- Volume 153, Issue 10, 2007

Osmoregulated periplasmic glucans (OPGs) are produced by many proteobacteria and are important for bacterial–host interactions. The opgG and opgH genes involved in the synthesis of OPGs are the most widely distributed genes in proteobacterial genomes. Two other non-homologous genes, both named ndvB, are also involved in OPG biosynthesis in several species. The Pseudomonas aeruginosa genome possesses two ORFs, PA5077 and PA5078, that show similarity to opgH and opgG of Pseudomonas syringae, respectively, and one ORF, PA1163, similar to ndvB of Sinorhizobium meliloti. Here, we report that the opgGH locus of P. aeruginosa PA14 is involved in the synthesis of linear polymers with β-1,2-linked glucosyl residues branched with a few β-1,6 glucosyl residues. Succinyl residues also substitute this glucose backbone. Transcription of opgGH is repressed by high osmolarity. Low osmolarity promotes the formation of highly structured biofilms, but biofilm development is slower and the area of biomass is reduced under high osmolarity. Biofilm development of an opgGH mutant grown under low osmolarity presents a similar phenotype to the wild-type biofilm grown under high osmolarity. These results suggest that OPGs are important for biofilm formation under conditions of low osmolarity. A previous study suggested that the P. aeruginosa ndvB gene is involved in the resistance of biofilms to antibiotics. We have shown that ndvB is not involved in the biosynthesis of the OPG described here, and opgGH do not appear to be involved in the resistance of P. aeruginosa PA14 biofilms to antibiotics.

The gene xynD (renamed pgdA) of Lactococcus lactis IL1403 was shown to encode a peptidoglycan N-acetylglucosamine deacetylase. Inactivation of pgdA in L. lactis led to fully acetylated peptidoglycan, whereas cloning of pgdA on a multicopy plasmid vector resulted in an increased degree of peptidoglycan deacetylation, as shown by analysis of peptidoglycan constituent muropeptides. An increased amount of N-unsubstituted glucosamine residues in peptidoglycan resulted in a reduction of the rate of autolysis of L. lactis cells. The activity of the L. lactis major autolysin AcmA was tested on L. lactis cells or peptidoglycan with different degrees of de-N-acetylation. Deacetylated peptidoglycan exhibited decreased susceptibility to AcmA hydrolysis. This reduced susceptibility to AcmA did not result from reduced AcmA binding to peptidoglycan with an increasing degree of de-N-acetylation. In conclusion, enzymic N-acetylglucosamine deacetylation protects peptidoglycan from hydrolysis by the major autolysin AcmA in L. lactis cells, and this leads to decreased cellular autolysis.

Invariant and highly conserved amino acids within a primase consensus sequence were targeted by site-specific mutations within the putative primase of Streptococcus thermophilus phage κ3. PCR products containing the desired mutation(s) within putative ATPase/helicase and/or oligomerization domains of the κ3-encoded primase gene were cloned into a high-copy-number vector and expressed in S. thermophilus NCK1125. The majority of the plasmid constructs failed to alter phage sensitivity; however, four of the constructs conferred strong phage resistance upon the host. Expression of the K238(A/T) and RR340-341AA mutant proteins in trans suppressed the function of the native phage primase protein in a dominant negative fashion via a proposed subunit poisoning mechanism. These constructs completely inhibited phage genome synthesis and reduced the efficiencies of plaquing and centre of infection formation by more than 9 and 3.5 logs, respectively. Amber mutations introduced upstream of the transdominant RR340-341AA and K238(A/T) mutations restored phage genome replication and sensitivity of the host, indicating that translation was required to confer phage resistance. Introduction of an E437A mutation in a putative oligomerization domain located downstream of the transdominant K238T mutation also completely suppressed phage resistance. This study appears to represent the first use of transdominant proteins to inhibit phages that are disruptive to cultures used in industrial fermentations.

