Analysis of transcription of the Staphylococcus aureus aerobic class Ib and anaerobic class III ribonucleotide reductase genes in response to oxygen.

Staphylococcus aureus is a gram-positive facultative aerobe that can grow in the absence of oxygen by fermentation or by using an alternative electron acceptor. To investigate the mechanism by which S. aureus is able to adapt to changes in oxygen concentration, we analyzed the transcriptional regulation of genes that encode the aerobic class Ib and anaerobic class III ribonucleotide reductase (RNR) systems that are responsible for the synthesis of deoxyribonucleotides needed for DNA synthesis. The S. aureus class Ib RNR nrdIEF and class III RNR nrdDG genes and their regulatory regions were cloned and sequenced. Inactivation of the nrdDG genes showed that the class III RNR is essential for anaerobic growth. Inhibition of aerobic growth by hydroxyurea showed that the class Ib RNR is an oxygen-dependent enzyme. Northern blot analysis and primer extension analysis demonstrated that transcription of class III nrdDG genes is regulated by oxygen concentration and was at least 10-fold higher under anaerobic than under aerobic conditions. In contrast, no significant effect of oxygen concentration was found on the transcription of class Ib nrdIEF genes. Disruption or deletion of S. aureus nrdDG genes caused up to a fivefold increase in nrdDG and nrdIEF transcription under anaerobic conditions but not under aerobic conditions. Similarly, hydroxyurea, an inhibitor of the class I RNRs, resulted in increased transcription of class Ib and class III RNR genes under aerobic conditions. These findings establish that transcription of class Ib and class III RNR genes is upregulated under conditions that cause the depletion of deoxyribonucleotide. Promoter analysis of class Ib and class III RNR operons identified several inverted-repeat elements that may account for the transcriptional response of the nrdIEF and nrdDG genes to oxygen.

Structure-function studies of the non-heme iron active site of isopenicillin N synthase: some implications for catalysis.

Isopenicillin N synthase (IPNS) is a non-heme ferrous iron-dependent oxygenase that catalyzes the ring closure of delta-(L-alpha-aminoadipoyl)-L-cysteinyl-D-valine (ACV) to form isopenicillin N. Spectroscopic studies and the crystal structure of IPNS show that the iron atom in the active species is coordinated to two histidine and one aspartic acid residues, and to ACV, dioxygen and H2O. We previously showed by site-directed mutagenesis that residues His212, Asp214 and His268 in the IPNS of Streptomyces jumonjinensis are essential for activity and correspond to the iron ligands identified by crystallography. To evaluate the importance of the nature of the protein ligands for activity, His214 and His268 were exchanged with asparagine, aspartic acid and glutamine, and Asp214 replaced with glutamic acid, histidine and cysteine, each of which has the potential to bind iron. Only the Asp214Glu mutant retained activity, approximately 1% that of the wild type. To determine the importance of the spatial arrangement of the protein ligands for activity, His212 and His268 were separately exchanged with Asp214; both mutant enzymes were completely defective. These findings establish that IPNS activity depends critically on the presence of two histidine and one carboxylate ligands in a unique spatial arrangement within the active site. Molecular modeling studies of the active site employing the S. jumonjinensis IPNS crystal structure support this view. Measurements of iron binding by the wild type and the Asp214Glu, Asp214His and Asp214Cys-modified proteins suggest that Asp214 may have a role in catalysis as well as in iron coordination.

Pore mutations affecting tetrameric assembly and functioning of the potassium channel KcsA from Streptomyces lividans.

Designed mutations within the Streptomyces lividans kcsA gene resulted in a set of mutant proteins, which were characterized in respect to their assembly and channel activities. (i) The amino acid residue leucine 81 located at the external side of KcsA was found to be exchangeable by a cysteine residue without affecting the channel characteristics. (ii) Substitution of the first glycine (G77) residue within the GYG motif by an alanine or substitution of the tyrosine (Y) residue 78 by a phenylalanine (F) led to mutant proteins which form tetramers of reduced stability. In contrast to the AYG mutant protein, GFG functions as an active K(+) channel whose characteristics correspond to those of the wild-type KcsA channel. (iii) The investigated mutant proteins, which carry different mutations (T72A, T72C, V76A, V76E, G77E, Y78A, G79A, G79D, G79E) within the signature sequence of the pore region, do not at all or only to a very small degree assemble as tetramers and lack channel activity.

