- Volume 145, Issue 3, 1999

Clusters of genes encoding enzymes for tetrapyrrole biosynthesis were cloned from Bacillus sphaericus, Bacillus stearothermophilus, Brevibacillus brevis and Paenibacillus macerans. The sequences of all hemX genes found, and of a 6·3 kbp hem gene cluster from P. macerans, were determined. The structure of the hem gene clusters was compared to that of other Gram-positive bacteria. The Bacillus and Brevibacillus species have a conserved organization of the genes hemAXCDBL, required for biosynthesis of uroporphrinogen III (Urolll) from glutamyl-tRNA. In P. macerans, the hem genes for Urolll synthesis are also closely linked but their organization is different: there is no hemX gene and the gene cluster also contains genes, cysG B and cysG A-hemD, encoding the enzymes required for synthesis of sirohaem from Urolll. Bacillus subtilis contains genes for three proteins, NasF, YlnD and YlnF, with sequence similarity to Escherichia coli CysG, which is a multi-functional protein catalysing sirohaem synthesis from Urolll. It is shown that YlnF is required for sirohaem synthesis and probably catalyses the precorrin-2 to sirohaem conversion. YlnD probably catalyses precorrin-2 synthesis from Uroll and NasF seems to be specific for nitrite reduction.

ε16, a temperate phage induced from Corynebacterium glutamicum ATCC 21792, lysogenizes its host via site-specific recombination. The phage attachment site, attP, was located to a 6·5 kb BamHI fragment of the ε16 genome. This fragment also contained ε16 integrative functions. The minimal phage DNA fragment required for integration was defined. This 1630 bp region contained a large open reading frame, int, encoding a protein of 416 amino acids with similarity in its carboxyl-terminal domain to tyrosine recombinases and particuliarly to the Xer recombinases. The comparison of the nucleotide sequences of attB, attL, attR, and attP identified a common 29 bp sequence, the core sequence. It lies 11 bp downstream of the 3′ end of the integrase gene. ε16 integrase was shown to catalyse site-specific integration in trans to attP with an efficiency of 5 x 103integrants per μg DNA. The integrating fragment catalysed integration in several Corynebacterium strains that are not infected by ε16, thus enlarging the host spectrum of integrating vectors derived from ε16. In these strains, the ε16 attB site was located in a conserved intergenic region and lies downstream of a clp gene.

During early stages of growth, Streptomyces reticuli synthesizes a hyphae-associated, haem-containing enzyme which exhibits catalase and peroxidase activities with broad substrate specificity (CpeB). The purified dimeric enzyme (160 kDa) consists of two identical subunits. Using anti-CpeB antibodies and an expression- as well as a mini-library, the corresponding cpeB gene was identified and sequenced. It encodes a protein of 740 aa with a molecular mass of 81·3 kDa. The deduced protein shares the highest level of amino acid identity with KatG from Caulobacter crescentus and Mycobacterium tuberculosis, and PerA from Bacillus stearothermophilus. Streptomyces lividans transformants carrying cpeB and the upstream-located furS gene with its regulatory region on the bifunctional vector pWHM3 produced low or enhanced levels of CpeB in the presence or absence of Fe ions, respectively. An in-frame deletion of the major part of furS induces increased CpeB synthesis. The data imply that FurS regulates the transcription of cpeB. The deduced FurS protein is rich in histidine residues, contains a putative N-terminally situated helix-turn-helix motif and has a molecular mass of 15·1 kDa. It shares only 29% amino acid identity with the Escherichia coli ferric uptake regulator (Fur) protein, but about 64% with FurA deduced from the genomic sequences of several mycobacteria. The predicated secondary structures of FurS and FurA are highly similar and considerably divergent from those of the E. coli Fur. In contrast to some Gram-negative bacteria, within several mycobacteria an intact furA gene or a furA pseudogene is upstream of a catalase-peroxidase (katG) gene predicted to encode a functional or a non-functional (Mycobacterium leprae) enzyme. Thus the data obtained for Streptomyces reticuli are expected to serve as an additional model to elucidate the regulation of mycobacterial catalase-peroxidase genes.

