- Volume 154, Issue 10, 2008

Cyclic di-GMP (c-di-GMP) plays an important role in bacterial adaptation to enable survival in changing environments. It orchestrates various pathways involved in biofilm formation, changes in the cell surface, host colonization and virulence. In this article, we report the presence of c-di-GMP in Mycobacterium smegmatis, and its role in the long-term survival of the organism. M. smegmatis has a single bifunctional protein with both GGDEF and EAL domains, which show diguanylate cyclase (DGC) and phosphodiesterase (PDE)-A activity, respectively, in vitro. We named this protein MSDGC-1. Deletion of the gene encoding MSDGC-1 did not affect growth and biofilm formation in M. smegmatis, but long-term survival under conditions of nutritional starvation was affected. Most of the proteins that contain GGDEF and EAL domains have been demonstrated to have either DGC or PDE-A activity. To gain further insight into the regulation of the protein, we cloned the individual domains, and tested their respective activities. MSDGC-1, the full-length protein, is required for activity, as its GGDEF and EAL domains are inactive when separated.

Cyanobacteria respond to environmental stress conditions by adjusting their photosynthesis machinery. In Synechococcus sp. PCC 7942, phycobilisome degradation and other acclimation responses after nutrient or high-light stress require activation by the orphan response regulator NblR, a member of the OmpR/PhoB family. Although NblR contains a putative phosphorylatable residue (Asp57), it lacks other conserved residues required to chelate the Mg2+ necessary for aspartic acid phosphorylation or to transduce the phosphorylation signal. In close agreement with these features, NblR was not phosphorylated in vitro by the low-molecular-mass phosphate donor acetyl phosphate and mutation of Asp57 to Ala had no impact on previously characterized NblR functions in Synechococcus. On the other hand, in vitro and in vivo assays show that the default state of NblR is monomeric, suggesting that, despite input differences, NblR activation could involve the same general mechanism of activation by dimerization present in known members of the OmpR/PhoB family. Structural and functional data indicate that the receiver domain of NblR shares similarities with other phosphorylation-independent response regulators such as FrzS and HP1043. To acknowledge the peculiarities of these atypical ‘two-component’ regulators with phosphorylation-independent signal transduction mechanisms, we propose the term PIARR, standing for phosphorylation-independent activation of response regulator.

The subcellular localization of the major type II secretion system of Pseudomonas aeruginosa, the Xcp system, was studied microscopically using a biarsenical ligand that becomes fluorescent upon binding to a tetracysteine motif (Lumio tag), which was fused to several Xcp components. Fusion of the Lumio tag to the C termini of the XcpR and XcpS proteins did not affect the functionality of these proteins. Fluorescence microscopy showed that they were predominantly localized to the poles of P. aeruginosa cells, when produced at levels comparable to chromosomally encoded XcpR and XcpS. In most labelled cells, the proteins were found at one of the poles, although bipolar localization was also observed. When produced in the absence of other Xcp components, labelled XcpS was still found to locate at the poles, whereas XcpR was evenly distributed in the cell. These data suggest that XcpS, but not XcpR, contains information required for polar localization. The polar location of the Xcp machinery was further confirmed by the visualization of protease secretion with an intramolecularly quenched casein conjugate.

The ability of each of the 11 Streptococcus pneumoniae division proteins to interact with itself and with each of the remaining proteins was studied in 66 combinations of protein pairs, using a bacterial two-hybrid system. Interactions (homo- or hetero-dimerizations) were detected between 37 protein pairs, whereas 29 protein pairs did not interact. In some cases, positive interactions of the S. pneumoniae proteins were confirmed by co-immunoprecipitation experiments in Escherichia coli. Comparison between the S. pneumoniae division protein interaction web and that of E. coli, the only micro-organisms for which the whole division interactome has been described systematically, was also performed. At least nine division proteins, ZapA, FtsZ, FtsA, FtsK, FtsQ/DivIB, FtsB/DivIC, FtsL, FtsI and FtsW, are believed to have a conserved function between these bacteria and thus we may say that a significant part of the interactions are conserved. Out of 45 protein pairs tested in both bacteria, 30 showed the same behaviour: 23 interacted while seven did not. In agreement with these results, cross-interactions between S. pneumoniae proteins and the corresponding E. coli orthologues were observed. Taken together, these results suggest a phylogenetically conserved minimal common interactome of the division proteins.

