- Volume 153, Issue 2, 2007

Pseudomonas aeruginosa is an important nosocomial opportunistic human pathogen and a major cause of chronic lung infections in individuals with cystic fibrosis. Serious infections by this organism are often treated with a combination of aminoglycosides and semi-synthetic penicillins. Subinhibitory concentrations of antibiotics are now being recognized for their role in microbial persistence and the development of antimicrobial resistance, two very important clinical phenomena. An extensive screen of a P. aeruginosa PAO1 luciferase gene fusion library was performed to identify genes that were differentially regulated during exposure to subinhibitory gentamicin. It was demonstrated that subinhibitory concentrations of gentamicin and tobramycin induced a set of genes that are likely to affect the interaction of P. aeruginosa with host cells, including the gene encoding Lon protease, which is known to play a major role in protein quality control. Studies with a lon mutant compared to its parent and a complemented strain indicated that this protein was essential for biofilm formation and motility in P. aeruginosa.

Yersinia ruckeri causes the enteric redmouth disease or yersiniosis, an important systemic fish infection. In an attempt to dissect the virulence mechanisms of this bacterium, a gene encoding a putative protein involved in the secretion/activation of a haemolysin (yhlB), which had been previously identified by in vivo expression technology, was further analysed. The gene yhlB precedes another ORF (yhlA) encoding a Serratia-type haemolysin. Other toxins belonging to this group have been identified in genomic analyses of human-pathogenic yersiniae, although their role and importance in pathogenicity have not been defined yet. In spite of its being an in vivo-induced gene, the expression of yhlA can be induced under certain in vitro conditions similar to those encountered in the host, as deduced from the results obtained by using a yhlB : : lacZY fusion. Thus, higher levels of expression were obtained at 18 °C, the temperature of occurrence of disease outbreaks, than at 28 °C, the optimal growth temperature. The expression of the haemolysin also increased under iron-starvation conditions. This confirmed the decisive role of iron and temperature as environmental cues that regulate and coordinate the expression of genes encoding extracellular factors involved in the virulence of Y. ruckeri. LD50 and cell culture experiments, using yhlB and yhlA insertional mutant strains, demonstrated the participation of the haemolysin in the virulence of Y. ruckeri and also its cytolytic properties against the BF-2 fish cell line. Finally, a screening for the production of haemolytic activity and the presence of yhlB and yhlA genes in 12 Y. ruckeri strains proved once more the genetic homogeneity of this species, since all possessed both haemolytic activity and the yhlB and yhlA genes.

The eis gene of Mycobacterium tuberculosis has been shown to play a role in the survival of the avirulent Mycobacterium smegmatis within the macrophage. In vitro and in vivo analysis of Δeis deletion mutants and complemented strains showed no effect on survival of M. tuberculosis in U-937 macrophages or in a mouse aerosol infection model, respectively. Further studies were done in an attempt to determine the role of eis in M. tuberculosis intracellular survival and to define a phenotypic difference between wild-type and the Δeis deletion mutant. Bioinformatic analysis indicated that Eis is an acetyltransferase of the GCN5-related family of N-acetyltransferases. Immunofluorescence microscopy and Western blot analysis studies demonstrated that Eis is released into the cytoplasm of M. tuberculosis-infected U-937 macrophages. Eis was also found in the extravesicular fraction and culture supernatant of M. tuberculosis-infected macrophages. The effect of Eis on human macrophage cytokine secretion was also examined. Eis modulated the secretion of IL-10 and TNF-α by primary human monocytes in response both to infection with M. tuberculosis and to stimulation with recombinant Eis protein. These results suggest that Eis is a mycobacterial effector that is released into the host cell to modulate inflammatory responses, possibly via transcriptional or post-translational means.

Campylobacter jejuni is a common cause of serious diarrhoeal disease in humans, in contrast to the avian population, where exposure results in prolonged colonization at high density without disease. Colonized poultry present a significant source of infection to humans worldwide. The aim of this work was to compare the interaction of Campylobacter with primary intestinal cells from humans and poultry to identify factors that account for the divergent outcome following Campylobacter exposure. A primary intestinal cell model of Campylobacter infection was developed using cells grown from human and chicken intestinal biopsies. The cultured cells were infected with a number of strains of Campylobacter. Invasion by C. jejuni and the influence of intestinal mucus on Campylobacter internalization were studied by fluorescence microscopy and gentamicin protection assays. C. jejuni invaded primary human intestinal cells in a microtubule-, microfilament- and caveolin-dependent manner. Entry of C. jejuni into primary chicken intestinal cells also occurred. Chicken mucus, but not intestinal mucus of human origin, significantly reduced infection of primary human intestinal cells. Avian mucus appears to inhibit Campylobacter from interacting with epithelial cell surfaces.