A new insertion sequence (IS2112) was identified in the genome of the 1-haloalkane-utilizing bacterium Rhodococcus rhodochrous NCIMB 13064. The insertion element is 1415 bp long, does not contain terminal inverted repeats, and is not flanked by directly repeated sequences. IS2112 belongs to the IS110 family of transposable elements, and forms a separate subfamily, along with IS116. Two copies of IS2112 were found in R. rhodochrous NCIMB 13064 and one, two or three copies of a similar sequence were detected in five other 1-haloalkane-degrading Rhodococcus strains. There were no sequences homologous to IS2112 found in the 1-haloalkane-degrading ‘Pseudomonas pavonaceae’ 170 and Rhodococcus sp. HA1 or in several Rhodococcus strains which do not utilize haloalkanes. IS2112 was originally found in plasmid pRTL1 of R. rhodochrous NCIMB 13064, which harbours genes encoding utilization of 1-haloalkanes, and was located 5 kbp upstream of the haloalkane dehalogenase gene (dhaA). Although the second copy of IS2112 in strain NCIMB 13064 was also present on the pRTL1 plasmid, these sequences do not apparently comprise a single composite transposon encoding haloalkane utilization. An analysis of derivatives of NCIMB 13064 revealed that IS2112 was involved in genome rearrangements. IS2112 appeared to change its location as a result of transposition and as a result of other rearrangements of the NCIMB 13064 genome.

The Escherichia coli glycine-cleavage enzyme system (gcvTHP and lpd gene products) provides C1 units for cellular methylation reactions. Both the GcvA and leucine-responsive regulatory (lrp) proteins are required for regulation of the gcv operon. One model proposed for gcv regulation is that Lrp plays a structural role, bending the DNA to allow GcvA to function as either an activator or a repressor in response to environmental signals. This hypothesis was tested by replacing all but the upstream 22 bp of the Lrp-binding region in a gcvT::lacZ fusion with the l1A site from phage λ. Integration host factor (IHF) binds the l1A site and bends the DNA about 140°. Shifting the l1A site by increments of 1 base around the DNA helix resulted in IHF-dependent activation and repression of gcvT::lacZ expression that were face-of-the-helix dependent. Activation was also dependent on the GcvA protein, and repression was dependent on both the GcvA and GcvR proteins, demonstrating that the roles for these proteins were not altered. The results are consistent with Lrp playing primarily a structural role in gcv regulation, although they do not completely rule out the possibility that Lrp also interacts with another gcv-regulatory protein or with RNA polymerase.

By gel retardation experiments with crude cell extracts of Paracoccus denitrificans it was demonstrated that a protein specifically binds to the promoter of the P. denitrificans recA gene. PCR mutagenesis of the recA promoter showed that the GAACN7GAAC motif is required for the formation of the DNA-protein complex. This protein also binds to the GTTCN7GTTC motif, which is present in the promoter of the P. denitrificans uvrA gene. Mutational analysis of the promoter regions of both P. denitrificans recA and uvrA genes indicated that the GAACN7GAAC and GTTCN7GTTC sequences are required for DNA-damage-mediated induction of these two genes in vivo. Furthermore, the P. denitrificans recA gene was DNA-damage-inducible when introduced into cells of the phylogenetically related phototrophic bacterium Rhodobacter sphaeroides, although this inducibility was lost in mutants in the GAACN7GAAC motif. These results indicate that P. denitrificans possesses the same SOS box as R. sphaeroides, which, in agreement with previous work, is proposed as being the GTTCN7GTTC motif.

The complete nucleotide sequence of a plasmid, pKJ50, isolated from an intestinal bacterium, Bifidobacterium longum KJ, has been determined. The plasmid was analysed and found to be 4960 bp in size with a G+C content of 61·7 mol%. Computer analysis of sequence data revealed three major ORFs encoding putative proteins of 31·5 (ORFI), 24·5 (ORFII) and 38·6 kDa (ORFIII). ORFI encodes a protein with a pl of 10·18 and shows relatively high amino acid sequence similarity (more than 60%) with several plasmid replication proteins from Gram-positive and -negative bacteria. Southern blot analysis showed that pKJ50 accumulates an ssDNA intermediate, suggesting that it replicates by a rolling-circle mechanism. Upstream of ORFI, three sets of repeated sequences resembling iteron structures of related plasmids were identified. ORFIII encodes a protein with a pl of 10·97. It also shows a high level of amino acid sequences similarity with some plasmid mobilization proteins. Upstream of ORFIII, a 12 bp stretch resembles an oriT DNA sequence with inverted repeats identical to those found in conjugative plasmids. Hydropathy plot analysis of ORFII, encoding an acidic protein (pl = 4·95), suggests it is a transmembrane protein. Several interesting palindromic sequences, repeat sequences and hairpin-loop structures around ORFI, which might confer regulatory effects on the replication of the plasmid, were also noted. Reverse transcriptase PCR (RT-PCR) and in vitro translation confirmed the expression of ORFI and ORFII. RT-PCR produced amplified DNA fragments of the expected sizes, corresponding to ORFI and ORFII. However, no RT-PCR product corresponding to ORFIII was obtained. In vitro translation showed protein bands of the expected sizes, corresponding to each ORF. A shuttle vector capable of transforming Bifidobacterium animalis MB209 was constructed by cloning pKJ50 and a chloramphenicol resistance gene into pBR322.

