- Volume 151, Issue 7, 2005

A putative prenyltransferase gene, ftmPT1, was identified in the genome sequence of Aspergillus fumigatus. ftmPT1 was cloned and expressed in Escherichia coli, and the protein FtmPT1 was purified to near homogeneity and characterized biochemically. This enzyme was found to catalyse the prenylation of cyclo-l-trp-l-Pro (brevianamide F) at the C-2 position of the indole nucleus. FtmPT1 is a soluble monomeric protein, which does not contain the usual prenyl diphosphate binding site (N/D)DXXD found in most prenyltransferases, and which does not require divalent metal ions for its enzymic activity. K m values for brevianamide F and dimethylallyl diphosphate were determined as 55 and 74 μM, respectively. The turnover number was 5·57 s−1. FtmPT1 showed a high substrate specificity towards dimethylallyl diphosphate, but accepted different tryptophan-containing cyclic dipeptides. Together with dimethylallyltryptophan synthase of ergot alkaloid biosynthesis, FtmPT1 belongs to a new group of prenyltransferases with aromatic substrates.

Cysteine replacement of Asp190, Glu192 and Ser201 residues in the cytoplasmic interdomain loop of the TetA(B) tetracycline efflux antiporter from Tn10 reduces tetracycline resistance [ Tamura, N., Konishi, S., Iwaki, S., Kimura-Someya, T., Nada, S. & Yamaguchi, A. (2001). J Biol Chem 276, 20330–20339 ]. It was found that these Cys substitutions altered the substrate specificity of TetA(B), increasing the relative resistance to doxycycline and minocycline over that to tetracycline by three- to sixfold. Substitutions of Asp190 and Glu192 by Ala, Asn and Gln also impaired the ability of TetA(B) to mediate tetracycline resistance while Ser201Ala and Ser201Thr substitutions did not. A Leu9Phe substitution in the first transmembrane helix of TetA(B) suppressed the Ser201Cys mutation, undoing the alterations in resistance and specificity. That the interdomain loop might contact substrate during transport, as is suggested from its role in substrate specificity, is unexpected considering that the primary sequence in the loop is not conserved among a group of otherwise homologous TetA proteins. However, in the interdomain loop of 11 of 14 homologous TetA efflux proteins, computational analysis revealed a short α-helix, which includes some residues affecting activity and substrate specificity. Perhaps this conserved secondary structure accounts for the role of the non-conserved interdomain loop in TetA function.

β-Glucuronidase activity (encoded by the gus gene) has been characterized for the first time from Ruminococcus gnavus E1, an anaerobic bacterium belonging to the dominant human gut microbiota. β-Glucuronidase activity plays a major role in the generation of toxic and carcinogenic metabolites in the large intestine, as well as in the absorption and enterohepatic circulation of many aglycone residues with protective effects, such as lignans, flavonoids, ceramide and glycyrrhetinic acid, that are liberated by the hydrolysis of the corresponding glucuronides. The complete nucleotide sequence of a 4537 bp DNA fragment containing the β-glucuronidase locus from R. gnavus E1 was determined. Five ORFs were detected on this fragment: three complete ORFs (ORF2, gus and ORF3) and two partial ORFs (ORF4 and ORF5). The products of ORF2 and ORF3 show strong similarities with many β-glucoside permeases of the phosphoenolpyruvate : β-glucoside phosphotransferase systems (PTSs), such as Escherichia coli BglC, Bacillus subtilis BglP and Bacillus halodurans PTS Enzyme II. The product of ORF5 presents strong similarities with the amino-terminal domain of Clostridium acetobutylicum β-glucosidase (bglA). The gus gene product presents similarities with several known β-glucuronidase enzymes, including those of Lactobacillus gasseri (69 %), E. coli (61 %), Clostridium perfringens (59 %) and Staphylococcus aureus (58 %). By complementing an E. coli strain in which the uidA gene encoding the enzyme was deleted, it was confirmed that the R. gnavus gus gene encodes the β-glucuronidase enzyme. Moreover, it was found that the gus gene was transcribed as part of an operon that includes ORF2, ORF3 and ORF5.