Recent advances in the structure and function of isopenicillin N synthase.

Isopenicillin N synthase is a key enzyme in the biosynthesis of penicillin and cephalosporin antibiotics, catalyzing the oxidative ring closure of delta-(L-alpha-aminoadipoyl)-L-cysteinyl-D-valine to form isopenicillin N. Recent advances in our understanding of the unique chemistry of this enzyme have come through the combined application of spectroscopic, molecular genetic and crystallographic approaches and led to important new insights into the structure and function of this enzyme. Here we review new information on the nature of the endogenous ligands that constitute the ferrous iron active site, sequence evidence for a novel structural motif involved in iron binding in this and related non-heme iron dependent dioxygenases, crystal structure studies on the enzyme and its substrate complex and the impact of these and site-directed mutagenesis studies for unraveling the mechanism of the isopenicillin N synthase reaction.

Gene organization in the trxA/B-oriC region of the Streptomyces coelicolor chromosome and comparison with other eubacteria.

The gene organization was determined in the trxA/B-rnpA region of the Streptomyces coelocolor chromosome, near to the origin of replication, oriC. Previously, we showed that the trxA and trxB genes, coding for thioredoxin and thioredoxin reductase, respectively, occur in S. coelicolor as a gene cluster and are contained on a cosmid H24 that carries oriC and several genes involved in DNA replication. Here we show that the trxA/B locus is positioned approx. 9.4kb from oriC, present the nucleotide sequence of the trxA/B-rnpA region and use sequence analysis to identify the nature of the intervening genes. Seven open reading frames were found, all oriented in the same direction, five of which were identified as the S. coelicolor homologs of SpoIIIJ, Jag, GidB, Soj and SpoOJ in Bacillus subtilis and which have been ascribed different functions in this and other bacteria for either DNA replication, chromosomal partitioning or morphological development. The arrangement of the genes coding for the above five proteins in the trxA/B-rnpA region in S. coelicolor resembles that in Mycobacterium leprae, Mycobacterium tuberculosis, B. subtilis and Pseudomonas putida, and supports the view that many of the genes necessary for development and cell division in bacteria are organized in a similar fashion. In B. subtilis and P. putida, however, the trxA/B genes are not present in the above gene arrangement.

The glutamine ligand in the ferrous iron active site of isopenicillin N synthase of Streptomyces jumonjinensis is not essential for catalysis.

Isopenicillin N synthase (IPNS) is a non-heme ferrous iron dependent dioxygenase that catalyses the ring closure of delta-(L-alpha-aminoadipoyl)-L-cysteinyl-D-valine (ACV) to isopenicillin N. We previously used site-directed mutagenesis to identify in the IPNS of Streptomyces jumonjinensis two histidines and one aspartic acid that are essential for activity. The recent crystal structure of the IPNS of Aspergillus nidulans establishes that these amino acids are iron ligands and reveals that the fourth ligand is the penultimate glutamine. The two histidines and one aspartic acid are conserved in several classes of non-heme ferrous iron dioxygenases, whereas the glutamine is present only in IPNSs. In this paper we show that the penultimate glutamine in S. jumonjinensis IPNS Gln-328 is not essential for catalysis. In contrast, Gln-230 which is highly conserved among the above dioxygenases and is proximal to the active site is crucial for activity.

Ferrous active site of isopenicillin N synthase: genetic and sequence analysis of the endogenous ligands.

