- Volume 154, Issue 6, 2008

A conserved ambient pH signal transduction pathway has been evidenced in both ascomycetous yeasts and filamentous fungi, called the Rim or Pal pathway, respectively. However, closely related PalC orthologues are found only in Yarrowia lipolytica and in filamentous fungi, where the Rim9p/PalI factor has a much longer C-terminal tail than in other yeasts. We show here that, like Aspergillus nidulans palI mutants, a Ylrim9Δ mutant has a less extreme phenotype than other mutants of the pathway, whereas rim9 mutants in Saccharomyces cerevisiae and Candida albicans reportedly exhibit a tight Rim phenotype. Deletion of the long C-terminal tail of YlRim9p/PalI had no phenotypic effect on ambient pH signalling. We also show that the Y. lipolytica PalC orthologue, named YlRim23p, is absolutely required for the alkaline pH response. Its only interactant identified in a genome-wide two-hybrid screen is YlSnf7/Vps32p, confirming the link between the Rim and the Vps pathways. YlRim13p and YlRim20p both interact with YlSnf7/Vps32p but not with YlRim23p. The long C-terminal tail of YlRim9p/PalI interacts neither with YlRim23p nor with YlSnf7/Vps32p. These results show that YlRim23p is a bona fide component of the Rim pathway in Y. lipolytica and that it participates in the complexes linking pH signalling and endocytosis.

Hydrophobins are a group of low-molecular-mass, cysteine-rich proteins that have unusual biophysical properties. They are highly surface-active and can self-assemble at hydrophobic–hydrophilic interfaces, forming surface layers that are able to reverse the hydropathy of surfaces. Here we describe a novel hydrophobin from the edible mushroom Grifola frondosa, which was named HGFI and belongs to class I. The hydrophobin gene was identified during sequencing of random clones from a cDNA library, and the corresponding protein was isolated as a hot SDS-insoluble aggregate from the cell wall. The purified HGFI was found to have 83 amino acids. The protein sequence deduced from the cDNA sequence had 107 amino acids, from which a 24 aa signal sequence had been cleaved off in the mature protein. This signal sequence was 5 aa longer than had been predicted on the basis of signal peptide analysis of the cDNA. Rodlet mosaic structures were imaged using atomic force microscopy (AFM) on mica surfaces after drying-down HGFI solutions. Using Langmuir films we were also able to take images of both the hydrophobic and hydrophilic sides of films formed at the air–water interface. No distinct structure was observed in films compressed once, but in films compressed several times rodlet structures could be seen. Most rodlets were aligned in the same direction, indicating that formation of rodlets may be promoted during compression of the monolayer.

For the microviruses and the leviviruses, bacteriophages with small single-stranded genomes, host lysis is accomplished by expression of a single gene that encodes an inhibitor of cell wall synthesis. In contrast, phages with double-stranded DNA genomes use a more complex system involving, at minimum, an endolysin, which degrades peptidoglycan, and a holin, which permeabilizes the membrane in a temporally programmed manner. To explore the basis of this difference, a chimera was created in which lysis gene E of the microvirus φX174 replaced the entire lysis cassette of phage λ, which includes the holin gene S and the endolysin gene R. The chimeric phage was viable but more variability was observed both in the distribution of plaque sizes and in the burst sizes of single cells, compared to the isogenic S+ parent. Using different alleles of E, it was found the average burst size increased with the duration of the latent period, just as observed with S alleles with different lysis times. Moreover, within a set of missense E alleles, it was found that variability in lysis timing was limited and almost exclusively derived from changes in the level of E accumulation. By contrast, missense mutations in S resulted in a wide variation in lysis times that was not correlated with levels of accumulation. We suggest that the properties of greater phenotypic plasticity and lesser phenotypic variation make the function of holin proteins more genetically malleable, facilitating rapid adaptation towards a lysis time that would be optimal for changed host and environmental conditions. The inferior malleability of single-gene systems like E would restrict their occurrence to phages in which coding capacity is the overriding evolutionary constraint.

