Abstract: Summary: A series of transposons are described which contain the gusA gene, encoding β-glucuronidase (GUS), expressed from a variety of promoters, both regulated and constitutive. The regulated promoters include the tac promoter which can be induced by IPTG, and nifH promoters which are symbiotically activated in legume nodules. One transposon contains gusA with a strong Shine-Dalgarno translation initiation context, but no promoter, and thus acts as a promoter-probe transposon. In addition, a gus operon deletion strain of Escherichia coli, and a transposon designed for use in chromosomal mapping using PFGE, are described. The GUS transposons are constructed in a mini-Tn5 system which can be transferred to Gram-negative bacteria by conjugation, and will form stable genomic insertions. Due to the absence of GUS activity in plants and many bacteria of economic importance, these transposons constitute powerful new tools for studying the ecology and population biology of bacteria in the environment and in association with plants, as well as for studies of the fundamental molecular basis of such interactions. The variety of assays available for GUS enable both quantitative assays and spatial localization of marked bacteria to be carried out.

Abstract: Scanning electron micrographs of the nematode-egg-parasitic fungus Paecilomyces lilacinus infecting eggs of the root-knot nematode Meloidogyne spp. suggested the involvement of lytic enzymes. When grown on a liquid mineral salts medium, supplemented with different substrates as the sole N-and C-source, the fungus produced an extracellular protease. Colloidal chitin, vitellin and intact eggs of the root-knot nematode Meloidogyne hapla induced proteolytic activity that was repressed by glucose. The protease was partially purified from the culture filtrate by affinity chromatography. It has a molecular mass of 33·5 kDa, a pH optimum of 10·3, a temperature optimum of 60°C and an isoelectric point above pH 10·2. The enzyme was completely inhibited by PMSF. The amino acid sequence, as derived from the nucleotide sequence of a cDNA clone, had high homology with several subtilisin-like serine proteases. It was shown that the purified enzyme degrades vitellin. The protease quantitatively bound to nematode eggs, and eggs incubated with the purified protease eventually floated. Incubation of the purified protease with nematode eggs significantly influenced their development as demonstrated by time-lapse microscopy. Immature eggs were highly vulnerable to protease treatments, whereas those containing a juvenile were more resistant. In addition, hatched larvae were not visibly affected by the protease. It can be concluded that the serine protease might play a role in penetration of the fungus through the egg-shell of nematodes.

Abstract: SUMMARY: Minimal requirements of amino acids and vitamins were determined in chemically defined medium for five strains of Clostridium difficile. Cysteine, isoleucine, leucine, proline, tryptophan and valine were essential amino acids for growth of C. difficile. Arginine, glycine, histidine, methionine and threonine enhanced growth. Biotin, pantothenate and pyridoxine were essential vitamins. A defined medium containing the minimal requirements of amino acids and vitamins produced a rapid and heavy growth which was comparable to that in modified brain heart infusion, a complex medium. Adenine was able to substitute for glycine and threonine, suggesting that the two amino acids may be utilized as precursors of purine nucleotides. The defined medium developed here will assist physiological and biochemical studies on C. difficile.

Abstract: Nucleotide sequencing of a region downstream from the Pseudomonas aeruginosa 1244 pilin structural gene, pilA, revealed an ORF potentially able to code for a protein of M(r) 50,862. This ORF, called pilO, was flanked by a tRNAthr gene, which was followed by a transcriptional termination sequence. The tRNAthr gene and the termination sequence were nearly identical to sequences found immediately adjacent to the pilA gene of several P. aeruginosa strains. A 2200 base mRNA strand, which contained both the pilO and pilA transcripts, was produced from this region, while a 650 base transcript containing only pilA was present in a 100-fold excess over the longer transcript. Hyperexpression of the pilA gene in a PilO- strain resulted in normal pilus-specific phage sensitivity and twitching motility. The pilin produced by this strain had a lower apparent M(r) and a more neutral pl compared to that produced by a strain containing a functional pilO gene. This pilin failed to react with a sugar-specific reagent which recognized pilin produced by the strain containing a functional pilO gene.

