- Volume 142, Issue 7, 1996

The complete nucleotide sequence of the surA gene, encoding a sucrase from Bacillus stearothermophilus NUB36, was determined. surA was composed of 1338 bp and encoded 445 amino acid residues. The deduced polypeptide of M r 51 519 showed strong sequence similarity to sucrose and sucrose phosphate hydrolases from Bacillus subtilis, Klebsiella pneumoniae and Vibrio alginolyticus, and contained the ‘sucrose box’ residues thought to be important for catalysis of the transfer of fructose from sucrose. The enzyme was partially purified using affinity chromotography from extracts of Escherichia coli containing the cloned surA. SurA displayed an optimum temperature for sucrose hydrolysis of 55 0C and high stability. The M r of SurA determined by gel filtration was 105000, which suggested that the active form of the enzyme is a dimer. SurA exhibited an apparent K m of 40 mM for sucrose but, unlike the homologous B. subtilis enzyme, had no detectable sucrose phosphate hydrolase activity.

Recently, the subunit composition of class A aspartate transcarbamoylases (ATCases) in fluorescent pseudomonads has been clarified. We present evidence that distribution of this type of ATCase may be more widespread than at first suspected. Bacterial ATCases exist in three forms: class A (molecular mass # 450-500 kDa); class B, typified by Escherichia coli ATCase (# 300 kDa); and class C, typified by Bacillus subtilis ATCase (# 100 kDa). Using gradient gel electrophoresis with activity-staining to scan bacterial sonicates, we report the existence of six more class A ATCases. We have purified one of these, Acinetobacter calcoaceticus ATCase, and found its subunit composition to be similar to that of the pseudomonad ATCases. Two of these ATCases come from bacteria outside the γ-subgroup of the Proteobacteria, one from the α-subgroup and one from Deinococcus radiophilus, a species phylogenetically remote from the Proteobacteria. Unexpectedly, three bacterial species, closely related to the fluorescent pseudomonads and acinetobacters, have ATCases of 100 kDa (class C). One of these, Stenotrophomonas (formerly Xanthomonas) maltophilia has been purified and found to be a homotrimer of 35 kDa polypeptide chains. We believe this is the first time that class C ATCases have been reported in Gram-negative bacteria. A distinctive cluster in the γ-3 subgroup of the Proteobacteria is formed by the enteric bacteria and their relatives. So far only class B ATCases have been reported in this group. The evolutionary implications of these findings are discussed.

A periplasmic arginine transport system that is a member of the ATP-dependent transport superfamily was identified in Pasteurella haemolytica. The gene encoding the periplasmic binding protein (lapT) was cloned and the protein overexpressed in Escherichia coli. LapT was purified to homogeneity using a modified osmotic shock procedure and anion-exchange column chromatography. Filter-binding assays established that LapT is an t-arginine-binding protein. Various amino acids were tested for their ability to inhibit L-arginine binding to LapT. When present in 100-fold excess, only L-arginine, D-arginine and citrulline competed with L-arginine for binding. Arginine transport in P. haemolytica whole cells was competitively inhibited by the same amino acids, suggesting that the LapT permease specifically transports L-arginine. The dissociation constant for the L-arginine-LapT complex was 170 nM and the stoichiometry of binding was approximately 0.8 mol L-arginine (mol LapT)-1. A polyclonal antibody raised against the purified protein permitted detection of LapT in P. haemolytica periplasmic fractions.

The kinetics, energetics and specificity of a general amino acid transporter were studied in the ectomycorrhizal fungus Paxillus involutus (Batsch) Fr. The uptake of amino acids showed features characteristic of active transport. After correction for a non-mediated transport component, the kinetics of glutamate, glutamine, alanine and aspartate uptake measured over a wide concentration range followed the simple Michaelis-Menten saturation curves. The apparent K m derived from the Eadie-Hofstee plots ranged from 7 μM for alanine to 27 μM for glutamate. Dinitrophenol, carbonyl cyanide m-chlorophenylhydrazone and NaN3 strongly inhibited amino acid uptake, whereas dicyclohexylcarbodiimide, vanadate and the ionophores monensin and nonactin had no effect on the uptake. Both pH dependence and inhibition by protonophores are consistent with a proton symport mechanism for amino acid uptake by P. involutus. Competition studies indicated a broad substrate recognition by the uptake system, which resembles the general amino acid permease of yeast. Dixon plots of the inhibition of glutamate uptake by alanine, lysine and methionine sulfoximine showed that inhibitions were competitive. The physiological importance of this transporter for the exchange of nitrogenous compounds between fungal and host plant cells in ectomycorrhizal associations is discussed.