Denitrification and arginine fermentation are major parts of the anaerobic metabolism of Pseudomonas aeruginosa, which is important for biofilm formation and infection. The two-component regulatory system NarX-NarL is part of the underlying network and is required for denitrifying growth. All target promoters identified so far are activated by NarL. In this study the effect of NarL on arginine fermentation was investigated using proteome, Northern blot and lacZ reporter gene analyses. NarL-dependent repression of the arcDABC operon was observed and the corresponding NarL-binding site in the arcD promoter region was functionally localized at −60 bp upstream of the transcriptional start site using site-directed promoter mutagenesis and reporter gene fusion experiments. The results clearly show that in the presence of nitrate NarL represses the arginine-dependent activation of the arcDABC operon mediated by ArgR. It does not influence the oxygen-tension-dependent activation via Anr. Thus, the anaerobic energy metabolism of P. aeruginosa is coordinated via NarX-NarL activity. In the presence of nitrate the highly efficient denitrification is preferred over the less attractive arginine fermentation.

The glyoxylate cycle, a metabolic pathway required for generating C4 units from C2 compounds, is an important factor in virulence, in both animal and plant pathogens. Here, we report the localization of the key enzymes of this cycle, isocitrate lyase (Icl1; EC 4.1.3.1) and malate synthase (Mls1; EC 2.3.3.9), in the human fungal pathogen Candida albicans. Immunocytochemistry in combination with subcellular fractionation showed that both Icl1 and Mls1 are localized to peroxisomes, independent of the carbon source used. Although Icl1 and Mls1 lack a consensus type I peroxisomal targeting signal (PTS1), their import into peroxisomes was dependent on the PTS1 receptor Pex5p, suggesting the presence of non-canonical targeting signals in both proteins. Peroxisomal compartmentalization of the glyoxylate cycle is not essential for proper functioning of this metabolic pathway because a pex5Δ/Δ strain, in which Icl1 and Mls1 were localized to the cytosol, grew equally as well as the wild-type strain on acetate and ethanol. Previously, we reported that a fox2Δ/Δ strain that is completely deficient in fatty acid β-oxidation, but has no peroxisomal protein import defect, displayed strongly reduced growth on non-fermentable carbon sources such as acetate and ethanol. Here, we show that growth of the fox2Δ/Δ strain on these carbon compounds can be restored when Icl1 and Mls1 are relocated to the cytosol by deleting the PEX5 gene. We hypothesize that the fox2Δ/Δ strain is disturbed in the transport of glyoxylate cycle products and/or acetyl-CoA across the peroxisomal membrane and discuss the possible relationship between such a transport defect and the presence of giant peroxisomes in the fox2Δ/Δ mutant.

The effect of different carbon sources on the expression of tricarboxylic acid (TCA) cycle genes, along with glyoxylate bypass genes, in Corynebacterium glutamicum was determined. All TCA cycle genes were coordinately expressed in medium containing acetate. Growth in the presence of acetate gave rise to abundant expression of most TCA cycle genes, with the level of gltA transcript being the highest. However, when the cells entered the stationary phase triggered by acetate exhaustion, all genes were repressed, except sucCD and mdhB, which were slightly induced. Acetate withdrawal from the growth medium during the exponential phase also led to rapid repression of most TCA cycle genes and a corresponding twofold increase in the expression of sucCD, which were strongly induced by citrate and succinate. In addition, glucose depletion during the stationary phase led to a corresponding 8–20-fold induction of the sucCD, aceA and aceB genes. Addition of glucose to acetate medium resulted in about 10-fold induction of sucCD. The strong dependence of TCA cycle sucCD and glyoxylate bypass aceA and aceB expression on carbon source availability was confirmed and the regulatory system will be studied precisely.