Phenylacetic acid (PA) degradation in bacteria involves an aerobic hybrid pathway encoded by the paa gene cluster. It is shown here that succinyl-CoA is one of the final products of this pathway in Pseudomonas putida and Escherichia coli. Moreover, in vivo and in vitro studies revealed that the paaE gene encodes the β-ketoadipyl-CoA thiolase that catalyses the last step of the PA catabolic pathway, i.e. the thiolytic cleavage of β-ketoadipyl-CoA to succinyl-CoA and acetyl-CoA. Succinyl-CoA is suggested as a common final product of aerobic hybrid pathways devoted to the catabolism of aromatic compounds.

Polyhydroxyalkanoic acids (PHAs) are synthesized by unspecific PHA synthases and deposited as energy and carbon storage granules in the cytoplasm of many prokaryotes. The number and size of the granules depend on the presence of phasins which are amphiphilic structural proteins occurring at the granule surface. Recently, it was shown that polythioesters (PTEs) are also synthesized by PHA synthases. To increase the yield of these polymers, the role of recombinant phasins was analysed in an artificial PHA-producing Escherichia coli strain. Overexpressed PhaP1 from Ralstonia eutropha H16 affected poly(3-mercaptopropionate) [poly(3MP)] and poly(3-hydroxybutyrate) [poly(3HB)] accumulation in recombinant E. coli, which expressed the non-natural BPEC pathway consisting of butyrate kinase and phosphotransbutyrylase from Clostridium acetobutylicum and PHA synthase from Thiococcus pfennigii. For this, BPEC-carrying E. coli with and without phaP1 was cultivated in presence of glucose as carbon source for growth plus 3-mercaptopropionate or 3-hydroxybutyrate as precursor substrates for poly(3MP) or poly(3HB) biosynthesis, respectively. In the presence of PhaP1, the recombinant E. coli produced about 50 or 68 % more poly(3MP) or poly(3HB), respectively. Therefore, coexpression of PhaP1 alongside the BPEC pathway is important for optimizing strains towards enhanced PHA or PTE production. Furthermore, in the absence of PhaP1, large amounts of the 16 kDa heat-shock protein HspA were synthesized and bound to the granule surface. Unusual small granules occurred in the cells of the recombinant E. coli strains. The diameter of the poly(3MP) granules was only 55±12 nm or 105±12 nm, and of the poly(3HB) granules only 56±10 or 110±22 nm in the presence or absence of PhaP1, respectively. This explains why no single granules capable of accumulating PHAs or PTEs occurred in the recombinant E. coli, unlike in PhaP1-negative mutants of R. eutropha. Obviously, HspA mimics the phasin, thereby preventing coalescence of granules into one single granule. However, the effect of PhaP1 on granule size and on amounts of accumulated polymers was more severe than that of HspA.

Using transposon mutagenesis, mutations have been isolated in several genes (ccdA, cycM, ccmC, ccmB and senC) that play a role in Sinorhizobium meliloti cytochrome metabolism. As in other bacteria, mutations in the S. meliloti ccdA, ccmB and ccmC genes resulted in the absence of all c-type cytochromes. However, the S. meliloti ccdA mutant also lacked cytochrome oxidase aa 3, a defect that does not appear to have been reported for other bacteria. The aa 3-type cytochromes were also missing from a mutant strain with an insertion into the gene encoding the haem-containing subunit (SU)I of aa 3 cytochrome c oxidase, but not in mutants unable to make SUII or SUIII, indicating that CcdA probably plays a role in assembling SUI. The cytochrome-deficient mutants also had other free-living phenotypes, including a significant decrease in growth rate on rich media and increased motility on minimal media. A senC mutant also had significantly decreased motility, but the motility and growth properties of the cycM mutant were unchanged. Unlike similar mutants in Bradyrhizobium japonicum and Rhizobium leguminosarum, an S. meliloti Rm1021 cycM mutant contained cytochrome oxidase aa 3. Cytochrome maturation in strain Rm1021 appeared to be similar to maturation in other rhizobia, but there were some differences in the cytochrome composition of the strain, and respiration chain function and assembly.

The ability of Hypocrea jecorina (Trichoderma reesei) to grow on lactose strongly depends on the formation of an extracellular glycoside hydrolase (GH) family 35 β-galactosidase, encoded by the bga1 gene. Previous studies, using batch or transfer cultures of pregrown cells, had shown that bga1 is induced by lactose and d-galactose, but to a lesser extent by galactitol. To test whether the induction level is influenced by the different growth rates attainable on these carbon sources, bga1 expression was compared in carbon-limited chemostat cultivations at defined dilution (=specific growth) rates. The data showed that bga1 expression by lactose, d-galactose and galactitol positively correlated with the dilution rate, and that galactitol and d-galactose induced the highest activities of β-galactosidase at comparable growth rates. To know more about the actual inducer for β-galactosidase formation, its expression in H. jecorina strains impaired in the first steps of the two d-galactose-degrading pathways was compared. Induction by d-galactose and galactitol was still found in strains deleted in the galactokinase-encoding gene gal1, which is responsible for the first step of the Leloir pathway of d-galactose catabolism. However, in a strain deleted in the aldose/d-xylose reductase gene xyl1, which performs the reduction of d-galactose to galactitol in a recently identified second pathway, induction by d-galactose, but not by galactitol, was impaired. On the other hand, induction by d-galactose and galactitol was not affected in an l-arabinitol 4-dehydrogenase (lad1)-deleted strain which is impaired in the subsequent step of galactitol degradation. These results indicate that galactitol is the actual inducer of Bga1 formation during growth on d-galactose in H. jecorina.