Azotobacter vinelandii is a nitrogen-fixing soil bacterium that undergoes differentiation to form cysts resistant to desiccation. Upon encystment, this bacterium becomes non-motile. As in enteric bacteria, motility in A. vinelandii occurs through the use of peritrichous flagella. Pseudomonas aeruginosa, a phylogenetically close relative of A. vinelandii, possesses a single polar flagellum. The FlhDC proteins are the master regulators of flagella and motility in enterobacteria, whereas FleQ is the master regulator in P. aeruginosa, and it is under AlgU (sigmaE) negative control. At present, nothing is known about the organization and expression of flagella genes in A. vinelandii. Here, we identified the flagella gene cluster of this bacterium. Homologues of the master regulatory genes flhDC and fleQ are present in A. vinelandii. Inactivation of flhDC, but not fleQ, impaired flagella biogenesis and motility. We present evidence indicating that a negative effect of the AlgU sigma factor on flhDC expression causes loss of motility in A. vinelandii, and that CydR (a homologue of Fnr) is under AlgU control and has a negative effect on flhDC expression. Taken together, these results suggest the existence of a cascade consisting of AlgU and CydR that negatively controls expression of flhDC; the results also suggest that the block in flagella synthesis under encystment conditions centres on flhDC repression by the AlgU–CydR cascade.

The Escherichia coli guaB promoter (P guaB ) regulates transcription of two genes, guaB and guaA, that are required for the synthesis of guanosine 5′-monophosphate (GMP), a precursor for the synthesis of guanine nucleoside triphosphates. Transcription from P guaB increases as a function of increasing cellular growth rate, and this is referred to as growth rate-dependent control (GRDC). Here we investigated the role of the factor for inversion stimulation (FIS) in the regulation of this promoter. The results showed that there are three binding sites for FIS centred near positions −11, +8 and +29 relative to the guaB transcription start site. Binding of FIS to these sites results in repression of P guaB in vitro but not in vivo. Deletion of the fis gene results in increased P guaB activity in vivo, but GRDC of P guaB is maintained.

The small heat-shock proteins IbpA/B are molecular chaperones that bind denatured proteins and facilitate their subsequent refolding by the ATP-dependent chaperones DnaK, DnaJ, GrpE and ClpB. In this report, we demonstrate that IbpA/B participate in the defence against copper-induced stress under aerobic conditions. In the presence of oxygen, ΔibpA/B cells exhibit increased sensitivity to copper ions and accumulate elevated amounts of oxidized proteins, while under oxygen depletion, the ΔibpA/B mutation has no effect on copper tolerance. This indicates that IbpA/B protect Escherichia coli cells from oxidative damage caused by copper. We show that AdhE, one of the proteins exposed to oxidation, is protected by IbpA/B against copper-mediated inactivation both in vivo and in vitro.

We have shown previously that Caulobacter crescentus grows on maltodextrins which are actively transported across the outer membrane by the MalA protein. Evidence for energy-coupled transport was obtained by deletion of the exbB exbD genes which abolished transport. However, removal of the TonB protein, which together with the ExbB ExbD proteins is predicted to form an energy-coupling device between the cytoplasmic membrane and the outer membrane, left transport unaffected. Here we identify an additional tonB gene encoded by the cc2334a ORF, which when deleted abolished maltose transport. MalA contains a TonB box that reads EEVVIT and is predicted to interact with TonB. Replacement of valine number 15 in the TonB box by proline abolished maltose transport. Maltose was transported across the cytoplasmic membrane by the MalY protein (CC2283). Maltose transport was induced by maltose and repressed by the MalI protein (CC2284). In addition to MalA, MalY and MalI, the mal locus encodes two predicted cytoplasmic α-amylases (CC2285 and CC2286) and a periplasmic glucoamylase (CC2282). The TonB dependence together with the previously described ExbB ExbD dependence demonstrates energy-coupled maltose transport across the outer membrane. MalY is involved in maltose transport across the cytoplasmic membrane by a presumably ion-coupled mechanism.

Nisin is a post-translationally modified antimicrobial peptide produced by Lactococcus lactis which binds to lipid II in the membrane to form pores and inhibit cell-wall synthesis. A nisin-resistant (NisR) strain of L. lactis, which is able to grow at a 75-fold higher nisin concentration than its parent strain, was investigated with respect to changes in the cell wall. Direct binding studies demonstrated that less nisin was able to bind to lipid II in the membranes of L. lactis NisR than in the parent strain. In contrast to vancomycin binding, which showed ring-like binding, nisin was observed to bind in patches close to cell-division sites in both the wild-type and the NisR strains. Comparison of modifications in lipoteichoic acid of the L. lactis strains revealed an increase in d-alanyl esters and galactose as substituents in L. lactis NisR, resulting in a less negatively charged cell wall. Moreover, the cell wall displays significantly increased thickness at the septum. These results indicate that shielding the membrane and thus the lipid II molecule, thereby decreasing abduction of lipid II and subsequent pore-formation, is a major defence mechanism of L. lactis against nisin.