A mutant of Rhizobium leguminosarum was isolated which fails to take up the siderophore vicibactin. The mutation is in a homologue of fhuB, which in Escherichia coli specifies an inner-membrane protein of the ferric hydroxamate uptake system. In Rhizobium, fhuB is in an operon fhuDCB, which specifies the cytoplasmic membrane and periplasmic proteins involved in siderophore uptake. fhuDCB mutants make vicibactin when grown in Fe concentrations that inhibit its production in the wild-type. Nodules on peas induced by fhuDCB mutants were apparently normal in N2 fixation. Transcription of an fhuDCB-lacZ fusion was Fe-regulated, being approximately 10-fold higher in Fe-depleted cells. Downstream of fhuB, in the opposite orientation, is a version of fhuA whose homologues in other bacteria specify hydroxamate outer-membrane receptors. This fhuA gene appears to be a pseudogene with stop codons and undetectable expression.

The soil bacterium Sinorhizobium meliloti (Rhizobium meliloti) has the ability to produce the alternative exopolysaccharide galactoglucan (EPS II) in addition to succinoglycan (EPS I). In the wild-type strain EPS II production is induced by phosphate-limiting conditions or by extra copies of the exp gene cluster. Based on similarities to transcriptional regulators of the MarR family, an additional putative regulatory gene, expG, was identified in the exp gene cluster. Using exp-lacZ transcriptional fusions, a stimulating effect of extra copies of this expG gene on the transcription of all exp complementation groups was determined. Phosphate limitation also resulted in increased expression of the exp-lacZ fusions. This increase was reduced in strains characterized by a deletion of expG. The previously reported high level of exp gene transcription in a mucR mutant was further elevated under phosphate-limiting conditions. The expA, expD, expG and expE promoters contain sequences with similarities to the PHO box known as the PhoB-binding site in phosphate-regulated promoters in Escherichia coli. The S. meliloti phoB gene was required for the activation of exp gene expression under phosphate limitation, but not for induction of exp expression by MucR or ExpG.

The Bacillus subtilis levanase structural gene sacC was expressed under the regulated control of sacR, the inducible levansucrase leader region, in a degU32(Hy) strain. In this genetic context, exocellular levanase is overproduced (0·5% of total protein) during the exponential phase of growth upon induction by sucrose at 37 ° and pH 7. No precursor form that comprised a signal peptide was detected in pulse-chase experiments. The subsequent release of the cell-associated processed protein is a slow event (t 1/2 = 80 · 10 s). The unfolding-folding transition of pure levanase monitored in vitro by the resistance to proteolysis was achieved within the same time range (t 1/2 = 50 s) under the same conditions of pH and temperature. Calcium ions, which modulate the rate and the yield of refolding, have a low affinity for the protein. Comparison of these results with those obtained previously with levansucrase and α-amylase overproduced in the same genetic and physiological context suggests that the precursor processing is more efficient in levanase and α-amylase than in levansucrase. This discrepancy could lie in information borne by the signal peptide sequence of these exoproteins. However, the rate of the ultimate stage of release of these three proteins, which includes the passage through the cell wall, is correlated with the rate of folding and appears to be independent of their molecular size.

In the yeast Saccharomyces cerevisiae, glucose or fructose represses the expression of a large number of genes. The phosphorylation of glucose or fructose is catalysed by hexokinase PI (Hxk1), hexokinase PII (Hxk2) and a specific glucokinase (Glk1). The authors have shown previously that either Hxk1 or Hxk2 is sufficient for a rapid, sugar-induced disappearance of catabolite-repressible mRNAs (short-term catabolite repression). Hxk2 is specifically required and sufficient for long-term glucose repression and either Hxk1 or Hxk2 is sufficient for long-term repression by fructose. Mutants lacking the TPS1 gene, which encodes trehalose 6-phosphate synthase, can not grow on glucose or fructose. In this study, suppressor mutations of the growth defect of a tps1Δ hxk1Δ double mutant on fructose were isolated and identified as novel HXK2 alleles. All six alleles studied have single amino acid substitutions. The mutations affected glucose and fructose phosphorylation to a different extent, indicating that Hxk2 binds glucose and fructose via distinct mechanisms. The mutations conferred different effects on long- and short-term repression. Two of the mutants showed very similar defects in catabolite repression, despite large differences in residual sugar-phosphorylation activity. The data show that the long- and short-term phases of catabolite repression can be dissected using different hexokinase mutations. The lack of correlation between in vitro catalytic hexokinase activity, in vivo sugar phosphate accumulation and the establishment of catabolite repression suggests that the production of sugar phosphate is not the sole role of hexokinase in repression. Using the set of six hxk2 mutants it was shown that there is a good correlation between the glucose-induced cAMP signal and in vivo hexokinase activity. There was no correlation between the cAMP signal and the short- or long-term repression of SUC2, arguing against an involvement of cAMP in either stage of catabolite repression.

