- Volume 153, Issue 12, 2007

Legionella pneumophila is a Gram-negative facultative intracellular pathogen, which multiplies in protozoa in its natural environment and can cause Legionnaires' disease in man, following infection of alveolar macrophages. In each of the different stages of infection of host cells, virulence proteins need to be delivered to their specific place of action and therefore must cross two barriers: the inner and the outer membrane. To date, several specialized secretion machineries for transport of proteins across the inner and outer membrane have been identified in L. pneumophila. Most of these secretion pathways have been shown to affect the virulence of this pathogen. An overview will be given of all the secretion pathways and the proteins transported by these secretion systems identified so far, with special attention paid to those that play a role in the pathogenicity of L. pneumophila.

dsrP, a gene that encodes a cell-associated dextransucrase produced by Leuconostoc mesenteroides IBT-PQ, was isolated, sequenced and expressed in Escherichia coli. From sequence analysis, seven repeat units in the N-terminal region were found, as well as five cell wall binding repeats in the C-terminal region. A model of the C-terminal domain of dextransucrase was built based on the solenoid structure of the cell wall binding domain already described in LytA. By experiments involving direct interactions of the enzyme with L. mesenteroides cells, as well as among the cells and the single C-terminal domain expressed in E. coli, evidence was obtained concerning the anchoring function of this region in cell-associated dextransucrase, a function which may be independent of its capacity to bind dextran.

Currently known fungal α-amylases are well-characterized extracellular enzymes that are classified into glycoside hydrolase subfamily GH13_1. This study describes the identification, and phylogenetic and biochemical analysis of novel intracellular fungal α-amylases. The phylogenetic analysis shows that they cluster in the recently identified subfamily GH13_5 and display very low similarity to fungal α-amylases of family GH13_1. Homologues of these intracellular enzymes are present in the genome sequences of all filamentous fungi studied, including ascomycetes and basidiomycetes. One of the enzymes belonging to this new group, Amy1p from Histoplasma capsulatum, has recently been functionally linked to the formation of cell wall α-glucan. To study the biochemical characteristics of this novel cluster of α-amylases, we overexpressed and purified a homologue from Aspergillus niger, AmyD, and studied its activity product profile with starch and related substrates. AmyD has a relatively low hydrolysing activity on starch (2.2 U mg−1), producing mainly maltotriose. A possible function of these enzymes in relation to cell wall α-glucan synthesis is discussed.

In Saccharomyces cerevisiae, the serine-threonine protein kinase activity of Dbf2p is required for tolerance to the weak organic acid sorbic acid. Here we show that Dbf2p is required for normal phosphorylation of the vacuolar H+-ATPase (V-ATPase) A and B subunits Vma1p and Vma2p. Loss of V-ATPase activity due to bafilomycin treatment or deletion of either VMA1 or VMA2 resulted in sorbic acid hypersensitivity and impaired vacuolar acidification, phenotypes also observed in both a kinase-inactive dbf2 mutant and cells completely lacking DBF2 (dbf2Δ). Crucially, VMA2 is a multicopy suppressor of both the sorbic acid-sensitive phenotype and the impaired vacuolar-acidification defect of dbf2Δ cells, confirming a functional interaction between Dbf2p and Vma2p. The yeast V-ATPase is therefore involved in mediating sorbic acid stress tolerance, and we have shown a novel and unexpected role for the cell cycle-regulated protein kinase Dbf2p in promoting V-ATPase function.

Bacterial chromosomes (though not Escherichia coli and some other γ-proteobacterial chromosomes) contain parS sequences and parAB genes encoding partitioning proteins, i.e. ParA (ATPase) and ParB (DNA-binding proteins) that are components of the segregation machinery. Here, mycobacterial parABS elements were characterized for the first time. parAB genes are not essential in Mycobacterium smegmatis; however, elimination or overexpression of ParB protein causes growth inhibition. Deletion of parB also leads to a rather severe chromosome segregation defect: up to 10 % of the cells were anucleate. Mycobacterial ParB protein uses three oriC-proximal parS sequences as targets to organize the origin region into a compact nucleoprotein complex. Formation of such a complex involves ParB–ParB interactions and is assisted by ParA protein.

