- Volume 152, Issue 12, 2006

Vfr, a global regulator of Pseudomonas aeruginosa virulence factors, is a homologue of the Escherichia coli cAMP receptor protein, CRP. Vfr is 91 % similar to CRP and maintains many residues important for CRP to bind cAMP, bind DNA, and interact with RNA polymerase at target promoters. While vfr can complement an E. coli crp mutant in β-galactosidase production, tryptophanase production and catabolite repression, crp can only complement a subset of Vfr-dependent phenotypes in P. aeruginosa. Using specific CRP binding site mutations, it is shown that Vfr requires the same nucleotides as CRP for optimal transcriptional activity from the E. coli lac promoter. In contrast, CRP did not bind Vfr target sequences in the promoters of the toxA and regA genes. Footprinting analysis revealed Vfr protected sequences upstream of toxA, regA, and the quorum sensing regulator lasR, that are similar to but significantly divergent from the CRP consensus binding sequence, and Vfr causes similar DNA bending to CRP in bound target sequences. Using a preliminary Vfr consensus binding sequence deduced from the Vfr-protected sites, Vfr target sequences were identified upstream of the virulence-associated genes plcN, plcHR, pbpG, prpL and algD, and in the vfr/orfX, argH/fimS, pilM/ponA intergenic regions. From these sequences the Vfr consensus binding sequence, 5′-ANWWTGNGAWNY : AGWTCACAT-3′, was formulated. This study suggests that Vfr shares many of the same functions as CRP, but has specialized functions, at least in terms of DNA target sequence binding, required for regulation of a subset of genes in its regulon.

Intrinsic resistance to drugs is one of the main determining factors in bacterial survival in the intestinal ecosystem. This is mediated by, among others, multidrug resistance (MDR) transporters, membrane proteins which extrude noxious compounds with very different chemical structures and cellular targets. Two genes from Bifidobacterium breve encoding hypothetical membrane proteins with a high homology with members of the ATP-binding cassette (ABC) family of multidrug efflux transporters, were expressed separately and jointly in Lactococcus lactis. Cells co-expressing both proteins exhibited enhanced resistance levels to the antimicrobials nisin and polymyxin B. Furthermore, the drug extrusion activity in membrane vesicles was increased when both proteins were co-expressed, compared to membranes in which the proteins were produced independently. Both proteins were co-purified from the membrane as a stable complex in a 1 : 1 ratio. This is believed to be the first study of a functional ABC-type multidrug transporter in Bifidobacterium and contributes to our understanding of the molecular mechanisms underlying the capacity of intestinal bacteria to tolerate cytotoxic compounds.

Meridamycin is a non-immunosuppressant, FKBP-binding macrocyclic polyketide, which has major potential as a neuroprotectant in a range of neurodegenerative disorders including dementia, Parkinson's disease and ischaemic stroke. A biosynthetic cluster predicted to encode biosynthesis of meridamycin was cloned from the prolific secondary-metabolite-producing strain Streptomyces sp. DSM 4137, not previously known to produce this compound, and specific gene deletion was used to confirm the role of this cluster in the biosynthesis of meridamycin. The meridamycin modular polyketide synthase consists of 14 extension modules distributed between three giant multienzyme proteins. The terminal module is flanked by a highly unusual cytochrome P450-like domain. The characterization of the meridamycin biosynthetic locus in this readily manipulated streptomycete species opens the way to the engineering of new, altered meridamycins of potential therapeutic importance.

Vibrio anguillarum serotype O2 strains produce a catechol siderophore named vanchrobactin, which has been identified as N-[N′-(2,3-dihydroxybenzoyl)-arginyl]-serine. This work describes a chromosomal region that harbours the genetic determinants necessary for the biosynthesis of vanchrobactin. The authors have identified the genes involved in 2,3-dihydroxybenzoic acid (DHBA) biosynthesis (vabA, vabB and vabC) and activation (vabE), and a gene (vabF) encoding a non-ribosomal peptide synthetase, which is putatively involved in the assembly of the siderophore components. Also described are the identification and characterization of genes encoding a putative vanchrobactin exporter (vabS) and a siderophore esterase (vabH). In-frame deletion mutants in vabA, vabB, vabC, vabE, vabF and vabH were impaired for growth under conditions of iron limitation, and the analysis of culture supernatants by chrome azurol-S and cross-feeding assays showed almost no production of siderophores in any of the vabABCEF mutants. In addition, deletion mutations of vabA, vabB and vabC abolished production of DHBA, as assessed by chemical and biological analyses. Complementation of each mutant with the corresponding gene provided in trans confirmed the involvement of this gene cluster in the biosynthesis of DHBA and vanchrobactin in V. anguillarum strain RV22. Based on chemical and genetic data, and on published models for other catechol siderophores, a model for vanchrobactin biosynthesis is proposed.

