[Cu,Zn]-Superoxide dismutase mutants of the swine pathogen Actinobacillus pleuropneumoniae are unattenuated in infections of the natural host.

Actinobacillus pleuropneumoniae, the causative agent of porcine pleuropneumonia, contains a periplasmic Cu- and Zn-cofactored superoxide dismutase ([Cu,Zn]-SOD, or SodC) which has the potential, realized in other pathogens, to promote bacterial survival during infection by dismutating host-defense-derived superoxide. Here we describe the construction of a site-specific, [Cu,Zn]-SOD-deficient A. pleuropneumoniae serotype 1 mutant and show that although the mutant is highly sensitive to the microbicidal action of superoxide in vitro, it remains fully virulent in experimental pulmonary infection in pigs.

The virulence plasmid of Salmonella typhimurium contains an autoregulated gene, rlgA, that codes for a resolvase-like DNA binding protein.

The virulence plasmid of Salmonella typhimurium contains a gene, rlgA, that shows strong homology to several reported resolvase-like proteins. This gene maps 5 kb upstream of spv locus, the major virulence determinant on the plasmid. Regulation of rlgA was studied using a lacZ transcriptional reporter fusion. The rlgA gene was found to be repressed at the level of transcription by its own product and to be expressed maximally in the late exponential phase of growth. The transcription start site of the rlgA gene was determined and the RlgA binding site was mapped and found to overlap with the transcription initiation signals. A derivative of the virulence plasmid was constructed with a knockout mutation in rlgA. This mutation did not alter the stability of the virulence plasmid nor did it affect the ability of S. typhimurium to cause systemic disease in mice.

Environmentally constrained mutation and adaptive evolution in Salmonella.

The relationship between environment and mutation is complex [1]. Claims of Lamarkian mutation [2] have proved unfounded [3-5]; it is apparent, however, that the external environment can influence the generation of heritable variation, through either direct effects on DNA sequence [6] or DNA maintenance and copying mechanisms [7-10], or as a consequence of evolutionary processes [11-16]. The spectrum of mutational events subject to environmental influence is unknown [6] and precisely how environmental signals modulate mutation is unclear. Evidence from bacteria suggests that a transient recombination-dependent hypermutational state can be induced by starvation [5]. It is also apparent that changes in the mutability of specific loci can be influenced by alterations in DNA topology [10,17]. Here we describe a remarkable instance of adaptive evolution in Salmonella which is caused by a mutation that occurs in intermediate-strength osmotic environments. We show that the mutation is not 'directed' and describe its genetic basis. We also present compelling evidence in support of the hypothesis that the mutational event is constrained by signals transmitted from the external environment via changes in the activity of DNA gyrase.

A role for the leucine-responsive regulatory protein and integration host factor in the regulation of the Salmonella plasmid virulence (spv ) locus in Salmonella typhimurium.

The Salmonella plasmid virulence (spv ) genes of Salmonella typhimurium are activated at the level of transcription as the bacteria enter stationary phase in vitro or in response to signals received during intracellular growth. Activation requires the LysR-like transcription factor SpvR and the alternative sigma factor RpoS. In this report, we show by biochemical and genetic analyses that two chromosomally encoded DNA-binding proteins contribute to the control of spv expression. These are the integration host factor (IHF), which binds to DNA sequences upstream of the spvR regulatory gene, and the leucine-responsive regulatory protein (Lrp), which binds to sequences upstream of the spvABCD operon. Under all conditions tested, inactivation of IHF expression reduces the level of spvR transcription by twofold. It also alters the response of the spv regulon to loss of DNA gyrase activity, consistent with a role for IHF in organizing DNA structure in the vicinity of the spvR promoter. Lrp represses spvA gene expression by up to fivefold and Lrp-mediated repression is antagonized by leucine. The Lrp binding site upstream of the spvA gene overlaps one of the binding sites for the positive regulator SpvR, suggesting a mechanism by which Lrp repression is exerted. This is a first demonstration of a role for Lrp in controlling genes that are also subject to intracellular regulation. These data show that the spv virulence genes belong simultaneously to several regulons in the cell, raising the possibility that spv expression can be fine-tuned in response to multiple environmental inputs.

In vivo analysis of the interactions of the LysR-like regulator SpvR with the operator sequences of the spvA and spvR virulence genes of Salmonella typhimurium.

The interaction of the Salmonella typhimurium virulence gene regulator, SpvR, with its operator sites upstream of the spvA and spvR genes was analysed in vivo by dimethyl sulphate (DMS) footprinting and site-directed mutagenesis. DMS methylation protection assays showed that, in vivo, SpvR forms direct protein-DNA contacts with nucleotides clustered in two regions (+1 to -27 and -51 to -71) of the spvA regulatory region. These regions were subjected to site-directed mutagenesis and the effects on SpvR binding and gene activation assessed. Mutations that prevented occupancy of the promoter distal site (-51 to -71) in vivo also prevented occupancy of the promoter proximal site (+1 to -27), whereas mutations in the proximal site affected binding only at the proximal site and not the distal site. SpvR binding at the promoter proximal site was an essential prerequisite for transcription activation. These findings demonstrated a hierarchy of SpvR binding in which the promoter distal site is dominant to the proximal. The spvR gene was found to possess an operator site that resembled closely the distal SpvR binding site of the spvA operator. Nonetheless, SpvR interaction with the spvR operator was difficult to detect in vivo. When the nucleotide sequence of the spvR operator was altered at two nucleotides so that it corresponded more precisely to that of the distal site of the spvA operator, strong SpvR-DNA interactions were detected, with nucleotides in the region -31 to -67 being protected from DMS methylation in vivo. However, despite the improved interaction with the transcriptional activator, the altered regulatory region was poorer at promoting spvR gene transcription than the wild type. We describe a two-step model for activation of the spvA promoter and discuss the possibility that a specific cofactor in addition to sigma factor RpoS is required for SpvR action at this promoter in vivo.

Osmotic and growth-phase dependent regulation of the eta gene of Staphylococcus aureus: a role for DNA supercoiling.

Transcriptional fusions were constructed between the promoter for the epidermolytic toxin A (eta) gene of Staphylococcus aureus and the luxAB and xylE reporter gene systems. The expression of the fusion products was found to be dependent upon the accessory gene regulator (agr) locus and was observed to increase significantly during the transition from the exponential to the stationary phase of growth. Furthermore the expression of the eta gene promoter was found to be osmotically regulated, with the expression levels of the eta fusions being inversely related to the osmolyte levels. The ability of environmental factors to influence DNA topology (and thence gene expression) was investigated. High osmolarity (0.7 M NaCl) resulted in an increase in the degree of negative supercoiling of plasmid DNA in the S. aureus strain 8325-4 (Agr+) but not in strain ISP546 (Agr-). Furthermore the eta promoter was strongly induced in S. aureus cultures grown in the presence of sub-inhibitory concentrations of novobiocin, a DNA gyrase inhibitor. However this induction was independent of agr, suggesting that the eta promoter is subject to both agr-dependent (osmolarity, growth phase) and-independent (DNA topology) regulatory processes.