Isopenicillin N synthase (IPNS) from Streptomyces jumonjinensis (M(r) 37,902) is a non-heme ferrous iron-containing enzyme that catalyzes the oxidative cyclization of the tripeptide delta-(L-alpha-aminoadipoyl)-L-cysteinyl-D-valine (ACV) to form isopenicillin N. Spectroscopic studies [reviewed in Cooper, R. D. (1993) Biomed. Chem. 1, 1-17] have led to a model for the coordination environment of the iron atom possessing three histidine and one aspartic acid endogenous ligands and a solvent molecule. A refinement of that model proposes that formation of the Fe(II) IPNS-ACV complex occurs with displacement of the H2O from the metal center and that one of the histidines is subsequently replaced by a solvent molecule on binding of dioxygen. Here we report genetic studies to determine the nature and location of the endogenous ligands in the S. jumonjinensis IPNS primary amino acid sequence that constitute the ferrous active site. Site-directed mutagenesis was used to exchange each of the seven histidines and the five aspartic acids that are conserved in bacterial and fungal IPNS proteins. Biochemical analysis of the alanine-substituted mutant proteins shows that two histidines, His212 and His268, and one aspartic acid, Asp214, are essential for enzyme activity. The other mutant enzymes have specific activities 5-68% that of wild type. Sequence analysis of 10 IPNS and 42 other non-heme ferrous iron-dependent dioxygenases reveal the presence of a common motif, HisXAsp(53-57)XHis, which in IPNS contains the identical two histidines and one aspartic acid essential for function. Accordingly, we have assigned residues His212, His268, and Asp214 as three of the four endogenous ligands postulated to form the IPNS ferrous active site. Compelling support for these conclusions comes from the recent crystal structure determination of the manganese form of a fungal IPNS [Roach et al. (1995) Nature 375, 700-704].

[Transposon Tn5 and its derivatives effectively transpose over a broad temperature range]. / Transpozon Tn5 i ego proizvodnye varianty éffektivno transpoziruiutsiav shirokom diapazone temperatur.

It was shown that the 1st class composite transposon Tn5 (5.8 Kb) and its synthetic derivatives--TnV (Tn5-ReppSC101; 6.1 Kb) and Tn5-MobRP4 (about 7.7 Kb) transpose in Escherichia coli K-12 cells (RecA strain HB101) with similar efficiency both at 28 and at 42 degrees C as well as at 37 degrees C. This property of Tn5-like elements distinguishes them from the class II transposons (such as Tn3, Tn21 etc.), whose transposition, as is well known, is strongly suppressed even at 37 degrees C. It was also demonstrated that transposition frequency of Tn5-derived elements depends on their copy number.

[Transposition of the composite synthetic transposon TnV (Tn5-Rep(pSC101)) is accompanied by the formation of the mini-plasmid pTnV, containing defective Is50-elements]. / Transpozitsiia sostavnogo sinteticheskogo transpozona TnV(Tn5-Rep(pSC101)) soprovozhdaetsia formirovaniem mini-plazmid pTnV, soderzhashchikh defektnye IS50-élementy.

Using thermoelimination (at 42 degrees C) of the thermoinducible coliphage P1tsCmr omega::TnV (TnV is a Tn5 derivative which contains the replication origin (Rep) of plasmid pSC101), more than 110 KmrCms Escherichia coli K12 clones were selected. It was supposed that the KmrCms phenotype could result only from insertion of TnV (Kmr) into E. coli chromosome and the loss of phage (Cms). It was found that the majority of KmrCms clones (35-90%) contained miniplasmids. Their molecular sizes did not exceed the TnV size (6.1 kb). The formation of miniplasmids called pTnV was observed both in RecA+ cells (C600) and in RecA- (HB101), more often in the latters. Interestingly, that miniplasmids of only several molecular sizes were detected: from 6.1 kb (pTnV60) to 4.35 kb (pTnV43). A restriction analysis showed that DNA of the majority of pTnV plasmids had varying deletions (0.3-1.3 kb) of mainly IS50L element which together with IS50R flank TnV. Very low transposition frequencies (approx. 10(-8) Kmr transconjugants per transferred R388) of all pTnV types (including pTnV60 plasmids containing probably microdeletions of the joining "outside" IS50's ends) suggest that pTnV plasmids are not intermediates in TnV transposition. Possibly the circularized TnV derivatives (pTnV's) are side products of the transposition resulting from the abortive attempts of an excised and autonomous transposon molecule to insert into itself. In the present paper the possible mechanisms of the origin of limited pTnV type numbers are also discussed.

[Plasmids of Azospirillum brasilense]. / Plazmidy Azospirillum brasilense.

The cells from natural isolates of A. Brasilense were found to harbour 1 to 4 plasmids with the molecular masses within the 27-300 Md range. 100 Md plasmids are specific for this bacterial species. Strains isolated from the roots of cereals (wheat, maize, barley) have more heterogeneous plasmid composition as compared to the strains isolated from soil.