- Volume 150, Issue 1, 2004

In several Gram-positive and Gram-negative bacteria glutamate decarboxylases play an important role in the maintenance of cellular homeostasis in acid environments. Here, new insight is brought to the regulation of the acid response in Escherichia coli. Overexpression of yhiE, similarly to overexpression of gadX, a known regulator of glutamate decarboxylase expression, leads to increased resistance of E. coli strains under high acid conditions, suggesting that YhiE is a regulator of gene expression in the acid response. Target genes of both YhiE (renamed GadE) and GadX were identified by a transcriptomic approach. In vitro experiments with GadE purified protein provided evidence that this regulator binds to the promoter region of these target genes. Several of them are clustered together on the chromosome and this chromosomal organization is conserved in many E. coli strains. Detailed structural (in silico) analysis of this chromosomal region suggests that the promoters of the corresponding genes are preferentially denatured. These results, along with the G+C signature of the chromosomal region, support the existence of a fitness island for acid adaptation on the E. coli chromosome.

Staphylococcus aureus has several extracellular proteases with proposed roles in virulence. SspA (serine protease), SspB (cysteine protease) and Aur (metalloprotease) have been characterized previously and SspA and SspB were found to be cotranscribed. The coding region for the cysteine protease ScpA has been identified and characterized. It is in a probable bi-cistronic operon with scpA located immediately upstream of a coding region for a 108 aa protein that is a specific inhibitor of ScpA. Using primer extension analysis promoters have been mapped and it was found that σ A is the only sigma factor involved in the transcription of scpA, sspABC and aur. The transcription of all the genes occurs maximally at post-exponential phase, being positively regulated by agr (accessory gene regulator) and negatively regulated by sarA (staphylococcal accessory regulator). Furthermore σ B represses transcription from the aur and scp operons similarly to the previously shown effect on ssp [ Horsburgh, M., Aish, J., White, I., Shaw, L., Lithgow, J. & Foster, S. (2002). J Bacteriol 184, 5457–5467 ]. Using mutations in each protease gene the proteolytic cascade of activation has been analysed. Aur, SspA, SspB and ScpA are all produced as zymogens, activated by proteolytic cleavage. Although the metalloprotease, Aur, does catalyse activation of the SspA zymogen, it is not the sole agent capable of conducting this process. Site-directed mutagenesis revealed that Aur is not capable of undergoing auto-proteolysis to achieve activation. The cysteine protease, ScpA, appears to reside outside this cascade of activation, as mature ScpA was observed in the aur, sspA and sspB mutant strains. Using a mouse abscess model, it has been shown that insertional inactivation of sspA or sspB results in significant attenuation of virulence, whilst mutations in aur or scpA do not. It is likely the attenuation observed in the sspA strain is due to polarity on the sspB gene.

Submission of wild-type Escherichia coli to heat shock causes an aggregation of cellular proteins. The aggregates (S fraction) are separable from membrane fractions by ultracentrifugation in a sucrose density gradient. In contrast, no protein aggregation was detectable in an E. coli grpE280 mutant either by this technique or by electron microscopy. In search of an explanation for this observation at a molecular level, two kinds of marker proteins were used: Fda (fructose-1,6-biphosphate aldolase), the previously identified S fraction component, and IbpA/B, small heat-shock proteins abundantly associated with the S fraction proteins. Both types of marker proteins, normally never found in the outer-membrane (OM) fraction of WT cells, were present in the OM fraction from grpE cells after heat shock. This pointed to the presence of aggregates smaller than those in WT cells that cosedimented with the OM fraction. The OM fraction was enlarged in grpE cells. Although not proven directly, the presence of still smaller aggregates, not exceeding the solubility level and containing inactive Fda, was noted in the soluble CP fraction containing the cytoplasmic and periplasmic proteins. Therefore, aggregation occurred in both strains, but in a different way. The autoregulation of the heat-shock response causes a greater increase of DnaK/DnaJ and IbpAB levels in grpE cells than in WT after temperature elevation. This may explain the prevalence of the small-sized aggregates in the grpE cells. Estimation of total Fda protein before and after heat shock did not show any loss. This indicated that renaturation rather than proteolysis underlies the final disappearance of the aggregates. Though surprising at first, this is not contradictory with the participation of heat-shock proteases in removal of protein components of the S fraction as shown before, since proteins that are irreversibly denatured are probably substrates for the proteases.

A gene that encodes a putative SecE protein, which is a component of the Sec protein-translocation system, was cloned from the onion yellows phytoplasma (OY). The identification of this gene and the previously reported genes encoding SecA and SecY provides evidence that the Sec system exists in phytoplasma. In addition, a gene encoding an antigenic membrane protein (Amp) (a type of immunodominant membrane protein) of OY was cloned and sequenced. The OY amp gene consisted of 702 nt encoding a protein of 233 aa which was highly similar to Amp of aster yellows phytoplasma (AY). Part of OY Amp was overexpressed in Escherichia coli, purified, and used to raise an anti-Amp polyclonal antibody. The anti-Amp antibody reacted specifically with an OY-infected plant extract in Western blot analysis and was therefore useful for the detection of OY as well as Amp. Amp has a conserved protein motif that is known to be exported by the Sec system of E. coli. A partial OY Amp protein expressed in E. coli was localized in the periplasm as a shorter, putatively processed form of the protein. It had probably been exported from the cytoplasm to the periplasm through the Sec system. Moreover, OY Amp protein expressed in OY and detected in OY-infected plants was apparently also processed. Because phytoplasmas cannot be cultured or transformed, little information is available regarding their protein secretion systems. This study suggests that the Sec system operates in this phytoplasma to export OY Amp.