The phylogenetically ancient genomic islands of the abundant PAGI-2/pKLC102 family are prone to horizontal gene transfer amongst proteobacteria, and account for most genomic diversity in Pseudomonas aeruginosa. The mRNA expression levels of the sequenced PAGI-2 and pKLC102 islands were determined in P. aeruginosa clone C strains C and SG17M during exponential and stationary growth in Luria broth or Vogel–Bonner mineral medium. Of the 111 ORFs of PAGI-2, only one gene was significantly expressed at a level of more than 0.0001 % of total RNA. The individual mRNA transcripts of the 103 pKLC102 ORFs, however, were present in the range of 0.001 % to more than 1 % in the bacterial RNA population, and amounted altogether to more than 10 % of cellular RNA. Homologous genes were strongly transcribed from pKLC102, but not at all from PAGI-2 under the tested conditions. Thus PAGI-2, which was stably captured by its host chromosome, was transcriptionally silent, whereas the mRNA transcripts derived from the mobile and episomally replicating pKLC102 were constitutively more abundant in the cell than the mRNA pool transcribed from the core genome.

The fur (ferric uptake regulator) gene of Burkholderia multivorans ATCC 17616 was identified by transposon mutagenesis analysis. The fur deletion mutant of strain ATCC 17616 (i) constitutively produced siderophores, (ii) was more sensitive to reactive oxygen species (ROS) than the wild-type strain, (iii) showed lower superoxide dismutase and catalase activities than the wild-type strain, (iv) was unable to grow on M9 minimal agar plates containing several substrates that can be used as sole carbon sources by the wild-type strain, and (v) was hypersensitive to nitrite and nitric oxide under microaerobic and aerobic conditions, respectively. These results clearly indicate that the Fur protein in strain ATCC 17616 plays pleiotropic roles in iron homeostasis, removal and/or resistance to ROS and nitrosative stress, and energy metabolism. Furthermore, employment of an in vivo Fur titration assay system led to the isolation from the ATCC 17616 genome of 13 Fur-binding DNA regions, and a subsequent electrophoretic mobility-shift assay confirmed the direct binding of Fur protein to all of these DNA regions. Transcriptional analysis of the genes located just downstream of the Fur-binding sites demonstrated that Fur acts as a repressor for these genes. Nine of the 13 regions were presumed to be involved in the acquisition and utilization of iron.

In this study, an MS-based proteomics approach to characterizing the virion structural proteins of the novel marine ‘photosynthetic’ phage S-PM2 is presented. The virus infects ecologically important cyanobacteria of the genus Synechococcus that make a substantial contribution to primary production in the oceans. The S-PM2 genome encodes 236 ORFs, some of which exhibit similarity to known phage virion structural proteins, but the majority (54 %) show no detectable homology to known proteins from other organisms. Using public and in-house bioinformatics tools the proteome of S-PM2 was predicted and a database compatible with MS-based search engines was constructed. S-PM2 virion proteins were resolved by SDS-PAGE, excised, tryptically digested and analysed by LC-ESI-MS/MS. The resulting MS data were searched against the database. A parallel control study was undertaken on the well-characterized coliphage T4 in order to assess the sensitivity and efficiency of this approach. In total, 11 of the 15 S-PM2 proteins, predicted to be virion proteins by bioinformatics approaches, were confirmed as such, together with the identification of a further 12 novel structural proteins. In the case of T4, 24 of the 39 known virion structural proteins were identified, including the major tail-fibre proteins. This approach has wide-ranging applicability and can be applied to any novel organism whose genome encodes ORFs with few detectable homologies in the public databases.

Bacteriocins are a large and functionally diverse family of toxins found in all major lineages of Bacteria. Colicins, those bacteriocins produced by Escherichia coli, serve as a model system for investigations of bacteriocin structure–function relationships, genetic organization, and their ecological role and evolutionary history. Colicin expression is often dependent on host regulatory pathways (such as the SOS system), is usually confined to times of stress, and results in death of the producing cells. This study investigates the role of the SOS system in mediating this unique form of toxin expression. A comparison of all the sequenced enteric bacteriocin promoters reveals that over 75 % are regulated by dual, overlapping SOS boxes, which serve to bind two LexA repressor proteins. Furthermore, a highly conserved poly-A motif is present in both of the binding sites examined, indicating enhanced affinity of the LexA protein for the binding site. The use of gene expression analysis and deletion mutations further demonstrates that these unique LexA cooperative binding regions result in a fine tuning of bacteriocin production, limiting it to times of stress. These results suggest that the evolution of dual SOS boxes elegantly accomplishes the task of increasing the amount of toxin produced by a cell while decreasing the rate of uninduced production, effectively reducing the cost of colicin production. This hypothesis may explain why such a promoter motif is present at such high frequencies in natural populations of bacteriocin-producing enteric bacteria.

