- Volume 153, Issue 4, 2007

The two proteins involved in the regulation of gas vesicle formation in Haloferax mediterranei, mcGvpE (activator) and mcGvpD (repressive function), are able to interact in vitro. It was also found that the respective proteins cGvpE and cGvpD of Halobacterium salinarum and the heterologous pairs mcGvpD–cGvpE and cGvpD–mcGvpE were able to interact. Previously constructed mcGvpD mutants with alterations in regions affecting the repressive function of GvpD (p-loop motif or the two arginine-rich regions bR1 and bR2) were tested for their ability to interact with GvpE, and all still bound GvpE. Even a deletion of or near the p-loop motif in GvpD did not affect this ability to interact. Further deletion variants lacking larger N- or C-terminal portions of mcGvpD yielded that neither the N-terminal region with the p-loop motif nor the C-terminal portion were important for the binding of GvpE, and suggested that the central portion is involved in GvpE binding. The GvpD protein also induces a reduction in the amount of GvpE in Haloferax volcanii transformants expressing both genes under fdx promoter control on a single plasmid. Such DEex transformants contain GvpD, but no detectable GvpE, whereas large amounts of GvpE are found in ΔDEex transformants that have incurred a deletion within the gvpD gene. A similar reduction was observed in Dex+Eex transformants harbouring both reading frames under fdx promoter control on two different plasmids. GvpD wild-type and also GvpD mutants were tested, and a significant reduction in the amount of GvpE was obtained in the case of GvpD wild-type and the super-repressor mutant GvpD3-AAA. In contrast, transformants harbouring GvpD mutants with alterations in the p-loop motif or the bR1 region still contained GvpE. Since the amount of gvpE transcript was not reduced, the reduction occurred at the protein level. These results underlined that a functional p-loop and the arginine-rich region bR1 of GvpD were required for the GvpD-mediated reduction in the amount of GvpE.

The GGDEF response regulator WspR couples the chemosensory Wsp pathway to the overproduction of acetylated cellulose and cell attachment in the Pseudomonas fluorescens SBW25 wrinkly spreader (WS) genotype. Here, it is shown that WspR is a diguanylate cyclase (DGC), and that DGC activity is elevated in the WS genotype compared to that in the ancestral smooth (SM) genotype. A structure–function analysis of 120 wspR mutant alleles was employed to gain insight into the regulation and activity of WspR. Firstly, 44 random and defined pentapeptide insertions were produced in WspR, and the effects determined using assays based on colony morphology, attachment to surfaces and cellulose production. The effects of mutations within WspR were interpreted using a homology model, based on the crystal structure of Caulobacter crescentus PleD. Mutational analyses indicated that WspR activation occurs as a result of disruption of the interdomain interface, leading to the release of effector-domain repression by the N-terminal receiver domain. Quantification of attachment and cellulose production raised significant questions concerning the mechanisms of WspR function. The conserved RYGGEEF motif of WspR was also subjected to mutational analysis, and 76 single amino acid residue substitutions were tested for their effects on WspR function. The RYGGEEF motif of WspR is functionally conserved, with almost every mutation abolishing function.

The significance of the soxS gene product on chemotrophic sulfur oxidation of Paracoccus pantotrophus was investigated. The thioredoxin SoxS was purified, and the N-terminal amino acid sequence identified SoxS as the soxS gene product. The wild-type formed thiosulfate-oxidizing activity and Sox proteins during mixotrophic growth with succinate plus thiosulfate, while there was no activity, and only traces of Sox proteins, under heterotrophic conditions. The homogenote mutant strain GBΩS is unable to express the soxSR genes, of which soxR encodes a transcriptional regulator. Strain GBΩS cultivated mixotrophically showed about 22 % of the specific thiosulfate-dependent O2 uptake rate of the wild-type, and when cultivated heterotrophically it produced 35 % activity. However, under both mixotrophic and heterotrophic conditions, strain GBΩS formed Sox proteins essential for sulfur oxidation in vitro at the same high level as the wild-type produced them during mixotrophic growth. Genetic complementation of strain GBΩS with soxS restored the activity upon mixotrophic and heterotrophic growth. Chemical complementation by reductants such as l-cysteine, DTT and tris(2-carboxyethyl)phosphine also restored the activity of strain GBΩS in the presence of chloramphenicol, which is an inhibitor of de novo protein synthesis. The data demonstrate that SoxS plays a key role in activation of the Sox enzyme system, and this suggests that SoxS is part of a novel type of redox control in P. pantotrophus.

