- Volume 158, Issue 5, 2012

Salmonella enterica serovars cause severe disease in humans, such as gastroenteritis and typhoid fever. The bacteria are able to invade and replicate within host cells, including epithelial cells and macrophages. Pathogenesis of Salmonella is facilitated by a type III secretion system (T3SS) encoded by genes of Salmonella pathogenicity island 2 (SPI-2). Intracellular replication occurs in a specialized membrane compartment, the Salmonella-containing vacuole (SCV), and depends on translocation of approximately 30 effector proteins via the SPI-2 T3SS into the host endomembrane system and cytoplasm. In this review we discuss the many different functions of these effectors, which range from maintaining the integrity of the SCV and its juxtanuclear location, to interference with the host cytoskeleton and immune signalling.

GlnR is the global transcriptional regulator of nitrogen assimilation in Streptomyces coelicolor. Under nitrogen starvation, GlnR controls the transcription of at least nine genes associated with nitrogen metabolism. In this study, we identified a new GlnR target gene, SCO2958, named nnaR (nitrate/nitrite assimilation regulator). In silico analysis of NnaR revealed the presence of two distinct domains: an N-terminal uroporphyrinogen-III synthase (HemD)-like enzymatic domain and a C-terminal DNA binding domain. Complementation experiments with a haemin auxotroph Escherichia coli ΔhemD mutant strain revealed that NnaR has no HemD activity. Physiological studies of an S. coelicolor nnaR : : Tn5062 mutant showed that NnaR is involved in regulating nitrite reduction. By electrophoretic mobility shift assays the functionality of the NnaR DNA binding domain was confirmed, and it was found that NnaR binds in front of the genes narK (putative nitrate extrusion protein), nirB (nitrite reductase), nirA (putative nitrite/sulphite reductase) and nasA (putative nitrate reductase), which are associated with nitrate/nitrite assimilation. Furthermore, a cooperative binding of NnaR together with GlnR to the nirB promoter was observed, suggesting that NnaR may act as a GlnR co-activator.

The ParB protein of Pseudomonas aeruginosa is important for growth, cell division, nucleoid segregation and different types of motility. To further understand its function we have demonstrated a vital role of the hydrophobic residues in the C terminus of ParB P.a. . By in silico modelling of the C-terminal domain (amino acids 242–290) the hydrophobic residues L282, V285 and I289 (but not L286) are engaged in leucine-zipper-like structure formation, whereas the charged residues R290 and Q266 are implicated in forming a salt bridge involved in protein stabilization. Five parB mutant alleles were constructed and their functionality was defined in vivo and in vitro. In agreement with model predictions, the substitution L286A had no effect on mutant protein activities. Two ParBs with single substitutions L282A or V285A and deletions of two or seven C-terminal amino acids were impaired in both dimerization and DNA binding and were not able to silence genes adjacent to parS, suggesting that dimerization through the C terminus is a prerequisite for spreading on DNA. The defect in dimerization also correlated with loss of ability to interact with partner protein ParA. Reverse genetics demonstrated that a parB mutant producing ParB lacking the two C-terminal amino acids as well as mutants producing ParB with single substitution L282A or V285A had defects similar to those of a parB null mutant. Thus so far all the properties of ParB seem to depend on dimerization.

Polynucleotide phosphorylase (PNPase), a multifunctional protein, is a 3′→5′ exoribonuclease or exoDNase in the presence of inorganic phosphate (Pi), and extends a 3′-OH of RNA or ssDNA in the presence of ADP or dADP. In Escherichia coli, PNPase is known to protect against H2O2- and mitomycin C-induced damage. Recent reports show that Bacillus subtilis PNPase is required for repair of H2O2-induced double-strand breaks. Here we show that absence of PNPase makes E. coli cells sensitive to UV, indicating that PNPase has a role in survival of UV radiation damage. Analyses of various DNA repair pathways show that in the absence of nucleotide excision repair, survival of UV radiation depends critically on PNPase function. Consequently, uvrA pnp, uvrB pnp and uvrC pnp strains show hypersensitivity to UV radiation. Whereas the pnp mutation is non-epistatic to recJ, recQ and recG mutations with respect to the UV-sensitivity phenotype, it is epistatic to uvrD, recB and ruvA mutations, implicating it in the recombinational repair process.