Abstract: Oligonucleotide sequences selected from the 16S rRNA genes of various species of ammonia-oxidizing bacteria were evaluated as specific PCR amplification primers and probes. The specificities of primer pairs for eubacterial, Nitrosospira and Nitrosomonas rRNA genes were established with sequence databases, and the primer pairs were used to amplify DNA from laboratory cultures and environmental samples. Eubacterial rRNA genes amplified from samples of soil and activated sludge hybridized with an oligonucleotide probe specific for Nitrosospira spp., but not with a Nitrosomonas-specific probe. Lakewater and sediment samples were analysed using a nested PCR technique in which eubacterial rRNA genes were subjected to a secondary amplification with Nitrosomonas or Nitrosospira specific primers. Again, the presence of Nitrosospira DNA, but not Nitrosomonas DNA, was detected and this was confirmed by hybridization of the amplified DNA with an internal oligonucleotide probe. Enrichments of lakewater and sediment samples, incubated for two weeks in the presence of ammonium, produced nitrite and were found to contain DNA from both Nitrosospira and Nitrosomonas as determined by nested PCR amplification and probing of 16S rRNA genes. This demonstrates that Nitrosospira spp. are widespread in the environment. The implications of the detection of Nitrosomonas DNA only after enrichment culture are discussed.

Abstract: The genes encoding the production of acidocin B, a bacteriocin produced by Lactobacillus acidophilus strain M46 which is active against Listeria monocytogenes, Clostridium sporogenes, Brochothrix thermosphacta, Lactobacillus fermentum and Lactobacillus delbrueckii subsp. bulgaricus, but inactive against most other Lactobacillus species, were previously localized on a 4 kb Xbal-HindIII fragment of plasmid pCV461. In the present work, DNA sequence analysis revealed the presence of three consecutive ORFs, which potentially code for hydrophobic peptides composed of 60, 91 and 114 amino acids, respectively, and a fourth ORF of opposite polarity which could potentially encode a peptide of 59 amino acids. The middle ORF (ORF-2; acdB) was identified as the gene encoding acidocin B by comparing the amino acid composition of highly purified acidocin B with the deduced amino acid sequence of ORF-2. Our results suggest that acidocin B is synthesized as a precursor which is processed at a site which conforms to the '-3, -1' rules of von Heijne to yield active acidocin B (59 amino acids). The presence of an immunity-protein-encoding gene on the 4 kb Xbal-BamHI fragment was deduced from the capacity of a plasmid vector harbouring this fragment to confer immunity upon transformation of L. fermentum NCK127. One of the three non-assigned ORFs may encode this immunity protein.

Abstract: A facultatively chemolithotrophic thiocyanate-degrading bacterium, strain THI 011T, which was previously isolated from activated sludge and tentatively named Thiobacillus sp., was studied taxonomically and phylogenetically. This bacterium utilizes thiocyanate as sole energy source and the specific growth rate for chemolithoautotrophic growth with thiocyanate was 0059 h-1. Molecular phylogenetic relationships of strain THI 011T to Thiobacillus versutus and members of the genus Paracoccus were elucidated by comparing 16S rRNA gene sequences. Binary sequence comparisons showed that strain THI 011T was most related to Paracoccus aminophilus, at a similarity level of 970%, and T. versutus was most similar to Paracoccus denitrificans, at a level of 991%. A neighbour-joining phylogenetic tree showed that strain THI 011T formed a cluster together with T. versutus and known species of the genus Paracoccus within the α-3 subclass of the Proteobacteria. DNA-DNA hybridization assays and phenotypic studies indicated that strain THI 011T differed from T. versutus and known species of the genus Paracoccus. On the basis of these results, we propose to classify strain THI 011T into a new species of the genus Paracoccus with the name Paracoccus thiocyanatus sp. nov. We also propose to transfer T. versutus to the genus Paracoccus and present an emended description of the genus.