Two unrelated protein families catalyse the oxidative decarboxylation of 2-oxoacids, i.e. the 2-oxoacid dehydrogenase complexes (OADHCs) and the 2-oxoacid ferredoxin oxidoreductases (OAFORs). In halophilic archaea, OAFORs were found to be responsible for decarboxylation of pyruvate and 2-oxoglutarate. Nevertheless, two gene clusters encoding OADHCs were found previously in Haloferax volcanii, but their biological function remained obscure. Here a third oadhc gene cluster of H. volcanii is presented. To characterize the function, the genes encoding the E1 subunit were inactivated in all three gene clusters by in-frame deletions. Under aerobic conditions none of the three mutants showed any phenotypic difference from the wild-type in various media. However, growth yields of two mutants were considerably lower than that of wild-type under nitrate-respirative conditions in complex medium. Northern blot analyses revealed (1) that polycistronic transcripts are formed and all three gene clusters are bona fide operons and (2) that transcription of all three operons is induced under anaerobic conditions compared to aerobic conditions. Taken together, the three H. volcanii enzymes do not fulfil one of the ‘usual’ aerobic functions of typical OADHCs, but decarboxylate an as-yet-unidentified novel substrate under anaerobic conditions. A survey of all 28 fully sequenced archaeal genomes revealed that nearly all archaea contain several OAFORs (three to four on average), suggesting that this protein family was already present in their last common ancestor. In contrast, only nine archaea encode one or two OADHCs, indicating that this protein family entered archaea by lateral transfer of the cognate genes from bacteria. This view is underscored by a phylogenetic tree of 33 archaeal and bacterial OADHCs.

Flavonoids comprise a large group of bioactive polyphenolic plant secondary metabolites. Several of these possess potent in vivo activity against Escherichia coli and Plasmodium falciparum, targeting enzymes involved in fatty acid biosynthesis, such as enoyl-ACP-reductase, β-ketoacyl-ACP reductase and β-hydroxyacyl-ACP dehydratase. Herein, we report that butein, isoliquirtigenin, 2,2′,4′-trihydroxychalcone and fisetin inhibit the growth of Mycobacterium bovis BCG. Furthermore, in vitro inhibition of the mycolic-acid-producing fatty acid synthase II (FAS-II) of Mycobacterium smegmatis suggests a mode of action related to those observed in E. coli and P. falciparum. Through a bioinformatic approach, we have established the product of Rv0636 as a candidate for the unknown mycobacterial dehydratase, and its overexpression in M. bovis BCG conferred resistance to growth inhibition by butein and isoliquirtigenin, and relieved inhibition of fatty acid and mycolic acid biosynthesis in vivo. Furthermore, after overexpression of Rv0636 in M. smegmatis, FAS-II was less sensitive to these inhibitors in vitro. Overall, the data suggest that these flavonoids are inhibitors of mycobacterial FAS-II and in particular Rv0636, which represents a strong candidate for the β-hydroxyacyl-ACP dehydratase enzyme of M. tuberculosis FAS-II.

DnaB and DnaI proteins conserved in low-GC content Gram-positive bacteria are apparently involved in helicase loading at the replication initiation site and during the restarting of stalled replication forks. In this study, we found five novel dnaB mutants and three novel dnaI mutants by screening 750 temperature-sensitive Gram-positive Staphylococcus aureus mutants. All of the mutants had a single amino acid substitution in either DnaB or DnaI that controlled temperature-sensitive growth, as confirmed by transduction experiments using phage 80α. DNA synthesis as measured by [3H]thymine incorporation, origin-to-terminus ratios and flow cytometric analysis revealed that the dnaB and dnaI mutants were unable to initiate DNA replication at restrictive temperatures, which is similar to previous findings in Bacillus subtilis. Furthermore, some of the mutants were found to exhibit asynchrony in the initiation of DNA replication. Also, a fraction of the dnaI mutant cells showed arrested replication, and the dnaI mutant tested was sensitive to mitomycin C, which causes DNA lesions. These results suggest that DnaB and DnaI are required not only for replication initiation and but also for regulation of its synchrony, and they provide support for the involvement of DnaI activity in the restart of arrested replication forks in vivo.