Lactobacillus casei is a lactic acid bacterium commonly found in the gastrointestinal tract of animals, and some strains are used as probiotics. The ability of probiotic strains to survive the passage through the gastrointestinal tract is considered a key factor for their probiotic action. Therefore, tolerance to bile salts is a desirable feature for probiotic strains. In this study we have characterized the response of L. casei BL23 to bile by a transcriptomic and proteomic approach. The analysis revealed that exposure to bile induced changes in the abundance of 52 proteins and the transcript levels of 67 genes. The observed changes affected genes and proteins involved in the stress response, fatty acid and cell wall biosynthesis, metabolism of carbohydrates, transport of peptides, coenzyme levels, membrane H+-ATPase, and a number of uncharacterized genes and proteins. These data provide new insights into the mechanisms that enable L. casei BL23 to cope with bile stress.

In Saccharomyces (Sacc.) cerevisiae, the final step of the complex sphingolipid biosynthetic pathway requires Ipt1p for synthesis of mannosyldiinositol phosphorylceramide [M(IP)2C]. No fission yeast equivalent to Ipt1p has been found in the Schizosaccharomyces (Schiz.) pombe genome, and the most abundant complex sphingolipid is mannosylinositol phosphorylceramide. To examine the effect of expressing Sacc. cerevisiae IPT1 (ScIPT1) in Schiz. pombe, the ScIPT1 gene was cloned into an inducible fission yeast integrative vector and expressed in wild-type Schiz. pombe. In the Schiz. pombe ScIPT1-expressing cells, M(IP)2C was detected, indicating that ScIpt1p functions in M(IP)2C synthesis in Schiz. pombe. Expression of ScIPT1 caused pleiotropic phenotypes, including aberrant morphology and mislocalization of ergosterols in the plasma membrane. Furthermore, growth of Schiz. pombe was severely impaired. We analysed the sphingolipid composition of ScIPT1-expressing cells following a prolonged lag phase, and found that M(IP)2C was not synthesized, indicating that Ipt1p had been inactivated. GFP-tagged ScIpt1 localized primarily in the Golgi apparatus in wild-type Schiz. pombe. Over time, ScIpt1p was eventually transported to the vacuolar lumen through the multivesicular body pathway. These results indicate that M(IP)2C is toxic to Schiz. pombe and that fission yeast possesses an unknown mechanism to effectively extrude toxic sphingolipids from cells.

The NblS-RpaB signalling pathway, the most conserved two-component system in cyanobacteria, regulates photosynthesis and acclimatization to a variety of environmental conditions and is involved in negative regulation of high-light-induced genes. However, relevant regulatory details of the NblS-RpaB signalling pathway remain to be elucidated. We recently showed that the response regulator RpaB is regulated by specific (de)phosphorylation from the histidine kinase NblS and that RpaB and its phosphorylatable residue Asp56 are both required for viability of Synechococcus elongatus PCC 7942. We show here that the phosphorylated form of RpaB is present in cells growing under standard laboratory conditions and that high light stress affected the ratio of phosphorylated to non-phosphorylated RpaB. It also decreased the amount of rpaB transcripts without appreciably changing the total levels of RpaB. Quantitative Western blotting and confocal microscopy analyses were consistent with RpaB being a very abundant regulator, with nucleoid localization. A genetically engineered RpaB-GFP (green fluorescent protein) fusion protein rescued lethality of the rpaB null mutant, indicating that it was functional. This is, to our knowledge, the first study demonstrating in a cyanobacterium, and for a two-component response regulator, that the in vivo ratio of phosphorylated to non-phosphorylated protein changes in response to environmental conditions.

The Rcs phosphorelay signal transduction system controls genes for capsule production and many other envelope-related functions and is implicated in biofilm formation. We investigated the activation of the Rcs system in a pgsA null mutant of Escherichia coli, which completely lacks the major acidic phospholipids phosphatidylglycerol and cardiolipin. We found that the Rcs activation, and consequent thermosensitivity, were suppressed by overexpression of the lgt gene, encoding diacylglyceryltransferase, which catalyses the modification of prolipoproteins that is the first step in the maturation and localization process of lipoproteins, and is a prerequisite for the later steps. The outer-membrane lipoprotein RcsF is an essential component of Rcs signalling. This lipoprotein was poorly localized to the outer membrane in the pgsA null mutant, probably because of the absence of phosphatidylglycerol, the major donor of diacylglycerol in the Lgt reaction. Even in a pgsA + background, the Rcs system was activated when RcsF was mislocalized to the inner membrane by alteration of the residues at positions 2 and 3 of its mature form to inner-membrane retention signals, or when it was mislocalized to the periplasm by fusing the mature form to maltose-binding protein. These results suggest that RcsF functions as a ligand for RcsC in activating Rcs signalling. Mislocalized versions of RcsF still responded to mutations pgsA, mdoH and tolB, further activating the Rcs system, although the rfaP mutation barely caused activation. It seems that RcsF must be localized in the outer membrane to respond effectively to stimuli from outside the cell.