Abstract: SUMMARY: 16S rDNA analysis was performed on 32 strains of 26 species of the genera Rhodococcus and Nocardia in order to investigate the phylogenetic structure of these genera within the radiation of other mycolic-acid-containing genera such as Corynebacterium, Dietzia, Gordona, Mycobacterium and Tsukamurella. The genus Rhodococcus shows a complex structure, consisting of six phylogenetically equidistant lineages. The genus Nocardia does not appear to be a sister taxon of Rhodococcus but branches off from within the radiation of Rhodococcus; thus its species can be considered to be derived from a Rhodococcus ancestor. The main known phenotypic feature that separates Nocardia species from those of Rhodococcus appears to be the presence of a cyclic component in menaquinone of the MK-8(H4) type.

Abstract: Increasing the expression of various glycolytic operons in Zymomonas mobilis caused a significant decrease rather than increase in the glycolytic flux and growth rate. Because the relative decrease depended on the amount of overexpressed protein, and was independent of which enzyme was overexpressed, we attributed it to a protein burden effect. More specifically, we examined if the decrease in glycolytic flux could be explained by a decreased concentration of other glycolytic enzymes (for which glucokinase was used as a marker enzyme). Using the summation theorem of metabolic control theory we predicted the extent of this protein burden effect. The predictions were in good agreement with the experimental observations. This suggests that the negative flux control is caused either by a simple competition of the overexpressed gene with the expression of all other genes or by simple dilution. Furthermore, we determined the implications of protein burden for the determination of the extent to which an enzyme limits a flux. We conclude that a protein burden can cause a significant underestimation of the flux control coefficient, especially if the enzyme under investigation is a highly expressed enzyme.

Abstract: Copper- and zinc-containing superoxide dismutases ([Cu,Zn]-SODs) are generally considered almost exclusively eukaryotic enzymes, protecting the cytosol and extracellular compartments of higher organisms from damage by oxygen free-radicals. The recent description of a few examples of bacterial forms of the enzyme, located in the periplasm of different Gram-negative micro-organisms, prompted a re-evaluation of this general perception. A PCR-based approach has been developed and used successfully to identify bacterial genes encoding [Cu,Zn]-SOD in a wide range of important human and animal pathogens - members of the Haemophilus, Actinobacillus and Pasteurella (HAP) group, and Neisseria meningitidis. Comparison of [Cu,Zn]-SOD peptide sequences found in Haemophilus ducreyi, Actinobacillus pleuropneumoniae, Actinobacillus actinomycetemcomitans, Pasteurella multocida, and N. meningitidis with previously described bacterial proteins and examples of eukaryotic [Cu,Zn]-SOD has shown that the bacterial proteins constitute a distinct family apparently widely separated in evolutionary terms from the eukaryotic examples. The widespread occurrence of [Cu,Zn]-SOD in the periplasm of bacterial pathogens, appropriately located to dismute exogenously derived superoxide radical anions, suggests that this enzyme may play a role in the interactive biology of organisms with their hosts and so contribute to their capacity to cause disease.

Abstract: A major limitation of rRNA-targeted group-specific probes is that they may cross-react with organisms of other physiological, or even phylogenetic groups when applied to environmental samples containing unknown sequences We have exploited the restricted physiology of methane-oxidizing bacteria to assess the specificity and efficiency of probes for this physiological type which target the 16S rRNA or genes involved in methanotroph physiology Seawater samples were enriched for methanotrophs by addition of methane and essential nutrients The changes in composition of the bacterial population were monitored by analysis of 16S rRNA gene libraries Methanotroph group-specific probes failed to give a signal with samples from these enrichments even though a methanol dehydrogenase structural gene was detected A 16S rDNA sequence that was abundant only after methane addition was recovered and found to show a close phylogenetic relationship to Methylomonas Organisms containing this sequence were observed in enrichments by in situ hybridization The combination of enrichment on methane and screening with the broad specificity methanol dehydrogenase probe allowed detection of novel methanotrophs that were not detected with the original suite of methanotroph group-specific probes