A Helicobacter pylori membrane fraction oxidized yeast and equine cytochrome c, and N,N,N’,N’-tetramethyl-p-phenylenediamine (TMPD). When ascorbate was used as reductant, the V max and apparent K m values were 612 nmol electron min-1 (mg protein)-1 and 14 μM for yeast, and 419 nmol electron min-1 (mg protein)-1 and 19 μM for equine cytochrome c, respectively. For TMPD oxidation, the V max and K m values were 640 nmol electron min-1 (mg protein)-1 and 182 μM, respectively. These oxidase activities showed a high affinity for oxygen. Inhibition of both cytochrome-c and TMPD oxidase activities by 50% was caused by about 4 μM cyanide and about 0.5 mM azide. Redox difference spectra of the membrane solubilized with Triton X-100 showed b- or c-type cytochromes but not aa 3-type cytochromes. c-type and a part of some b-type cytochromes were reduced with ascorbate plus TMPD. A CO difference spectrum revealed that protohaem, but not an aa 3-type cytochrome, may be interacting with CO/oxygen. Only protohaem was detected in the haem fraction extracted from the membrane. Three polypeptides (60, 38 and 29 kDa) were found to be bearing haem c after SDS-PAGE of the membrane. From these results, it was suggested that the cbb 3-type cytochrome-c oxidase, having a haem-copper binuclear centre like the cytochrome aa 3-type oxidase, but differing in a few other properties, functions as a terminal oxidase in the respiratory chain of H. pylori.

Cell surface hydrophobicity (CSH) of Aspergillus fumigatus grown both in complex medium (yeast extract/peptone/dextrose; YPD) and minimal (Vogel's N) medium was monitored by assessing attachment of polystyrene microspheres to the cell surface. It was found that mature mycelium was hydrophobic. Treatment of intact mycelium with β-mercaptoethanol (βME) abolished binding of the microspheres to hyphal elements, and coating of the microspheres with βME extracts from mycelium inhibited their attachment to intact mycelial cells. A. fumigatus mycelium was tagged in vivo with biotin and treated with βME. The βME extracts were analysed by SDS-PAGE and Western blotting with both peroxidase-conjugated-ExtrAvidin and concanavalin A (ConA). This procedure allowed identification of cell wall surface proteins and glycoproteins. Rabbit polyclonal antisera were raised against βME extracts obtained from cells grown in YPD and Vogel's N media. These antisera defined some major cell-wall-bound antigens. SDS-PAGE and Western blotting analysis of the cell wall material released by βME and adsorbed on polystyrene microspheres revealed about 19 protein species with apparent molecular masses ranging from 20 to 70 kDa, and two high-molecular-mass glycoproteins of 115 and 210 kDa. Treatment of cells grown in YPD, but not those grown in Vogel's N medium, with βME released a 55 kDa polypeptide able to adsorb to polystyrene microspheres that was detectable with the antisera. The ability to bind to polystyrene particles exhibited by several protein and glycoprotein species released by βME treatment suggested that these cell wall moieties possess exposed hydrophobic domains that could be responsible for the CSH of mycelium.