Multidrug-resistant staphylococci often harbour plasmids that carry genes conferring resistance to several antimicrobial compounds. Many of these multiresistance plasmids appear to utilize a related theta-type replication system for which multiresistance plasmid pSK1 serves as a prototype. Essential pSK1 replication elements were identified by cloning segments of the replication region and testing the resulting plasmids for replication proficiency. An iterated region within rep and a DNA segment of up to 68 bp upstream of the rep promoter were both found to be essential for origin activity. The Rep protein was overexpressed as a 6×His-tagged C-terminal fusion protein and was shown to bind in vitro to four Rep boxes located within the rep coding region. Inactivation of a divergently oriented promoter upstream of rep, designated P rnaI , resulted in an elevated plasmid copy number. Comparative analyses suggest that the replication systems of many staphylococcal multiresistance plasmids share a similar genetic organization and utilize an antisense-RNA-mediated regulatory mechanism for copy number control.

Carbon catabolite repression is an important mechanism allowing efficient carbon source utilization. In the soil bacterium Acinetobacter baylyi, this mechanism has been shown to apply to the aromatic degradative pathways for the substrates protocatechuate, p-hydroxybenzoate and vanillate. In this investigation, transcriptional fusions with the gene for luciferase in the gene clusters for the degradation of benzyl esters, anthranilate, benzoate, hydroxycinnamates and dicarboxylates (are, ant, ben, hca and dca genes) were constructed and established in the chromosome of A. baylyi. The respective strains revealed the presence of strong carbon catabolite repression at the transcriptional level. In all cases, succinate and acetate in combination had the strongest repressing effect, and pyruvate (or lactate in case of the ben and hca genes) allowed the highest expression when these carbon sources were supplied together with the respective inducer. The pattern of repression for the different cosubstrates was similar for all operons investigated and was also observed in the absence of the respective inducing compounds, indicating a mechanism that is independent of the respective specific regulators. Repression by acetate and succinate varied between 88 % for the hca genes and 99 % for the pca genes.

The zoonotic pathogen Streptococcus equi subsp. zooepidemicus (S. zooepidemicus) is commonly found harmlessly colonizing the equine nasopharynx. Occasionally, strains can invade host tissues or cross species barriers, and S. zooepidemicus is associated with numerous different diseases in a variety of hosts, including inflammatory airway disease and abortion in horses, pneumonia in dogs and meningitis in humans. A biovar of S. zooepidemicus, Streptococcus equi subsp. equi, is the causative agent of strangles, one of the most important infections of horses worldwide. We report here the development of the first multilocus sequence typing (MLST) scheme for S. zooepidemicus and its exploitation to define the population genetic structure of these related pathogens. A total of 130 unique sequence types were identified from 277 isolates of diverse geographical and temporal origin. Isolates of S. equi shared a recent evolutionary ancestor with isolates of S. zooepidemicus that were significantly associated with cases of uterine infection or abortion in horses (P<0.001). Isolates of S. zooepidemicus from three UK outbreaks of acute fatal haemorrhagic pneumonia in dogs during 1999, 2001 and 2008 were found to be related to isolates from three outbreaks of this disease in the USA during 2005, 1993 and 2006, respectively. Our data provide strong evidence that S. equi evolved from an ancestral S. zooepidemicus strain and that certain related strains of S. zooepidemicus have a greater propensity to infect particular hosts and tissues.

Using a parallel-plate flow chamber, the hydrodynamic shear forces to prevent bacterial adhesion (Fprev ) and to detach adhering bacteria (Fdet ) were evaluated for hydrophilic glass, hydrophobic, dimethyldichlorosilane (DDS)-coated glass and six different bacterial strains, in order to test the following three hypotheses. 1. A strong hydrodynamic shear force to prevent adhesion relates to a strong hydrodynamic shear force to detach an adhering organism. 2. A weak hydrodynamic shear force to detach adhering bacteria implies that more bacteria will be stimulated to detach by passing an air–liquid interface (an air bubble) through the flow chamber. 3. DLVO (Derjaguin, Landau, Verwey, Overbeek) interactions determine the characteristic hydrodynamic shear forces to prevent adhesion and to detach adhering micro-organisms as well as the detachment induced by a passing air–liquid interface. Fprev varied from 0.03 to 0.70 pN, while Fdet varied from 0.31 to over 19.64 pN, suggesting that after initial contact, strengthening of the bond occurs. Generally, it was more difficult to detach bacteria from DDS-coated glass than from hydrophilic glass, which was confirmed by air bubble detachment studies. Calculated attractive forces based on the DLVO theory (FDLVO ) towards the secondary interaction minimum were higher on glass than on DDS-coated glass. In general, all three hypotheses had to be rejected, showing that it is important to distinguish between forces acting parallel (hydrodynamic shear) and perpendicular (DLVO, air–liquid interface passages) to the substratum surface.