Streptomycetes belong to the ecologically important bacterial population within soil, which is also inhabited by many fungi. The highly chitinolytic Streptomyces olivaceoviridis and the ascomycete Aspergillus proliferans were chosen as models to test for interactions among bacteria and fungi. In medium lacking a soluble carbon source, individually cultivated spores of the bacterium S. olivaceoviridis and the fungus A. proliferans do not germinate. However, as shown by viability tests, cultivation of a mixture of both spore types provokes successive events: (i) stimulation of the germination of S. olivaceoviridis spores, (ii) initiation of the outgrowth of some fungal spores to which the S. olivaceoviridis chitinase ChiO1 adheres, (iii) massive extension of viable networks of S. olivaceoviridis hyphae at the expense of fungal hyphae and (iv) balanced proliferation of closely interacting fungal and S. olivaceoviridis hyphae. The replacement of the S. olivaceoviridis wild-type strain by a chromosomal disruption mutant (ΔC), lacking production of the extracellular chitin-binding protein CHB1 but still secreting the chitinase ChiO1, provokes (v) germination of each spore type, (vi) retarded development of both partners, followed by (vii) preferential proliferation of the fungus. Together with biochemical and immunomicroscopy studies, the data support the conclusion that CHB1 molecules aggregate to an extracellular matrix, maintaining a close contact, followed by several concerted responses of the bacterium and the fungus.

Poly-3-hydroxybutyrate (PHB) and glycogen are major carbon storage compounds in Sinorhizobium meliloti. The roles of PHB and glycogen in rhizobia–legume symbiosis are not fully understood. Glycogen synthase mutations were constructed by in-frame deletion (glgA1) or insertion (glgA2). These mutations were combined with a phbC mutation to make all combinations of double and triple mutants. PHB was not detectable in any of the mutants containing the phbC mutation; glycogen was not detectable in any of the mutants containing the glgA1 mutation. PHB levels were significantly lower in the glgA1 mutant, while glycogen levels were increased in the phbC mutant. Exopolysaccharide (EPS) was not detected in any of the phbC mutants, while the glgA1 and glgA2 mutants produced levels of EPS similar to the wild-type. Symbiotic properties of these strains were investigated on Medicago truncatula and Medicago sativa. The results indicated that the strains unable to synthesize PHB, or glycogen, were still able to form nodules and fix nitrogen. However, phbC mutations caused greater nodule formation delay on M. truncatula than on M. sativa. Time-course studies showed that (1) the ability to synthesize PHB is important for N2 fixation in M. truncatula nodules and younger M. sativa nodules, and (2) the blocking of glycogen synthesis resulted in lower levels of N2 fixation on M. truncatula and older nodules on M. sativa. These data have important implications for understanding how PHB and glycogen function in the interactions of S. meliloti with Medicago spp.

It is becoming recognized that leghaemoglobin constitutes an important buffer for the cytotoxic nitric oxide radical (NO•) in root nodules, although the sources of this NO• within nodules are unclear. In Bradyrhizobium japonicum bacteroids, NO• can be produced through the denitrification process, during which nitrate is reduced to nitrite by the periplasmic nitrate reductase Nap, and nitrite is reduced to NO• by the respiratory nitrite reductase NirK. To assess the contribution of bacteroidal denitrification to the NO• within nitrate-treated soybean nodules, electron paramagnetic resonance and UV–visible spectroscopy were employed to study the presence of nitrosylleghaemoglobin (LbNO) within nodules from plants inoculated with wild-type, napA or nirK B. japonicum strains. Since it has been found that hypoxia induces NO• production in plant root tissue, and that plant roots can be subjected to hypoxic stress during drought and flooding, the effect of hypoxic stress on the formation of LbNO complexes within nodules was also investigated. Maximal levels of LbNO were observed in nodules from plants treated with nitrate and subjected to hypoxic conditions. It is shown that, in the presence of nitrate, all of the LbNO within normoxic nodules arises from nitrate reduction by the bacteroidal periplasmic nitrate reductase, whereas Nap activity is only responsible for half of the LbNO within hypoxic nodules. In contrast to Nap, NirK is not essential for LbNO formation under any condition tested.