Previous studies identified five nifH-like genes (nifH2 through nifH6) in Clostridium pasteurianum (strain W5), where the nifH1 gene encodes the nitrogenase iron protein. Transcripts of these nifH genes, with the exception of nifH3, were detected in molybdenum-sufficient nitrogen-fixing cells. However, the size of the transcripts, the level of transcription and the presence of polypeptides encoded by the nifH-like genes were not reported. The nifH2 and nifH6 genes were extremely similar, as they seemed to differ by only two bases in a span of 2481 bp, one in the coding region and another in the upstream region. Re-examination of the DNA sequences revealed that the coding region of nifH2 and nifH6 was identical, whereas the difference in the upstream region was confirmed. Results from the authors' ongoing study of the nif genes of single-colony isolates of C. pasteurianum suggest that the nifH6 designation should be eliminated. Here the size of mRNA from nifH2 and the detection of the NifH2 polypeptide in nitrogen-fixing cells of C. pasteurianum are reported. Northern blot analysis of periodically collected nitrogen-fixing cells showed that the nifH1 and nifH2 mRNAs were present throughout growth. Addition of ammonium acetate repressed the transcription of both these genes similarly. Using an antiserum raised against NifH of Azotobacter vinelandii, two NifH-related bands were detected by Western blot analysis after electrophoretic separation of proteins in extracts of nitrogen-fixing C. pasteurianum cells. After separation of proteins by preparative SDS-PAGE, the NifH polypeptides were characterized by MALDI-TOF-MS (matrix-assisted laser desorption/ionization time-of-flight mass spectrometry) and by ES-MS/MS (electrospray tandem mass spectrometry) analyses. The results confirmed the presence of NifH2, in addition to NifH1, in nitrogen-fixing C. pasteurianum cells.

The siderophore salmochelin is produced under iron-poor conditions by Salmonella and many uropathogenic Escherichia coli strains. The production of salmochelin, a C-glucosylated enterobactin, is dependent on the synthesis of enterobactin and the iroBCDEN gene cluster. An E. coli IroD protein with an N-terminal His-tag cleaved cyclic salmochelin S4 to the linear trimer salmochelin S2, the dimer salmochelin S1, and the monomers dihydroxybenzoylserine and C-glucosylated dihydroxybenzoylserine (salmochelin SX, pacifarinic acid). The periplasmic IroE protein was purified as a MalE–IroE fusion protein. This enzyme degraded salmochelin S4 and ferric-salmochelin S4 to salmochelin S2 and ferric-salmochelin S2, respectively. In E. coli, uptake of ferric-salmochelin S4 was dependent on the cleavage by IroE, and independent of the FepBDGC ABC transporter in the cytoplasmic membrane. IroC, which has similarities to ABC-multidrug-resistance proteins, was necessary for the uptake of salmochelin S2 from the periplasm into the cytoplasm. IroE did not function as a classical binding protein since salmochelin S2 was taken up in the absence of a functional IroE protein. IroC mediated the uptake of iron via enterobactin in a fepB mutant. IroE was also necessary in this case for the uptake of ferric-enterobactin, which indicated that only the linear degradation products of enterobactin were taken up via IroC. PfeE, the Pseudomonas aeruginosa IroE homologue, was cloned, and its enzymic activity was shown to be very similar to that of IroE. It is suggested that homologues in other bacteria are also periplasmic IroE-type esterases of siderophores.

The presence of a polar species of glycopeptidolipid (GPL) in carbon-starved Mycobacterium smegmatis has been reported previously. In this study, the complete structure of this GPL is established with the help of MALDI-TOF (matrix assisted laser desorption/ionization time of flight) and ESI (electrospray ionization) -MS, 13C-SEFT (spin echo Fourier transform) -NMR spectroscopy, and HPLC analysis. In the molecule, two units of a 3,4-di-O-methyl derivative of rhamnose are attached to l-alaninol via a 1→2 linkage. Various methyl derivatives of rhamnose and 6-deoxytalose were synthesized as standards to establish this structure. The accumulation of this polar GPL in M. smegmatis is sigB dependent, as a SigB-overproducing strain of M. smegmatis shows the presence of this spot in the exponential phase, and a sigB-knockout strain of M. smegmatis does not show the presence of any polar GPLs.