The twin-arginine translocation (Tat) pathway transports folded proteins across bacterial cytoplasmic membranes. The Tat system of Streptomyces lividans consists of TatA, TatB and TatC, unlike most Gram-positive bacteria, which only have TatA and TatC subunits. Interestingly, in S. lividans TatA and TatB are localized in both the cytoplasm and the membrane. In the cytoplasm soluble TatA and TatB were found as monomers or as part of a hetero-oligomeric complex. Further analysis showed that specific information for recognition of the precursor by the soluble Tat components is mainly present in the twin-arginine signal peptide. Study of the role of the Tat subunits in complex assembly and stability in the membrane and cytoplasm showed that TatB stabilizes TatC whereas a key role in driving Tat complex assembly is suggested for TatC. Finally, by analysis of the oligomeric properties of TatA in the membrane of S. lividans and study of the affinity of membrane-embedded TatA for Tat/Sec precursors, a role for TatA as a translocator is postulated.

The bacterial genus Streptomyces has long been appreciated for its ability to produce various kinds of medically important secondary metabolites, such as antibiotics, anti-tumour agents, immunosuppressants and enzyme inhibitors. Tautomycetin (TMC), which is produced by Streptomyces sp. CK4412, is a novel activated T cell-specific immunosuppressive compound with an ester bond linkage between a terminal cyclic anhydride moiety and a linear polyketide chain bearing an unusual terminal alkene. Using a Streptomyces polyketide methylmalonyl-CoA acyltransferase gene as a probe, three overlapping cosmids were isolated from the genomic library of TMC-producing Streptomyces sp. CK4412. Sequence information of an approximately 70 kb contiguous DNA region revealed two multi-modular type I polyketide synthases (PKSs), and 12 additional gene products presumably involved in TMC biosynthesis. The deduced roles for most of the TMC PKS catalytic domains were consistent with the expected functions necessary for TMC chain elongation and processing. In addition, disruption of a putative TMC acyl-CoA transferase gene, located upstream of the PKS gene locus, completely abolished TMC biosynthesis. Taken together, these data provide strong supporting evidence that the cloned gene cluster identified in this study is responsible for TMC biosynthesis in Streptomyces sp. CK4412, and set the stage for detailed genetic and biochemical studies of the biosynthesis of this important metabolite.

Iron is essential for the survival of almost all organisms, although excess iron can result in the generation of free radicals which are toxic to cells. To avoid the toxic effects of free radicals, the concentration of intracellular iron is generally regulated by the ferric uptake regulator Fur in bacteria. The 150 aa fur ORF from Listeria monocytogenes was cloned into pRSETa, and the His-tagged fusion protein was purified by nickel affinity column chromatography. DNA binding activity of this protein was studied by an electrophoretic mobility shift assay using the end-labelled promoters PfhuDC and Pfur. The results showed a decrease in migration for both promoter DNAs in the presence of the Fur protein, and the change in migration was competitively inhibited with an excess of the same unlabelled promoters. No shift in migration was observed when a similar assay was performed using non-specific end-labelled DNA. The assay showed that binding of Fur to Pfur or PfhuDC was independent of iron or manganese ions, and was not inhibited in the presence of 2 mM EDTA. Inductively coupled plasma MS of the Fur protein showed no iron or manganese, but 0.48 mole zinc per mole protein was detected. A DNase I protection assay revealed that Fur specifically bound to and protected a 19 bp consensus Fur box sequence located in the promoters of fur and fhuDC. There was no requirement for iron or manganese in this assay also. However, Northern blot analysis showed an increase in fur transcription under iron-restricted compared to high-level conditions. Thus, the study suggests that under in vitro conditions, the affinity of the Fur protein for the 19 bp Fur box sequence does not require iron, but iron availability regulates fur transcription in vivo. Thus, the regulation by Fur in this intracellular pathogen may be dependent on either the structure of the DNA binding domain or other intracellular factors yet to be identified.