When grown anaerobically on a succinate+nitrate (SN) medium, Paracoccus denitrificans forms the membrane-bound, cytoplasmically oriented, chlorate-reducing nitrate reductase Nar, while the periplasmic enzyme Nap is expressed during aerobic growth on butyrate+oxygen (BO) medium. Preincubation of SN cells with chlorate produced a concentration-dependent decrease in nitrate utilization, which could be ascribed to Nar inactivation. Toluenization rendered Nar less sensitive to chlorate, but more sensitive to chlorite, suggesting that the latter compound may be the true inactivator. The Nap enzyme of BO cells was inactivated by both chlorate and chlorite at concentrations that were at least two orders of magnitude lower than those shown to affect Nar. Partial purification of Nap resulted in insensitivity to chlorate and diminished sensitivity to chlorite. Azide was specific for SN cells in protecting nitrate reductase against chlorate attack, the protective effect of nitrate being more pronounced in BO cells. The results are discussed in terms of different metabolic activation of chlorine oxoanions in both types of cells, and limited permeation of chlorite across the cell membrane.

Pseudomonas stutzeri strain A1501 isolated from rice fixes nitrogen under microaerobic conditions in the free-living state. This paper describes the properties of nifL and nifA mutants as well as the physical interaction between NifL and NifA proteins. A nifL mutant strain that carried a mutation non-polar on nifA expression retained nitrogenase activity. Complementation with a plasmid containing only nifL led to a decrease in nitrogenase activity in both the wild-type and the nifL mutant, suggesting that NifL acts as an antiactivator of NifA activity. Using the yeast two-hybrid system and purified protein domains of NifA and NifL, an interaction was shown between the C-terminal domain of NifL and the central domain of NifA, suggesting that NifL antiactivator activity is mediated by direct protein interaction with NifA.

Nisin-producing Lactococcus lactis protects its own cell membrane against the bacteriocin with the ABC transporter NisFEG, and the immunity lipoprotein NisI. In this study, in order to localize a site for specific nisin interaction in NisI, a C-terminal deletion series of NisI was constructed, and the C-terminally truncated NisI proteins were expressed in L. lactis. The shortest deletion (5 aa) decreased the nisin immunity capacity considerably in the nisin-negative strain MG1614, resulting in approximately 78 % loss of immunity function compared with native NisI. A deletion of 21 aa decreased the immunity level even more, but longer deletions, up to 74 aa, provided the same level of nisin immunity as the 21 aa deletion, i.e. approximately 14 % of the immunity provided by native NisI. Similar to native NisI, all the C-terminally truncated NisI proteins provided higher immunity to nisin in the NisFEG-expressing strain NZ9840 than in MG1614, i.e. approximately 40–50 % of the immunity capacity of native NisI. Then, it was determined whether the NisI C-terminal 21 aa fragment could protect cells against nisin. To target the 21 aa fragment to its natural location, 21 C-terminal amino acids from the subtilin-specific immunity lipoprotein SpaI were replaced by 21 C-terminal amino acids from NisI. The expression of the SpaI′–′NisI fusion in L. lactis strains significantly increased their nisin immunity. This is the first time the immunity function of a lantibiotic immunity protein has been transferred to another protein. However, unlike native NisI, and the C-terminally truncated NisI fragments, the increase in nisin immunity conferred by the SpaI′–′NisI fusion was the same in both the NisFEG strain NZ9840 and MG1614. In conclusion, the SpaI′–′NisI fusion could not enhance nisin immunity by interacting with NisFEG, whereas the C-terminally truncated NisI fragments and native NisI were able to enhance nisin immunity, probably by co-operation with NisFEG. The results made it evident that the C terminus of NisI is involved in specific interaction with nisin, and that it confers specificity for the NisI immunity lipoprotein.