Abstract: The growth of Listeria monocytogenes ATCC 23074 in defined medium is sensitive to high osmolarity when compared with its growth in complex media, such as brain heart infusion (BHI). The two major contributors to this difference in growth rate are the availability in BHI of the osmoprotectant glycine betaine and peptides. Peptone plays two major roles: firstly as a nutritional supplement for protein synthesis, and secondly as a source of amino acids and peptides that serve as a mechanism of maintaining turgor. In the presence of peptone the total amino acid pool at high osmolarity is substantial and even in the presence of glycine betaine the amino acid pool makes a major contribution to turgor maintenance. At high osmolarity there is a general increase in amino acid pools, with particularly substantial pools of glutamate, aspartate, proline, hydroxyproline and glycine. Peptides are also accumulated by cells from the peptone supplied in the medium. Glycine-containing peptides are accumulated in the cytoplasm under all conditions. Specific glycine- and proline-containing peptides stimulate growth at high osmolarity. The peptide prolyl-hydroxyproline accumulates in cells to high levels in response to growth at high osmolarity, and the pools of the derived amino acids also show a dependence on the external osmotic pressure. However, proline only confers significant osmoprotection when supplied as peptides. The significance of these data in the context of the occurrence of L. monocytogenes in foods with high peptide content is discussed.

Abstract: SUMMARY: Vibrio cholerae cells were incubated at 4 °C in nutrient-limited artificial seawater (ASW) microcosms. Plate counts declined from 8 × 105 to less than 2 c.f.u. ml−1 in about 23 d. When samples of microcosms were shifted to 30 °C, plate counts increased to 2-2 × 105 c.f.u. ml−1 in 72 h. An experiment was performed to determine whether culturable cells obtained after temperature upshifts were the result of “resuscitation”, or outgrowth, of nonculturable cells or of cell division and growth of the few culturable cells that remained in samples. Prior to temperature upshift, samples from the microcosms were diluted 10- and 100-fold in filter-sterilized (0-1 μm) ASW from the microcosms. Undiluted, 1/10, and 1/100 diluted samples recovered culturability to about 2-2 × 105 c.f.u. ml−1 within 72 h of temperature upshift. If resuscitation of nonculturable cells had occurred, the resultant number of culturable cells in diluted samples would have been 1/10 and 1/100 that of undiluted samples, respectively. In microcosms where plate counts had declined to less than 1 c.f.u. ml−1, 1/100 diluted samples did not regain culturability, i.e. no culturable cells remained from which growth could occur. Our conclusions are that in the experiments reported here, recovery of culturable cells on temperature upshifts resulted from growth and that there were no growthinhibiting factors in the spent growth medium, supported by the finding that about 102 recovered V. cholerae cells ml−1 inoculated into filter-sterilized microcosm ASW grew to about 6-2 × 105 c.f.u. ml−1 in 24 h, confirming that V. cholerae is capable of significant growth in ASW.