Arcanobacterium pyogenes, an opportunistic pathogen of economically important food animals, is the causative agent of liver abscesses in feedlot cattle, osteomyelitis in turkeys, and pneumonia and arthritis in pigs. Previous studies identified the first A. pyogenes adhesin, CbpA, a protein located on the bacterial surface which has the ability to bind collagen and promotes adhesion to the host cells. The protein has an N-terminal ligand-binding region (region A) and a C-terminal repetitive domain (region B). In this study we found that CbpA bound to almost all the collagen types tested but not to other proteins, and it displayed a propensity to interact with several collagenous peptides derived by CNBr cleavage of type I and II collagens. The K D values of CbpA for type I and II collagens and collagen peptides determined by solid-phase binding assay and intrinsic tryptophan fluorescence were in the range of 1–15 nM. It was also found that CbpA and its A region bound fibronectin, and that collagen and fibronectin interacted with distinct subsites. Anti-CbpA antibodies were effective at inhibiting both binding of isolated CbpA and bacterial adhesion to immobilized collagen, suggesting that CbpA is a functional collagen-binding adhesin. Analysis of the immunological cross-reactivity of CbpA with antibodies against other bacterial collagen-binding proteins indicated that CbpA is immunologically related to ACE from Enterococcus faecalis but not to CNA from Staphylococcus aureus or Acm from Enterococcus faecium. Far-UV and near-UV circular dichroism spectra showed that full-length CbpA and its region A are mainly composed of β-sheet with only a minor α-helical component and that both the proteins have a well-defined tertiary structure.

The life cycle of the pathogen Leptospira interrogans involves stages outside and inside the host. Entry of L. interrogans from moist environments into the host is likely to be accompanied by the induction of genes encoding virulence determinants and the concomitant repression of genes encoding products required for survival outside of the host. The expression of the adhesin LigA, the haemolysin Sph2 (Lk73.5) and the outer-membrane lipoprotein LipL36 of pathogenic Leptospira species have been reported to be regulated by mammalian host signals. A previous study demonstrated that raising the osmolarity of the leptospiral growth medium to physiological levels encountered in the host by addition of various salts enhanced the levels of cell-associated LigA and LigB and extracellular LigA. In this study, we systematically examined the effects of osmotic upshift with ionic and non-ionic solutes on expression of the known mammalian host-regulated leptospiral genes. The levels of cell-associated LigA, LigB and Sph2 increased at physiological osmolarity, whereas LipL36 levels decreased, corresponding to changes in specific transcript levels. These changes in expression occurred irrespective of whether sodium chloride or sucrose was used as the solute. The increase of cellular LigA, LigB and Sph2 protein levels occurred within hours of adding sodium chloride. Extracellular Sph2 levels increased when either sodium chloride or sucrose was added to achieve physiological osmolarity. In contrast, enhanced levels of extracellular LigA were observed only with an increase in ionic strength. These results indicate that the mechanisms for release of LigA and Sph2 differ during host infection. Thus, osmolarity not only affects leptospiral gene expression by affecting transcript levels of putative virulence determinants but also affects the release of such proteins into the surroundings.

The anaerobic utilization of l-ascorbate by gene products of the ula regulon in Escherichia coli has been widely documented. Under aerobic conditions, we have shown that this metabolism is only functional in the presence of casein acid hydrolysate. Transcriptional fusions and proteomic analysis indicated that both the ula regulon and the yiaK-S operon are required for the aerobic utilization of this compound. The aerobic dissimilation of l-ascorbate shares the function of three paralogous proteins, UlaD/YiaQ, UlaE/YiaR and UlaF/YiaS, which encode a decarboxylase, a 3-epimerase and a 4-epimerase, respectively. In contrast, l-ascorbate enters the cells through the ula-encoded phosphotransferase transport system, but it is not carried by the yiaMNO-encoded ABC transporter. Proteomic analysis also indicated enhanced expression of the alkyl hydroperoxide reductase encoded by the ahpC gene, suggesting a response to oxidative stress generated during the aerobic metabolism of l-ascorbate. Control of ahpC expression by the OxyR global regulator in response to l-ascorbate concentration is consistent with the formation of hydrogen peroxide under our experimental conditions. The presence of certain amino acids such as proline, threonine or glutamine in the culture medium allowed aerobic l-ascorbate utilization by Escherichia coli cells. This effect could be explained by the ability of these amino acids to allow yiaK-S operon induction by l-ascorbate, thus increasing the metabolic flux of l-ascorbate dissimilation. Alternatively, these amino acids may slow the rate of l-ascorbate oxidation.