Lysine biosynthesis of Thermus thermophilus proceeds in a similar way to arginine biosynthesis, and some lysine biosynthetic enzymes from T. thermophilus so far investigated have the potential to function in arginine biosynthesis. These observations suggest that arginine might regulate the expression of genes for lysine biosynthesis. To test this hypothesis, the argR gene encoding the regulator of arginine biosynthesis was cloned from T. thermophilus and its function in lysine biosynthesis was analysed. The addition of arginine to the culture medium inhibited the growth of an arginase gene knockout mutant of T. thermophilus, which presumably accumulates arginine inside the cells. Arginine-dependent growth inhibition was not alleviated by the addition of ornithine, which is a biosynthetic intermediate of arginine and serves as a peptidoglycan component of the cell wall in T. thermophilus. However, the growth inhibition was cancelled either by the simultaneous addition of lysine and ornithine or by a knockout of the argR gene, suggesting the involvement of argR in regulation of lysine biosynthesis in T. thermophilus. Electrophoretic mobility shift assay and DNase I footprinting revealed that the ArgR protein specifically binds to the promoter region of the major lysine biosynthetic gene cluster. Furthermore, an α-galactosidase reporter assay for this promoter indicated that arginine repressed the promoter in an argR-dependent manner. These results indicate that lysine biosynthesis is regulated by arginine in T. thermophilus.

Turnover of damaged molecules is considered to play a key role in housekeeping of cells exposed to oxidative stress, and during the progress of ageing. In this work, global changes in the transcriptome were analysed during recovery of yeast cells after H2O2 stress. Regarding induced genes, those associated with protein fate were the most significantly over-represented. In addition to genes encoding subunits of the 20S proteasome, genes related to vacuolar proteolysis (PEP4 and LAP4), protein sorting into the vacuole, and vacuolar fusion were found to be induced. The upregulation of PEP4 gene expression was associated with an increase in Pep4p activity. The induction of genes related to proteolysis was correlated with an increased protein turnover after H2O2-induced oxidation. Furthermore, protein degradation and the removal of oxidized proteins decreased in Pep4p-deficient cells. Pep4p activity also increased during chronological ageing, and cells lacking Pep4p displayed a shortened lifespan associated with higher levels of carbonylated proteins. PEP4 overexpression prevented the accumulation of oxidized proteins, but did not increase lifespan. These results indicate that Pep4p is important for protein turnover after oxidative damage; however, increased removal of oxidized proteins is not sufficient to enhance lifespan.

Secondary and tertiary amino groups were introduced into polymer chains grafted onto a polyethylene flat-sheet membrane to evaluate the effects of surface properties on the adhesion and viability of a strain of the Gram-negative bacterium Escherichia coli and a strain of the Gram-positive bacterium Bacillus subtilis. The characterization of the surfaces containing amino groups, i.e. ethylamino (EA) and diethylamino (DEA) groups, revealed that the membrane potentials are proportional to amino-group densities and contact angle hysteresis. A high bacterial adhesion rate constant k was observed at high membrane potential, which indicates that membrane potential could be used as an indicator for estimating bacterial adhesion to the EA and DEA sheets, especially in B. subtilis. The bacterial adhesion rate constant of E. coli markedly increased at a membrane potential higher than −7.8 mV, whereas that of B. subtilis increased at a membrane potential higher than −8.3 mV, at which the dominant effect on bacterial adhesion is expected to change. The viability experiments revealed that approximately 80 % of E. coli cells adhering to the sheets with high membrane potential were inactivated after a contact time of 8 h, whereas 60 % of B. subtilis cells were inactivated. Furthermore, E. coli viability significantly decreased at a membrane potential higher than −8 mV, whereas B. subtilis viability decreased as membrane potential increased, which reflects differences in cell wall structure between E. coli and B. subtilis.

All strains of the moderately thermophilic, acidophilic, sulphur-oxidizing bacterium Acidithiobacillus caldus that have been tested contain a set of chromosomal arsenic resistance genes. Highly arsenic-resistant strains isolated from commercial arsenopyrite bio-oxidation tanks contain additional transposon-located (TnAtcArs) arsenic resistance genes. The chromosomal At. caldus ars genes were cloned and found to consist of arsR and arsC genes transcribed in one direction, and arsB in the opposite direction. The arsRC genes were co-transcribed with ORF1, and arsB with ORF5 in both At. caldus and Escherichia coli, although deletion of ORFs 1 and 5 did not appear to affect resistance to arsenate or arsenite in E. coli. ORFs 1 and 5 have not previously been reported as part of the ars operons, and had high amino acid identity to hypothetical proteins from Polaromonas naphthalenivorus (76 %) and Legionella pneumophila (60 %), respectively. Reporter-gene studies showed that the arsenic operon of transposon origin (TnAtcArs) was expressed at a higher level, and was less tightly regulated in E. coli than were the At. caldus ars genes of chromosomal origin. Plasmid pSa-mediated conjugal transfer of TnAtcArs from E. coli to At. caldus strains lacking the transposon was successful, and resulted in greatly increased levels of resistance to arsenite.