Information on the molecular determinants of receptor recognition, membrane insertion and toxin pore-formation was sought by making 42 single and multiple substitutions of residues 312-314 (GYY), 367-370 (YRRP) and 438-441 (SGFS) in the Bacillus thuringiensis insecticidal CrylAc δ-endotoxin by site-directed mutagenesis. These three regions correspond to three putative surface-exposed loops (loops 1, 2 and 3, respectively) in domain II of the δ-endotoxin, forming the molecular apex of the structure. All except mutants GFY (loop 1), YKRA, SRRA, YRKA (loop 2) and TGFS (loop 3) expressed δ-endotoxin protein at wild-type levels which was stable upon activation by Pieris brassicae gut extract or trypsin. Toxicity assays for all the fully stable mutants using Manduca sexta larvae showed that G312, Y367, R368, R369, S438 and G439 are important for activity. Wild-type toxin was then labelled in vivo with [35S]methionine and heterologous competition binding assays were carried out for all the mutants using brush border membrane vesicles prepared from Manduca sexta midgut. Most and least conservative mutations of G439 and least conservative substitutions of Y367, R368 and R369 reduced the ability of the toxin to bind competitively. The most conservative mutation, S441T, gave significantly increased binding. These results suggested that these four residues play a role in the initial receptor binding step in the toxin mechanism. As no significant effect on binding affinity was observed in relatively non-toxic mutants in which residues G312 and S438 were mutated, we suggest that these residues are involved in the subsequent steps of membrane insertion and pore-formation.

The expression of the Bacillus subtilis lysC operon, which encodes the first specific enzyme of lysine biosynthesis, is controlled by the availability of the end product, lysine. The question of whether lysine exerts its control by inducing premature termination of transcription was addressed using Northern blot analysis. Whereas lys-C-specific RNA from lysine-starved B. subtilis consisted primarily of the expected full-length mRNA (1.6 kb), that from bacteria grown with an excess of lysine consisted of a truncated 0.27 kb RNA in place of the full-length 1.6 kb transcript. On the other hand, a B. subtilis aecA mutant, in which the lysC operon was derepressed owing to a single nucleotide substitution in the region corresponding to the lysC leader transcript, produced full-length lysC mRNA, but no 0.27 kb RNA, even during growth with excess lysine. Mapping of the truncated 0.27 kb lysC RNA by hybridization with oligonucleotide probes showed that it corresponded to the upstream portion of the lysC leader transcript, extending from the transcription initiation site to a putative rho-independent terminator element. Quantitative transcript analysis by hybridization with specific oligonucleotides showed that lysine did not affect the number of lysC-specific RNA molecules but promoted the stoichiometric replacement of full-length mRNA with truncated 0.27 kb molecules. These results indicate that lysine regulates the expression of the lysC operon by effecting the premature termination of transcription at a rho-independent terminator site in the lysC leader region and that the site of the aecA mutation, far upstream of the putative terminator element, must play an essential role in premature transcription termination by a mechanism which is not yet understood.

A small-scale functional analysis screen has revealed several new phenotypes associated with a large deletion of GDH3, one of two Saccharomyces cerevisiae genes known to encode NADP-linked glutamate dehydrogenase. Diploids heterozygous for the deletion are able to sporulate in rich media, while haploid deletants produce dark, wrinkled colonies containing pseudohyphal cells. The haploid cells rapidly lose viability upon starvation.

The induction of the heat shock response in Lactococcus lactis subsp. cremoris strain MG1363 was analysed at the RNA level using a novel RNA isolation procedure to prevent degradation. Cloning of the dnaJ and groEL homologues was carried out. Northern blot analysis showed a similar induction pattern for dnaK, dnaJ and groELS after transfer from 30 0C to 43 0C when MG1363 was grown in defined medium. The dnaK gene showed a 100-fold induction level 15 min after temperature shifting. Induction of the first two genes in the dnaK operon, orf1 and grpE, resembled the pattern observed for the above genes, although maximum induction was observed earlier for orf1 and grpE. Novel transcript sizes were detected in heat-shocked cells. The induction kinetics observed for ftsH suggested a different regulation for this gene. Experimental evidence for a pronounced transcriptional regulation being involved in the heat shock response in L. lactis MG1363 is presented. A gene located downstream of the dnaK operon in strain MG1363, named orf4, was shown not to be regulated by heat shock.