A putative prenyltransferase gene, Afu3g12930, was identified in the genome sequence of Aspergillus fumigatus. EAL92290, encoded by Afu3g12930, consists of 472 aa, with a molecular mass of about 53 kDa. The coding sequence of Afu3g12930 was cloned in pQE60, and overexpressed in Escherichia coli. The soluble His6-fusion protein was purified to apparent homogeneity, and characterized biochemically. The enzyme was found to catalyse the prenylation of Trp at the C-7 position of the indole moiety, in the presence of dimethylallyl diphosphate (DMAPP); therefore, it functions as a 7-dimethylallyltryptophan synthase (7-DMATS). The structure of the enzymic product was elucidated by NMR and MS analysis. K m values were 67 μM for DMAPP, and 137 μM for l-Trp. Geranyl diphosphate was not accepted as prenyl donor, while Trp-containing dipeptides were found to be aromatic substrates of 7-DMATS. 7-DMATS did not need divalent metal ions for its enzymic reaction, although Ca2+ enhanced the reaction velocity slightly. The enzyme is the second dimethylallyltryptophan synthase identified in A. fumigatus. Interestingly, it shares a sequence identity of only 31 % at the amino acid level with another known dimethylallyltryptophan synthase, FgaPT2, from the same fungus; FgaPT2 prenylates l-Trp at the C-4 position of the indole ring. Afu3g12930 belongs to a putative biosynthetic gene cluster consisting of eight genes. Orthologous clusters were also identified in the genome sequences of Neosartorya fischeri and Aspergillus terreus. The putative roles of the genes in the cluster are discussed.

Hydrophobins are small amphipathic proteins that function in a broad range of growth and developmental processes in fungi. They are involved in the formation of aerial structures, the attachment of fungal cells to surfaces, and act in signalling in response to surface cues and pathogenesis. Beauveria bassiana is an important entomopathogenic fungus used as an arthropod biological control agent. To examine the feasibility of using phage display technology to clone cDNAs encoding hydrophobins, biopanning experiments were performed using a variety of affinity resins, including N,N′-diacetylchitobiose-, fucose-, lactose-, maltose- and melibiose-coupled agarose beads. After five rounds of iterative biopanning, cDNAs corresponding to two B. bassiana (class I) hydrophobins were selectively enriched using melibiose- or lactose-coupled agarose beads. Expression analysis revealed that the hyd1 gene was expressed in all samples tested, including aerial conidia, in vitro blastospores, submerged conidia, and cells sporulating on chitin and insect cuticle, with hyd1 expression peaking in growing mycelia. In contrast, the hyd2 gene was not appreciably expressed in any of the single-cell types (aerial conidia, blastospores and submerged conidia), but was constitutively expressed in growing mycelia and when cells were sporulating on chitin and insect cuticle. MS fingerprinting of an ∼10 kDa protein found in boiling SDS-insoluble, trifluoroacetic acid-soluble extracts from aerial conidia identified the major component of the B. bassiana rodlet layer to be the hyd2 gene product. These results reveal the differential regulation of the isolated hydrophobins and indicate that phage display represents a novel approach to cDNA cloning of hydrophobins.

‘Endomicrobia’, a distinct and diverse group of uncultivated bacteria in the candidate phylum Termite Group I (TG-1), have been found exclusively in the gut of lower termites and wood-feeding cockroaches. In a previous study, we had demonstrated that the ‘Endomicrobia’ clones retrieved from Reticulitermes santonensis represent intracellular symbionts of the two major gut flagellates of this termite. Here, we document that ‘Endomicrobia’ are present also in many other gut flagellates of lower termites. Phylogeny and host specificity of ‘Endomicrobia’ were investigated by cloning and sequencing of the small subunit rRNA genes of the flagellate and the symbionts, which originated from suspensions of individual flagellates isolated by micropipette. Each flagellate harboured a distinct phylogenetic lineage of ‘Endomicrobia’. The results of fluorescent in situ hybridization with ‘Endomicrobia’-specific oligonucleotide probes corroborated that ‘Endomicrobia’ are intracellular symbionts specifically affiliated with their flagellate hosts. Interestingly, the ‘Endomicrobia’ sequences obtained from flagellates belonging to the genus Trichonympha formed a monophyletic group, suggesting co-speciation between symbiont and host.