The ssr3341 locus was previously suggested to encode an orthologue of the RNA chaperone Hfq in the cyanobacterium Synechocystis sp. strain PCC 6803. Insertional inactivation of this gene resulted in a mutant that was not naturally transformable and exhibited a non-phototactic phenotype compared with the wild-type. The loss of motility was complemented by reintroduction of the wild-type gene, correlated with the re-establishment of type IV pili on the cell surface. Microarray analyses revealed a small set of genes with drastically reduced transcript levels in the knockout mutant compared with the wild-type cells. Among the most strongly affected genes, slr1667, slr1668, slr2015, slr2016 and slr2018 stood out, as they belong to two operons that had previously been shown to be involved in motility, controlled by the cAMP receptor protein SYCRP1. This suggests a link between cAMP signalling, motility and possibly the involvement of RNA-based regulation. This is believed to be the first report demonstrating a functional role of an Hfq orthologue in cyanobacteria, establishing a new factor in the control of motility.

Biocides and dyes are commonly employed in hospital and laboratory settings. Many of these agents are substrates for multiple-drug resistance (MDR)-conferring efflux pumps of both Gram-positive and Gram-negative organisms. Several such pumps have been identified in Staphylococcus aureus, and mutants overexpressing the NorA and MepA MDR pumps following exposure to fluoroquinolones have been identified. The effect of exposure to low concentrations of biocides and dyes on the expression of specific pump genes has not been evaluated. Using quantitative reverse-transcription PCR we found that exposure of clinical isolates to low concentrations of a variety of biocides and dyes in a single step, or to gradually increasing concentrations over several days, resulted in the appearance of mutants overexpressing mepA, mdeA, norA and norC, with mepA overexpression predominating. Overexpression was frequently associated with promoter-region or regulatory protein mutations. Mutants having significant increases in MICs of common pump substrates but no changes in expression of studied pump genes were also observed; in these cases changes in expression of as-yet-unidentified MDR pump genes may have occurred. Strains of S. aureus that exist in relatively protected environments and are repeatedly exposed to sublethal concentrations of biocides can develop efflux-related resistance to those agents, and acquisition of such strains poses a threat to patients treated with antimicrobial agents that are also substrates for those pumps, such as ciprofloxacin and moxifloxacin.

During applications of 5-fluorocytosine (5FC) and fluconazole (FLC), additive or synergistic action may even occur when primary resistance to 5FC is established. Here, we analysed conjoint drug action in Saccharomyces cerevisiae strains deficient in genes known to be essential for 5FC or FLC function. Despite clear primary resistance, residual 5FC activity and additive 5FC+FLC action in cells lacking cytosine permease (Fcy2p) or uracil phosphoribosyl transferase (Fur1p) were detected. In contrast, Δfcy1 mutants, lacking cytosine deaminase, became entirely resistant to 5FC, concomitantly losing 5FC+FLC additivity. Disruption of the orotate phosphoribosyltransferase gene (URA5) in the wild-type led to low-level 5FC tolerance, while an alternative orotate phosphoribosyltransferase, encoded by URA10, contributed to 5FC toxicity only in the Δura5 background. Remarkably, combination of Δura5 and Δfur1 resulted in complete 5FC resistance. Thus, yeast orotate phosphoribosyltransferases are involved in 5FC metabolism. Similarly, disruption of the ergosterol Δ5,6-desaturase-encoding gene ERG3 resulted only in partial resistance to FLC, and concomitantly a synergistic effect with 5FC became evident. Full resistance to FLC occurred in Δerg3 Δerg11 double mutants and, simultaneously, synergism or even an additive effect with FLC and 5FC was no longer discernible. Since the majority of spontaneously occurring resistant yeast clones displayed residual sensitivity to either 5FC or FLC and those strains responded to combined drug treatment in a predictable manner, careful resistance profiling based on the findings reported here may help to address yeast infections by combined application of antimycotic compounds.