In Pseudomonas putida A ATCC 12633 cells grown with tetradecyltrimethylammonium bromide and exposed to Al3Cl, phosphatidylcholine (PC) levels increased, which alleviated stress caused by the Al3+. Here we cloned and sequenced a gene from this strain that encodes a phosphatidylcholine synthase (PCS) and characterized a pcs-deficient mutant. In the pcs-deficient mutant, PC could not be detected, whereas the mutant could be successfully complemented and expressed the enzyme, indicating that PC synthesis occurs exclusively via the PCS pathway in this organism. Although under non-stressing growth conditions the pcs-deficient mutant showed growth like that of the wild-type strain, the mutant was much more sensitive when challenged with Al3+, which strongly supports the supposition that PC is involved in the response of P. putida to Al3+ and acts as a temporary reservoir of available ions through the formation of Al3+ : PC complexes.

The opportunistic pathogen Candida albicans has a single protein phosphatase Z (PPZ) candidate gene termed CaPPZ1, which shows significant allele variability. We demonstrate here that bacterially expressed CaPpz1 protein exhibits phosphatase activity which can be inhibited by recombinant Hal3, a known inhibitor of Saccharomyces cerevisiae Ppz1. Site-directed mutagenesis experiments based on natural polymorphisms allowed the identification of three amino acid residues that affect enzyme activity or stability. The expression of CaPPZ1 in ppz1 S. cerevisiae and pzh1 Schizosaccharomyces pombe cells partially rescued the salt and caffeine phenotypes of the deletion mutants. CaPpz1 also complemented the slt2 S. cerevisiae mutant, which is crippled in the mitogen-activated protein (MAP) kinase that mediates the cell wall integrity signalling pathway. Collectively, our results suggest that the orthologous PPZ enzymes have similar but not identical functions in different fungi. The deletion of the CaPPZ1 gene in C. albicans resulted in a mutant that was sensitive to salts such as LiCl and KCl, to caffeine, and to agents that affect cell wall biogenesis such as Calcofluor White and Congo red, but was tolerant to spermine and hygromycin B. Reintegration of the CaPPZ1 gene into the deletion mutant alleviated all of the mutant phenotypes tested. Thus CaPpz1 is involved in cation homeostasis, cell wall integrity and the regulation of the membrane potential of C. albicans. In addition, the germ tube growth rate, and virulence in the BALB/c mouse model, were reduced in the null mutant, suggesting a novel function for CaPpz1 in the yeast to hypha transition that may have medical relevance.

Proteolytic control can govern the levels of specific regulatory factors, such as Spx, a transcriptional regulator of the oxidative stress response in Gram-positive bacteria. Under oxidative stress, Spx concentration is elevated and upregulates transcription of genes that function in the stress response. When stress is alleviated, proteolysis of Spx catalysed by ClpXP reduces Spx concentration. Proteolysis is enhanced by the substrate recognition factor YjbH, which possesses a His–Cys-rich region at its N terminus. However, mutations that generate H12A, C13A, H14A, H16A and C31/34A residue substitutions in the N terminus of Bacillus subtilis YjbH (BsYjbH) do not affect functionality in Spx proteolytic control in vivo and in vitro. Because of difficulties in obtaining soluble BsYjbH, the Geobacillus thermodenitrificans yjbH gene was cloned, which yielded soluble GtYjbH protein. Despite its lack of a His–Cys-rich region, GtYjbH complements a B. subtilis yjbH null mutant, and shows high activity in vitro when combined with ClpXP and Spx in an approximately 30 : 1 (ClpXP/Spx : GtYjbH) molar ratio. In vitro interaction experiments showed that Spx and the protease-resistant SpxDD (in which the last two residues of Spx are replaced with two Asp residues) bind to GtYjbH, but deletion of 12 residues from the Spx C terminus (SpxΔC) significantly diminished interaction and proteolytic degradation, indicating that the C terminus of Spx is important for YjbH recognition. These experiments also showed that Spx, but not GtYjbH, interacts with ClpX. Kinetic measurements for Spx proteolysis by ClpXP in the presence and absence of GtYjbH suggest that YjbH overcomes non-productive Spx–ClpX interaction, resulting in rapid degradation.

Reactive oxygen species (ROSs) affect several macromolecules and cellular components in eukaryotic and prokaryotic cells. In this work, the effect of various ROS-generating compounds on the Escherichia coli membrane was studied. Membrane fatty acid profiles, oxidative damage levels and bacterial resistance to these toxicants were determined. Studies included wild-type cells as well as a strain exhibiting a modified monounsaturated fatty acid (MUFA) profile (accomplished by overexpressing the β-hydroxyacyl acyl carrier protein dehydratase-encoding gene, fabA). Levels of membrane MUFAs and oxidative damage markers decreased slightly upon toxicant exposure with a concomitant increase in cell resistance to these ROS-generating compounds. A direct relationship between MUFAs and lipid peroxidation was observed. The lower the MUFA the lower the peroxide levels, suggesting that MUFAs are targets for membrane lipid oxidation.