The abundant proteolytic plasminogen (Plg)/plasmin system is important in several physiological functions in mammals and also engaged by a number of pathogenic microbial species to increase tissue invasiveness or to obtain nutrients. This paper reports that a commensal bacterium, Lactobacillus crispatus, interacts with the Plg system. Strain ST1 of L. crispatus enhanced activation of human Plg by the tissue-type Plg activator (tPA), whereas enhancement of the urokinase-mediated Plg activation was lower. ST1 cells bound Plg, plasmin and tPA only poorly, and the Plg-binding and activation-enhancing capacities were associated with extracellular material released from the bacteria into buffer. The extracellular proteome of L. crispatus ST1 contained enolase and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as major components. The enolase and the GAPDH genes of ST1 were cloned, sequenced and expressed in recombinant Escherichia coli as His6-fusion proteins, which bound Plg and enhanced its activation by tPA. Variable levels of secretion of enolase and GAPDH proteins as well as of the Plg activation cofactor function were detected in strains representing major taxonomic groups of the genus Lactobacillus. So far, interference with the Plg system has been addressed with pathogenic microbes. The results reported here demonstrate a novel interaction between a member of the microbiota and a major proteolytic system in humans.

The lead enzymes of polyamine biosynthesis, i.e. ornithine decarboxylase (ODC) and arginine decarboxylase (ADC), were not detected in Toxoplasma gondii [the limit of detection for ODC and ADC was 5 pmol min−1 (mg protein)−1], indicating that T. gondii lacks a forward-directed polyamine biosynthetic pathway, and is therefore a polyamine auxotroph. The biochemical results were supported by results obtained from data-mining the T. gondii genome. However, it was possible to demonstrate the presence of a highly active backconversion pathway that formed spermidine from spermine, and putrescine from spermidine, via the combined action of spermidine/spermine N 1-acetyltransferase (SSAT) or spermidine N 1-acetyltransferase (SAT) and polyamine oxidase (PAO). With spermine as the substrate, T. gondii SSAT had a specific activity of 1.84 nmol min−1 (mg protein)−1, and an apparent K m for spermine of 180 mM; with spermidine as the substrate, the SAT had a specific activity of 3.95 nmol min−1 (mg protein)−1, and a K m for spermidine of 240 mM. T. gondii PAO had a specific activity of 10.6 nmol min−1 (mg protein)−1, and a K m for acetylspermine of 36 mM. Furthermore, the results demonstrated that T. gondii SSAT was 50 % inhibited by 30 mM di(ethyl)norspermine. The parasite actively transported arginine and ornithine, which were converted via the arginine dihydrolase pathway to citrulline and carbamoyl phosphate, resulting in the formation of ATP via carbamate kinase. The lack of polyamine biosynthesis by T. gondii is contrasted with polyamine metabolism by other apicomplexans.