Dermatophytes are keratinophilic fungi able to infect keratinized tissues of human or animal origin. Among them, Trichophyton mentagrophytes is known to be a species complex composed of several species or variants, which occur in both human and animals. Since the T. mentagrophytes complex includes both anthropophilic and zoophilic pathogens, accurate molecular identification is a critical issue for comprehensive understanding of the clinical and epidemiological implications of the genetic heterogeneity of this complex. Here, 41 T. mentagrophytes isolates from either human patients (14 isolates) or animals (27 isolates) with dermatophytosis were prospectively isolated by culture and identified on morphological bases at the University Hospital Centres of Lille and Poitiers, and the Veterinary School of Alfort, respectively. The isolates were differentiated by DNA sequencing of the variable internal transcribed spacer (ITS) regions flanking the 5.8S rDNA, and of the housekeeping gene encoding the manganese-containing superoxide dismutase (MnSOD), an enzyme which is involved in defence against oxidative stress and has previously provided interesting insight into both fungal taxonomy and phylogeny. ITS1-ITS2 regions and MnSOD sequences successfully differentiate between members of the T. mentagrophytes complex and the related species Trichophyton rubrum. Whatever the phylogenetic marker used, members of this complex were classified into two major clades exhibiting a similar topology, with a higher variability when the ITS marker was used. Relationships between ITS/MnSOD sequences and host origin, clinical pattern and phenotypic characteristics (macroscopic and microscopic morphologies) were analysed.

Dissimilatory adenosine-5′-phosphosulfate (APS) reductase (AprBA) is a key enzyme of the dissimilatory sulfate-reduction pathway. Homologues have been found in photo- and chemotrophic sulfur-oxidizing prokaryotes (SOP), in which they are postulated to operate in the reverse direction, oxidizing sulfite to APS. Newly developed PCR assays allowed the amplification of 92–93 % (2.1–2.3 kb) of the APS reductase locus aprBA. PCR-based screening of 116 taxonomically divergent SOP reference strains revealed a distribution of aprBA restricted to photo- and chemotrophs with strict anaerobic or at least facultative anaerobic lifestyles, including Chlorobiaceae, Chromatiaceae, Thiobacillus, Thiothrix and invertebrate symbionts. In the AprBA-based tree, the SOP diverge into two distantly related phylogenetic lineages, Apr lineages I and II, with the proteins of lineage II (Chlorobiaceae and others) in closer affiliation to the enzymes of the sulfate-reducing prokaryotes (SRP). This clustering is discordant with the dissimilatory sulfite reductase (DsrAB) phylogeny and indicates putative lateral aprBA gene transfer from SRP to the respective SOB lineages. In support of lateral gene transfer (LGT), several beta- and gammaproteobacterial species harbour both aprBA homologues, the DsrAB-congruent ‘authentic’ and the SRP-related, LGT-derived gene loci, while some relatives possess exclusively the SRP-related apr genes as a possible result of resident gene displacement by the xenologue. The two-gene state might be an intermediate in the replacement of the resident essential gene. Collected genome data demonstrate the correlation between the AprBA tree topology and the composition/arrangement of the apr gene loci (occurrence of qmoABC or aprM genes) from SRP and SOP of lineages I and II. The putative functional role of the SRP-related APS reductases in photo- and chemotrophic SOP is discussed.

Marked differences in surface characteristics were observed among three types of single-cell propagules produced by the entomopathogenic fungus Beauveria bassiana. Atomic force microscopy (AFM) revealed the presence of bundles or fascicles in aerial conidia absent from in vitro blastospores and submerged conidia. Contact angle measurements using polar and apolar test liquids placed on cell layers were used to calculate surface tension values and the free energies of interaction of the cell types with surfaces. These analyses indicated that the cell surfaces of aerial conidia were hydrophobic, whereas those of blastospores and submerged conidia were hydrophilic. Zeta potential determinations of the electrostatic charge distribution across the surface of the cells varied from +22 to −30 mV for 16-day aerial conidia at pH values ranging from 3 to 9, while the net surface charge ranged from +10 to −13 mV for submerged conidia, with much less variation observed for blastospores, +4 to −4 mV, over the same pH range. Measurements of hydrophobicity using microbial adhesion to hydrocarbons (MATH) indicated that the surfaces of aerial conidia were hydrophobic, and those of blastospores hydrophilic, whereas submerged conidia displayed cell surface characteristics on the borderline between hydrophobic and hydrophilic. Insect pathology assays using tobacco budworm (Heliothis virescens) larvae revealed some variation in virulence among aerial conidia, in vitro blastospores and submerged conidia, using both topical application and haemocoel injection of the fungal cells.