Sortase A (SrtA) is required for cell-wall anchoring of LPXTG-containing Gram-positive surface proteins. It was hypothesized, therefore, that disruption of the srtA gene would alter surface anchoring and functions of target LPXTG motif-bearing SspA and SspB proteins of Streptococcus gordonii. Mutant strains in srtA (V288srtA −, DL1srtA− ) were constructed in S. gordonii V288 (wtV288) and DL1 (wtDL1). When compared to wtV288, the V288srtA− mutant showed decreased biofilm formation on polystyrene, and reduced binding to immobilized purified salivary agglutinin (BIAcore analysis). The wtV288 and V288srtA− strains were similar in ultrastructure, but immunogold-labelled SspA/SspB surface expression was reduced on the V288srtA− mutant. DL1srtA− was also complemented to obtain DL1srtA+ . From the wild-type strains (wtV288, wtDL1), srtA− mutants (V288srtA− , DL1srtA− ), and the complemented mutant (DL1srtA+ ), cytoplasmic, cell-wall and released extracellular protein fractions were isolated. Each fraction was analysed by SDS-PAGE and immunoblotting with anti-P1. Spent medium from srtA− mutant cells contained over-represented proteins, including SspA/SspB (P1 antigen). Mutants showed less P1 on the cell surface than wild-types, as estimated using whole-cell ELISA, and no P1 appeared in the cytoplasmic fractions. Expression of several adhesin genes (sspA/B, cshA/B, fbpA) was generally upregulated in the mutants (V288srtA− , DL1srtA− ), but restored to wild-type levels in DL1srtA+ . These data therefore imply that in addition to its role in processing LPXTG-containing adhesins, sortase A has the novel function of contributing to transcriptional regulation of adhesin gene expression.

The key virulence factor in Shiga-toxigenic Escherichia coli is the expression of Shiga toxin (Stx), which is conferred by Stx-encoding temperate lambdoid phages (Stx-phages). It had been assumed that Stx-phages would behave similarly to λ phage. However, contrary to the λ superinfection immunity model, it has been demonstrated that double lysogens can be produced with the Stx-phage Φ24B. Here, the Φ24B integrase gene is identified, and the preferred site of integration defined. Although an E. coli int gene was identified close to the Φ24B integration site, it was shown not to be involved in the phage integration event. An additional six potential integration sites were identified in the E. coli genome, and three of these were confirmed experimentally. Two of the other potential sites lie within genes predicted to be essential to E. coli and are therefore unlikely to support phage integration. A Φ24B gene, possessing similarity to the well-characterized P22 ant gene, was identified. RT-PCR was used to demonstrate that ant is transcribed in a Φ24B E. coli lysogen, and expression of an anti-repressor is the likely explanation for the absence of immunity to superinfection. Demonstration of the ability of Φ24B to form multiple lysogens has two potentially serious impacts. First, multiple integrated prophages will drive the evolution of bacterial pathogens as novel Stx-phages emerge following intracellular mutation/recombination events. Second, multiple copies of the stx gene may lead to an increase in toxin production and consequently increased virulence.

Spiramycin, a 16-membered macrolide antibiotic used in human medicine, is produced by Streptomyces ambofaciens; it comprises a polyketide lactone, platenolide, to which three deoxyhexose sugars are attached. In order to characterize the gene cluster governing the biosynthesis of spiramycin, several overlapping cosmids were isolated from an S. ambofaciens gene library, by hybridization with various probes (spiramycin resistance or biosynthetic genes, tylosin biosynthetic genes), and the sequences of their inserts were determined. Sequence analysis showed that the spiramycin biosynthetic gene cluster spanned a region of over 85 kb of contiguous DNA. In addition to the five previously described genes that encode the type I polyketide synthase involved in platenolide biosynthesis, 45 other genes have been identified. It was possible to propose a function for most of the inferred proteins in spiramycin biosynthesis, in its regulation, in resistance to the produced antibiotic or in the provision of extender units for the polyketide synthase. Two of these genes, predicted to be involved in deoxysugar biosynthesis, were inactivated by gene replacement, and the resulting mutants were unable to produce spiramycin, thus confirming their involvement in spiramycin biosynthesis. This work reveals the main features of spiramycin biosynthesis and constitutes a first step towards a detailed molecular analysis of the production of this medically important antibiotic.

The adaptation of Bacillus subtilis to elevated levels of copper ions requires the copper-inducible copZA operon encoding a copper chaperone and efflux ATPase. Here we identify CsoR (formerly YvgZ) as the copper-sensing repressor that regulates the copZA operon. CsoR binds with high affinity to an operator site overlapping the copZA promoter and its binding is specifically inhibited by copper salts. As previously described, the YhdQ (CueR) protein also binds to the copZA regulatory region, but genetic experiments indicate that this protein is not responsible for the copper-dependent regulation of this operon.