Abstract: SUMMARY To develop a rapid and accurate method of typing large numbers of clinical isolates of Staphylococcus aureus, the spacer region C of the rRNA operon [1391-507 (16S-23S)] was enzymically amplified from 322 strains. When the products were separated by denaturing PAGE, 15 variable-length rrn alleles were demonstrated, ranging in size from 906 to 1223 bp. The variable-length HpaII-digested region C [(region E; 1446-196 (16S-23S)] amplification products were cloned into M13mp18RF to sequence separate variable-length alleles. A total of 17 region E inserts were sequenced, aligned and divided into nine alleles by length (938-1174) and sequence properties. The 16S-23S spacer rDNA varied in length (303-551 bp) and in properties; three alleles contained a tRNAlle gene alone, two alleles contained a tRNAlle and a tRNAAla gene, and four alleles lacked tRNA genes. The sequences of two alleles showed less than 1% variation when isolated from two or three S. aureus strains. The 48 penicillin-and methicillin-sensitive strains were divided into 26 ribotypes; in contrast, the 274 methicillin-resistant S. aureus (MRSA) strains were divided into nine ribotypes (A-I) with 97% typing as either ribotype A or B (rrnL was missing in B). The sequence conservation of the rrn operons argues for the use of the 16S-23S spacer region as a stable and direct indicator of the evolutionary divergence of S. aureus strains.

Abstract: To facilitate the differential identification of the genus Streptomyces, the 16S rRNA genes of 17 actinomycetes were sequenced and screened for the existence of streptomycete-specific signatures The 16S rDNA of the Streptomyces strains and Amycolatopsis orientalis subsp lurida exhibited 95-100% similarity, while that of the 16S rDNA of Actinoplanes utahensis showed only 88% similarity to the streptomycete 16S rDNAs Potential genus-specific sequences were found in regions located around nucleotide positions 120, 800 and 1100 Several sets of primers derived from these characteristic regions were investigated as to their specificity in PCR-mediated amplifications Most sets allowed selective amplification of the streptomycete rDNA sequences studied RFLPs in the 16S rDNA permitted all strains to be distinguished

Abstract: Summary: The induction of chitinolytic enzymes in the biocontrol agent Trichoderma harzianum during parasitism on Sclerotium rolfsii and the role of fungal-fungal recognition in this process were studied. A change in the chitinolytic enzyme profile was detected during the interaction between the fungi, grown in dual culture on synthetic medium. Before coming into contact with each other, both fungi contained a protein with constitutive 1,4-†bT-N-acetylglucosaminidase activity. As early as 12 h after contact, the chitinolytic activity in S. rolfsii disappeared, while that of T. harzianum (a protein with a molecular mass of 102 kDa, CHIT 102) greatly increased. After 24 h of interaction, the activity of CHIT 102 diminished concomitantly with the appearance of a 73 kDa 1,4-β-N-acetylglucosaminidase, which became clear and strong at 48 h. This phenomenon did not occur if the S. rolfsii mycelium was autoclaved prior to incubation with T. harzianum, suggesting its dependence on vital elements from the host. Cycloheximide inhibited this phenomenon, indicating that de novo synthesis of enzymes is taking place in Trichoderma during these stages of the parasitism. A biomimetic system based on the binding of a purified surface lectin from the host S. rolfsii to nylon fibres was used to dissect the effect of recognition. An increase in CHIT 102 activity was detected, suggesting that the induction of chitinolytic enzymes in Trichoderma is an early event which is elicited by the recognition signal (i.e. lectin-carbohydrate interactions). It is postulated that recognition is the first step in a cascade of antagonistic events which triggers the parasitic response in Trichoderma.

Abstract: A gene encoding catalase (hydrogen-peroxide: hydrogen-peroxide oxidoreductase; EC 1.11.1.6) from Campylobacter jejuni was cloned by functional complementation of a catalase-deficient mutant of Escherichia coli. The catalase structural gene, designated katA, was assigned by subcloning and its nucleotide sequence determined. The deduced protein product of 508 amino acids, which had a calculated molecular mass of 58346 Da, was found to be structurally and enzymically similar to hydrogen-peroxidases from other bacterial species. The region of DNA containing the structural catalase gene was disrupted by insertion of a tetracycline-resistance marker and the modified sequence then introduced into a strain of Campylobacter coli via natural transformation. Genetic and enzymic analyses of a tetracycline-resistant C. coli transformant confirmed that catalase-deficient mutants had arisen via interspecific allelic exchange. Compared to the isogenic parental strain the mutant was more sensitive to killing by H2O2.