Yeast cells respond to a shift to higher osmolarity by increasing the cellular content of the osmolyte glycerol. This response is accompanied by a stimulation of the expression of genes encoding enzymes in the glycerol production pathway. In this study the osmotic induction of one of those genes, GPD1, which encodes glycerol-3-phosphate dehydrogenase, was monitored in time course experiments. The response is independent of the osmolyte and consists of four apparent phases: a lag phase, an initial induction phase, a feedback phase and a sustained long-term induction. Osmotic shock with progressively higher osmolyte concentrations caused a prolonged lag phase. Deletion of HOG1, which encodes the terminal protein kinase of the high osmolarity glycerol (HOG) response pathway, led to an even longer lag phase and drastically lower basal and induced GPD1 mRNA levels. However, the induction was only moderately diminished. Overstimulation of Hog1p by deletion of the genes for the protein phosphatases PTP2 and PTP3 led to higher basal and induced mRNA levels and a shorter lag phase. The protein phosphatase calcineurin, which mediates salt-induced expression of some genes, does not appear to contribute to the control of GPD1 expression. Although GPD1 expression has so far not been reported to be controlled by a general stress response mechanism, heat-shock induction of the GPD1 mRNA level was observed. However, unregulated protein kinase A activity, which strongly affects the general stress response, only marginally altered the mRNA level of GPD1. The osmotic stimulation of GPD1 expression does not seem to be mediated by derepression, since deletion of the SSN6 gene, which encodes a general repressor, did not significantly alter the induction profile. A hypo-osmotic shock led to a transient 10-fold drop of the GPD1 mRNA level. Neither the HOG nor the protein kinase C pathway, which is stimulated by a decrease in external osmolarity, is involved in this effect. It was concluded that osmotic regulation of GPD1 expression is the result of an interplay between different signalling pathways, some of which remain to be identified.

Using a DNA fragment containing the Aspergillus niger abfB gene as a probe, the homologous Aspergillus nidulans gene, designated abfB, has been cloned from a genomic library containing size-selected HindIII fragments. The nucleotide sequence of the A. nidulans abfB gene shows strong homology with the A. niger abfB, Trichoderma reesei abf-1 and Trichoderma koningii α-L-arabinofuranosidase/β-xylosidase genes. Regulation of abfB expression has been investigated in cultures induced with L-arabitol. The accumulation of abfB mRNA, total α-L-arabinofuranosidase activity and AbfB protein levels have been determined in a wild-type A. nidulans strain as well as in different mutant strains. These strains are affected either in their response to ambient pH (palA1 and pacCc14 mutants), carbon catabolite repression (creAd4 mutant), the ability to utilize L-arabitol as a carbon source (araA1 mutant) or a combination of both latter genotypes (araA1 creAd4). The results obtained indicate that the expression of the A. nidulans abfB gene was higher at acidic pHs and was superinduced in this double mutant. Furthermore, disruption of the abfB gene demonstrated that in A. nidulans AbfB is the major p-nitrophenyl α-L-arabinofuranoside-hydrolysing activity but at least one minor activity is expressed, which is involved in the release of L-arabinose from polysaccharides.

Sixteen nif and ‘nif-associated’ genes (expressed only under conditions of nitrogen fixation) in Synechococcus sp. strain RF-1 have been cloned and sequenced. All of the nif and nif-associated genes identified in Synechococcus RF-1 were arranged in a continuous cluster spanning approximately 18 kb and containing seven operons. The nifH operon (nifH-nifD-nifK) has been reported previously. nifB, fdxN, nifS, nifU and nifP were found to be located upstream of the nifH operon. nifB-fdxN-nifS-nifU were expressed as an operon. A nifP-like gene was found to be located just upstream of nifB. nifE, nifN, nifX, nifW and the nif-associated hesA, hesB and ‘fdx’ were found to be located downstream from nifK. The genes located downstream from nifK are arranged nifE-nifN-nifX-orf-nifW-hesA-hesB- fdx’ and span approximately 7 kb. The function of the ORF situated between nifX and nifW is not known. However, it was identified as a counterpart of ORF-2 in Anabaena sp. strain PCC 7120 based on the deduced amino acid sequence. Northern hybridization and primer extension analysis indicated that the nif and nif-associated genes are organized in nifE-nifN, nifX-orf, nifW-hesA-hesB and ‘fdx’-containing operons, respectively. According to the results of this study and previous reports, the genes are expressed in a rhythmic pattern with peaks during the dark phase when the culture is grown in a 12 h light/12 h dark regimen. The rhythm persisted after the culture was transferred to continuous illumination.