Salmonella thin aggregative fimbriae (Tafi; curli) are important in pathogenesis and biofilm formation; however, less is known of their structure and morphogenesis. In the Salmonella agfBAC Tafi operon, the transcription and role of agfC have been elusive. In this study, agfBAC transcripts were detected using a sensitive reverse transcriptase technique. Native AgfC was not detected using polyclonal antibodies generated against purified hexahistidine-tagged AgfC; however, in trans expression revealed that AgfC was localized to the periplasm as a mature form. An isogenic ΔagfC mutant displayed an abundance of 20 nm fibres, in addition to native Tafi (5–7 nm), and had an increase in cell surface hydrophobicity. Purified 20 nm fibres were depolymerized under exceptionally stringent conditions to release what proved to be AgfA subunits. This revealed that the 20 nm fibres represented a different form of Tafi. The role of AgfC in Tafi assembly was investigated further using an antibody-capture assay of isogenic Δagf mutants. A soluble antibody-accessible form of AgfA was captured in wild-type (wt), ΔagfB and ΔagfF strains, in support of the extracellular nucleation–precipitation pathway of Tafi assembly, but not in ΔagfC or ΔagfE mutants. This indicates that AgfC and AgfE are important for AgfA extracellular assembly, facilitating the synthesis of Tafi.

Nitriles are important environmental compounds, both as natural products and industrial pollutants. Until now, there have been no data on the possibility of microbial nitrile degradation at high pH/salt conditions. Acetonitrile (CH3C≡N) is the simplest organic nitrile. Here, evidence is provided of microbial utilization of acetonitrile as a carbon, energy and nitrogen source at extremely high pH and moderate salinity. Positive enrichment cultures with acetonitrile at pH 10 and salt content equivalent to 0.6 M total Na+ were obtained from mixed sediment samples from soda lakes, but not from soda soils. Purification of these cultures resulted in the isolation of two bacterial strains capable of growth with acetonitrile as sole carbon, energy and nitrogen source under haloalkaline conditions. Apart from acetonitrile, the bacteria also grew with propionitrile. Nitrile hydrolysis to acetamide was identified as the rate-limiting step of acetonitrile degradation via the nitrile hydratase/amidase pathway. The new bacteria belonged to moderately salt-tolerant obligate alkaliphiles with optimum growth at pH 10 and 0.5 M total Na+. The cells were yellow-coloured due to a high concentration of carotenoids dominated by zeaxanthin. Phylogenetic analysis placed the isolates into a new lineage within the family Ectothiorhodospiraceae in the Gammaproteobacteria. On the basis of unique phenotypic properties and their separate phylogenetic position, the new bacteria are placed into a new genus and species for which the name Natronocella acetinitrilica gen. nov., sp. nov is proposed.

The RcsCDB phosphorelay was originally identified as the main regulator of colanic acid biosynthesis in Escherichia coli K-12. However, recent transcriptomic analyses have identified more than 150 genes belonging to the Rcs regulon, including yjbE, yjbF, yjbG and yjbH. These genes are clustered on the genome and oriented in the same direction but their function remains unknown. In this work it is shown that yjbE, yjbF, yjbG and yjbH are transcribed as a single operon and it is confirmed that the expression of this operon is controlled by the Rcs phosphorelay, in a manner that is dependent on the auxiliary regulatory protein RcsA. Interestingly, Northern blot analysis revealed that the amount of yjbE transcripts in the cell is higher than the amount of yjbEFGH transcripts and it is proposed that this differential expression is mediated by the presence of a strong stem–loop structure in the yjbE-yjbF intergenic region. Finally, evidence is provided that the overexpression of yjbEFGH affects colony morphology and leads to the production of an extracellular polysaccharide that binds Congo red and toluidine blue-O.

