorylated form of BvgA.

In vivo characterization of the unorthodox BvgS two-component sensor protein of Bordetella pertussis.

Two-component sensor proteins are typically composed of an amino-acid sensory and a carboxy-terminal transmitter domain containing a kinase activity which catalyses the autophosphorylation of a histidine residue. In a second step, the phosphate is transferred to aspartic acid residues located in the receiver domain of the second component, the response regulator. A few sensor proteins such as the BvgS protein of Bordetella pertussis have a more complex structure. BvgS possesses additional C-terminal domains, including receiver and output modules usually found only in the response regulators. The function of these BvgS domains was investigated by mutation and complementation analysis in vivo. BvgS derivatives were constructed lacking the C-terminal domains or containing mutations in conserved amino acids. All mutations caused the inactivation of BvgS as measured by the expression of virulence factors at the transcriptional and translational level after integration of the mutated alleles in the B. pertussis chromosome. However, some of these mutants could be complemented to the wild-type phenotype by the separate expression of various C-terminal BvgS domains in trans indicating a direct interaction of the truncated and complete BvgS proteins. Therefore, the dimerization capacity of the cytoplasmic BvgS domains was analysed using a lambda repressor based dimerization probe system. These results indicated that BvgS has two dimerization regions, one in the transmitter and a second in the C-terminal receiver/output domains. Furthermore, several BvgS hybrid proteins were constructed which contained substitutions of the BvgS receiver and output domains with similar domains of two-component response regulators and of the sensor protein EvgS. It was found that the receiver domain does not carry BvgS-specific functions and can be exchanged by a heterologous receiver domain. However, the BvgS output domain could not be substituted with output domains of the related proteins without inactivation of BvgS.

Effect of mutations causing overexpression of RNA polymerase alpha subunit on regulation of virulence factors in Bordetella pertussis.

In Bordetella pertussis, expression of virulence factors is controlled by the Bvg proteins, which comprise a sensor-regulator two-component signal transduction system. Previously, we described a mutant strain of B. pertussis that had reduced transcription of pertussis toxin and adenylate cyclase toxin genes, while other virulence factors were relatively unaffected. We obtained a B. pertussis clone that repaired the defect in both this strain and an independent mutant strain with a similar phenotype when introduced onto the chromosome by allelic exchange. Further analysis revealed that the mutations were just upstream of the translational start site of the rpoA gene encoding the alpha subunit of RNA polymerase. We confirmed that these mutations were responsible for the mutant phenotype by site-directed mutagenesis. Our hypothesis that these mutations cause an overexpression of rpoA was confirmed by Western immunoblotting and translational fusion analysis. Corroboration of this effect was obtained by overexpressing rpoA on a plasmid in wild-type B. pertussis, which caused the same phenotype as the mutants showed. Conclusions in regard to the identity of the transcription activator of the toxin genes are discussed.