Abstract: Summary: We have cloned and expressed the gp63 gene of Leishmania major in BCG to develop a recombinant vaccine against zoonotic cutaneous leishmaniasis. Two different expression systems were investigated. The first system consists of pAN, a Mycobacterium paratuberculosis promoter, which drives expression of ORF2, an open reading frame in IS900. This system allows the production of heterologous polypeptides as hybrids with the ORF2 gene product. The second expression system relies on the production of antigenic fragments as fusion proteins with the N-terminal region of Mycobacterium fortuitum β-lactamase. Both constructs resulted in the production of Gp63 in BCG. The ability of the two recombinant BCG strains to induce protective immunity against a challenge with L. major amastigotes was evaluated after vaccination of susceptible (BALB/c), and resistant (C57BL/6) mice. Recombinant BCG producing Gp63 as a hybrid protein with the N-terminal region of the β-lactamase elicited significant protection against a challenge with L. major in BALB/c-immunized mice.

Abstract: The fliD genes of Salmonella typhimurium and Escherichia coli encode the filament-cap protein of the flagellar apparatus, which facilitates the polymerization of endogenous flagellin at the tips of the growing filaments. Previous sequence analysis of this operon in both organisms has revealed that the fliD gene constitutes an operon together with two additional genes, fliS and fliT. Based on the gene-disruption experiment in E. coli, both the fliS and fliT genes have been postulated to be necessary for flagellation. In the present study, we constructed S. typhimurium mutants in which either fliS or fliT on the chromosome was specifically disrupted. Both mutants were found to produce functional flagella, indicating that these genes are dispensable for motility development in S. typhimurium. However, flagellar filaments produced by the fliS mutant were much shorter than those produced by the wild-type strain. This indicates that the fliS mutation affects the elongation step of filament assembly. The excretion efficiency of flagellin was examined in the fliD-mutant background, where the exported flagellin molecules cannot assemble onto the hooks, resulting in their excretion into the culture media. We found that the amount of flagellin excreted was much reduced by the fliS mutation. Based on these results, we conclude that FliS facilitates the export of flagellin through the flagellum-specific export pathway.

Abstract: pUS933, a bifunctional Mycobacterium-Escherichia coli translational fusion vector containing an amino-terminally truncated E. coli lacZ reporter gene, was constructed. Derivatives of pUS933, containing the promoter, RBS and start codon of the Mycobacterium bovis BCG hsp60 gene, the Mycobacterium leprae 28 kDa gene and the M. leprae 18 kDa gene were constructed and introduced into E. coli, Mycobacterium smegmatis and M. bovis BCG. β-Galactosidase activity was measured for mycobacteria grown in liquid culture. Primerextension analysis was used to determine the transcriptional start point for the 18 kDa promoter in M. smegmatis. Murine macrophages were infected with recombinant BCG containing the pUS933 derivatives and expression levels were examined, by fluorescence microscopy and fluorometry, during intracellular growth of BCG. Both the BCG hsp60 gene promoter and the M. leprae 28 kDa gene promoter gave high levels of β-galactosidase expression in all situations examined. In contrast, the M. leprae 18 kDa promoter fragment gave very low levels of expression in M. smegmatis and BCG grown in liquid culture, but in BCG growing within macrophages it was induced to levels almost as high as the other promoters. This indicated that the 18 kDa gene is specifically activated during intracellular growth and may therefore be involved in survival of M. leprae within macrophages. This pattern of regulation may be useful for controlling expression of foreign genes in recombinant BCG strains.