Pseudomonas aeruginosa is an opportunistic pathogen that causes serious respiratory disease in the immune-compromised host. Using an aerosol infection model, 11 inbred mouse strains (129/Sv, A/J, BALB/c, C3H/HeN, C57BL/6, DBA/2, FVB, B10.D2/oSnJ, B10.D2/nSnJ, AKR/J and SWR/J) were tested for increased susceptibility to P. aeruginosa lung colonization. DBA/2 was the only mouse strain that had increased bacterial counts in the lung within 6 h post-infection. This deficiency incited a marked inflammatory response with reduced bacterial lung clearance and a mortality rate of 96.7 %. DBA/2 mice displayed progressive deterioration of lung pathology with extensive alveolar exudate and oedema formation at 48–72 h post-infection. The neutrophil-specific myeloperoxidase activity remained elevated throughout infection, suggesting that the increased leukocyte infiltration into alveoli caused acute inflammatory lung injury. DBA/2 mice lack the haemolytic complement; however, three additional mouse strains (AKR/J, SWR/J and A/J) with the same defect effectively cleared the infection, indicating that other host factors are involved in defence. Bone marrow-derived macrophages of DBA/2 showed an initial increase in phagocytosis, while their bactericidal activity was reduced compared to that of C57BL/6 macrophages. Comparison of pulmonary cytokine profiles of DBA/2 versus C57BL/6 or C3H/HeN indicated that DBA/2 had similar increases in tumour necrosis factor (TNF)-α, KC and interleukin (IL)-1a as C3H/HeN, but showed specific induction of IL-17, monocyte chemotactic protein (MCP)-1 and vascular endothelial growth factor (VEGF). Together, DBA/2 mice have a defect in the initial lung defence against P. aeruginosa colonization, which causes the host to produce a greater, but damaging, inflammatory response. Such a response may originate from the reduced antimicrobial activity of DBA/2 macrophages.

The sequencing of prfA, encoding the transcriptional regulator of virulence genes, in 26 low-virulence field Listeria monocytogenes strains showed that eight strains exhibited the same single amino-acid substitution: PrfAK220T. These strains exhibited no expression of PrfA-regulated proteins and thus no virulence. This substitution inactivated PrfA, since expression of the PrfAK220T mutant gene in an EGDΔprfA strain did not restore the haemolytic and phosphatidylcholine phospholipase C activities, in contrast to the wild-type prfA gene. The substitution of the lysine at position 220 occurred in the helix αH. However, the data showed that the PrfAK220T protein is dimerized just as well as its wild-type counterpart, but does not bind to PrfA-boxes. PrfAK220T did not form a PrfA–DNA complex in electrophoretic mobility shift assays, but low concentrations of CI complexes (PrfAK220T–RNA polymerase–DNA complex) were formed by adding RNA polymerase, suggesting that PrfA interacted with RNA polymerase in solution in the absence of DNA. Formation of some transcriptionally active complexes was confirmed by in vitro runoff transcription assays and quantitative RT-PCR. Crystallographic analyses described the structure of native PrfA and highlighted the key role of allosteric changes in the activity of PrfA and especially the role of the Lys220 in the conformation of the helix–turn–helix (HTH) motif.

Helicobacter hepaticus colonizes the murine intestine and has been associated with hepatic inflammation and neoplasia in susceptible mouse strains. In this study, the catalase of an enterohepatic Helicobacter was characterized for the first time. H. hepaticus catalase is a highly conserved enzyme that may be important for bacterial survival in the mammalian intestine. Recombinant H. hepaticus catalase was expressed in Escherichia coli in order to verify its enzymic activity in vitro. H. hepaticus catalase comprises 478 amino acids with a highly conserved haem-ligand domain. Three conserved motifs (R-F-Y-D, RERIPER and VVHAKG) in the haem-ligand domain and three surface-predicted motifs were identified in H. hepaticus catalase and are shared among bacterial and mammalian catalases. H. hepaticus catalase is present in the cytoplasmic and periplasmic compartments. Mice infected with H. hepaticus demonstrated immune responses to murine and H. hepaticus catalase, suggesting that Helicobacter catalase contains conserved structural motifs and may contribute to autoimmune responses. Antibodies to H. hepaticus catalase recognized murine hepatocyte catalase in hepatic tissue from infected mice. Antibodies from sera of H. hepaticus-infected mice reacted with peptides comprising two conserved surface-predicted motifs in H. hepaticus catalase. Catalases are highly conserved enzymes in bacteria and mammals that may contribute to autoimmune responses in animals infected with catalase-producing pathogens.