The catabolic potential for sterol degradation of fast-growing mycobacteria is well known. However, no genes or enzymes responsible for the steroid degradation process have been identified as yet in these species. One of the key enzymes required for degradation of the steroid ring structure is 3-ketosteroid Δ1-dehydrogenase (KsdD). The recent annotation of the Mycobacterium smegmatis genome (TIGR database) revealed six KsdD homologues. Targeted disruption of the MSMEG5898 (ksdD-1) gene, but not the MSMEG4855 (ksdD-2) gene, resulted in partial inactivation of the cholesterol degradation pathway and accumulation of the intermediate 4-androstene-3,17-dione. This effect was reversible by the introduction of the wild-type ksdD-1 gene into M. smegmatis ΔksdD-1 or overexpression of ksdD-2. The data indicate that KsdD1 is the main KsdD in M. smegmatis, but that KsdD2 is able to perform the cholesterol degradation process when overproduced.

Corynebacterium callunae and Corynebacterium efficiens are close relatives of the glutamate-producing mycolata species Corynebacterium glutamicum. The properties of the pore-forming proteins, extracted by organic solvents, were studied. The cell extracts contained channel-forming proteins that formed ion-permeable channels with a single-channel conductance of about 2 to 3 nS in 1 M KCl in a lipid bilayer assay. The corresponding proteins from both corynebacteria were purified to homogeneity and were named PorHC.call and PorHC.eff. Electrophysiological studies of the channels suggested that they are wide and water-filled. Channels formed by PorHC.call are cation-selective, whereas PorHC.eff forms slightly anion-selective channels. Both proteins were partially sequenced. A multiple sequence alignment search within the known chromosome of C. efficiens demonstrated that it contains a gene that fits the partial amino acid sequence of PorHC.eff. PorHC.call shows high homology to PorHC.eff. PorHC.eff is encoded in the bacterial chromosome by a gene that is localized within the vicinity of the porA gene of C. efficiens. PorHC.eff has no signal sequence at the N terminus, which means that it is not exported by the Sec-secretion pathway. The structure of PorH in the cell wall of the corynebacteria is discussed.

Vectors have been developed for inducible gene expression in Lactobacillus sakei and Lactobacillus plantarum in which expression of the gene of interest is driven by strong, regulated promoters from bacteriocin operons found in L. sakei strains. The activity of these promoters is controlled via a two-component signal transduction system, which responds to an externally added peptide pheromone. The vectors have a modular design, permitting easy exchange of all essential elements: the inducible promoter, the cognate regulatory system, the gene of interest, the antibiotic resistance marker and the replicon. Various variants of these so-called ‘pSIP’ vectors were constructed and tested, differing in terms of the bacteriocin regulon from which the regulatory elements were derived (sakacin A or sakacin P), the regulated promoter selected from these regulons, and the replicon (derived from p256 or pSH71). Using β-glucuronidase (GusA) and aminopeptidase N (PepN) as reporters, it was shown that the best vectors permitted inducible, pheromone-dose-dependent gene expression at very high levels, while displaying moderate basal activities when not induced. The most effective set-up was obtained using a vector containing the pSH71 replicon, the orfX promoter from the sakacin P regulon, and the cognate regulatory genes, in a L. sakei host. GusA levels obtained with this set-up were approximately ten times higher than the levels obtained with prototype pSIP versions, whereas PepN levels amounted to almost 50 % of total cellular protein.