The lvh region of the Legionella pneumophila genome, which encodes a type IV secretion system, is located on a plasmid-like element in strains Paris (pP36) and Philadelphia (pLP45). The pP36 element has been described either integrated in the chromosome or excised as a multi-copy plasmid, in a similar manner to pLP45. In this paper, the chromosomal integration of pP36 in the Paris strain genome was described, occurring through site-specific recombination at the 3′ end of a transfer-messenger RNA gene by recombination between attachment sites, in a similar manner to pathogenicity islands. This integration was growth-phase dependent, occurring during the exponential phase. Several pP36-borne genes were expressed during the lag phase of bacterial growth, coinciding with the peak amount of the episomal form of pP36. Expression of the same genes decreased during the exponential and stationary phases, owing to the integration phenomenon and a loss of episomal copies of pP36. A similar plasmid-like element was described in the Lens strain genome, suggesting that the mobility of the lvh region is a phenomenon widespread among Legionella sp.

The cholera toxin (CT) is a critical determinant of the virulence of epidemic Vibrio cholerae strains. The ctxAB operon encoding CT is part of the genome of a filamentous bacteriophage CTXΦ, which may integrate as a single copy or as multiple copies in the genome of V. cholerae. The CTXΦ genome is composed of RS2 (2.4 kb) and core (4.5 kb) regions. In the present study extensive genetic mapping analyses indicated that two copies of tandemly arrayed CTX prophages are integrated in the small chromosome of an environmental V. cholerae strain, VCE232, belonging to serogroup O4. Further mapping revealed that the integration of prophages has occurred in the same genetic locus of the small chromosome of VCE232 as that of V. cholerae O1 biotype El Tor strains. Interestingly, a new type of RS2-like element 3.5 kb in size was found in the CTX prophage genome in the small chromosome of VCE232. Cloning followed by sequencing of the new RS2-like element of VCE232 revealed the presence of three ORFs, which probably encode highly divergent types of phage regulatory proteins. Furthermore, the strain VCE232 also harbours two copies of a tandemly arranged CTX prophage devoid of the ctxAB genes, called pre-CTX prophage, in its large chromosome. The presence of multiple copies of diverse CTX prophages in both the chromosomes of VCE232 suggests that toxigenic environmental V. cholerae non-O1, non-O139 strains could play a role in the emergence of new epidemic clones.

During infection, the opportunistic fungal pathogen Candida albicans grows invasively into the tissues of its host, forming filaments that penetrate the host tissue. To search for genes that are important for invasive filamentation, a screen for mutants that were defective in invasion of agar medium was conducted. A mutant carrying an insertion mutation in the locus of a gene, termed here DRG1, was identified. DRG1 encodes a highly conserved cytoplasmic G protein, with orthologues in the genomes of organisms from humans to yeast and archaea. C. albicans strains lacking Drg1p were defective in producing filaments that penetrated agar media, but produced filaments normally under other conditions, such as during liquid growth. When inoculated intravenously into mice, the drg1 null mutant caused delayed lethality accompanied by delayed invasive growth in the kidneys of the host, in comparison with those of the wild-type strain. These results implicate Drg1p in the control of invasive filamentation in the laboratory, and in the progression of invasive disease in the host.

To better understand Neisseria meningitidis genomes and virulence, microarray comparative genome hybridization (mCGH) data were collected from one Neisseria cinerea, two Neisseria lactamica, two Neisseria gonorrhoeae and 48 Neisseria meningitidis isolates. For N. meningitidis, these isolates are from diverse clonal complexes, invasive and carriage strains, and all major serogroups. The microarray platform represented N. meningitidis strains MC58, Z2491 and FAM18, and N. gonorrhoeae FA1090. By comparing hybridization data to genome sequences, the core N. meningitidis genome and insertions/deletions (e.g. capsule locus, type I secretion system) related to pathogenicity were identified, including further characterization of the capsule locus, bioinformatics analysis of a type I secretion system, and identification of some metabolic pathways associated with intracellular survival in pathogens. Hybridization data clustered meningococcal isolates from similar clonal complexes that were distinguished by the differential presence of six distinct islands of horizontal transfer. Several of these islands contained prophage or other mobile elements, including a novel prophage and a transposon carrying portions of a type I secretion system. Acquisition of some genetic islands appears to have occurred in multiple lineages, including transfer between N. lactamica and N. meningitidis. However, island acquisition occurs infrequently, such that the genomic-level relationship is not obscured within clonal complexes. The N. meningitidis genome is characterized by the horizontal acquisition of multiple genetic islands; the study of these islands reveals important sets of genes varying between isolates and likely to be related to pathogenicity.