Fur (ferric uptake regulator) is an iron-responsive transcriptional regulator in many bacterial species, and the fur mutant of Burkholderia multivorans ATCC 17616 exhibits pleiotropic phenotypes, such as an inability to efficiently use several carbon sources, as well as high sensitivity to hydrogen peroxide (H2O2), paraquat (a superoxide-producing compound) and nitric oxide (NO). To gain more insight into the pleiotropic role of the Fur protein of ATCC 17616, spontaneous suppressor mutants of the ATCC 17616 fur mutant that restored tolerance to NO were isolated and characterized in this study. The microarray-based comparative genomic analysis and subsequent sequencing analysis indicated that such suppressor mutants had a 2 bp deletion in the oxyR gene, whose orthologues encode H2O2-responsive transcriptional regulators in other bacterial species. The suppressor mutants and the reconstructed fur–oxyR double-deletion mutant showed indistinguishable phenotypes in that they were all (i) more resistant than the fur mutant to H2O2, superoxide, NO and streptonigrin (an iron-activated antibiotic) and (ii) able to use carbon sources that cannot efficiently support the growth of the fur mutant. These results clearly indicate that the oxyR mutation suppressed the pleiotropic effect of the B. multivorans fur mutant. The fur–oxyR double mutants were found to overexpress the KatG (catalase/peroxidase) and AhpC1 and AhpD (alkyl hydroperoxide reductase subunits C and D) proteins, and their enzymic activities to remove reactive oxygen and nitrogen species were suggested to be responsible for the suppression of phenotypes caused by the fur mutation.

Components of the bacterial phosphoenolpyruvate (PEP) : carbohydrate phosphortransferase system (PTS) have multiple regulatory roles in addition to PEP-dependent transport/phosphorylation of numerous carbohydrates. We have recently shown that, in an opportunistic human pathogen, Vibrio vulnificus, enzyme IIAGlc (EIIAGlc) interacts with a peptidase that has high sequence similarity to mammalian insulin-degrading enzymes, called Vibrio insulin-degrading enzyme (vIDE). Although the vIDE–EIIAGlc interaction is independent of the phosphorylation state of EIIAGlc, vIDE shows no peptidase activity unless complexed with the unphosphorylated form of EIIAGlc. A deletion mutant of ideV, the gene encoding vIDE, shows remarkably lower degrees of survival and virulence than the wild-type strain in mice, implying that vIDE is a virulence factor. In this study, we investigated regulation of ideV expression at the transcriptional level. Primer extension analysis identified two different transcriptional start sites of ideV: PL for the longer transcript and PS for the shorter transcript. We performed ligand fishing experiments by using the promoter region of ideV and found that the cAMP receptor protein (CRP) specifically binds to the promoter. DNase I footprinting experiments revealed that CRP binds to a region between the two promoters. In vitro transcription assays showed that CRP activates ideV PS transcription in the presence of cAMP whose concentration is regulated by EIIAGlc. These results suggest that EIIAGlc regulates the expression level of vIDE as well as its activity.

The Gram-negative plant-pathogenic bacterium Xanthomonas campestris pv. vesicatoria employs a type III secretion (T3S) system to translocate effector proteins into plant cells. T3S depends on HrpB2, which is essential for assembly of the extracellular T3S pilus and is itself weakly secreted. To characterize the role of HrpB2, we used a transposon mutagenesis approach, which led to the insertion of pentapeptide-encoding sequences into hrpB2. Complementation studies with HrpB2 mutant derivatives revealed that the N-terminal region of HrpB2 tolerates pentapeptide insertions, whereas insertions in the regions spanning amino acids 60–74 and 93–130, respectively, resulted in a loss of bacterial pathogenicity and T3S, including secretion of HrpB2 itself. The C-terminal region (amino acids 93–130) of HrpB2 contains a conserved VxTLxK amino acid motif that is also present in predicted inner rod proteins from animal-pathogenic bacteria and is required for the contribution of HrpB2 to pilus assembly and T3S. Electron microscopy and fractionation studies revealed that HrpB2 is not a component of the extracellular pilus structure but localizes to the bacterial periplasm and the outer membrane. We therefore propose that the essential contribution of HrpB2 to T3S and pilus assembly is linked to its possible function as a periplasmic component of the T3S system at the base of the pilus.