Curli are necessary for the adherence of Escherichia coli to surfaces, and to each other, during biofilm formation, and the csgBA and csgDEFG operons are both required for their synthesis. A recent survey of gene expression in Pseudomonas aeruginosa biofilms has identified tolA as a gene activated in biofilms. The tol genes play a fundamental role in maintaining the outer-membrane integrity of Gram-negative bacteria. RcsC, the sensor of the RcsBCD phosphorelay, is involved, together with RcsA, in colanic acid capsule synthesis, and also modulates the expression of tolQRA and csgDEFG. In addition, the RcsBCD phosphorelay is activated in tol mutants or when Tol proteins are overexpressed. These results led the authors to investigate the role of the tol genes in biofilm formation in laboratory and clinical isolates of E. coli. It was shown that the adherence of cells was lowered in the tol mutants. This could be the result of a drastic decrease in the expression of the csgBA operon, even though the expression of csgDEFG was slightly increased under such conditions. It was also shown that the Rcs system negatively controls the expression of the two csg operons in an RcsA-dependent manner. In the tol mutants, activation of csgDEFG occurred via OmpR and was dominant upon repression by RcsB and RcsA, while these two regulatory proteins repressed csgBA through a dominant effect on the activator protein CsgD, thus affecting curli synthesis. The results demonstrate that the Rcs system, previously known to control the synthesis of the capsule and the flagella, is an additional component involved in the regulation of curli. Furthermore, it is shown that the defect in cell motility observed in the tol mutants depends on RcsB and RcsA.

Highly expressed genes in bacteria often have a stronger codon bias than genes expressed at lower levels, due to translational selection. In this study, a comparative analysis of predicted highly expressed (PHX) genes in the Streptomyces coelicolor and Streptomyces avermitilis genomes was performed using the codon adaptation index (CAI) as a numerical estimator of gene expression level. Although it has been suggested that there is little heterogeneity in codon usage in G+C-rich bacteria, considerable heterogeneity was found among genes in these two G+C-rich Streptomyces genomes. Using ribosomal protein genes as references, ∼10 % of the genes were predicted to be PHX genes using a CAI cutoff value of greater than 0·78 and 0·75 in S. coelicolor and S. avermitilis, respectively. The PHX genes showed good agreement with the experimental data on expression levels obtained from proteomic analysis by previous workers. Among 724 and 730 PHX genes identified from S. coelicolor and S. avermitilis, 368 are orthologue genes present in both genomes, which were mostly ‘housekeeping’ genes involved in cell growth. In addition, 61 orthologous gene pairs with unknown functions were identified as PHX. Only one polyketide synthase gene from each Streptomyces genome was predicted as PHX. Nevertheless, several key genes responsible for producing precursors for secondary metabolites, such as crotonyl-CoA reductase and propionyl-CoA carboxylase, and genes necessary for initiation of secondary metabolism, such as adenosylmethionine synthetase, were among the PHX genes in the two Streptomyces species. The PHX genes exclusive to each genome, and what they imply regarding cellular metabolism, are also discussed.

The availability of the complete genome sequence of Streptomyces coelicolor A3(2) has allowed the prediction of the Tat-exported proteins of this Gram-positive bacterium. To predict secreted proteins that potentially use the Tat pathway for their secretion, the TATscan program was developed. This program identified 129 putative Tat substrates. To test the validity of these predictions, nine signal sequences, including three which were not identified by existing prediction programs, were selected and fused to the structural xlnC gene in place of its native signal sequence. Xylanase C (XlnC) is a cofactorless enzyme which is secreted in an active form exclusively through the Tat-dependent pathway by Streptomyces lividans. Among the nine chosen signal sequences, seven were shown to be Tat-dependent, one was Sec-dependent and one was probably not a signal sequence. The seven Tat-dependent signal sequences comprised two lipoprotein signal sequences and three sequences not predicted by previous programs. Pulse–chase experiments showed that the precursor-processing rate in the seven transformants was generally slower than wild-type XlnC, indicating that these signal peptides were not equivalent in secretion. This suggested that there might be some incompatibility between the signal peptide and the reporter protein fused to it. To test this possibility, the signal peptides were fused to a cofactorless chitosanase (SCO0677), a Tat-dependent protein validated in this work but structurally different from XlnC. With some fluctuations, similar results were obtained with this enzyme, indicating that the type of folding of the reporter protein had little effect on the Tat secretion process.