Chronic lung infections caused by Pseudomonas aeruginosa are the leading cause of morbidity and mortality for cystic fibrosis (CF) patients. Adaptation of P. aeruginosa to the CF lung results in the loss of acute virulence determinants and appears to activate chronic virulence strategies in this pathogen. In order to identify such strategies, a random transposon mutagenesis was performed and 18 genes that were required for optimal infection of alfalfa seedlings by FRD1, a CF isolate of P. aeruginosa, were recognized. The largest subset of genes (seven of the 18), were associated with central carbon metabolism, including the gene that encodes isocitrate lyase (ICL), aceA. Because FRD1 is avirulent in animal infection models, we constructed an ICL mutant in P. aeruginosa strain PAO1 in order to assess the requirement of ICL in mammalian infection. The PAO1 ICL mutant was less virulent in the rat lung infection model, indicating that ICL is required for the pathogenesis of P. aeruginosa in mammals. Furthermore, FRD1 showed increased ICL activity and expression of an aceA : : lacZ fusion compared to PAO1. We suggest that upregulation of ICL occurred during adaptation of FRD1 to the CF lung and that some of the novel virulence mechanisms employed by FRD1 to infect alfalfa seedlings may be the same mechanisms P. aeruginosa relies upon to persist within human niches.

Several fungal pathogens are able to enter and persist within eukaryotic cells as part of their infectious life cycle. Metarhizium anisopliae is a saprophytic entomopathogenic fungus virulent towards numerous tick species, including those within the genera Ixodes and Amblyomma. Infection of the target organism by this fungus proceeds via several steps, including adhesion and penetration of the host cuticle, proliferation within tissues and the haemolymph, and eventual eruption through the host cadaver. To determine whether M. anisopliae could enter and persist within tick cells, we examined the uptake of wild-type and green fluorescent protein (GFP)-expressing fungal strains into two different tick cells lines, IDE12 and AAE2, derived from Ixodes scapularis and Amblyomma americanum, respectively. Uptake by tick cells was monitored by confocal fluorescent microscopy, as well as by scanning and transmission electron microscopy. M. anisopliae-specific antibodies were used to discriminate between extracellular and internalized fungal cells and to quantify the rate of uptake. Both cell lines internalized fungal conidia, and quantitative studies using the AAE2 cell line indicated that almost 70 % of the AAE2 cells contained internalized conidia after 6 h incubation. Internalization of conidia by AAE2 cells was time and temperature dependent and could be inhibited by 80 % with 1 mM cytochalasin D. Internalized conidia remained viable within the AAE2 cells, where they were able to germinate and grow, eventually erupting from the host cells. These data provide evidence that M. anisopliae conidia can be internalized, survive and grow within phagocytic cells in vitro and indicate that phagocytosis may serve as an alternative invasion route facilitating fungal nutrient acquisition, immune system evasion and dissemination throughout the host.

Flavobacterium psychrophilum is an important infectious Gram-negative bacterium causing cold-water disease (CWD) and rainbow trout fry syndrome. Outer-membrane proteins (OMPs) are key molecules with regard to the interface between the cell and its environment. Therefore, we sought to define the outer-membrane (OM) subproteome of F. psychrophilum in order to gain insight into the biology and pathogenesis of this bacterium and to identify the dominant antigens targeted by the rainbow trout (Oncorhynchus mykiss) immune system during infection. First, OMs were prepared from a cell-envelope suspension by differential Sarkosyl (sodium lauryl sarcosinate) solubility. We then isolated the OMPs and identified 36 proteins from 34 spots resolved by two-dimensional electrophoresis and LC-MS/MS. An immunoproteomic approach using antibodies from CWD-convalescent rainbow trout was then used to identify 25 immunoreactive F. psychrophilum antigens that may be relevant in pathogenesis and diagnosis. These included the previously characterized surface-exposed OMPs OmpA, OmpH/P18 and FspA, as well as newly described antigenic proteins. This study provides a number of novel candidate proteins for developing vaccine(s) against flavobacteriosis infection in aquaculture.