Homologues of the plasmid replicator gene repC were detected and characterized in a sample of Rhizobium leguminosarum strains. Conserved PCR primers were designed from published sequences of repC; they amplified a fragment of about 750 bp from 39 out of 41 strains tested, and also from several Sinorhizobium strains, including S. meliloti. Restriction endonuclease digestion showed that the PCR product from individual strains, though uniform in size, was often heterogeneous in sequence. PCR products from 24 field isolates of R. leguminosarum from France, Germany and the UK were cloned and partially sequenced from both ends. Phylogenies constructed from the 5′ and 3′ ends (200 bp each) were largely congruent and demonstrated four clearly defined groups plus several unique strains. Published Agrobacterium repC sequences fall within the phylogeny of R. leguminosarum sequences, though not within any of the four groups. Specific pairs of PCR primers were designed for each of the four groups; 29 out of 41 R. leguminosarum strains gave a PCR product of the expected size with more than one group-specific primer pair. We hypothesize that the sequence groups correspond to incompatibility groups of Rhizobium plasmids.

The transcription of the 14 gvp genes of the gas-vesicle-encoding mc-vac region was investigated, using RNA from 25% and 15% (w/v) salt cultures of the moderately halophilic archaeon Haloferax mediterranei. Transcription occurred only from two promoters, located in front of the mc-gvpA and mc-gvpD genes. In both cultures transcripts spanning the entire mc-gvpDEFGHIJKLM transcription unit were formed only during the exponential growth phase. Amounts of these transcripts were larger in the 25% salt culture, in which the 2.0 kb mc-gvpD mRNA and large amounts of 1.3 kb and 0.45 kb partial mc-gvpD transcripts were also synthesized during the stationary phase. The levels of the mc-gvpD transcripts and of the 324 nt mc-gvpA mRNA increased in parallel during the stationary phase of the 25% salt culture. Only under these conditions were mRNAs spanning the entire mc-gvpACNO transcription unit observed, and gas-vesicles were formed. Investigation of the influence of the mc-gvpDE genes on both mc-vac promoters in transformants revealed that by themselves they were nearly inactive. The addition of mc-gvpE, however, resulted in a high level of constitutively produced mc-gvpA and mc-gvpD mRNA, indicating a transcriptional activator function for the mc-gvpE product.

In Escherichia coli, the MetR and PurR proteins positively and negatively regulate glyA gene expression, respectively. A DNase I footprint analysis showed that both proteins bind independently to the glyA control region. The PurR protein blocks RNA polymerase (RNAP) from binding to the glyA promoter. The presence of hypoxanthine, the co-repressor of PurR, increases the ability of PurR to prevent RNAP binding, providing a model for repression of the glyA gene by PurR. In contrast, MetR alters the RNAP footprint pattern of the glyA control region. In addition, the MetR footprint is increased in the presence of RNAP, suggesting that the two proteins might interact.

Analysis of spontaneous mutations in Acinetobacter calcoaceticus revealed a 1237 bp insertion sequence named IS 1236 and possessing a nucleotide sequence resembling those of members of the IS3 family. The chromosome of A. calcoaceticus strain ADP1 contains seven copies of IS 1236 which appears to insert preferentially into pobR, the transcriptional activator of the structural gene for p-hydroxybenzoate hydroxylase. IS 1236 creates tandem 3 bp DNA duplications flanking the sites of its insertion in pobR. Different duplication patterns are found following insertion of IS 1236 into pcaH, a structural gene for protocatechuate 3,4-dioxygenase. Therefore the insertion properties of IS 1236 appear to be influenced by its DNA target. Amino acid sequences associated with the apparent transposase function have been conserved in ORFB of IS 1236 whereas the presumed DNA-binding helix-turn-helix region of IS 1236 ORFA exhibits substantial amino acid sequence divergence from its IS3 counterparts. IS 1236 ORFA and ORFB coding sequences overlap considerably, and sequence evidence indicates mechanisms for ORFB expression in IS 1236 may resemble those employed by other members of the IS3 family. Portions of the IS 1236 terminal repeats exhibit substantial sequence divergence from other members of the IS3 family, but evolution appears to have conserved a mechanism preventing expression of the insertion sequence genes as a consequence of transcriptional readthrough.