Abstract: Colonization of the oral cavity by Candida albicans involves adherence of yeast cells to oral surfaces. An assay was developed to measure the attachment of C. albicans cells, metabolically labelled with [35S]methionine, to saliva-coated hydroxylapatite (SHA) beads--a model for the tooth surface. Using this assay approximately 1.26 x 10(6) C. albicans cells (50.5% of input cells) attached to the SHA beads (12 mg). Different strains of C. albicans adhered to varying degrees to SHA beads, but in general adherence was promoted by growth of cells at 28 degrees C and by starvation of cells for glucose. Proteins in human whole, or parotid, saliva samples were fractionated by gel filtration (Sephacryl S-200 column) and fractions were adsorbed to hydroxylapatite beads. Fractions that contained proline-rich proteins or statherin promoted attachment of C. albicans ATCC 10261 cells. Neuraminidase treatment of SHA beads, but not of cells, significantly increased yeast cell binding to the SHA beads. Neuraminidase activity in the oral cavity may unmask glycoprotein receptors involved in the adhesion of C. albicans to saliva-coated surfaces.

Abstract: Siderophore-mediated iron uptake systems play a central role in the pathogenesis of infection for many bacterial pathogens. Campylobacter species are not thought to produce siderophores, yet they are able to utilize both ferrichrome and enterochelin as sources of iron. Part of an operon named ceuBCDE, encoding components of a periplasmic binding-protein-dependent transport (PBT) system for the uptake of a ferric siderophore from Campylobacter coli, was cloned directly into Escherichia coli using a plasmid rescue technique. Phenotypic and genetic analyses of this system showed it to comprise two hydrophobic integral membrane proteins, CeuB (35-5 kDa) and CeuC (34-8 kDa), which may form the cytoplasmic membrane permease, an ATP-binding protein, CeuD (28-8 kDa), and a periplasmic substrate-binding protein, CeuE (34-5 kDa). In vivo labelling studies using [3H]palmitate demonstrated that CeuE, the periplasmic binding protein, is expressed as a lipoprotein in C. coli, which is unusual for a Gram-negative PBT system. Mutants of C. coli, defective in components of the transport mechanism, were severely impaired in the ability to utilize enterochelin as an iron source suggesting that this siderophore is a substrate for the transport system. This is the first molecular characterization of a PBT system in Campylobacter species.

Abstract: SUMMARY: The nucleotide sequence of the 3-chlorobenzoate 3,4-dioxygenase genes, designated cbaAB,from the transposon Tn5271 was determined. The function of the two sequenced open reading frames was evaluated by mutagenesis and expressionin vivoto show that thecbaAandcbaBgenes code for dioxygenase and reductase proteins, respectively. Comparison of the deduced amino acid sequences of thecbaAB genes with sequences for other oxygenases revealed a clearly defined lineage among the class IA oxygenases that shows several unique features. This lineage includes phthalate 4,5-dioxygenase (pht23), and based on the available NH3-terminal sequence of component A, also includes 4-sulphobenzoate 3,4-dioxygenase. Vanillate demethylase, encoded by the vanAB genes and formally a monooxygenase enzyme catalysing an oxidative demethylation, is also included in this lineage. The terminal chlorobenzoate dioxygenase (CbaA) component is characterized by a conserved Rieske-type [2Fe-2S]R ligand centre. The reductase component (CbaB) contains a plant-type ferredoxin [2Fe-2S]Fd” FMN-isoalloxazine and NAD-ribose-binding domains and the orientation of these domains is conserved in all known class IA reductases. These results support the hypothesis that alternative fusions of the electron transfer modules of the reductases arose early in the divergence of oxygenase systems. The over-riding evolutionary constraint acting on the divergence of the class IA oxygenases would appear to be the requirement for a carboxyl group para to the site of oxygen insertion into the aromatic ring.