Genes vmeA and vmeB, encoding a multidrug efflux transporter in the halophilic bacterium Vibrio parahaemolyticus, have been cloned using a drug-hypersusceptible Escherichia coli strain as the host. Cells of E. coli KAM33 (ΔacrAB ΔydhE) carrying the vmeAB region from V. parahaemolyticus conferred much higher MICs for a variety of antimicrobial agents than did control cells. Cells possessing VmeAB under energized conditions maintained very low intracellular concentrations of ethidium. This was as expected for an energy-dependent efflux system, and supports the notion – based on sequence homology – that VmeAB belongs to the resistance nodulation cell division (RND) family of multidrug efflux transporters. It is likely that VmeAB forms functional complexes with the outer-membrane protein TolC in E. coli, because introduction of vmeAB into cells of E. coli KAM43, which lacks the tolC gene, failed to elevate the MICs for any of the antimicrobial agents tested. Therefore, a V. parahaemolyticus homologue of tolC was also cloned, designated vpoC, and was introduced together with vmeAB into cells of E. coli KAM43. The MICs of all agents tested were raised and were comparable to the values observed in E. coli KAM33 harbouring a plasmid carrying vmeAB. Finally, a vmeAB-deficient mutant of V. parahaemolyticus was constructed (designated TM3). TM3 showed slightly higher susceptibility than the parental V. parahaemolyticus to some antimicrobial agents. Survival rate of the TM3 when exposed to deoxycholate decreased compared with that of the parent.

FimB and FimE are site-specific recombinases, part of the λ integrase family, and invert a 314 bp DNA switch that controls the expression of type 1 fimbriae in Escherichia coli. FimB and FimE differ in their activity towards the fim switch, with FimB catalysing inversion in both directions in comparison to the higher-frequency but unidirectional on-to-off recombination catalysed by FimE. Previous work has demonstrated that FimB, but not FimE, recombination is completely inhibited in vitro and in vivo by a regulator, PapB, expressed from a distinct fimbrial locus. The aim of this work was to investigate differences between FimB and FimE activity by exploiting the differential inhibition demonstrated by PapB. The research focused on genetic changes to the fim switch that alter recombinase binding and its structural context. FimB and FimE still recombined a switch in which the majority of fimS DNA was replaced with a larger region of non-fim DNA. This demonstrated a minimal requirement for FimB and FimE recombination of the Fim binding sites and associated inverted repeats. With the original leucine-responsive regulatory protein (Lrp) and integration host factor (IHF)-dependent structure removed, PapB was now able to inhibit both recombinases. The relative affinities of FimB and FimE were determined for the four ‘half sites’. This analysis, along with the effect of extensive swaps and duplications of the half sites on recombination frequency, demonstrated that FimB recruitment and therefore subsequent activity was dependent on a single half site and its context, whereas FimE recombination was less stringent, being able to interact initially with two half sites with equally high affinity. While increasing FimB recombination frequencies failed to overcome PapB repression, mutations made in recombinase binding sites resulted in inhibition of FimE recombination by PapB. Overall, the data support a model in which the recombinases differ in loading order and co-operative interactions. PapB exploits this difference and FimE becomes susceptible when its normal loading is restricted or changed.

Giardia lamblia is a common intestinal-dwelling protozoan and causes diarrhoea in humans and animals worldwide. For several years, a small number of drugs such as the 5-nitroimidazole metronidazole (MET) or the thiazolide nitazoxanide (NTZ) have been used for chemotherapy against giardiasis. However, various pre-clinical and clinical investigations revealed that antigiardial chemotherapy may be complicated by emergence of giardial resistance to these drugs. The present study addressed the question if isoflavones with antigiardial activity, such as daidzein (DAI) or formononetin (FOR), may serve as alternative compounds for treatment of giardiasis. For this purpose, the potential of G. lamblia clone WB C6 to form resistance to FOR and related isoflavones was tested in vitro. In the line of these experiments, a clone (C3) resistant to isoflavones, but sensitive to MET and NTZ, was generated. Affinity chromatography on DAI-agarose using cell-free extracts of G. lamblia trophozoites resulted in the isolation of a polypeptide of approximately 40 kDa, which was identified by mass spectrometry as a nucleoside hydrolase (NH) homologue (EAA37551.1). In a nucleoside hydrolase assay, recombinant NH hydrolysed all nucleosides with a preference for purine nucleosides and was inhibited by isoflavones. Using quantitative RT-PCR, the expression of genes that are potentially involved in resistance formation was analysed, namely NH and genes encoding variant surface proteins (VSPs, TSA417). The transcript level of the potential target NH was found to be significantly reduced in C3. Moreover, drastic changes were observed in VSP gene expression. This may indicate that resistance formation in Giardia against isoflavones is linked to, and possibly mediated by, altered gene expression. Taken together, our results suggest FOR or related isoflavones as an alternative antigiardial agent to overcome potential problems of resistance to drugs like MET or NTZ. However, the capacity of Giardia to develop resistance to isoflavones can potentially interfere with this alternative treatment of the disease.