Abstract: Summary: Because of their potential usefulness as natural food preservatives, increased interest has focused on bacteriocins from lactic acid bacteria. Mesentericin Y105 is a small non-lantibiotic bacteriocin (class II) encoded within a 35 kb plasmid from Leuconostoc mesenteroides Y105 and it is active against Listeria monocytogenes. Using reverse genetic methodologies, an 8 kb Drall fragment has been cloned that contains the mesentericin Y105 structural gene, mesY, which encodes a precursor of the bacteriocin with a 24 amino acid N-terminal extension ending with a Gly-Gly motif upstream of the cleavage site, which is typical of class II bacteriocins. Four other putative genes are associated with mesY within two divergent putative operons. In addition to mesY, the first putative operon is predicted to encode a protein, similar to that encoded by ORF2 in the leucocin A operon, whose function remains to be elucidated. The second putative operon contains three ORFs, two of which, mesD and mesE, encode proteins that resemble ATP-dependent transporters and accessory factors, respectively. For three other class II bacteriocin systems (lactococcin A, pediocin PA-1, colicin V), these proteins have been shown to be involved in bacteriocin secretion independently of the general sec-dependent secretion pathway. The last putative gene (mesC) does not resemble any previously characterized gene. Results concerning the heterologous expression of the cloned mesY in Lactobacillus johnsonii NCK64 suggest that the maturation and secretion functions dedicated to lactacin F (another class II bacteriocin) are efficient for mesentericin Y105 as well. This characteristic may be of great interest in the development of industrial fermentation starters producing multiple bactericidal activities.

Abstract: The reduction of exogenous ferric iron by Listeria monocytogenes, a Gram-positive food-borne pathogen, was investigated. Using an assay incorporating the ferrous iron chelator ferrozine, we showed that intact cells of L. monocytogenes, when exposed to ferric iron, were able to rapidly reduce and solubilize the iron to the ferrous form. Reduction occurred only after direct contact between the bacteria and the iron source. A number of different ferric iron chelates, including transferrin and lactoferrin-bound iron, haemoglobin, ferritin, and iron complexed to siderophores, could be reduced. The ferric reductase activity was expressed by both reference strains and clinical isolates of L. monocytogenes and by all other species of Listeria, although significant quantitative differences were observed. In L. monocytogenes, the expression of ferric reductase was not affected by the growth phase of the bacteria nor by the presence or absence of iron in the growth medium. However, expression was greatly reduced in bacteria grown anaerobically and when cultured in media of reduced pH. In addition, bacteria grown at a cold temperature displayed greater ferric reductase activity than cells grown at higher temperatures. A surface-associated ferric reductase system may be one component of a general iron scavenging mechanism which can be used by Listeria growing in a variety of environments.

Abstract: SUMMARY: The relationship between genes and enzymes in the methionine biosynthetic pathway has been studied in Pseudomonas aeruginosa. The first step is catalysed by an O-succinylhomoserine synthase, the product of the metA gene mapped at 20 min on the chromosome. The second step is achieved by direct sulfhydrylation, involving the enzyme encoded by a metZ gene that we have identified and sequenced, located at 40 min. Thus Pseudomonas appears to be the only organism so far described that uses O-succinylhomoserine as substrate for a direct sulfhydrylation. As in yeast, the two transsulfuration pathways between cysteine and homocysteine, with cystathionine as an intermediate, probably exist in parallel in this organism.

Abstract: SUMMARY: A gene encoding a chitinase from Serratia marcescens BJL200 was cloned and expressed in Escherichia coli and S. marcescens. Nucleotide sequencing revealed an open reading frame encoding a 55·5 kDa protein of 499 amino acids without a typical signal peptide for export. The cellular localization of the chitinase was studied, using two types of cell fractionation methods and immunocytochemical techniques. These analyses showed that the chitinase is located in the cytoplasm in E. coli, whereas it is exported to the periplasm in S. marcescens. Analysis of chitinase isolated from periplasmic fractions of S. marcescens carrying the cloned gene showed that export of the enzyme is not accompanied by processing at the N-terminus. The chitinase did not show any of the characteristics that have been proposed to direct the export of other non-processed extracellular proteins such as the E. coli haemolysin and might therefore be secreted via a hitherto unknown mechanism.