Structural analysis of mycolic acids from Mycobacterium simiae (including some ‘habana’ strains) was carried out using 1H-NMR and MS. Results indicated that this species presents a general pattern of α-, α′- and keto-mycolates. α-Mycolates were composed of a complex mixture of 82 to 89 carbon atoms (C82–C89), with the predominant molecular species containing two di-substituted cyclopropane rings. Among keto-mycolates (C84–C89), those containing one trans di-substituted cyclopropane ring were the most abundant. The α′-mycolates were monounsaturated (C64, C66). According to MS and 1H-NMR data, the strains studied differed in fine structural details of α-mycolates and keto-mycolates. Notably, strain ‘habana’ TMC 5135 (belonging to the ‘habana’ group, and considered as highly immunogenic in tuberculosis and leprosy) presented a particular composition of α-mycolates, with a major component (C87) containing one cis plus one trans di-substituted cyclopropane ring, unlike the type strain of M. simiae and other strains of the ‘habana’ group (IPK-220 and IPK-337R), in which the major component (C84) contained two cis di-substituted cyclopropane rings. In spite of this finding, the ‘habana’ strains were closely related to each other and mainly differed from the type strain of M. simiae in some details of the fine structure of keto-mycolates. The present work indicated that within an identical general pattern of mycolic acids, there is a complex composition in M. simiae and structural variation among different strains, as reported for pathogenic species of the genus. Noteworthy was the particular composition of α-mycolates in strain ‘habana’ TMC 5135.

Mycolic acids are vital components of the Mycobacterium tuberculosis cell wall and are essential for survival. While most components of the fatty acid synthase-II (FAS-II) enzymic machinery that synthesizes these long chain α-alkyl, β-hydroxy fatty acids have been identified, the gene encoding the β-hydroxyacyl-acyl carrier protein (ACP) dehydratase activity has remained elusive. Recent bioinformatics-based studies and drug inhibition experiments have identified the M. tuberculosis gene Rv0636 as a promising candidate for this role. Using a recently described, specialized transduction-based genetic tool we now demonstrate that MSMEG1341, the Mycobacterium smegmatis homologue of Rv0636, is an essential gene; null mutants of the gene could only be generated in a merodiploid strain which contained a second integrated acetamide-inducible copy of MSMEG1341. Growth of the conditional mutant in the absence of acetamide resulted in loss of mycolic acid biosynthesis and eventually loss of viability due to cell lysis. Null MSMEG1341 mutants could also be generated in a M. smegmatis strain containing an integrated copy of Rv0636, indicating that Rv0636 was the functional counterpart of MSMEG1341 in M. tuberculosis. Our results demonstrate that MSMEG1341 is an essential gene involved in mycolic acid biosynthesis and encodes the FAS-II β-hydroxyacyl-ACP dehydratase.

Salmonella enterica is one of the most extensively studied bacterial species in terms of physiology, genetics, cell culture and development. As a very diverse group, the serovars of S. enterica display a spectrum of host specificities ranging from a broad host range to strictly host-adapted variants. This study utilized a classic proteomic approach combining 2D gel electrophoresis and mass spectrometry for the comparative analysis of the proteomes of serovars Typhimurium, Enteritidis, Choleraesuis, Pullorum and Dublin. The comparative analysis revealed species-specific protein factors with no significant change in expression amongst all isolates, as well as proteins with fluctuating expression levels between serovars and strains. Examples include an isoform of SodA specific for serovar Typhimurium, the third isoform of the lysine arginine ornithine (LAO)-binding amino acid transporter specific for serovar Pullorum, and the enzyme GabD found to be unique to serovar Choleraesuis. Overall the study demonstrated the importance of using multiple isolates when characterizing the expression patterns of bacteria in order to account for the intrinsic diversity of a bacterial population and revealed several factors with potential roles in host adaptation and pathogenicity of the serovars of S. enterica.